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  Section: Plant Lab Protocols
 
 
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Methodology for Vitamins

 
     
 
Niacin
 
An important water-soluble vitamin is niacin. It is also called nicotinamide or nicotinic acid depending upon the presence or absence of the amide group. It is physiologically very important since two coenzymes, NAD+, and NADP+ are derived from this vitamin. It is present in all living organisms. A chemical method for the estimation of niacin content is described below.
 



Principle

Niacin reacts with cyanogen bromide to give a pyridinium compound which undergoes rearrangement yielding derivatives. These derivatives couple with aromatic amines to give yellow colored pigment. Under proper conditions, the intensity of the yellow color produced is proportional to the amount of niacin present.
 
Materials
Extraction
Grind the sample (5g) in 4N sulphuric acid (30mL) and steam it for 30 min. Cool and make up to 50mL with distilled water. Filter through Whatman No.1 filter paper. Add 60% basic lead acetate to the filtrate (5mL to 25mL of filtrate). Adjust to pH 9.5 with a pH meter or thymol blue indicator, using ION sodium hydroxide. Centrifuge. To the supernatant add 2mL Cone. H2SO4 and allow to stand for 1h. Centrifuge. Collect the supernatant. Add 5mL 40% ZnSO4 and adjust to pH 8.4 with 10N NaOH. Centrifuge and collect the supernatant. Adjust to pH 7. If precipitate appears, centrifuge and use the supernatant as the source material.
 
Cyanogen Bromide
Keep the bromine bottle in an ice bath for a few minutes and then using a vacupet, pipette out 25mL of bromine into a beaker or conical flask containing 500mL distilled water. Take a 10% solution of sodium cyanide in a burette and deliver slowly to the bromine water with constant shaking until the solution becomes colorless.
 
Redistilled 4% Aniline in Absolute Alcohol
Standard
Dissolve 10mg niacin and make up to 100mL with distilled water in a volumetric flask. Alternatively 100mg may be dissolved in 100mL and then 10mL of this is again diluted to 100mL. One mL of the standard contains 100mg niacin.
 
 
Procedure
1.
Into a series of test tubes pipette out 0.1mL to 0.5mL of the standard niacin solution.
2.
Take two or three different volumes of samples solution in other test tubes (0.1-0.5mL).
3.
Make up the volume to 6mL with distilled water in all the tubes.
4.
Add 3mL of cyanogen bromide using a burette and shake the contents in the tube well. After 10 min add 1mL of 4% aniline to each tube.
5.
Read the yellow color developed after 5 min at 420nm against a reagent blank.
 
 
Calculation
Draw a standard graph with the readings of the standard niacin. Find out the niacin content in the sample volume taken and calculate for g weight of the sample.
 



Notes

1. Handle the liquor bromine very carefully. If any spill occurs, wash the affected part of body with plenty of water, thoroughly.
2. Never mouth-pipette bromine, sodium cyanide or cyanogen bromide as they are poisonous.
 
     
 
 
     




     
 
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