Colorimetric Proportioning

A colorimetric proportioning is possible when that a chemical reaction gives coloured products and if the intensity of coloring is proportional to the concentration of the element to proportion. Proportioning colorimetric rests on the Loi of Beer-Lambert.

Law of Beer-lambert

The form used is the following one: With = ε × L × C

a: absorptance of the solution without unit
ε: molar coefficient of extinction in L×mol^-1×cm^-1 (sometimes noted with a ξ)
L: length of tank crossed by the light in cm
C: molar concentration in mol×L^-1

The measurement of the Absorbance is given by a Spectrophotomètre which measures the optical density. The more one solution is coloured, the more the light has evil to cross, therefore more the absorptance of the solution increases.

General protocol of colorimetric proportionings

General terms of realization

To carry out a colorimetric proportioning, certain conditions must be filled:

the reaction must give a coloring proportional to the concentration
coloring must be stable time to make measurements
the compound to be analyzed must be in very weak concentrations
the range standard must be realized under the same conditions physicochemical as the tests
the wavelengths of the spectrophotometer must be that which allows the strongest possible absorptance

if these first conditions are met, a colorimetric proportioning can be possible.

Realization of a range of calibration

To regulate the spectrophotometer, the realization of a range of calibration is essential (except for the kits sold in the trade). The range of calibration makes it possible to determine an absorptance with a wavelength given for a concentration in required compound. It is thus necessary to prepare a solution of the element to proportion weak concentration. At the time of the realization of the range, the volume of solution must be identical in all the tubes. However volumes of reagents must remain constant to allow the reaction and the quantities of compound to be proportioned must vary in each tube. It will thus be necessary to supplement with water distilled with the same volume so that the tubes is on the same level of solution. A tube 0 or white must imperatively be carried out to cancel the absorptance due to the reagents themselves. This tube does not contain a compound to proportion the realization of the ranges of calibration requires much precision, and must be preferably, realized under the same conditions as the tests

Realization of a colorimetric table

For you help, the realization of a colorimetric table makes it possible to avoid error at the time of the realization of tiresome protocol and to note your results. Here in example the colorimetric table of the Proportioning of glucose by the DNS.

Reading of the results

During obtaining the values of absorptances, it is necessary to trace the right absorption according to the quantities of composed by tubes . It must pass by the origin of the reference mark and all the values if proportioning indeed were carried out. Defer on this line the values of absorptance of your tests to determine the quantity of compound present of it. To go back to the concentration in compound present in the product, do not forget possible dilutions or the quantities of matter which were used.

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