Colorimetry Spectrophotometry

A number of EA exhibit a strong fluorescence upon irradiation. This feature is extensively used for the detection of EA after their separation by HPLC or TLC. Without any preceding separation, the fluorescence of ergot derivatives can also be used for the development of some fluorimetric assays (Gyenes and Bayer, 1961 Hooper et al., 1974). The limitation of all direct methods of EA determination including colorimetry, spectrophotometry or fluorimetry is the lack of specificity regarding related alkaloids or their own degradation products. Consequently, the majority of these methods has now been abandoned, but the specific features or reactions of EA are further employed for their detection by more sophisticated HPLC and TLC methods. 

By mass spectrometry Quantition can be performed by several methods, such as Colorimetry spectrophotometry Spectrofluorimetry HPLC... 

Cahbration with standard reflectance and transmittance samples should be routinely used for optimum results in spectrophotometry and colorimetry. Cahbration of the wavelength (32) and photometric (33) scales is also advisable. The cahbration of a white reflectance standard in terms of the perfect reflecting diffuse, T, has been discussed (34), as have diagnostic tiles for tristimulus colorimetry (35). A collaborative reference program is available on instmment performance (36). 

Spectrophotometry-Colorimetry One of most useful and versatile methods but can be time consuming 10- to 10- M (10- with pre-concentration)... 

Optical methods of analysis are dependent either upon (i) measurement of the amount of radiant energy of a particular wavelength absorbed by the sample, or (ii) the emission of radiant energy and measurement of the amount of energy of a particular wavelength emitted. Absorption methods are usually classified according to the wavelength involved as (a) visible spectrophotometry (colorimetry), (b) ultraviolet spectrophotometry, and (e) infrared spectrophotometry. 

Spectrophotometry proper is mainly concerned with the following regions of the spectrum ultraviolet, 185-400 nm visible 400-760 nm and infrared, 0.76-15 /tm. Colorimetry is concerned with the visible region of the spectrum. In this chapter attention will be confined largely to the visible and near ultraviolet region of the spectrum. 

This is the fundamental equation of colorimetry and spectrophotometry, and is often spoken of as the Beer-Lambert Law. The value of a will clearly depend upon the method of expression of the concentration. If c is expressed in mole h 1 and / in centimetres then a is given the symbol and is called the molar absorption coefficient or molar absorptivity (formerly the molar extinction coefficient). 

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