Detectors

INTRODUCTION

Detectors equipped with the flow-through cell was a major breakthrough in the development of modern liquid chromatography. Such detection was first applied by the group of Tiselius, in Sweden in 1940, by continuously measuring the refractive index of the column effluent. Current LC detectors have wide dynamic range normally allowing both analytical and preparative scale runs on the same instrument. They have high sensitivities often allowing the detection of nanograms of material, and the better models are very flexible, allowing rapid conversion from one mobile phase to another and from one mode to another.

Almost all LC detectors are the on-stream monitors. The only relatively successful off-line detector is FTIR spiral disk monitor, which require sample transfer on the germanium disk and the following scanning in FTIR instrument. HPLC detectors always used under continuous flow conditions and the sample is always dissolved in the eluent during detection. Actual sample is only present in a ng quantity in the detector, but in trace analysis, this quantity could be fg and even the single molecule! The mobile phase is a factor which must always be considered.

In the last decade there is a significant progress in the development of LC/MS interfacing systems. MS as an on-line HPLC detector is said to be the most sensitive, selective and in the same time the most universal detector. But it is still the most expensive one.

Actually, now we could not recommend any universal detector, so the liquid chromatographer must expect to eventually use more than one type of detector.

Here we will discuss the most common HPLC detectors:

	Refractive index
	UV/Vis Fixed wavelength Variable wavelength Diode array
	Fluorescence

