The Variable Wavelength UV Detector

Distinguish between the fixed-wavelength UV detector and the variable-wavelength UV detector in terms of design and use. 

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. 

Sensitivity for the eluted solute peak is often much larger than with analytical SFC. In fact, as often happens in preparative HPLC, the variable wavelength UV detector must be de-tuned away from the wavelength of maximum absorption of the eluting species to prevent overload of absorption. For the vast majority of cases when using modified carbon dioxide mobile phases there is no need to look further than the simple UV detector. 

In order to calculate particle size distributions in the adsorption regime and also to determine the relative effects of wavelength on the extinction cross section and imaginary refractive index of the particles, a series of turbidity meas irements were made on the polystyrene standards using a variable wavelength UV detector. More detailed discussions are presented elsewhere (23) > shown here is a brief summary of some of the major results and conclusions. 

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. 

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. 

High Performance Size Exclusion Chromatography (HPSEC) A Varian Model 5000 liquid chromatograph equipped with a variable wavelength UV detector, two columns in series (PL gel, 300 x7.5 mm, particle size 5/im, porosity of 50 and 500A), was used, THF being the eluent. 

Appropriate detectors for the analyses, preferably a variable wavelength UV detector as well as a refractive index detector... 

Equipment. A LDC (Laboratory Data Control) Constametric III pump was used together with a Rheodyne 7120 20 yl loop injection valve and two LDC Spectromonitor III, variable wavelength UV-detectors. A Stanstead constant pressure pump was used for packing the columns. 

All experiments utilized a variable wavelength uv detector. Analysis of the hexane fraction was carried out using both the UV detector and an on-line Nicolet 6000 FTIR. The FTIR flow cell interface has been described in detail elsewhere.(9) This same FTIR was also used to gather static spectra on the various fractions, utilizing a liquid cell with KBr windows. 

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). 

Figure 10.2 Urinary creatinine assay. The chromatographic conditions were as follows column, 150 mm X 4.6 mm (5 pm) cation-exchange silica (SCX HPLC Technology, UKI eluent, 50mM sodium formate adjusted to ca. pH 6-methanol (80 20) flow rate, 1 mimin, injection, 20 pi temperature, ambient detector, variable wavelength UV detector (Cecil) set at 230 nm. Qiroraatogram courtesy Dr Ian James and the author. Department of Medicine St Barts. London.

Fixed and variable wavelength UV detectors. This is probably the HPLC detector found in most labs. Configurations vary from fixed wavelength (commonly 254 nm) to multi-wavelength and stop run scan-... 

Liu et al. separated polyhydroxylflavonoids, quercetin, and risetin by packed-column SFC with a ternary mobile phase. They designed an SFC apparatus with two syringe pumps and a variable-wavelength UV detector. A manual back-pressure regulator was also used to control the flow rate. This experiment showed that there are several factors affecting the result. 

HPLC on pBondapak Ph column with UV detection at 280 nm has been used for the determination of sulfacetamide, sulfabenzamide and sulfathiazole in a triple sulfa cream. The mobile phase consists of a 7 3 mixture of 0.01 M ammonium hydrogen phosphate and methanol (pH 7.2). The proposed method has been shown to give better results for sulfacetamide assay compared with those of the USP method (97). Sulfacetamide, sulfadiazine, sulfamerazine and sulfamethazine in mixtures and in pharmaceuticals have been determined by HPLC using a nonpolar column with methanol-0.02 M potassium dihydrogen phosphate in water as the mobile phase and a variable wavelength UV detector. The method is precise with relative standard deviations of 2.1, 0.6,1.9 and 1.6% respectively for the four compounds. The preservatives do not interfere with the method (98). 

A variable wavelength UV detector of this type, a spectrophotometer, produces light of a preselected wavelength, directs it through the sample cell, and measures the light transmitted by the cell. 

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