Indian Journal of Chemistry Vol. 40A. October 200 1. pp. 1130-1134

Separation of amino acids on alumina layer developed with oil-in-water microemulsion

Ali Mohammad* & Vineeta Agrawa l

Department of Appli ed Chemistry. Faculty of Engineering and Technology. Ali garh Muslim Uni versity.

Ali garh 202 002. (U. P). Indi a

I?eceived 17 May 200 I .. revised 16 Jllly 200 I

Oil-in-water microemulsion systems have been used as mobile phase in thin layer chromatographic separat ion of aliphatic and aromatic amino acids on plain (or non-impregnated) alumina and Li+. Na+ or NI-I / - impregnated alumina layers. The results obtained with water and oil-in-water microemulsions have been compared. The lower limits of detecti on of amino acids have also been determined. Proline. dI-valine and dl-asparti c acid have been selecti vely separated from other amino acids.

Due to the phys iologica l importance, chromato­graphic techniques such as gas and high-pressure liquid chromatography (GC and HPLC) have been generally used for the separation of amino acids . However, these methods depend upon costly instrumentation and sometime require sample derivatization making the procedure more complicated. On the other hand, thin layer chromatographic (TLC) systems are specially suited for economical routine ana lysis of large number of test samples. For separation of amino acids by TLC, three main approaches have been reported ' .6. In literature:

(i) Separation on traditional stationary phases with chiral additi ves in the eluent.

(ii) Separation on stat ionary phases impregnated with transition metal ions and their anions via complexation between sample and the impregnant.

(iii) Direct separation on chiral stationary phases.

Few reports7•9 on the use of reversed-phase

stationary phases and the stationary phases impregnated with organic complexing agents for the separation of amino acids are also available in literature. All the reported TLC methods mainly involve the use of traditional aqueous, non-aqueous and mixed-aqueous organic solvents as mobile phase and the use of microemulsion systems has been

neglected . In continuation of our earl ier work on the use of surfactant mediated systems as mobile phase in organic TLC'o" " we report here (probably first time) use of oil-in-water microemulsion as mobile phase in TLC analysis of amino acids.

Experimental A thin layer chromatographic apparatus

(Toshniwal, India), 20 x 3 cm glass plates, 24 x 6 cm glass jars were used for the development of chromatographic plates . A glass sprayer was used to spray reagent on the plates to detect the spot.

Aluminium oxides 'G' , amino acids, heptane, inorganic salts [CDH, India] ; sodium dodecyl sulphate (SDS) [ Qualigens , India] and pentanol [Fluca AG, Buchs, SG, made in Switzerl and] . All reagents were of Analar reagent grade.

Glycine (AI), dl-alanine (A2), dl-serine (A3), 1-proline (A4), dl-valine (AS), dl-threonine (A6), 1-cysteine hydrochloride (A 7), I-leucine (A8), dl­isoleucine (A9), I-isoleucine (A 10), dl-norleucine (All), dl-methionine (AI2), I-tyrosine (Al3), dl ­tryptophan (AI4), I-cystine (AIS) , dl-phenyl alanine (A 16), I-ornithine monohydrochloride (A 17), 1-histadine monohydrochloride (AI8), I-arg inine monohydrochloride (AI9), I-Iycine monohydro­chloride (A20), dl-aspartic acid (A21), I-glutamic ac id (A22) and dl-2-amino-n-butyric acid (A23). The test solution (l %) of all amino acids were prepared in distilled water. A 0.4% ninhydrin soluti on in acetone was used to detect all the amino acids. The appearance of a dark violet spots shows the presence of amino acids.

Chromatographic system The various sorbent used as stationary phases as

follows:

S,: Aluminum oxide'G'

S2: Aluminum oxide impregnated with aqueous solution (2-8%) of NaCI

S3: Aluminum oxide impregnated with aqueous solution (2-8 %) of LiCi

S4: Aluminum oxide impregnated with aqueous solution (2-8 %) of (NIL)2S04

S5: Aluminum oxide impregnated with aqueous solution (2-8%) of NH4Cl

...."