and the less common, but important

	Conductivity
	Mass-spectrometric (LC/MS)
	Evaporative light scattering BASIC DETECTOR REQUIREMENTS Regardless of the principle of operation, an ideal LC detector should have the following properties: Low drift and noise level (particularly crucial in trace analysis). High sensitivity. Fast response. Wide linear dynamic range (this simplifies quantitation). Low dead volume (minimal peak broadening). Cell design which eliminates remixing of the separated bands. Insensitivity to changes in type of solvent, flow rate, and temperature. Operational simplicity and reliability. It should be tuneable so that detection can be optimized for different compounds. It should be non-destructive. Noise and drift In HPLC we deal with the time-dependent process. The appearance of the component from the column in the detector represented by the deflection of the recorder pen from the baseline. It is a problem to distinguish between the actual component and artifact caused by the pressure fluctuation, bubble, compositional fluctuation, etc. If the peaks are fairly large, one has no problem in distinguishing them. However, the smaller the peaks, the more important that the baseline be smooth, free of noise, and drift. Sensitivity Detector sensitivity is one of the most important properties of a LC detector. Sensitivity of the detector is a measure of its ability to discriminate between small differences in analyte concentration. So, it is actually the slope of the calibration curve. It is also dependent on the standard deviation of the measurements. The higher the slope of your calibration curve the higher the sensitivity of your detector for that particular component, but high fluctuations of your measurements will decrease the sensitivity. Sensitivity of a detector is not the minimum amount that can be detected. This value is influenced by the chromatographic conditions. Early eluting peaks are usually sharp, whereas the ones with long retention times are broad and sometimes difficult to discern from the noise. Selectivity Selectivity is another highly desirable property of HPLC detectors. A selective detector allows one to see only components of interest despite of their co-elution with any others. Refractive index is an example of almost nonselective detector. Any component could make a response, but in case of poorly resolved mixture analyst will not be able to distinguish components. Fluorescence and electrochemical detectors are the most selective among the common detectors. Only about 10% of organic compounds are able to fluoresce, and by choosing excitation and emission wavelength specific for the particular compound one can detect only this compound. Usually, the more selective the detection, the lower signal noise, and the higher the sensitivity. Baseline noise is the short time variation of the baseline from a straight line caused by electric signal fluctuations, lamp instability, temperature fluctuations and other factors. Noise usually has much higher frequency than actual chromatographic peak. Noise is normally measured "peak-to-peak": i.e., the distance from the top of one such small peak to the bottom of the next. Sometimes, noise is averaged over a specified period of time. Noise is the factor which limits detector sensitivity. In trace analysis, the operator must be able to distinguish between noise spikes and component peaks. A practical limit for this is a 3 x signal-to-noise ratio, but only for qualitative purposes. Practical quantitative detection limit better be chosen as 10x signal-to-noise ratio. This ensures correct quantification of the trace amounts with less than 2% variance. Figure below illustrates this, indicating the noise level of a baseline(measured at highest detector sensitivity) and the smallest peak which can be unequivocally detected. Definition of noise, drift, and smallest detectable peak. Another parameter related to the detector signal fluctuation is drift. Noise is a short-time characteristic of a detector, an additional requirement is that the baseline should deviate as little as possible from a horizontal line. It is usually measured for a specified time, e.g., 1/2 hour or one hour. Drift usually associated to the detector heat-up in the first hour after power-on. Figure also illustrates the meaning of drift. Reflective detectors The refractive index detector based on the Fresnel principle is relatively rear. There was only one or two commercial models and they are not in the production any more. Figure below shows the optical schematic of this type of detector. Here, the light beam is reflected from the liquid-glass interface in the detecting photocell. As the introduction of sample into one cell causes light to be refracted at a different angle. The deflection of the light beam from the photoresistor cause the appearance of the electrical signal. Here, too, this difference between sample cell signal and reference-cell signal is output to a recorder or data handling system as peak. The major advantage of this type of detector is a very high sensitivity since the optics allow a higher concentration of signal in a particular RI range than is possible in other wide-range detectors. Other advantages include the ability to operate at extremely low flow rates with very low-volume cells, easy cell accessibility, and low cost. Its disadvantages are the incredible sensitivity to the flow and pressure fluctuations, and the need for changing prisms to accommodate either high or low RI solvents and the need to manually adjust the optical path when making solvent changes. Optical schematic of the Fresnel-type refractive index detector. The refractive index of a analyte is a function of its