Đề tài về Vitamin A

VITAMINS
Vitamin A

H3C

CH3
CH3

CH3

CH3
CH2OH


β - Carotene
CH3
CH3

CH3

CH3

H3 C

CH3

CH3

CH3

CH3

Oxidation

H3 C

CH3

CH3

CH3

O
C

H

Retainal

CH3
- 2H

H3 C

CH3
CH3

CH3

CH3
CH2 OH
Retinol (Vitamin A)

CH3


Vitamin A and β - Carotene Determination

Food
KOH (Alcoholic)
Saponification

3 hrs. at room temperature
Ether for Extraction
Extract (vit.A and carotenoids)

Total Carotenoids
only at 440 nm

Vitamin A + Carotenes
Carr-Price Reagent
Measure at 620 nm


A at 440 nm
and 620 nm

β-CAROTENE STANDARD ABSORBANCES AT 440 AND 620 nm

A at 440 nm

A at 620 nm

Use absorbances at 440 nm and then convert this to absorbance at
620 nm and subtract from the absorbance at 620 nm to determine
the absorbance at 620 due to Vitamin A.

Vitamin A Absorbance at 620 nm

Absorbance at 440nm

x
x
x
x
x
Carotenoid (µg/ml)

x

x

Absorbance at 62 nm
0

Carotenoid Absorbance at 440nm

x
x
x
x

Vitamin A µg/cuvette (sample)


THIAMIN DETERMINATION

pyrimidine
H3C

N
N

thiazole
NH 2
CH 2

S
N

CH 2CH 2OH
CH 3

K3 Fe(CN) 6
Oxidation

H3C

N
N

N

S

CH2CH 2OH

N
CH3
CH 2
THIOCHROME (Fluorescent)

Excite thiochrome at 365 nm and measure the absorbance at 435 nm


Thiamin Determination in Foods
 
5 g enriched flour
75 ml 0.1 N HCl
Make volume to 100 ml
Digest at 1000C for 30 min
Centrifuge and filter


Oxidation of Thiamine
Tube 1
5 ml Sample
2.5 g NaCl
3 ml K3FeCN6
13 ml Isobutanol
Centrifuge

Tube 2
5 ml Sample
2.5 g NaCl
3 ml NaOH
13 ml Isobutanol
Centrifuge

Tube 3
5 ml Standard (thiamin)
2.5 g NaCl
3 ml K3FeCN6
13 ml Isobutanol
Centrifuge

Tube 4
5 ml Standard (thiamin)
2.5 g NaCl
3 ml NaOH
13 ml Isobutanol
Centrifuge

 

 
Thiamin Standard Solution = 0.2 µg/ml
Calculation of B1 (µg of B1/5 ml assay sample) = (Tube 1 – Tube) / (Tube 3 - Tube 4 )


RIBOFLAVIN DETERMINATION

CH 2OH
HOCH
HOCH
HOCH
CH 3
CH 3

7
6

8
5

HCH
N
9

10

N

N
1
4

O
2
3

O

6,7 Dimethyl-9-D-1-Ribitylisoalloxazine


Fluorometric Method
Autoclave in 0.1 N HCl for 30 min
Adjust pH to isoelectric point to precipitate proteins
Filter
 Determination: Test Tubes
10 ml sample,

+ 1 ml of riboflavin standard or 1 ml H2O

+ 1 ml acetic acid, + 0.5 ml KMnO4 (4%)
+ 0.5 ml 3% H2O2 -- KMnO4 color should disappear in 10 seconds, + 20 mg
powdered Na2S2O4
Calculation
 mg Riboflavin/ml Final Sample Solution

[F(sample) - F(blank)] x 0.1 x 0.001
=
F(sample + standard) - F(sample)


NIACIN

O
C
N

O
OH

C
N

NH 2


Niacin Determination 
1. Digestion an Hydrolysis
Sample + Ca(OH) 2

Autoclave
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 OHREARRANGEMENT

-

N

REARRANGED
RING IS OPENED DERIVATIVE

N
Br CN

2
RERRANGED
DERIVATIVE

+ H2 N

O
S OH
O
O
C-OH
R-N-CH=CH-CH=C-CH=N

O
S OH
O


A at 470 nm

NIACINE STANDARD CURVE

µ g Niacin


VITAMIN C
Ascorbic Acid

Dehydroascorbic Acid

CH2OH
O
HOCH
O

-2 H

CH2OH
O
HOCH
O

+2H
HO

OH

O

HSCH2CH2(SH)CH2OH 2,3-DIMETHYLPROPANOL
Reducing agent or converting dehydroascorbic acid to ascorbic acid

O


Titrimetric 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).

 


Calculation for Reduced Vitamin C

F
V
x
mg Ascorbic Acid/g = (A-B) 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