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Author Topic: SPECTROPHOTOMETRIC TECHNIQUES- using the spec, blank solutions, readings...  (Read 19739 times)
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Francis Umeoguaju
Expert in Bioscience Issues
Posts: 657

« on: April 10, 2010, 07:45:47 am »


Using spectrophotometer to determine the concentration of compounds

Usually in a Biochemical lab, the concentration of a sample are determined principally either spectrophotometrically or with titration. Most often spectrophotometric determination is the technique of choice because the results obtained are usually more precise and specific.

The use of spectrophotometer to determine the concentration of compound or group of compounds stems from the ability of the spectrophotometer to measure the intensity of colored solution or the intensity of specific chemical groups such as peptides, tryptophan and tyrosine content of protein in colorless solution.

Many compounds are not themselves colored but can be made to absorb light in the visible region by reacting them with suitable reagent. The reactions are often very specific and in most cases very sensitive, so that quantities of material in the region of millimolar concentrations can be measured.

Since the spectrophotometer is used to compare the intensities of colored sample with that of known standard, it is necessary to treat the unknown sample in the same manner as the standards. Usually a calibration curve which comprise varying concentration of known standard is included in the protocol that also includes the sample.

When the coloured solutions of the standards and sample are fully developed, the absorbance is then read off from the spec.

Preparations of blank solutions

Before the absorbance of any sample is read off from the spec, there is need to zero the equipment with the aid of a
blank which may be water or reagent blank. The use of water blank simply involves taking the absorbance of the water and then subsequently adjusting the knob until it gets to the zero mark. (ie for equipments that uses analog display, newer equipments has a zeroing button that sets the value of the blanks to zero)

Reagent blank solution contains all the reagents and chemical used in the chemical development of the colour but lacks the substance being assayed. It is used to correct for any absorption of light by the solvents used to develop the colour. It must be included in every protocol that would be used to estimate the concentration of sample.

Extrapolation of result from spectrophotometric assay experiments

After taking the absorbance reading of both the standards and sample, a calibration curve of absorbance against the concentration of the standards are plotted. The concentration of the unknown sample is then extrapolated from this graph.

If the graph were to be linear, then the formular;

Concn (test) = Absorbance (test) x conc (standard)/   Absorbance (standard)

Can be used to determine the concentration of sample in subsequent analysis. In using the formula it necessary to repeat a standard together with each set of unknown samples. The limitation of using the formula is the inherent error since only a standard sample is used as a basis of comparison with that of unknown sample. For all analysis that I conducts, the concentration of the unknown sample were extrapolated from the calibration curve. Errors are minimized since about five standards were used to create the line of best fit of the graph.

When the reading of the test solution is beyond, the limits of the graph or the range of the instrument, the solution can be diluted with an appropriate diluents and the absorbance of the diluted solution is read. In most cases the whole process is repeated with a more diluted sample.

The concentration extrapolated for the diluted samples is multiplied with the dilution factor.

Chances favours the trained minds
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