Ultraviolet/visible absorbance detector

Owing to the varied structures of various food dyes, they can often be differentiated from one another by their characteristic ultraviolet/visible absorbance spectra. Using HPLC coupled with a diode array detector (HPLC-DAD) it is possible to collect a compound s absorbance spectrum as it elutes from the HPLC column, which greatly assists in identification. At Reading Scientific Services Ltd (RSSL) this type of detector is routinely used in a range of analyses of such substances as patulin, a mycotoxin found in apple juice, and in the analysis of colours and vitamins, which allows a more certain assignment of a particular peak to a specific compound to be made. 

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. 

The ultraviolet-visible spectrophotometer is the most widely used detector for HPLC. The basis of UV-VIS detection is the difference in the absorbance of light by the analyte and the solvent. A number of functional groups absorb... 

For ultraviolet and visible spectroscopic detectors, a standard solution of a compound whose molar absorption constant is known must be prepared, and placed in the flow cell. The absorbance obtained is then compared with the value measured by a standard spectrophotometer. 

The single most useful and versatile physicochemical detectors in drug residue analysis are probably those based on ultraviolet-visible (UV-Vis) spectrophotometry. These detectors allow a wide selection of detection wavelengths, thus offering high sensitivity for analytes that exhibit absorbance in either the ultraviolet or the visible region of the electromagnetic radiation. 

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the usual method of choice for the separation of anthocyanins combined with an ultraviolet-visible (UV-Vis) or diode-array detector (DAD)(Hebrero et al., 1988 Hong et ah, 1990). With reversed-phase columns the elution pattern of anthocyanins is mainly dependent on the partition coefficients between the mobile phase and the Cjg stationary phase, and on the polarity of the analytes. The mobile phase consists normally of an aqueous solvent (water/carboxylic acid) and an organic solvent (methanol or acetonitrile/carboxylic acid). Typically the amount of carboxylic acid has been up to 10%, but with the addition of a mass spectrometer as a detector, the amount of acid has been decreased to as low as 1 % with a shift from trifluoroacetic acid to formic acid to prevent quenching of the ionization process that may occur with trifluoroacetic acid. The acidic media allows for the complete displacement of the equilibrium to the fiavylium cation, resulting in better resolution and a characteristic absorbance between 515 and 540 nm. HPLC separation methods, combined with electrochemical or DAD, are effective tools for anthocyanin analysis. The weakness of these detection methods is a lack of structural information and some nonspecificity leading to misattribution of peaks, particularly with electrochemical... 

Analysis is an integral part of research, clinical, and industrial laboratory methodology. The determination of the components of a substance or the sample in question can be qualitative, quantitative, or both. Techniques that are available to the analyst for such determinations are abundant. In absorption spectroscopy, the molecular absorption properties of the analyte are measured with laboratory instruments that function as detectors. Those that provide absorbance readings over the ultraviolet-visible (UV-vis) light spectrum are commonly used in high-performance liquid chromatography (HPLC). The above method is sufficiently sensitive for quantitative analysis and it has a broader application than other modes of detection. 

Experimental. Absorbances of selected polymer and copolymer samples were measured with a Waters 440 ultraviolet absorbence detector equipped with a 254 nm filter, and a Beckman 25 ultraviolet/visible spectrophotometer. The Water s instrument was used on-line with the size exclusion chromatograph (16). The Beckman 25 was used to calibrate and standardize the Water s 440 UV detector. THF was used as standard solvent. The extinction coefficients were estimated from absorbance measurements at several polymer concentrations. The standard deviation for the absorbance measurements was typically . 001 Au for replicates on the same sample. All measurements were done at room temperature. 

The detector can be considered as the "soul" of a HPLC system. Connected to the outlet end of the column, its role is to monitor the column effluent in real time. Detectors can be the most sophisticated and expensive component of the system. Classification of detectors is of two sorts, selective detectors which give different responses depending on the molecular structure of the sample under analysis, or universal detectors, for whom the response is similar for most compounds. Absorbance and fluorescence detectors are termed selective detectors, while the refractive index (RI) is a "universal detector". The Ultraviolet-Visible (UV-Vis) detector is more selective and sensitive, being able to detect amounts as low as lO g/mL, while the RI detector s sensitivity is in the range of lO g/mL. Therefore selective detectors can be used to minimise interference from unwanted components. As for fluorescence detectors, their sensitivity is in the range of lO i g/mL for... 

Solute-specific detectors , these are based upon the characteristic nature of the solute. For example the ability a solute might have to absorb fight at a given frequency whereas the solvent system does not. Among detectors of this type we note Ultraviolet-Visible (UV-Vis), fluorescence, electrochemical (EC) and conductivity detectors. 

The ultraviolet-visible, UV-Vis, detector is considered the "workhorse" of detectors for HPLC systems. Since UV principles are well-described in several undergraduate textbooks, only a limited description of the principles involved in UV-absorbance will be given in here. 

UV detector HPLC detector based on an ultraviolet-visible spectrophotometer using microsample flow cells (10 pi). Response to components depends on their absorption spectrum and absorptivity coefficient as defined by the Beer-Lambert law. Detector wavelength is set to give maximum sensitivity ideally at Amax however an optimum wavelength, Aopt at which all the components have a satisfactory absorbance, may be used. 

Detection methods for CE analysis are as diverse as those used for HPLC. The most widely used detection methods used in CE include ultraviolet/visible (UVA is) absorbance, LIE, mass spectrometry, conductivity, amperometry, radiometric, and refractive index. When deciding which detection method is best suited to an intended application, the analyst must first know if the compounds to be separated can be detected using a certain type of detector, that is, does the analyte have a chromophore. 

Organic compounds that possess an ultraviolet- or visible-absorbing chromophore obey the Beer Lambert or Beer-Bouguer law of spectrophotometry. In what is generally termed molecular absorption spectrophotometry, a cuvette (in the case of stand-alone UV-vis spectrophotometers) or a micro-flow cell (in the case of flow through HPLC UV-vis detectors). We now proceed to derive the fundamental equation that relates absorbance as measured on an UV-vis HPLC detector to concentration because this relationship is important to the practice... 

Diode array detector (DAD) DAD is used to measure molecular absorption of at a certain ultraviolet-visible (UV/Vis) wavelength. The amount of light absorbed will depend on the amount of a particular compound that is passing through the beam at the time and the absorbance of that compound at a particular UV wavelength. The difference between a DAD and a fixed wavelength UV detector is that a DAD can scan across a predetermined wavelength. 

The detector based in the absorbance of ultraviolet—visible (UV—Vis) is currently the most commrm technique. Diode array detector (DAD) is a detector that can scan a variety of wavelengths, and it is widely used. A number of stmcmrally related... 

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