NOTES 1131

Mobile phase The oil-in-water microemulsion systems used as

mobile phase were prepared at 40°C following the method reported by Berthode l2

. The SOS/pentanol ratio (l :2, w/v) was kept constant for all compositions.

Mobile SDS plus pentanol Heptane Water(mL) phase (I :2. w/v) (mL) (mL)

MI 5.1 0.68 94.30

M2 10.2 1.4 88.4

M3 10.0 2.2 88.S

M4 14.1 00 85.9

Ms 20.0 00 80.0

Preparation of TLC plates

(a) Plaill alumillium oxide thilllayer plates The aluminium oxide plates were prepared by

mixing a luminium oxide with water in a 1:3 ratio with constant shaking until homogeneous slurry was obtained. The resultant slurry was applied on the glass plates with the help of an applicator to give a 0.2S mm-thick layer. The plates were first dried at room temperature and then activated at 100 ± SoC by heating in an electrically controlled oven for 1 h. The activated plates were stored in a close chamber at room temperature until used .

(b) Impregnated alumillium oxide thin layer plates Impregnated plates were prepared by mixing

aluminium oxide with a mixture comprising of 2-8% aq. NaCI, aq. LiCl, aq. (NH4hS04 and aq. NH4C1 solution in 1:3 ratio by shaking for S-IO minute. Thin layer of the resultant slurry was prepared by following the procedure as described above in (a).

Procedure About 10 ilL of test sol ution was spotted on thin

layer plates with the help of micropipet. The plates were developed in the chosen solvent system by the ascending technique. The solvent ascent was fixed to 10 em in all cases. After development was complete, the plate was withdrawn from glass jars, dried at room temperature, sprayed with freshly prepared ninhydrin solution and heated at 90-100°C for IS-20 minutes. The amino acids were detected as violet spots. The RL (RF of leading front) and RT (RF of trailing front) values for each spot were determined and the RF value was calculated.

The limits of detection of amino acids were determined by spotting different amounts of amino acids on the TLC plates, developing the plates and detecting the spot. The method was repeated with and successive lowering of the amount of amino acids until no spots were detected. The lowest amount of amino acids detected on the TLC plates was taken as the limit of detection.

For separation, equal amounts of different types of

amino acids were mixed and 20 ilL of the resultant mixture was loaded on the TLC plates. The plates were developed , the spot were detected and the RF values of the separated amino acids were determined.

Results and discussion The unique features of the present study include (a)

the use of oil-in-water microemulsion as eluent ; (b) the use of Li+, Na+ and NH/ - impregnated alumina layers as stationary phase; (c) the salting out effect of lithium - impregnated layer on the mobility of neutral amino acids; and (d) selective separation of I-leucine from I-isoleucine and dl-norleucine.

In all five oil-in-water microemulsion systems (M 1-

Ms) were prepared from different combinat ions of heptane, water and 1:2 (w/v) mixture of SOS and pentano!. These systems were used as mobile phase in normal phase TLC to investigate the mobility trend of amino acids on thin layers prepared from plain alumina and sod ium, ammonium or lithium ion impregnated alumina. The resulted microemulsion systems were transparent and optically stable.

The results of the present study have been summarized in Table 1 and Figs 1-3.

( I )Plain alumina - microemulsioll system Amino acids were chromatographed on plain

alumina using distilled water, microemulsion systems (M I-MS), pure heptane and pentanol as mobile phases. The results obtained with MI-Ms are listed in Table l. It is evident from these data that the mobility of amino acids is controlled by the proportional composition of microemulsion systems. The SDS­pentanol-water-heptane system consisting of 0.68% heptane (M 1) was found most effective for realizing quick separations of amino ac ids. Interestingly, heptaneless SOS-pentanol-water systems (M4-MS) were found inferior to heptane containing systems (M I-M3) as mobile phase for analysis of amino acids. Thus, upon addition of heptane, the oil-in-water

1132 INDIAN J CHEM , SEC A, OCTOBER 2001

structure becomes more organized providing unique selectivity and better separations.

