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SummaryThin-layer chromatography (TLC) of fifteen amino acids was per-

formed using silica gel and alumina impregnated with micellar solu-

tions of cetrimide and cetylpyridinium chloride as stationary phas-

es and aqueous solutions of dextrose as mobile phases. TLC system

comprising of silica gel impregnated with micellar solution of

cetrimide (5.0 mM) as stationary phase and 40% (w/v) aqueous solu-

tion of dextrose as mobile phase was found the most favourable for

the separation of amino acids. Impregnation of silica gel with the

micellar solution of cetrimide brings about a substantial change in

the mobility of lysine. Separation of lysine (ketogenic) from arginine

(glucogenic) is important physiologically. Surface modification of

silica gel on impregnation, as indicated by FTIR and SEM studies,

was responsible for improved chromatographic performance. The

effect on the separation of the presence in the sample of heavy metal

cations, as impurities, was examined. Limits of detection for lysine

and arginine were 0.17 μg and 0.12 μg, respectively. For validation,

the stability of the mixture and the reproducibility of the chromato-

graphic properties ΔRF, separation factor (k), and resolution (R

S)

were calculated. The proposed method is simple, rapid, and free

from use of volatile organic solvents.

1 Introduction

Because of the biochemical and physiological importance of

amino acids, several analytical techniques, for example high-

performance liquid chromatography [1], gas chromatogra-

phy [2], thin-layer chromatography [3], electrophoresis [4], vis-

cometry [5] and micellar electrokinetic chromatography [6],

have been used for their analysis. Among chromatographic tech-

niques, thin-layer chromatography (TLC) has been the most

popular for routine analysis of amino acids because of several

attractive features, for example wider choice of mobile and sta-

tionary phases, flexibility in sample detection, the open and dis-

posable nature of thin layer chromatographic plates, low solvent

consumption, minimum sample clean up, reasonable resolving

power, and the ability to handle a large number of samples

simultaneously.

To achieve chromatography with improved selectivity, resolu-

tion, and reproducibility, a variety of layer materials as station-

ary phases in TLC had been successfully used in the analysis of

amino acids [7–19]. Surfactant modified TLC is a promising

method and has found wide application. Surfactants were first

used in paper chromatography in 1963 [20]. Later, surfactants

were proposed for preliminary impregnation of different adsor-

bents in thin-layer chromatography [21]. Different versions of

TLC with surfactant modification expanded the potential of the

method, and, in some cases, enabled efficient separation of mix-

tures, especially those containing neutral and charged organic

compounds. Adsorption of the surfactant by the stationary phase

could occur in at least two ways:

– hydrophobic adsorption, in which the alkyl tail is adsorbed so

the ionic head group would be in contact with the polar solution;

and

– silanophilic adsorption, in which the ionic head group is

adsorbed and the stationary phase becomes more hydropho-

bic [22].

According to Ref. [23] the main mechanisms of separation on an

impregnated stationary phase are ion-exchange (cationic and

anionic species) and distribution (neutral species).

To our surprise, despite several advantageous features, for

example non-toxicity, non volatility, non-inflammability, non-

aggressiveness, high-biodegradability and cost effectiveness,

the full potential of surfactants as reagents for impregnation of

stationary phase for TLC analysis of amino acids has not been

exploited.

The objective of this work was to develop new stationary phas-

es by surface modification of silica gel with aqueous solution of

surfactants at surfactant concentration levels below and above

the critical micelle concentration (CMC). Surfactants at these

two concentrations were deliberately used because below the

CMC non-ionic surfactants behave as monomers whereas

cationic and anionic surfactants acts hydrophobic counter ions.

Above the CMC, however, surfactants form micelles which

selectively solubilize hydrophobic and hydrophilic compounds.

A. Mohammad and N. Haq, Analytical Research Laboratory, Department ofApplied Chemistry, Faculty of Engineering and Technology, Aligarh MuslimUniversity, Aligarh-202 002, India.E-mail: alimohammad08@gmail.com

TLC Separation of Amino Acids with a Green Mobile Phase

Ali Mohammad* and Nazrul Haq

Key Words

Thin-layer chromatographySurface modificationSeparationAmino acidsDextrose

Journal of Planar Chromatography 23 (2010) 4, 260–264 DOI: 10.1556/JPC.23.2010.4.40933-4173/$ 20.00 © Akadémiai Kiadó, Budapest