Chapter 11
Vitamins Analysis
Introduction
1. What is vitamins?
VITAMIN： VITA (LIFE)-AMINE
2. Vitamins classification
Hydrosoluble vitamins: B, C,
Liposoluble Vitamins: A, D, E, K
3. Vitamins and health
Vitamins determination
The general techniques for determining vitamins can be
used for biological liquids and plant or animal tissues.
The general principles are the same, but the extraction
methods differ depending on the matter being
analyzed. For many decades, the determination of
vitamins has been based on the evolution of diverse
technologies from microbiological methods to more
sophisticated techniques like liquid or gaseous phase
chromatography coupled with specific detection
systems(ultraviolet or fluorimetric).
The real problem at present is no longer the methods of
determination; rather, it is extraction procedures. The
current change in legislation that permits vitamin
supplement food substances necessitates constant
analytical control, and underlines the importance of
the methods for determining these vitamins.
CH3 CH3 CH3
CH 2 CH 3 CH 3 H3C
CH 2OH
CH 3
CH2OH
CH3
CH 3
VA1 VA2
CH3
CH3 H3C
CH3 CH3
CH3 CH3 CH3
CH
CH3 3
β－胡萝卜素
R3
H3C
R3 R2 O CH3
H3C
R2 O CH3
CH3 CH3 CH3
HO
CH3 CH3 CH3
HO R1
R1 生育三烯酚
生育酚 H3C
H3C
CH3
H3C
CH3
H3C HO H3C
C H3 H3C
C H3 CH3
C H3 麦角固醇 H3C
CH3
C H3
HO
HO
7－脱氢胆固醇 维生素D3(胆钙化醇)
Global Extraction Tech for Liposoluble Vitamins
Weigh proper amount of the Add 10 ml of 33% KOH and
food substance and transfer to 40 ml ethanol
a 150 ml round flask
Cool very rapidly, then add 17
Boil at reflux for 30 mins ml of 25% HCl, and cool again
Add 50 ml of petroleum ether Wait to obtain complete
and shake vigorously for 3 separation of the two phases
mins
Evaporate to dry in a vacuum
Remove and filter the organic at a temperature of 35 degree
phase through anhydrous C
sodium phases
Redissolve the residue with a
know volume of hexane for
HPLC analysis
Extraction of Vitamin A
1. Weigh 5-10 g of the previously crushed food substance into a 1 L round
flask.
2. Add 20 ml of a 50% NaOH solution and warm the mixture in a water
bath.
3. Then, add 100 ml of diethyl alcohol and 2 ml of a hydroquinone
solution that was obtained by dissolving 20 g in 100 ml of pure alcohol.
4. Maintain the water bath at 90℃ for 30 minutes.
5. Pour the contents of the round flask into a decanting vial and add 100
ml of water.
6. Add 50 ml of ethylic ether and shake.
7. Add 50 ml of petroleum ether. Shake and allow it to decant.
8. Extract once or twice with 50 ml of petrol ether.
9. Wash the ether phase three times with 100 ml of water.
10. Filter, evaporate, and concentrate until 1 ml is obtained.
It must be noted that all of these steps are conducted away from light.
Moreover, saponification with NaOH is not useful with nonfatty products.
Extraction of Vitamin D2 or D3
1. Weigh between 5 and 10 g of the sample food
substance.
2. Add 1 g of pyropanol, 90 ml of a mixture of 60 ml
absolute ethanol, and 30 ml of a 50% potash solution.
3. Extract three times, each time with 50 ml of
petroleum ether.
4. Wash the ether extract and material three times
with water.
5. Filter, evaporate, and concentrate until 1 ml is
obtained.
Saponification with the alcoholic potash mixture is not necessary if the
products to be analyzed do not contain fats.
Extract of Vitamin E
1. Weigh between 5 and 10 g of the food substance that you c
rush.
2. Add 100 ml of ascorbic acid methanol solution obtained by
mixing 0.5 g of ascorbic acid, 4 ml of water, and 20 ml of
ethanol brought to 100 ml with methanol.
3. Keep in boiling water for 15-20 minutes.
Add 15 ml of a 70% KOH solution Place again in the wa
ter bath for 40 minutes.
4. Decant the contents of the flask into a separation flask vial,
washing the flask with 50 ml of water.
5. Add 120 ml of ethylic ether and stir the mixture. Decant an
d filter on Na2SO4.
6. Extract again with 120 ml of ethyl ether.
7. Filter, evaporate, and concentrate to 1 ml.
Saponification with potash is not necessary for nonfat pro
ducts.
Other Extraction Techniques
Other Extraction Techniques Specific to Ea
ch Liposoluble Vitamin and the Product Be
ing Analyzed
These techniques are detailed in the official a
nalysis methods proposed by the Official As
sociation of Analytical Chemists (OAAC).
Determination Methods
Numerous determination techniques can be proposed:
● determination by fluorimetry and by colorimetry;
● determination by liquid phase chromatography,
with ultraviolet or fluorimetric detection;
● determination by gas phase chromatography
(vitamin E);
● microbiological determination.
Determination of Vitamin A
After extraction, the determination is carried out on the solvent of
the liquid extraction.
