Colorimetric

methods in biochemistry

Basma Ahmed Eltonsy
201816890












 Colorimetric methods in biochemistry

colorimetry or colourimetry is a technique "used to determine the
concentration of colored compounds in solution." A colorimeter is a
device used to test the concentration of a solution by measuring its
absorbance of a specific wavelength of light (not to be confused with
the tristimulus colorimeter used to measure colors in general).
To use the colorimeter, different solutions must be made, including a
control or reference of known concentration. With a visual colorimeter,
for example the Duboscq colorimeter illustrated, the length of the light
path through the solutions can be varied while filtered light transmitted
through them is compared for a visual match. The concentration times
path length is taken to be equal when the colors match, so the
concentration of the unknown can be determined by simple
proportions. Nessler tubes work on the same principle.

There are also electronic automated colorimeters; before these
machines are used, they must be calibrated with a cuvette containing
the control solution. The concentration of a sample can be calculated
from the intensity of light before and after it passes through the sample
by using the Beer–Lambert law. Photoelectric analyzers came to
dominate in the 1960s.

The color or wavelength of the filter chosen for the colorimeter is
extremely important, as the wavelength of light that is transmitted by
the colorimeter has to be the same as that absorbed by the substance
being measured. For example, the filter on a colorimeter might be set
to red if the liquid is blue.


Absorption colorimeter

A colorimeter is a device used to test the concentration of a solution by
measuring its absorbance of a specific wavelength of light. To use this
device, different solutions must be made, and a control (usually a
mixture of distilled water and another solution) is first filled into a
cuvette and placed inside a colorimeter to calibrate the machine. Only
after the device has been calibrated you can use it to find the densities
and/or concentrations of the other solutions. You do this by repeating
the calibration, except with cuvettes filled with the other solutions. The
filter on a colorimeter must be set to red if the liquid is blue. The size of
the filter initially chosen for the colorimeter is extremely important, as
the wavelength of light that is transmitted by the colorimeter has to be
same as that absorbed by the substance.

Types of tests depends on colourimetric method

There are many tests based on colourimetric method:
1-Urine Report “Analysis”
2-Cholesterol
3-Determination of Proteins
4-Blood Glocuse test
Every test have it’s own method and calculates.
-Urine Report “Analysis”:
Physical properties:
-Color: Amber yellow / yellow
-Odor: characteristic Odor / Acetone Odor in case of keton bodies
-Appearnce: Clear / Turpid (in case of protien)
-Aspect: No deposit
-Reaction: Slightly Acidic
-Specific Gravity: 1025
 Chemical properties:
1-Fehilng Test
Observation: Brick Red (ppt)
Result: Glucosuria
2-Rothera’s Test “Ketone Body Test”
Observation: permngnate color
Result: ketonuria
3-Heat Coagulation
Observation: White coagulation
Result: protenuria
4-Hye’s sulfer
Observation: Sulfur Sink
Result: Bile Salts are present

-Cholesterol”Enzymatic colorimetric method”
The cholesterol is determined aftor enzymatic hydrolysis and oxldation. The
quinonelmine is formed from hydrogen peroxide and 4 aminoantipyrine in
the presence of phenol and peroxidase.

- Determination of Proteins
A new colorimetric method for the determination of proteins was investigated using
the local dye “Uri isi”. Serial dilution of a solution of bovine serum albumin (BSA) was
made. Protein assay was carried out photo-metrically with BSA using both the biuret
reagent and the local dye. The results showed that the optical density decreased
from 1.269 to 0.189 with an increase in the concentration of protein in the case of
biuret reagent and also decreased from 0.276 to 0.174 with increase in the
concentration of protein in the case of the local dye. The results suggest that the
local dye was more sensitive than biuret reagent for assaying proteins since the
bovine serum albumin was diluted 100 times before the local dye could be used for
the assay.