IDENTIFICATIONS OF VITAMINS AND HORMONES

1. Identification of cholesterol by Salkowski’s reaction

Add 1 ml of cholesterol dissolved in chloroform. Next add carefully 1 ml concentrated H 2SO4. In the
presence of cholesterol observe green fluorescence of acid layer under UV lamp and red color in chloroform
layer.

2. Identification of vitamin A
a) Add to glass tube about 1 ml of chloroform solution of vitamin A. Next add 1 ml concentrated H 2SO4. In
the presence of vitamin A observe formation of blue color.
b) Add to glass tube about 1 ml of chloroform solution of vitamin A. Next add 1 ml Carr-Price reagent . In
the presence of vitamin A observe formation of blue or violet-red color.

3. Identification of vitamin D3 and cholesterol

Add to the two separate glass tubes chloroform solution of vitamin D 3 and cholesterol. Next add to
both probes 0,5 ml anhydride acetic acid and 1 drop concentrated H 2SO4. In the presence of cholesterol and
vitamin D3 observe the formation of red color passing into green-blue.

4. Identification of vitamin D3
Add to glass tube about 2 ml of chloroform solution of vitamin D3. Next add 1 ml of aniline and 2-3
drops of concentrated H2SO4. Observe the colors of both layers. Intense red color of the lower layer indicates
the presence of vitamin D3 in the sample.

5. Identification of vitamin C
Add to glass tube about 1 ml of aquatic solution of vitamin C. Next add 3 drops of 0,1% methylene
blue. Thanks reducing properties of vitamin C blue color of dye decolorizes (blue → white). As a result of
shaking technique blue color of dye is backing by oxidation with oxygen from the air.

6. Oxidation of vitamin C by K4Fe(CN)6

Add to glass tube about 1 ml of aquatic solution of vitamin C. Next add the drops of 2M NaOH and
10% K4Fe(CN)6. Mix. Next add a few drops of 2M HCl and 1% FeCl3.
In an alkaline medium vitamin C reduces the potassium hexacyanoferrate (III) to potassium
hexacyanoferrate (II). This compound may be detected by the addition of FeCl3 in acidic (HCl) medium by
formation of dye Berlin blue.

7. Examination of reducing properties of vitamin C (iodine clock reaction)

During the experiment, follow these steps in order:
1. add to beaker (with capacity 100 ml) 0,5 ml of I2 in KI (iodine),
2. Add appropriate amounts of vitamin C (by drops) until discoloration of solution (about 2 ml),
3. Add 1 ml 1% starch solution
4. Add 10 ml water,
5. Add 15 ml 3% H2O2.
6. Mix obtained solution without color and observe changes. At first there is no visible reaction.
After a short time delay, the liquid suddenly turns to a shade of dark blue.
It is very important to add all reagents in the presented above order. Explain this reaction.

8. Reaction of vitamin C with I2

Prepare two glass tubes. To both add a few drops of 1% starch and iodine solution. To the first tube
add aquatic solution of vitamin C, and to second lemon juice by drops. Note the number of drops which you
need to discoloration of iodine solution. Next prepare new 2 glass tubes with some milliliters of Vitamin C
and lemon juice and heat in water bath during 5 minutes. Repeat reactions with iodine using boiled vitamin
C and lemon juice and note also the number of drops used to discoloration of iodine.

9. Determination of vitamin B1
Add 1 ml solution of vitamin B 1 to glass tube. Add 3 ml 2M NaOH and 2-3 drops 10% K 4Fe(CN)6.
Next add isobutyl alcohol and mix. In the presence of vitamin B 1 observe the fluorescence of alcohol layer
under UV.

10. Determination of vitamin B2

Add 1 ml solution of vitamin B 1 to glass tube. Add a few drops of 2M NaOH. Mix. Keep the mixture
under UV lamp during 1 minute. Next add some drops of 5% CH 3COOH and mix with an equal amount of
chloroform. Observe green-yellow fluorescence of chloroform layer.

11. Determination of auxin (IAA)

Prepare reagent: Mix in the tube 1 ml of sulphanilic acid and sodium nitrite. Shake the tube with
reagent for a few minutes while cooling it under running tap water.
Reaction: Add to glass tube 1 ml of auxin solution and 1 ml of prepared reagent. Mix. Add by drops
15% Na2CO3 to obtain red color.

12. Determination of insulin

Add to glass tube 1 ml of insulin. Add 4-5 pastilles of NaOH and 2 ml 5% lead acetate. Heat in water
bath during 2-3 minutes. Observe the formation of brown color in the presence of hormone.

DETERMINATION OF CONCENTRATION OF VITAMIN C

a) Weigh about 0,1 g of vitamin C. Dissolve in 25 ml of water. Add 5 ml 5 % H 2SO4 and 1 ml 2 % starch
solution. Titrate the probe using 0,1N iodine solution.

1 ml iodine used to titrate the probe respond to 8,806 mg vitamin C

b) Filter 5 ml of juice (orange, or apple) using paper filter. Make up to the 25 ml by water. Titrate following
point a (next steps are the same as point a).