Heavy Metals | S |
31
HEAVY METALS
HEAVY METALS This test is provided to demonstrate that the content of metallic impurities that are colored by sulfide ion, under the specified test conditions, does not exceed the Heavy metals limit specified in the individual monograph in percentage (by weight) of lead in the test substance, as determined by concomitant visual comparison (see Visual Comparison in the section Procedure under Spectrophotometry and Light-Scattering 
851) with a control prepared from a Standard Lead Solution. [NOTE—Substances that typically will respond to this test are lead, mercury, bismuth, arsenic, antimony, tin, cadmium, silver, copper, and molybdenum.]
851) with a control prepared from a Standard Lead Solution. [NOTE—Substances that typically will respond to this test are lead, mercury, bismuth, arsenic, antimony, tin, cadmium, silver, copper, and molybdenum.] Determine the amount of heavy metals by Method I, unless otherwise specified in the individual monograph. Method I is used for substances that yield clear, colorless preparations under the specified test conditions. Method II is used for substances that do not yield clear, colorless preparations under the test conditions specified for Method I, or for substances that, by virtue of their complex nature, interfere with the precipitation of metals by sulfide ion, or for fixed and volatile oils. Method III, a wet-digestion method, is used only in those cases where neither Method I nor Method II can be used.
Special Reagents
Lead Nitrate Stock Solution— Dissolve 159.8 mg of lead nitrate in 100 mL of water to which has been added 1 mL of nitric acid, then dilute with water to 1000 mL. Prepare and store this solution in glass containers free from soluble lead salts.
Standard Lead Solution— On the day of use, dilute 10.0 mL of Lead Nitrate Stock Solution with water to 100.0 mL. Each mL of Standard Lead Solution contains the equivalent of 10 µg of lead. A comparison solution prepared on the basis of 100 µL of Standard Lead Solution per g of substance being tested contains the equivalent of 1 part of lead per million parts of substance being tested.
Method I
pH 3.5 Acetate Buffer— Dissolve 25.0 g of ammonium acetate in 25 mL of water, and add 38.0 mL of 6 N hydrochloric acid. Adjust, if necessary, with 6 N ammonium hydroxide or 6 N hydrochloric acid to a pH of 3.5, dilute with water to 100 mL, and mix.
Standard Preparation— Into a 50-mL color-comparison tube pipet 2 mL of Standard Lead Solution (20 µg of Pb), and dilute with water to 25 mL. Using a pH meter or short-range pH indicator paper as external indicator, adjust with 1 N acetic acid or 6 N ammonium hydroxide to a pH between 3.0 and 4.0, dilute with water to 40 mL, and mix.
Test Preparation— Into a 50-mL color-comparison tube place 25 mL of the solution prepared for the test as directed in the individual monograph; or, using the designated volume of acid where specified in the individual monograph, dissolve in and dilute with water to 25 mL the quantity, in g, of the substance to be tested, as calculated by the formula:
2.0/(1000L),
in which L is the Heavy metals limit, as a percentage. Using a pH meter or short-range pH indicator paper as external indicator, adjust with 1 N acetic acid or 6 N ammonium hydroxide to a pH between 3.0 and 4.0, dilute with water to 40 mL, and mix. Monitor Preparation— Into a third 50-mL color-comparison tube place 25 mL of a solution prepared as directed for Test Preparation, and add 2.0 mL of Standard Lead Solution. Using a pH meter or short-range pH indicator paper as external indicator, adjust with 1 N acetic acid or 6 N ammonium hydroxide to a pH between 3.0 and 4.0, dilute with water to 40 mL, and mix.
Procedure— To each of the three tubes containing the Standard Preparation, the Test Preparation, and the Monitor Preparation, add 2 mL of pH 3.5 Acetate Buffer, then add 1.2 mL of thioacetamide–glycerin base TS, dilute with water to 50 mL, mix, allow to stand for 2 minutes, and view downward over a white surface *: the color of the solution from the Test Preparation is not darker than that of the solution from the Standard Preparation, and the color of the solution from the Monitor Preparation is equal to or darker than that of the solution from the Standard Preparation. [NOTE—If the color of the Monitor Preparation is lighter than that of the Standard Preparation, use Method II instead of Method I for the substance being tested.]
Method II
NOTE—This method does not recover mercury.
pH 3.5 Acetate Buffer— Prepare as directed under Method I.
Standard Preparation— Pipet 4 mL of the Standard Lead Solution into a suitable test tube, and add 10 mL of 6 N hydrochloric acid.
Test Preparation— Use a quantity, in g, of the substance to be tested as calculated by the formula: 
to 600, until the carbon is completely burned off (no longer than 2 hours). If carbon remains, allow the residue to cool, add a few drops of sulfuric acid, evaporate, and ignite again. Cool, add 5 mL of 6 N hydrochloric acid, cover, and digest on a steam bath for 10 minutes. Cool, and quantitatively transfer the solution to a test tube. Rinse the crucible with a second 5-mL portion of 6 N hydrochloric acid, and transfer the rinsing to the test tube.
4.0/(1000L),
in which L is the Heavy metals limit, as a percentage. Transfer the weighed quantity of the substance to a suitable crucible, add sufficient sulfuric acid to wet the substance, and carefully ignite at a low temperature until thoroughly charred. (The crucible may be loosely covered with a suitable lid during the charring.) Add to the carbonized mass 2 mL of nitric acid and 5 drops of sulfuric acid, and heat cautiously until white fumes no longer are evolved. Ignite, preferably in a muffle furnace, at 500 to 600, until the carbon is completely burned off (no longer than 2 hours). If carbon remains, allow the residue to cool, add a few drops of sulfuric acid, evaporate, and ignite again. Cool, add 5 mL of 6 N hydrochloric acid, cover, and digest on a steam bath for 10 minutes. Cool, and quantitatively transfer the solution to a test tube. Rinse the crucible with a second 5-mL portion of 6 N hydrochloric acid, and transfer the rinsing to the test tube. Monitor Preparation— Pipet 4 mL of the Standard Lead Solution into a crucible identical to that used for the Test Preparation and containing a quantity of the substance under test that is equal to 10% of the amount required for the Test Preparation. Evaporate on a steam bath to dryness. Ignite at the same time, in the same muffle furnace, and under the same conditions used for the Test Preparation. Cool, add 5 mL of 6 N hydrochloric acid, cover, and digest on a steam bath for 10 minutes. Cool, and quantitatively transfer to a test tube. Rinse the crucible with a second 5-mL portion of 6 N hydrochloric acid, and transfer the rinsing to the test tube.
Procedure— Adjust the solution in each of the tubes containing the Standard Preparation, the Test Preparation, and the Monitor Preparation with ammonium hydroxide, added cautiously and dropwise, to a pH of 9. Cool, and adjust with glacial acetic acid, added dropwise, to a pH of 8, then add 0.5 mL in excess. Using a pH meter or short-range pH indicator paper as external indicator, check the pH, and adjust, if necessary, with 1 N acetic acid or 6 N ammonium hydroxide to a pH between 3.0 and 4.0. Filter, if necessary, washing the filter with a few mL of water, into a 50-mL color-comparison tube, and then dilute with water to 40 mL. Add 2 mL of pH 3.5 Acetate Buffer, then add 1.2 mL of thioacetamide–glycerin base TS, dilute with water to 50 mL, mix, allow to stand for 2 minutes, and view downward over a white surface*: the color of the solution from the Test Preparation is not darker than that of the solution from the Standard Preparation, and the color of the solution from the Monitor Preparation is equal to or darker than that of the solution from the Standard Preparation. [NOTE—If the color of the solution from the Monitor Preparation is lighter than that of the solution from the Standard Preparation, proceed as directed for Method III for the substance being tested.]
Method III
pH 3.5 Acetate Buffer— Prepare as directed under Method I.
Standard Preparation— Transfer a mixture of 8 mL of sulfuric acid and 10 mL of nitric acid to a clean, dry, 100-mL Kjeldahl flask, and add a further volume of nitric acid equal to the incremental volume of nitric acid added to the Test Preparation. Heat the solution to the production of dense, white fumes; cool; cautiously add 10 mL of water; and, if hydrogen peroxide was used in treating the Test Preparation, add a volume of 30 percent hydrogen peroxide equal to that used for the substance being tested. Boil gently to the production of dense, white fumes. Again cool, cautiously add 5 mL of water, mix, and boil gently to the production of dense, white fumes and to a volume of 2 to 3 mL. Cool, dilute cautiously with a few mL of water, add 2.0 mL of Standard Lead Solution (20 µg of Pb), and mix. Transfer to a 50-mL color-comparison tube, rinse the flask with water, adding the rinsing to the tube until the volume is 25 mL, and mix.
Test Preparation— Unless otherwise indicated in the individual monograph, use a quantity, in g, of the substance to be tested as calculated by the formula:
2.0/(1000L),
in which L is the Heavy metals limit, as a percentage. If the substance is a solid— Transfer the weighed quantity of the test substance to a clean, dry, 100-mL Kjeldahl flask. [NOTE—A 300-mL flask may be used if the reaction foams excessively.] Clamp the flask at an angle of 45, and add a sufficient quantity of a mixture of 8 mL of sulfuric acid and 10 mL of nitric acid to moisten the substance thoroughly. Warm gently until the reaction commences, allow the reaction to subside, and add portions of the same acid mixture, heating after each addition, until a total of 18 mL of the acid mixture has been added. Increase the amount of heat, and boil gently until the solution darkens. Cool, add 2 mL of nitric acid, and heat again until the solution darkens. Continue the heating, followed by addition of nitric acid until no further darkening occurs, then heat strongly to the production of dense, white fumes. Cool, cautiously add 5 mL of water, boil gently to the production of dense, white fumes, and continue heating until the volume is reduced to a few mL. Cool, cautiously add 5 mL of water, and examine the color of the solution. If the color is yellow, cautiously add 1 mL of 30 percent hydrogen peroxide, and again evaporate to the production of dense, white fumes and a volume of 2 to 3 mL. If the solution is still yellow, repeat the addition of 5 mL of water and the peroxide treatment. Cool, dilute cautiously with a few mL of water, and rinse into a 50-mL color-comparison tube, taking care that the combined volume does not exceed 25 mL.
, and add a sufficient quantity of a mixture of 8 mL of sulfuric acid and 10 mL of nitric acid to moisten the substance thoroughly. Warm gently until the reaction commences, allow the reaction to subside, and add portions of the same acid mixture, heating after each addition, until a total of 18 mL of the acid mixture has been added. Increase the amount of heat, and boil gently until the solution darkens. Cool, add 2 mL of nitric acid, and heat again until the solution darkens. Continue the heating, followed by addition of nitric acid until no further darkening occurs, then heat strongly to the production of dense, white fumes. Cool, cautiously add 5 mL of water, boil gently to the production of dense, white fumes, and continue heating until the volume is reduced to a few mL. Cool, cautiously add 5 mL of water, and examine the color of the solution. If the color is yellow, cautiously add 1 mL of 30 percent hydrogen peroxide, and again evaporate to the production of dense, white fumes and a volume of 2 to 3 mL. If the solution is still yellow, repeat the addition of 5 mL of water and the peroxide treatment. Cool, dilute cautiously with a few mL of water, and rinse into a 50-mL color-comparison tube, taking care that the combined volume does not exceed 25 mL. If the substance is a liquid— Transfer the weighed quantity of the test substance to a clean, dry, 100-mL Kjeldahl flask. [NOTE—A 300-mL flask may be used if the reaction foams excessively.] Clamp the flask at an angle of 45, and cautiously add a few mL of a mixture of 8 mL of sulfuric acid and 10 mL of nitric acid. Warm gently until the reaction commences, allow the reaction to subside, and proceed as directed for If the substance is a solid, beginning with “add portions of the same acid mixture.”
, and cautiously add a few mL of a mixture of 8 mL of sulfuric acid and 10 mL of nitric acid. Warm gently until the reaction commences, allow the reaction to subside, and proceed as directed for If the substance is a solid, beginning with “add portions of the same acid mixture.” Monitor Preparation— Proceed with the digestion, using the same amount of sample and the same procedure as directed in the subsection If the substance is a solid in the section Test Preparation, until the step “Cool, dilute cautiously with a few mL of water.” Add 2.0 mL of Lead Standard Solution (20 µg of lead), and mix. Transfer to a 50-mL color comparison tube, rinse the flask with water, adding the rinsing to the tube until the volume is 25 mL, and mix.
Procedure— Treat the Test Preparation, the Standard Preparation, and the Monitor Preparation as follows. Using a pH meter or short-range pH indicator paper as external indicator, adjust the solution to a pH between 3.0 and 4.0 with ammonium hydroxide (a dilute ammonia solution may be used, if desired, as the specified range is approached), dilute with water to 40 mL, and mix.
To each tube add 2 mL of pH 3.5 Acetate Buffer, then add 1.2 mL of thioacetamide–glycerin base TS, dilute with water to 50 mL, mix, allow to stand for 2 minutes, and view downward over a white surface*: the color of the Test Preparation is not darker than that of the Standard Preparation, and the color of the Monitor Preparation is equal to or darker than that of the Standard Preparation.
* In those countries or jurisdictions where thioacetamide cannot be used, add 10 mL of freshly prepared hydrogen sulfide TS to each of the tubes, mix, allow to stand for 5 minutes, and view downward over a white surface.
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197 SPECTROPHOTOMETRIC IDENTIFICATION TESTS Spectrophotometric tests contribute meaningfully toward the identification of many compendial chemical substances. The test procedures that follow are applicable to substances that absorb IR and/or UV radiation (see Spectrophotometry and Light-Scattering 851).
851).The IR absorption spectrum of a substance, compared with that obtained concomitantly for the corresponding USP Reference Standard, provides perhaps the most conclusive evidence of the identity of the substance that can be realized from any single test. The UV absorption spectrum, on the other hand, does not exhibit a high degree of specificity. Conformance with both IR absorption and UV absorption test specifications, as called for in a large proportion of compendial monographs, leaves little doubt, if any, regarding the identity of the specimen under examination.
INFRARED ABSORPTION
Six methods are indicated for the preparation of previously dried test specimens and Reference Standards for analysis. The reference 197K in a monograph signifies that the substance under examination is mixed intimately with potassium bromide. The reference 197M in a monograph signifies that the substance under examination is finely ground and dispersed in mineral oil. The reference 197F in a monograph signifies that the substance under examination is suspended neat between suitable (for example, sodium chloride or potassium bromide) plates. The reference 197S signifies that a solution of designated concentration is prepared in the solvent specified in the individual monograph, and the solution is examined in 0.1-mm cells unless a different cell path length is specified in the individual monograph. The reference 197A signifies that the substance under examination is intimately in contact with an internal reflection element for attenuated total reflectance (ATR) analysis. The reference 197E signifies that the substance under examination is pressed as a thin sample against a suitable plate for IR microscopic analysis. The ATR 197A and the 197E techniques can be used as alternative methods for 197K, 197M, 197F, and 197S where testing is performed qualitatively and the Reference Standard spectra are similarly obtained.
197K in a monograph signifies that the substance under examination is mixed intimately with potassium bromide. The reference 197M in a monograph signifies that the substance under examination is finely ground and dispersed in mineral oil. The reference 197F in a monograph signifies that the substance under examination is suspended neat between suitable (for example, sodium chloride or potassium bromide) plates. The reference 197S signifies that a solution of designated concentration is prepared in the solvent specified in the individual monograph, and the solution is examined in 0.1-mm cells unless a different cell path length is specified in the individual monograph. The reference 197A signifies that the substance under examination is intimately in contact with an internal reflection element for attenuated total reflectance (ATR) analysis. The reference 197E signifies that the substance under examination is pressed as a thin sample against a suitable plate for IR microscopic analysis. The ATR 197A and the 197E techniques can be used as alternative methods for 197K, 197M, 197F, and 197S where testing is performed qualitatively and the Reference Standard spectra are similarly obtained.Record the spectra of the test specimen and the corresponding USP Reference Standard over the range from about 2.6 µm to 15 µm (3800 cm–1 to 650 cm–1) unless otherwise specified in the individual monograph. The IR absorption spectrum of the preparation of the test specimen, previously dried under conditions specified for the corresponding Reference Standard unless otherwise specified, or unless the Reference Standard is to be used without drying, exhibits maxima only at the same wavelengths as that of a similar preparation of the corresponding USP Reference Standard.
Differences that may be observed in the spectra so obtained sometimes are attributed to the presence of polymorphs, which are not always acceptable (see Procedure under Spectrophotometry and Light-Scattering 851). Unless otherwise directed in the individual monograph, therefore, continue as follows. If a difference appears in the IR spectra of the analyte and the standard, dissolve equal portions of the test specimen and the Reference Standard in equal volumes of a suitable solvent, evaporate the solution to dryness in similar containers under identical conditions, and repeat the test on the residues.
851). Unless otherwise directed in the individual monograph, therefore, continue as follows. If a difference appears in the IR spectra of the analyte and the standard, dissolve equal portions of the test specimen and the Reference Standard in equal volumes of a suitable solvent, evaporate the solution to dryness in similar containers under identical conditions, and repeat the test on the residues.ULTRAVIOLET ABSORPTION
The reference 197U in a monograph signifies that a test solution and a Standard solution are examined spectrophotometrically, in 1-cm cells, over the spectral range from 200 to 400 nm unless otherwise specified in the individual monograph.
197U in a monograph signifies that a test solution and a Standard solution are examined spectrophotometrically, in 1-cm cells, over the spectral range from 200 to 400 nm unless otherwise specified in the individual monograph.Dissolve a portion of the substance under examination in the designated Medium to obtain a test solution having the concentration specified in the monograph for Solution. Similarly prepare a Standard solution containing the corresponding USP Reference Standard.
Record and compare the spectra concomitantly obtained for the test solution and the Standard solution. Calculate absorptivities and/or absorbance ratios where these criteria are included in an individual monograph. Unless otherwise specified, absorbances indicated for these calculations are those measured at the maximum absorbance at about the wavelength specified in the individual monograph. Where the absorbance is to be measured at about the specified wavelength other than that of maximum absorbance, the abbreviations (min) and (sh) are used to indicate a minimum and shoulder, respectively, in an absorption spectrum. The requirements are met if the UV absorption spectra of the test solution and the Standard solution exhibit maxima and minima at the same wavelengths and absorptivities and/or absorbance ratios are within specified limits.
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281 RESIDUE ON IGNITION The Residue on Ignition / Sulfated Ash test utilizes a procedure to measure the amount of residual substance not volatilized from a sample when the sample is ignited in the presence of sulfuric acid according to the procedure described below. This test is usually used for determining the content of inorganic impurities in an organic substance.
Procedure— Weigh accurately 1 to 2 g of the substance, or the amount specified in the individual monograph, in a suitable crucible (silica, platinum, quartz, or porcelain) that previously has been ignited at 600 ± 50
for 30 minutes, cooled in a desiccator (silica gel or other suitable desiccant), and weighed. Moisten the sample with a small amount (usually 1 mL) of sulfuric acid. Heat, gently at first, at a temperature as low as practicable until the substance is thoroughly charred, cool, then, unless otherwise directed in the individual monograph, moisten the residue with a small amount (usually 1 mL) of sulfuric acid, heat gently until white fumes are no longer evolved, and ignite at 600 ± 50, unless another temperature is specified in the individual monograph, until the carbon is consumed. Ensure that flames are not produced at any time during the procedure. Cool in a desiccator (silica gel or other suitable desiccant), weigh, and calculate the percentage of residue. Unless otherwise specified, if the amount of the residue so obtained exceeds the limit specified in the individual monograph, repeat the moistening with sulfuric acid, heating and igniting as before, until constant weight is attained or until the percentage of residue complies with the limit in the individual monograph. Conduct the ignition in a well-ventilated hood, but protected from air currents, and at as low a temperature as is possible to effect the complete combustion of the carbon. A muffle furnace may be used, if desired, and its use is recommended for the final ignition at 600 ± 50.
. Calibration of the muffle furnace may be carried out using an appropriate digital temperature meter and a working thermocouple probe calibrated against a standard thermocouple traceable to the National Institute of Standards and Technology.
Verify the accuracy of the measuring and controlling circuitry of the muffle furnace by checking the positions in the furnace at the control set point temperature of intended use. Select positions that reflect the eventual method of use with respect to location of the specimen under test. The tolerance is ±25 at each position measured.
at each position measured.Sulphated Ash tests found in the European and Japanese Pharmacopoeias are considered equivalent to this test, except where noted.
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467 ORGANIC VOLATILE IMPURITIES RESIDUAL SOLVENTS LIMITS
For pharmacopeial purposes, residual solvents in pharmaceuticals are defined as organic volatile chemicals that are used or produced in the manufacture of drug substances or excipients, or in the preparation of drug products. The residual solvents are not completely removed by practical manufacturing techniques. Appropriate selection of the solvent for the synthesis of a drug substance or an excipient may enhance the yield, or determine characteristics such as crystal form, purity, and solubility. Therefore, the solvent may sometimes be a critical element in the synthetic process. This General Chapter does not address solvents deliberately used as excipients nor does it address solvates. However, the content of solvents in such products should be evaluated and justified.
Because residual solvents do not provide therapeutic benefit, they should be removed, to the extent possible, to meet ingredient and product specifications, good manufacturing practices, or other quality-based requirements. Drug products should contain no higher levels of residual solvents than can be supported by safety data. Solvents that are known to cause unacceptable toxicities (Class 1, Table 1) should be avoided in the production of drug substances, excipients, or drug products unless their use can be strongly justified in a risk-benefit assessment. Solvents associated with less severe toxicity (Class 2, Table 2) should be limited in order to protect patients from potential adverse effects. Ideally, less toxic solvents (Class 3, Table 3) should be used where practical. The complete list of solvents included in this General Chapter is given in Appendix 1. These tables and the list are not exhaustive. Where other solvents have been used, based on approval by the competent regulatory authority, such solvents may be added to the tables and list.
Testing of drug substances, excipients, and drug products for residual solvents should be performed when production or purification processes are known to result in the presence of such residual solvents. It is only necessary to test for residual solvents that are used or produced in the manufacture or purification processes.
Although manufacturers may choose to test the drug product, a cumulative procedure may be used to calculate the residual solvent levels in the product from the levels in its ingredients. If the calculation results in a level equal to or below that recommended in this General Chapter, no testing of the drug product for residual solvents needs to be considered. If, however, the calculated levels are above the recommended level, the drug product should be tested to ascertain whether the formulation process has reduced the relevant solvent levels to within acceptable amounts. A drug product should also be tested if a residual solvent is used during its manufacture.
See Appendix 2 for additional background information related to residual solvents.
CLASSIFICATION OF RESIDUAL SOLVENTS BY RISK ASSESSMENT
The term “tolerable daily intake” (TDI) is used by the International Program on Chemical Safety (IPCS) to describe exposure limits of toxic chemicals and the term “acceptable daily intake” (ADI) is used by the World Health Organization (WHO) and other national and international health authorities and institutes. The term “permitted daily exposure” (PDE) is defined as a pharmaceutically acceptable intake of residual solvents to avoid confusion of differing values for ADIs of the same substance.
Residual solvents specified in this General Chapter are listed in Appendix 1 by common names and structures. They were evaluated for their possible risk to human health and placed into one of three classes as follows:
| Class 1 | Residual Solvents: Solvents to be Avoided Known human carcinogens Strongly suspected human carcinogens Environmental hazards |
| Class 2 | Residual Solvents: Solvents to be Limited Nongenotoxic animal carcinogens or possible  causative agents of other irreversibletoxicity, such as neurotoxicity or teratogenicity.Solvents suspected of other significant but rever- sible toxicities. |
| Class 3 | Residual Solvents: Solvents with Low Toxic Po-tential Solvents with low toxic potential to humans; no health-based exposure limit is needed.[NOTE—Class 3 residual solvents may have PDEs of up to 50 mg or more per day.]* |
| * For residual solvents with PDEs of more than 50 mg per day, see the discussion in the section Class 3 under Limits of Residual Solvents. |
PROCEDURES FOR ESTABLISHING EXPOSURE LIMITS
The procedure used to establish permitted daily exposures for residual solvents is presented in Appendix 3.
OPTIONS FOR DETERMINING LEVELS OF CLASS 2 RESIDUAL SOLVENTS
Two options are available to determine levels of Class 2 residual solvents.
Option 1
The concentration limits in ppm stated in Table 2 are used. They were calculated using equation (1) below by assuming a product weight of 10 g administered daily.
Here, PDE is given in terms of mg per day, and dose is given in g per day.
These limits are considered acceptable for all drug substances, excipients, and drug products. Therefore, this option may be applied if the daily dose is not known or fixed. If all drug substances and excipients in a formulation meet the limits given in Option 1, these components may be used in any proportion. No further calculation is necessary provided the daily dose does not exceed 10 g. Products that are administered in doses greater than 10 g per day are to be considered under Option 2.
Option 2
It is not necessary for each component of the drug product to comply with the limits given in Option 1. The PDE in terms of mg per day as stated in Table 2 can be used with the known maximum daily dose and equation (1) above to determine the concentration of residual solvent allowed in a drug product. Such limits are considered acceptable provided that it has been demonstrated that the residual solvent has been reduced to the practical minimum. The limits should be realistic in relation to analytical precision, manufacturing capability, and reasonable variation in the manufacturing process. The limits should also reflect contemporary manufacturing standards.
Option 2 may be applied by adding the amounts of a residual solvent present in each of the components of the drug product. The sum of the amounts of solvent per day should be less than that given by the PDE.
Consider an example of the application of Option 1 and Option 2 to acetonitrile concentration in a drug product. The permitted daily exposure to acetonitrile is 4.1 mg per day; thus, the Option 1 limit is 410 ppm. The maximum administered daily weight of a drug product is 5.0 g, and the drug product contains two excipients. The composition of the drug product and the calculated maximum content of residual acetonitrile are given in the following table.
| Component | Amount in Formulation (g) | Acetonitrile Content (ppm) | Daily Exposure (mg) |
| Drugsubstance | 0.3 | 800 | 0.24 |
| Excipient 1 | 0.9 | 400 | 0.36 |
| Excipient 2 | 3.8 | 800 | 3.04 |
| Drug product | 5.0 | 728 | 3.64 |
Excipient l meets the Option 1 limit, but the drug substance, excipient 2, and drug product do not meet the Option 1 limit. Nevertheless, the drug product meets the Option 2 limit of 4.1 mg per day and thus conforms to the acceptance criteria in this General Chapter.
Consider another example using acetonitrile as the residual solvent. The maximum administered daily weight of a drug product is 5.0 g, and the drug product contains two excipients. The composition of the drug product and the calculated maximum content of residual acetonitrile are given in the following table.
| Component | Amount in Formulation (g) | Acetonitrile Content (ppm) | Daily Exposure (mg) |
| Drugsubstance | 0.3 | 800 | 0.24 |
| Excipient 1 | 0.9 | 2000 | 1.80 |
| Excipient 2 | 3.8 | 800 | 3.04 |
| Drug product | 5.0 | 1016 | 5.08 |
In this example, the drug product meets neither the Option 1 nor the Option 2 limit. The manufacturer could test the drug product to determine if the formulation process reduced the level of acetonitrile. If the level of acetonitrile was not reduced to the allowed limit during formulation, the product fails the requirements of the test.
