Amino acids thin-layer chromatography separation

Exercises in thin-layer chromatography. Separation of amino acids. Prepare solutions of DL-alanine, L-leucine and L-lysine hydrochloride by dissolving 5 mg of each separately in 0.33 ml of distilled water, measured with a graduated 1 ml pipette (leucine may require warming to effect solution). Mix one drop of each solution to provide a mixture of the three amino acids and dilute the remainder of each solution to 1 ml to give solutions of the respective amino acids. The latter will contain about 5 pg of each amino acid per pi. Apply approximately 0.5 pi of each of the solutions to a Silica Gel G plate and allow to dry in the air (i.e. until the spots are no longer visible). 

The 2-D TLC was successfully applied to the separation of amino acids as early as the beginning of thin-layer chromatography. Separation efficiency is, by far, best with chloroform-methanol-17% ammonium hydroxide (40 40 20, v/v), n-butanol-glacial acetic acid-water (80 20 20, v/v) in combination with phenol-water (75 25, g/g). A novel 2-D TLC method has been elaborated and found suitable for the chromatographic identification of 52 amino acids. This method is based on three 2-D TLC developments on cellulose (CMN 300 50 p) using the same solvent system 1 for the first dimension and three different systems (11-IV) of suitable properties for the second dimension. System 1 n-butanol-acetone -diethylamine-water (10 10 2 5, v/v) system 11 2-propanol-formic acid-water (40 2 10, v/v) system 111 iec-butanol-methyl ethyl ketone-dicyclohexylamine-water (10 10 2 5, v/v) and system IV phenol-water (75 25, g/g) (h- 7.5 mg Na-cyanide) with 3% ammonia. With this technique, all amino acids can be differentiated and characterized by their fixed positions and also by some color reactions. Moreover, the relative merits of cellulose and silica gel are discussed in relation to separation efficiency, reproducibility, and detection sensitivity. Two-dimensional TLC separation of a performic acid oxidized mixture of 20 protein amino acids plus p-alanine and y-amino-n-butyric acid was performed in the first direction with chloroform-methanol-ammonia (17%) (40 40 20, v/v) and in the second direction with phenol-water (75 25, g/g). Detection was performed via ninhydrin reagent spray. 

EXPERIMENT 31 THIN-LAYER CHROMATOGRAPHY SEPARATION OF AMINO ACIDS... 

D.W. Armstrong and M. McNeely,fAe of Micelles in the Thin Layer Chromatography Separation of Polynuclear Aromatic Compouruk and Amino-Acids, Anal. Lett., 12 1285 (1979). 

De Vault, GL. and Sepaniak, M.J., Two-dimensional capillary eleclrophoresis-thin layer chromatography separations of amino acid enantiomers using electrofilament transfer, J. Micro. Sep., 12 419, 2000. 

Thin-Layer Chromatography (tic). Tic (126) is used widely for quahtative analysis and micro-quantity separation of amino acid mixtures. The amino acids detected are developed by ninhydrin coloring, except for proline and hydroxyproline. Isatia has been recommended for specific coloring of pToline (127). 

Pereira, A., Pereira, M, M., Free amino acids in green coffee from Huambo (Angola). Separation and identification by electrophoresis and thin layer chromatography, Coll. Sc 1. Int. Cafe, 8, 545, 1977. (CA92 196460s)... 

Procedure 10.1 Preparation of dansyl derivatives of amino acids for sub e quest separation by thin layer chromatography... 

Reagents used for the visualisation of amino acids on the dried chromatogram may be applied either by spraying or dipping. Those commonly used produce intensely coloured bands with approximately 20 nmol of each amino acid for paper chromatography and 5 nmol for thin-layer separations, although smaller amounts can be detected. 

It has been demonstrated that ILMs are suitable for qualitative and quantitative analyses of low-molecular weight compounds of biological interest, for example, carbohydrates, vitamins and amino acids [38], and glycolipids [40]. ILMs were further used for fhe direct analysis of alkaloids, anesthetics and antibiotics, separated by thin-layer chromatography (TLC) [46]. For this purpose, the ILM was spotted onto the fractions on the TLC-plates and the complete plate was measured in MALDI MS without the need for additional pretreatment of the TLC-samples. The mass deviation inherently caused by the inhomogeneous surface of fhe TLC-plafe was balanced by using the... 

Thin-layer chromatography of amino acid-phenylthiohydantoin derivatives on silica gel plates, (a) Separation is done in a 98 2 mixture of chloroform and ethanol. (b) This is followed by further separation using an 88 2 10 mixture of chloroform, ethanol, and methanol. More sophisticated procedures, using column chromatography, give superior resolution and improved sensitivity. Automated sequencers always use such procedures. A general description of the use of columns is given in chapter 6. 

How can you determine which amino acids are present in a particular protein This involves hydrolysis of the peptide (amide) bonds in the protein so that the individual amino acids are released. This can only be done by heating the protein with dilute hydrochloric acid. The mixture of amino acids is then separated by thin layer chromatography (TLC) (Figure 15.24) or electrophoresis. In both cases, a locating agent (Chapter 2, p. 23), such as ninhydrin, is used. This ensures that the spots of amino acid are visible. 

Following adjustment to pH 6.0, the solution is applied to a SP-Sephadex C-25 column in the sodium form. Amino acids are then eluted with 0.2 M citrate phosphate buffer, pH 8.0, and the effluent evaporated to dryness at 50 °C. The residue is dissolved in 0.1 N hydrochloric acid and applied to the amino acid analyser. Amino acids are separated by passing 0.2 M, pH 8 sodium citrate solution down the column. The S-methylmethionine content can then be obtained from the chromatogram, as illustrated in Fig. 8.1. The results obtained agree reasonably well with those obtained by thin-layer chromatography [13]. 

Paper Chromatography. This more primitive version of thin-layer chromatography (see Fig. 10-24) separated compounds based on their chemical properties, allowing identification of single amino acids and, in some cases, dipeptides. Thin-layer chromatography also separates larger peptides. 

The amino acids in a protein hydrolysate can be conveniently separated for qualitative analysis by paper or thin-layer chromatography or by elec-... 

Furfural (18) was oxidized to give the butenolide 19, which on Michael condensation with diethyl ethylmalonate afforded lactone 20. Hydrolysis of 20 yielded the dilactone 21. This was treated with ethanol in the presence of sulfuric acid. The isomer mixture obtained was separated by preparative thin-layer chromatography, yielding 4-ethoxy-3-ethoxycarbonylmethyl-2-ethyl-4-butanolide (22). Elimination of ethanol, with the aid of p-amino-benzenesulfonic acid, gave 2-(3//)-furanone 23 the 2-(5//)-furanone isomer 24 was obtained with the aid of orthophosphoric acid. Both isomeric esters gave the acid 25 after hydrolysis, which on catalytic hydrogenation afforded m-3-carboxymethyl-2-ethyl-4-butanolide 26, identical to homopilopic acid. This synthesis of homopilopic acid differs from earlier syntheses because the less stable cw-2,3-disubstituted butanolide (26) is formed in the last step. 

Thin-layer chromatography is very widely used, mainly for qualitative purposes almost any mixture can be at least partially resolved. Inorganic applications, such as the separation of metals in alloys, soil and geological samples, and polar organic systems, such as mixtures of amino acids or... 

Chitin has been used as the stationary phase to separate mixtures of phenols, amino acids, nucleic acid derivatives, and inorganic ions by thin layer chromatography. Also chitin has been used to prepare affinity chromatography columns to isolate lectins and determine their structure. 

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