Russian Journal of Applied Chemistry, 2010, Vol. 83, No. 5, pp. 869−873.
Pleiades Publishing, Ltd., 2010.
Original Russian Text
I.I. Malakhova, B.V. Tyaglov, A.S. Mironov, V.D. Krasikov, 2010, published in Zhurnal Prikladnoi Khimii, 2010, Vol. 83, No. 5,
AND POLYMERIC MATERIALS
Quantitative High-Performance Thin-Layer
Chromatography of Nucleosides
I. I. Malakhova
, B. V. Tyaglov
, A. S. Mironov
, and V. D. Krasikov
State Research Institute of Genetics and Selection of Industrial Microorganisms,
Federal State Unitary Enterprise, Moscow, Russia
Institute of Macromolecular Compounds, Russian Academy of Sciences, St. Petersburg, Russia
Received December 10, 2009
Abstract—A procedure was developed for express analysis of thymidine by high-performance thin-layer
chromatography in the cultural liquid in the course of microbiological synthesis.
Thymidine is a raw material for the production
of a drug, thymidine azide, used for inhibiting the
growth of human immunodeﬁ ciency virus (HIV).
Today thymidine is produced in industry by enzymatic
hydrolysis of DNA  and by chemical synthesis .
At the same time, active studies aimed to develop
a biotechnological procedure for thymidine production
are being performed at the State Research Institute of
Genetics and Selection of Industrial Microorganisms.
This procedure seems to be advantageous from the
economical and environmental viewpoints.
In the course of fermentation in thymidine bio-
synthesis, it is necessary to monitor the target product
concentration. However, fermentation solutions of
thymidine producers contain a number of concomitant
nucleosides (inosine, uridine, etc.), which makes it
necessary to use express analytical methods such as
quantitative high-performance thin-layer chromato-
graphy (HPTLC) (or planar chromatography, PC) or
high-performance liquid chromatography (HPLC).
Numerous original papers and monographs deal
with the separation of nitrogen bases, nucleosides,
and nucleotides by planar methods [3, 4]. In the
overwhelming majority of these studies, the stationary
phase was cellulose or polyethylenimine–cellulose
applied onto a support, or chromatographic paper.
Recently the separation of mixtures of nucleosides and
bases has been performed on cyano-N-propyl, amino-
N-propyl, and ethylenediamine-N-propyl silica gels
. As for unmodiﬁ ed silica gel, it was used, as a rule,
for separation of nucleosides from their derivatives
with various substituents in the heteroring or in the
carbohydrate moiety .
The goal of this study was the development of
an express procedure for analysis of nucleosides in
the cultural liquid (CL) by HPTLC using chromato-
densitometry of Sorbﬁ l thin-layer plates (Russia).
Thymidine was prepared by microbiological
synthesis using KGM-5 culture (State Research
Institute of Genetics and Selection of Industrial
Microorganisms). The following chemically pure and
analytically pure grade chemicals were used: methanol,
ethanol, 2-propanol, 1-butanol, chloroform, ethyl
acetate, 25% aqueous ammonia, acetonitrile, potassium
dihydrogen phosphate, formic acid, and ammonium
hydrogen carbonate. Organic solvents were puriﬁ ed by
standard procedures . Water was puriﬁ ed on a Super Q
installation (Millipore, USA).
Nucleosides (main substance content 99.0 wt %)