HPLC detectors, types

Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

A number of different types of HPLC detector have been discussed in the previous chapter. In comparison to these, a mass spectrometer is a relatively expensive detector and there need to be considerable advantages associated with its use to make the significant financial investment worthwhile. What are these advantages In order to answer this question, we must first consider what it is we are trying to achieve when using chromatography ... 

Figure 4.11 Schematic classification of different types of HPLC detectors...

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. 

The ideal HPLC detector should have the same characteristics as those required for GC detectors, i.e. rapid and reproducible response to solutes, a wide range of linear response, high sensitivity and stability of operation. No truly universal HPLC detector has yet been developed but the two most widely applicable types are those based on the absorption of UV or visible radiation by the solute species and those which monitor refractive index differences between solutes dissolved in the mobile phase and the pure mobile phase. Other detectors which are more selective in their response rely on such solute properties as fluorescence, electrical conductivity, diffusion currents (amperometric) and radioactivity. The characteristics of the various types of detector are summarized in Table 4.14. 

Even though detectors used for GC are generally more sensitive and provide unique selectivity for many types of samples, the available HPLC detectors offer unique advantages in a variety of applications. In short, it is a good idea to recognize the fact that HPLC detectors are favored for some samples, whereas GC detectors are better for others. It should be noted that mass spectrometric detectors have been used effectively for both GC and HPLC. 

Reversed-phase HPLC has been used to analyze the oxidation products of triacylglycerols in edible oils. The detection is often based on monitoring the conjugated dienes with an ultraviolet detector (234-235 nm). However, the UV detector provides no information about oxidation products without a conjugated diene structure, e.g., products of oleic acid. Information about these compounds is important when oils with a high oleic acid content are studied. The most common universal detector types—refractive index and flame ionization detectors—are not sensitive enough to detect small amounts of oxidation products. 

Three types of inline HPLC detector have been used to measure fat-soluble vitamin concentrations in food sample extracts absorbance, fluorescence, and electrochemical detectors. Each of these detectors provides a continuous electrical output that is a function of the concentration of solute in the column effluent passing through the flow cell. 

There are many types of HPLC detectors available today with the most popular ones including UV and UV-photodiode array (PDA), fluorescence, refractive index, evaporative light scattering (ELSD), charged aerosol (CAD), and the mass spectrometer. Of these, the most commonly used detector for pharmaceutical analytical methods is the UV detector since a majority of pharmaceutical compounds have some type of chromophore. Multiple detectors in series can also be utilized in order to obtain more information per chromatographic run. For example, a PDA detector can... 

Artemisinin was produced by some A. annua cultures, as were three other electrochemically reducible compounds, based on the type of HPLC detector used for analysis of the peroxide-bridged, functionally active materials in the... 

Generally, HPLC detectors are classified as either of two types (1) Those which monitor bulk properties of the mobile phase and (2) Those which are sensitive to solute properties. 

Derivatization of solute molecules can be utilized to modify properties of analytes of interest such that they may be more readily identified by a specific type of HPLC detector. 

Current IPC detectors are on-stream monitors. HPLC detectors range from (1) non selective or universal (bulk property detectors such as the refractive index (RI) detector), characterized by limited sensitivity, (2) selective (discriminating solute property detectors such as UV-Vis detectors) to (3) specific (specific solute property detectors such as fluorescence detectors). Traditional detection techniques are based on analyte architecture that gives rise to high absorbance, fluorescence, or electrochemical activity. Mass spectrometry (MS) and evaporative light scattering detectors (ELSDs), can be considered universal types in their own right... 

A listing of the most common types of HPLC detector is given in Table 6.1, along with some of their properties. By far the most widely used HPLC detector is the LTV absorbance detector. This is due to a combination of factors firstly, although sensitivity is not good compared to other detector types (e.g. fluorescence, electrochemical), sensitivity is adequate for the majority of HPLC analyses secondly, a great many compounds are detectable by UV absorbance measurements thirdly, simplicity of construction and economies of scale mean that UV absorbance detectors... 

The performance of all HPLC detectors can be characterised by certain parameters such as sensitivity, noise, drift, limit of detection, linear and dynamic range, and detection volume. Other factors are more specific to individual types of detectors, and are discussed in their respective sections. 

Virtually every type of high-performance liquid chromatography (HPLC) detector can be combined with SCIC refractive index, UV absorbance (direct and indirect), electrochemical, and so forth. 

In contrast to the cumbersome and time-consuming tasks of conventional electrophoresis, CE is well suited to automation. Samples are easily applied to the capillary, a variety of detector types can be used, and the resulting electrophore-tograms can be analyzed and manipulated in much the same manner as chromatograms. Commercial instruments resemble many HPLC instruments in terms of automated sample loading and data analysis. Traditional serum protein electrophoresis, for example, can be fully automated with CE. 

Other detectors. The above discussions have been concerned with those detectors most commonly employed in routine HPLC analysis and which are commercially available. Many other detectors have been developed to monitor specific solute properties in column effluents and new detection systems continue to be reported in the literature, many directed to meeting the requirements imposed by microbore and capillary separation technologies. The interested reader is directed to the reviews of Yeung [46] and Fielden [47] for a more detailed discussion of detector types employed in HPLC. 

Infrared Absorption Detectors. Two types of infrared detectors have been offered commercially. The first is a filter instrument similar in design to that shown in Figure 16-13. The second, and more sophisticated, type of infrared detector is based on Fourier transform instruments similar to those discussed in Section I6B-1. Several of the manufacturers of Fourier transform infrared (FTIR) instruments offer accessories that permit their use as HPLC detectors. Infrared detector cells are similar in construction to those used with ultraviolet radiation except that windows arc constructed of sodium chloride or calcium fluoride. Cell path lengths range from 0.2 to 1.0 mm and volumes from 1.5 to 10 pL. 

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