BIOCHEMISTRY - Laboratory Practical - NAU 2015

MODULE I

BIOCHEMICAL COMPONENTS OF THE CELL

Laboratory Work 5

IDENTIFICATION OF VITAMINS IN MODEL SOLUTIONS

Objective: to master the Methods for identifying Water- and Fat-soluble Vitamins.

Basic Theoretical Background

Vitamins are low-molecular-weight Organic compounds. They must be supplied to the Organism in small quantities yet play a vital role in METABOLISM. Water-Soluble Vitamins do not accumulate in the body, whereas fat-soluble Vitamins can be stored, typically in the Liver.

Water-soluble vitamins form Coenzymes that function alongside Enzymes and are essential for specific biochemical reactions. For instance, vitamin B₁ (thiamine) forms the coenzyme Thiamine diphosphate (thiamine pyrophosphate), which participates in The oxidative decarboxylation of α-keto acids. Vitamins B₂ (riboflavin) and B₅ or PP (nicotinamide) form the coenzymes of dehydrogenases: FAD (flavin adenine dinucleotide phosphate) and FMN (flavin mononucleotide) are based on riboflavin (its Structure is built upon an isoalloxazine ring), while NAD(P) (nicotinamide adenine dinucleotide (phosphate)) is based on nicotinamide (its structure contains a pyridine ring). Vitamin B₆ (pyridoxal and pyridoxamine) serves as the basis for the coenzymes PLP (Pyridoxal phosphate) and PMP (pyridoxamine phosphate), which are involved in transferase reactions. Its structure is also based on pyridine. Vitamin C exists in oxidized and reduced forms (ascorbic acid and dehydroascorbic acid), participates in redox reactions, and can be synthesized from
L-sorbose, which is a ketohexose. All water-soluble vitamins are sometimes collectively referred to as B-complex vitamins.

Fat-soluble vitamins do not form coenzymes. Their Functions are highly diverse. They influence organism GROWTH AND REPRODUCTION, tissue differentiation, skeletal development, ensure the integrity of Introduction/36.html">Biological Membranes and Cell Division capacity, protect the organism against hemorrhages, participate in visual information Processing, and so forth. Fat-soluble vitamins include vitamins A, D, E, K, and F (a complex of higher polyunsaturated Fatty acids).

Equipment: test tube rack, dropper bottles, pipettes, spatula, incubator, water bath.

5.1. Colorimetric Tests for Vitamin P (Rutin, Permeability Vitamin, Citrin)

Materials and Reagents: rutin (powder and saturated aqueous solution), 1% iron(III) chloride solution, concentrated sulfuric acid, 0.5% Hydrochloric acid solution, 10% sodium hydroxide solution, Fehling's reagent (see Laboratory Work 2).

Procedure

Reaction of Rutin with Iron(III) Chloride

Add a few drops of FeCl₃ solution to 2 ml of a saturated aqueous solution of rutin. Iron(III) chloride reacts with rutin to form emerald-green coordination complexes.

Observe the appearance of a green coloration.

Reaction of Rutin with Concentrated Sulfuric Acid

Carefully add 1 ml of concentrated sulfuric acid down the side of the test tube to 2 ml of a saturated aqueous solution of rutin. Concentrated sulfuric acid reacts with flavones and flavonols, which include vitamin P, to yield flavylium salts whose solutions exhibit a bright yellow color.

A yellow ring forms at the liquid-liquid interface.

Reaction of Rutin with Fehling's Reagent

Add 5 ml of hydrochloric acid solution to 0.5 g of rutin, boil for 1 min, and then filter. Acid Hydrolysis of rutin cleaves the rutinose molecule. Subsequently, rutinose breaks down into glucose and rhamnose, which possess reducing properties and can therefore be identified using Tollen's, Trommer's, or Fehling's tests. Add 3 ml of sodium hydroxide solution and 3 ml of freshly prepared Fehling's reagent to 5 ml of the filtrate, and reheat to boiling.

