Question

PROCEDURE Table 4.1: Dilutions of Albumin for Standard Curve Determination. SAMPLE NUMBER VOLUME OF 0.9% NACL (mL) VOLUME OF 10 mg/ml ALBUMIN STOCK SOLUTION (mL) VOLUME OF BIURET REAGENT (mL) TOTAL VOLUME IN EACH TUBE ABSORBANCE READING ALBUMIN CONCENTRATION IN SOLUTION 1 (Blank) 1.0 mL OmL 4.0 mL 0.8 mL 0.2 mL 4.0 mL .4 0.6 mL 0.4 mL 4.0 mL 5 mL 5ml sme 5mL 5ml 5mL .228 .088 1 0.271 773 1.444 11827 0.4 mL 0.6 mL 4.0 mL 1.2 1.6 0.2 mL 0.8 mL 4.0 mL O mL 1.0 mL 4.0 mL 2 Table 4.2 SAMPLE NUMBER VOLUME OF 0.9% NaCl (mL) VOLUME OF UNKNOWN SAMPLE PROVIDED IN A TUBE ON YOUR TABLE VOLUME OF BIURET REAGENT (ML) ABSORBANCE Inknown 1 6 ml (4 ml 40 ml 22

Answer 1

1. Ans

Spectrophotometer is sophisticated device used to measure the absorbance of a solution while a light of specific wave length pass through it. It is based on Beer Lambert’s law. This law correlates the concentration of a liquid with absorbance or transmittance of a light having particular wave length.

In spectrophotometry we use three kinds of solutions

1. Blank solution
2. Standard solution
3. Test solution

-A blank solution should contain all the contents in the test and standard solution except the one which we want to measure. In some cases, distilled water also used as blank.

-Standard solution contains known concentration of the particular ligand.

-Test solution contains unknown concentration of the ligand (that is to be measured)

Standard solution gives the absorbance of known concentration and a blank solution gives the absorbance of solution in the absence of the ligand. Values obtained from the absorbance of standard we can plot a graph of absorbance Vs concentration. From this graph we can find out the concentration of the test solution.

2. Ans

Absorbance and transmittance

When a solution is subjected to a particular wave length of light, the amount of light passes through the solution is called transmittance and the amount of light absorbed by the solution is called absorbance. The sum of transmitted light and absorbance is equal to the amount of light subjected to the solution.

3. Ans

The given data is not fit for a standard graph as the absorbance values are not directly proportional with the concentration. The image of graph plotted using the data is attached

ax Om 0.2 y axis un ABSORBANce RENDING One vom od 0.6 08 to 1.2 16 ALBUMIN CONCENTRATION op SHARE

4. Ans

Yes, it is possible to generate standard curves of other substances like carbohyrates, lipids etc. But the reagents and wave length of light used will be different