Absorption detectors variable wavelength

Figure 5.8 Scheaatic optical diagram of a variable wavelength dual beam absorption detector. A, and a photodiode array detector with reverse optics, B.

Stewart, J. E., Spectral-bandwidth effects of variable-wavelength absorption detectors in liquid chromatography, ]. Chromatogr., 174, 283, 1979. 

Omura et al.21 used a reverse phase high performance liquTcT cEromatographic column, JASCO PACK SV-02-500, for macrolide antibiotics with methanol, M/15 acetate buffer pH 4.9, and acetonitrile (35 60 5) as solvent. A variable wavelength UV detector using the absorption of the individual compounds gave the required sensitivity. Alterations of buffer pH and the composition ratio of the mobile phase gave selectivity for separation of individual macrolide antibiotics. 

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. 

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. 

High-Performance Liquid Chromatography. A Varian 5060 delivery system was used for this work with detection by UV absorption. Either a Varian UV-50 variable wavelength detector or a Hewlett Packard 1040A scanning diode array detector was used. All HPLC columns were packed in our laboratory (10) with 5-/um particle size Spherisorb-ODS, Spherisorb-CN (Phase Separations), or 8-pm particle size Zorbax-CN (Dupont Ltd). HPLC columns (20 or 25 cm X 4.6 mm i.d.) were coupled via short lengths of stainless steel capillary tubing (5 cm X 0.25 mm i.d.). Separation conditions were as follows ... 

These detectors are often used to detect components of a fluorescent derivative prepared to increase the detection sensitivity of compounds with poor UV absorptions. Both variable and filter variable, fixed-wavelength fluorom-eters are available for HPLC, with the same limits of lamp life and sensitivity seen in comparable UV detectors. 

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). 

An example of the use of the variable wavelength UV detector to select a specific wavelength to give a high sensitivity for a certain group of compounds is afforded by the separation of some carboxylic acids that is monitored by UV absorption at 210 nm. The separation is shown in figure 6. The separation of a series of common fatty acids was carried out on a reversed phase column using water buffered with phosphoric acid as the mobile phase. 

The basic components of spectrophotometers are a light source, wavelength selector, absorption cell (cuvette), and photodetector. Colorimeters or absorptiometers commonly use nondispersive wavelength selection (a filter with bandwidth 4 -40 nm) and solid state or simple phototube detectors, while spectrophotometers employ a prism or grating monochromator (with bandwidth down to 0.2 nm) and a photomultiplier. Colorimeters are inexpensive and most appropriate for repetitive measurements of absorption at a fixed wavelength. The more expensive spectrophotometer can also fulfill this function, but its main purpose, by virtue of its accurate and variable wavelength control, is the measurement of absorption spectra. 

Because of the short optical path length defined by the capillary, the optimal detection wavelength is frequently much lower into the UV compared to HPLC. In HPCE with a variable-wavelength absorption detector, the optimal signal-to-noise (S/N) ratio for peptides is found at 200 nm. To optimize the detector wavelength, it is best to plot the S/N ratio at various wavelengths. The optimal S/N is then easily selected. 

Most of the steroidal alkaloids have UV absorption only at about 200 nm. Using UV detectors with variable wavelength, it is possible to analyze microgram amounts of such alkaloids. 

Detection of the sorbate peak after elution from the adsorption column was accomplished using a variable wavelength detector. With the exception of 2,4-decadienal, many of the solutes utilized in this research had small molar absorptivities and could only be detected in the far ultraviolet region of the spectrum. Convenient wavelengths for monitoring purposes were 215-230 nm for the carbonyl-bearing compounds and 200 nm for the n-alcohols. 

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