concentration. Change in concentration is reflected as a change in the RI. A refractive index detector for liquid chromatography should be sensitive to changes as small as 10-7 RI units (corresponding to a concentration change of 1 ppm). Presence of dissolved air, changes in solvent composition, improper mixing and column bleed will contribute to baseline drift. Eluent pressure change of 15 psi will cause the change of 1 x 10-6 RI unit and 1°C temperature variation will be equivalent to the change of 600 x 10-6 RI units. Thus it is obvious that both of these parameters must be closely controlled, especially temperature. To operate at high sensitivities, a RI detector must usually be thermostated (± 0.01°C), actually the using of the water bath connected to the detector head does not give required temperature stability, alternately, passive thermostabilisation with massive metallic block usually gives much better results. Reflective detectors The refractive index detector based on the Fresnel principle is relatively rear. There was only one or two commercial models and they are not in the production any more. Figure below shows the optical schematic of this type of detector. Here, the light beam is reflected from the liquid-glass interface in the detecting photocell. As the introduction of sample into one cell causes light to be refracted at a different angle. The deflection of the light beam from the photoresistor cause the appearance of the electrical signal. Here, too, this difference between sample cell signal and reference-cell signal is output to a recorder or data handling system as peak. The major advantage of this type of detector is a very high sensitivity since the optics allow a higher concentration of signal in a particular RI range than is possible in other wide-range detectors. Other advantages include the ability to operate at extremely low flow rates with very low-volume cells, easy cell accessibility, and low cost. Its disadvantages are the incredible sensitivity to the flow and pressure fluctuations, and the need for changing prisms to accommodate either high or low RI solvents and the need to manually adjust the optical path when making solvent changes. Optical schematic of the Fresnel-type refractive index detector. The refractive index of a analyte is a function of its concentration. Change in concentration is reflected as a change in the RI. A refractive index detector for liquid chromatography should be sensitive to changes as small as 10-7 RI units (corresponding to a concentration change of 1 ppm). Presence of dissolved air, changes in solvent composition, improper mixing and column bleed will contribute to baseline drift. Eluent pressure change of 15 psi will cause the change of 1 x 10-6 RI unit and 1°C temperature variation will be equivalent to the change of 600 x 10-6 RI units. Thus it is obvious that both of these parameters must be closely controlled, especially temperature. To operate at high sensitivities, a RI detector must usually be thermostated (± 0.01°C), actually the using of the water bath connected to the detector head does not give required temperature stability, alternately, passive thermostabilisation with massive metallic block usually gives much better results. ULTRAVIOLET/VISIBLE SPECTROSCOPIC DETECTORS Principles Any chemical compound could interact with the electromagnetic field. Beam of the electromagnetic radiation passed through the detector flow-cell will experience some change in its intensity due to this interaction. Measurement of this changes is the basis of the most optical HPLC detectors. Radiation absorbance depends on the radiation wavelength and the functional groups of the chemical compound. Electromagnetic field depending on its energy (frequency) can interact with electrons causing their excitation and transfer onto the higher energy level, or it can excite molecular bonds causing their vibration or rotation of the functional group. The intensity of the beam which energy corresponds to the possible transitions will decrease while it is passing through the flow-cell. According to the Lambert-Bear law absorbance of the radiation is proportional to the compound concentration in the cell and the length of the cell. The electromagnetic spectrum is traditionally divided into several regions: infrared (IR) 2,500 - 50,000 nm near infrared 800 - 2,500 nm visible 400 - 800 nm ultraviolet (UV) 190 - 400 nm Three major regions (IR, visible, and UV) are used in the spectroscopy. In liquid chromatography, IR spectrophotometers have found only limited use. There are few transparent polar liquids which can be used as the mobile phase. On the other hand, spectrophotometers working in the range (200 - 600 nm) are used widely as LC detectors. UV and visible region of the electromagnetic radiation corresponds to the excitation of the relatively low energy electrons such as pi-electrons, or non-paired electrons of some functional groups. For example, n-alkanes could absorb in the UV region below 180 nm. s-electrons require high energy radiation to get excited and to show absorption of the radiation. But any compounds which have benzene ring will show absorbance at 205-225 and 245-265 nm. The last corresponds to the excitation of conjugated p-electrons of the benzene ring. The majority of organic compounds can be analyzed by UV/VIS detectors. Almost 70% of published HPLC analyses were performed with UV/VIS detectors. This fact, plus the relative ease of its operation, makes the UV detector the most useful and the most widely used LC detector. UV Absorbance Absorbance is the logarithm of the ratio of the intensities of the incident light (Io) and the transmitted light (I). It is related according to the Beer-Lambert