LIMITS OF RESIDUAL SOLVENTS
Ethylene Oxide
[NOTE—The test for ethylene oxide is conducted only where specified in the individual monograph.] The standard solution parameters and the procedure for determination are described in the individual monograph. Unless otherwise specified in the individual monograph, the limit is 10 µg per g.
Class 1
Class 1 residual solvents (Table 1) should not be employed in the manufacture of drug substances, excipients, and drug products because of the unacceptable toxicities or deleterious environmental effects of these residual solvents. However, if Class 1 residual solvents are used, their levels should be restricted as shown in Table 1, unless otherwise stated in the individual monograph. The solvent 1,1,1-trichloroethane is included in Table 1 because it is an environmental hazard. The stated limit of 1500 ppm is based on safety data.
When Class 1 residual solvents are used in the manufacture of a drug substance, excipient, or drug product, the methodology described in the Identification, Control, and Quantification of Residual Solvents section of this General Chapter is to be applied wherever possible. Otherwise an appropriate validated procedure is to be employed. Such procedure shall be submitted to the USP for inclusion in the relevant individual monograph.
Table 1. Class 1 Residual Solvents
| Solvent | Concentration Limit (ppm) | Concern |
| Benzene | 2 | Carcinogen |
| Carbon tetrachloride | 4 | Toxic and environmental hazard |
| 1,2-Dichloroethane | 5 | Toxic |
| 1,1-Dichloroethene | 8 | Toxic |
| 1,1,1-Trichloroethane | 1500 | Environmentalhazard |
Class 2
Class 2 residual solvents (Table 2) should be limited in drug substances, excipients, and drug products because of the inherent toxicities of the residual solvents. PDEs are given to the nearest 0.1 mg per day, and concentrations are given to the nearest 10 ppm. The stated values do not reflect the necessary analytical precision of the determination procedure. Precision should be determined as part of the procedure validation.
If Class 2 residual solvents are present at greater than their Option 1 limits, they should be identified and quantified. The procedures described in the Identification, Control, and Quantification of Residual Solvents section of this General Chapter are to be applied wherever possible. Otherwise an appropriate validated procedure is to be employed. Such procedure shall be submitted to the USP for inclusion in the relevant individual monograph.
NOTE—The following Class 2 residual solvents are not readily detected by the headspace injection conditions described in the Identification, Control, and Quantification of Residual Solvents section of this General Chapter: formamide, 2-ethoxyethanol, 2-methoxyethanol, ethylene glycol, N-methylpyrrolidone, and sulfolane. Other appropriate validated procedures are to be employed for the control of these residual solvents. Such procedures shall be submitted to the USP for inclusion in the relevant individual monograph.
Table 2. Class 2 Residual Solvents
| Solvent | PDE (mg/day) | Concentration limit (ppm) |
| Acetonitrile | 4.1 | 410 |
| Chlorobenzene | 3.6 | 360 |
| Chloroform | 0.6 | 60 |
| Cyclohexane | 38.8 | 3880 |
| 1,2-Dichloroethene | 18.7 | 1870 |
| 1,2-Dimethoxyethane | 1.0 | 100 |
| N,N-Dimethylacetamide | 10.9 | 1090 |
| N,N-Dimethylformamide | 8.8 | 880 |
| 1,4-Dioxane | 3.8 | 380 |
| 2-Ethoxyethanol | 1.6 | 160 |
| Ethylene glycol | 6.2 | 620 |
| Formamide | 2.2 | 220 |
| Hexane | 2.9 | 290 |
| Methanol | 30.0 | 3000 |
| 2-Methoxyethanol | 0.5 | 50 |
| Methylbutylketone | 0.5 | 50 |
| Methylcyclohexane | 11.8 | 1180 |
| Methylene chloride | 6.0 | 600 |
| N-Methylpyrrolidone | 5.3 | 530 |
| Nitromethane | 0.5 | 50 |
| Pyridine | 2.0 | 200 |
| Sulfolane | 1.6 | 160 |
| Tetrahydrofuran | 7.2 | 720 |
| Tetralin | 1.0 | 100 |
| Toluene | 8.9 | 890 |
| Trichloroethene | 0.8 | 80 |
| Xylene* | 21.7 | 2170 |
| * Usually 60% m-xylene, 14% p-xylene, 9% o-xylene with 17% ethyl benzene | ||
Class 3
Class 3 residual solvents (Table 3) may be regarded as less toxic and of lower risk to human health than Class 1 and Class 2 residual solvents. Class 3 includes no solvent known as a human health hazard at levels normally accepted in pharmaceuticals. However, there are no long-term toxicity or carcinogenicity studies for many of the residual solvents in Class 3. Available data indicate that they are less toxic in acute or short-term studies and negative in genotoxicity studies.
Unless otherwise stated in the individual monograph, Class 3 residual solvents are limited to not more than 50 mg per day (corresponding to 5000 ppm or 0.5% under Option 1). If a Class 3 solvent limit in an individual monograph is greater than 50 mg per day, that residual solvent should be identified and quantified. The procedures described in the Identification, Control, and Quantification of Residual Solvents section of this General Chapter are to be applied wherever possible. Otherwise an appropriate validated procedure is to be employed. Such procedure shall be submitted to the USP for inclusion in the relevant individual monograph.
Table 3. Class 3 Residual Solvents
(limited by GMP or other quality-based requirements in drug substances, excipients, and drug products)
(limited by GMP or other quality-based requirements in drug substances, excipients, and drug products)
| Acetic acid | Heptane |
| Acetone | Isobutyl acetate |
| Anisole | Isopropyl acetate |
| 1-Butanol | Methyl acetate |
| 2-Butanol | 3-Methyl-1-butanol |
| Butyl acetate | Methylethylketone |
| tert-Butylmethyl ether | Methylisobutylketone |
| Cumene | 2-Methyl-l-propanol |
| Dimethyl sulfoxide | Pentane |
| Ethanol | 1-Pentanol |
| Ethyl acetate | 1-Propanol |
| Ethyl ether | 2-Propanol |
| Ethyl formate | Propyl acetate |
| Formic acid |
Other Residual Solvents
The residual solvents listed in Table 4 may also be of interest to manufacturers of drug substances, excipients, or drug products. However, no adequate toxicological data on which to base a PDE was found. Specifications for these residual solvents will be provided in the respective individual monograph.
Table 4. Other Residual Solvents
(for which no adequate toxicological data was found)
(for which no adequate toxicological data was found)
| 1,1-Diethoxypropane | Methyl isopropyl ketone |
| 1,1-Dimethoxymethane | Methyltetrahydrofuran |
| 2,2-Dimethoxypropane | Solvent Hexane |
| Isooctane | Trichloroacetic acid |
| Isopropyl ether | Trifluoroacetic acid |
IDENTIFICATION, CONTROL, AND QUANTIFICATION OF RESIDUAL SOLVENTS
NOTE—The organic-free water specified in the following procedures produces no significantly interfering peaks when chromatographed.
Class 1 and Class 2 Residual Solvents
WATER-SOLUBLE ARTICLES
Procedure A—
Class 1 Standard Stock Solution— Transfer 1.0 mL of USP Class 1 Residual Solvents Mixture RS to a 100-mL volumetric flask, add 9 mL of dimethyl sulfoxide, dilute with water to volume, and mix. Transfer 1.0 mL of this solution to a 100-mL volumetric flask, dilute with water to volume, and mix. Transfer 1.0 mL of this solution to a 10-mL volumetric flask, dilute with water to volume, and mix.
Class 1 Standard Solution— Transfer 1.0 mL of Class 1 Standard Stock Solution to an appropriate headspace vial, add 5.0 mL of water, apply stopper, cap, and mix.
Class 2 Standard Stock Solution— Transfer 1.0 mL of USP Class 2 Residual Solvents Mixture RS to a 100-mL volumetric flask, dilute with water to volume, and mix.
Class 2 Standard Solution— Transfer 1.0 mL of Class 2 Standard Stock Solution to an appropriate headspace vial, add 5.0 mL of water, apply stopper, cap, and mix.
Test Stock Solution— Transfer about 250 mg of the article under test, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with water to volume, and mix.
Test Solution— Transfer 5.0 mL of Test Stock Solution to an appropriate headspace vial, add 1.0 mL of water, apply stopper, cap, and mix.
Class 1 System Suitability Solution— Transfer 1.0 mL of Class 1 Standard Stock Solution to an appropriate headspace vial, add 5.0 mL of Test Stock Solution, apply stopper, cap, and mix.
Chromatographic System (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector, a 0.32-mm × 30-m fused-silica column coated with a 1.8-µm layer of phase G43 or a 0.53-mm × 30-m wide-bore column coated with a 3.0-µm layer of phase G43. The carrier gas is nitrogen or helium with a linear velocity of about 35 cm per second, and a split ratio of 1:5. The column temperature is maintained at 40 for 20 minutes, then raised at a rate of 10 per minute to 240, and maintained at 240 for 20 minutes. The injection port and detector temperatures are maintained at 140 and 250, respectively. Chromatograph the Class 1 Standard Solution, Class 1 System Suitability Solution, and Class 2 Standard Solution, and record the peak responses as directed for Procedure: the signal-to-noise ratio of 1,1,1-trichloroethane in the Class 1 Standard Solution is not less than 5; the signal-to-noise ratio of each peak in the Class 1 System Suitability Solution is not less than 3; and the resolution, R, between acetonitrile and methylene chloride in the Class 2 Standard Solution is not less than 1.0.
621)— The gas chromatograph is equipped with a flame-ionization detector, a 0.32-mm × 30-m fused-silica column coated with a 1.8-µm layer of phase G43 or a 0.53-mm × 30-m wide-bore column coated with a 3.0-µm layer of phase G43. The carrier gas is nitrogen or helium with a linear velocity of about 35 cm per second, and a split ratio of 1:5. The column temperature is maintained at 40 for 20 minutes, then raised at a rate of 10 per minute to 240, and maintained at 240 for 20 minutes. The injection port and detector temperatures are maintained at 140 and 250, respectively. Chromatograph the Class 1 Standard Solution, Class 1 System Suitability Solution, and Class 2 Standard Solution, and record the peak responses as directed for Procedure: the signal-to-noise ratio of 1,1,1-trichloroethane in the Class 1 Standard Solution is not less than 5; the signal-to-noise ratio of each peak in the Class 1 System Suitability Solution is not less than 3; and the resolution, R, between acetonitrile and methylene chloride in the Class 2 Standard Solution is not less than 1.0. Procedure— Separately inject (following one of the headspace operating parameter sets described in the table below) equal volumes of headspace (about 1.0 mL) of the Class 1 Standard Solution, Class 2 Standard Solution, and the Test Solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. If a peak response of any peak in the Test Solution is greater than or equal to a corresponding peak in either the Class 1 Standard Solution or the Class 2 Standard Solution, proceed to Procedure B to verify the identity of the peak; otherwise the article meets the requirements of this test.
Table 5. Headspace Operating Parameters
| Headspace OperatingParameter Sets | |||
| 1 | 2 | 3 | |
| Equilibration temperature () | 80 | 105 | 80 |
| Equilibration time (min.) | 60 | 45 | 45 |
| Transfer-line temperature () | 85 | 110 | 105 |
| Carrier gas: nitrogen or helium at an appropriate pressure | |||
| Pressurization time (s) | 30 | 30 | 30 |
| Injection volume (mL) | 1 | 1 | 1 |
Procedure B—
Class 1 Standard Stock Solution, Class 1 Standard Solution, Class 2 Standard Stock Solution, Class 2 Standard Solution, Test Stock Solution, Test Solution, and Class 1 System Suitability Solution— Prepare as directed for Procedure A.
Chromatographic System (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector, a 0.32-mm × 30-m fused-silica column coated with a 0.25-µm layer of phase G16, or a 0.53-mm × 30-m wide-bore column coated with a 0.25-µm layer of phase G16. The carrier gas is nitrogen or helium with a linear velocity of about 35 cm per second and a split ratio of 1:5. The column temperature is maintained at 50 for 20 minutes, then raised at a rate of 6 per minute to 165, and maintained at 165 for 20 minutes. The injection port and detector temperatures are maintained at 140 and 250, respectively. Chromatograph the Class 1 Standard Solution, the Class 1 System Suitability Solution, and the Class 2 Standard Solution, and record the peak responses as directed for Procedure: the signal-to-noise ratio of benzene in the Class 1 Standard Solution is not less than 5; the signal-to-noise ratio of each peak in the Class 1 System Suitability Solution is not less than 3; the resolution, R, between acetonitrile and trichloroethylene in the Class 2 Standard Solution is not less than 1.0.
621)— The gas chromatograph is equipped with a flame-ionization detector, a 0.32-mm × 30-m fused-silica column coated with a 0.25-µm layer of phase G16, or a 0.53-mm × 30-m wide-bore column coated with a 0.25-µm layer of phase G16. The carrier gas is nitrogen or helium with a linear velocity of about 35 cm per second and a split ratio of 1:5. The column temperature is maintained at 50 for 20 minutes, then raised at a rate of 6 per minute to 165, and maintained at 165 for 20 minutes. The injection port and detector temperatures are maintained at 140 and 250, respectively. Chromatograph the Class 1 Standard Solution, the Class 1 System Suitability Solution, and the Class 2 Standard Solution, and record the peak responses as directed for Procedure: the signal-to-noise ratio of benzene in the Class 1 Standard Solution is not less than 5; the signal-to-noise ratio of each peak in the Class 1 System Suitability Solution is not less than 3; the resolution, R, between acetonitrile and trichloroethylene in the Class 2 Standard Solution is not less than 1.0. Procedure— Separately inject (following one of the headspace operating parameter sets described in Table 5) equal volumes of headspace (about 1.0 mL) of the Class 1 Sandard Solution, the Class 2 Standard Solution, and the Test Solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. If the peak response(s) in the Test Solution of the peak(s) identified in Procedure A is/are greater than or equal to a corresponding peak(s) in either the Class 1 Standard Solution or the Class 2 Standard Solution, proceed to Procedure C to quantify the peak; otherwise the article meets the requirements of this test.
Procedure C—
Class 1 Standard Solution, Class 2 Standard Solution, Test Stock Solution, Test Solution, and Class 1 System Suitability Solution— Prepare as directed for Procedure A.
Standard Solution— Transfer an accurately measured volume of the USP Reference Standard for each peak identified and verified by Procedures A and B to a suitable container, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a final concentration of 1/100 of the value stated in Table 1 or 2 (under Concentration limit). Transfer 5.0 mL of this solution to an appropriate headspace vial, add 1.0 mL of water, apply stopper, cap, and mix.
Chromatographic System (see Chromatography 621)— [NOTE—If the results of the chromatography from Procedure A are found to be inferior to those found with Procedure B, the Chromatographic System from Procedure B may be substituted.] The gas chromatograph is equipped with a flame-ionization detector, a 0.32-mm × 30-m fused-silica column coated with a 1.8-µm layer of phase G43 or a 0.53-mm × 30-m wide-bore column coated with a 3.0-µm layer of phase G43. The carrier gas is nitrogen or helium with a linear velocity of about 35 cm per second, and a split ratio of 1:5. The column temperature is maintained at 40 for 20 minutes, then raised at a rate of 10 per minute to 240, and maintained at 240 for 20 minutes. The injection port and detector temperatures are maintained at 140 and 250, respectively. Chromatograph the Class 1 Standard Solution, the Class 1 System Suitability Solution, and the Class 2 Standard Solution, and record the peak responses as directed for Procedure: the signal-to-noise ratio of 1,1,1-trichloroethane in the Class 1 Standard Solution is not less than 5; the signal-to-noise ratio of each peak in the Class 1 System Suitability Solution is not less than 3; and the resolution, R, between acetonitrile and methylene chloride in the Class 2 Standard Solution is not less than 1.0.
621)— [NOTE—If the results of the chromatography from Procedure A are found to be inferior to those found with Procedure B, the Chromatographic System from Procedure B may be substituted.] The gas chromatograph is equipped with a flame-ionization detector, a 0.32-mm × 30-m fused-silica column coated with a 1.8-µm layer of phase G43 or a 0.53-mm × 30-m wide-bore column coated with a 3.0-µm layer of phase G43. The carrier gas is nitrogen or helium with a linear velocity of about 35 cm per second, and a split ratio of 1:5. The column temperature is maintained at 40 for 20 minutes, then raised at a rate of 10 per minute to 240, and maintained at 240 for 20 minutes. The injection port and detector temperatures are maintained at 140 and 250, respectively. Chromatograph the Class 1 Standard Solution, the Class 1 System Suitability Solution, and the Class 2 Standard Solution, and record the peak responses as directed for Procedure: the signal-to-noise ratio of 1,1,1-trichloroethane in the Class 1 Standard Solution is not less than 5; the signal-to-noise ratio of each peak in the Class 1 System Suitability Solution is not less than 3; and the resolution, R, between acetonitrile and methylene chloride in the Class 2 Standard Solution is not less than 1.0. Procedure— Separately inject (following one of the headspace operating parameters described in Table 5) equal volumes of headspace (about 1.0 mL) of the Standard Solution and Test Solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the amount, in ppm, of each residual solvent found in the article under test by the formula:
4(C/W)(rU / rS),
in which C is the concentration, in ppm, of the appropriate USP Reference Standard in the Standard Solution; W is the weight, in g, of the article under test taken to prepare the Test Stock Solution; and rU and rS are the peak responses of each residual solvent obtained from the Test Solution and the Standard Solution, respectively. WATER-INSOLUBLE ARTICLES
Procedure A—
Class 1 Standard Stock Solution, Class 1 Standard Solution, Class 1 System Suitability Solution, Class 2 Standard Stock Solution, Class 2 Standard Solution, and Chromatographic System— Proceed as directed for Procedure A under Water-Soluble Articles.
Test Stock Solution— Transfer about 250 mg of the article under test, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with dimethylformamide to volume, and mix.
Test Solution 1— Transfer 5.0 mL of Test Stock Solution to an appropriate headspace vial, add 1.0 mL of dimethylformamide, apply stopper, cap, and mix.
Test Solution 2— Transfer about 250 mg of the article under test, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with 1,3-dimethyl-2-imidazolidinone to volume, and mix. Transfer 5.0 mL of this solution to an appropriate headspace vial, add 1.0 mL of 1,3-dimethyl-2-imidazolidinone, apply stopper, cap, and mix.
Procedure— Separately inject (following one of the headspace operating parameters described in Table 5) equal volumes of headspace (about 1.0 mL) of the Class 1 Standard Solution, the Class 2 Standard Solution, Test Solution 1, and Test solution 2 into the chromatograph, record the chromatograms, and measure the responses for the major peaks. If a peak response of any peak in Test solution 1 is greater than or equal to a corresponding peak in either the Class 1 Standard Solution or the Class 2 Standard Solution, proceed to Procedure B to verify the identity of the peak; otherwise the article meets the requirements of this test. If the peak response for dimethylformamide or N,N-dimethylacetamide in Test Solution 2 is greater than or equal to the corresponding peak in the Class 2 Standard Solution, proceed to Procedure B to verify the identity of the peak; otherwise the article meets the requirements of this test.
Procedure B—
Class 1 Standard Stock Solution, Class 1 Standard Solution, Class 2 Standard Stock Solution, Class 2 Standard Solution, and Class 1 System Suitability Solution— Prepare as directed for Procedure A under Water-Soluble Articles.
Test Stock Solution, Test Solution 1, and Test Solution 2— Proceed as directed for Procedure A.
Chromatographic System— Proceed as directed for Procedure B under Water-Soluble Articles.
Procedure— Separately inject (following one of the headspace operating parameters described in Table 5) equal volumes of headspace (about 1.0 mL) of the Class 1 Standard Solution, Class 2 Standard Solution, Test Solution 1, and/or Test Solution 2 into the chromatograph, record the chromatograms, and measure the responses for the major peaks. If the peak response(s) in Test Solution 1 of the peak(s) identified in Procedure A is/are greater than or equal to a corresponding peak(s) in either the Class 1 Standard Solution or the Class 2 Standard Solution, proceed to Procedure C to quantify the peak; otherwise the article meets the requirements of this test. If the peak response for dimethylformamide or N,N-dimethylacetamide in Test Solution 2 is greater than or equal to the corresponding peak in the Class 2 Standard Solution, proceed to Procedure C to quantify the peak; otherwise the article meets the requirements of this test.
Procedure C—
Class 1 Standard Solution, Class 1 System Suitability Solution, and Class 2 Standard Solution— Proceed as directed for Procedure A under Water-Soluble Articles.
Test Stock Solution, Test Solution 1, and Test Solution 2— Proceed as directed for Procedure A.
Standard Solution, and Chromatographic System— Proceed as directed for Procedure C under Water-Soluble Articles.
Procedure— Separately inject (following one of the headspace operating parameters described in Table 5) equal volumes of headspace (about 1.0 mL) of the Standard Solution, Test Solution 1, and/or Test Solution 2 into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the amount, in ppm, of each residual solvent found in the article under test by the formula:
4(C/W)(rU / rS),
in which C is the concentration, in ppm, of the appropriate USP Reference Standard in the Standard Solution; W is the weight, in g, of the article under test taken to prepare the Test Stock Solution; and rU and rS are the peak responses of each residual solvent obtained from Test Solution 1 or Test Solution 2 and the Standard Solution, respectively. Class 3 Residual Solvents
If only Class 3 solvents are present, the level of residual solvents is to be determined as directed under Loss on Drying 731. If the loss on drying value is greater than 0.5%, a water determination should be performed on the test sample as directed under Water Determination 921. Determine the water by Method Ia, unless otherwise specified in the individual monograph. If a Class 3 solvent limit in an individual monograph is greater than 50 mg per day (corresponding to 5000 ppm or 0.5% under Option 1), that residual solvent should be identified and quantified, and the procedures as described above are to be applied wherever possible. Otherwise an appropriate validated procedure is to be employed. Such procedure shall be submitted to the USP for inclusion in the relevant individual monograph. A flow diagram for the application of residual solvent limit tests is shown in Figure 1.
731. If the loss on drying value is greater than 0.5%, a water determination should be performed on the test sample as directed under Water Determination 921. Determine the water by Method Ia, unless otherwise specified in the individual monograph. If a Class 3 solvent limit in an individual monograph is greater than 50 mg per day (corresponding to 5000 ppm or 0.5% under Option 1), that residual solvent should be identified and quantified, and the procedures as described above are to be applied wherever possible. Otherwise an appropriate validated procedure is to be employed. Such procedure shall be submitted to the USP for inclusion in the relevant individual monograph. A flow diagram for the application of residual solvent limit tests is shown in Figure 1.
Fig. 1. Diagram relating to the identification of residual solvents and the application of limit tests.
OTHER ANALYTICAL PROCEDURES
The following procedures, with any necessary variations, are used where specified in the individual monographs.
Method I
A gas chromatograph capable of temperature programming and equipped with a wide-bore, wall-coated open tubular column and a flame-ionization detector is used in the following procedure.
Standard Solution— Prepare a solution, in organic-free water, or the solvent specified in the monograph, containing in each mL, 12.0 µg of methylene chloride, 7.6 µg of 1,4-dioxane, 1.6 µg of trichloroethylene, and 1.2 µg of chloroform. [NOTE—Prepare fresh daily.]
Test Solution— Dissolve in organic-free water, or the solvent specified in the monograph, an accurately weighed portion of the material to be tested to obtain a final solution having a known concentration of about 20 mg of the test material per mL.
Chromatographic System (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector, a 0.53-mm × 30-m fused silica analytical column coated with a 5-µm chemically cross-linked G27 stationary phase and a 0.53-mm × 5-m silica guard column deactivated with phenylmethyl siloxane. The carrier gas is helium with a linear velocity of about 35 cm per second. [NOTE—When a makeup gas is used, nitrogen is recommended.] The injection port temperature and the detector temperature are maintained at 70 and 260, respectively. The column temperature is programmed as follows. Initially, the column temperature is maintained at 35 for 5 minutes, then increased at a rate of 8 per minute to 175, followed by an increase at a rate of 35 per minute to 260, and maintained at 260 for at least 16 minutes.
621)— The gas chromatograph is equipped with a flame-ionization detector, a 0.53-mm × 30-m fused silica analytical column coated with a 5-µm chemically cross-linked G27 stationary phase and a 0.53-mm × 5-m silica guard column deactivated with phenylmethyl siloxane. The carrier gas is helium with a linear velocity of about 35 cm per second. [NOTE—When a makeup gas is used, nitrogen is recommended.] The injection port temperature and the detector temperature are maintained at 70 and 260, respectively. The column temperature is programmed as follows. Initially, the column temperature is maintained at 35 for 5 minutes, then increased at a rate of 8 per minute to 175, followed by an increase at a rate of 35 per minute to 260, and maintained at 260 for at least 16 minutes. Inject the Standard Solution, and record the peak responses as directed for Procedure: a suitable system is one that yields chromatograms in which all of the components in the Standard Solution are resolved; the resolution, R, between any two components is not less than 1.0; and the relative standard deviation of the individual peak responses from replicate injections is not more than 15%.
Procedure— Separately inject equal volumes (about 1 µL) of the Standard Solution and the Test Solution into the chromatograph, record the chromatograms, and measure the peak responses.
Identify, on the basis of retention time, any peaks present in the chromatogram of the Test Solution. The identity and peak response in the chromatogram may be established as being from any of the organic volatile impurities listed in the table shown below or from some other volatile impurity eluting with a comparable retention time as determined by mass spectrometric relative abundance procedures or by the use of a second validated column containing a different stationary phase.
Unless otherwise specified in the individual monograph, the amount of each organic volatile impurity present in the material does not exceed the limit given in the table shown below.
| Organic Volatile Impurity | Limit (µg per g) |
| Chloroform | 60 |
| 1,4-Dioxane | 380 |
| Methylene Chloride | 600 |
| Trichloroethylene | 80 |
Method IV
Standard Solution— Prepare as directed for Standard Solution in Method I. Pipet 5 mL of the solution into a vial fitted with a septum and crimp cap, containing 1 g of anhydrous sodium sulfate, and seal. Heat the sealed vial at 80 for 60 minutes.
for 60 minutes. Test Solution— Transfer 100 mg, accurately weighed, of the material under test to a vial, add 5.0 mL of water, or the solvent specified in the monograph, and 1 g of anhydrous sodium sulfate, and seal with a septum and crimp cap. Heat the sealed vial at 80 for 60 minutes, or as specified in the individual monograph.
for 60 minutes, or as specified in the individual monograph. Chromatographic System and Procedure— [NOTE—The use of headspace apparatuses that automatically transfer a measured amount of headspace is allowed. Also, the use of a guard column in this headspace procedure is not necessary.] Proceed as directed for Method V, except to inject, using a heated gas-tight syringe, 1 mL of the headspace.
Method V
Standard Solution and Test Solution— Prepare as directed for Method I.
Chromatographic System (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector, a 0.53-mm × 30-m fused silica analytical column coated with a 3.0-µm G43 stationary phase, and a 0.53-mm × 5-m silica guard column deactivated with phenylmethyl siloxane. The carrier gas is helium with a linear velocity of about 35 cm per second. The injection port and detector temperatures are maintained at 140 and 260, respectively. The column temperature is programmed according to the following steps. It is maintained at 40 for 20 minutes, then increased rapidly to 240, and maintained at 240 for 20 minutes.
