This document describes methods for estimating various vitamins and acetic acid from samples. It discusses estimating vitamins A, B, and C using different techniques like HPLC, colorimetric, and spectrophotometric methods. For vitamin A, it provides details on the Carr-Price colorimetric method. It also outlines procedures for determining vitamins B using fluorimetric and spectrophotometric methods. Finally, it summarizes a titration process for estimating the concentration of acetic acid in vinegar using sodium hydroxide and a phenolphthalein indicator.

1. Laboratory Methods for Estimating
Vitamins(A,B,C) & Estimation of
Acetic Acid from Sample
Md. Rakib Hossain
ID: 1805481
Course Code: FSN-510

2. What are vitamins?
Vitamins are a group of organic substances that are needed
for normal cell function, growth and development.
 There are 13 essential vitamins that means they are required for
the body to work properly.
 Vitamins are two types. Fat soluble and water soluble.
Fat soluble vitamin are A,D,E,K and water soluble are vitamin C
and vitamin B into 8 classes are :
 B1 (Thiamin)  B6
 B2 (Riboflavin)  B7 (Biotin)
 B3 (Niacin)  B9 (Folic acid)
 B5 (Pantothenic)  B12

3. Vitamin A Determination Method
 Vitamin A is a group of unsaturated nutritional organic compounds
that includes retinol, retinal, retinoic acid, and pro-vitamin
beta-carotene.
 Vitamin A has multiple functions: it is important for growth and
development, for the maintenance of the immune system and
good vision.
.

5. Vitamin A Determination Method
Types of method:
 High Performance Liquid Chromatography (HPLC)
 Colorimetric method/ Carr-price method.
 UV-spectrophotometric method.
Colorimetric method:
 Anhydrous Antimony Tri chloride (SbCl3) in chloroform
reacts with a dilute solution of vitamin A to form a
transient blue color.
 Reaction occurs between Antimony tri chloride and
unsaturated side chain of vitamin A. The carotene, polyene
acids and various other material present in natural oils
produce same color.
 The color rapidly reaches maximum intensity and just as
rapidly it fades. The reading must be taken within 10-
15seconds.

6. Vitamin A determination by HPLC
Principle: Vitamin A is assayed from the biological sample by HPLC method after
processing and separation of samples & this method is very specific, accurate and
sensative. The instrument is set at 100% transmittance with a solution containing 1.0
ml of dry chloroform and 9.0 ml of antimony trichloride reagent ( a 25% solution in
dry chloroform ). Filter the solution, if the solution is turbid. In the experimental tube
(sample tube ) sample will be in dry chloroform and the maximum color intensity is
measured by adding 9ml of antimony trichloride reagent.
Materials and Reagent:
• Dry chloroform
• Saturated antimony trichloride solution in chloroform
• Standard vitamin A solution
• Biological sample
• Glassware
• 10ml test tube
• Spectrophotometer

7. Procedure:
 Dissolve 50,00 IU (15000 μg) of vitamin A in a 10ml dry chloroform
(1500μg/ml).
 Take 1ml of this solution and dilute to 5 ml in dry CHCl3 (300 μg/ml).
 Take 0.10,0.25,0.50 and 1.0 ml of this solution(300 μg/ml) in four
different clean and dry test tubes to make the final volume 1.0 ml by
adding 0.90,0.75,0.50 and 0.0 ml of dry chloroform (test tube no 1-4)
 Take 9.0ml of 25% antimony trichloride solution in 10ml test tubes
( four large tubes 1-4).
 Take 1.0ml CHCl3 plus 9.0ml SbCl3 in test tube no 5 to use it as blank. The
instrument is set 100% transmittance with this solution.
 Add the content 1.0ml of test tube to 9.0 ml of 25% SbCl3 solution test
tube. Immediately mix and take the maximum OD at 620 nm of the
mixed solution within 15 second as the color is time sensitive and fade
gradually.

8. Determination of vitamin A content in the supplied sample
The supplied sample is diluted to 5ml in dry CHCl3
and take 1.0ml of this solution (induplicate test
tubes). The total content of each of this solution
is added to reagents of large test tube containing
9.0 ml of 25% SbCl3 solution in dry chloroform.
Take the maximum OD immediately of these
solutions as described in the procedure of
standard solution. Plot the OD in the standard
curve and calculate the amount of Vitamin A in
the supplied sample.

9. Tube no Volume
of
standard
sample
(μl)
CHCl3
added
(μl)
SbCl3
added
(μl)
Volume of
total
solution
(ml)
Absorbance
at 500 nm
Average
absorbance
Blank 10 0.0
1 100 900 9 10 1.0921
2 200 800 9 10 1.525
3 300 700 9 10 2.04
4 5000 500 9 10 2.541
5 600 400 9 10 2.422
Unknown
sample 1
600 9 10 2.081
2.082
Unknown
sample 2
600 9 10 2.083
Table: Estimation of the absorbance of standard, supplied and unknown sample
solutions at 500 nm wavelength.

