SummaryAqueous micellar bile salt, sodium deoxycholate (NaDC) solution as

additive in acetonitrile (ACN) in the ratio (1:5 v/v) was identified as

the most favorable mobile phase for on-plate identification and res-

olution of three series of common sugars (disaccharide, pentose and

hexose) on commercially available high performance silica gel

plates. Effect of mobile phase composition and the presence of heavy

metal cations as impurities in the analyte sample have been investi-

gated to optimize experimental conditions for the separation. The

lowest possible amount of all sugar on high-performance thin-layer

chromatography (HPTLC) plate has been determined at nanogram

level. This method was successfully applied for identification and

separation of sugars in pharmaceutical formulations [cough syrup

(Honitus), multivitamin syrup (Becozinc)] and biological matrix

(human blood).

1 Introduction

Among the natural substances, carbohydrates are most widely

distributed with diverse roles. Determination of carbohydrates

occurs frequently in food, beverages, and pharmaceutical fer-

mentations and in clinical analysis with the aid of thin-layer

chromatography (TLC) [1–3]. In most cases, mixture of organic

solvents, and mixed aqueous–organic eluents comprising of

chloroform, pyridine, benzene etc. as one of the components

have been used for the analysis of polar sugars [4, 5]. Mixtures

of acetonitrile (ACN) with water as eluent in combination with

silica gel or amino-bonded layers have been more frequently

used to resolve complex mixture of sugars on TLC plates [6, 7].

The use of micellar bile salt mobile phases in chromatographic

separation of isomeric compounds has been reported by Wil-

liams et al. [8]. This study has opened a novel route for replac-

ing conventional hydro-organic mobile phase systems by bile

salt surfactant solutions with judicious use of organic additives.

It has generated the renewed interest of chromatographers in the

use of bile salt as mobile phase or in the stationary phase to

achieve the separations of analytical importance [9–11].

Bile salts being naturally occurring steroid-based surfactants

play an important role in biological systems and have been sub-

ject of numerous studies [12, 13]. Sodium salt of cholic acid

form helical aggregates in aqueous solutions, which has lateral

surface with backbone of steroid having angular methyl and

hydroxyl groups protruding outside and inside the helix respec-

tively. More recently, the use of bile salt instead of long-chain

surfactants for separating a range of neutral analytes as well as

for chiral separations has been reported [14]. Surprisingly, salts

of bile acids have not been used in high-performance thin-layer

chromatography (HPTLC) of sugars whereas limited use of tra-

ditional linear surfactants (sodium dodecyl sulfate (SDS)) has

been reported [15]. Thus, it is worthwhile to utilize the analyti-

cal potential of physiologically important surfactant in chro-

matographic analysis of biomolecules. Micellar mobile phase is

expected to be very effective in the resolution of disaccharide

from monosaccharide.

Hydrophilic interaction chromatography (HILIC) is a variant of

normal phase chromatography, important for the separation of

highly polar substances including biologically active com-

pounds, such as pharmaceutical drugs, neurotransmitters, nucle-

osides, nucleotides, amino acids, peptides, proteins, oligosac-

charides, carbohydrates, etc. [16]. The mechanism of separation

is liquid–liquid partition that is by passing a hydrophobic or

mostly organic mobile phase across a neutral hydrophilic sta-

tionary phase (bare silica) causing solutes to elute in the order of

increasing hydrophilicity [17]. The retention of polar analytes

often requires a highly aqueous mobile phase to achieve reten-

tion which sometimes lead to problems such as phase collapse

(dewetting) [18]. In HILIC, the mobile phase forms a water-rich

layer on the surface of the polar stationary phase vs. the water-

deficient mobile phase, creating a liquid/liquid extraction sys-

tem. The analyte is distributed between these two layers. How-

ever, HILIC is more than just simple partitioning and includes

hydrogen donor interactions between neutral polar species as

A. Mohammad and S. Laeeq, Analytical Research Laboratory, Department ofApplied Chemistry, Faculty of Engineering & Technology, Aligarh MuslimUniversity, Aligarh, India.E-mail: alimohammad08@gmail.com

Journal of Planar Chromatography 24 (2011) 6, 491–496 DOI: 10.1556/JPC.24.2011.6.7 4910933-4173/$ 20.00 © Akadémiai Kiadó, Budapest

Identification of Coexisting Pentose, Hexose, andDisaccharides with Preliminary Separation throughHydrophilic Interaction on Silica HPTLC Plate Using Aqueous Sodium Deoxycholate–Acetonitrile Mobile Phase System

Ali Mohammad* and Sameen Laeeq

Key Words

BiosurfactantSugarsSeparationHigh-performance thin-layer chromatography (HPTLC)Micelles