621)— The gas chromatograph is equipped with a flame-ionization detector, a 0.53-mm × 30-m fused silica analytical column coated with a 3.0-µm G43 stationary phase, and a 0.53-mm × 5-m silica guard column deactivated with phenylmethyl siloxane. The carrier gas is helium with a linear velocity of about 35 cm per second. The injection port and detector temperatures are maintained at 140 and 260, respectively. The column temperature is programmed according to the following steps. It is maintained at 40 for 20 minutes, then increased rapidly to 240, and maintained at 240 for 20 minutes. Inject the Standard Solution, and record the peak responses as directed for Procedure: a suitable system is one that yields chromatograms in which all of the components in the Standard Solution are resolved; the resolution, R, between any two components is not less than 3; and the relative standard deviation of the individual peak responses from replicate injections is not more than 15%.
Procedure— Proceed as directed for Method I, the injection volume being about 1 µL.
Method VI
Standard Solution and Test Solution— Prepare as directed for Method I.
Chromatographic System (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector. The column and column temperature conditions, as chosen from the list below (see Table 6), are specified in the individual monograph. The carrier gas, linear velocity or flow rate, and detector and injection port temperatures are appropriate to the column dimensions and column temperatures chosen from the list below.
621)— The gas chromatograph is equipped with a flame-ionization detector. The column and column temperature conditions, as chosen from the list below (see Table 6), are specified in the individual monograph. The carrier gas, linear velocity or flow rate, and detector and injection port temperatures are appropriate to the column dimensions and column temperatures chosen from the list below. Inject the Standard Solution, and record the peak responses as directed for Procedure: a suitable system is one that yields the chromatograms in which all of the components in the Standard Solution are resolved; the resolution, R, between any two components is not less than 1.0; and the relative standard deviation of the individual peak responses from replicate injections is not more than 15%.
Procedure— Proceed as directed for Method I, the injection volume being about 1 µL.
Table 6. Chromatographic Conditions for Method VI
| Chromatographic Conditions | USP Column Designation | Column Size | Column Temperature |
| A | S3 | 3-mm × 2-m | 190 |
| B | S2 | 3-mm × 2.1-m | 160 |
| C | G16 | 0.53-mm × 30-m | 40 |
| D | G39 | 3-mm × 2-m | 65 |
| E | G16 | 3-mm × 2-m | 70 |
| F | S4 | 2-mm × 2.5-m | Hold 120 (35 min.) Gradient 120 –200(2/min.)Hold 20 min. |
| H | G14 | 2-mm × 2.5-m | Hold 45 (3 min.) Gradient 45 –120 (8/min.)Hold 15 min. |
| I | G27 | 0.53-mm × 30-m | Hold 35 (5 min.) 35 –175 (8 /min.)175 –260(35/min.)Hold 16 min. |
| J | G16 | 0.33-mm × 30-m | Hold 50 (20 min.) 50 –165 (6/min.) |
GLOSSARY
Genotoxic carcinogens: Carcinogens that produce cancer by affecting genes or chromosomes.
Lowest-observed-effect level (LOEL): The lowest dose of a substance in a study or group of studies that produces biologically significant increases in frequency or severity of any effects in exposed humans or animals.
Modifying factor: A factor determined by professional judgment of a toxicologist and applied to bioassay data so that the data can be safely related to humans.
Neurotoxicity: The ability of a substance to cause adverse effects on the nervous system.
No-observed-effect level (NOEL): The highest dose of a substance at which there are no biologically significant increases in frequency or severity of any effects in exposed humans or animals.
Permitted daily exposure (PDE): The maximum acceptable intake per day of a residual solvent in pharmaceutical products.
Reversible toxicity: The occurrence of harmful effects that are caused by a substance and that disappear after exposure to the substance ends.
Strongly suspected human carcinogen: A substance for which there is no epidemiological evidence of carcinogenesis but for which there are positive genotoxicity data and clear evidence of carcinogenesis in rodents.
Teratogenicity: The occurrence of structural malformations in a developing fetus when a substance is administered during pregnancy.
APPENDIX 1. LIST OF RESIDUAL SOLVENTS INCLUDED IN THIS GENERAL CHAPTER
| Solvent | Other Names | Structure | Class |
| Acetic acid | Ethanoic acid | CH3COOH | Class 3 |
| Acetone | 2-Propanone Propan-2-one | CH3COCH3 | Class 3 |
| Acetonitrile | CH3CN | Class 2 | |
| Anisole | Methoxybenzene |  | Class 3 |
| Benzene | Benzol |  | Class 1 |
| 1-Butanol | n-Butyl alcohol Butan-1-ol | CH3(CH2)3OH | Class 3 |
| 2-Butanol | sec-Butyl alcohol Butan-2-ol | CH3CH2CH(OH)CH3 | Class 3 |
| Butyl acetate | Acetic acid butyl ester | CH3COO(CH2)3CH3 | Class 3 |
| tert-Butylmethyl ether | 2-Methoxy-2-methylpropane | (CH3)3COCH3 | Class 3 |
| Carbon tetrachloride | Tetrachloromethane | CCl4 | Class 1 |
| Chlorobenzene |  | Class 2 | |
| Chloroform | Trichloromethane | CHCl3 | Class 2 |
| Cumene | Isopropylbenzene (1-Methylethyl)benzene |  | Class 3 |
| Cyclohexane | Hexamethylene |  | Class 2 |
| 1,2-Dichloroethane | sym-Dichloroethane Ethylene dichloride Ethylene chloride | CH2ClCH2Cl | Class 1 |
| 1,1-Dichloroethene | 1,1-Dichloroethylene Vinylidene chloride | H2C=CCl2 | Class 1 |
| 1,2-Dichloroethene | 1,2-Dichloroethylene Acetylene dichloride | ClHC=CHCl | Class 2 |
| 1,2-Dimethoxyethane | Ethyleneglycol dimethyl ether Monoglyme Dimethyl cellosolve | H3COCH2CH2OCH3 | Class 2 |
| N,N-Dimethylacetamide | DMA | CH3CON(CH3)2 | Class 2 |
| N,N-Dimethylformamide | DMF | HCON(CH3)2 | Class 2 |
| Dimethyl sulfoxide | Methylsulfinylmethane Methyl sulfoxide DMSO | (CH3)2SO | Class 3 |
| 1,4-Dioxane | p-Dioxane [1,4]Dioxane |  | Class 2 |
| Ethanol | Ethyl alcohol | CH3CH2OH | Class 3 |
| 2-Ethoxyethanol | Cellosolve | CH3CH2OCH2CH2OH | Class 2 |
| Ethyl acetate | Acetic acid ethyl ester | CH3COOCH2CH3 | Class 3 |
| Ethylene glycol | 1,2-Dihydroxyethane 1,2-Ethanediol | HOCH2CH2OH | Class 2 |
| Ethyl ether | Diethyl ether Ethoxyethane 1,1¢-Oxybisethane | CH3CH2OCH2CH3 | Class 3 |
| Ethyl formate | Formic acid ethyl ester | HCOOCH2CH3 | Class 3 |
| Formamide | Methanamide | HCONH2 | Class 2 |
| Formic acid | HCOOH | Class 3 | |
| Heptane | n-Heptane | CH3(CH2)5CH3 | Class 3 |
| Hexane | n-Hexane | CH3(CH2)4CH3 | Class 2 |
| Isobutyl acetate | Acetic acid isobutyl ester | CH3COOCH2CH(CH3)2 | Class 3 |
| Isopropyl acetate | Acetic acid isopropyl ester | CH3COOCH(CH3)2 | Class 3 |
| Methanol | Methyl alcohol | CH3OH | Class 2 |
| 2-Methoxyethanol | Methyl cellosolve | CH3OCH2CH2OH | Class 2 |
| Methyl acetate | Acetic acid methyl ester | CH3COOCH3 | Class 3 |
| 3-Methyl-1-butanol | Isoamyl alcohol Isopentyl alcohol 3-Methylbutan-1-ol | (CH3)2CHCH2CH2OH | Class 3 |
| Methylbutylketone | 2-Hexanone Hexan-2-one | CH3(CH2)3COCH3 | Class 2 |
| Methylcyclohexane | Cyclohexylmethane |  | Class 2 |
| Methylene chloride | Dichloromethane | CH2Cl2 | Class 2 |
| Methylethylketone | 2-Butanone MEK Butan-2-one | CH3CH2COCH3 | Class 3 |
| Methyl isobutyl ketone | 4-Methylpentan-2-one 4-Methyl-2-pentanone MIBK | CH3COCH2CH(CH3)2 | Class 3 |
| 2-Methyl-1-propanol | Isobutyl alcohol | (CH3)2CHCH2OH | Class 3 |
| 2-Methylpropan-1-ol | |||
| N-Methylpyrrolidone | 1-Methylpyrrolidin-2-one 1-Methyl-2-pyrrolidinone |  | Class 2 |
| Nitromethane | CH3NO2 | Class 2 | |
| Pentane | n-Pentane | CH3(CH2)3CH3 | Class 3 |
| 1-Pentanol | Amyl alcohol Pentan-1-ol Pentyl alcohol | CH3(CH2)3CH2OH | Class 3 |
| 1-Propanol | Propan-1-ol Propyl alcohol | CH3CH2CH2OH | Class 3 |
| 2-Propanol | Propan-2-ol Isopropyl alcohol | (CH3)2CHOH | Class 3 |
| Propyl acetate | Acetic acid propyl ester | CH3COOCH2CH2CH3 | Class 3 |
| Pyridine |  | Class 2 | |
| Sulfolane | Tetrahydrothiophene 1,1-diox-ide |  | Class 2 |
| Tetrahydrofuran | Tetramethylene oxide |  | Class 2 |
| Oxacyclopentane | |||
| Tetralin | 1,2,3,4-Tetrahydronaphthalene |  | Class 2 |
| Toluene | Methylbenzene |  | Class 2 |
| 1,1,1-Trichloroethane | Methylchloroform | CH3CCl3 | Class 1 |
| Trichloroethene | 1,1,2-Trichloroethene | HClC=CCl2 | Class 2 |
| Xylene* | Dimethybenzene Xylol |  | Class 2 |
| * Usually 60% m-xylene, 14% p-xylene, 9% o-xylene with 17% ethyl benzene. | |||
APPENDIX 2. ADDITIONAL BACKGROUND
A2.1. Environmental Regulation of Organic Volatile Solvents
Several of the residual solvents frequently used in the production of pharmaceuticals are listed as toxic chemicals in Environmental Health Criteria (EHC) monographs and in the Integrated Risk Information System (IRIS). The objectives of such groups as the International Programme on Chemical Safety (IPCS), the United States Environmental Protection Agency (EPA), and the United States Food and Drug Administration (FDA) include the determination of acceptable exposure levels. The goal is maintenance of environmental integrity and protection of human health against the possible deleterious effects of chemicals resulting from long-term environmental exposure. The procedures involved in the estimation of maximum safe exposure limits are usually based on long-term studies. When long-term study data are unavailable, shorter term study data can be used with modification of the approach, such as use of larger safety factors. The approach described therein relates primarily to long-term or lifetime exposure of the general population in the ambient environment (i.e., ambient air, food, drinking water, and other media).
A2.2. Residual Solvents in Pharmaceuticals
Exposure limits in this General Chapter are established by referring to methodologies and toxicity data described in EHC and IRIS monographs. However, the following specific assumptions about residual solvents to be used in the synthesis and formulation of pharmaceutical products should be taken into account in establishing exposure limits.
- Patients (not the general population) use pharmaceuticals to treat their diseases or for prophylaxis to prevent infection or disease.
- The assumption of lifetime patient exposure is not necessary for most pharmaceutical products but may be appropriate as a working hypothesis to reduce risk to human health.
- Residual solvents are unavoidable components in pharmaceutical production and will often be a part of medicinal products.
- Residual solvents should not exceed recommended levels except in exceptional circumstances.
- Data from toxicological studies that are used to determine acceptable levels for residual solvents should have been generated using appropriate protocols such as those described, for example, by the Organization for Economic Cooperation and Development (OECD), EPA, and the FDA Red Book.
APPENDIX 3. PROCEDURES FOR ESTABLISHING EXPOSURE LIMITS
The Gaylor-Kodell method of risk assessment (Gaylor, D. W. and Kodell, R. L. Linear Interpolation Algorithm for Low Dose Assessment of Toxic Substance. Journal of Environmental Pathology and Toxicology, 4:305, 1980) is appropriate for Class 1 carcinogenic solvents. Only in cases where reliable carcinogenicity data are available should extrapolation by the use of mathematical models be applied to setting exposure limits. Exposure limits for Class 1 residual solvents could be determined with the use of a large safety factor (i.e., 10,000 to 100,000) with respect to the no-observed-effect level (NOEL). Detection and quantification of these residual solvents should be performed by state-of-the-art analytical techniques.
Acceptable exposure levels in this General Chapter for Class 2 residual solvents were established by calculation of PDE values according to the procedures for setting exposure limits in pharmaceuticals (page 5748 of PF 15(6) [Nov.–Dec. 1989]), and the method adopted by IPCS for Assessing Human Health Risk of Chemicals (Environmental Health Criteria 170, WHO, 1994). These procedures are similar to those used by the U.S. EPA (IRIS) and the U.S. FDA (Red Book) and others. The method is outlined here to give a better understanding of the origin of the PDE values. It is not necessary to perform these calculations in order to use the PDE values presented in Table 2 of this document.
PDE is derived from the no-observed-effect level (NOEL), or the lowest-observed effect level (LOEL), in the most relevant animal study as follows:
The PDE is derived preferably from a NOEL. If no NOEL is obtained, the LOEL may be used. Modifying factors proposed here, for relating the data to humans, are the same kind of “uncertainty factors” used in Environmental Health Criteria (Environmental Health Criteria 170, WHO, Geneva, 1994) and “modifying factors” or “safety factors” in Pharmacopeial Forum. The assumption of 100 percent systemic exposure is used in all calculations regardless of route of administration.
The modifying factors are as follows:
| F1 = | A factor to account for extrapolation between species | |
| F1 = | 2 for extrapolation from dogs to humans | |
| F1 = | 2.5 for extrapolation from rabbits to humans | |
| F1 = | 3 for extrapolation from monkeys to humans | |
| F1 = | 5 for extrapolation from rats to humans | |
| F1 = | 10 for extrapolation from other animals to humans | |
| F1 = | 12 for extrapolation from mice to humans | |
F1 takes into account the comparative surface area to body weight ratios for the species concerned and for man. Surface area (S) is calculated as:
S = kM 0.67,(2)
(2)in which M = body weight, and the constant k has been taken to be 10. The body weights used in the equation are those shown below in Table A3.-1.
| F2 = | A factor of 10 to account for variability between individuals. A factor of 10 is generally given for all organic solvents, and 10 is used consistently in this General Chapter. |
| F3 = | A variable factor to account for toxicity studies of short-term exposure. | |
| F3 = | 1 for studies that last at least one half-lifetime (1 year for rodents or rabbits; 7 years for cats, dogs, and monkeys). | |
| F3 = | 1 for reproductive studies in which the whole period of organogenesis is covered. | |
| F3 = | 2 for a 6-month study in rodents, or a 3.5-year study in nonrodents. | |
| F3 = | 5 for a 3-month study in rodents, or a 2-year study in nonrodents. | |
| F3 = | 10 for studies of a shorter duration. | |
In all cases, the higher factor has been used for study durations between the time points (e.g., a factor of 2 for a 9-month rodent study).
| F4 = | A factor that may be applied in cases of severe toxicity, e.g., nongenotoxic carcinogenicity, neurotoxicity, or teratogenicity. In studies of reproductive toxicity, the following factors are used: | |
| F4 = | 1 for fetal toxicity associated with maternal toxicity | |
| F4 = | 5 for fetal toxicity without maternal toxicity | |
| F4 = | 5 for a teratogenic effect with maternal toxicity | |
| F4 = | 10 for a teratogenic effect without maternal toxicity | |
| F5 = | A variable factor that may be applied if the no-effect level was not established. |
When only a LOEL is available, a factor of up to 10 can be used depending on the severity of the toxicity. The weight adjustment assumes an arbitrary adult human body weight for either sex of 50 kilograms (kg). This relatively low weight provides an additional safety factor against the standard weights of 60 kg or 70 kg that are often used in this type of calculation. It is recognized that some adult patients weigh less than 50 kg; these patients are considered to be accommodated by the built-in safety factors used to determine a PDE. If the solvent was present in a formulation specifically intended for pediatric use, an adjustment for a lower body weight would be appropriate.
As an example of the application of this equation, consider a toxicity study of acetonitrile in mice that is summarized in Pharmeuropa, Vol. 9, No. 1, Supplement, April 1997, page S24. The NOEL is calculated to be 50.7 mg kg–1 day–l. The PDE for acetonitrile in this study is calculated as follows:
In this example,
| F1 = | 12 to account for the extrapolation from mice to humans |
| F2 = | 10 to account for differences between individual humans |
| F3 = | 5 because the duration of the study was only 13 weeks |
| F4 = | 1 because no severe toxicity was encountered |
| F5 = | 1 because the no-effect level was determined |
A3.-1. - Values Used in the Calculations in This Document
| Rat body weight | 425 g |
| Pregnant rat body weight | 330 g |
| Mouse body weight | 28 g |
| Pregnant mouse body weight | 30g |
| Guinea-pig body weight | 500 g |
| Rhesus monkey body weight | 2.5 kg |
| Rabbit body weight (pregnant or not) | 4 kg |
| Beagle dog body weight | 11.5 kg |
| Rat respiratory volume | 290 L/day |
| Mouse respiratory volume | 43 L/day |
| Rabbit respiratory volume | 1440 L/day |
| Guinea-pig respiratory volume | 430 L day |
| Human respiratory volume | 28,800 L/day |
| Dog respiratory volume | 9000 L/day |
| Monkey respiratory volume | 1150 L/day |
| Mouse water consumption | 5 mL/day |
| Rat water consumption | 30 mL/day |
| Rat food consumption | 30 g/day |
The equation for an ideal gas, PV = nRT, is used to convert concentrations of gases used in inhalation studies from units of ppm to units of mg/L or mg/m3. Consider as an example the rat reproductive toxicity study by inhalation of carbon tetrachloride (molecular weight 153.84) summarized in Pharmeuropa, Vol. 9, No. 1, Supplement, April 1997, page S9.
The relationship 1000 L = 1 m3 is used to convert to mg/ m3.
541 TITRIMETRY Direct Titrations— Direct titration is the treatment of a soluble substance, contained in solution in a suitable vessel (the titrate), with an appropriate standardized solution (the titrant), the endpoint being determined instrumentally or visually with the aid of a suitable indicator.
The titrant is added from a suitable buret and is so chosen, with respect to its strength (normality), that the volume added is between 30% and 100% of the rated capacity of the buret. [NOTE—Where less than 10 mL of titrant is required, a suitable microburet is to be used.] The endpoint is approached directly but cautiously, and finally the titrant is added dropwise from the buret in order that the final drop added will not overrun the endpoint. The quantity of the substance being titrated may be calculated from the volume and the normality or molarity factor of the titrant and the equivalence factor for the substance given in the individual monograph.
Residual Titrations— Some Pharmacopeial assays require the addition of a measured volume of a volumetric solution, in excess of the amount actually needed to react with the substance being assayed, the excess of this solution then being titrated with a second volumetric solution. This constitutes a residual titration and is known also as a “back titration.” The quantity of the substance being titrated may be calculated from the difference between the volume of the volumetric solution originally added, corrected by means of a blank titration, and that consumed by the titrant in the back titration, due allowance being made for the respective normality or molarity factors of the two solutions, and the equivalence factor for the substance given in the individual monograph.
Complexometric Titrations— Successful complexometric titrations depend on several factors. The equilibrium constant for formation of the titrant-analyte complex must be sufficiently large that, at the endpoint, very close to 100% of the analyte has been complexed. The final complex must be formed rapidly enough that the analysis time is practical. When the analytical reaction is not rapid, a residual titration may sometimes be successful.
In general, complexometric indicators are themselves complexing agents. The reaction between metal ion and indicator must be rapid and reversible. The equilibrium constant for formation of the metal-indicator complex should be large enough to produce a sharp color change but must be less than that for the metal-titrant complex. Indicator choice is also restricted by the pH range within which the complexation reaction must be carried out and by interference of other ions arising from the sample or the buffer. Interfering ions may often be masked or “screened” via addition of another complexing agent. (The masking technique is also applicable to redox titrations.)
Oxidation-Reduction (Redox) Titrations— Determinations may often be carried out conveniently by the use of a reagent that brings about oxidation or reduction of the analyte. Many redox titration curves are not symmetric about the equivalence point, and thus graphical determination of the endpoint is not possible; but indicators are available for many determinations, and a redox reagent can often serve as its own indicator. As in any type of titration, the ideal indicator changes color at an endpoint that is as close as possible to the equivalence point. Accordingly, when the titrant serves as its own indicator, the difference between the endpoint and the equivalence point is determined only by the analyst's ability to detect the color change. A common example is the use of permanganate ion as an oxidizing titrant since a slight excess can easily be detected by its pink color. Other titrants that may serve as their own indicators are iodine, cerium (IV) salts, and potassium dichromate. In most cases, however, the use of an appropriate redox indicator will yield a much sharper endpoint.
It may be necessary to adjust the oxidation state of the analyte prior to titration through use of an appropriate oxidizing or reducing agent; the excess reagent must then be removed, e.g., through precipitation. This is nearly always the practice in the determination of oxidizing agents since most volumetric solutions of reducing agents are slowly oxidized by atmospheric oxygen.
Titrations in Nonaqueous Solvents— Acids and bases have long been defined as substances that furnish, when dissolved in water, hydrogen and hydroxyl ions, respectively. This definition, introduced by Arrhenius, fails to recognize the fact that properties characteristic of acids or bases may be developed also in other solvents. A more generalized definition is that of Brönsted, who defined an acid as a substance that furnishes protons, and a base as a substance that combines with protons. Even broader is the definition of Lewis, who defined an acid as any material that will accept an electron pair, a base as any material that will donate an electron pair, and neutralization as the formation of a coordination bond between an acid and a base.
The apparent strength of an acid or a base is determined by the extent of its reaction with a solvent. In water solution all strong acids appear equally strong because they react with the solvent to undergo almost complete conversion to oxonium ion and the acid anion (leveling effect). In a weakly protophilic solvent such as acetic acid the extent of formation of the acetate acidium ion shows that the order of decreasing strength for acids is perchloric, hydrobromic, sulfuric, hydrochloric, and nitric (differentiating effect).
Acetic acid reacts incompletely with water to form oxonium ion and is, therefore, a weak acid. In contrast, it dissolves in a base such as ethylenediamine, and reacts so completely with the solvent that it behaves as a strong acid. The same holds for perchloric acid.
This leveling effect is observed also for bases. In sulfuric acid almost all bases appear to be of the same strength. As the acid properties of the solvent decrease in the series sulfuric acid, acetic acid, phenol, water, pyridine, and butylamine, the bases become progressively weaker until all but the strongest have lost their basic properties. In order of decreasing strength, the strong bases are sodium 2-aminoethoxide, potassium methoxide, sodium methoxide, and lithium methoxide.
Many water-insoluble compounds acquire enhanced acidic or basic properties when dissolved in organic solvents. Thus the choice of the appropriate solvent permits the determination of a variety of such materials by nonaqueous titration. Furthermore, depending upon which part of a compound is the physiologically active moiety, it is often possible to titrate that part by proper selection of solvent and titrant. Pure compounds can be titrated directly, but it is often necessary to isolate the active ingredient in pharmaceutical preparations from interfering excipients and carriers.
The types of compounds that may be titrated as acids include acid halides, acid anhydrides, carboxylic acids, amino acids, enols such as barbiturates and xanthines, imides, phenols, pyrroles, and sulfonamides. The types of compounds that may be titrated as bases include amines, nitrogen-containing heterocyclic compounds, oxazolines, quaternary ammonium compounds, alkali salts of organic acids, alkali salts of weak inorganic acids, and some salts of amines. Many salts of halogen acids may be titrated in acetic acid or acetic anhydride after the addition of mercuric acetate, which removes halide ion as the unionized mercuric halide complex and introduces the acetate ion.
For the titration of a basic compound, a volumetric solution of perchloric acid in glacial acetic acid is preferred, although perchloric acid in dioxane is used in special cases. The calomel-glass electrode system is useful in this case. In acetic acid solvent, this electrode system functions as predicted by theory.
For the titration of an acidic compound, two classes of titrant are available: the alkali metal alkoxides and the tetraalkylammonium hydroxides. A volumetric solution of sodium methoxide in a mixture of methanol and toluene is used frequently, although lithium methoxide in methanol-benzene solvent is used for those compounds yielding a gelatinous precipitate on titration with sodium methoxide.
The alkali error limits the use of the glass electrode as an indicating electrode in conjunction with alkali metal alkoxide titrants, particularly in basic solvents. Thus, the antimony-indicating electrode, though somewhat erratic, is used in such titrations. The use of quaternary ammonium hydroxide compounds, e.g., tetra-n-butylammonium hydroxide and trimethylhexadecylammonium hydroxide (in benzene-methanol or isopropyl alcohol), has two advantages over the other titrants in that (a) the tetraalkylammonium salt of the titrated acid is soluble in the titration medium, and (b) the convenient and well-behaved calomel-glass electrode pair may be used to conduct potentiometric titrations.
Because of interference by carbon dioxide, solvents for acidic compounds need to be protected from excessive exposure to the atmosphere by a suitable cover or by an inert atmosphere during the titration. Absorption of carbon dioxide may be determined by performing a blank titration. The blank should not exceed 0.01 mL of 0.1 N sodium methoxide VS per mL of solvent.
The endpoint may be determined visually by color change, or potentiometrically, as indicated in the individual monograph. If the calomel reference electrode is used, it is advantageous to replace the aqueous potassium chloride salt bridge with 0.1 N lithium perchlorate in glacial acetic acid for titrations in acidic solvents or potassium chloride in methanol for titrations in basic solvents.
Where these or other mixtures are specified in individual monographs, the calomel reference electrode is modified by first removing the aqueous potassium chloride solution and residual potassium chloride, if any, by rinsing with water, then eliminating residual water by rinsing with the required nonaqueous solvent, and finally filling the electrode with the designated nonaqueous mixture.
In nearly all cases, except those where silver ion might interfere, a silver-silver chloride reference electrode may be substituted for the calomel electrode. The silver-silver chloride electrode is more rugged, and its use helps to eliminate toxic mercury salts from the laboratory. Generally, a salt bridge may be used to circumvent interference by silver ion.
The more useful systems for titration in nonaqueous solvents are listed in Table 1.