Law to the molar absorptivity (molar extinction coefficient, e), the thickness of the substance (i.e., the path length of the cell, b) and the molar concentration of the substance (c): In HPLC, the photodetector measures transmitted light I, but the electronics converts this signal to a logarithmic relationship (A) which is proportional to concentration. The ordinate of the chromatogram represents the detector signal, which in general, is proportional to the analyte concentration in the cell. Since chromatographic systems permit the quantitative analysis of sample components representing many orders of magnitude - from ppm to percent concentrations - one may select, various amplification ranges so that the visual display of components (both small and large). Table. Molar Absorptivity (e) Values of Various Compound Types at Specified Wavelengths. Name Chromophore Wavelength [nm] Molar extinction, e acetylide -C=C 175-180 6,000 Aldehyde -CHO 210 1,500 amine -NH2 195 2,800 azo -N=N- 285-400 3-25 bromide -Br 208 300 carboxyl -COOH 200-210 50 - 70 disulphide -S-S- 194 5,500 ester -COOR 205 50 ether -O- 185 1,000 ketone >C=O 195 1,000 nitrate -ONO2 270 12 nitrile -C=N 160 - nitrite -ONO 220 - 230 1000-2000 nitro -NO2 210 strong In UV detection, one expresses the detector range in absorbance units (A). One absorbance unit correspond to the depreciation of the light intensity by 90% of the incident light. Molar Absorptivity. This term (e) - also called the molar extinction coefficient - corresponds to the absorbance for a molar concentration of the substance with a path length of 1 cm. Molar adsorptivity is dependent on the wavelength and chromatographic conditions, (solvent, pH and temperature). It is a constant at a specified wavelength. Table above lists the molar absorptivities of a number of compounds at specified wavelengths. The knowledge of these values is important because they indicate the wavelengths to be selected for maximum response. Fixed wavelength detectors HPLC detectors which does not allow to change the wavelength of the radiation called fixed-wavelength detectors. They are usually very cheap, in the nowadays you probably wont be able to find that type of the detector on the market. low-pressure mercury vapor lamp emit very intense light at 253.7 nm. By filtering out all other emitted wavelengths, manufacturers have been able to utilize this 254 nm line to provide stable, highly sensitive detectors capable of measuring subnanogram quantities of any components which contains aromatic ring. The 254 nm was chosen since the most intense line of mercury lamp is 254 nm, and most of UV absorbing compounds have some absorbance at 254 nm. Variable-wavelength detectors Detectors which allow the selection of the operating wavelength called variable wavelength detectors and they are are particularly useful in three cases: offer best sensitivity for any absorptive component by selecting an appropriate wavelength; individual sample components have high absorptivity at different wavelengths and thus, operation at a single wavelength would reduce the system's sensitivity; Depending on the sophistication of the detector, wavelength change is done manually or programmed on a time basis into the memory of the system. Diode-array detectors As already mentioned, a special feature of some variable wavelength UV detectors is the ability to perform spectroscopic scanning and precise absorbance readings at a variety of wavelengths while the peak is passing though the flow cell. Diode array adds a new dimension of analytical capability to liquid chromatography because it permits qualitative information to be obtained beyond simple identification by retention time. There are two major advantages of diode array detection. In the first, it allows for the best wavelength(s) to be selected for actual analysis. This is particularly important when no information is available on molar absorptivities at different wavelengths. The second major advantage is related to the problem of peak purity. Often, the peak shape in itself does not reveal that it actually corresponds to two (or even more) components. In such a case, absorbance rationing at several wavelengths is particularly helpful in deciding whether the peak represents a single compound or, is in fact, a composite peak. In absorbance rationing, the absorbance is measured at two or more wavelengths and ratios are calculated for two selected wavelengths. Simultaneous measurement at several wavelengths allows one to calculate the absorbance ratio. Evaluation can be carried out in two ways: In the first case, the ratios at chosen wavelength are continuously monitored during the analysis: if the compound under the peak is pure, the response will be a square wave function (rectangle),. If the response is not rectangle, the peak is not pure. FLUORESCENCE DETECTORS Principles Fluorescence detectors are probably the most sensitive among the existing modern HPLC detectors. It is possible to detect even a presence of a single analyte molecule in the flow cell. Typically, fluorescence sensitivity is 10 -1000 times higher than that of the UV detector for strong UV absorbing materials. Fluorescence detectors are very specific and selective among the others optical detectors. This is normally used as an advantage in the measurement of specific fluorescent species in samples. When compounds having specific functional groups are excited by shorter wavelength energy and emit higher wavelength radiation which called fluorescence. Usually, the emission is measured at right angles to the excitation. Roughly about 15% of all compounds have a natural fluorescence. The presence of conjugated pi-electrons especially in the aromatic components