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  Section: Plant Lab Protocols
 
 
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Methodology for Vitamins

 
     
 
Thiamine
 
Thiamine is one of the B group vitamins whose deficiency causes beri-beri. It occurs in outer layers of grains of many plants including cereals. Thus unpolished rice and foods made of whole wheat are good sources of this vitamin. Thiamine is water-soluble, and overcooking may leach out or destroy thiamine originally present in the food sources. Among a number of chemical and micro-biological methods available for the estimation of thiamine fluorimetric method is easier and popular.
 
 



Principle

Alkaline potassium ferricyanide oxidizes thiamine to thiochrome which is a fluorescent compound. The thiochrome is extracted in isobutyl alcohol and measured in a fluorimeter.
 
 
Materials
15%NaOH
1% Potassium Ferricyanide
Isobutyl Alcohol
Sodium Sulphate (anhydrous)
Approximately 0.1N H2SO4
Standard Thiamine Hydrochloride
Stock Solution
Dissolve 50mg thiamine hydrochloride in 500mL of about 0.1N sulphuric acid containing 25% alcohol (100mg/mL). Store this solution in a brown bottle in a refrigerator.
Working Standard Solution
Prior to experiment, dilute 5mL of the stock solution to 100mL with 0.1N sulphuric acid and again dilute 5mL of the second solution to 100mL with 0.1N H2SO4 (0.25mg/mL) and use.
 
 
Procedure
Extraction of Thiamine
1.
Weigh accurately 5g finely ground sample into a 250mL conical flask in duplicate.
2.
Slowly add 100mL 0.1N H2SO4 without shaking, stopper the flask and let it stand overnight.
3.
Next morning shake vigorously and filter through Whatman No.1 filter paper, discarding the first 10 to 15mL of filtrate.
 
Oxidation
4.
Pipette out 10mL of the extract in duplicate into 100mL separating funnels.
5.
Pipette out 10mL of working standard (in 4-5 replicates).
6.
Add 3mL of 15% NaOH into each separating funnel immediately followed by four drops (0.2mL) of ferricyanide solution.
7.
Shake gently for exactly 30 seconds.
8.
Add 15mL of isobutanol rapidly from a quick delivery burette or a measuring cylinder.
9.
Stopper immediately, shake vigorously for 60 sec and allow the layers to separate.
10.
Drain off the bottom layer carefully and discard it.
11.
Add one spatula-full of sodium sulphate directly into the separating funnel, stopper and swirl gently to clarify the extract. If the extract is not clear, add a little more Na2SO4 and clarify.
12.
Collect the clear extract from the top using a Pasteur pipette into a clean dry test tube.
13.
Prepare a set of sample blanks by pipetting out 10mL of the extract and follow the above procedure but omit addition of ferricyanide.
14.
Prepare a blank for the standard (in duplicate) separately.
15.
Select suitable primary (366nm) and secondary filters as per the make of the fluorimeter.
16.
Set fluorimeter by initially adjusting the standard blank to 0 reading and standard to 100. Then read the sample blank and sample. Since the light intensity sometimes changes progressively it is advised to take 5-6 readings.
 
 
Calculation
mg thiamine content in 100g sample
=
0.25 x 10
x
(x – x1) x 100
x
10
a – a1
10
5

where  
a              = reading of standard = 100
a1                   = reading of standard blank = 0
x              = reading of standard sample and
x1                   = reading of standard sample blank
 
 



Notes

1.
Since the standard has to be read a number of times during the measurement it is convenient to oxidise the standard in 4 or 5 replicates and to combine all the oxidized extracts in one conical flask. Portions of this may be read at a time and discarded.
2.
Thiochrome is rapidly destroyed by UV light, so the solution once exposed should not be read again. It should be discarded.
3.
Follow the instructions given in the manual for operating the fluorimeter.
 
 
     
 
 
     




     
 
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