Colorimetric Determination
Through this method, carotenoids and vitamin A are determined.
Determination of carotenoids.
Carotenoids are determined at 450 nm. After evaporating the
ether phase of the extracted solution, dissolved the extraction
with 1 ml of hexane. Determine the O.D. of this phase at 450
nm.
Determination of vitamin A.
The hexane phase obtained earlier is taken again and
concentrated in a vacuum. Redissolve the extract in a
chloroform. Then, to the volume of chloroform, add four
volumes of the trifluoroacetic acid reagent prepared by mixing
1 v of trifluoroacetic acid with three volumes of chloroform.
Then, observe the DO at 620 nm
Determination of Vitamins D
Determination of Vitamins D2,D3,and Their Metabolites
If the sample contains all the vitamian D metabolites, then
you can carry out a liquid chromatography under the
following conditions:
Column : fatty acid analysis column
Solvent : MeCN (acetonitrile), 55%
Mixture of water/acetic acid
(4 ml of acetic per liter) 45%
Flow rate : 1 ml/min
Wavelength : 265 nm
Solvent temperature : 25℃
Temperature of oven : 40℃
Determination of Vitamin E
A number of methods can be used to determine this vitamin.
1. Colorimetric Determination
After extraction and evaporation, re-dissolve the residue using n-heptan
e. Add 1 ml of dipyridil solution, then determine the absorbance at 460 n
m. Methods derived from this one have been recommended for use with f
erric chloride with a reading at 510 nm.
2. Determination by Liquid Chromatography
Using the extract prepared as described earlier, proceed with an HPLC d
etermination under the following conditions:
Column : Lichrosorb R P 8,25 cm ×, 4.6 mm, 5 µm
Solvent : methanol/water (92:8)
Flow rate : 1.5 ml/min
Wavelength : 288 nm
Solvent temperature : 25℃
Temperature of oven : 40℃
*For some foods, all three vitamins (A, D, E) can be determined, or only
vitamins A and E simultaneously.
Extraction of Hydrosoluble Vitamins
1. General principle is that the sample
must be crashed as finely as possible.
2. Enzymatic extraction is conducted w
ith amylolytic or proteolytic enzymes.
Tech of extracting the ensemble of
hydrosoluble vitamins
homogenize the sample ►► Weigh 5-10 g sample into a
after crushing it finely and 250 ml flask and
rapidly (if necessary) add 65 ml 0.1 M HCl
Heat at 100 deg C for 30 Cool and adjust to pH4.5
mins in a water bath ►►
with 2.5 M NaOAc
Add 50mg β-amylase and
Decant quantitatively into a
50 mg takadiastase, ►► 100 ml volumetric flask and
incubate at 37 oven for
adjust volume with water
overnight
►
►
►
Filter the supernatant and do further
treatment to the filtrate if necessary
Ascorbic Acid Extraction
1. In a breaker weigh to about 0.1 mg of a certain product quantity as
a function of the assumed vitamin C content of the sample food
substance.
2. Decant into a 50 ml volumetric flask using 0.4% metaphosphoric
acid.
3. Bring the volume to 50 ml with this solution.
*. For the analysis of a liquid, pippet the sample directly into the flask
and adjust the volume to 50 ml with the 0.4% metaphosphoric acid
solution.
4. Filter the solution an a cellulose acetate membrane (0.2 µg), then
pass the filtered substance through a SEP-PAK C18 cartridge
(supplied by Waters, a division of Millipore). Eliminate the first 2
ml, then collect 5 ml for analysis by RP-HPLC.
(Note: It must be pointed out that some authors may propose a
determination of ascorbic and dehydro-L-sacorbic acids for which
the extraction technique is different from the preceding one).
Determination of Water Soluble Vitamins
1. Determination of Vitamin B1 Thiamine
1.1 Fluorimetric Determination
After the action of potassium ferricyanide in the presence of potash, dete
rmine fluorescence using a 360-365 nm primary filter and a 460-480 nm se
condary filter.
1.2 Microbiological Determination
A number of lactobacilli can be used, such as Lactobacillus fermentum a
nd Lactobacillus viridiceus ATCC 1270 C, depending on the chemical met
hods of determination. The statistical calculation of activity is based on th
e six-point method.
1.3 Determination by Liquid Chromatography
Chromatographic conditions
Column : C 18, 25 cm × 4.6 mm, 5µm
Solvent : methanol
0.005 M sodium acetate
adjusted to 4.5 pH (30-70)
Flow rate : 1 ml/min
Detection : excitation: 366 nm
emission: 435 nm
* Principle of NP-C and RP-C
流动相: 正己 流动相: 乙氰/
烷 水
SiOH Si-O-Si-C 18
的极 非物
SiOH HO 物性 Si-O-Si - CH 2(CH 2)16CH 3 极质
质较 性
SiOH 大
固定相: 普通硅 H 流 动 相 :C18 改 性 CH 3
胶 H 3C CH 3 硅胶 的极
物性
HO 物弱 质较
质极 大
性
Normal phase chromatography Reverse phase chromatography
So much for this lesson
and see you next time !