Detectors variable wavelength absorbance

Thompson and Hatina (135) showed that the sensitivity of a fluorescence detector toward unesterified vitamin E compounds under normal-phase conditions was at least 10 times greater than that of a variable-wavelength absorbance detector. The relative fluorescence responses of the tocopherols at 290 nm (excitation) and 330 nm (emission), as measured by HPLC peak area, were a-T, 100 /3-T, 129 y-T, 110 and 5-T, 122. The fluorescence responses of the corresponding to-cotrienols were very similar to those of the tocopherols, and therefore tocotrienol standards were not needed for calibration purposes. The fluorescence detector also allows the simultaneous monitoring of ubiquinone derivatives for example ubiquinone-10 has been detected in tomato (136). 

Classical LC detectors (refractive index, fixed wavelength UV absorbance at 254 or 280 nm) have lacked the sensitivity to allow direct analysis of cannabi-noids in biological fluids. However, recent development of variable wavelength absorbance detectors extending into the 195-220nm UV region and of fluorescence detectors for HPLC led the authors to initiate... 

An HPLC detector measures the concentration (or mass) of eluting analytes4 by monitoring one of their inherent properties, such as UV absorbance. A detector can be universal to all analytes or specific to particular classes of analytes. Common detectors and their attributes are listed in the Table 4.2. Early HPLC detectors are spectrometers equipped with small flow cells however, most modern units are compact and designed solely for HPLC. The ubiquitous UV/visible variable wavelength absorbance and the photodiode array detectors (PDA) are covered in more depth in this section. Note that mass spectrometers (MS) and nuclear magnetic resonance spectrometers (NMR) are discussed in the section on hyphenated systems. 

Both fixed and variable wavelength uv/visible detectors are available. The variable types use a deuterium and/or a tungsten filament lamp as the radiation source and can operate between about 190-700 nm. They will have a number of absorbance ranges (ranges are given... 

FIGURE 13.8 The HPLC variable-wavelength UV detector—a UV spectrophotometer with a flow cell. A peak appears when a mixture component that absorbs the set wavelength elutes from the column. 

A continuously monitoring detector of high sensitivity is required and those that measure absorption in the ultraviolet are probably the most popular. These may operate at fixed wavelengths selected by interference filters but the variable wavelength instruments with monochromators are more useful. Wavelengths in the range of 190-350 nm are frequently used and this obviously means that a mobile phase must not absorb at those wavelengths. 

With a modern variable-wavelength or PDA detector, it is safe to use a maximum absorbance of up to 1.0-1.5 absorbance units (AU). A typical UV detector today should have a baseline noise in the order of 10 micro absorbance units (10 AU). Therefore, modern UV detectors should have a dynamic range of 4-5 orders of magnitude. The sample concentration and injection volume can be adjusted to make it possible to quantify the major component and impurities at the 0.05% level in the same chromatographic run without changing detection wavelength, sample concentration, or injection volume. 

HPLC has transformed quality control in the pharmaceutical and chemical industries, as it provides a rapid means of checking the purity of samples and even allows for the purification of small amounts of samples by preparative HPLC. The majority of such systems use L V to detect and quantify substances as they elute from the separative column. UV detectors are usually variable wavelength and can be used to detect molecules with absorption maxima above 210 nm by measuring the absorbance of the eluent. When a UV-absorbing substance is eluted from the HPLC column, it absorbs UV radiation at the appropriate wavelength for its chromophore. The amount of UV absorbed is proportional to the quantity of substance being eluted, and is converted into a peak on a chart recorder. Integration of each peak allows the relative quantities of the components of the solute to be determined. 

The most commonly used detector for pesticide residue analysis by LC is UV-VIS. It includes fixed wavelength and variable wavelength. Most OCPs and OPPs absorb appreciably only at wavelengths below 250 nm, the same spectral region where many solvents, solvent impurities, and matrix-derived interferences absorb. Analysis of these compounds by HPLC is still possible with very clean environmental substrates such as water. 

Several absorbance detectors which may be adapted to liquid chromatography are available from Cecil, Cambridge, Great Britain. A model which uses the CE212 variable-wavelength monitor has a wavelength range of 200—400 nm. Cells of 10-pl and 5-pl capacity are available and may be easily coupled to the system. 

Analysis of Tween 80 was performed using a Hewlett Packard 1100 series HPLC equipped with a Sedex 55 Evaporative Light Scattering Detector (ELSD). The mobile phase consisted of 80% acetonitrile and 20% water. Duplicate injections (5 pL) of each sample were evaluated by HPLC. Potassium iodide, used for the 1-D column and 2-D box tracer studies, was analyzed with a continuous flow Isco V4 variable UV wavelength absorbance detector equipped with an EZChrom Chromatography data acquisition system. 

The choice of detectors in LC is often a trade-off between wide scope and high sensitivity. For instance, the refractometer is readily available and easy to operate it can detect most compounds (wide scope), but it often has a lack of sensitivity for many compounds. A variable-wavelength UV detector offers a good choice for solutes that have some UV absorbance capability. Absorption at a specific wavelength results in a more selective and sensitive detection mode than a refractometer, but only for UV-absorbing compounds. In other specific cases the fluorescence or electrochemical detector can be used. These have a high sensitivity for individual compounds but are also limited in the number and type of compounds they can detect (narrow scope). 

Many organic compounds absorb at 254 nm and hence a fixed-wavelength detector has many uses. However, a variable-wavelength detector can be invaluable to increase the sensitivity of detection... 

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