Leucine
One of three branched-chain amino acids that enhance energy, increase endurance, and aid in muscle tissue recovery and repair. This group also lowers elevated blood sugar levels and increases growth hormone production. Supplemental valine should always be combined with isoleucine and valine at a respective milligram ratio of 2:1:2.
Lysine
Lysine is one of the 20 amino acids normally found in proteins. With its 4-aminobutyl side-chain, it is classified as a basic amino acid, along with arginine and histidine. It is an essential amino acid, and the human nutritional requirement is 1–1.5 g daily. A deficiency in lysine can result in a deficiency in niacin this can cause the disease pellagra. Lysine can also be used as a nutritional supplement to help against herpes.
Equipment
- Safety spectacles and gloves
- Measuring cylinder
- Beaker
- Watch glass
- Ethanol
- Wash bottle of distilled water
- Ammonia solution
- Square of chromatography paper
- Pencil and ruler
- Sheet of the paper
- 4 melting – point tubes
- Aspartic acid
- Leucine solution
- Lysine solution
- Mixture of the three amino acids above
- 2 retort stand, bosses and clamps
- Hair dryer
- Ninhydrin aerosol spray
- Oven
Method
- In the fume cupboard the solvent mixture was poured into a 400cm3 beaker.
- The mixture solution was swirl to mix, and the beaker was covered with a watch glass and left to stand.
- Making sure by handing the square of chromatography paper by the top edge and place it on a clean sheet of paper and draw the lines and labels shown on the diagram below
- By using a clean melting point tube was dip into the solution of mixed amino acids and then touched briefly on the paper
- Using a fresh tubes each time step 4 was repeat with the three other solution of single amino acids
- There was a clean ruler placed with its edge along one of the dashed lines and it was held firmly in place with one hand. Without it being touched by fingers, the chromatography paper was folded by sliding a hand under the file paper and lifting.
- The following procedure was repeated for the other two lines so that the opposite edges of the paper almost met to form a square cross-section.
- The paper was held by the edge furthest from the start line, and was placed in a beaker so that it does not touch the sides. The covers were placed and it was left to stand.
- The ruler was clamped horizontally at a height of 20-30cm between two retort stands in a fume-cupboard. This supported the chromatography paper for the drying when the run had finished.
- When the solvent nearly reached the top of the paper (30-40 minutes) the paper was removed from the beaker> It was opened out and was clipped on the ruler to dry.
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When the paper was dry, it was sprayed evenly with ninhdrin solution. It was dried again and then it was heated in a oven at 105oC for 5 minutes.
- The paper was removed from the oven, and then it was marked with a pencil of the positions of the coloured spots.
- The distances from the origin line to the centres of the spots were measured and were recorded in a table.
Interpreting the Data
The Rf value for each spot should be calculated. Rf stands for "ratio of fronts" and is characteristic for any given compound. Hence, known Rf values can be compared to those of unknown substances to aid in their identifications.
(Note: Rf values often depend on the temperature, solvent, and type of paper used in the experiment; the most effective way to identify a compound is to spot known substances next to unknown substances on the same chromatogram.)
In addition, the purity of a sample may be estimated from the chromatogram. An impure sample will often develop as two or more spots.
Purity of the sample
After the chromatography experiment, if a sample develops as only one spot it may or may not be pure. The sample may contain another compound which did not separate under the conditions of the experiment. Purity of samples is often determined in conjunction with other techniques, such as measuring a sample's melting point or recording its nuclear magnetic resonance spectrum
Risk assessment
In the experiment I made the solvent mixture with ethanol I must be aware that it is highly flammable and when being handle to keep it away from a naked flame also it is harmful to the skin and eyes so you should wear safety spectacles and gloves, also in the solvent in the mixture there will ammonia I must be aware that It is very toxic and if inhaled it could be fatal this is why the solvent must be made in a fume cupboard it is also corrosive so safety spectacles and gloves had to been worn
In the experiment I used melting – point tubes to put the spot of amino acids on the chromatography paper I must be aware not to press down to hard on the melting – point tubes as there might break and cut my fingers
Coming to the end of the experiment ninhydrin sprays will be used to see the amino acids I must be aware that it gives off toxic fumes and if inhaled it could be fatal that is why a fume cupboard used when using this spray also it is harmful to the skin and eyes Safety spectacles and gloves had to been worn.
