SI No.

CONTENTS

1 Scope of the Document

2 Principle and Reagent preparation

3 Selenite reduction

4 Procedure

5 Calculation

6 Reference
1. The scope of the document: This documentation is applicable for the estimation of selenium in sodium Selenite
samples.

2. Principle: The principle is based on the reaction between DAB and tetravalent selenium and spectrophotometrically
estimating the selenium content.

2.1. Reagents: Glycine buffer – 0.1 M –Take 0.75 g of glycine dissolve in 100 ml water, then set the pH to 2.6 with
0.1 N HCl
DAB: 50 mg in 50 ml of water, 6N HCl -Take 52.6 ml of conc HCL makeup to 100 ml with water.
Sodium Selenite standard – 100 mg of standard in 20 ml of 6 N HCL. Sample weight is 100 mg.

3. Reduction of Selenite: Reduction is not applicable; the sample is taken directly to 20 ml of water.

4. Procedure for Selenite Estimation: From the above take aliquot of 0.01,0.02 and 0.03 and make up the volume
to 5ml with glycine buffer and add 1 ml DAB reagent, Keep aside for half an hour at room temperature. Set the pH to
6 using 1 N NaOH and add 10 ml of toluene, vortex until the lower layer is clear, read the OD at 420 nm.

5. Calculation:
OD of sample X Conc of standard X Dilution Factor X 100
in microgram
OD of Standard
Weight in mg X 1000

DF=Dilution Factor

Numerator 100 = to convert into percentages

Denominator 100 = weight taken in mg

1000= convert microgram to mille gram

 Concentration Vs OD
0.4
0.35 y = 0.0147x + 0.0695
R² = 0.9995
0.3
Optical Density

0.25
0.2
OD
0.15
Linear (OD )
0.1
0.05
0
0 5 10 15 20 25
Concentration in microgram

6. Reference: Spectrophotometric determination of trace elements, Z. Marczenko .