gives the most intense fluorescent activity. Also, aliphatic and alicyclic compounds with carbonyl groups and compounds with highly conjugated double bonds fluoresce, but usually to a lesser degree. Most unsubstituted aromatic hydrocarbons fluoresce with quantum yield increasing with the number of rings, their degree of condensation and their structural rigidity. Fluorescence intensity depends on both the excitation and emission wavelength, allowing selectively detect some components while suppressing the emission of others. The detection of any component significantly depends on the chosen wavelength and if one component could be detected at 280 ex and 340 em., another could be missed. Most of the modern detectors allow fast switch of the excitation and emission wavelength, which offer the possibility to detect all component in the mixture.. For example, in the analysis of very important polynuclear aromatic compounds the excitation and emission wavelengths were 280 and 340 nm, respectively, for the first 6 components, and then changed to the respective values of 305 and 430 nm; the latter values represent the best compromise to allow sensitive detection of compounds. Fluorescence detectors Figure below shows the optical schematic of a typical fluorescence detector for liquid chromatography. The detectors available on the market differ in the method in which the wavelengths are controlled. Less expensive instruments utilize filters; medium priced units offer monochromator control of at least emission wavelength, and full capability research-grade instruments provide monochromator control of both excitation and emission wavelengths. Optical schematic of a typical fluorescence detector for liquid chromatography. ELECTROCHEMICAL DETECTORS The electrochemical detector is also a popular liquid chromatographic detector. It should be considered by the chromatographer because of the additional selectivity and sensitivity for some compounds. This detector is based on the measurements of the current resulting from oxidation/reduction reaction of the analyte at a suitable electrode. Since the level of the current is directly proportional to the analyte concentration, this detector could be used for quantification.. The eluent should contain electrolyte and be electrically conductive. Most of the analytes to be successfully detected require the pH adjustments. The areas of application of electrochemical detection are not large, but the compounds for which it does apply, represent some of the most important drug, pollutant and natural product classes. For these, the specificity, and sensitivity make it very useful for monitoring these compounds in complex matrices such as body fluids and natural products. Sensitivities for compounds such as phenol, catecholamines, nitrosamines, and organic acids are in the picomole (nanogram) range. The purity of the eluent is very important, because the presence of oxygen, metal contamination and halides may cause significant background current and therefore, noise and drift in the base line. ELECTROLYTIC CONDUCTIVITY DETECTOR The conductivity of the column effluent is continuously measured and the appearance of the analyte in the cell is indicated by a change in conductivity. Usually this is a very low volume flow-through capillary equipped with two electrodes and variations in conductivity of the mobile phase due to the eluted sample components are continuously recorded. Response is linear with concentration over a wide range, quantitation of the output signal is possible with suitable preliminary calibration. Best use is made of this detector in isocratic analysis since solvent gradients will cause a proportional shift in the baseline. Such detectors have been used most successfully in ion-exchange chromatography of anions and cation but generally, they have found only limited popular acceptance. EVAPORATIVE LIGHT SCATTERING Evaporative Light Scattering Detectors involves nebulization of the column effluent to an aerosol, followed by solvent vaporization to produce a small solute droplets, and then these droplets detected in the light scattering cell. System consists of three parts the nebulizer, the drift tube, and the light scattering cell. Courtesy of Alltech Associates, Inc. Analytical column outlet is connected directly to the nebulizer . The column effluent is mixed with a stream of nebulizing gas to form an aerosol. The aerosol consists of a uniform dispersion of droplets. The lower the mobile phase flow rate, the less gas and heat are needed to nebulize and evaporate it. Reduction of flow rate by using 2.1mm I.D. column should be considered when sensitivity is important. The gas flow rate will also regulate the size of the droplets in the aerosol. Larger droplets will scatter more light and increase the sensitivity of the analysis. The lower the gas flow used, the larger the droplets will be. It is also important to remember that the larger the droplet, the more difficult it will be to vaporize in the drift tube. Unevaporized mobile phase will increase baseline noise. There will be an optimum gas flow rate for each method which will produce the highest signal-to-noise ratio. Volatile components of the aerosol are evaporated in the drift tube . LIGHT SCATTERING CELL The nebulized column effluent enters the light scattering cell. In the cell, the sample particles scatter the laser light, but the evaporated mobile phase does not. The scattered light is detected by a silicone photodiode located at a 90º angle from the laser. The photodiode produces a signal which is sent to the analog outputs for collection. A light trap is located 180º from the laser to collect any light not scattered by particles in the aerosol stream.