Observe The formation of a red copper(I) oxide precipitate.

5.2. Colorimetric Tests for Vitamin C

Materials and Reagents: 2,6-dichlorophenolindophenol solution (0.1%), 10% hydrochloric acid solution, 0.1% iodine solution in potassium iodide, 0.01% methylene blue solution, 10% Na₂CO₃ solution, 1% K₃[Fe(CN)₆] solution, 1% FeCl₃ solution, 0.1% ascorbic acid solution, rosehip extract, distilled water.

Procedure

Reduction of 2,6-dichlorophenolindophenol by ascorbic acid

Add 0.5 ml of 2,6-dichlorophenolindophenol solution, 1–2 drops of HCl solution, and drops of ascorbic acid solution or rosehip extract to each of three test tubes (add 0.5 ml of distilled water to the control tube). Ascorbic acid reduces 2,6-dichlorophenolindophenol, converting it into a colorless leuko compound.

The 2,6-dichlorophenolindophenol solution decolorizes.

Reduction of methylene blue by ascorbic acid

Add one drop each of methylene blue solution and sodium carbonate solution to three test tubes. Add 5 drops of ascorbic acid solution to the first tube, rosehip extract to the second, and an equal amount of distilled water to the third. Heat all test tubes simultaneously at 37–40 °C. Ascorbic acid is capable of reducing methylene blue, converting it into a colorless leuko compound.

The liquid in the test tubes containing ascorbic acid and rosehip extract decolorizes.

Reduction of potassium hexacyanoferrate(II) by ascorbic acid

Add one drop each of potassium hexacyanoferrate(III) and iron(III) chloride solutions to three test tubes. To the resulting green-brown liquid, add 5–10 drops of ascorbic acid solution in the first tube, 5–10 drops of rosehip extract In the second, and 5–10 drops of distilled water in the third (control) tube. Ascorbic acid reduces potassium hexacyanoferrate(III) to potassium hexacyanoferrate(II), which reacts with iron(III) chloride to form Prussian blue, a blue-colored compound.

The liquid in the First and Second test tubes turns a greenish-blue color, and a blue precipitate of Prussian blue settles out. Upon careful layering of distilled water, the precipitate at the bottom of the tube becomes more distinct. In the control tube, the green-brown liquid shows no change in color.

Reduction of molecular iodine by ascorbic acid

Add 10 drops of distilled water and 1–2 drops of iodine solution in potassium iodide to three test tubes. Add 10 drops of ascorbic acid to the first tube, 10 drops of rosehip extract to the second, and 10 drops of distilled water to the third (control) tube. Ascorbic acid reduces molecular iodine, converting it into colorless hydriodic acid.

Decolorization of the iodine solution is observed in the test tubes with ascorbic acid and rosehip extract.

5.3. Reaction for thiamine (vitamin B₁) with diazo reagent

Materials and reagents: sulfanilic acid solution (1%), sodium nitrate solution (5%), sodium bicarbonate solution (10%), thiamine (powder or its 5% solution).

Procedure

Pour 1 ml of sulfanilic acid solution and
1 ml of sodium nitrate solution into a test tube (forming the diazo reagent). Then add a small amount (on the tip of a spatula) of thiamine powder or 0.5 ml of thiamine solution to the tube, and carefully layer
1 ml of Na₂CО₃ solution down the wall of the tube. In an alkaline medium, vitamin B₁ reacts with the diazo reagent to form a complex orange or red coordination compound.

The formation of a colored ring is observed at the interface of the two liquids.

5.4. Reduction reactions of riboflavin (vitamin B₂)

Materials and reagents: concentrated hydrochloric acid, zinc metal, 0.025% vitamin B₂ solution (riboflavin suspension in water).