In order to illustrate the separation effic iency of microemulsion system, a plot of f':..RF Vs amino acids was constructed (Fig. 1). It is evident from this figure that the mobility trend is drastically modified in

microemulsion media compared to purely aqueous medium (i.e. distilled water). On the other hand, all amino acids behave in almost identical fashion (RF "'" 0.05) when alumina plates are developed with pure pentanol (development time, several hours) thus, no separation is possible with pentanol or heptane as

Table I-Mobility pattern of amino acids on plain alumina layer developed with oil-in-water microemulsion systems

Amino acid M) M2 MJ M4 M5

Glycine 0.46 0.45 0.45 0.76 0.38

dl-alanine 0.69 0.60 0.57 0.58 0.66

dl-serine 0.37 0.38 0.45 0.48 0.33

I-proline 0.91 0.90 0.93 0.85 0.87

dl-valine 0.78 0.57 0.64 0.64 0.71

dl-threonine 0.51 0.51 0.43 0.27 0.32

I-cysteine hydrochloride 0.31 0.15 0.16 0. 11 O. IS

I-leucine 0.71T 0.62 0.62 0.72 0.53

dl-isoleucine 0.74 0.77 0.78 0.76 0.71

I-isoleucine 0.80 0.69 0.79 0.80 0.87

dl-norleucine 0.85 0.64 0.64 0.64 0.77

dl-methionine 0.63 0.57 0.48 0.76 0.52

I-tyrosine NO NO 0.69 0.79 NO

dl-tryptophan 0.60 0.75 0.80 0.76 0.48

I-cystine NO NO NO NO NO

dl-phenyl alanine 0.56 0.60 0.6 1 0.69 0 .78

I-ornithine monohydrochloride 0.30T 0.32 0.22T 0.20 0.22

I-histadine monohydrochloride 0.40 0.22 0.35 0.29 0.25

I-arginine monohydrochloride 0.34 0.27 0.19 NO ND

I-lysine monohydrochloride 0.45 0.31 0.23T 0.31 0.40

dl-aspartic acid 0.14 0.08 0.12 0.11 0.10

I-glutamic acid 0.34 0.21 0.18 0. 17 0.09

dl-2-amino-n-butyric acid 0.67 0.55 0.65 0.72 0.69

ND = Not detected, T= Tailed spot

',- j 0,4

0,2 ,

u. ~ ° ':::;] > > > > ~ > ~ ~ .... 00 'C

0 N '" V. '" .... ·O~ r Amino acids

-0,4

-0,6 I

Fig. I-Plot of !1RF vs amino acids. -.-!1RF in microemulsion-RF in water

NOTES 1133

mobile phase in the absence of water or surfactant. In spite of differential migration of amino acids on alumina layers developed with oil-in-water microemulsion or pure water, separations of closely related amino acids could not be realized .

(2) Impregnated alumina - microellluision system To explore further improved separation

possibilities, the nature of stationary phase was altered by treating alumina with different concentrations of aqueous salt solutions (chloride of lithium, sodium or ammonium and ammonium sulphate). When alumina is brought in contact of aqueous salt solutions, a cation exchange

m AI - 0 - H + M+n ~ M (AI - 0 -)"Ill-n + mH+

takes place at the surface of alumina to provide a new

sorbent phase with altered selectivity. The TLC plates coated with impregnated alumina were of good quality and the most effective concentration levels of impregnation for resolution of amino acids were 2-8%.

