THIN-LAYER CHROMATOGRAPHY OF AMINO ACIDS: A REVIEW PDF

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This article was downloaded by: [Universiti Sains Malaysia] On: 09 December 2013, At: 19:37 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Liquid Chromatography & Related Technologies Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljlc20

THIN-LAYER CHROMATOGRAPHY OF AMINO ACIDS: A REVIEW a

a

b

S. A. Bhawani , M. N. Mohamad Ibrahim , O. Sulaiman , R. b

c

Hashim , A. Mohammad & S. Hena

b

a

School of Chemical Sciences, University Sains Malaysia , Pulau Pinang , Malaysia b

School of Industrial Technology, University Sains Malaysia , Pulau Pinang , Malaysia c

Analytical Research Laboratory, Department of Applied Chemistry, Faculty of Engineering and Technology , Aligarh Muslim University , Aligarh , India Accepted author version posted online: 23 Mar 2012.Published online: 22 Jun 2012.

To cite this article: S. A. Bhawani , M. N. Mohamad Ibrahim , O. Sulaiman , R. Hashim , A. Mohammad & S. Hena (2012) THIN-LAYER CHROMATOGRAPHY OF AMINO ACIDS: A REVIEW, Journal of Liquid Chromatography & Related Technologies, 35:11, 1497-1516 To link to this article: http://dx.doi.org/10.1080/10826076.2011.619039

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Journal of Liquid Chromatography & Related Technologies, 35:1497–1516, 2012 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6076 print/1520-572X online DOI: 10.1080/10826076.2011.619039

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THIN-LAYER CHROMATOGRAPHY OF AMINO ACIDS: A REVIEW

S. A. Bhawani,1 M. N. Mohamad Ibrahim,1 O. Sulaiman,2 R. Hashim,2 A. Mohammad,3 and S. Hena2 1 School of Chemical Sciences, University Sains Malaysia, Pulau Pinang, Malaysia 2 School of Industrial Technology, University Sains Malaysia, Pulau Pinang, Malaysia 3 Analytical Research Laboratory, Department of Applied Chemistry, Faculty of Engineering and Technology, Aligarh Muslim University, Aligarh, India

& Thin-layer chromatography is a versatile separation technique and is compatible to almost all types of compounds available in nature. This review presents numerous chromatographic systems developed for the analysis of amino acids. The stationary phases, solvent systems, and detection reagents used by the various chromatographers are incorporated in this review. From the available literature, it is obvious that amino acids have been greatly analyzed by thin-layer chromatography. Keywords amino acids, analysis, detection, quantification, separation, thin-layer chromatography

INTRODUCTION Amino acids are generally considered the building blocks of proteins. Amino acids also serve as an energy source, especially in times of starvation. Proline is one of the amino acid used as an energy reserve in blood-feeding insects and in certain blood-feeding protozoan parasites. In the case of plants, these compounds are essential for the biosynthesis of numerous endogenous nitrogenous compounds. Amino acids are used in the manufacturing of many industrial products such as in food technology as a flavor enhancer, as well as in production of biodegradable plastics, drugs, and chiral catalysts. The determination of amino acids from biological samples is very important because it highlights some anomalies correlated to various metabolic illnesses. Several chromatographic methods have been developed to meet the challenges of environmental mutations. Because of the simplicity of the technique for the analysis of various useful and unwanted materials in the Address correspondence to S. A. Bhawani, School of Chemical Sciences, University Sains Malaysia, 11800, Pulau Pinang, Malaysia. E-mail: [email protected]

