Absorbance detector

Accuracy is a measure of the closeness of a measurement to the true value. Precision is a measure of how reproducible the measurements are. For many detectors, the accuracy of a measurement is maintained by user calibration. For some detectors, however, such as photodiode array detectors, accuracy relies on internal calibration. The linear dynamic range of the detector is the maximum linear response, divided by the detector noise. The detector response is said to be linear if the difference in response for two concentrations of a given compound is proportional to the difference in concentration between the two samples. Most detectors become nonlinear as the sample concentration increases. 

With direct UV detection the eluent exhibits little or no absorbance at the wavelength to be monitored. When a solute is exposed to UV radiation, the radiation is absorbed by particular electronic configurations of 

Chapter 3 Instrumentation for High-Performance Liquid Chromatography 

If a solute of interest does not contain a chromophore, it may be detected by indirect UV detection. Indirect detection is a technically simple and sensitive method for the detection of compounds with little inherent detector response. Indirect UV detection is a nondestructive technique, in which the analyte is characterized in native form. Indirect detection is a universal detection mode, with few requirements as to the exact nature of the analyte. The properties of indirect detection have been reviewed by Yeung.22 Indirect detection is particularly attractive for the analysis of biological compounds. Optical systems are the same for direct or indirect detection the only difference is that, in indirect detection, the mobile phase, rather than the analyte, contains a UV chromophore. 

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A UV/Vis absorbance detector can also be used if the solute ions absorb ultraviolet or visible radiation. Alternatively, solutions that do not absorb in the UV/Vis range can be detected indirectly if the mobile phase contains a UV/Vis-absorbing species. In this case, when a solute band passes through the detector, a decrease in absorbance is measured at the detector. 

Limits of detection become a problem in capillary electrophoresis because the amounts of analyte that can be loaded into a capillary are extremely small. In a 20 p.m capillary, for example, there is 0.03 P-L/cm capillary length. This is 1/100 to 1/1000 of the volume typically loaded onto polyacrylamide or agarose gels. For trace analysis, a very small number of molecules may actually exist in the capillary after loading. To detect these small amounts of components, some on-line detectors have been developed which use conductivity, laser Doppler effects, or narrowly focused lasers (qv) to detect either absorbance or duorescence (47,48). The conductivity detector claims detection limits down to lO molecules. The laser absorbance detector has been used to measure some of the components in a single human cell (see Trace AND RESIDUE ANALYSIS). 

The method of detecting dimethylterephthalate (DMTP), dibuthyl-phthalate (DBP) and diocthylphthalate (DOP) in aqueous extract is based on their extraction with an organic solvent (hexane) and subsequent concentration using gas-liquid chromatography and an electron-absorbing detector. The detection limit is 0.05 mg/dirf for DMTP and DBP, and 0,01 mg/dm for DOP. 

Accordingly GPC analysis of dried latex samples were carried out. The carrier solvent was tetrahydrofuran and peaks were monitored by a Waters dual absorbance detector at wavelengths of 2 h and 3U0 nm. The latter detection was the closest to 350 nm available. 

The determination of acifluorfen in soybean was performed using CE," under the following conditions capillary column (total length 83 cm, 65 cm to the detector, with a 3-mm pathlength, 75-p.m i.d.) absorbance detector, 240 nm capillary oven temperature, 20 °C mnning buffer, 50 mM ammonium acetate buffer (pH, 4.75) applied voltage, 17 kV injection, 0.4 min at 4 mbar migration time, 20 min. 

Absorbance detectors are also commonly used in combination with postcolumn reactors. Here, most issues of detector linearity and detection limit have to do with optimization of the performance of the reactor. In a typical application, organophosphorus compounds with weak optical absorbances have been separated, photolyzed to orthophosphate, and reacted with molybdic acid, with measurement being performed by optical absorbance.58... 

