Spectrophotometry PDF

SPECTROPHOTOME TRY  1 INTRO It is known that a particular substance, owing to its molecular structure, will only absorb light of certain wavelengths and reflect or transmit the rest. Chlorophyll, for instanc...

SPECTROPHOTOME TRY  1 INTRO It is known that a particular substance, owing to its molecular structure, will only absorb light of certain wavelengths and reflect or transmit the rest. Chlorophyll, for instance, absorbs a significant amount of red light but not green light, which is transmitted back; chlorophyll- bearing plants therefore appear green (assuming, of course, that other pigments are absent or are in negligible amounts).  2 INTRO Note further that though two substances may absorb light of the same wavelengths, it is very probable that their absorption of such light will be in different degrees, because of intrinsic variation in molecular structure.  3 INTRO On these facts lie the basis of spectrophotometry, a technique of prime importance in biochemical analysis. Spectrophotometry is the quantitative measurement of the absorption and transmission of light by a solution. If the solutions being spectrophotometrically determined are colored, the technique is alternatively referred to as colorimetry.  4 ABSORPTION SPECTRUM A particular substance may be characterized by a graph showing the amount of light it absorbs (absorbance) at different wavelengths. This is known as the absorption spectrum of the compound. As an example, Figure 23- represents the absorption spectrum of riboflavin.  5 ABSORPTION SPECTRUM  6 STANDARD CURVE A standard curve is a graph indicating the amount of light absorbed by different known concentrations of a substance. Ideally linear, it may then be used to determine the unknown concentration of a compound by first measuring the absorbance of the unknown at the wavelength used in preparing the standard curve. The concentration is interpolated from the standard curve using the absorb-ance of the unknown. However, a more accurate way of determining the unknown concentration would be through the use of the Lambert-Beer Law equation.  7 LAMBERT-BEER LAW Lambert-Beer Law derivation helps us define the relationship between the intensity of visible UV radiation and the exact quantity of substance present. The derivation of Beer- Lambert Law has many applications in modern-day science. Used in modern-day labs for testing of medicines, organic chemistry and tests with quantification. These are some fields in which this law finds its uses.  8 LAMBERT-BEER LAW The Lambert-Beer law states that: for a given material sample path length and concentration of the sample are directly proportional to the absorbance of the light.  9 SPECTROPHOTOMETER The instrument, which measures the absorption or transmission of light by a solution, is called a spectrophotometer.  10 SPECTROPHOTOMETER In place of a monochromator and a selector, some colorimeters use filters. Appropriate filters absorb those wavelengths of light ordinarily transmitted by the solution while at the same time permitting those wavelengths of light, which are maximally absorbed by the solution to pass through the filter and strike the solution. The percent change in the amount of transmitted or absorbed light caused by a given change in concentration of the solution is thus made maximal; needless to say, this increases the sensitivity of colorimetric measurements. Two different sizes of cuvette used in a spectrophotometer  10 SPECTROPHOTOMETER Spectrometers are generally classified based on the wavelength of light the source is. There are two main classifications: UV-Vis Spectrophotometer: The UV-Vis spectrophotometer is commonly referred to as just ‘UV-Vis’. This instrument measures absorbance in the ultraviolet and visible range. Generally, that means 200 nm – 700 nm. IR Spectrophotometer: The IR spectrophotometer measures samples using infrared light, 700-15000 nm.  10 SPECTROPHOTOMETER Applications Some of the major applications of spectrophotometers include the following: Detection of concentration of substances Detection of impurities Structure elucidation of organic compounds Monitoring dissolved oxygen content in freshwater and marine ecosystems Characterization of proteins Detection of functional groups Respiratory gas analysis in hospitals Molecular weight determination of compounds The visible and UV spectrophotometer may be used to identify classes of compounds in both the pure state and in biological preparations.