Procedure

Pour 1 ml of vitamin B₂ solution, 0.5 ml of concentrated hydrochloric acid, and a small piece of zinc metal into a test tube. When zinc metal mixes with concentrated hydrochloric acid, hydrogen is generated, which reduces yellow riboflavin first to rhodoflavin (an intermediate red compound) and then to colorless leukoflavin.

The liquid in the test tube gradually turns pink and then decolorizes. Upon shaking, the decolorized leukoflavin solution is reoxidized back to riboflavin by atmospheric oxygen.

Observe the color change.

5.5. Reaction for Vitamin PP (antipellagra factor, B₅) with copper acetate

Materials and reagents: vitamin PP powder, 10% acetic acid solution, 5% copper acetate solution.

Procedure

Place 5–10 mg of vitamin PP into a test tube and dissolve it in 1–2 ml of acetic acid solution upon heating. Add an equal volume of copper acetate solution to the boiling-hot mixture. Upon heating vitamin PP with the copper acetate solution, a poorly soluble blue precipitate of the copper salt of vitamin PP is formed.

The liquid becomes turbid and acquires a blue tint. After a short time, a blue precipitate appears.

5.6. Reaction with iron chloride for pyridoxine (vitamin B₆)

Materials and reagents: aqueous solution (1%) of vitamin B₆,
1% FeCl₃ solution.

Procedure

Mix 1 ml of the aqueous pyridoxine solution and two drops of iron chloride solution in a test tube. When pyridoxine interacts with the iron chloride solution, the liquid turns red due to the formation of a complex iron phenolate-type salt.

Observe the coloration of the liquid.

5.7. Qualitative reactions for fat-soluble vitamins

Qualitative reactions for vitamin A

Materials and reagents: chloroform solution of antimony trichloride (33%); chloroform; 0.05% oil solution of vitamin A in chloroform or fish oil solution in chloroform at a 1:5 ratio; concentrated sulfuric acid saturated with iron(II) sulfate; glacial acetic acid.

Procedure

Reaction with antimony trichloride

To one or two drops of the vitamin A solution in chloroform or fish oil solution in chloroform in a dry test tube, add four to five drops of antimony trichloride solution in chloroform and mix. The chloroform solution of vitamin A or fish oil turns a specific blue color upon The addition of antimony trichloride.

Observe the coloration of the liquid.

Drummond's reaction

Add two to three drops of concentrated sulfuric acid to two to three drops of vitamin A solution in a test tube. Vitamin A forms a blue-colored complex with concentrated sulfuric acid in a benzene solution.

The Contents of the test tube turn blue, which after some time changes to violet and brown.

Observe the color change.

Reaction with iron(II) sulfate

To two to three drops of fish oil solution in chloroform or an oil solution of vitamin A in chloroform in a test tube, add five to ten drops of concentrated sulfuric acid saturated with iron(II) sulfate and one to two drops of glacial acetic acid. Vitamin A reacts with iron(II) sulfate in an acidic medium within a chloroform solution to form a red-pink compound.

Observe the appearance of a blue color, which gradually transforms into red-pink.

Carotenes exhibit a green color in this reaction.

Qualitative reactions for vitamin D

Materials and Reagents: saturated SbCl₅ solution, chloroform solution of vitamin D, chloroform, aniline reagent — aniline with concentrated HCl (15:1), bromine solution in chloroform (1:60).

Procedure

Reaction with Antimony(V) Chloride

Add 0.2 mL of saturated SbCl₅ solution to 2 mL of a chloroform solution of vitamin D in a dry test tube. Vitamin D in a chloroform solution forms a yellow color upon reacting with a saturated solution of antimony(V) chloride.

Observe The Development of color.

Reaction with Aniline

Place one to two drops of fish oil or a chloroform solution of vitamin D into a dry test tube and add one drop of the aniline reagent. Mixing produces a yellow emulsion. Upon heating the emulsion with a mixture of aniline and concentrated hydrochloric acid, the solution turns red.