10. Calculation
For Standard solution
• Concentration of working standard solution is 300 μg/ ml
• Test tube 1, amount of vitamin A = 300 μg/ ml x 0.1 ml = 30 μg
• Test tube 2, amount of vitamin A = 300 μg/ ml x 0.2 ml = 60 μg
• Test tube 3, amount of vitamin A = 300 μg/ ml x 0.3 ml = 90 μg
• Test tube 4, amount of vitamin A = 300 μg/ ml x 0.5 ml = 150 μg
• Test tube 5, amount of vitamin A = 300 μg/ ml x 0.6 ml = 180 μg
Sample
• From Standard curve, The unknown sample contain 90 μg vitamin A
• So, the total amount of Vitamin A in 1 ml unknown sample = 90 x 12.5
μg = 1125 μg or 1.125mg

11. Result & Precaution
Result:
Amount of given sample = 1.125 mg
Precausion:
 Vitamin A destroyed by exposure to UV light thus it was
covered from light during experiment.
 SbCl3 was handled using gloves.
 Chloroform should handled carefully.

12. Determination of vitamin C
Source:
• Lemon
• Amlok
• Cauliflower
• Apple
• Mango
• Banana
• Potato
• Pomegranate
• Carrot
• Green Chili
• Cucumber
• Grapes

13. Chemicals
Ascorbic Acid
 Iodine
 Potassium iodide
 Starch solution
 Distilled Water

14. Prepared Solution & Equipment
 Iodine Solution
 2% Starch Solution
 Lemonade, Apple, and Orange Juice
 250 mL Erlenmeyer Flask
 Buret
 Distilled H2O
 Electronic Scale
 Pipet

15. Procedure
 Pour 50 mL of juice into 250 mL Erlenmeyer flask.
 Add 15 drops of starch indicator solution.
 Dissolve in 25 mL of Distilled H2O
 Record initial level reading of iodine solution in the
buret.
 Add iodine solution from the buret to the Vitamin C
solution until a deep-blue color is obtained, or any
color change is obtained.
 Recorded the final reading of buret.

16. Reading Apple
(ml)
Orange
(ml)
Lemonade
(ml)
Initial I2 Soln .3 5.9 33.2
Final I2 Soln 2.8 33.2 34.9
Reading Apple
(ml)
Orange
(ml)
Lemonade
(ml)
Initial I2 Soln 2.8 1.2 34.9
Final I2 Soln 5.9 28.7 36.3
Trial 1
Trial 2

17. Results Table
Juice Trial Vitamin C
mg
Lemonade 1 1.8
Orange 1 29.5
Apple 1 2.7
Lemonade 2 1.5
Orange 2 29.7
Apple 2 3.4

18. Estimation Methods of Vitamin B
FLUORIMETRIC METHOD
• Direct determination
• Direct additive determination
• Adsorptive additive determination
 SPECTROPHOTOMETRIC METHOD

19. Determination of Acetic acid
 Determination of acetic acid concentration in commercially
available white vinegar is one of the simplest and easiest
titrations.
 It is also possible to determine concentration of acetic acid in
other types of vinegar. The only problem is that the color of the
vinegar can make it difficult to spot the end point.
 Reaction
This is a simple neutralization reaction:
CH3COOH + NaOH → CH3COONa + H2O

20.  Solutions used
• To perform titration we will need
 titrant - 0.2 M or 0.1 M sodium hydroxide solution.
 indicator - phenolphthalein solution and
 some amount of distilled water to dilute acetic acid sample.
 procedure
 Pipette aliquot of acetic acid solution into 250mL Erlenmeyer
flask.
 Dilute with distilled water to about 100 mL.
 Add 2-3 drops of phenolphthalein solution.
 Titrate with NaOH solution till the first color change.

21. End point detection
 Depending on the concentration equivalence point of acetic acid titration lies
somewhere between pH 8 and pH 9.
 When titrating with base phenolphthalein starts to change its color around pH
8.2, which makes it a perfect candidate for the end point detection.
 Titrate against phenolphthalein to the first visible color change.
 Color change of phenolphthalein during titration –
 on the left, colorless solution before end point,
 on the right - pink solution after end point.
 Note we have to end titration at first sight of color change, before color gets
saturated.

22. Result calculation
 According to the reaction equation
CH3COOH + NaOH → CH3COONa + H2O
 Acetic acid reacts with NaOH on the 1:1 basis. That makes
calculation especially easy - Number of moles of sodium
hydroxide used it will be number of moles of acetic acid.