Table 1. Systems for Nonaqueous Titrations
| Type of Solvent | Acidic (for titrationof bases and their salts) | Relatively Neutral (for differential titration of bases) | Basic (for titrationof acids) | Relatively Neutral (for differential titration of acids) |
Solvent1 | Glacial Acetic Acid Acetic Anhydride Formic Acid Propionic Acid Sulfuryl Chloride | AcetonitrileAlcoholsChloroformBenzeneTolueneChlorobenzeneEthyl AcetateDioxane | Dimethylformamide n-Butylamine Pyridine Ethylenediamine Morpholine | Acetone Acetonitrile Methyl Ethyl Ketone Methyl Isobutyl Ketone tert-Butyl Alcohol |
| Indicator | Crystal Violet Quinaldine Red p-Naphtholbenzein Alphezurine 2-G Malachite Green | Methyl RedMethyl Orangep-Naphtholbenzein | Thymol Blue Thymolphthalein Azo Violet o-Nitroaniline p-Hydroxyazobenzene | Azo Violet Bromothylmol Blue p-Hydroxyazobenzene Thymol Blue |
| Electrodes | Glass–calomel Glass–silver–silver chlorideMercury–mercuric acetate | Glass–calomelCalomel–silver–silverchloride | Antimony–calomel Antimony–glass Antimony–antimony2 Platinum–calomel Glass–calomel | Antimony–calomel Glass–calomel Glass–platinum2 |
| 1 Relatively neutral solvents of low dielectric constant such as benzene, toluene, chloroform, or dioxane may be used in conjunction with any acidic or basic solvent in order to increase the sensitivity of the titration end-points. | ||||
| 2 In titrant. | ||||
Indicator and Potentiometric Endpoint Detection— The simplest and most convenient method by which the equivalence point, i.e., the point at which the stoichiometric analytical reaction is complete, may be determined is with the use of indicators. These chemical substances, usually colored, respond to changes in solution conditions before and after the equivalence point by exhibiting color changes that may be taken visually as the endpoint, a reliable estimate of the equivalence point.
A useful method of endpoint determination results from the use of electrochemical measurements. If an indicator electrode, sensitive to the concentration of the species undergoing titrimetric reaction, and a reference electrode, whose potential is insensitive to any dissolved species, are immersed in the titrate to form a galvanic cell, the potential difference between the electrodes may be sensed by a pH meter and used to follow the course of the reaction. Where such a series of measurements is plotted correctly (i.e., for an acid-base titration, pH versus mL of titrant added; for a precipitimetric, complexometric, or oxidation-reduction titration, mV versus mL of titrant added), a sigmoid curve results with a rapidly changing portion (the “break”) in the vicinity of the equivalence point. The midpoint of this linear vertical portion or the inflection point may be taken as the endpoint. The equivalence point may also be determined mathematically without plotting a curve. However, it should be noted that in asymmetrical reactions, which are reactions in which the number of anions reacting is not the same as the number of cations reacting, the endpoint as defined by the inflection of the titration curve does not occur exactly at the stoichiometric equivalence point. Thus, potentiometric endpoint detection by this method is not suitable in the case of asymmetric reactions, examples of which are the precipitation reaction,
2Ag+ + CrO4–2
and the oxidation-reduction reaction,
5Fe+2 + MnO4–.
All acid-base reactions, however, are symmetrical. Thus, potentiometric endpoint detection may be employed in acid-base titrations and in other titrations involving symmetrical reversible reactions where an indicator is specified, unless otherwise directed in the individual monograph.
Two types of automatic electrometric titrators are available. The first is one that carries out titrant addition automatically and records the electrode potential differences during the course of titration as the expected sigmoid curve. In the second type, titrant addition is performed automatically until a preset potential or pH, representing the endpoint, is reached, at which point the titrant addition ceases.
Several acceptable electrode systems for potentiometric titrations are summarized in Table 2.
Table 2. Potentiometric Titration Electrode Systems
| Titration | Indicating Electrode | Equation1 | Reference Electrode | Applicability2 |
| Acid-base | Glass | E = k + 0.0591 pH | Calomel or silver–silver Chloride | Titration of acids and bases |
| Precipitimetric (silver) | Silver | E = E + 0.0591 log [Ag +] | Calomel (withpotassium nitratesalt bridge) | Titration with or of silverinvolving halidesor thiocyanate |
| Complexometric | Mercury–mercury(II) | E = E + 0.0296(log k¢ pM) | Calomel | Titration of various metals(M), e.g., Mg+2, Ca+2Al+3, Bi+3, with EDTA |
| Oxidation–reduction | Platinum | E = E + (0.0591/n)×log [ox]/[red] | Calomel or silver–silverchloride | Titrations with arsenite,bromine, cerate,dichromate,exacyonoferrate(III),iodate, nitrite,permanganate,thiosulfate |
| 1 Appropriate form of Nernst equation describing the indicating electrode system: k = glass electrode constant; k¢ = constant derived from Hg–Hg(II)–EDTA equilibrium; M = any metal undergoing EDTA titration; [ox] and [red] from the equation, ox + ne  red. 2 Listing is repesentative but not exhaustive. | ||||
Blank Corrections— As previously noted, the endpoint determined in a titrimetric assay is an estimate of the reaction equivalence point. The validity of this estimate depends upon, among other factors, the nature of the titrate constituents and the concentration of the titrant. An appropriate blank correction is employed in titrimetric assays to enhance the reliability of the endpoint determination. Such a blank correction is usually obtained by means of a residual blank titration, wherein the required procedure is repeated in every detail except that the substance being assayed is omitted. In such instances, the actual volume of titrant equivalent to the substance being assayed is the difference between the volume consumed in the residual blank titration and that consumed in the titration with the substance present. The corrected volume so obtained is used in calculating the quantity of the substance being titrated, in the same manner as prescribed under Residual Titrations. Where potentiometric endpoint detection is employed, the blank correction is usually negligible.
| | |
The bulk density of a solid is often very difficult to measure since the slightest disturbance of the bed may result in a new bulk density. Moreover, it is clear that the bulking properties of a powder are dependent on the “history” of the powder (e.g., how it was handled), and that it can be packed to have a range of bulk densities. Thus, it is essential in reporting bulk density to specify how the determination was made.
Because the interparticulate interactions that influence the bulking properties of a powder are also the interactions that interfere with powder flow, a comparison of the bulk and tapped densities can give a measure of the relative importance of these interactions in a given powder. Such a comparison is often used as an index of the ability of the powder to flow. The bulk density often is the bulk density of the powder “as poured” or as passively filled into a measuring vessel. The tapped density is a limiting density attained after “tapping down,” usually in a device that lifts and drops a volumetric measuring cylinder containing the powder a fixed distance.
BULK DENSITY
Bulk density is determined by measuring the volume of a known mass of powder sample that has been passed through a screen into a graduated cylinder (Method I) or through a volume-measuring apparatus into a cup (Method II).
Method I—Measurement in a Graduated Cylinder
Procedure— Unless otherwise specified, pass a quantity of material sufficient to complete the test through a 1.00-mm (No. 18) screen to break up agglomerates that may have formed during storage. Into a dry 250-mL cylinder introduce, without compacting, approximately 100 g of test sample, M, weighed with 0.1% accuracy. If it is not possible to use 100 g, the amount of the test sample and the volume of the cylinder may be modified and the test conditions specified with the results. Select a sample mass having an untapped apparent volume of 150 to 250 mL. A 100-mL cylinder is used for apparent volumes between 50 mL and 100 mL. Carefully level the powder without compacting, if necessary, and read the unsettled apparent volume, Vo, to the nearest graduated unit. Calculate the bulk density, in g per mL1 , by the formula:
(M) / (Vo).
Generally replicate determinations are desirable for the determination of this property. Method II—Measurement in a Volumeter
The apparatus (Fig. 1),
Fig. 1. Scott Volumeter.
conforming to the dimensions in ASTM B 329-90 (Scott Volumeter)2 , consists of a top funnel fitted with a 1.00-mm (No. 18) screen or the screen opening specified in the individual monograph. The funnel is mounted over a baffle box containing four glass baffle plates over which the powder slides and bounces as it passes. At the bottom of the baffle box is a funnel that collects the powder and allows it to pour into a cup of specified capacity mounted directly below it. The cup may be cylindrical (25.00 ± 0.05 mL volume with an inside diameter of 30.00 ± 2.00 mm) or a square (16.39 ± 0.05 mL volume with inside dimensions of 25.4 ± 0.076 mm).
Procedure— Allow an excess of powder to flow through the apparatus into the sample receiving cup until it overflows, using a minimum of 25 cm3 of powder with the square cup and 35 cm3 of powder with the cylindrical cup. Carefully scrape excess powder from the top of the cup by smoothly moving the edge of the blade of a spatula perpendicular to and in contact with the top surface of the cup, taking care to keep the spatula perpendicular to prevent packing or removal of powder from the cup. Remove any material from the sides of the cup, and determine the weight, M, of the powder to the nearest 0.1%. Calculate the bulk density, in g per mL, by the formula:
(M ) / (Vo),
in which Vo is the volume, in mL, of the cup. Generally replicate determinations are desirable for the determination of this property. TAPPED DENSITY
Tapped density is achieved by mechanically tapping a measuring cylinder containing a powder sample. After observing the initial volume, the cylinder is mechanically tapped, and volume readings are taken until little further volume change is observed. The mechanical tapping is achieved by raising the cylinder and allowing it to drop under its own weight a specified distance by either of two methods as described below. Devices that rotate the cylinder during tapping may be preferred to minimize any possible separation of the mass during tapping down.
Method I
Procedure— Unless otherwise specified, pass a quantity of material sufficient to complete the test through a 1.00-mm (No. 18) screen to break up agglomerates that may have formed during storage. Into a dry 250-mL glass graduated cylinder (readable to 2 mL) weighing 220 ± 44 g and mounted on a holder weighing 450 ± 10 g introduce, without compacting, approximately 100 g of test sample, M, weighed with 0.1% accuracy. If it is not possible to use 100 g, the amount of the test sample may be reduced and the volume of the cylinder may be modified by using a suitable 100-mL graduated cylinder (readable to 1 mL) weighing 130 ± 16 g and mounted on a holder weighing 240 ± 12 g. The modified test conditions are specified with the results. Carefully level the powder without compacting, if necessary, and read the unsettled apparent volume, Vo, to the nearest graduated unit.
Mechanically tap the cylinder containing the sample by raising the cylinder and allowing it to drop under its own weight using a suitable mechanical tapped density tester that provides a fixed drop of 14 ± 2 mm at a nominal rate of 300 drops per minute. Unless otherwise specified, tap the cylinder 500 times initially and measure the tapped volume, Va, to the nearest graduated unit. Repeat the tapping an additional 750 times and measure the tapped volume, Vb, to the nearest graduated unit. [NOTE—Fewer taps may be appropriate, if validated, for some powders.] If the difference between the two volumes is less than 2%, Vb is the final tapped volume, Vf . Repeat in increments of 1250 taps, as needed, until the difference between succeeding measurements is less than 2%. Calculate the tapped density, in g per mL, by the formula:
(M ) / (Vf).
Generally replicate determinations are desirable for the determination of this property.
Method II
Proceed as directed under Method I except that a suitable mechanical tapped density tester that provides a fixed drop of 3 mm (±10%) at a nominal rate of 250 drops per minute is used.
MEASURES OF POWDER COMPRESSIBILITY
The Compressibility Index and Hausner Ratio are measures of the propensity of a powder to be compressed. As such, they are measures of the relative importance of interparticulate interactions. In a free-flowing powder, such interactions are generally less significant, and the bulk and tapped densities will be closer in value. For poorer flowing materials, there are frequently greater interparticle interactions, and a greater difference between the bulk and tapped densities will be observed. These differences are reflected in the Compressibility Index and the Hausner Ratio.
Compressibility Index— Calculate by the formula: 
Hausner Ratio— Calculate by the formula: 
1 The density of solids normally is expressed in g per cm3 and that of liquids is normally expressed in g per mL; however, because powder volumes are measured in cylinders graduated in mL, bulk and tapped densities will be expressed in g per mL. By definition, the mL and the cm3 are equivalent volumes.
2 Apparatus may be purchased from scientific supply companies and is usually described as a “Scott, Schaeffer and White Paint Pigment Volumeter.”
INTRODUCTION
This chapter defines the terms and procedures used in chromatography and provides general information. Specific requirements for chromatographic procedures for drug substances and dosage forms, including adsorbent and developing solvents, are given in the individual monographs.
Chromatography is defined as a procedure by which solutes are separated by a dynamic differential migration process in a system consisting of two or more phases, one of which moves continuously in a given direction and in which the individual substances exhibit different mobilities by reason of differences in adsorption, partition, solubility, vapor pressure, molecular size, or ionic charge density. The individual substances thus separated can be identified or determined by analytical procedures.
The general chromatographic technique requires that a solute undergo distribution between two phases, one of them fixed (stationary phase), the other moving (mobile phase). It is the mobile phase that transfers the solute through the medium until it eventually emerges separated from other solutes that are eluted earlier or later. Generally, the solute is transported through the separation medium by means of a flowing stream of a liquid or a gaseous solvent known as the “eluant.” The stationary phase may act through adsorption, as in the case of adsorbents such as activated alumina and silica gel, or it may act by dissolving the solute, thus partitioning the latter between the stationary and mobile phases. In the latter process, a liquid coated onto an inert support, or chemically bonded onto silica gel, or directly onto the wall of a fused silica capillary, serves as the stationary phase. Partitioning is the predominant mechanism of separation in gas–liquid chromatography, paper chromatography, in forms of column chromatography and in thin-layer chromatography designated as liquid-liquid separation. In practice, separations frequently result from a combination of adsorption and partitioning effects. Other separation principles include ion exchange, ion-pair formation, size exclusion, hydrophobic interaction, and chiral recognition.
The types of chromatography useful in qualitative and quantitative analysis that are employed in the USP procedures are column, gas, paper, thin-layer, (including high-performance thin-layer chromatography), and pressurized liquid chromatography (commonly called high-pressure or high-performance liquid chromatography). Paper and thin-layer chromatography are ordinarily more useful for purposes of identification, because of their convenience and simplicity. Column chromatography offers a wider choice of stationary phases and is useful for the separation of individual compounds, in quantity, from mixtures. Modern high-performance thin-layer chromatography, gas chromatography, and pressurized liquid chromatography require more elaborate apparatus but usually provide high resolution and identify and quantitate very small amounts of material.
Use of Reference Substances in Identity Tests— In paper and thin-layer chromatography, the ratio of the distance (this distance being measured to the point of maximum intensity of the spot or zone) traveled on the medium by a given compound to the distance traveled by the front of the mobile phase, from the point of application of the test substance, is designated as the RF value of the compound. The ratio between the distances traveled by a given compound and a reference substance is the RR value. RF values vary with the experimental conditions, and thus identification is best accomplished where an authentic specimen of the compound in question is used as a reference substance on the same chromatogram.
For this purpose, chromatograms are prepared by applying on the thin-layer adsorbent or on the paper in a straight line, parallel to the edge of the chromatographic plate or paper, solutions of the substance to be identified, the authentic specimen, and a mixture of nearly equal amounts of the substance to be identified and the authentic specimen. Each sample application contains approximately the same quantity by weight of material to be chromatographed. If the substance to be identified and the authentic specimen are identical, all chromatograms agree in color and RF value and the mixed chromatogram yields a single spot; i.e., RR is 1.0.
Location of Components— The spots produced by paper or thin-layer chromatography may be located by: (1) direct inspection if the compounds are visible under white or either short-wavelength (254 nm) or long-wavelength (360 nm) UV light, (2) inspection in white or UV light after treatment with reagents that will make the spots visible (reagents are most conveniently applied with an atomizer), (3) use of a Geiger-Müller counter or autoradiographic techniques in the case of the presence of radioactive substances, or (4) evidence resulting from stimulation or inhibition of bacterial growth by the placing of removed portions of the adsorbent and substance on inoculated media.
In open-column chromatography, in pressurized liquid chromatography performed under conditions of constant flow rate, and in gas chromatography, the retention time, t, defined as the time elapsed between sample injection and appearance of the peak concentration of the eluted sample zone, may be used as a parameter of identification. Solutions of the substance to be identified or derivatives thereof, of the reference compound, and of a mixture of equal amounts of these two are chromatographed successively on the same column under the same chromatographic conditions. Only one peak should be observed for the mixture. The ratio of the retention times of the test substance, the reference compound, and a mixture of these, to the retention time of an internal standard is called the relative retention time RR and is also used frequently as a parameter of identification.
The deviations of RR, RF, or t values measured for the test substance from the values obtained for the reference compound and mixture should not exceed the reliability estimates determined statistically from replicate assays of the reference compound.
Chromatographic identification by these methods under given conditions strongly indicates identity but does not constitute definitive identification. Coincidence of identity parameters under three to six different sets of chromatographic conditions (temperatures, column packings, adsorbents, eluants, developing solvents, various chemical derivatives, etc.) increases the probability that the test and reference substances are identical. However, many isomeric compounds cannot be separated. Specific and pertinent chemical, spectroscopic, or physicochemical identification of the eluted component combined with chromatographic identity is the most valid criterion of identification. For this purpose, the individual components separated by chromatography may be collected for further identification.
PAPER CHROMATOGRAPHY
In paper chromatography the adsorbent is a sheet of paper of suitable texture and thickness. Chromatographic separation may proceed through the action of a single liquid phase in a process analogous to adsorption chromatography in columns. Since the natural water content of the paper, or selective imbibition of a hydrophilic component of the liquid phase by the paper fibers, may be regarded as a stationary phase, a partitioning mechanism may contribute significantly to the separation.
Alternatively, a two-phase system may be used. The paper is impregnated with one of the phases, which then remains stationary (usually the more polar phase in the case of unmodified paper). The chromatogram is developed by slow passage of the other, mobile phase over the sheet. Development may be ascending, in which case the solvent is carried up the paper by capillary forces, or descending, in which case the solvent flow is also assisted by gravitational force.
Differences in the value of RF have been reported where chromatograms developed in the direction of the paper grain (machine direction) are compared with others developed at right angles to the grain; therefore, the orientation of paper grain with respect to solvent flow should be maintained constant in a series of chromatograms. (The machine direction is usually designated by the manufacturer on packages of chromatography paper.)
Descending Chromatography
In descending chromatography, the mobile phase flows downward on the chromatographic sheet.
Apparatus— The essential equipment for descending chromatography consists of the following:
A vapor-tight chamber provided with inlets for addition of solvent or for releasing internal pressure. The chamber is constructed preferably of glass, stainless steel, or porcelain and is so designed as to permit observation of the progress of the chromatographic run without opening of the chamber. Tall glass cylinders are convenient if they are made vapor-tight with suitable covers and a sealing compound.
A rack of corrosion-resistant material about 5 cm shorter than the inside height of the chamber. The rack serves as a support for solvent troughs and for antisiphon rods which, in turn, hold up the chromatographic sheets.
One or more glass troughs capable of holding a volume of solvent greater than that needed for one chromatographic run. The troughs must also be longer than the width of the chromatographic sheets.
Heavy glass antisiphon rods to be supported by the rack and running outside of, parallel to, and slightly above the edge of the glass trough.
Chromatographic sheets of special filter paper at least 2.5 cm wide and not wider than the length of the troughs are cut to a length approximately equal to the height of the chamber. A fine pencil line is drawn horizontally across the filter paper at a distance from one end such that, when the sheet is suspended from the antisiphon rods with the upper end of the paper resting in the trough and the lower portion hanging free into the chamber, the line is located a few centimeters below the rods. Care is necessary to avoid contaminating the filter paper by excessive handling or by contact with dirty surfaces.
Procedure— The substance or substances to be analyzed are dissolved in a suitable solvent. Convenient volumes, delivered from suitable micropipets, of the resulting solution, normally containing 1 to 20 µg of the compound, are placed in 6- to 10-mm spots not less than 3 cm apart along the pencil line. If the total volume to be applied would produce spots of a diameter greater than 6 to 10 mm, it is applied in separate portions to the same spot, each portion being allowed to dry before the next is added.
The spotted chromatographic sheet is suspended in the chamber by use of the antisiphon rod, which holds the upper end of the sheet in the solvent trough. The bottom of the chamber is covered with the prescribed solvent system. Saturation of the chamber with solvent vapor is facilitated by lining the inside walls with paper that is wetted with the prescribed solvent system. It is important to ensure that the portion of the sheet hanging below the rods is freely suspended in the chamber without touching the rack or the chamber walls or the fluid in the chamber. The chamber is sealed to allow equilibration (saturation) of the chamber and the paper with the solvent vapor. Any excess pressure is released as necessary. For large chambers, equilibration overnight may be necessary.
A volume of the mobile phase in excess of the volume required for complete development of the chromatogram is saturated with the immobile phase by shaking. After equilibration of the chamber, the prepared mobile solvent is introduced into the trough through the inlet. The inlet is closed and the mobile solvent phase is allowed to travel the desired distance down the paper. Precautions must be taken against allowing the solvent to run down the sheet when opening the chamber and removing the chromatogram. The location of the solvent front is quickly marked, and the sheets are dried.
The chromatogram is observed and measured directly or after suitable development to reveal the location of the spots of the isolated drug or drugs. The paper section(s) predetermined to contain the isolated drug(s) may be cut out and eluted by an appropriate solvent, and the solutions may be made up to a known volume and quantitatively analyzed by appropriate chemical or instrumental techniques. Similar procedures should be conducted with various amounts of similarly spotted reference standard on the same paper in the concentration range appropriate to prepare a valid calibration curve.
Ascending Chromatography
In ascending chromatography, the lower edge of the sheet (or strip) is dipped into the mobile phase to permit the mobile phase to rise on the chromatographic sheet by capillary action.
Apparatus— The essential equipment for ascending chromatography is substantially the same as that described under Descending Chromatography.
Procedure— The test materials are applied to the chromatographic sheets as directed under Descending Chromatography, and above the level to which the paper is dipped into the developing solvent. The bottom of the developing chamber is covered with the developing solvent system. If a two-phase system is used, both phases are added. It is also desirable to line the walls of the chamber with paper and to saturate this lining with the solvent system. Empty solvent troughs are placed on the bottom of the chamber, and the chromatographic sheets are suspended so that the end on which the spots have been added hangs free inside the empty trough.
The chamber is sealed, and equilibration is allowed to proceed as described under Descending Chromatography. Then the developing solvent (mobile phase) is added through the inlet to the trough in excess of the solvent required for complete moistening of the chromatographic sheet. The chamber is resealed. When the solvent front has reached the desired height, the chamber is opened and the sheet is removed and dried.
Quantitative analyses of the spots may be conducted as described under Descending Chromatography.
THIN-LAYER CHROMATOGRAPHY
In thin-layer chromatography, the adsorbent is a relatively thin, uniform layer of dry, finely powdered material applied to a glass, plastic, or metal sheet or plate, glass plates being most commonly employed. The coated plate can be considered an “open chromatographic column” and the separations achieved may be based upon adsorption, partition, or a combination of both effects, depending on the particular type of stationary phase, its preparation, and its use with different solvents. Thin-layer chromatography on ion-exchange layers can be used for the fractionation of polar compounds. Presumptive identification can be effected by observation of spots or zones of identical RF value and about equal magnitude obtained, respectively, with an unknown and a reference sample chromatographed on the same plate. A visual comparison of the size or intensity of the spots or zones may serve for semiquantitative estimation. Quantitative measurements are possible by means of densitometry (absorbance or fluorescence measurements), or the spots may be carefully removed from the plate, followed by elution with a suitable solvent and spectrophotometric measurement. For two-dimensional thin-layer chromatography, the chromatographed plate is turned at a right angle and again chromatographed, usually in another chamber equilibrated with a different solvent system.
Apparatus— Acceptable apparatus and materials for thin-layer chromatography consist of the following.
A TLC or HPTLC plate. The chromatography is generally carried out using precoated plates or sheets (on glass, aluminum, or polyester support) of suitable size. It may be necessary to clean the plates prior to separation. This can be done by migration of, or immersion in, an appropriate solvent. The plates may also be impregnated by procedures such as development, immersion, or spraying. At the time of use, the plates may be activated, if necessary, by heating in an oven at 120 for 20 minutes. The stationary phase of TLC plates has an average particle size of 10–15 µm, and that of HPTLC plates an average particle size of 5 µm. Commercial plates with a preadsorbant zone can be used if they are specified in a monograph. Sample applied to the preabsorbant region develops into sharp, narrow bands at the preabsorbant-sorbent interface. Alternatively, flat glass plates of convenient size, typically 20 cm × 20 cm can be coated as described under Preparation of Chromatographic Plates.
for 20 minutes. The stationary phase of TLC plates has an average particle size of 10–15 µm, and that of HPTLC plates an average particle size of 5 µm. Commercial plates with a preadsorbant zone can be used if they are specified in a monograph. Sample applied to the preabsorbant region develops into sharp, narrow bands at the preabsorbant-sorbent interface. Alternatively, flat glass plates of convenient size, typically 20 cm × 20 cm can be coated as described under Preparation of Chromatographic Plates. A suitable manual, semiautomatic, or automatic application device can be used to ensure proper positioning of the plate and proper transfer of the sample, with respect to volume and position, onto the plate. Alternatively, a template can be used to guide in manually placing the test spots at definite intervals, to mark distances as needed, and to aid in labeling the plates. For the proper application of the solutions, micropipets, microsyringes, or calibrated disposable capillaries are recommended.
For ascending development, a chromatographic chamber made of inert, transparent material and having the following specifications is used: a flat bottom or twin trough, a tightly fitted lid, and a size suitable for the plates. For horizontal development, the chamber is provided with a reservoir for the mobile phase, and it also contains a device for directing the mobile phase to the stationary phase.
Devices for transfer of reagents onto the plate by spraying, immersion, or exposure to vapor and devices to facilitate any necessary heating for visualization of the separated spots or zones.
A UV light source suitable for observations under short (254 nm) and long (365 nm) wavelength UV light.
A suitable device for documentation of the visualized chromatographic result.
Procedure— Apply the prescribed volume of the test solution and the standard solution in sufficiently small portions to obtain circular spots of 2 to 5 mm in diameter (1 to 2 mm on HPTLC plates) or bands of 10 to 20 mm by 1 to 2 mm (5 to 10 mm by 0.5 to 1 mm on HPTLC plates) at an appropriate distance from the lower edge—during chromatography the application position must be 3 mm (HPTLC) to 5 mm (TLC) above the level of the developing solvent—and from the sides of the plate. Apply the solutions on a line parallel to the lower edge of the plate with an interval of at least 10 mm (5 mm on HPTLC plates) between the centers of spots or 4 mm (2 mm on HPTLC plates) between the edges of bands, and allow to dry.
Ascending Development— Line at least one wall of the chromatographic chamber with filter paper. Pour into the chromatographic chamber a quantity of the mobile phase sufficient for the size of the chamber to give, after impregnation of the filter paper, a level of depth appropriate to the dimension of the plate used. For saturation of the chromatographic chamber, close the lid, and allow the system to equilibrate. Unless otherwise indicated, the chromatographic separation is performed in a saturated chamber.
Place the plate in the chamber, ensuring that the plate is as vertical as possible and that the spots or bands are above the surface of the mobile phase, and close the chamber. The stationary phase faces the inside of the chamber. Remove the plate when the mobile phase has moved over the prescribed distance. Dry the plate, and visualize the chromatograms as prescribed. For two-dimensional chromatography, dry the plates after the first development, and carry out a second development in a direction perpendicular to that of the first development.
Horizontal Development— Introduce a sufficient quantity of the developing solvent into the reservoir of the chamber using a syringe or pipet. Place the plate horizontally in the chamber, connect the mobile phase direction device according to the manufacturer's instructions, and close the chamber. If prescribed, develop the plate starting simultaneously at both ends. Remove the plate when the mobile phase has moved over the distance prescribed in the monograph. Dry the plate, and visualize the chromatograms as prescribed.
For two-dimensional chromatography, dry the plates after the first development, and carry out a second development in a direction perpendicular to that of the first development.
Detection— Observe the dry plate first under short-wavelength UV light (254 nm) and then under long-wavelength UV light (365 nm) or as stated in the monograph. If further directed, spray, immerse, or expose the plate to vapors of the specified reagent, heat the plate when required, observe, and compare the test chromatogram with the standard chromatogram. Document the plate after each observation. Measure and record the distance of each spot or zone from the point of origin, and indicate for each spot or zone the wavelength under which it was observed. Determine the RF values for the principal spots or zones (see Glossary of Symbols).