After 1–2 minutes, the emulsion separates into two layers. The lower layer exhibits an intense red coloration.

Reaction with Bromine

Add four to five drops of a bromine solution in chloroform to a test tube containing two to four drops of a chloroform solution of vitamin D or fish oil.

Observe the mixture in the test tube gradually turning green-blue.

Color Reactions for Vitamin K

Materials and Reagents: diethyl malonate solution (1%), KOH solution (1%), alcoholic solution of vitamin K, diethyldithiocarbamate solution (5%), alcoholic NaOH solution (4%), aniline solution, menadione sodium bisulfite (vikasol) alcoholic solution (0.05%).

Procedure

Reaction with Diethyl Malonate

Add 0.5 mL of diethyl malonate solution and 0.1 mL of KOH solution to 2 mL of an alcoholic solution of vitamin K in a test tube. In an alkaline medium, the alcoholic solution of vitamin K yields a red-violet color with diethyl malonate.

Observe the appearance of a red-violet color.

Reaction with Diethyldithiocarbamate

Add 2 mL of diethyldithiocarbamate solution and 0.5 mL of ethanolic NaOH solution to 2 mL of an alcoholic solution of vitamin K in a test tube. In an alkaline medium, the alcoholic solution of vitamin K reacts with diethyldithiocarbamate to form a blue-colored compound.

Observe the development of color.

Reaction with Aniline

Add two drops of aniline to 1 mL of a vikasol alcoholic solution in a test tube. In the presence of aniline, the alcoholic solution of vitamin K turns red due to the formation of 1-methyl-2-phenylaminonaphthoquinone.

Mix and observe the appearance of color.

Color Reactions for Vitamin E

Materials and Reagents: alcoholic solution (0.1%) of α-tocopherol, concentrated nitric acid, iron(III) chloride solution (1%).

Procedure

Reaction with nitric acid

Place five drops of an alcoholic solution of vitamin E into a dry test tube, add 1 ml of concentrated HNО₃, and mix thoroughly. The interaction of α-tocopherol with concentrated nitric acid turns the reaction mixture red. This occurs because the oxidation product of α-tocopherol features a quinoid structure.

A gradual appearance of a red color is observed.

Reaction with ferric chloride

Place 0.5 ml of an alcoholic solution of
α-tocopherol into a dry test tube, then add 0.5 ml of a FeCl₃ solution and mix thoroughly. Upon interaction with iron(III) chloride, α-tocopherol is oxidized to α-tocopherylquinone, which is a red compound:

The appearance of coloration is observed.

Processing of experimental data

Construct a table detailing: the water-soluble vitamins you know; their corresponding coenzymes; reactions involving these coenzymes; identification reactions for these vitamins; dietary sources of the vitamins; and the recommended daily allowance.

Construct a table specifying fat-soluble vitamins, their biological role, dietary sources, and the recommended daily allowance.

Selection/41.html">Review Questions and tasks

1. What is the Biological Role of water-soluble vitamins? Write the chemical formulas for vitamins В₁, В₂, В₅, В₆, and С.

2. Write the reaction of phenol and vitamin В₆ with FeCl₃. Demonstrate the formation of three covalent and three coordinate bonds. Indicate the phenolic hydroxyl groups in the rutin molecule.

3. What property of ascorbic acid is the basis for its detection?

4. Draw the isoalloxazine ring, which forms the core of riboflavin, in its oxidized and reduced forms. Write the coenzymes of vitamins В₅ and В₂ in their oxidized and reduced forms.

5. What fat-soluble vitamins do you know? What is The Role of vitamins A, D, E, K, and F in metabolism?

6. What substance serves as a precursor for the synthesis of vitamin D in The Human Body? Which hormone is synthesized from vitamin D?

7. Identify the quinone moiety within The structure of vitamin E.

8. Explain the principles underlying the identification reactions for fat-soluble vitamins. Can these reactions be used for quantitative determination?

References: [1; 2; 4 —7].