AMINO ACIDS PTT 202: ORGANIC CHEMISTRY FOR BIOTECHNOLOGY PREPARED BY: NOR HELYA IMAN KAMALUDIN helya@unimap.edu.my

Introduction of Amino Acids Amino acids are organic molecules of low relative molecular mass (approximately 100-200) Contain at least one carboxyl (COOH) and one amino (NH2) group. Essential constituents of plant and animal tissues. There are several methods available for the quantitation of amino acids Mostly only give information about the total amino acid content of the sample regardless of whether one or several amino acids are present. Cannot differentiate between the individual components. However, for detection a particular amino acid in the presence of others , the most useful methods are employed This include chromatographic of electrophoretic technique for the separation of various amino acids in the sample. Calorimetric or fluorimetric methods for the qualitative and quantitative determination of each component.

Separation of Amino Acid Mixtures Techniques/ Methods Thin-layer chromatography Electrophoresis Gas-liquid chromatography Amino acid analyzer

Paper and thin-layer chromatography Paper chromatography has been used successfully for many years and is still a useful tool due to: Larger volumes of sample can be applied to paper, permitting the subsequent elution of a particular amino acid for further purification and analysis. This is importance in the identification of an unknown sample constituent. Prior to chromatography, it may be necessary to remove interfering substances such as protein, carbohydrates and salts. This may be done using an ion-exchange resin, solvent extraction, dialysis or protein precipitation.

Paper and thin-layer chromatography The identification of an amino acid is achieved by comparison of RF values with those of reference solutions. The use of at least three different solvent systems is recommended. The nature of the amino acids is an important factor in the choice of a solvent and different solvent will permit better resolution of acidic, basic and neutral components (Table 1). In general, increasing the proportion of water in the solvent will increase all RF values and the introduction of small amounts of ammonia will increase the RF values of the basic amino acids.

Solvents for both paper and thin-layer chromatography Table 1: Solvents for both paper and thin-layer chromatography

Paper and Thin-Layer Chromatography The resolving power of paper or thin-layer chromatography can be increased by the use of two-dimensional techniques, which involve the use of two different solvent systems. Two-dimensional separations permit the resolution of large numbers of amino acids present in a sample and those having a similar mobility in one dimension will usually be separated from each other in the second. This is especially useful in the detection of components that are present only in low concentrations and might be obscured in one dimension by other amino acids that are present in higher concentrations.

Electrophoresis The fact that different amino acids carry different net charges at any particular pH permits mixture to be separated using low or high voltage electrophoresis. Separations at high voltages can be achieved more quickly than at low voltages. One of the principal advantages of the former is that salts and other substances that may be present in the sample affect the quality of electrophoretogram to a less extent. This permits the separation of amino acids in relatively crude extracts and untreated fluids.

Electrophoresis Although electrophoretic separations can be achieved using buffers over a wide range of pH values, in practice the pH values chosen are either pH 2.0 or pH 5.3. At pH 2.0 all amino acids will carry a positive charge and the basic amino acids, having the highest positive charge, will migrate furthest toward the cathode. At pH 5.3 migration will occur towards both electrodes depending on the charge carried. Separations at ph 5.3 are particularly useful to determine the acidic or basic nature of an unknown amino acid or dipeptode. For separating similar amino acids and short peptides A two-dimensional technique involving initial separation by high voltage electrophoresis at pH 2.0 followed by chromatography is a useful means. Does not require desalting or excessive purification of the sample.

Gas-Liquid Chromatography Derivatives of amino acids are required because Amino acids are not themselves sufficiently volatile for gas-liquid chromatography Difficulties may be encountered in the choice and method of derivatization. Several techniques involves for derivatization are as follows: The derivatization of trimethylsilyl (TMS) derivatives is carried out by the addition of N,O-bis(trimetylsilyl)trifluoroacetamide (BSTFA) in acetinitrile and heating for aprrox. 2 h at 150˚C under anhydrous conditions in a sealed tube. The formation of n-butyl esters of the amino acids and their subsequent trimethylsilylation. The n-butyl esters are formed by heating the amino acids for 15 min in n-butanol and HCl and these are then converted to the N-TMS-n-butyl ester derivatives. Acetylation of the butyl, metyl or propyl esters of amino acids, to give trifluorocetyl (TFA) or heptafluorobutryl (HFB) derivatives can be performed by reacting them with either TFA or HFB anhydrides in methylene dichloride at 150˚C for 15 min.

Amino Acid Analyser Spackman, Stein and Moore developed the first amino acid analyser based on separation by ion-exchange chromatography and quantitation of each component in the column effluent using the ninhydrin reaction. The instruments currently available are based on their original design, permitting buffers of varying pH or ionic strength to be pumped through a thermostatically controlled resin column (Figure 1).

Figure 1: Schematic diagram of an amino acid analyser using the ninhydrin reagent for quantitation.

Principles of separation of amino acid analyser The separation take place in a column of sulphonated cross-linked polystyrene resin, which is a strong cationic exchanger. The matrix of the resin is strongly anionic in nature (SO3-) and at low pH used initially, the amino acids will be positively charged and will be attracted to the negatively charged sulphonate groups. As the pH of the buffer passing through the column is raised, the amino acids will be differentially eluted as their net positive charge diminishes and they are less strongly attracted to the sulphonate sites on the resin matrix.

Principles of separation of amino acid analyser At pH 3.25, the amino acids with an extra acidic group in their side chain will have very little affinity for the resin and will be the first to emerge from the column. At the same pH value, those amino acids whose side chain contains an extra ionizable group capable of carrying a positive charge, will be strongly held on the resin and will be eluted from the column only as the pH is raised substantially and their net positive charge reduced.

Principles of separation of amino acid analyser The determination of the relative elution position of the amino acids, not only depends on the pH of the eluting buffer but also the cation concentration of the buffer. Sodium citrate buffer solutions are commonly used and the positive sodium ions compete with the positively charged amino acids for the sulphonic acid on the resin.

Principles of separation of amino acid analyser Although the amino acids have a considerable affinity for the resin, the sodium ions are constantly present in a much higher concentration, and as a result, the equilibrium of the equation is shifted to the right and the amino acids are displaced from the resin. Thus, the molarity of the eluting buffer affects elution and when the ionic concentration of the buffer is increased, the amino acids are eluted more rapidly from the column.

Principles of separation of amino acid analyser The quantitative resolution of amino acid mixtures can be achieved by varying the composition of the buffer flowing through the column by either: Increasing the pH and maintaining a constant molarity (cationic concentration). Keeping the pH constant but varying the molarity. Using combination of both of the above.

Quantitation The colour or fluorescence produced per mole of amino acids varies slightly for different amino acids and this must be determined for each one to be quantitated. This is done by loading a mixture of amino acids containing the same concentration of each amino acids including the chosen internal standard and from the areas of the peaks on the recorder trace calculating each response factor . An internal standard should always be used for every analysis carried out. This is an amino acid that is known to be absent from the sample under investigation. If the amount of internal standard which was added to the sample is known, the concentration of the unknown amino acid can be determined using peak area relationships.

Figure 2: Amino acid analyser trace Quantitation Figure 2: Amino acid analyser trace

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