Quantitative Measurement— Using appropriate instrumentation, substances separated by TLC and responding to ultraviolet-visible (UV-Vis) irradiation prior to or after derivatization can be determined directly on the plate. While moving the plate or the measuring device, the plate is examined by measuring the reflectance of the incident light. Similarly, fluorescence may be measured using an appropriate optical system. Substances containing radionuclides can be quantified in three ways: (1) directly by moving the plate alongside a suitable counter or vice versa; (2) by cutting the plates into strips and measuring the radioactivity on each individual strip using a suitable counter; or (3) by scraping off the stationary phase, dissolving it in a suitable scintillation cocktail, and measuring the radioactivity using a liquid scintillation counter (see Radioactivity 821).
821). The apparatus for direct quantitative measurement on the plate is a densitometer that is composed of a mechanical device to move the plate or the measuring device along the x-axis and the y-axis, a recorder, a suitable integrator or a computer; and, for substances responding to UV-Vis irradiation, a photometer with a source of light, an optical device capable of generating monochromatic light, and a photo cell of adequate sensitivity, all of which are used for the measurement of reflectance. In the case where fluorescence is measured, a suitable filter is also required to prevent the light used for excitation from reaching the photo cell while permitting the emitted light or specific portions thereof to pass. The linearity range of the counting device must be verified.
For quantitative tests, it is necessary to apply to the plate not fewer than three standard solutions of the substance to be examined, the concentrations of which span the expected value in the test solution (e.g., 80%, 100%, and 120%). Derivatize with the prescribed reagent, if necessary, and record the reflectance or fluorescence in the chromatograms obtained. Use the measured results for the calculation of the amount of substance in the test solution.
Preparation of Chromatographic Plates—
Apparatus—
Flat glass plates of convenient size, typically 20 cm × 20 cm.
An aligning tray or a flat surface upon which to align and rest the plates during the application of the adsorbent.
A storage rack to hold the prepared plates during drying and transportation. The rack holding the plates should be kept in a desiccator or be capable of being sealed in order to protect the plates from the environment after removal from the drying oven.
The adsorbent consists of finely divided adsorbent materials, normally 5 to 40 µm in diameter, suitable for chromatography. It can be applied directly to the glass plate or can be bonded to the plate by means of plaster of Paris [calcium sulfate hemihydrate (at a ratio of 5% to 15%)] or with starch paste or other binders. The plaster of Paris will not yield as hard a surface as will the starch, but it is not affected by strongly oxidizing spray reagents. The adsorbent may contain fluorescing material to aid in the visualization of spots that absorb UV light.
A spreader, which, when moved over the glass plate, will apply a uniform layer of adsorbent of desired thickness over the entire surface of the plate.
Procedure— [NOTE—In this procedure, use Purified Water that is obtained by distillation.] Clean the glass plates scrupulously, using an appropriate cleaning solution (see Cleaning Glass Apparatus 1051), rinsing them with copious quantities of water until the water runs off the plates without leaving any visible water or oily spots, then dry. It is important that the plates be completely free from lint and dust when the adsorbent is applied.
1051), rinsing them with copious quantities of water until the water runs off the plates without leaving any visible water or oily spots, then dry. It is important that the plates be completely free from lint and dust when the adsorbent is applied. Arrange the plate or plates on the aligning tray, place a 5- × 20-cm plate adjacent to the front edge of the first square plate and another 5- × 20-cm plate adjacent to the rear edge of the last square, and secure all of the plates so that they will not slip during the application of the adsorbent. Position the spreader on the end plate opposite the raised end of the aligning tray. Mix 1 part of adsorbent with 2 parts of water (or in the ratio suggested by the supplier) by shaking vigorously for 30 seconds in a glass-stoppered conical flask, and transfer the slurry to the spreader. Usually 30 g of adsorbent and 60 mL of water are sufficient for five 20- × 20-cm plates. Complete the application of adsorbents using plaster of Paris binder within 2 minutes of the addition of the water, because thereafter the mixture begins to harden. Draw the spreader smoothly over the plates toward the raised end of the aligning tray, and remove the spreader when it is on the end plate next to the raised end of the aligning tray. (Wash away all traces of adsorbent from the spreader immediately after use.) Allow the plates to remain undisturbed for 5 minutes, then transfer the square plates, layer side up, to the storage rack, and dry at 105 for 30 minutes. Preferably place the rack at an angle in the drying oven to prevent the condensation of moisture on the back sides of plates in the rack. When the plates are dry, allow them to cool to room temperature, and inspect the uniformity of the distribution and the texture of the adsorbent layer; transmitted light will show uniformity of distribution, and reflected light will show uniformity of texture. Store the satisfactory plates over silica gel in a suitable chamber.
for 30 minutes. Preferably place the rack at an angle in the drying oven to prevent the condensation of moisture on the back sides of plates in the rack. When the plates are dry, allow them to cool to room temperature, and inspect the uniformity of the distribution and the texture of the adsorbent layer; transmitted light will show uniformity of distribution, and reflected light will show uniformity of texture. Store the satisfactory plates over silica gel in a suitable chamber.COLUMN CHROMATOGRAPHY
Apparatus— The apparatus required for column chromatographic procedures is simple, consisting only of the chromatographic tube itself and a tamping rod, which may be needed to pack a pledget of glass wool or cotton, if needed, in the base of the tube and compress the adsorbent or slurry uniformly within the tube. In some cases a porous glass disk is sealed at the base of the tube in order to support the contents. The tube is cylindrical and is made of glass, unless another material is specified in the individual monograph. A smaller-diameter delivery tube is fused or otherwise attached by a leakproof joint to the lower end of the main tube. Column dimensions are variable; the dimensions of those commonly used in pharmaceutical analysis range from 10 to 30 mm in uniform inside diameter and 150 to 400 mm in length, exclusive of the delivery tube. The delivery tube, usually 3 to 6 mm in inside diameter, may include a stopcock for accurate control of the flow rate of solvents through the column. The tamping rod, a cylindrical ram firmly attached to a shaft, may be constructed of plastic, glass, stainless steel, or aluminum, unless another material is specified in the individual monograph. The shaft of the rod is substantially smaller in diameter than the column and is not less than 5 cm longer than the effective length of the column. The ram has a diameter about 1 mm smaller than the inside diameter of the column.
Column Adsorption Chromatography
The adsorbent (such as activated alumina or silica gel, calcined diatomaceous silica, or chromatographic purified siliceous earth) as a dry solid or as a slurry is packed into a glass or quartz chromatographic tube. A solution of the drug in a small amount of solvent is added to the top of the column and allowed to flow into the adsorbent. The drug principles are quantitatively removed from the solution and are adsorbed in a narrow transverse band at the top of the column. As additional solvent is allowed to flow through the column, either by gravity or by application of air pressure, each substance progresses down the column at a characteristic rate resulting in a spatial separation to give what is known as the chromatogram. The rate of movement for a given substance is affected by several variables, including the adsorptive power of the adsorbent and its particle size and surface area; the nature and polarity of the solvent; the hydrostatic head or applied pressure; and the temperature of the chromatographic system.
If the separated compounds are colored or if they fluoresce under UV light, the adsorbent column may be extruded and, by transverse cuts, the appropriate segments may then be isolated. The desired compounds are then extracted from each segment with a suitable solvent. If the compounds are colorless, they may be located by means of painting or spraying the extruded column with color-forming reagents. Chromatographed radioactive substances may be located by means of Geiger-Müller detectors or similar sensing and recording instruments. Clear plastic tubing made of a material such as nylon, which is inert to most solvents and transparent to short-wavelength UV light, may be packed with adsorbent and used as a chromatographic column. Such a column may be sliced with a sharp knife without removing the packing from the tubing. If a fluorescent adsorbent is used, the column may be marked under UV light in preparation for slicing.
A “flowing” chromatogram, which is extensively used, is obtained by a procedure in which solvents are allowed to flow through the column until the separated drug appears in the effluent solution, known as the “eluate.” The drug may be determined in the eluate by titration or by a spectrophotometric or colorimetric method, or the solvent may be evaporated, leaving the drug in more or less pure form. If a second drug principle is involved, it is eluted by continuing the first solvent or by passing a solvent of stronger eluting power through the column. The efficiency of the separation may be checked by obtaining a thin-layer chromatogram on the individual fractions.
A modified procedure for adding the mixture to the column is sometimes employed. The drug, in a solid form, and, as in the case of a powdered tablet, without separation from the excipients, is mixed with some of the adsorbent and added to the top of a column. The subsequent flow of solvent moves the drug down the column in the manner described.
Column Partition Chromatography
In partition chromatography the substances to be separated are partitioned between two immiscible liquids, one of which, the immobile phase, is adsorbed on a Solid Support, thereby presenting a very large surface area to the flowing solvent or mobile phase. The exceedingly high number of successive liquid-liquid contacts allows an efficiency of separation not achieved in ordinary liquid-liquid extraction.
The Solid Support is usually polar, and the adsorbed immobile phase more polar than the mobile phase. The Solid Support that is most widely used is chromatographic siliceous earth having a particle size suitable to permit proper flow of eluant.1 In reverse-phase partition chromatography the adsorbed immobile phase is less polar than the mobile phase and the solid adsorbent is rendered nonpolar by treatment with a silanizing agent, such as dichlorodimethylsilane, to give silanized chromatographic siliceous earth.
The sample to be chromatographed is usually introduced into the chromatographic system in one of two ways: (a) a solution of the sample in a small volume of the mobile phase is added to the top of the column; or, (b) a solution of the sample in a small volume of the immobile phase is mixed with the Solid Support and transferred to the column as a layer above a bed of a mixture of immobile phase with adsorbent.
Development and elution are accomplished with flowing solvent as before. The mobile solvent usually is saturated with the immobile solvent before use.
In conventional liquid-liquid partition chromatography, the degree of partition of a given compound between the two liquid phases is expressed by its partition or distribution coefficient. In the case of compounds that dissociate, distribution can be controlled by modifying the pH, dielectric constant, ionic strength, and other properties of the two phases. Selective elution of the components of a mixture can be achieved by successively changing the mobile phase to one that provides a more favorable partition coefficient, or by changing the pH of the immobile phase in situ with a mobile phase consisting of a solution of an appropriate acid or base in an organic solvent.
Unless otherwise specified in the individual monograph, assays and tests that employ column partition chromatography are performed according to the following general methods.
Solid Support— Use purified siliceous earth. Use silanized chromatographic siliceous earth for reverse-phase partition chromatography.
Stationary Phase— Use the solvent or solution specified in the individual monograph. If a mixture of liquids is to be used as the Stationary Phase, mix them prior to the introduction of the Solid Support.
Mobile Phase— Use the solvent or solution specified in the individual monograph. Equilibrate it with water if the Stationary Phase is an aqueous solution; if the Stationary Phase is a polar organic fluid, equilibrate with that fluid.
Preparation of Chromatographic Column— Unless otherwise specified in the individual monograph, the chromatographic tube is about 22 mm in inside diameter and 200 to 300 mm in length, without porous glass disk, to which is attached a delivery tube, without stopcock, about 4 mm in inside diameter and about 50 mm in length. Pack a pledget of fine glass wool in the base of the tube. Place the specified volume of Stationary Phase in a 100- to 250-mL beaker, add the specified amount of Solid Support, and mix to produce a homogeneous, fluffy mixture. Transfer this mixture to the chromatographic tube, and tamp, using gentle pressure, to obtain a uniform mass. If the specified amount of Solid Support is more than 3 g, transfer the mixture to the column in portions of approximately 2 g, and tamp each portion. If the assay or test requires a multisegment column, with a different Stationary Phase specified for each segment, tamp after the addition of each segment, and add each succeeding segment directly to the previous one.
If a solution of the analyte is incorporated in the Stationary Phase, complete the quantitative transfer to the chromatographic tube by scrubbing the beaker used for the preparation of the test mixture with a mixture of about 1 g of Solid Support and several drops of the solvent used to prepare the test solution.
Pack a pledget of fine glass wool above the completed column packing. The Mobile Phase flows through a properly packed column as a moderate stream or, if reverse-phase chromatography is applied, as a slow trickle.
Procedure— Transfer the Mobile Phase to the column space above the column packing, and allow it to flow through the column under the influence of gravity. Rinse the tip of the chromatographic column with about 1 mL of Mobile Phase before each change in composition of Mobile Phase and after completion of the elution. If the analyte is introduced into the column as a solution in the Mobile Phase, allow it to pass completely into the column packing, then add Mobile Phase in several small portions, allowing each to drain completely, before adding the bulk of the Mobile Phase. Where the assay or test requires the use of multiple chromatographic columns mounted in series and the addition of Mobile Phase in divided portions is specified, allow each portion to drain completely through each column, and rinse the tip of each with Mobile Phase prior to the addition of each succeeding portion.
GAS CHROMATOGRAPHY
The distinguishing features of gas chromatography are a gaseous mobile phase and a solid or immobilized liquid stationary phase. Liquid stationary phases are available in packed or capillary columns. In the packed columns, the liquid phase is deposited on a finely divided, inert solid support, such as diatomaceous earth, porous polymer, or graphitized carbon, which is packed into a column that is typically 2 to 4 mm in internal diameter and 1 to 3 m in length. In capillary columns, which contain no packing, the liquid phase is deposited on the inner surface of the column and may be chemically bonded to it. In gas-solid chromatography, the solid phase is an active adsorbent, such as alumina, silica, or carbon, packed into a column. Polyaromatic porous resins, which are sometimes used in packed columns, are not coated with a liquid phase.
When a vaporized compound is introduced into the carrier gas and carried into the column, it is partitioned between the gas and stationary phases by a dynamic countercurrent distribution process. The compound is carried down the column by the carrier gas, retarded to a greater or lesser extent by sorption and desorption on the stationary phase. The elution of the compound is characterized by the partition ratio, k¢, a dimensionless quantity also called the capacity factor (see Glossary of Symbols for the definition of symbols). It is equivalent to the ratio of the time required for the compound to flow through the column (the retention time) to the elution time of an unretained compound. The value of the capacity factor depends on the chemical nature of the compound, the nature, amount, and surface area of the liquid phase, the column temperature, and the gas flow rate. Under a specified set of experimental conditions, a characteristic capacity factor exists for every compound. Separation by gas chromatography occurs only if the compounds concerned have different capacity factors.
Apparatus— A gas chromatograph consists of a carrier gas source, an injection port, column, detector, and recording device. The injection port, column, and detector are temperature-controlled. The typical carrier gas is helium, nitrogen, or hydrogen, depending on the column and detector in use. The gas is supplied from a high-pressure cylinder or high-purity gas generator and passes through suitable pressure-reducing valves and a flow meter to the injection port and column. Compounds to be chromatographed, either in solution or as gases, are injected into the gas stream at the injection port. Depending upon the configuration of the apparatus, the test mixture may be injected directly into the column or be vaporized in the injection port and mixed into the flowing carrier gas prior to entering the column.
Once in the column, compounds in the test mixture are separated by virtue of differences in their capacity factors, which in turn depend upon vapor pressure and degree of interaction with the stationary phase. The capacity factor, which governs resolution, retention times, and column efficiencies of components of the test mixture, is also temperature-dependent. The use of temperature-programmable column ovens takes advantage of this dependence to achieve efficient separation of compounds differing widely in vapor pressure.
As resolved compounds emerge separately from the column, they pass through a differential detector, which responds to the amount of each compound present. The type of detector to be used depends upon the nature of the compounds to be analyzed and is specified in the individual monograph. Detectors are heated to prevent condensation of the eluting compounds.
Detector output is recorded as a function of time, producing a chromatogram, which consists of a series of peaks on a time axis. Each peak represents a compound in the vaporized test mixture, although some peaks may overlap. The elution time is a characteristic of an individual compound; and the instrument response, measured as peak area or peak height, is a function of the amount present.
Injectors—Sample injection devices range from simple syringes to fully programmable automatic injectors. The amount of sample that can be injected into a capillary column without overloading is small compared to the amount that can be injected into packed columns, and may be less than the smallest amount that can be manipulated satisfactorily by syringe. Capillary columns, therefore, often are used with injectors able to split samples into two fractions, a small one that enters the column and a large one that goes to waste. Such injectors may be used in a splitless mode for analyses of trace or minor components.
Purge and trap injectors are equipped with a sparging device by which volatile compounds in solution are carried into a low-temperature trap. When sparging is complete, trapped compounds are desorbed into the carrier gas by rapid heating of the temperature-programmable trap.
Headspace injectors are equipped with a thermostatically controlled sample heating chamber. Solid or liquid samples in tightly closed containers are heated in the chamber for a fixed period of time, allowing the volatile components in the sample to reach an equilibrium between the nongaseous phase and the gaseous or headspace phase.
After this equilibrium has been established, the injector automatically introduces a fixed amount of the headspace in the sample container into the gas chromatograph.
Columns—Capillary columns, which are usually made of fused silica, are typically 0.2 to 0.53 mm in internal diameter and 5 to 60 m in length. The liquid or stationary phase, which is sometimes chemically bonded to the inner surface, is 0.1 to 1.0 µm thick, although nonpolar stationary phases may be up to 5 µm thick. A list of liquid phases in current use is given in the section Chromatographic Reagents.
Packed columns, made of glass or metal, are 1 to 3 m in length with internal diameters of 2 to 4 mm. Those used for analysis typically are porous polymers or solid supports with liquid phase loadings of about 5% (w/w). High-capacity columns, with liquid phase loadings of about 20% (w/w), are used for large test specimens and for the determination of low molecular weight compounds such as water. The capacity required influences the choice of solid support.
Supports for analysis of polar compounds on low-capacity, low-polarity liquid phase columns must be inert to avoid peak tailing. The reactivity of support materials can be reduced by silanizing prior to coating with liquid phase. Acid-washed, flux-calcined diatomaceous earth is often used for drug analysis. Support materials are available in various mesh sizes, with 80- to 100-mesh and 100- to 120-mesh being most commonly used with 2- to 4-mm columns. Supports and liquid phases are listed in the section Chromatographic Reagents.
Retention time and the peak efficiency depend on the carrier gas flow rate; retention time is also directly proportional to column length, while resolution is proportional to the square root of the column length. For packed columns, the carrier gas flow rate is usually expressed in mL per minute at atmospheric pressure and room temperature. It is measured at the detector outlet with a flowmeter while the column is at operating temperature. The linear flow rate through a packed column is inversely proportional to the square of the column diameter for a given flow volume. Flow rates of 60 mL per minute in a 4-mm column and 15 mL per minute in a 2-mm column give identical linear flow rates and thus similar retention times. Unless otherwise specified in the individual monograph, flow rates for packed columns are about 30 to 60 mL per minute. For capillary columns, linear flow velocity is often used instead of flow rate. This is conveniently determined from the length of the column and the retention time of a dilute methane sample, provided a flame-ionization detector is in use. At high operating temperatures there is sufficient vapor pressure to result in a gradual loss of liquid phase, a process called bleeding.
Detectors—Flame-ionization detectors are used for most pharmaceutical analyses, with lesser use made of thermal conductivity, electron-capture, nitrogen-phosphorus, and mass spectrometric detectors. For quantitative analyses, detectors must have a wide linear dynamic range: the response must be directly proportional to the amount of compound present in the detector over a wide range of concentrations. Flame-ionization detectors have a wide linear range and are sensitive to most organic compounds. Detector response depends on the structure and concentration of the compound and on the flow rates of the combustion, air, makeup, and carrier gases. Unless otherwise specified in individual monographs, flame-ionization detectors with either helium or nitrogen carrier gas are to be used for packed columns, and helium or hydrogen is used for capillary columns.
The thermal conductivity detector employs a heated wire placed in the carrier gas stream. When an analyte enters the detector with the carrier gas, the difference in thermal conductivity of the gas stream (carrier and sample components) relative to that of a reference flow of carrier gas alone is measured. In general, the thermal conductivity detector responds uniformly to volatile compounds regardless of structure; however, it is considerably less sensitive than the flame-ionization detector.
The alkali flame-ionization detector, sometimes called an NP or nitrogen-phosphorus detector, contains a thermionic source, such as an alkali-metal salt or a glass element containing rubidium or other metal, that results in the efficient ionization of organic nitrogen and phosphorus compounds. It is a selective detector that shows little response to hydrocarbons.
The electron-capture detector contains a radioactive source of ionizing radiation. It exhibits an extremely high response to compounds containing halogens and nitro groups but little response to hydrocarbons. The sensitivity increases with the number and atomic weight of the halogen atoms.
Data Collection Devices—Modern data stations receive the detector output, calculate peak areas and peak heights, and print chromatograms, complete with run parameters and peak data. Chromatographic data may be stored and reprocessed, with integration and other calculation variables being changed as required. Data stations are used also to program the chromatograph, controlling most operational variables and providing for long periods of unattended operation.
Data can also be collected for manual measurement on simple recorders or on integrators whose capabilities range from those providing a printout of peak areas to those providing chromatograms with peak areas and peak heights calculated and data stored for possible reprocessing.
Procedure— Packed and capillary columns must be conditioned before use until the baseline and other characteristics are stable. This may be done by operation at a temperature above that called for by the method or by repeated injections of the compound or mixture to be chromatographed. The column or packing material supplier provides instructions for the recommended conditioning procedure. In the case of thermally stable methyl- and phenyl-substituted polysiloxanes, a special sequence increases inertness and efficiency; maintain the column at a temperature of 250 for 1 hour, with helium flowing, to remove oxygen and solvents. Stop the flow of helium, heat at about 340 for 4 hours, then reduce the heating to a temperature of 250, and condition with helium flowing until stable.
for 1 hour, with helium flowing, to remove oxygen and solvents. Stop the flow of helium, heat at about 340 for 4 hours, then reduce the heating to a temperature of 250, and condition with helium flowing until stable. Most drugs are reactive polar molecules. Successful chromatography may require conversion of the drug to a less polar and more volatile derivative by treatment of reactive groups with appropriate reagents. Silylating agents are widely used for this purpose and are readily available.
Assays require quantitative comparison of one chromatogram with another. A major source of error is irreproducibility in the amount of sample injected, notably when manual injections are made with a syringe. The effects of variability can be minimized by addition of an internal standard, a noninterfering compound present at the same concentration in test and standard solutions. The ratio of peak response of the analyte to that of the internal standard is compared from one chromatogram to another. Where the internal standard is chemically similar to the substance being determined, there is also compensation for minor variations in column and detector characteristics. In some cases, the internal standard may be carried through the sample preparation procedure prior to gas chromatography to control other quantitative aspects of the assay. Automatic injectors greatly improve the reproducibility of sample injections and reduce the need for internal standards.
Many monographs require that system suitability requirements be met before samples are analyzed (see System Suitability and Interpretation of Chromatograms).
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
High-pressure liquid chromatography (HPLC), sometimes called high-performance liquid chromatography, is a separation technique based on a solid stationary phase and a liquid mobile phase. Separations are achieved by partition, adsorption, or ion-exchange processes, depending upon the type of stationary phase used. HPLC has distinct advantages over gas chromatography for the analysis of organic compounds. Compounds to be analyzed are dissolved in a suitable solvent, and most separations take place at room temperature. Thus, most drugs, being nonvolatile or thermally unstable compounds, can be chromatographed without decomposition or the necessity of making volatile derivatives. Most pharmaceutical analyses are based on partition chromatography and are completed within 30 minutes.
As in gas chromatography, the elution time of a compound can be described by the capacity factor, k¢ (see Glossary of Symbols), which depends on the chemical nature of the analyte, the composition and flow rate of the mobile phase, and the composition and surface area of the stationary phase. Column length is an important determinant of resolution. Only compounds having different capacity factors can be separated by HPLC.
Apparatus— A liquid chromatograph consists of a reservoir containing the mobile phase, a pump to force the mobile phase through the system at high pressure, an injector to introduce the sample into the mobile phase, a chromatographic column, a detector, and a data collection device such as a computer, integrator, or recorder. Short, small-bore columns containing densely packed particles of stationary phase provide for the rapid exchange of compounds between the mobile and stationary phases. In addition to receiving and reporting detector output, computers are used to control chromatographic settings and operations, thus providing for long periods of unattended operation.
Pumping Systems—HPLC pumping systems deliver metered amounts of mobile phase from the solvent reservoirs to the column through high-pressure tubing and fittings. Modern systems consist of one or more computer-controlled metering pumps that can be programmed to vary the ratio of mobile phase components, as is required for gradient chromatography, or to mix isocratic mobile phases (i.e., mobile phases having a fixed ratio of solvents). However, the proportion of ingredients in premixed isocratic mobile phases can be more accurately controlled than in those delivered by most pumping systems. Operating pressures up to 5000 psi or higher, with delivery rates up to about 10 mL per minute are typical. Pumps used for quantitative analysis should be constructed of materials inert to corrosive mobile phase components and be capable of delivering the mobile phase at a constant rate with minimal fluctuations over extended periods of time.
Injectors—After dissolution in mobile phase or other suitable solution, compounds to be chromatographed are injected into the mobile phase, either manually by syringe or loop injectors, or automatically by autosamplers. The latter consist of a carousel or rack to hold sample vials with tops that have a pierceable septum or stopper and an injection device to transfer sample from the vials to a loop from which it is loaded into the chromatograph. Some autosamplers can be programmed to control sample volume, the number of injections and loop rinse cycles, the interval between injections, and other operating variables.
A syringe can be used for manual injection of samples through a septum when column head pressures are less than 70 atmospheres (about 1000 psi). At higher pressures an injection valve is essential. Some valve systems incorporate a calibrated loop that is filled with test solution for transfer to the column in the mobile phase. In other systems, the test solution is transferred to a cavity by syringe and then switched into the mobile phase.
Columns—For most pharmaceutical analyses, separation is achieved by partition of compounds in the test solution between the mobile and stationary phases. Systems consisting of polar stationary phases and nonpolar mobile phases are described as normal phase, while the opposite arrangement, polar mobile phases and nonpolar stationary phases, is called reverse-phase chromatography. Partition chromatography is almost always used for hydrocarbon-soluble compounds of molecular weight less than 1000. The affinity of a compound for the stationary phase, and thus its retention time on the column, is controlled by making the mobile phase more or less polar. Mobile phase polarity can be varied by the addition of a second, and sometimes a third or even a fourth, component.
Stationary phases for modern, reverse-phase liquid chromatography typically consist of an organic phase chemically bound to silica or other materials. Particles are usually 3 to 10 µm in diameter, but sizes may range up to 50 µm or more for preparative columns. Small particles thinly coated with organic phase provide for low mass transfer resistance and, hence, rapid transfer of compounds between the stationary and mobile phases. Column polarity depends on the polarity of the bound functional groups, which range from relatively nonpolar octadecyl silane to very polar nitrile groups. Liquid, nonbound stationary phases must be largely immiscible in the mobile phase. Even so, it is usually necessary to presaturate the mobile phase with stationary phase to prevent stripping of the stationary phase from the column. Polymeric stationary phases coated on the support are more durable.
