Amino Acids From A Protein By Paper Chromatography Biology Essay

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Chromatography is a large range of methods used to separate or analyze (or both) complex mixtures. The components that are to be separated are split between two stages, a stationary phase and a mobile phase.

The mobile phase may be a gas, liquid or solid, and the stationary phase may be solid, liquid or gel. These two phases must be immiscible with each other, and the components of the mixture must not react with either phase.

The mobile phase, which is usually a solvent, carries the substance to be analysed through the stationary phase, which is usually a liquid supported on an inert solid. This process is called elution.

To monitor the separated components, a detection system is used.

A component is attracted to both mobile and stationary phases, and the relative strength of attraction for each phase is important. A component which is strongly attracted to the stationary phase will be held back, and a component which is strongly attracted to the mobile phase will pass through the stationary phase and move further along the inert solid. Separation is achieved when components have different relative attraction for each of the two phases.

Different types of chromatography

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There are four types of chromatography:

Thin layer

Paper

Gas-layer

High-performance liquid

Chromatography

Diagram

Mobile phase

Stationary phase

Thin-layer

Liquid Solvent

Aluminium oxide (Al₂O₃) or Silicon oxide (SiO₂) often bonded together with Calcium Sulphate (CaSO₄) and supported on a plastic sheet or glass plate, called a tic plate.

Paper

Liquid Solvent

Water (Hâ‚‚O) on the surface of absorbent chromatography paper and therefore, it is supported by the cellulose fibres in the paper.

Gas-layer

Inert gas (usually nitrogen or argon)

Liquid, often a silicone gum, supported on suitable solid particles and contained in a glass or metal tube

High-performance liquid

Liquid Solvent

Small solid particles packed tightly in a tube, usually stainless steel

The role of silica/aluminia

different absorption of the components

Examples of uses of chromatography in industry

Over 150 years ago, chromatography was used in the dyeing industry. Workers would test a dyes quality by placing a drop onto absorbent cloth or paper.

These days, chromatography is an important tool for analysis which is used in industry and medicine. Is used to detect, separate or purify different substances. Food, blood, soil, water, drugs, fuel and petroleum and air are all analysed using chromatography. For example in medicine, chromatography can determine the presence of drugs in a person's blood.

Chromatography is also used to synthesize new products, as it can separate pure substances from complete mixtures very precisely. The techniques are not damaging, and so the most delicate of products can be treated.

Chromatography is used in the semiconductor industry as it is capable of determining contaminates on the surface of a water, die or package. Ionic contamination is a major source of corrosion issues in the industry, and ion chromatography is considered to be a crucial tool when analysing water samples suspected of causing corrosion problems.

Chromatography is also used in the petroleum industry to separate and purify products and to make petroleum jelly. It is also used in the pharmaceutical industry.

Rf values

Rf values are the distance travelled by the sample divided by distance travelled by the solvent in a chromatography experiment.

For example, if a compound travels 3.5 cm and the solvent front travels 3.8 cm, the Rf is 0.92

The expression for calculating Rf values is

Distance moved by amino acid

Rf value =

Distance moved by solvent

The Rf for a compound is a constant from one experiment to the next only if 5 important conditions for the chromatography procedure remain the same. These are:

adsorbent

thickness of the adsorbent

amount of material spotted

temperature

Method

We put spots of 0.01M amino acids in aqueous solution 1.5cm from the bottom edge of the chromatography paper cut to the correct dimensions. To do this, we dipped a clean capillary tube in the stock solution and applied a small drop to the chromatography paper.

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We applied 3 drops of each amino acid, one on top of the other to make the drops concentrated. The drops were then labelled A, B, C and D.

A fresh solvent mixture of butan-1-ol (12cm3), pure ethanoic acid (3cm3), and water (6cm3) was produced in a covered 1dm3 beaker to produce a saturated atmosphere.

We then put the chromatography paper into the covered solvent beaker, secured it with sellotape and left it until the solvent had risen to nearly the top of the paper.

Once this had happened, we removed the chromatography paper from the beaker and marked the solvent level.

The amino acids were detected by spraying the paper sparingly with 0.02M ninhydrin solution in a fume cupboard, and then heating in an oven at 110°c for 10 minutes. Purple spots then appeared at the positions occupied by the amino acids.

These spots were preserved by spraying with a mixture made up of methanol (19cm3), 1M aqueous copper (II) nitrate (1cm3), and 2M nitric acid (a drop) and then were exposed in a fume cupboard to the fumes from 8M ammonia. We were then able to determine the Rf values for each of the amino acids.

Results Table

Conclusion

Evaluation

Risk assessment

Chemical/

Apparatus/

Procedure

Hazard

Risk (what could go wrong?)

Emergency

action

Safety

precautions

Disposal

Storage

Risk

Ammonia solution

Corrosive - causes severe burns, turns skin yellow on contact. Skin will peel off in due cause. Vapour is dangerous to eyes and respiratory system.

Swallowed

Wash out mouth with water. Seek medical attention.

