Detectors in HPLC

The most common detectors in HPLC are ultraviolet, fluorescence, electrochemical detector and diffractometer. However, despite all improvements of these techniques it seems necessary to have a more selectivity and sensitivity detector for the purposes of the medical analysis. It should be therefore improvements to couple analytical techniques like infrared IR, MS, nuclear magnetic resonance (NMR), inductively coupled plasma-MS (ICP-MS) or biospecific detectors to the LC-system and many efforts have been made in this field. 

Detectors. The function of the detector in HPLC is to monitor the mobile phase as it emerges from the column. The detection process in liquid chromatography has presented more problems than in gas chromatography there is, for example no equivalent to the universal flame ionisation detector of gas chromatography for use in liquid chromatography. Suitable detectors can be broadly divided into the following two classes ... 

Mobile phase That part of a chromatographic system which causes the analyte to move from the point of injection to the detector - in HPLC, this is a liquid. 

The second most widely used detector in HPLC is the differential refractometer (RI). Being a bulk property detector, the RI responds to all substances. As noted in Table 3 the detection limits are several orders of magnitude higher than obtained with the UV detector. Thus, one turns to the RI detector in those cases in which substances are non-UV active, e.g. lipids, prostaglandins. In addition, the RI detector finds use in preparative scale operation. Finally, relative to the UV detector, the RI is significantly more temperature and flow sensitive and cannot be used in gradient elution. 

Specifications for modem detectors in HPLC are given by Hanai [538] and comprise spectroscopic detectors (UV, F, FUR, Raman, RID, ICP, AAS, AES), electrochemical detectors (polarography, coulometry, (pulsed) amperometry, conductivity), mass spectromet-ric and other devices (FID, ECD, ELSD, ESR, NMR). None of these detectors meets all the requirement criteria of Table 4.40. The four most commonly used HPLC detectors are UV (80%), electrochemical, fluorescence and refractive index detectors. As these detectors are several orders of magnitude less sensitive than their GC counterparts, sensor contamination is not so severe, and... 

After passing through the column, the separated solutes are sensed by an in-line detector. The output of the detector is an electrical signal, the variation of which is displayed on a potentiometric recorder, a computing integrator or a vdu screen. Most of the popular detectors in hplc are selective devices, which means that they may not respond to all of the solutes that are present in a mixture. At present there is no universal detector for hplc that can compare with the sensitivity and performance of the flame ionisation detector used in gas chromatography. Some solutes are not easy to detect in hplc, and have to be converted into a detectable form after they emerge from the column. This approach is called post-column derivatisation. 

The function of the detector in hplc is to monitor the mobile phase emerging from the column. The output of the detector is an electrical signal that is proportional to some property of the mobile phase and/or the solutes. Refractive index, for example, is a property of both the solutes and the mobile phase. A detector that measures such a property is called a bulk property detector. Alternatively, if the property is possessed essentially by the solute, such as absorption of uv/visible radiation or electrochemical activity, the detector is called a solute property detector. Quite a large number of devices, some of them rather complicated and tempremental, have been used as hplc detectors, but only a few have become generally useful, and we will examine five such types. Before doing this, it is helpful to have an idea of the sort of characteristics that are required of a detector. 

These are by far the most popular detectors in hplc. The principle is that the mobile phase from the column is passed through a small flow cell held in the radiation beam of a uv/visible photometer or spectrophotometer. These detectors are selective in the sense that they will detect only those solutes that absorb uv (or visible) radiation. Such solutes include alkenes, aromatics and compounds having multiple bonds between C and O, N or S. The mobile phase we use, on the other hand, should absorb little or no radiation. 

Identification (ID) tests in Category IV require only specificity for their validation. Identification by HPLC usually involves comparison of the retention time (%) or relative retention time (RRT) of a sample and standard injection. The increasing use of photodiode array (PDA) detectors in HPLC methods also allows identification by comparison of UV spectra for standards and samples, in addition to retention characteristics. The information required for either ID test by HPLC can be gathered while performing any other HPLC method for a given sample. Identification tests are often incorporated into the assay method and the satisfactory completion of specificity for the assay will meet the requirements for ID as well. 

For comparison of impurity levels quoted as % area/area, the normalized PA [area divided by the respective MTs, often stated as corrected PA (Ac)] must be used in CE to compensate for the residence time difference of the species in the detector. In HPLC, the separation takes place on the column. After the column, all analytes travel through the detector at the same speed (that of the mobile phase) and hence have the same residence time in the detector cell. However, in CE, the electrical field also takes effect in the detection cell. Therefore, the residence time of the species that have a higher apparent mobility (as shorter Jm) will give a lower response than species with a lower mobility, for species with the same absorptivity and concentration. ... 

Fuel cell polymer battery photoelectric cell capacitor Storage element liquid crystal display device electrochromic display device electrochemiluminescence device photoelectric transducer Biosensor ion sensor detector in HPLC and FIA gas sensor voltam-metric indicator electrode reference electrode... 

Mass spectrometer can be used as a detector in HPLC analysis. A special interface between the column and the ion source removes the mobile phase solvent and vaporizes the nonvolatile analytes without destroying or rearranging them. The molecules are ionized, and the ions are directed into a quadrupole mass analyzer or into a magnetic sector spectrometer. 

The detector converts a change in column effluent into an electrical signal that is recorded by the data station. Detectors in HPLC include... 

Schematic of a fluorescence monitor in series with an absorbance detector in HPLC... 

Fig.l Block diagram of electrical conductivity detector in HPLC. 

More generally, the term refers to any device used to sense or measure the amount or kind of radiation or substance in an analytical system. The term is also used to refer to any device that can respond to a particular signal or substance and provide qualitative or quantitative information about it, e.g., electrochemical detectors in HPLC, thermal conductivity detectors in GC, etc. 

Diode-Array detection systems become more and more standard detectors in HPLC-analysis. Based on the principle of UVA/lS-spectrophotometers, DAD facilitates the simultaneous detection and registration of UV-chromatograms at different wavelength and the spectra of the single substances. This additional spectrum information is often required for the definite identification of the single substance peaks in UV/ VIS-chromatograms [15]. 

Detection in HPTLC, unlike that in HPLC, is a static process, being completely separated from chromatographic development. Consequently, the selection of the mobile phase does not limit the choice of the detector. For example, UV absorbing solvents cannot be used with UV detectors in HPLC. In HPTLC the solvent is completely evaporated between development and measurement so that it does not influence the detection process. 

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