Visible spectrophotometric detector

This technique is only of value when the identity of the compoimd to be determined is known. There are also limitations on the sensitivity that can be achieved, usually milligrams per litre or occasionally, microgreuns per litre. 

Visible spectrophotometers are commonly used in the water industry for the estimation of colour in a sample or for the estimation of coloured products produced by reacting a colourless compound of the sample with a reagent, which produces a colour that can be evaluated spectrophoto-metrically. 

Some commercially available instruments, in addition to visible spectrophotometers, can also perform measurements in the ultraviolet and near IR regions of the spectrum. These have not yet found extensive application in the field of water analysis. 

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Ultraviolet-visible (UV-Vis) spectrophotometric detectors are used to monitor chromatographic separations. However, this type of detection offers very little specificity. Element specific detectors are much more useful and important. Atomic absorption spectrometry (AAS), inductively coupled plasma-atomic emission spectroscopy (ICPAES) and inductively coupled plasma-mass spectrometry (ICP-MS) are often used in current studies. The highest sensitivity is achieved by graphite furnace-AAS and ICP-MS. The former is used off-line while the latter is coupled to the chromatographic column and is used on-line . 

Besides the spectrophotometric detectors based on absorbance or fluorescence of UV/visible radiation, and used when the mobile phase does not absorb appreciably, another mode of detection exists based upon electrolyte conductivity. Thus, at the outlet of the column, the conductance (the inverse of the resistance) of the mobile phase is measured between two microelectrodes. The measuring cell should be of a very small volume (approx. 2pL). The difficulty is to recognize in the total signal the part due to ions or ionic substances present in the sample. In order to do direct measurements, the ionic charge of the mobile phase has to be as low as possible and the measuring cell requires strict temperature control to within 0.01 °C because of the high dependence of conductance on temperature ( 5%/°C). 

Depending on the light source, the UV detector can operate from 190 nm and well into the visible area. The UV detector can be of various kinds, for example, filter photometric detectors, spectrophotometric detectors, and diode array detectors. 

With phototubes and photomultiplier-type detectors (photoemissive detectors, ultraviolet to visible range), thermal noise becomes insignificant as compared to shot noise. Shot noise is the random fluctuation of the electron current from an electron-emitting surface (i.e., across a junction from cathode to anode), and in PM mbes that is amplified and becomes the noise-limiting fluctuation. In instruments with these detectors, the absolute error is not constant at all values of T, and the expressions for the spectrophotometric error become more complicated. It has been calculated that, for these cases, the minimal error should occur at 0.136 or A = 0.87. These instruments have a working range of about 0.1 to 1.5 A. 

Colorimetry refers to the determination of a substance from its ability to absorb visible light. Visual colorimetric methods are based on the comparison of a colored solution of unknown concentration with one or more colored solutions of known concentration. In spectrophotometric methods, the ratio of the intensities of the incident and the transmitted beams of light is measured at a specific wavelength by means of a detector such as a photocell. 

Spectrophotometric analyses were carried out using a Biochrom Model Libra S12 UV/Visible Spectrophotometer, equipped with tungsten halogen and deuterium arc light sources with a single solid state silicon photodiode detector, and operating software. 

A spectrophotometric UV-Vis detector is selective, yet its selectivity can be changed simply by changing the wavelength monitored by the detector. Versatility of the detector can be increased by adding a color-forming reagent to the eluent or the column effluent. The fundamental law under which ultraviolet-visible (UV-VIS) detectors operate is the Lambert-Beer law. It can be stated in the following form ... 

Sample ions that absorb sufficiently in the UV or visible spectral region may be detected by direct spectrophotometry. Indirect spectrophotometric detection is commonly used for ions that do not absorb. An absorptive reagent is added to the BGE, and this gives a peak in the direction of reduced absorbance when a sample ion passes through the detector. The absorbing reagent, which is sometimes called a visualization reagent, should have a mobility that matches those of the sample ions as closely as possible. Chromate is often used for the indirect detection of anions and a protonated amine cation, such as benzylamine, for detection of cations. 

For routine HPLC analysis, the detection of flavins is carried out either spectro-photometrically, using variable- or fixed-wavelength HPLC detectors in the ultraviolet (e.g., 254 nm) or visible (e.g., 405 nm) region, or fluorimetrically. For riboflavin, the excitation wavelength for fluorimetric detection is usually 440 to 450 nm, and the emission wavelength 530 nm. The detection limit for fluorescence detectors is >1 pmol (0.38 ng) riboflavin, whereas <30 pmol (11 ng) can be detected spectrophotometrically at 254 nm. Photodiode array detectors are significantly less sensitive than normal HPLC spectrophotometers (38). 

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