Vitamins
Vitamin A (Retinol)
CH3 CH3
H3C CH3
OH
Retinol
CH3
b - Carotene and Retinol
CH 3
CH 3 CH 3 CH 3
H3 C
CH 3
CH 3 CH 3 CH 3
CH 3
Oxidation
CH 3 CH 3 O
H3 C CH 3
C H
CH 3 Retainal
- 2H
CH 3 CH 3
H3 C CH 3
CH 2 OH
CH 3 Retinol (Vitamin A)
Vitamin A Activity Determnination in Foods
Vitamin A Activity = b-Carotene + Retinol
Vitamin A and b - Carotene Determination
Food
KOH (Alcoholic)
Saponification
3 hrs. at room temperature
Ether for Extraction
Extract (retinol and b-carotene)
HPLC
Vitamin A
• Column: Nova-Pack C18; 150 x 3.9mm
• Eluent : 95:5 Methanol: CH3CN
• Detection wavelength: 285 nm
Thiamin Determination
Pyrimidine Thiazole
H3C N NH2 S CH2CH2OH Mono-, pyro-,
N N or triphosphate.
CH2 CH3
Protein
K 3Fe(CN) 6
Oxidation
H3C N N S CH2CH 2OH
N N
CH2 CH3
measure the absorbance at 435 nm
THIOCHROME (Fluorescent)
Thiamin Determination in Flour
1. 5 g flour
2. 75 ml 0.1 N HCl
3. Make volume to 100 ml
4. Digest at 100oC for 30 min
5. Centrifuge and filter
Thiamine
• Column : 250 x 4.6 mm, Spherisorb
• Eluent : 90:10 chloroform:methanol
• Wavelength: 435 nm
Water-Soluble Vitamins
2
Pyridoxin
Riboflavin
3
Niacin Thiamin
Inject 4
1
Column: u Bondapak C18
Solvent: MeOH
Sample: Water-Soluble Vitamins
0 5 10 15 20
Riboflavin Determination
Flavinmononucleotide(FMN)
CH2 OH
Flavoadeninedinucleotide (FAD)
HOCH Flavoprotein
HOCH
HOCH
HCH
CH 3
N N O
8 9 1
7 2
6 10 3
5 4
CH 3 N
O
6,7 Dimethyl-9-D-1-Ribitylisoalloxazine
Sample Preparation
Autoclave in 0.1 N HCl for 30 min
1. Adjust pH to isoelectric point to precipitate proteins
2. Filter
3. Dilute to 100 ml in volumetric flask
Riboflavin Analysis by HPLC
• Column: 250 x 4.6 mm 5µm; Spherisorb
• Eluent : 50:50 methanol : water
• Wavelength: 510 nm
Niacin
O O
C OH C NH2
N N
Ribose Ribose
Nicotinamideadeninedincleotide (NAD)
Niacin Determination
1. Digestion an Hydrolysis
Autoclave
Sample + Ca(OH) 2 2 Hours
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)
Niacine Determination
1 O
O
C OH + CNBr C OH REARRANGEMENT
REARRANGED
RING IS OPENED DERIVATIVE
N N
Br CN
2
O
RERRANGED +
H2 N S OH
DERIVATIVE
O
O
C-OH O
R-N-CH=CH-CH=C-CH=N S OH
O
Niacine Standard Plot
mg Niacin
Vitamin C
Ascorbic Acid Dehydroascorbic Acid
CH2 OH CH2 OH
HOCH O HOCH O
-2 H
O O
+2 H
HO OH O O
HSCH2CH(SH)CH2OH : 2, 3-dithiol-Propanol
Reducing agent and converts dehydroascorbic acid to ascorbic acid
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).
Oxidation and Reduction of Ascorbic Acid
2,6-dichloroindophenol
HO OH Cl
+ O N OH
H
H O H 2C O O Cl
Blue Color
Cl
O O
HO NH OH
+
H
O Cl
H O H 2C O
Pink color in acidic condition
Extra oxidized indophenol form in the absence of ascorbic acidic gives pink color
at end point
Calculation for Reduced Vitamin C
F V
mg Ascorbic Acid/g = (A-B) x x
E X
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