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Paper chromatography of amino acids 1. Take a sheet of chromatographic paper (18 x 18 cm). Find out a centre of the sheet. Draw

a circle around the centre about 3 cm in diameter. You can use prepared template. This is

the "base-line", the start position. On the base-line make 6 marks evenly spaced and

number them 1-6.

Chromatogram

2. Using labelled capillaries make small spots (less than 0.5 cm in diameter) of about 5 μl of

the standards and your unknown sample. The application should be repeated 2 times.

Before the further application, the wet spots must be dried under the infra-red lamp or

a ventilator.

3. Use your pencil to perforate the paper exactly in the centre to draw through the hole

a paper "wick". Place the paper into a Petri dish partly filled with a solvent mixture

(n-butanol, acetic acid, water – 4:1:1) and cover the paper with a cap. Do not take the

Petri dish out from the fume chamber! All following procedures must be done in a fume

chamber!

Petri dish with solvent chromatogram paper wick

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4. The chromatograms are left to develop until the head of a solvent overlaps the Petri dish

cap. The paper is removed, dried, and sprayed with ninhydrine reagent. Heat the paper at

80°C in order to develop the formation of blue complexes.

5. Calculate the individual Rf according to the equation.

Site Amino acid Rf

1 Lysine

2 Glycine

3 Alanine

4 Isoleucine

5 The mixture of 1-4 - - -

6 Unknown sample

Sample identification:

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Separation of plant pigments by thin-layer chromatography

There are a few lipophilic pigments in green parts of plants. They have different chemical and

functional properties. The primary function of pigments in plants is photosynthesis which uses

the green pigment chlorophyll along with several red and yellow pigments. These pigments

can be separated using Thin Layer Chromatography (TLC).

Procedure:

1. There are prepared samples of green leaves or needles on the working place. Cut it in

pieces in a mortar. Then add a small spoon of calcium carbonate, a little bit of glass

shards and crush it with a pestle in the presence of 1-2 ml of acetone. Protect your

eyes using glasses.

2. Take two TLC plates. After the separation, one is for you to be attached to the

protocol, the second will be used further and destroyed by procedure of extraction of

one of the pigments.

3. Draw the base-line about 2 cm from the shorter edge of the plate. Use the thin pencil.

4. Dip the glass capillary into the acetone extract and touch the tip of the capillary to the

plate at the position you’ve marked. Gently and quickly make spot according to the

picture. Try to make as small spots as possible and avoid scratching of the thin layer.

For higher concentration repeat the process of application for several times.

5. Prepared separation chamber should be kept close. There is approximately 1cm thick

layer of liquid solvent mixture on the bottom and the rest volume is filled with its

vapors.

6. Place the thin layer plate carefully in the chamber (chromatography tank), containing a

solvent at the bottom. Cover the vessel with a glass plate and allow to separate about

15 min. The base – line must be situated above the level of a solvent.

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7. When the front of a solvent almost overlaps the top of the TLC plate, remove the

plate. Evaluate the dry plate for the presence of pigments and enclose to laboratory

protocol.

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Measurement of absorption spectrum of one of the pigments After separation of the plant pigments, take one of the TLC plates and select one of the green strips. Use the scissors to cut out the strip from the TLC plate. Put the piece with the strip into a test tube and add 2 ml of ethanol. Wait until complete discoloration of TLC plate piece.

Measure absorption spectrum of this fraction using a spectrophotometer in a range of wavelengths from 350 to 700 nm. As a blank solution, use distilled water. By comparing the spectrum with spectra of individual pigments, determine whether the fraction contains chlorophyll a or chlorophyll b.

wavelength nm

A wavelength

nm A

wavelength nm

A

350 470 590

360 480 600

370 490 610

380 500 620

390 510 630

400 520 640

410 530 650

420 540 660

430 550 670

440 560 680

450 570 690

460 580 700

Name of the pigment

0

0.05

0.1

0.15

0.2

0.25

350 400 450 500 550 600 650 700 nm

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Separation of dye mixture by gel chromatography

Task: Separate dextran blue and potassium chromate.

Dextran blue

What are dextrans?

Colour Molar mass

~ 2 × 106 g/mol

Potassium chromate

formula:

Colour Molar mass

g/mol

There is a chromatographic column filled with Sephadex gel on the working place. Sephadex is polysaccharide of dextran type.

Procedure:

(1) Uncap the chromatographic column, open the tap and allow carefully run out the excess of fluid until the surface of the gel bed is reached. Pipette on the gel slowly 0.5 ml of the coloured mixture (potassium chromate and dextran blue). Use the pipette with fixed volume (=500 µl) and proper tip (blue). Pipette carefully! Not to whirl the surface of the gel!