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environmental samples, thin-layer chromatography (TLC) has a privileged position, especially in the field of biochemical analysis. These analyses provide solutions for some questions in the diagnostic therapy and, on the other hand, detect some biochemical modifications of diseases at the cellular level. Thin-layer chromatography is a fast, inexpensive, and versatile separation technique with many practical considerations that contribute to its effectiveness. R. Bhushan and G.P. Reddy[1] provided an extensive review on thin-layer chromatography of dansyl and dinitrophenyl derivatives of amino acids. Later, in 1991, R. Bhushan[2] published one more review on thin-layer chromatography of amino acids and their derivatives. Jain[3] reviewed the analysis of amino acids in biological fluids and tissues by TLC. R. Bhushan and J. Martens[4] contributed a book chapter on the amino acids and their derivatives and also W. Gołkiewicz and B. Polak[5] provided a chapter on chiral separation of amino acid enantiomers. This review presents the contribution of thin-layer chromatography (1995–July 2011) for the analysis of amino acids. Almost all the aspects, including stationary phases, solvent systems, and detection reagents, are incorporated in this review. SAMPLE PREPARATION (EXTRACTION/ISOLATION) The isolation of amino acids can be carried out by different extraction methods from dry materials. The following solvents have been previously utilized for the extraction of amino acids: 5% NaCl solution, 75% ethyl alcohol, 0.25% NaOH, 0.25 M HCl, metasiliconic acid or a CH3COOH
HCl
H2O (18:1:1 v:v:v) mixture. Hodisan et al.[6] extracted 0.5 g dry plant in 10 mL 1% HCl solution. The proteins were then removed from the extract by precipitation with a Na3P(W3O10)4 solution. After centrifugation, the solution was passed through an ion exchange Amberlite IR 120H column. The column was eluted with 40 mL 10% ammonia solution. The solution obtained was evaporated to dryness and the residue was redissolved in 1 mL aqueous 30% iso-propanol (v:v). Males et al.[7] have extracted amino acids from air-dried, powdered flowering tops of Hypericum taxa (1.0 g) by heating under refluxing with water (10.0 mL) for 60 min and then filtered. The filtrate was concentrated under reduced pressure and the residue was dissolved in water (5.0 mL). THIN-LAYER CHROMATOGRAPHY OF AMINO ACIDS Major portion of the review is presented in the form of Table 1. Our main concern was to make things easily accessible to readers and researchers. The data presented in this review indicates that amino acids have been

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Tin(IV) selenoarsenate layers, silica-tin(IV) selenium arsenate (1:1) layers and silica-calcium sulfate layers Zeolite-ellulose (1:1)

Silica

cellulose-silica (5:2)

Amino acids

L-tryptophan

Amino acids

Amino acids

Silica

Stationary Phase

Amino acids

Analyte

Mobile Phase

Isopropanol-ethyl acetateacetone-methanolisopentanol-NH3-water (9:3:3:1:1:3:3) and butanol-acetoneisopropanol-formic acid-water (18:8:8:3:6)

2-propanol-NH3 (25%) (7:3)

Water-saturated phenolethanol-water-acetic acid (12:4:4:1)

Dimethyl sulfoxide

1-butanol-acetone-acetic acid-water (7:7:2:4)

TABLE 1 Thin-Layer Chromatographic Analysis of Amino Acids Remarks

[12]

[11]

[11]

[9]

[8]

Ref.

(Continued )

Investigation of the precision, detection limit and limit of quantification for each method was carried out on laboratory prepared plates. Detection by spraying with 1% ninhydrin solution in i-butanol and heating at 80 C for 20 min. Determination by visible reflectance spectrometry, image analysis and densitometry. Quantitative analysis of L-tryptophan was performed by densitometry at 625 or 276 nm. Tryptophan was detected by spraying or dipping with=into 4-(dimethylamino)benzaldehyde solution. Study of 17 amino acids in Bos grunniens linnaeus horn hydrolysate was performed by Two-dimensional TLC. Detection by spraying with 0.5% ninhydrin in acetone.

Analysis of the resulting third-order data using a direct trilinear decomposition (TLD). Monitoring the kinetic profile of the reaction with a charge-coupled device camera by taking sequential images of the separation medium after the reaction starts. Detection by dipping into a solution of 0.05% o-phthalaldehyde in methanol, containing 0.2% 2-mercaptoethanol and 0.09% Brij-35, and under UV 366 nm. Thin-layer chromatography of a -amino acids was studied on different layers. Detection by spraying with ninhydrin.

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Silica gel

Lysine, homoserine, threonine, and tryptophan

Silicic impregnated glass fiber sheets

Silica gel impregnated with (1 R,3 R, 5 R)-2-azabicyclo[3,3,0]

Proline and hydroxyproline

Enantiomers of amino acids and dansyl derivatives

Thirteen DABS-amino acids

Silica impregnated with erythromycin as the chiral selector Silica

Cu(II)-impregnated silica

Amino acids

Ten dansyl-DL-amino acids

Silica

Stationary Phase

Amino acids (enantiomers)

Analyte

TABLE 1 Continued

0.5 M NaCl-acetonitrile in different ratios and addition of methanol in some cases

0.5 M aqueous NaCl-cetonitrile-methanol in varying proportions Eluents containing increasing concentrations of ethyl acetate in heptane and chloroform by multiple gradient development Isopropanol-water (7:3)