Columns used for analytical separations usually have internal diameters of 2 to 5 mm; larger diameter columns are used for preparative chromatography. Columns may be heated to give more efficient separations, but only rarely are they used at temperatures above 60 because of potential stationary phase degradation or mobile phase volatility. Unless otherwise specified in the individual monograph, columns are used at ambient temperature.
because of potential stationary phase degradation or mobile phase volatility. Unless otherwise specified in the individual monograph, columns are used at ambient temperature.Ion-exchange chromatography is used to separate water-soluble, ionizable compounds of molecular weight less than 1500. The stationary phases are usually synthetic organic resins; cation-exchange resins contain negatively charged active sites and are used to separate basic substances such as amines, while anion-exchange resins have positively charged active sites for separation of compounds with negatively charged groups, such as phosphate, sulfonate, or carboxylate groups. Water-soluble ionic or ionizable compounds are attracted to the resins, and differences in affinity bring about the chromatographic separation. The pH of the mobile phase, temperature, ion type, ionic concentration, and organic modifiers affect the equilibrium, and these variables can be adjusted to obtain the desired degree of separation.
In size-exclusion chromatography, columns are packed with a porous stationary phase. Molecules of the compounds being chromatographed are filtered according to size. Those too large to enter the pores pass unretained through the column. Smaller molecules enter the pores and are increasingly retained as molecular size decreases. These columns are typically used to measure aggregation and degradation of large molecules (see Size-Exclusion Chromatography section).
Detectors—Many compendial HPLC methods require the use of spectrophotometric detectors. Such a detector consists of a flow-through cell mounted at the end of the column. A beam of UV radiation passes through the flow cell and into the detector. As compounds elute from the column, they pass through the cell and absorb the radiation, resulting in measurable energy level changes.
Fixed, variable, and multi-wavelength detectors are widely available. Fixed wavelength detectors operate at a single wavelength, typically 254 nm, emitted by a low-pressure mercury lamp. Variable wavelength detectors contain a continuous source, such as a deuterium or high-pressure xenon lamp, and a monochromator or an interference filter to generate monochromatic radiation at a wavelength selected by the operator. The wavelength accuracy of a variable-wavelength detector equipped with a monochromator should be checked by the procedure recommended by its manufacturer; if the observed wavelengths differ by more than 3 nm from the correct values, recalibration of the instrument is indicated. Modern variable wavelength detectors can be programmed to change wavelength while an analysis is in progress. Multi-wavelength detectors measure absorbance at two or more wavelengths simultaneously. In diode array multi-wavelength detectors, continuous radiation is passed through the sample cell, then resolved into its constituent wavelengths, which are individually detected by the photodiode array. These detectors acquire absorbance data over the entire UV-visible range, thus providing the analyst with chromatograms at multiple, selectable wavelengths and spectra of the eluting peaks. Diode array detectors usually have lower signal-to-noise ratios than fixed or variable wavelength detectors, and thus are less suitable for analysis of compounds present at low concentrations.
Differential refractometer detectors measure the difference between the refractive index of the mobile phase alone and that of the mobile phase containing chromatographed compounds as it emerges from the column. Refractive index detectors are used to detect non-UV absorbing compounds, but they are less sensitive than UV detectors. They are sensitive to small changes in solvent composition, flow rate, and temperature, so that a reference column may be required to obtain a satisfactory baseline.
Fluorometric detectors are sensitive to compounds that are inherently fluorescent or that can be converted to fluorescent derivatives either by chemical transformation of the compound or by coupling with fluorescent reagents at specific functional groups. If derivatization is required, it can be done prior to chromatographic separation or, alternatively, the reagent can be introduced into the mobile phase just prior to its entering the detector.
Potentiometric, voltametric, or polarographic electrochemical detectors are useful for the quantitation of species that can be oxidized or reduced at a working electrode. These detectors are selective, sensitive, and reliable, but require conducting mobile phases free of dissolved oxygen and reducible metal ions. A pulseless pump must be used, and care must be taken to ensure that the pH, ionic strength, and temperature of the mobile phase remain constant. Working electrodes are prone to contamination by reaction products with consequent variable responses.
Electrochemical detectors with carbon-paste electrodes may be used advantageously to measure nanogram quantities of easily oxidized compounds, notably phenols and catechols.
New detectors continue to be developed in attempts to overcome the deficiencies of those being used.
Data Collection Devices—Modern data stations receive and store detector output and print out chromatograms complete with peak heights, peak areas, sample identification, and method variables. They are also used to program the liquid chromatograph, controlling most variables and providing for long periods of unattended operation.
Data also may be collected on simple recorders for manual measurement or on stand-alone integrators, which range in complexity from those providing a printout of peak areas to those providing chromatograms with peak areas and peak heights calculated and data stored for possible subsequent reprocessing.
Procedure— The mobile phase composition significantly influences chromatographic performance and the resolution of compounds in the mixture being chromatographed. For accurate quantitative work, high-purity reagents and “HPLC grade” organic solvents must be used. Water of suitable quality should have low conductivity and low UV absorption, appropriate to the intended use.
Reagents used with special types of detectors (e.g., electrochemical, mass spectrometer) may require the establishment of additional tolerances for potential interfering species. Composition has a much greater effect than temperature on the capacity factor, k¢.
In partition chromatography, the partition coefficient, and hence the separation, can be changed by addition of another component to the mobile phase. In ion-exchange chromatography, pH and ionic strength, as well as changes in the composition of the mobile phase, affect capacity factors. The technique of continuously changing the solvent composition during the chromatographic run is called gradient elution or solvent programming. It is sometimes used to chromatograph complex mixtures of components differing greatly in their capacity factors. Detectors that are sensitive to change in solvent composition, such as the differential refractometer, are more difficult to use with the gradient elution technique.
The detector must have a broad linear dynamic range, and compounds to be measured must be resolved from any interfering substances. The linear dynamic range of a compound is the range over which the detector signal response is directly proportional to the amount of the compound. For maximum flexibility in quantitative work, this range should be about three orders of magnitude. HPLC systems are calibrated by plotting peak responses in comparison with known concentrations of a reference standard, using either an external or an internal standardization procedure.
Reliable quantitative results are obtained by external calibration if automatic injectors or autosamplers are used. This method involves direct comparison of the peak responses obtained by separately chromatographing the test and reference standard solutions. If syringe injection, which is irreproducible at the high pressures involved, must be used, better quantitative results are obtained by the internal calibration procedure where a known amount of a noninterfering compound, the internal standard, is added to the test and reference standard solutions, and the ratios of peak responses of drug and internal standard are compared.
Because of normal variations in equipment, supplies, and techniques, a system suitability test is required to ensure that a given operating system may be generally applicable. The main features of system suitability tests are described below.
For information on the interpretation of results, see the section Interpretation of Chromatograms.
Size-Exclusion Chromatography
Size-exclusion chromatography is a high-pressure liquid chromatographic technique that separates molecules in solution according to their size. Methods for size-exclusion chromatography are divided into gel permeation chromatographic methods, which utilize nonpolar organic mobile phases and hydrophilic packings, and gel filtration chromatographic methods, which utilize aqueous mobile phases and hydrophobic packings. The sample is introduced into a column, which is filled with a gel or a porous particle packing material and is carried by the mobile phase through the column. The size separation takes place by repeated exchange of the solute molecules between the solvent of the mobile phase and the same solvent in the stationary liquid phase within the pores of the packing material. The pore-size range of the packing material determines the molecular-size range within which separation can occur.
Molecules small enough to penetrate all the pore spaces elute at the total permeation volume, VT. On the other hand, molecules apparently larger than the maximum pore size of the packing material migrate along the column only through the spaces between the particles of the packing material without being retained and elute at the exclusion volume, VO (void volume). Separation according to molecular size occurs between the exclusion volume and the total permeation volume, useful separation usually occurring in the first two-thirds of this range.
Apparatus— The components of the chromatograph are described under High-Pressure Liquid Chromatography.
Column—If necessary, the column is temperature-controlled. It is packed with a separation material that is capable of fractionation in the appropriate range of molecular sizes and through which the eluant is passed at a constant rate. One end of the column is usually fitted with a suitable device for applying the sample, such as a flow adaptor, a syringe through a septum or an injection valve, and it may also be connected to a suitable pump for controlling the flow of the eluant. Alternatively, the sample may be applied directly to the drained bed surface, or, where the sample is denser than the eluant, it may be layered beneath the eluant. The packing material may be a soft support such as a swollen gel or a rigid support composed of a material such as glass, silica, or a solvent-compatible, cross-linked organic polymer. Rigid supports usually require pressurized systems giving faster separations. The mobile phase is chosen according to sample type, separation medium, and method of detection.
Detector—The outlet of the column is usually connected to a suitable detector fitted with an automatic recorder that enables the monitoring of the relative concentrations of separated components of the sample. Detectors are usually based on photometric, refractometric, or luminescent properties (see Detectors under High-Pressure Liquid Chromatography). An automatic fraction collector may be attached, if necessary.
Procedure— Before carrying out the separation, the packing material is treated and the column is packed, as described in the individual monograph or according to the manufacturer's instructions. Where necessary, procedures for verifying the suitability of the system are described in the individual monograph. The column efficiency may be evaluated from the number of theoretical plates, N (see the section Interpretation of Chromatograms). The elution characteristics of a compound in a particular column may be described by the distribution coefficient, KD, which is calculated by the formula:
(VI – VO) / (VT – VO)
in which VO, V T, and VI are the retention volumes for the nonretained component, the component that has full access to all the pores in the support, and the compound under test, respectively. Each retention volume is measured from the time of application to the time of the peak maximum. Determination of Relative Component Composition of Mixture—Carry out the separation as directed in the individual monograph. Monitor the elution of the components continuously, and measure the corresponding peak areas. If all the components under test exhibit equivalent responses to the physicochemical property being monitored (for example, if they exhibit corresponding absorptivities), calculate the relative amount of each component by dividing the respective peak area by the sum of the peak areas of all the components under test. If the responses to the property used for detection of the components under test are not equivalent, calculate the content using calibration curves obtained from the calibration procedure specified in the individual monograph.
Determination of Molecular Weights—Size-exclusion chromatography is used to determine molecular weights of components under test by comparison to calibration standards specified in the individual monograph. Plot the retention volumes of the calibration standards versus the logarithm of their molecular weights. Draw the line that best fits the plotted points within the exclusion and total permeation limits for the particular separation medium. From the calibration curve, molecular weights of components under test are estimated. This calibration is valid only for the particular macromolecular solute-solvent system used under the specified experimental conditions.
Determination of Molecular Weight Distribution of Polymers—The material used for calibration and the methods for determination of the distribution of molecular weights of polymers are specified in the individual monograph. However, sample comparison is valid only for results obtained under identical experimental conditions.
INTERPRETATION OF CHROMATOGRAMS
Figure 1 represents a typical chromatographic separation of two substances, 1 and 2, where t1 and t2 are the respective retention times, h, h/2, and Wh/2 are the height, the half-height, and the width at half-height, respectively, for peak 1. W1 and W2 are the respective widths of peaks 1 and 2 at the baseline. Air peaks are a feature of gas chromatograms and correspond to the solvent front in liquid chromatography.
Figure 1.Chromatographic separation of two substances
Chromatographic separation of two substancesChromatographic retention times are characteristic of the compounds they represent but are not unique. Coincidence of retention times of a test and a reference substance can be used as a feature in construction of an identity profile but is insufficient on its own to establish identity. Absolute retention times of a given compound vary from one chromatogram to the next. Comparisons are normally made in terms of relative retention, 
, which is calculated by the equation:
, which is calculated by the equation: 
in which t2 and t1 are the retention times, measured from the point of injection, of the test and reference substances, respectively, determined under identical experimental conditions on the same column, and ta is the retention time of a nonretained substance, such as methane in the case of gas chromatography.
In this and the following expressions, the corresponding retention volumes or linear separations on the chromatogram, both of which are directly proportional to retention time, may be substituted in the equations. Where the value of ta is small, RR may be estimated from the retention times measured from the point of injection (t2/t1).
The number of theoretical plates, N, is a measure of column efficiency. For Gaussian peaks, it is calculated by the equation:
in which t is the retention time of the substance and W is the width of the peak at its base, obtained by extrapolating the relatively straight sides of the peak to the baseline. Wh/2 is the peak width at half-height, obtained directly by electronic integrators. The value of N depends upon the substance being chromatographed as well as the operating conditions such as mobile phase or carrier gas flow rates and temperature, the quality of the packing, the uniformity of the packing within the column and, for capillary columns, the thickness of the stationary phase film, and the internal diameter and length of the column.
The separation of two components in a mixture, the resolution, R, is determined by the equation:
in which t2 and t1 are the retention times of the two components, and W2 and W1 are the corresponding widths at the bases of the peaks obtained by extrapolating the relatively straight sides of the peaks to the baseline.
Where electronic integrators are used, it may be convenient to determine the resolution, R, by the equation:
and to determine the number of theoretical plates, N, by the equation:
however, in the event of dispute, only equations based on peak width at baseline are to be used.
Peak areas and peak heights are usually proportional to the quantity of compound eluting. These are commonly measured by electronic integrators but may be determined by more classical approaches. Peak areas are generally used but may be less accurate if peak interference occurs. For manual measurements, the chart should be run faster than usual, or a comparator should be used to measure the width at half-height and the width at the base of the peak, to minimize error in these measurements. For accurate quantitative work, the components to be measured should be separated from any interfering components. Peak tailing and fronting and the measurement of peaks on solvent tails are to be avoided.
Chromatographic purity tests for drug raw materials are sometimes based on the determination of peaks due to impurities, expressed as a percentage of the area due to the drug peak. It is preferable, however, to compare impurity peaks to the chromatogram of a standard at a similar concentration. The standard may be the drug itself at a level corresponding to, for example, 0.5% impurity, or in the case of toxic or signal impurities, a standard of the impurity itself.
Change to read:
SYSTEM SUITABILITY
System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that the 
detection sensitivity,USP29 (Official June 1, 2006) resolution, and reproducibility of the chromatographic system are adequate for the analysis to be done. The tests are based on the concept that the equipment, electronics, analytical operations, and samples to be analyzed constitute an integral system that can be evaluated as such.
detection sensitivity,USP29 (Official June 1, 2006) resolution, and reproducibility of the chromatographic system are adequate for the analysis to be done. The tests are based on the concept that the equipment, electronics, analytical operations, and samples to be analyzed constitute an integral system that can be evaluated as such.The detection sensitivity is a measure used to ensure the suitability of a given chromatographic procedure for the complete detection of the impurities in the Chromatographic purity or Related compounds tests by injecting a volume of a quantitation limit solution equal to that of the Test solution. Unless otherwise specified in the individual monograph, the quantitation limit solution may be prepared by dissolving the drug substance Reference Standard in the same solvent as that used for the Test solution at a 0.05% concentration level relative to the amount of drug substance in the Test solution for drug substances, and a 0.1% level relative to the amount of drug substance in the Test solution for drug products. The signal-to-noise ratio for the drug substance peak obtained with the quantitation limit solution should be not less than 10.USP29 (Official June 1, 2006)The resolution, R, [NOTE—All terms and symbols are defined in the Glossary of Symbols] is a function of column efficiency, N, and is specified to ensure that closely eluting compounds are resolved from each other, to establish the general resolving power of the system, and to ensure that internal standards are resolved from the drug. Column efficiency may be specified also as a system suitability requirement, especially if there is only one peak of interest in the chromatogram; however, it is a less reliable means to ensure resolution than direct measurement. Column efficiency is a measure of peak sharpness, which is important for the detection of trace components.
Replicate injections of a standard preparation used in the assay or other standard solution are compared to ascertain whether requirements for precision are met. Unless otherwise specified in the individual monograph, data from five replicate injections of the analyte are used to calculate the relative standard deviation, SR, if the requirement is 2.0% or less; data from six replicate injections are used if the relative standard deviation requirement is more than 2.0%.
The tailing factor, T, a measure of peak symmetry, is unity for perfectly symmetrical peaks and its value increases as tailing becomes more pronounced (see Figure 2). In some cases, values less than unity may be observed. As peak asymmetry increases, integration, and hence precision, becomes less reliable.
Figure 2.Asymmetrical chromatographic peak
Asymmetrical chromatographic peakThese tests are performed by collecting data from replicate injections of standard or other solutions as specified in the individual monograph. The specification of definitive parameters in a monograph does not preclude the use of other suitable operating conditions (see Procedures under Tests and Assays in the General Notices). Adjustments of operating conditions to meet system suitability requirements may be necessary.
Unless otherwise directed in the monograph, system suitability parameters are determined from the analyte peak.
To ascertain the effectiveness of the final operating system, it should be subjected to suitability testing. Replicate injections of the standard preparation required to demonstrate adequate system precision may be made before the injection of samples or may be interspersed among sample injections. System suitability must be demonstrated throughout the run by injection of an appropriate control preparation at appropriate intervals. The control preparation can be a standard preparation or a solution containing a known amount of analyte and any additional materials useful in the control of the analytical system, such as excipients or impurities. Whenever there is a significant change in equipment or in a critical reagent, suitability testing should be performed before the injection of samples. No sample analysis is acceptable unless the requirements of system suitability have been met. Sample analyses obtained while the system fails requirements are unacceptable.
GLOSSARY OF SYMBOLS
To promote uniformity of interpretation, the following symbols and definitions are employed where applicable in presenting formulas in the individual monographs. [NOTE—Where the terms W and t both appear in the same equation they must be expressed in the same units.]
| relative retention, | |
 | ||
| cR, cI, cU | concentrations of Reference Standard, internal standard, and analyte in a particular solution. | |
| CA | concentration ratio of analyte and internal standard in test solution or Assay preparation, | |
 | ||
| CS | concentration ratio of Reference Standard and internal standard in Standard solution, | |
 | ||
| f | distance from the peak maximum to the leading edge of the peak, the distance being measured at a point 5% of the peak height from the baseline. | |
| k¢ | capacity factor, | |
 | ||
 | ||
| N | number of theoretical plates in a chromatographic column, | |
 | ||
| qR, qI, qU | total quantities (weights) of Reference Standard, internal standard, and analyte in a particular solution. | |
| QA | quantity ratio of analyte and internal standard in test solution or Assay preparation, | |
 | ||
| QS | quantity ratio of Reference Standard and internal standard in Standard solution, | |
 | ||
| rS | peak response of the Reference Standard obtained from a chromatogram. | |
| rU | peak response of the analyte obtained from a chromatogram. | |
| R | resolution between two chromatographic peaks, | |
 | ||
| RF | chromatographic retardation factor equal to the ratio of the distance from the origin to the center of a zone divided by the distance from the origin to the solvent front. | |
| RR | relative retention | |
 | ||
| RR | relative retention time | |
 | ||
| RS | peak response ratio for Standard preparation containing Reference Standard and internal standard, | |
 | ||
| RU | peak response ratio for Assay preparation containing the analyte and internal standard, | |
 | ||
| SR (%) | relative standard deviation in percentage, | |
 | ||
| where Xi is an individual measurement in a set of N measurements and X is the arithmetic mean of the set. | ||
| T | tailing factor, | |
 | ||
| t | retention time measured from time of injection to time of elution of peak maximum. | |
| ta | retention time of nonretarded component, air with thermal conductivity detection. | |
| W | width of peak measured by extrapolating the relatively straight sides to the baseline. | |
| Wh/2 | width of peak at half height. | |
| W0.05 | width of peak at 5% height. |
CHROMATOGRAPHIC REAGENTS
The following list of packings (L), phases (G), and supports (S) is intended to be a convenient reference for the chromatographer. [NOTE—Particle sizes given in this listing are those generally provided. Where other, usually finer, sizes are required, the individual monograph specifies the desired particle size. Within any category of packings or phases listed below, there may be a wide range of columns available. Where it is necessary to define more specifically the chromatographic conditions, the individual monograph so indicates.]
Packings
L1—Octadecyl silane chemically bonded to porous silica or ceramic micro-particles, 3 to 10 µm in diameter.
L2—Octadecyl silane chemically bonded to silica gel of a controlled surface porosity that has been bonded to a solid spherical core, 30 to 50 µm in diameter.
L3—Porous silica particles, 5 to 10 µm in diameter.
L4—Silica gel of controlled surface porosity bonded to a solid spherical core, 30 to 50 µm in diameter.
L5—Alumina of controlled surface porosity bonded to a solid spherical core, 30 to 50 µm in diameter.
L6—Strong cation-exchange packing–sulfonated fluorocarbon polymer coated on a solid spherical core, 30 to 50 µm in diameter.
L7—Octylsilane chemically bonded to totally porous silica particles, 3 to 10 µm in diameter.
L8—An essentially monomolecular layer of aminopropylsilane chemically bonded to totally porous silica gel support, 3 to 10 µm in diameter.
L9—10-µm irregular or spherical, totally porous silica gel having a chemically bonded, strongly acidic cation-exchange coating.
L10—Nitrile groups chemically bonded to porous silica particles, 3 to 10 µm in diameter.
L11—Phenyl groups chemically bonded to porous silica particles, 5 to 10 µm in diameter.
L12—A strong anion-exchange packing made by chemically bonding a quaternary amine to a solid silica spherical core, 30 to 50 µm in diameter.
L13—Trimethylsilane chemically bonded to porous silica particles, 3 to 10 µm in diameter.
L14—Silica gel having a chemically bonded, strongly basic quaternary ammonium anion-exchange coating, 5 to 10 µm in diameter.
L15—Hexylsilane chemically bonded to totally porous silica particles, 3 to 10 µm in diameter.
L16—Dimethylsilane chemically bonded to porous silica particles, 5 to 10 µm in diameter.
L17—Strong cation-exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in the hydrogen form, 7 to 11 µm in diameter.
L18—Amino and cyano groups chemically bonded to porous silica particles, 3 to 10 µm in diameter.
L19—Strong cation-exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in the calcium form, about 9 µm in diameter.
L20—Dihydroxypropane groups chemically bonded to porous silica particles, 5 to 10 µm in diameter.
L21—A rigid, spherical styrene-divinylbenzene copolymer, 5 to 10 µm in diameter.
L22—A cation-exchange resin made of porous polystyrene gel with sulfonic acid groups, about 10 µm in size.
L23—An anion-exchange resin made of porous polymethacrylate or polyacrylate gel with quaternary ammonium groups, about 10 µm in size.
L24—A semi-rigid hydrophilic gel consisting of vinyl polymers with numerous hydroxyl groups on the matrix surface, 32 to 63 µm in diameter.2
L25—Packing having the capacity to separate compounds with a molecular weight range from 100–5000 (as determined by polyethylene oxide), applied to neutral, anionic, and cationic water-soluble polymers. A polymethacrylate resin base, cross-linked with polyhydroxylated ether (surface contained some residual carboxyl functional groups) was found suitable.
L26—Butyl silane chemically bonded to totally porous silica particles, 5 to 10 µm in diameter.
L27—Porous silica particles, 30 to 50 µm in diameter.
L28—A multifunctional support, which consists of a high purity, 100 
, spherical silica substrate that has been bonded with anionic exchanger, amine functionality in addition to a conventional reversed phase C8 functionality.
, spherical silica substrate that has been bonded with anionic exchanger, amine functionality in addition to a conventional reversed phase C8 functionality.L29—Gamma alumina, reverse-phase, low carbon percentage by weight, alumina-based polybutadiene spherical particles, 5 µm in diameter with a pore volume of 80 .
.L30—Ethyl silane chemically bonded to totally porous silica particles, 3 to 10 µm in diameter.
L31—A strong anion-exchange resin-quaternary amine bonded on latex particles attached to a core of 8.5-µm macroporous particles having a pore size of 2000 and consisting of ethylvinylbenzene cross-linked with 55% divinylbenzene.
and consisting of ethylvinylbenzene cross-linked with 55% divinylbenzene.L32—A chiral ligand-exchange packing–L-proline copper complex covalently bonded to irregularly shaped silica particles, 5 to 10 µm in diameter.
L33—Packing having the capacity to separate dextrans by molecular size over a range of 4,000 to 500,000 Da. It is spherical, silica-based, and processed to provide pH stability.3
L34—Strong cation-exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in the lead form, about 9 µm in diameter.
L35—A zirconium-stabilized spherical silica packing with a hydrophilic (diol-type) molecular monolayer bonded phase having a pore size of 150 .
.L36—A 3,5-dinitrobenzoyl derivative of L-phenylglycine covalently bonded to 5-µm aminopropyl silica.
L37—Packing having the capacity to separate proteins by molecular size over a range of 2,000 to 40,000 Da. It is a polymethacrylate gel.
L38—A methacrylate-based size-exclusion packing for water-soluble samples.
L39—A hydrophilic polyhydroxymethacrylate gel of totally porous spherical resin.
L40—Cellulose tris-3,5-dimethylphenylcarbamate coated porous silica particles, 5 to 20 µm in diameter.
L41—Immobilized 1-acid glycoprotein on spherical silica particles, 5 µm in diameter.
1-acid glycoprotein on spherical silica particles, 5 µm in diameter.L42—Octylsilane and octadecylsilane groups chemically bonded to porous silica particles, 5 µm in diameter.
L43—Pentafluorophenyl groups chemically bonded to silica particles by a propyl spacer, 5 to 10 µm in diameter.
L44—A multifunctional support, which consists of a high purity, 60 , spherical silica substrate that has been bonded with a cationic exchanger, sulfonic acid functionality in addition to a conventional reversed phase C8 functionality.
, spherical silica substrate that has been bonded with a cationic exchanger, sulfonic acid functionality in addition to a conventional reversed phase C8 functionality.L45—Beta cyclodextrin bonded to porous silica particles, 5 to 10 µm in diameter.
L46—Polystyrene/divinylbenzene substrate agglomerated with quaternary amine functionalized latex beads, about 10 µm in diameter.
L47—High-capacity anion-exchange microporous substrate, fully functionalized with trimethlyamine groups, 8 µm in diameter.4
L48—Sulfonated, cross-linked polystyrene with an outer layer of submicron, porous, anion-exchange microbeads, 15 µm in diameter.
L49—A reversed-phase packing made by coating a thin layer of polybutadiene onto spherical porous zirconia particles, 3 to 10 µm in diameter.5
L50—Multifunction resin with reversed-phase retention and strong anion-exchange functionalities. The resin consists of ethylvinylbenzene, 55% cross-linked with divinylbenzene copolymer, 3 to 15 µm in diameter, and a surface area not less than 350 m2 per g. Substrate is coated with quaternary ammonium functionalized latex particles consisting of styrene cross-linked with divinylbenzene.6
L51—Amylose tris-3,5-dimethylphenylcarbamate-coated, porous, spherical, silica particles, 5 to 10 µm in diameter.7
L52—A strong cation exchange resin made of porous silica with sulfopropyl groups, 5 to 10 µm in diameter.8
L53—Weak cation-exchange resin consisting of ethylvinylbenzene, 55% cross-linked with divinylbenzene copolymer, 3 to 15 µm diameter. Substrate is surface grafted with carboxylic acid and/or phosphoric acid functionalized monomers. Capacity not less than 500 µEq/column.9
L54—A size exclusion medium made of covalent bonding of dextran to highly cross-linked porous agarose beads, about 13 µm in diameter.10
L55—A strong cation-exchange resin made of porous silica coated with polybutadiene–maleic acid copolymer, about 5 µm in diameter.11
L56—Isopropyl silane chemically bonded to totally porous silica particles, 3 to 10 µm in diameter.12
L57—A chiral-recognition protein, ovomucoid, chemically bonded to silica particles, about 5 µm in diameter, with a pore size of 120 .