Ninhydrin Spray
Flammable harmful if swollen irritating to respiteratory system and skin. When I did the experiment in the class I use Ninhydrin Spray to spray the chromatography paper in the fume cupboard because to respiteratory system
Observation
When we did the experiment with chromatography paper. All the amino acids solution were colourless. It took two days for the solvent to reached the top of the chromatography paper, when the chromatography paper was dried we could not see the spots so we sprayed Ninhydrin Spray over the chromatography paper and the put it in the oven so we can see the spots clearly. By doing I was able to measure the Rf value.
Calculation
Conclusion
As you can see for my result Aspartic Acid - alone was 1.44 this due to poor solubility this also suggest it is less polar
Evaluation
As I was carry out my experiment ii found out that certain things which thought would be easy to do was not for example the ninhydrin sprays was use in a fume cupboards but I need to have a box in the fume cupboard in the box I will place the chromatography paper inside the box then I will used the ninhydrin sprays in the box inside the fume cupboard this is due to health and safety reason so the ninhydrin sprays dose not defused out of the fume cupboard.
If I do the experiment again I would use a method of chromatography may be Thin-Layer Chromatography which is Using a absorbent material on flat glass plates. This is a simple and rapid method to check the purity of the organic compound also the is Gas Chromatography which is Used to analyze volatile gases. Helium is used to move the gaseous mixture through a column of absorbent material and Liquid Chromatography which is used to analyze metal ions and organic compounds in solutions. It uses liquids which may incorporate hydrophilic, insoluble molecules
Column Chromatography
Column chromatography uses a mobile phase to move a mixture of substances through a stationary phase. The different components of the sample have different affinities for the mobile and stationary phases, and emerge from the stationary phase at different times. The stationary phase and mobile phase are chosen based on the nature of the sample mixture in order to achieve the best possible separation of its components.
The stationary phase is either silica (SiO2) or alumina (Al2O3), which is mixed with the solvent being used as the mobile phase to yield a thick white slurry. The mobile phase is a liquid that is chosen to maximize the separation of the sample. This can be water or any organic solvent.
The stationary phase is pre-loaded into the column above a plug of glass wool (to prevent solid material from contaminating products) and a thin layer of sand (to provide a uniform bed for the stationary phase).
Careless addition of sample can disturb the stationary phase and lead to poor separation. For this reason, a second bed of sand is added above the column as a “shock absorber.” Nevertheless, you must be very careful when adding sample or mobile phase to the top of the column. Most importantly, no part of the stationary phase must ever be dry! Air bubbles trapped in the stationary phase can severely impair your separation. To avert disaster, always keep the stationary phase covered with the mobile phase!
Because the silica or alumina gel that makes up the stationary phase is quite dense, column chromatography tends proceed very slowly if gravity is the only force pulling the mobile phase through the gel. The process can be sped up if high gas pressure at the top of the column or a vacuum at the bottom of the column is used to push or pull the mobile phase more quickly. This method is called flash column chromatography. In your food dye experiment, you will have the option of performing flash column chromatography by using a syringe attached to the bottom of the column to provide vacuum suction and thereby quicken elution.
A final note is necessary about the versatility of column chromatography. While most organic chemistry laboratories restrict themselves to the usual silica or alumina stationary phase, this is not the case in biochemical applications. Biochemists have been incredibly creative in adapting the column technique for separating macromolecules. For example, by coating the stationary phase with anionic groups, it is possible to selectively adsorb positively charged sample molecules to the column. Or, in an even more advanced application, a stationary phase of cellulose coated with antibodies against a particular molecule can be used to isolate that molecule from a cell extract. There are few separations that column chromatography can't perform!