Butanol-acetate buffer (0.3 M pH 6.0)-acetonitrile (10:12:5) 1-propanol-25% NH3 (11:9), 2-propanol-acetone-water25% NH3 (25:25:7:6), 2-propanol-ethyl acetate-25% NH3-water (40:40:3:50) and 2-propanol-25% NH3 (7:3)

Acetonitrile-water (2:5) containing 2-o-[(R)2-hydroxypropyl]-CD as additive

Mobile Phase

Remarks

Separation of proline and hydroxyproline from biological samples. Detection by spraying with 0.25% solution of ninhydrin in 95% ethanol and heating at 105 C for 5 min. Also, separation of radioactive labeled amino acids. (1 R,3 R, 5 R)-2-azabicyclo[3,3,0]octan-3-carboxylic acid was used as impregnating reagent for the resolution of enantiomers of amino acids and

Separation of enantiomers of six selected amino acids using 2-o-[(R)-2-hydroxypropyl]- -CD as the mobile phase additive. Detection by spraying with a solution of 1.5 g salicylaldehyde in 100 mL toluene and heating at 50 C for 10 min. Comparative study of results obtained by HPLC and TLC. Detection by spraying with 0.2% ninhydrin in acetone and heating at 60 C for 30 min. TLC separation of lysine, homoserine, threonine, and tryptophan from accompanying amino acids in culture liquids. Detection of tryptophan after drying for 15 min and heating at 120 C for 15 min, dipping into a solution of 0.5% 4-DMABA in ethanol containing 5% conc. sulfuric acid, and heating for 5–7 min at 110 C. Quantification was done by densitometry. Macrocyclic antibiotic (erythromycin) was used as a chiral selector for the separation of enantiomeric dansylamino acids. Detection under 254 nm. Multiple gradient development was used for the separation of DABS derivatives of amino acids. Detection by densitometry at 485 nm.

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[19]

[18]

[17]

[16]

[15]

[14]

[13]

Ref.

1501

Cellulose

Silica gel plates impregnated with vancomycin (0.34 mM)

Silica gel

Dansyl-DL-amino acids

L-tryptophan

Silica gel

Amino acids

Amino acids

Silica gel

Amino acids

octan-3-carboxylic acid

2-propanol-25% NH3 (7:3)

Acetonitrile-0.5 M aq. NaCl (5:2) and (14:3)

1,Chloroform-methanol-NH3 conc. (2:2:1) 2, phenol-water (3:1) n-propanol-water (7:3)

for dansyl-DL-amino acids and different combinations of acetonitrile-methanolwater for other amino acids Ethanol-water (4:1), (8:1), (2:1) and propanol-acetic acid-water (3:1:1), (6:2:1), (1:1:1).

[24]

[23]

[22]

[21]

[20]

(Continued )

dansyl derivatives. Dansyl-DL-amino acids were visualized under UV 254 nm. Other amino acids were detected by spraying with 0.2% ninhydrin in acetone. Correlation analysis was conducted and fifteen parameters are discussed, including topological indexes and physicochemical properties; three of which were selected with both topological and physicochemical significance for construction of multi-parameter regression equations for prediction of the Rf values of the amino acids. Densitometry was carried out at 460 nm after visualization (with Ninhydrin) and heating for 1.5–2 min at 105 C. Identification of tryptophan and histidine by FT-SERS. Detection under UV 254 nm. Detection limit was 8 mg. Determination of amino acids in snail-conditioned water from Biomphalaria glabrata, two strains of Helisoma trivolvis, and Lymnaea elodes. Detection with ninhydrin reagent (0.3 g in 100 mL of n-butanol and 3 mL of acetic acid). Densitometry was done at 610 nm. Direct racemic resolution of dansyl-DL-amino acids. Detected with a fixed dual-wavelength UV light at 254 nm. Detection limit was 2.1 mg. Determination of L-tryptophan in fermentation broth. Visualization of amino acids by dipping into a solution of 300 mg ninhydrin in acetone= acetic acid 97:3. Detection of L-tryptophan by treatment with 4-dimethylaminobenzaldehyde and heating at 110 C for 7–10 min. Quantitation by densitometry at 625 nm.