.L58—Strong cation-exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in the sodium form, about 7 to 11 µm in diameter.13
L59—Packing having the capacity to separate proteins by molecular weight over the range of 10 to 500 kDa. It is spherical (10 µm), silica-based, and processed to provide hydrophilic characteristics and pH stability.14
L60—Spherical, porous silica gel, 3 or 5 µm in diameter, the surface of which has been covalently modified with palmitamidopropyl groups and endcapped with acetamidopropyl groups to a ligand density of about 6 µmoles per m2.15
L61—A hydroxide selective strong anion-exchange resin consisting of a highly cross-linked core of 13 µm microporous particles having a pore size less than 10 units and consisting of ethylvinylbenzene cross-linked with 55% divinylbenzene with a latex coating composed of 85 nm diameter microbeads bonded with alkanol quaternary ammonium ions (6%).16
units and consisting of ethylvinylbenzene cross-linked with 55% divinylbenzene with a latex coating composed of 85 nm diameter microbeads bonded with alkanol quaternary ammonium ions (6%).16 L62—C30 silane bonded phase on a fully porous spherical silica, 3 to 15 µm in diameter.
Phases
G1—Dimethylpolysiloxane oil.
G2—Dimethylpolysiloxane gum.
G3—50% Phenyl-50% methylpolysiloxane.
G4—Diethylene glycol succinate polyester.
G5—3-Cyanopropylpolysiloxane.
G6—Trifluoropropylmethylpolysiloxane.
G7—50% 3-Cyanopropyl-50% phenylmethylsilicone.
G8—80% Bis(3-cyanopropyl)-20% 3-cyanopropylphenylpolysiloxane (percentages refer to molar substitution).
G9—Methylvinylpolysiloxane.
G10—Polyamide formed by reacting a C36 dicarboxylic acid with 1,3-di-4-piperidylpropane and piperidine in the respective mole ratios of 1.00:0.90:0.20.
G11—Bis(2-ethylhexyl) sebacate polyester.
G12—Phenyldiethanolamine succinate polyester.
G13—Sorbitol.
G14—Polyethylene glycol (av. mol. wt. of 950 to 1050).
G15—Polyethylene glycol (av. mol. wt. of 3000 to 3700).
G16—Polyethylene glycol compound (av. mol. wt. about 15,000). A high molecular weight compound of polyethylene glycol with a diepoxide linker. Available commercially as Polyethylene Glycol Compound 20M, or as Carbowax 20M, from suppliers of chromatographic reagents.
G17—75% Phenyl-25% methylpolysiloxane.
G18—Polyalkylene glycol.
G19—25% Phenyl-25% cyanopropyl-50% methylsilicone.
G20—Polyethylene glycol (av. mol. wt. of 380 to 420).
G21—Neopentyl glycol succinate.
G22—Bis(2-ethylhexyl) phthalate.
G23—Polyethylene glycol adipate.
G24—Diisodecyl phthalate.
G25—Polyethylene glycol compound TPA. A high molecular weight compound of a polyethylene glycol and a diepoxide that is esterified with terephthalic acid. Available commercially as Carbowax 20M-TPA from suppliers of chromatographic reagents.
G26—25% 2-Cyanoethyl-75% methylpolysiloxane.
G27—5% Phenyl-95% methylpolysiloxane.
G28—25% Phenyl-75% methylpolysiloxane.
G29—3,3¢-Thiodipropionitrile.
G30—Tetraethylene glycol dimethyl ether.
G31—Nonylphenoxypoly(ethyleneoxy)ethanol (av. ethyleneoxy chain length is 30); Nonoxynol 30.
G32—20% Phenylmethyl-80% dimethylpolysiloxane.
G33—20% Carborane-80% methylsilicone.
G34—Diethylene glycol succinate polyester stabilized with phosphoric acid.
G35—A high molecular weight compound of a polyethylene glycol and a diepoxide that is esterified with nitroterephthalic acid.
G36—1% Vinyl-5% phenylmethylpolysiloxane.
G37—Polyimide.
G38—Phase G1 containing a small percentage of a tailing inhibitor.17
G39—Polyethylene glycol (av. mol. wt. about 1500).
G40—Ethylene glycol adipate.
G41—Phenylmethyldimethylsilicone (10% phenyl-substituted).
G42—35% phenyl-65% dimethylpolysiloxane (percentages refer to molar substitution).
G43—6% cyanopropylphenyl-94% dimethylpolysiloxane (percentages refer to molar substitution).
G44—2% low molecular weight petrolatum hydrocarbon grease and 1% solution of potassium hydroxide.
G45—Divinylbenzene-ethylene glycol-dimethylacrylate.
G46—14% Cyanopropylphenyl-86% methylpolysiloxane.
G47—Polyethylene glycol (av. mol. wt. of about 8000).
G48—Highly polar, partially cross-linked cyanopolysiloxane.
G49—Proprietary derivatized phenyl groups on a polysiloxane backbone.18
Supports
NOTE—Unless otherwise specified, mesh sizes of 80 to 100 or, alternatively, 100 to 120 are intended.
S1A—Siliceous earth for gas chromatography has been flux-calcined by mixing diatomite with Na2CO3 flux and calcining above 900. The siliceous earth is acid-washed, then water-washed until neutral, but not base-washed. The siliceous earth may be silanized by treating with an agent such as dimethyldichlorosilane19 to mask surface silanol groups.
. The siliceous earth is acid-washed, then water-washed until neutral, but not base-washed. The siliceous earth may be silanized by treating with an agent such as dimethyldichlorosilane19 to mask surface silanol groups.S1AB—The siliceous earth as described above is both acid- and base-washed.19
S1C—A support prepared from crushed firebrick and calcined or burned with a clay binder above 900 with subsequent acid-wash. It may be silanized.
with subsequent acid-wash. It may be silanized.S1NS—The siliceous earth is untreated.
S2—Styrene-divinylbenzene copolymer having a nominal surface area of less than 50 m2 per g and an average pore diameter of 0.3 to 0.4 µm.
S3—Copolymer of ethylvinylbenzene and divinylbenzene having a nominal surface area of 500 to 600 m2 per g and an average pore diameter of 0.0075 µm.
S4—Styrene-divinylbenzene copolymer with aromatic –O and –N groups, having a nominal surface area of 400 to 600 m2 per g and an average pore diameter of 0.0076 µm.
S5—40- to 60-mesh, high-molecular weight tetrafluorethylene polymer.
S6—Styrene-divinylbenzene copolymer having a nominal surface area of 250 to 350 m2 per g and an average pore diameter of 0.0091 µm.
S7—Graphitized carbon having a nominal surface area of 12 m2 per g.
S8—Copolymer of 4-vinyl-pyridine and styrene-divinylbenzene.
S9—A porous polymer based on 2,6-diphenyl-p-phenylene oxide.
S10—A highly polar cross-linked copolymer of acrylonitrite and divinylbenzene.
S11—Graphitized carbon having a nominal surface area of 100 m2 per g modified with small amounts of petrolatum and polyethylene glycol compound.20
S12—Graphitized carbon having a nominal surface area of 100 m2 per g.
1 A suitable grade is acid-washed Celite 545, available from Johns-Manville Corp., 22 East 40th St., New York, NY 10016.
2 Available as YMC-Pack PVA-SIL manufactured by YMC Co., Ltd. and distributed by Waters Corp. (www.waters.com).
3 Available as TSKgel G4000 SWXL from Tosoh Biosep (www.tosohbiosep.com).
5 Available as Zirchrom PBD, manufactured by ZirChrom Separations, Inc., distributed by Alltech, www.Alltechweb.com.
6 Available as OmniPac PAX-500 and distributed by Dionex Corp. (www.dionex.com).
7 Available as Chiralpak AD from Chiral Technologies, Inc., (www.chiraltech.com).
8 Available as TSK IC SW Cation from Tosoh Biosep (www.tosohbiosep.com).
9 Available as IonPac CS14 distributed by Dionex Corp. (www.dionex.com).
10 Available as Superdex Peptide HR 10/30 from Amersham Pharmacia Biotech (www.amershambiosciences.com).
11 Available as IC-Pak C M/D from Waters Corp. (www.waters.com).
12 Available as Zorbax SB-C3 from Agilent Technologies (www.agilent.com/chem).
13 Available as Aminex HPX-87N from Bio-Rad Laboratories, (2000/01 catalog, #125-0143) (www.bio-rad.com).
14 Available as TSKgel G3000SW Column (analytical column) and TSKgel Guard (guard column) from Tosoh Biosep (part numbers 05789 and 05371, respectively). (www.tosohbiosep.com)
15 Available as Supelcosil ABZ from Supelco. (www.sigma-aldrich.com/supelco)
16 Available as Ion Pac AS 11 and Ag 11 from Dionex (www.dionex.com).
17 A suitable grade is available commercially as “SP2100/0.1% Carbowax 1500” from Supelco, Inc., (www.sigma-aldrich.com/supelco).
18 A suitable grade is available commercially as “Optima Delta 3” from Machery-Nagel, Inc., 215 River Vale Road, River Vale, NJ 07675.
19 Unless otherwise specified in the individual monograph, silanized support is intended.
20 Commercially available as SP1500 on Carbopack B from Supelco.
| | |
THIN-LAYER CHROMATOGRAPHY
In thin-layer chromatography, the adsorbent is a relatively thin, uniform layer of dry, finely powdered material applied to a glass, plastic, or metal sheet or plate, glass plates being most commonly employed. The coated plate can be considered an “open chromatographic column” and the separations achieved may be based upon adsorption, partition, or a combination of both effects, depending on the particular type of stationary phase, its preparation, and its use with different solvents. Thin-layer chromatography on ion-exchange layers can be used for the fractionation of polar compounds. Presumptive identification can be effected by observation of spots or zones of identical RF value and about equal magnitude obtained, respectively, with an unknown and a reference sample chromatographed on the same plate. A visual comparison of the size or intensity of the spots or zones may serve for semiquantitative estimation. Quantitative measurements are possible by means of densitometry (absorbance or fluorescence measurements), or the spots may be carefully removed from the plate, followed by elution with a suitable solvent and spectrophotometric measurement. For two-dimensional thin-layer chromatography, the chromatographed plate is turned at a right angle and again chromatographed, usually in another chamber equilibrated with a different solvent system.
Apparatus— Acceptable apparatus and materials for thin-layer chromatography consist of the following.
A TLC or HPTLC plate. The chromatography is generally carried out using precoated plates or sheets (on glass, aluminum, or polyester support) of suitable size. It may be necessary to clean the plates prior to separation. This can be done by migration of, or immersion in, an appropriate solvent. The plates may also be impregnated by procedures such as development, immersion, or spraying. At the time of use, the plates may be activated, if necessary, by heating in an oven at 120 for 20 minutes. The stationary phase of TLC plates has an average particle size of 10–15 µm, and that of HPTLC plates an average particle size of 5 µm. Commercial plates with a preadsorbant zone can be used if they are specified in a monograph. Sample applied to the preabsorbant region develops into sharp, narrow bands at the preabsorbant-sorbent interface. Alternatively, flat glass plates of convenient size, typically 20 cm × 20 cm can be coated as described under Preparation of Chromatographic Plates.
for 20 minutes. The stationary phase of TLC plates has an average particle size of 10–15 µm, and that of HPTLC plates an average particle size of 5 µm. Commercial plates with a preadsorbant zone can be used if they are specified in a monograph. Sample applied to the preabsorbant region develops into sharp, narrow bands at the preabsorbant-sorbent interface. Alternatively, flat glass plates of convenient size, typically 20 cm × 20 cm can be coated as described under Preparation of Chromatographic Plates. A suitable manual, semiautomatic, or automatic application device can be used to ensure proper positioning of the plate and proper transfer of the sample, with respect to volume and position, onto the plate. Alternatively, a template can be used to guide in manually placing the test spots at definite intervals, to mark distances as needed, and to aid in labeling the plates. For the proper application of the solutions, micropipets, microsyringes, or calibrated disposable capillaries are recommended.
For ascending development, a chromatographic chamber made of inert, transparent material and having the following specifications is used: a flat bottom or twin trough, a tightly fitted lid, and a size suitable for the plates. For horizontal development, the chamber is provided with a reservoir for the mobile phase, and it also contains a device for directing the mobile phase to the stationary phase.
Devices for transfer of reagents onto the plate by spraying, immersion, or exposure to vapor and devices to facilitate any necessary heating for visualization of the separated spots or zones.
A UV light source suitable for observations under short (254 nm) and long (365 nm) wavelength UV light.
A suitable device for documentation of the visualized chromatographic result.
Procedure— Apply the prescribed volume of the test solution and the standard solution in sufficiently small portions to obtain circular spots of 2 to 5 mm in diameter (1 to 2 mm on HPTLC plates) or bands of 10 to 20 mm by 1 to 2 mm (5 to 10 mm by 0.5 to 1 mm on HPTLC plates) at an appropriate distance from the lower edge—during chromatography the application position must be 3 mm (HPTLC) to 5 mm (TLC) above the level of the developing solvent—and from the sides of the plate. Apply the solutions on a line parallel to the lower edge of the plate with an interval of at least 10 mm (5 mm on HPTLC plates) between the centers of spots or 4 mm (2 mm on HPTLC plates) between the edges of bands, and allow to dry.
Ascending Development— Line at least one wall of the chromatographic chamber with filter paper. Pour into the chromatographic chamber a quantity of the mobile phase sufficient for the size of the chamber to give, after impregnation of the filter paper, a level of depth appropriate to the dimension of the plate used. For saturation of the chromatographic chamber, close the lid, and allow the system to equilibrate. Unless otherwise indicated, the chromatographic separation is performed in a saturated chamber.
Place the plate in the chamber, ensuring that the plate is as vertical as possible and that the spots or bands are above the surface of the mobile phase, and close the chamber. The stationary phase faces the inside of the chamber. Remove the plate when the mobile phase has moved over the prescribed distance. Dry the plate, and visualize the chromatograms as prescribed. For two-dimensional chromatography, dry the plates after the first development, and carry out a second development in a direction perpendicular to that of the first development.
Horizontal Development— Introduce a sufficient quantity of the developing solvent into the reservoir of the chamber using a syringe or pipet. Place the plate horizontally in the chamber, connect the mobile phase direction device according to the manufacturer's instructions, and close the chamber. If prescribed, develop the plate starting simultaneously at both ends. Remove the plate when the mobile phase has moved over the distance prescribed in the monograph. Dry the plate, and visualize the chromatograms as prescribed.
For two-dimensional chromatography, dry the plates after the first development, and carry out a second development in a direction perpendicular to that of the first development.
Detection— Observe the dry plate first under short-wavelength UV light (254 nm) and then under long-wavelength UV light (365 nm) or as stated in the monograph. If further directed, spray, immerse, or expose the plate to vapors of the specified reagent, heat the plate when required, observe, and compare the test chromatogram with the standard chromatogram. Document the plate after each observation. Measure and record the distance of each spot or zone from the point of origin, and indicate for each spot or zone the wavelength under which it was observed. Determine the RF values for the principal spots or zones (see Glossary of Symbols).
Quantitative Measurement— Using appropriate instrumentation, substances separated by TLC and responding to ultraviolet-visible (UV-Vis) irradiation prior to or after derivatization can be determined directly on the plate. While moving the plate or the measuring device, the plate is examined by measuring the reflectance of the incident light. Similarly, fluorescence may be measured using an appropriate optical system. Substances containing radionuclides can be quantified in three ways: (1) directly by moving the plate alongside a suitable counter or vice versa; (2) by cutting the plates into strips and measuring the radioactivity on each individual strip using a suitable counter; or (3) by scraping off the stationary phase, dissolving it in a suitable scintillation cocktail, and measuring the radioactivity using a liquid scintillation counter (see Radioactivity 821).
821). The apparatus for direct quantitative measurement on the plate is a densitometer that is composed of a mechanical device to move the plate or the measuring device along the x-axis and the y-axis, a recorder, a suitable integrator or a computer; and, for substances responding to UV-Vis irradiation, a photometer with a source of light, an optical device capable of generating monochromatic light, and a photo cell of adequate sensitivity, all of which are used for the measurement of reflectance. In the case where fluorescence is measured, a suitable filter is also required to prevent the light used for excitation from reaching the photo cell while permitting the emitted light or specific portions thereof to pass. The linearity range of the counting device must be verified.
For quantitative tests, it is necessary to apply to the plate not fewer than three standard solutions of the substance to be examined, the concentrations of which span the expected value in the test solution (e.g., 80%, 100%, and 120%). Derivatize with the prescribed reagent, if necessary, and record the reflectance or fluorescence in the chromatograms obtained. Use the measured results for the calculation of the amount of substance in the test solution.
Preparation of Chromatographic Plates—
Apparatus—
Flat glass plates of convenient size, typically 20 cm × 20 cm.
An aligning tray or a flat surface upon which to align and rest the plates during the application of the adsorbent.
A storage rack to hold the prepared plates during drying and transportation. The rack holding the plates should be kept in a desiccator or be capable of being sealed in order to protect the plates from the environment after removal from the drying oven.
The adsorbent consists of finely divided adsorbent materials, normally 5 to 40 µm in diameter, suitable for chromatography. It can be applied directly to the glass plate or can be bonded to the plate by means of plaster of Paris [calcium sulfate hemihydrate (at a ratio of 5% to 15%)] or with starch paste or other binders. The plaster of Paris will not yield as hard a surface as will the starch, but it is not affected by strongly oxidizing spray reagents. The adsorbent may contain fluorescing material to aid in the visualization of spots that absorb UV light.
A spreader, which, when moved over the glass plate, will apply a uniform layer of adsorbent of desired thickness over the entire surface of the plate.
Procedure— [NOTE—In this procedure, use Purified Water that is obtained by distillation.] Clean the glass plates scrupulously, using an appropriate cleaning solution (see Cleaning Glass Apparatus 1051), rinsing them with copious quantities of water until the water runs off the plates without leaving any visible water or oily spots, then dry. It is important that the plates be completely free from lint and dust when the adsorbent is applied.
1051), rinsing them with copious quantities of water until the water runs off the plates without leaving any visible water or oily spots, then dry. It is important that the plates be completely free from lint and dust when the adsorbent is applied. Arrange the plate or plates on the aligning tray, place a 5- × 20-cm plate adjacent to the front edge of the first square plate and another 5- × 20-cm plate adjacent to the rear edge of the last square, and secure all of the plates so that they will not slip during the application of the adsorbent. Position the spreader on the end plate opposite the raised end of the aligning tray. Mix 1 part of adsorbent with 2 parts of water (or in the ratio suggested by the supplier) by shaking vigorously for 30 seconds in a glass-stoppered conical flask, and transfer the slurry to the spreader. Usually 30 g of adsorbent and 60 mL of water are sufficient for five 20- × 20-cm plates. Complete the application of adsorbents using plaster of Paris binder within 2 minutes of the addition of the water, because thereafter the mixture begins to harden. Draw the spreader smoothly over the plates toward the raised end of the aligning tray, and remove the spreader when it is on the end plate next to the raised end of the aligning tray. (Wash away all traces of adsorbent from the spreader immediately after use.) Allow the plates to remain undisturbed for 5 minutes, then transfer the square plates, layer side up, to the storage rack, and dry at 105 for 30 minutes. Preferably place the rack at an angle in the drying oven to prevent the condensation of moisture on the back sides of plates in the rack. When the plates are dry, allow them to cool to room temperature, and inspect the uniformity of the distribution and the texture of the adsorbent layer; transmitted light will show uniformity of distribution, and reflected light will show uniformity of texture. Store the satisfactory plates over silica gel in a suitable chamber.
GAS CHROMATOGRAPHY
GAS CHROMATOGRAPHY
for 30 minutes. Preferably place the rack at an angle in the drying oven to prevent the condensation of moisture on the back sides of plates in the rack. When the plates are dry, allow them to cool to room temperature, and inspect the uniformity of the distribution and the texture of the adsorbent layer; transmitted light will show uniformity of distribution, and reflected light will show uniformity of texture. Store the satisfactory plates over silica gel in a suitable chamber. | |
GAS CHROMATOGRAPHY
The distinguishing features of gas chromatography are a gaseous mobile phase and a solid or immobilized liquid stationary phase. Liquid stationary phases are available in packed or capillary columns. In the packed columns, the liquid phase is deposited on a finely divided, inert solid support, such as diatomaceous earth, porous polymer, or graphitized carbon, which is packed into a column that is typically 2 to 4 mm in internal diameter and 1 to 3 m in length. In capillary columns, which contain no packing, the liquid phase is deposited on the inner surface of the column and may be chemically bonded to it. In gas-solid chromatography, the solid phase is an active adsorbent, such as alumina, silica, or carbon, packed into a column. Polyaromatic porous resins, which are sometimes used in packed columns, are not coated with a liquid phase.
When a vaporized compound is introduced into the carrier gas and carried into the column, it is partitioned between the gas and stationary phases by a dynamic countercurrent distribution process. The compound is carried down the column by the carrier gas, retarded to a greater or lesser extent by sorption and desorption on the stationary phase. The elution of the compound is characterized by the partition ratio, k¢, a dimensionless quantity also called the capacity factor (see Glossary of Symbols for the definition of symbols). It is equivalent to the ratio of the time required for the compound to flow through the column (the retention time) to the elution time of an unretained compound. The value of the capacity factor depends on the chemical nature of the compound, the nature, amount, and surface area of the liquid phase, the column temperature, and the gas flow rate. Under a specified set of experimental conditions, a characteristic capacity factor exists for every compound. Separation by gas chromatography occurs only if the compounds concerned have different capacity factors.
Apparatus— A gas chromatograph consists of a carrier gas source, an injection port, column, detector, and recording device. The injection port, column, and detector are temperature-controlled. The typical carrier gas is helium, nitrogen, or hydrogen, depending on the column and detector in use. The gas is supplied from a high-pressure cylinder or high-purity gas generator and passes through suitable pressure-reducing valves and a flow meter to the injection port and column. Compounds to be chromatographed, either in solution or as gases, are injected into the gas stream at the injection port. Depending upon the configuration of the apparatus, the test mixture may be injected directly into the column or be vaporized in the injection port and mixed into the flowing carrier gas prior to entering the column.
Once in the column, compounds in the test mixture are separated by virtue of differences in their capacity factors, which in turn depend upon vapor pressure and degree of interaction with the stationary phase. The capacity factor, which governs resolution, retention times, and column efficiencies of components of the test mixture, is also temperature-dependent. The use of temperature-programmable column ovens takes advantage of this dependence to achieve efficient separation of compounds differing widely in vapor pressure.
As resolved compounds emerge separately from the column, they pass through a differential detector, which responds to the amount of each compound present. The type of detector to be used depends upon the nature of the compounds to be analyzed and is specified in the individual monograph. Detectors are heated to prevent condensation of the eluting compounds.
Detector output is recorded as a function of time, producing a chromatogram, which consists of a series of peaks on a time axis. Each peak represents a compound in the vaporized test mixture, although some peaks may overlap. The elution time is a characteristic of an individual compound; and the instrument response, measured as peak area or peak height, is a function of the amount present.
Injectors—Sample injection devices range from simple syringes to fully programmable automatic injectors. The amount of sample that can be injected into a capillary column without overloading is small compared to the amount that can be injected into packed columns, and may be less than the smallest amount that can be manipulated satisfactorily by syringe. Capillary columns, therefore, often are used with injectors able to split samples into two fractions, a small one that enters the column and a large one that goes to waste. Such injectors may be used in a splitless mode for analyses of trace or minor components.
Purge and trap injectors are equipped with a sparging device by which volatile compounds in solution are carried into a low-temperature trap. When sparging is complete, trapped compounds are desorbed into the carrier gas by rapid heating of the temperature-programmable trap.
Headspace injectors are equipped with a thermostatically controlled sample heating chamber. Solid or liquid samples in tightly closed containers are heated in the chamber for a fixed period of time, allowing the volatile components in the sample to reach an equilibrium between the nongaseous phase and the gaseous or headspace phase.
After this equilibrium has been established, the injector automatically introduces a fixed amount of the headspace in the sample container into the gas chromatograph.
Columns—Capillary columns, which are usually made of fused silica, are typically 0.2 to 0.53 mm in internal diameter and 5 to 60 m in length. The liquid or stationary phase, which is sometimes chemically bonded to the inner surface, is 0.1 to 1.0 µm thick, although nonpolar stationary phases may be up to 5 µm thick. A list of liquid phases in current use is given in the section Chromatographic Reagents.
Packed columns, made of glass or metal, are 1 to 3 m in length with internal diameters of 2 to 4 mm. Those used for analysis typically are porous polymers or solid supports with liquid phase loadings of about 5% (w/w). High-capacity columns, with liquid phase loadings of about 20% (w/w), are used for large test specimens and for the determination of low molecular weight compounds such as water. The capacity required influences the choice of solid support.
Supports for analysis of polar compounds on low-capacity, low-polarity liquid phase columns must be inert to avoid peak tailing. The reactivity of support materials can be reduced by silanizing prior to coating with liquid phase. Acid-washed, flux-calcined diatomaceous earth is often used for drug analysis. Support materials are available in various mesh sizes, with 80- to 100-mesh and 100- to 120-mesh being most commonly used with 2- to 4-mm columns. Supports and liquid phases are listed in the section Chromatographic Reagents.
Retention time and the peak efficiency depend on the carrier gas flow rate; retention time is also directly proportional to column length, while resolution is proportional to the square root of the column length. For packed columns, the carrier gas flow rate is usually expressed in mL per minute at atmospheric pressure and room temperature. It is measured at the detector outlet with a flowmeter while the column is at operating temperature. The linear flow rate through a packed column is inversely proportional to the square of the column diameter for a given flow volume. Flow rates of 60 mL per minute in a 4-mm column and 15 mL per minute in a 2-mm column give identical linear flow rates and thus similar retention times. Unless otherwise specified in the individual monograph, flow rates for packed columns are about 30 to 60 mL per minute. For capillary columns, linear flow velocity is often used instead of flow rate. This is conveniently determined from the length of the column and the retention time of a dilute methane sample, provided a flame-ionization detector is in use. At high operating temperatures there is sufficient vapor pressure to result in a gradual loss of liquid phase, a process called bleeding.
Detectors—Flame-ionization detectors are used for most pharmaceutical analyses, with lesser use made of thermal conductivity, electron-capture, nitrogen-phosphorus, and mass spectrometric detectors. For quantitative analyses, detectors must have a wide linear dynamic range: the response must be directly proportional to the amount of compound present in the detector over a wide range of concentrations. Flame-ionization detectors have a wide linear range and are sensitive to most organic compounds. Detector response depends on the structure and concentration of the compound and on the flow rates of the combustion, air, makeup, and carrier gases. Unless otherwise specified in individual monographs, flame-ionization detectors with either helium or nitrogen carrier gas are to be used for packed columns, and helium or hydrogen is used for capillary columns.
The thermal conductivity detector employs a heated wire placed in the carrier gas stream. When an analyte enters the detector with the carrier gas, the difference in thermal conductivity of the gas stream (carrier and sample components) relative to that of a reference flow of carrier gas alone is measured. In general, the thermal conductivity detector responds uniformly to volatile compounds regardless of structure; however, it is considerably less sensitive than the flame-ionization detector.
The alkali flame-ionization detector, sometimes called an NP or nitrogen-phosphorus detector, contains a thermionic source, such as an alkali-metal salt or a glass element containing rubidium or other metal, that results in the efficient ionization of organic nitrogen and phosphorus compounds. It is a selective detector that shows little response to hydrocarbons.