Eye protection

Cover skin

Gloves

Dust mask

Dilute with water to less than 0.5moldm-3 and wash down drain.

In glass containers in small quantities. Keep container on tray to hold spillages.

High

Spilt on skin or clothes

Flood affected area with large quantities of water. Remove contaminated clothing and rinse thoroughly. If large area affected or blistering occurs, seek medical attention.

Solution or vapour gets into eyes

Flood eyes with gently running tap water for over 10 minutes. Seek medical attention. Take steps to ventilate the area.

Spilt in laboratory

Open all windows and doors. Consider evacuation if people are affected. Wear eye protection, gloves and rubber boots if spill is large.

Butan-1-ol

Flammable.

Harmful if swallowed. Irritating to respiratory system and skin. Absorbed by the skin.

Vapours may cause drowsiness and dizziness.

Swallowed

Wash out mouth with water. Seek medical attention.

Wear eye protection

Mixtures should be prepared before experiments in a fume cupboard by a technician or teacher wearing eye protection.

Dilute to less than 10% (w/v), before pouring down a foul-water drain.

Used solvent mixtures should be added to a bucket of water in a fume cupboard before washing to waste.

Vapours inhaled

Spilt on skin

Flood affected area with large amounts of water. If large area affected or blistering occurs, seek medical attention.

Copper nitrate

Harmful.

Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.

Swallowed

Wash out mouth with water and seek medical attention

Eye protection

Cover skin

Wear gloves

Dilute to less than 0.4 mol dm-3 or dissolve 90g in 1litre of water. This disposal procedure should be kept to a minimum

The substance absorbs water from the atmosphere

Spilt into eyes

Flood eyes with gently running tap water for over 10 minutes. Seek medical attention. Take steps to ventilate the area

Spilt on skin

Flood the area with large amounts of water. If large area affected or blistering occurs, seek medical attention

Ethanoic acid

Corrosive - causes severe burns.

Irritant when weaker than 4 mol dm-3

Flammable

Dangerous with Chromium (VI) Oxide, Magnates (VII), Nitric(V)acid, peroxides. Violent or explosive reactions may occur

Spilt on skin

Flood the large area with large amounts of cold water for at least 10minutes. Seek medical attention

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Goggles

Do not inhale

Cover skin

Wear gloves

Use a fume cupboard, dilute to less than 0.1mol dm-3 and pour the solution down a foul-water drain

On cold days, the liquid solidifies. The melting point is 17°c

Swallowed

Wash out mouth with water. Seek medical attention

Gets into eyes

Flood eyes with gently running tap water for over 10 minutes. Seek medical attention. Take steps to ventilate the area.

Vapour inhaled

Methanol

Highly flammable

Toxic by inhalation, in contact with skin and if swallowed. Danger of very serious irreversible effects.

Vapour inhaled

Eye protection

Keep skin covered

Wear gloves

Dilute to a 1% (v/v) solution before pouring it down a foul-water drain

Bottles used regularly in the laboratory should not be more than 500cm3 in capacity

Spilt on skin

Flood affected area with large quantities of water. If large area affected or blistering occurs, seek medical attention

Swallowed

Wash out mouth with water. Seek medical attention.

Ninhydrin

Flammable

Harmful if swallowed

Irritating to respiratory system and skin. Risk of serious damage to eyes. Vapours may cause drowsiness and dizziness.

Contact with skin produces a violet stain which can persist for several days.

Swallowed

Wash out mouth with water. Seek medial attention

Wear eye protection

Use a fume cupboard

Wear disposable nitrile gloves

Windows must be open near the oven during the heating process.

Dissolve no more than 100g in1litre of water before pouring down a foul-water drain

Vapours inhaled

Split on skin

Flood affected area with water. If large area affected or blistering occurs, seek medical attention

Spilt into eyes

Flood the eye with gently running tap water for over 10 minutes. Seek medical attention

Nitric Acid

Oxidising - contact with combustible materials may cause fire.

Corrosive - causes severe burns. Solutions weaker than 0.1mol dm-3 should be labelled irritant.

Spilt on skin - Skin turns yellow on contact and peel off in due course.

If the splashed area is treated correctly with water, peeling will happen gradually over several days. If the contamination is not treated quickly, peeling may be severe and painful. Seek medical attention.

Wear eye protection

Keep skin covered

Wear goggles

Wear chemical resistant gloves

Add no more than 50cm3 of concentrated nitric(V) acid slowly to 1litre of 1mol dm-3 sodium carbonate. Do not attempt to dispose of large volumes of fuming nitric(V) acid by this method.

Bottles should not be stored in direct sunlight; dark glass bottles or opaque containers are best. Light decomposes the colourless acid. If there is a brown colouration, decomposition has occurred and the liquid should be disposed of.

Spilt in laboratory

Keep others away. Wear eye protection and chemical resistant gloves (and rubber boots if spill is large). Ventilate the area of the spill as much as possible. Cover with mineral absorbent and clear up into a bucket. Wash the area of the spill thoroughly.