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1502

RP-18 impregnated with a proline derivative and copper(II) ions Silica gel with pre-adsorbent sample application zone and on cellulose

9 amino acids (Ala, Phe, Val, Leu, Ile, Trp, Tyr, Asp, Gln) Histidine, lysine, alanine, methionine, threonine,

RP-18

Phosphorylated amino acids

Silica gel

Alumina and Li-impregnated alumina layers

Amino acids

Lysine, threonine, homoserine, tryptophan, and phenylalanine

Silica gel and RP-18

Stationary Phase

a -amino acids

Analyte

TABLE 1 Continued

n-butanol-acetic acid-water (3:1:1)

Methanol-water-acetonitrile (1:1:4)

1-propanol-25% NH3 (11:9), 2-propanol-acetone-water25% NH3 (25:25:7:6), 2-propanol-ethyl acetate-25% NH3-water (40:40:3:50), and 2-propanol-25% NH3 (7:3)

Ethanol-NH3 conc.-water (65:10:16)

Toluene-pyridine-acetic acid (40:10:1) for purification and aqueous solution of cyclodextrin containing different proportions of either acetonitrile or methanol for stereochemical analysis 1% cetyl trimethyl ammonium bromide in water-butanol (19:1)

Mobile Phase

[30]

[29]

[28]

[27]

[26]

[25]

TLC of a -amino acids in small peptides; after sequential acid hydrolysis of a peptide, derivatization of the free amino acids with dansyl chloride. Detection by densitometry at 366 nm.

L-proline was selectively separated from other aliphatic and aromatic amino acids. Detection by spraying with a 0.4% solution of ninhydrin in acetone and heating for 15–20 min at 90–100 C. Reversed-phase thin-layer chromatographic analysis of phosphorylated amino acids (phospholysine, phosphoserine, phosphothreonine, phosphoarginine, phosphotyrosine, phosphohistidine). Detection by spraying with 0.2% ninhydrin in ethanol and heating at 37 C for 30 min. Determination of industrial essential amino acids in fermentation solution. Detection of tryptophan by immersion for 40 s in a 0.5% solution of 4-dimethylamino-benzaldehyde in ethanol containing 5% conc. sulfuric acid and heating at 110 C for 5–7 min. Amino acid spots were scanned with two different densitometers. Enantioseparation of amino acids on sorbents containing copper complexes of l-hydroxyproline derivatives. Analysis of amino acids in Biomphalaria glabrata infected with Schistosoma mansoni. Detection of the zones was achieved by spraying the developed

Ref.

Remarks

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1503

Silica gel, cellulose, strong acid cation-exchange sheets, and RP-18

Alanine, arginine, asparagine, aspartic acid, glycine,

8 binary phases (methanol-water, ethanol-water, propanol-water, acetonitrile-water, THF-water, acetonitrile-buffer, and methanol-buffer), in which the concentration of organic modifier varied from 0 to 100% Propanol-0.5 M NaCl 2:3 was used as eluent for the RP phase, pH 3.3 citrate buffer

Chloroform-methanol (1:1), isopropanol-NH3 (7:3), ethanol-water (7:3), chloroform-methanolNH3(20:20:9) and butanol-acetic acid-water (4:1:5)

Silica gel or cellulose

Amino-modified silica gel

Dimethyl sulfoxide-1 M HCl (1:1)

Untreated and on triaryl phosphate (TAP)impregnated talc, starch, silica gel and alumina

Rosmarinic and valerenic acids

24 amino acids (Gly, Hyd, Ala, n-But, Ser, Pro, Val, Thr, Cyst, Leu, Ile, n-Leu, Met, Tyr, -Phe, Trp, DOPA, Cys, Orn, Lys, His, Arg, Asp, Glu) Glycine, alanine, aspartic acid

asparagine, proline, and leucine= isoleucine

[34]

[33]

[32]

[31]

(Continued )

Free pool amino acids in cercariae, rediae, encysted metacercariae, and excysted metacercariae of Echinostoma caproni were analyzed by TLC.

New selective and sensitive method was developed for the detection of Glycine, alanine, and aspartic acid. Detection by the improved iodine-azide procedure (the developed plates were sprayed with a freshly prepared mixture of 4% sodium azide adjusted to a proper pH with 0.5% starch solution 10:1 and were exposed to iodine vapor for 5 s). White spots developed on a violet-gray background which were stable for 20 min. Retention properties of rosmarinic and valerenic acids are studied. Visualization by spraying wit...