The electron-capture detector contains a radioactive source of ionizing radiation. It exhibits an extremely high response to compounds containing halogens and nitro groups but little response to hydrocarbons. The sensitivity increases with the number and atomic weight of the halogen atoms.
Data Collection Devices—Modern data stations receive the detector output, calculate peak areas and peak heights, and print chromatograms, complete with run parameters and peak data. Chromatographic data may be stored and reprocessed, with integration and other calculation variables being changed as required. Data stations are used also to program the chromatograph, controlling most operational variables and providing for long periods of unattended operation.
Data can also be collected for manual measurement on simple recorders or on integrators whose capabilities range from those providing a printout of peak areas to those providing chromatograms with peak areas and peak heights calculated and data stored for possible reprocessing.
Procedure— Packed and capillary columns must be conditioned before use until the baseline and other characteristics are stable. This may be done by operation at a temperature above that called for by the method or by repeated injections of the compound or mixture to be chromatographed. The column or packing material supplier provides instructions for the recommended conditioning procedure. In the case of thermally stable methyl- and phenyl-substituted polysiloxanes, a special sequence increases inertness and efficiency; maintain the column at a temperature of 250 for 1 hour, with helium flowing, to remove oxygen and solvents. Stop the flow of helium, heat at about 340 for 4 hours, then reduce the heating to a temperature of 250, and condition with helium flowing until stable.
for 1 hour, with helium flowing, to remove oxygen and solvents. Stop the flow of helium, heat at about 340 for 4 hours, then reduce the heating to a temperature of 250, and condition with helium flowing until stable. Most drugs are reactive polar molecules. Successful chromatography may require conversion of the drug to a less polar and more volatile derivative by treatment of reactive groups with appropriate reagents. Silylating agents are widely used for this purpose and are readily available.
Assays require quantitative comparison of one chromatogram with another. A major source of error is irreproducibility in the amount of sample injected, notably when manual injections are made with a syringe. The effects of variability can be minimized by addition of an internal standard, a noninterfering compound present at the same concentration in test and standard solutions. The ratio of peak response of the analyte to that of the internal standard is compared from one chromatogram to another. Where the internal standard is chemically similar to the substance being determined, there is also compensation for minor variations in column and detector characteristics. In some cases, the internal standard may be carried through the sample preparation procedure prior to gas chromatography to control other quantitative aspects of the assay. Automatic injectors greatly improve the reproducibility of sample injections and reduce the need for internal standards.
Many monographs require that system suitability requirements be met before samples are analyzed (see System Suitability and Interpretation of Chromatograms).
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GAS CHROMATOGRAPHY
The distinguishing features of gas chromatography are a gaseous mobile phase and a solid or immobilized liquid stationary phase. Liquid stationary phases are available in packed or capillary columns. In the packed columns, the liquid phase is deposited on a finely divided, inert solid support, such as diatomaceous earth, porous polymer, or graphitized carbon, which is packed into a column that is typically 2 to 4 mm in internal diameter and 1 to 3 m in length. In capillary columns, which contain no packing, the liquid phase is deposited on the inner surface of the column and may be chemically bonded to it. In gas-solid chromatography, the solid phase is an active adsorbent, such as alumina, silica, or carbon, packed into a column. Polyaromatic porous resins, which are sometimes used in packed columns, are not coated with a liquid phase.
When a vaporized compound is introduced into the carrier gas and carried into the column, it is partitioned between the gas and stationary phases by a dynamic countercurrent distribution process. The compound is carried down the column by the carrier gas, retarded to a greater or lesser extent by sorption and desorption on the stationary phase. The elution of the compound is characterized by the partition ratio, k¢, a dimensionless quantity also called the capacity factor (see Glossary of Symbols for the definition of symbols). It is equivalent to the ratio of the time required for the compound to flow through the column (the retention time) to the elution time of an unretained compound. The value of the capacity factor depends on the chemical nature of the compound, the nature, amount, and surface area of the liquid phase, the column temperature, and the gas flow rate. Under a specified set of experimental conditions, a characteristic capacity factor exists for every compound. Separation by gas chromatography occurs only if the compounds concerned have different capacity factors.
Apparatus— A gas chromatograph consists of a carrier gas source, an injection port, column, detector, and recording device. The injection port, column, and detector are temperature-controlled. The typical carrier gas is helium, nitrogen, or hydrogen, depending on the column and detector in use. The gas is supplied from a high-pressure cylinder or high-purity gas generator and passes through suitable pressure-reducing valves and a flow meter to the injection port and column. Compounds to be chromatographed, either in solution or as gases, are injected into the gas stream at the injection port. Depending upon the configuration of the apparatus, the test mixture may be injected directly into the column or be vaporized in the injection port and mixed into the flowing carrier gas prior to entering the column.
Once in the column, compounds in the test mixture are separated by virtue of differences in their capacity factors, which in turn depend upon vapor pressure and degree of interaction with the stationary phase. The capacity factor, which governs resolution, retention times, and column efficiencies of components of the test mixture, is also temperature-dependent. The use of temperature-programmable column ovens takes advantage of this dependence to achieve efficient separation of compounds differing widely in vapor pressure.
As resolved compounds emerge separately from the column, they pass through a differential detector, which responds to the amount of each compound present. The type of detector to be used depends upon the nature of the compounds to be analyzed and is specified in the individual monograph. Detectors are heated to prevent condensation of the eluting compounds.
Detector output is recorded as a function of time, producing a chromatogram, which consists of a series of peaks on a time axis. Each peak represents a compound in the vaporized test mixture, although some peaks may overlap. The elution time is a characteristic of an individual compound; and the instrument response, measured as peak area or peak height, is a function of the amount present.
Injectors—Sample injection devices range from simple syringes to fully programmable automatic injectors. The amount of sample that can be injected into a capillary column without overloading is small compared to the amount that can be injected into packed columns, and may be less than the smallest amount that can be manipulated satisfactorily by syringe. Capillary columns, therefore, often are used with injectors able to split samples into two fractions, a small one that enters the column and a large one that goes to waste. Such injectors may be used in a splitless mode for analyses of trace or minor components.
Purge and trap injectors are equipped with a sparging device by which volatile compounds in solution are carried into a low-temperature trap. When sparging is complete, trapped compounds are desorbed into the carrier gas by rapid heating of the temperature-programmable trap.
Headspace injectors are equipped with a thermostatically controlled sample heating chamber. Solid or liquid samples in tightly closed containers are heated in the chamber for a fixed period of time, allowing the volatile components in the sample to reach an equilibrium between the nongaseous phase and the gaseous or headspace phase.
After this equilibrium has been established, the injector automatically introduces a fixed amount of the headspace in the sample container into the gas chromatograph.
Columns—Capillary columns, which are usually made of fused silica, are typically 0.2 to 0.53 mm in internal diameter and 5 to 60 m in length. The liquid or stationary phase, which is sometimes chemically bonded to the inner surface, is 0.1 to 1.0 µm thick, although nonpolar stationary phases may be up to 5 µm thick. A list of liquid phases in current use is given in the section Chromatographic Reagents.
Packed columns, made of glass or metal, are 1 to 3 m in length with internal diameters of 2 to 4 mm. Those used for analysis typically are porous polymers or solid supports with liquid phase loadings of about 5% (w/w). High-capacity columns, with liquid phase loadings of about 20% (w/w), are used for large test specimens and for the determination of low molecular weight compounds such as water. The capacity required influences the choice of solid support.
Supports for analysis of polar compounds on low-capacity, low-polarity liquid phase columns must be inert to avoid peak tailing. The reactivity of support materials can be reduced by silanizing prior to coating with liquid phase. Acid-washed, flux-calcined diatomaceous earth is often used for drug analysis. Support materials are available in various mesh sizes, with 80- to 100-mesh and 100- to 120-mesh being most commonly used with 2- to 4-mm columns. Supports and liquid phases are listed in the section Chromatographic Reagents.
Retention time and the peak efficiency depend on the carrier gas flow rate; retention time is also directly proportional to column length, while resolution is proportional to the square root of the column length. For packed columns, the carrier gas flow rate is usually expressed in mL per minute at atmospheric pressure and room temperature. It is measured at the detector outlet with a flowmeter while the column is at operating temperature. The linear flow rate through a packed column is inversely proportional to the square of the column diameter for a given flow volume. Flow rates of 60 mL per minute in a 4-mm column and 15 mL per minute in a 2-mm column give identical linear flow rates and thus similar retention times. Unless otherwise specified in the individual monograph, flow rates for packed columns are about 30 to 60 mL per minute. For capillary columns, linear flow velocity is often used instead of flow rate. This is conveniently determined from the length of the column and the retention time of a dilute methane sample, provided a flame-ionization detector is in use. At high operating temperatures there is sufficient vapor pressure to result in a gradual loss of liquid phase, a process called bleeding.
Detectors—Flame-ionization detectors are used for most pharmaceutical analyses, with lesser use made of thermal conductivity, electron-capture, nitrogen-phosphorus, and mass spectrometric detectors. For quantitative analyses, detectors must have a wide linear dynamic range: the response must be directly proportional to the amount of compound present in the detector over a wide range of concentrations. Flame-ionization detectors have a wide linear range and are sensitive to most organic compounds. Detector response depends on the structure and concentration of the compound and on the flow rates of the combustion, air, makeup, and carrier gases. Unless otherwise specified in individual monographs, flame-ionization detectors with either helium or nitrogen carrier gas are to be used for packed columns, and helium or hydrogen is used for capillary columns.
The thermal conductivity detector employs a heated wire placed in the carrier gas stream. When an analyte enters the detector with the carrier gas, the difference in thermal conductivity of the gas stream (carrier and sample components) relative to that of a reference flow of carrier gas alone is measured. In general, the thermal conductivity detector responds uniformly to volatile compounds regardless of structure; however, it is considerably less sensitive than the flame-ionization detector.
The alkali flame-ionization detector, sometimes called an NP or nitrogen-phosphorus detector, contains a thermionic source, such as an alkali-metal salt or a glass element containing rubidium or other metal, that results in the efficient ionization of organic nitrogen and phosphorus compounds. It is a selective detector that shows little response to hydrocarbons.
The electron-capture detector contains a radioactive source of ionizing radiation. It exhibits an extremely high response to compounds containing halogens and nitro groups but little response to hydrocarbons. The sensitivity increases with the number and atomic weight of the halogen atoms.
Data Collection Devices—Modern data stations receive the detector output, calculate peak areas and peak heights, and print chromatograms, complete with run parameters and peak data. Chromatographic data may be stored and reprocessed, with integration and other calculation variables being changed as required. Data stations are used also to program the chromatograph, controlling most operational variables and providing for long periods of unattended operation.
Data can also be collected for manual measurement on simple recorders or on integrators whose capabilities range from those providing a printout of peak areas to those providing chromatograms with peak areas and peak heights calculated and data stored for possible reprocessing.
Procedure— Packed and capillary columns must be conditioned before use until the baseline and other characteristics are stable. This may be done by operation at a temperature above that called for by the method or by repeated injections of the compound or mixture to be chromatographed. The column or packing material supplier provides instructions for the recommended conditioning procedure. In the case of thermally stable methyl- and phenyl-substituted polysiloxanes, a special sequence increases inertness and efficiency; maintain the column at a temperature of 250 for 1 hour, with helium flowing, to remove oxygen and solvents. Stop the flow of helium, heat at about 340 for 4 hours, then reduce the heating to a temperature of 250, and condition with helium flowing until stable.
for 1 hour, with helium flowing, to remove oxygen and solvents. Stop the flow of helium, heat at about 340 for 4 hours, then reduce the heating to a temperature of 250, and condition with helium flowing until stable. Most drugs are reactive polar molecules. Successful chromatography may require conversion of the drug to a less polar and more volatile derivative by treatment of reactive groups with appropriate reagents. Silylating agents are widely used for this purpose and are readily available.
Assays require quantitative comparison of one chromatogram with another. A major source of error is irreproducibility in the amount of sample injected, notably when manual injections are made with a syringe. The effects of variability can be minimized by addition of an internal standard, a noninterfering compound present at the same concentration in test and standard solutions. The ratio of peak response of the analyte to that of the internal standard is compared from one chromatogram to another. Where the internal standard is chemically similar to the substance being determined, there is also compensation for minor variations in column and detector characteristics. In some cases, the internal standard may be carried through the sample preparation procedure prior to gas chromatography to control other quantitative aspects of the assay. Automatic injectors greatly improve the reproducibility of sample injections and reduce the need for internal standards.
Many monographs require that system suitability requirements be met before samples are analyzed (see System Suitability and Interpretation of Chromatograms).
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
Size-Exclusion Chromatography
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HIGH-PRESSURE LIQUID CHROMATOGRAPHY
High-pressure liquid chromatography (HPLC), sometimes called high-performance liquid chromatography, is a separation technique based on a solid stationary phase and a liquid mobile phase. Separations are achieved by partition, adsorption, or ion-exchange processes, depending upon the type of stationary phase used. HPLC has distinct advantages over gas chromatography for the analysis of organic compounds. Compounds to be analyzed are dissolved in a suitable solvent, and most separations take place at room temperature. Thus, most drugs, being nonvolatile or thermally unstable compounds, can be chromatographed without decomposition or the necessity of making volatile derivatives. Most pharmaceutical analyses are based on partition chromatography and are completed within 30 minutes.
As in gas chromatography, the elution time of a compound can be described by the capacity factor, k¢ (see Glossary of Symbols), which depends on the chemical nature of the analyte, the composition and flow rate of the mobile phase, and the composition and surface area of the stationary phase. Column length is an important determinant of resolution. Only compounds having different capacity factors can be separated by HPLC.
Apparatus— A liquid chromatograph consists of a reservoir containing the mobile phase, a pump to force the mobile phase through the system at high pressure, an injector to introduce the sample into the mobile phase, a chromatographic column, a detector, and a data collection device such as a computer, integrator, or recorder. Short, small-bore columns containing densely packed particles of stationary phase provide for the rapid exchange of compounds between the mobile and stationary phases. In addition to receiving and reporting detector output, computers are used to control chromatographic settings and operations, thus providing for long periods of unattended operation.
Pumping Systems—HPLC pumping systems deliver metered amounts of mobile phase from the solvent reservoirs to the column through high-pressure tubing and fittings. Modern systems consist of one or more computer-controlled metering pumps that can be programmed to vary the ratio of mobile phase components, as is required for gradient chromatography, or to mix isocratic mobile phases (i.e., mobile phases having a fixed ratio of solvents). However, the proportion of ingredients in premixed isocratic mobile phases can be more accurately controlled than in those delivered by most pumping systems. Operating pressures up to 5000 psi or higher, with delivery rates up to about 10 mL per minute are typical. Pumps used for quantitative analysis should be constructed of materials inert to corrosive mobile phase components and be capable of delivering the mobile phase at a constant rate with minimal fluctuations over extended periods of time.
Injectors—After dissolution in mobile phase or other suitable solution, compounds to be chromatographed are injected into the mobile phase, either manually by syringe or loop injectors, or automatically by autosamplers. The latter consist of a carousel or rack to hold sample vials with tops that have a pierceable septum or stopper and an injection device to transfer sample from the vials to a loop from which it is loaded into the chromatograph. Some autosamplers can be programmed to control sample volume, the number of injections and loop rinse cycles, the interval between injections, and other operating variables.
A syringe can be used for manual injection of samples through a septum when column head pressures are less than 70 atmospheres (about 1000 psi). At higher pressures an injection valve is essential. Some valve systems incorporate a calibrated loop that is filled with test solution for transfer to the column in the mobile phase. In other systems, the test solution is transferred to a cavity by syringe and then switched into the mobile phase.
Columns—For most pharmaceutical analyses, separation is achieved by partition of compounds in the test solution between the mobile and stationary phases. Systems consisting of polar stationary phases and nonpolar mobile phases are described as normal phase, while the opposite arrangement, polar mobile phases and nonpolar stationary phases, is called reverse-phase chromatography. Partition chromatography is almost always used for hydrocarbon-soluble compounds of molecular weight less than 1000. The affinity of a compound for the stationary phase, and thus its retention time on the column, is controlled by making the mobile phase more or less polar. Mobile phase polarity can be varied by the addition of a second, and sometimes a third or even a fourth, component.
Stationary phases for modern, reverse-phase liquid chromatography typically consist of an organic phase chemically bound to silica or other materials. Particles are usually 3 to 10 µm in diameter, but sizes may range up to 50 µm or more for preparative columns. Small particles thinly coated with organic phase provide for low mass transfer resistance and, hence, rapid transfer of compounds between the stationary and mobile phases. Column polarity depends on the polarity of the bound functional groups, which range from relatively nonpolar octadecyl silane to very polar nitrile groups. Liquid, nonbound stationary phases must be largely immiscible in the mobile phase. Even so, it is usually necessary to presaturate the mobile phase with stationary phase to prevent stripping of the stationary phase from the column. Polymeric stationary phases coated on the support are more durable.
Columns used for analytical separations usually have internal diameters of 2 to 5 mm; larger diameter columns are used for preparative chromatography. Columns may be heated to give more efficient separations, but only rarely are they used at temperatures above 60 because of potential stationary phase degradation or mobile phase volatility. Unless otherwise specified in the individual monograph, columns are used at ambient temperature.
because of potential stationary phase degradation or mobile phase volatility. Unless otherwise specified in the individual monograph, columns are used at ambient temperature.Ion-exchange chromatography is used to separate water-soluble, ionizable compounds of molecular weight less than 1500. The stationary phases are usually synthetic organic resins; cation-exchange resins contain negatively charged active sites and are used to separate basic substances such as amines, while anion-exchange resins have positively charged active sites for separation of compounds with negatively charged groups, such as phosphate, sulfonate, or carboxylate groups. Water-soluble ionic or ionizable compounds are attracted to the resins, and differences in affinity bring about the chromatographic separation. The pH of the mobile phase, temperature, ion type, ionic concentration, and organic modifiers affect the equilibrium, and these variables can be adjusted to obtain the desired degree of separation.
In size-exclusion chromatography, columns are packed with a porous stationary phase. Molecules of the compounds being chromatographed are filtered according to size. Those too large to enter the pores pass unretained through the column. Smaller molecules enter the pores and are increasingly retained as molecular size decreases. These columns are typically used to measure aggregation and degradation of large molecules (see Size-Exclusion Chromatography section).
Detectors—Many compendial HPLC methods require the use of spectrophotometric detectors. Such a detector consists of a flow-through cell mounted at the end of the column. A beam of UV radiation passes through the flow cell and into the detector. As compounds elute from the column, they pass through the cell and absorb the radiation, resulting in measurable energy level changes.
Fixed, variable, and multi-wavelength detectors are widely available. Fixed wavelength detectors operate at a single wavelength, typically 254 nm, emitted by a low-pressure mercury lamp. Variable wavelength detectors contain a continuous source, such as a deuterium or high-pressure xenon lamp, and a monochromator or an interference filter to generate monochromatic radiation at a wavelength selected by the operator. The wavelength accuracy of a variable-wavelength detector equipped with a monochromator should be checked by the procedure recommended by its manufacturer; if the observed wavelengths differ by more than 3 nm from the correct values, recalibration of the instrument is indicated. Modern variable wavelength detectors can be programmed to change wavelength while an analysis is in progress. Multi-wavelength detectors measure absorbance at two or more wavelengths simultaneously. In diode array multi-wavelength detectors, continuous radiation is passed through the sample cell, then resolved into its constituent wavelengths, which are individually detected by the photodiode array. These detectors acquire absorbance data over the entire UV-visible range, thus providing the analyst with chromatograms at multiple, selectable wavelengths and spectra of the eluting peaks. Diode array detectors usually have lower signal-to-noise ratios than fixed or variable wavelength detectors, and thus are less suitable for analysis of compounds present at low concentrations.
Differential refractometer detectors measure the difference between the refractive index of the mobile phase alone and that of the mobile phase containing chromatographed compounds as it emerges from the column. Refractive index detectors are used to detect non-UV absorbing compounds, but they are less sensitive than UV detectors. They are sensitive to small changes in solvent composition, flow rate, and temperature, so that a reference column may be required to obtain a satisfactory baseline.
Fluorometric detectors are sensitive to compounds that are inherently fluorescent or that can be converted to fluorescent derivatives either by chemical transformation of the compound or by coupling with fluorescent reagents at specific functional groups. If derivatization is required, it can be done prior to chromatographic separation or, alternatively, the reagent can be introduced into the mobile phase just prior to its entering the detector.
Potentiometric, voltametric, or polarographic electrochemical detectors are useful for the quantitation of species that can be oxidized or reduced at a working electrode. These detectors are selective, sensitive, and reliable, but require conducting mobile phases free of dissolved oxygen and reducible metal ions. A pulseless pump must be used, and care must be taken to ensure that the pH, ionic strength, and temperature of the mobile phase remain constant. Working electrodes are prone to contamination by reaction products with consequent variable responses.
Electrochemical detectors with carbon-paste electrodes may be used advantageously to measure nanogram quantities of easily oxidized compounds, notably phenols and catechols.
New detectors continue to be developed in attempts to overcome the deficiencies of those being used.
Data Collection Devices—Modern data stations receive and store detector output and print out chromatograms complete with peak heights, peak areas, sample identification, and method variables. They are also used to program the liquid chromatograph, controlling most variables and providing for long periods of unattended operation.
Data also may be collected on simple recorders for manual measurement or on stand-alone integrators, which range in complexity from those providing a printout of peak areas to those providing chromatograms with peak areas and peak heights calculated and data stored for possible subsequent reprocessing.
Procedure— The mobile phase composition significantly influences chromatographic performance and the resolution of compounds in the mixture being chromatographed. For accurate quantitative work, high-purity reagents and “HPLC grade” organic solvents must be used. Water of suitable quality should have low conductivity and low UV absorption, appropriate to the intended use.
Reagents used with special types of detectors (e.g., electrochemical, mass spectrometer) may require the establishment of additional tolerances for potential interfering species. Composition has a much greater effect than temperature on the capacity factor, k¢.
In partition chromatography, the partition coefficient, and hence the separation, can be changed by addition of another component to the mobile phase. In ion-exchange chromatography, pH and ionic strength, as well as changes in the composition of the mobile phase, affect capacity factors. The technique of continuously changing the solvent composition during the chromatographic run is called gradient elution or solvent programming. It is sometimes used to chromatograph complex mixtures of components differing greatly in their capacity factors. Detectors that are sensitive to change in solvent composition, such as the differential refractometer, are more difficult to use with the gradient elution technique.
The detector must have a broad linear dynamic range, and compounds to be measured must be resolved from any interfering substances. The linear dynamic range of a compound is the range over which the detector signal response is directly proportional to the amount of the compound. For maximum flexibility in quantitative work, this range should be about three orders of magnitude. HPLC systems are calibrated by plotting peak responses in comparison with known concentrations of a reference standard, using either an external or an internal standardization procedure.
Reliable quantitative results are obtained by external calibration if automatic injectors or autosamplers are used. This method involves direct comparison of the peak responses obtained by separately chromatographing the test and reference standard solutions. If syringe injection, which is irreproducible at the high pressures involved, must be used, better quantitative results are obtained by the internal calibration procedure where a known amount of a noninterfering compound, the internal standard, is added to the test and reference standard solutions, and the ratios of peak responses of drug and internal standard are compared.
Because of normal variations in equipment, supplies, and techniques, a system suitability test is required to ensure that a given operating system may be generally applicable. The main features of system suitability tests are described below.
For information on the interpretation of results, see the section Interpretation of Chromatograms.
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Size-Exclusion Chromatography
Size-exclusion chromatography is a high-pressure liquid chromatographic technique that separates molecules in solution according to their size. Methods for size-exclusion chromatography are divided into gel permeation chromatographic methods, which utilize nonpolar organic mobile phases and hydrophilic packings, and gel filtration chromatographic methods, which utilize aqueous mobile phases and hydrophobic packings. The sample is introduced into a column, which is filled with a gel or a porous particle packing material and is carried by the mobile phase through the column. The size separation takes place by repeated exchange of the solute molecules between the solvent of the mobile phase and the same solvent in the stationary liquid phase within the pores of the packing material. The pore-size range of the packing material determines the molecular-size range within which separation can occur.
Molecules small enough to penetrate all the pore spaces elute at the total permeation volume, VT. On the other hand, molecules apparently larger than the maximum pore size of the packing material migrate along the column only through the spaces between the particles of the packing material without being retained and elute at the exclusion volume, VO (void volume). Separation according to molecular size occurs between the exclusion volume and the total permeation volume, useful separation usually occurring in the first two-thirds of this range.
Apparatus— The components of the chromatograph are described under High-Pressure Liquid Chromatography.
Column—If necessary, the column is temperature-controlled. It is packed with a separation material that is capable of fractionation in the appropriate range of molecular sizes and through which the eluant is passed at a constant rate. One end of the column is usually fitted with a suitable device for applying the sample, such as a flow adaptor, a syringe through a septum or an injection valve, and it may also be connected to a suitable pump for controlling the flow of the eluant. Alternatively, the sample may be applied directly to the drained bed surface, or, where the sample is denser than the eluant, it may be layered beneath the eluant. The packing material may be a soft support such as a swollen gel or a rigid support composed of a material such as glass, silica, or a solvent-compatible, cross-linked organic polymer. Rigid supports usually require pressurized systems giving faster separations. The mobile phase is chosen according to sample type, separation medium, and method of detection.
Detector—The outlet of the column is usually connected to a suitable detector fitted with an automatic recorder that enables the monitoring of the relative concentrations of separated components of the sample. Detectors are usually based on photometric, refractometric, or luminescent properties (see Detectors under High-Pressure Liquid Chromatography). An automatic fraction collector may be attached, if necessary.
Procedure— Before carrying out the separation, the packing material is treated and the column is packed, as described in the individual monograph or according to the manufacturer's instructions. Where necessary, procedures for verifying the suitability of the system are described in the individual monograph. The column efficiency may be evaluated from the number of theoretical plates, N (see the section Interpretation of Chromatograms). The elution characteristics of a compound in a particular column may be described by the distribution coefficient, KD, which is calculated by the formula:
(VI – VO) / (VT – VO)
in which VO, V T, and VI are the retention volumes for the nonretained component, the component that has full access to all the pores in the support, and the compound under test, respectively. Each retention volume is measured from the time of application to the time of the peak maximum. Determination of Relative Component Composition of Mixture—Carry out the separation as directed in the individual monograph. Monitor the elution of the components continuously, and measure the corresponding peak areas. If all the components under test exhibit equivalent responses to the physicochemical property being monitored (for example, if they exhibit corresponding absorptivities), calculate the relative amount of each component by dividing the respective peak area by the sum of the peak areas of all the components under test. If the responses to the property used for detection of the components under test are not equivalent, calculate the content using calibration curves obtained from the calibration procedure specified in the individual monograph.
Determination of Molecular Weights—Size-exclusion chromatography is used to determine molecular weights of components under test by comparison to calibration standards specified in the individual monograph. Plot the retention volumes of the calibration standards versus the logarithm of their molecular weights. Draw the line that best fits the plotted points within the exclusion and total permeation limits for the particular separation medium. From the calibration curve, molecular weights of components under test are estimated. This calibration is valid only for the particular macromolecular solute-solvent system used under the specified experimental conditions.
Determination of Molecular Weight Distribution of Polymers—The material used for calibration and the methods for determination of the distribution of molecular weights of polymers are specified in the individual monograph. However, sample comparison is valid only for results obtained under identical experimental conditions.
