Vitamin
B6 (Finished product) Powder form Assay (By UV method)
Preparation of Standard solution (1) :Take 25
mg of pyridoxine Hydrochloride WS in50
ml volumetric flask, add 25 ml 0.1 N hydrochloric acid shake to dissolve &
dilute to 50 ml with same solvent.
(Stock solution A): Take 5
ml of Standard solution (1) in 100 ml volumetric flask & Dilute to 100 ml
with distilled water.
Preparation of Sample solution (Stock Solution B): Take sample
equivalent to 25 mg pyridoxine
Hydrochloride in 100 ml volumetric flask
& dilute to 100 ml with distilled water.
Reagents:
1. Chlorimide Solution (0.04% w/v) : Dissolve
40 mg of 2,6-dichloroquinone- Chlorimide in 100 ml of isopropyl alcohol.
2. Ammonia ammonium chloride buffer solution: Dissolve
16.0 gm of Ammonium chloride in 70 ml distilled water,add 16 ml of ammonia
solution (about 25%) and dilute with distilled water to 100 ml.
3. Sodium Acetate (20.0% w/v) : Dissolve
10.0 gm of sodium acetate in 50 ml of Distilled Water.
4.0 Boric Acid (5.0 % w/v): Dissolve
2.5 gm of Boric acid in 50 ml of distilled water.
Procedure
A] Standard solution: Take 5 ml
of stock solution A in 50 ml volumetric flask & add 25 ml isopropyl
alcohol.
B] Sample solution: Take 5ml
of stock solution B in 50 ml volumetric flask &add 25 ml isopropyl alcohol.
Standard preparation (a)
Pipette out 5 ml of standard
solution in 50 ml volumetric flask (shake the flask after every addition of
solution) add 1 ml of Ammonia ammonium chloride buffer solution, 1 ml Sodium
Acetate (20.0% w/v), 1 ml distilled water & 2 ml Chlorimide Solution (0.04%
w/v) after addition of Chlorimide solution shake vigorously for accurately
timed 1 min& Take the absorbance at the wavelength of maximum absorbance at
650 nm accurately timed 2 min.
Using water as the blank. Designate the Absorbance as (S)
Standard Blank preparation (b): Repeat
the procedure standard preparation (a)
but substitute the 1.0 ml of Boric Acid (5.0% w/v) for the 1.0 ml of water.
Designate the Absorbance as (S1)
Sample preparation (c): Pipette
out 5 ml of sample solution in 50 ml
volumetric flask (shake the flask after every addition of solution) add 1 ml of
Ammonia ammonium chloride buffer solution, 1 ml Sodium Acetate (20.0% w/v), 1
ml distilled water & 2 ml Chlorimide Solution (0.04% w/v) after addition of
Chlorimide solution shake vigorously for accurately timed 1 min& Take the
absorbance at the wavelength of maximum absorbance at 650 nm accurately timed 2
min.
Using water as the blank. Designate the Absorbance as (T)
Sample Blank preparation (d): Repeat
the procedure sample preparation (c)
but substitute the 1.0 ml of Boric Acid (5.0% w/v) for the 1.0 ml of water.
Designate the Absorbance as (T1)
|
Sr. No. |
Cont. |
Quantity |
|||
|
Std Blk Prepn |
Std Prepn |
Spl Blk Prepn |
Spl Blk
Prepn |
||
|
1 |
Std sol |
5.0 ml |
5.0 ml |
------------- |
-------------- |
|
2 |
Spl sol |
---------- |
----------- |
5.0 ml |
5.0 ml |
|
3 |
Ammonia Ammonium Chloride buffer solution |
1.0 ml |
1.0 ml |
1.0 ml |
1.0 ml |
|
4 |
Sodium Acetate (20% w/v) |
1.0 ml |
1.0 ml |
1.0 ml |
1.0 ml |
|
5 |
Water |
----------- |
1.0 ml |
------------- |
1.0 ml |
|
6 |
Boric acid (5.0 % w/v) |
1.0 ml |
--------------- |
1.0 ml |
------------- |
|
7 |
Chlorimide solution (0.04%w/v) |
2.0 ml |
2.0 ml |
2.0 ml |
2.0 ml |
Calculation :
(T – T1) x std wt. (mg) x 5 x 100 x purity of B6
= -------------------------------------------------------x Av. Net Cont
(S – S1) x 50 x 100 x sample wt. gm x 100
mg x 100
i. e = -------------------------------
Label claim