The results obtained on impregnated and non­impregnated (i.e. plain) alumina layers have been compared in Fig. 2. For this purpose tlRF (RF on impregnated alumina minus RF on plain alumina) was calculated for 2% impregnant concentration level of NH4CI, NaCI, (NH4)2S04 or LiCI and plotted in Fig. 2. The positive and negative tlRF values of amino acids clearly demonstrate the detrimental effect of impregnation on the mobility of amino acids. The similar trend in variation of RF values were noticed with alumina layers impregnated at concentration levels of 5, 7 and 8% of the impregnant. However, the magnitude of RF value in certain cases was found to

Fig. 2-Plot of 6.RF vs amino acids. - 0- 6.RF=RF on NH4Ci illlpregnated-RF on plain alumina; -A.- 6.RF=RF on (NH4)zS04 impregnated-RF on plain alumina; - 0 - 6.RF=RF on Nnel impregnated-RF on plain alumina; --- 6.RF=RF on Liel

imprcgnatcd-RF on plain alumina;

0.8 I

::: 1 0.5 I

14-II: 0.4

0.3

0.2

0.1

o ~-------.-----1 2 3

----, .. 6 7 8 9 Concentration of Liel (%)

Fig. 3-Effcct of Liel concentra ti on present in stationary phase on thc R value of amino acids. Mobile phase MI. -.- Gl ycine ; - 0 -

dl-alaninc; -+-I-proline; -A. - dl-valine; -e- I-i soleucine; -6.- dI-norieucine ; - 0- dl-methionine

1134 INDIAN J CHEM, SEC A, OCTOBER 2001

depend on the degree of concentration of the impregnant. It was always possible to separate proline (RF = 0.87) and dl-valine (RF = 0.75) from glycine, dl­serine, dl-threonine, I-cysteine hydrochloride, dl­methionine, dl-aspartic acid and I-histadine monohydrochloride on plain as well as impregnated alumina stationary phases (SI - Ss) with the mobile phase MI' Similarly with the same TLC system dl­aspartic acid (RF = 0.10) was successfully separated from I-histadine monohydrochloride, I-arginine monohydrochloride, dl-methionine, dl-norleucine, 1-isoleucine, I-cysteine hydrochloride, dl-threonine, dl­serine, glycine and dl-2-amino-n-butyric acid. However, the important separations worth mentioning include I-leucine (0.43) from I-isoleucine (0.73) or dl­isoleucine (0.69) and dl-norleucine (0.68) on 2% NH4C1 impregnated alumina and dl-aspartic acid

-(0.09) from I-glutamic acid (0.30) and dl-2-amino-n­butyric acid (0.65) on plain alumina layer. The reported separations of amino acids are important because these include the separation of non-polar (proline and valine) from polar (glycine, serine, threonine and histadine), of non-polar (leucine) from non-polar (isoleucine) and of polar (aspartic acid) from polar (glutamic acid) amino acids.

An interesting aspect of this investigation is that LiCI impregnated alumina shows a salting out effect (i .e. decrease in mobility (or RF value) with the increase in salt concentration) in the case of neutral amino acids (Fig. 3). This important phenomenon can be utilized to distinguish neutral amino acids from basic or acidic amino acids which do not respond to the salting out effect. The present study is probably the first showing salting out effect when the variable concentrations of salt is taken in stationary phase instead of in the mobile phase. The available literature on salting-out TLC of organic and inorganic compounds concerned with the use of aqueous solutions of salts as mobile phases I3-18

•

The results of lowest possible detectable microgram amount and dilution limits of amino acids (given in parenthesis) on plain alumina layer were dl­aspartic acid (2.0, 1: 5 x 10\ dl-alanine (4.0, 1: 2.5xlO\ I-cysteine hydrochloride (4.0, 1 : 2.5 x 10\ dl-methionine (3.9, 1 : 2.5 x 10\ dl-phenyl alanine (2.8, 1 : 3.5 x 103) and I-argenine monohydrochloride (2.9, 1 : 3.4 x 10\

Acknowledgement The authors are thankful to Prof. K G Varshney,

Chairman, Department of Applied Chemistry for providing research facilities.

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