Colour of the mixture:

(2) By turning the tap let the sample pass into the column.

(3) Pipette (using glass pipette and rubber suction bulb) about 3 ml of elution solution (physiologic solution; 0.9% NaCl) and let it penetrate the gel in the same way.

(4) Elute the column with sufficient amount of elution solution until both fractions leave the outflow.

(5) Collect both fractions in calibration test tubes.

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What is happening in the columne? Describe the changes:

Fraction order Colour Substance Fraction volume

1

2

Explain the order of separated dyes:

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Vitamin C - demonstration of reducing properties

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-Carotene – extraction and estimation

Extraction procedure:

(1) For disintegrating the tissue put 1g of grated fresh carrot into a mortar and grind it

carefully using a pestle. Add 20 ml of 96% ethanol and mix the content. The

suspension is then quantitatively transferred on a cellulose filter, and washed with

acetone collected in a low-lying vessel until the sediment turns completely colorless.

(2) The acetone filtrate is then poured into a separatory funnel. Make sure that the

lower stopcock of the funnel is closed. Add 40 ml of petroleum ether (benzine) and in

addition water to fill the funnel to the ¾ of the total volume. Gently swirl the funnel

for about a minute to allow carotene entering the upper petroleum ether (p.e.) layer.

Place the funnel in a ring stand, remove the tap, and wait until the two layers are

fully separated. Open both top and bottom taps and release the lower water phase.

This liquid is to be discarded.

(3) Add 20ml of 20% ethanol to the funnel and adjust the volume again to ¾ of the total

with water. Gently swirl the funnel again and repeat the washing 2-3 more times,

until the ethanol-water phase remains colorless. Transfer the p.e. extract to a bottle

equipped with a stopper, add a spoonful of waterless sodium sulfate and shake the

content for 20 s.

(4) Clarify the extract by filtration into a 50 ml volumetric flask. Combine with the filter

and salt p.e. wash before filling the flask with p.e. up to the mark. Mix, and put an

aliquott to the photometric cuvette. Read the optical density (absorbance) at

450 477 nm. The beta-carotene content (mg/kg of carrot) is determined from the

calibration graph. The beta-carotene purity is assessed by the difference between

absorbance values at 450 and 477 nm. For a pure beta-carotene, the proportions of

the two values ranges between 1.16-1.18.

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Note: The separatory funnel runs on the concept of "like dissolves like". This means that if a compound is polar, then it can only be dissolved in a polar solvent. Following this logic, a non-polar compound can only be dissolved in a non-polar solvent. In a separatory funnel, there are two phases: the aqueous layer is polar, and the organic layer is non-polar. Because these two layers share a surface, any polar substances that were initially in the solution will be pulled into the polar/aqueous layer, and any non-polar substances will be pulled into the non-polar/organic layer. The mixtures to be separated are added through the top with the stopcock at the bottom closed. The funnel is then closed and shaken gently by inverting the funnel multiple times, temporarily releasing the upper tap to reduce the vapor pressure. Following the shaking, the separating funnel is set aside to allow for the complete separation of the phases. The denser of the two layers is then removed via the stopcock. Biological value of carotenoids and vitamin A:

Carotenoids are yellow, orange, or red tetraterpenes (40C). Carotenoids are found in plants and are known as provitamin A, as they can be cleaved to yield retinaldehyde and hence retinol and retinoic acid, collectively called vitamin A. The alpha-, beta- and gamma-carotenes are the most important provitamin A carotenoids. These carotenoids are cleaved in the intestinal mucosa by carotene dioxygenase, yielding retinaldehyde, which is reduced to retinol. The intestinal activity of carotene dioxygenase is low, so that a relatively large proportion of ingested beta-carotene may appear in the circulation unchanged and beta-carotene is only one-sixth as effective a source of vitamin A as retinol. However, beta-carotene, unlike vitamin A, is an antioxidant due to its conjugated double bonds.

Vitamin A plays an important role in vision. In retina, retinaldehyde functions as prosthetic group of the light-sensitive opsin proteins, forming rhodopsin in rods. In vitamin A deficiency, both the time taken to adapt to darkness and the ability to see in poor light are impaired. A more prolonged deficiency leads to xerophthalmia: keratinization of the cornea and skin and blindness. Vitamin A is toxic in excess, affecting the central nervous system, the liver, the bones and the skin.

Retinoic acid regulates growth, development, and tissue differentiation. Like the steroid hormones regulate the transcription of specific genes.