Fluorescence detectors can be made much more sensitive than uv absorbance detectors for favourable solutes (such as anthracene) the noise equivalent concentration can be as low as 10 12 g cm-3. Because both the excitation wavelength and the detected wavelength can be varied, the detector can be made highly selective, which can be very useful in trace analysis. The response of the detector is linear provided that no more than about 10% of the incident radiation is absorbed by the sample. This results in a linear range of 103-104. 

One type of ec detector (the coulometric detector) reacts all of the electroactive solute passing through it. This type has never become very popular (there is only one on the market at the moment). Another type (the amperometric detector) reacts a much smaller quantity of the solute, less than 1%. The currents observed with these detectors are very small (nanoamps), but such currents are not too difficult to measure and the detector has a high sensitivity, considerably higher than that of uv/visible absorbance detectors although not as good as fluorescence detectors. Noise equivalent concentrations of about 10, 0g cm-3 have been obtained in favourable cases. Another advantage of these detectors is that they can be made with a very small internal volume. 

Although they are more sensitive (and cheaper) than uv absorbance detectors, ec detectors are not as easy to use, and have a more limited range of applications. They are chosen for trace analyses where the uv detector does not have a high enough sensitivity. Fig. 2.4k shows some examples of compounds for which ec detection has been used. 

Refractive index detectors are not as sensitive as uv absorbance detectors. The best noise levels obtainable are about 1CT7 riu (refractive index units), which corresponds to a noise equivalent concentration of about 10-6 g cmT3 for most solutes. The linear range of most ri detectors is about 104. If you want to operate them at their highest sensitivity you have to have very good control of the temperature of the instrument and of the composition of the mobile phase. Because of their sensitivity to mobile phase composition it is very difficult to do gradient elution work, and they are generally held to be unsuitable for this purpose. 

With uv absorbance detectors, we have to consider the uv absorption of the mobile phase, which always increases as the wavelength decreases. The uv cut-off of solvents indicates the useful wavelength range of the solvent and means the wavelength below which the solvent has an absorbance of 1 or more when measured in a 1 cm cell. Aliphatic hydrocarbons cut off at about 210 nm the best polar solvents for low wavelength work are methanol and acetonitrile, which cut off at 205 and 190 nm, respectively, provided they are pure. Acetonitrile is difficult to purify, and is consequently expensive. 

High performance liquid chromatography is used to determine the purity of calcitriol, and to separate it from related compounds. Using a 10 micron silica column of 25 cm length, and a mobile phase of spectroquality heptane ethyl acetate. methanol (50 50 1) at a flow rate of 1.7 ml/ minute, separation and quantitation are achieved. p-Dimethyl-aminobenzaldehyde may be used as an internal standard to compensate for variations in injection technique and instrumental conditions. With a 254 nm ultraviolet absorbance detector, 0.01 ug of calcitriol may be detected (3). 

An HPLC detector is often a modified spectrophotometer equipped with a small flow cell, which monitors the concentration (or mass) of eluting sample components. A number of detectors used in HPLC are discussed below. Most applications utilize absorbance detectors such as UV/Vis or... 

FIGURE 13.9 The HPLC diode array UV absorbance detector. When a mixture component elutes from the column, not only the chromatography peak but the entire UV absorption spectrum for that component can be recorded. 

In Chapter 12, we discussed the need to calculate response factors, specifically when a TCD detector is used (Section 12.8.2). Would response factors need to be calculated in HPLC when a UV absorbance detector is used Explain. 

Molecular weights were determined using a Waters high-pressure GPC instrument (Model 6000 A pymp, a series of five p-Styragel columns (10s, 10s, 10, 103, 500 A), Differential Refractometer 2401 and UV Absorbance Detector Model (440) and a calibration curve made by well fractionated polyisobutylene standards. 

Recently, comparatively inexpensive, very reliable, and stable single quadrupole mass spectrometers have entered the market. These spectrometers can be coupled to GC, LC, and CE separation methods simply by modifying the sampling interfaces. Although these detectors are more expensive than most conventional detectors including the versatile electron capture and diode array absorbance detectors used for GC and LG respectively, the reduction in sample preparation effort and their increased specificity can often rapidly... 

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