Vitamins

1
Vitamins
2
Vitamin A (Retinol)
Retinol
3
b - Carotene and Retinol
CH
3
CH
CH
CH
3
3
3
H
C
3
CH
3
CH
CH
CH
3
3
3
CH
3
Oxidation
CH
CH
O
3
3
H
C
CH
3
3
C H
Retainal
CH
3
- 2H
CH
CH
3
3
H
C
CH
3
3
CH
OH
2
Retinol (Vitamin A)
CH
3
4
Vitamin A Activity Determnination in Foods
Vitamin A Activity b-Carotene Retinol
5
Vitamin A and b - Carotene Determination
Food
KOH (Alcoholic)
Saponification
3 hrs. at room temperature
Ether for Extraction
Extract (retinol and b-carotene)
HPLC

6
Vitamin A

• Column Nova-Pack C18 150 x 3.9mm
• Eluent 955 Methanol CH3CN
• Detection wavelength 285 nm

7
Thiamin Determination
Pyrimidine Thiazole

Mono-, pyro-, or triphosphate. Protein
S
H
C
NH
CH
CH
OH
N
3
2
2
2
N
N
CH
CH
3
2
K
Fe(CN)
3
6
Oxidation
N
S
H
C
CH
CH
OH
N
3
2
2
N
N
CH
CH
3
2
measure the absorbance at 435 nm
THIOCHROME (Fluorescent)
8
Thiamin Determination in Flour

• 5 g flour
• 75 ml 0.1 N HCl
• Make volume to 100 ml
• Digest at 100oC for 30 min
• Centrifuge and filter

9
Thiamine

• Column 250 x 4.6 mm, Spherisorb
• Eluent 9010 chloroformmethanol
• Wavelength 435 nm

10
Water-Soluble Vitamins
Pyridoxin
Riboflavin
Thiamin
Niacin
11
Riboflavin Determination

Flavinmononucleotide(FMN) Flavoadeninedinucleotide
(FAD) Flavoprotein
OH
CH
2
HOCH
HOCH
HOCH
HCH
N
N
O
CH
3
1
8
9
2
7
6
10
3
5
4
N
CH
3
O
6,7 Dimethyl-9-D-1-Ribitylisoalloxazine
12

Sample Preparation

• Autoclave in 0.1 N HCl for 30 min
• Adjust pH to isoelectric point to precipitate
  proteins
• Filter
• Dilute to 100 ml in volumetric flask

13
Riboflavin Analysis by HPLC

• Column 250 x 4.6 mm 5µm Spherisorb
• Eluent 5050 methanol water
• Wavelength 510 nm

14
Niacin
Ribose
Ribose
Nicotinamideadeninedincleotide (NAD)
15
Niacin Determination

• 1. Digestion an Hydrolysis
• 2. PPT Protein with (NH4)2SO4
• 3. Centrifuge and Filter
• 4. Rupture of Pyridine Ring with CNBR (Cyanogen
  Bromide)
• 5. Color Formation with Sulfanilic Acid
• 6. Determine the Absorption at 470 nm
• 7. Plot the Standard Curve of Niacin vs.
  Concentration
• 8. (Straight Line of Best Fit)

Autoclave
Ca(OH)
Sample
2 Hours
2
16
Niacine Determination
1
O
O
C
OH
C
OH
CNBr
REARRANGEMENT
REARRANGED
RING IS OPENED
DERIVATIVE
N
N

Br
CN
2

O
RERRANGED

H
N
S
OH
2
DERIVATIVE
O
O
C-OH
O
R-N-CHCH-CHC-CHN
OH
S
O
17
Niacine Standard Plot
Absorbance at 470 nm
m
g Niacin
18
Vitamin C
Ascorbic Acid Dehydroascorbic Acid
HSCH2CH(SH)CH2OH 2, 3-dithiol-Propanol
Reducing agent and converts dehydroascorbic
acid to ascorbic acid
19
Titration Method for Reduced Vitamin C
1. Extract with metaphosphoric acid (HPO3) in
HOAC. 2. Titrate with 2, 6-dichloroindophenol
(blue). At end point, rose pink (2,
6-dichloroindophenol in acidic condition).  
20
Oxidation and Reduction of Ascorbic Acid
2,6-dichloroindophenol
O
H
H
O
Cl
N
O
OH

H
O
O
Cl
H
O
H
C
2
Blue Color
C
l
O
O
O
H
N
H
H
O

H
C
l
O
O
H
O
H
C
2
Pink color in acidic condition
Extra oxidized indophenol form in the absence of
ascorbic acidic gives pink color at end point
21
Calculation for Reduced Vitamin C
A ml of 2,6-dichloroindophenol for sample
titration B ml of 2,6-dichloroindophenol for
blank F mg of ascorbic acid equivalent to 1
ml of indophenol standard solution V Initial
assay solution volume E Number of grams X
Volume sample aliquot titrated
22
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