1070-4272/04/7706-0905 C 2004 MAIK [Nauka/Interperiodica]
Russian Journal of Applied Chemistry, Vol. 77, No. 6, 2004, pp. 905!914. Translated from Zhurnal Prikladnoi Khimii, Vol. 77, No. 6, 2004,
Original Russian Text Copyright + 2004 by Krasikov, Malakhova.
AND ION-EXCHANGE PROCESSES
Planar Chromatography for Quantitative Analysis
of Amino Acids in Fermentation Solutions
of Industrial Producer Cultures
V. D. Krasikov and I. I. Malakhova
Lenkhrom Scientific and Technical Center, St. Petersburg, Russia
Received December 25, 2003; in final form, February 2004
Abstract-Proximate procedures for quantitative analysis of amino acids using high-performance thin-layer
chromatography were developed.
Amino acids (hereinafter, natural L-amino acids)
are the main building material in synthesis of specific
tissue proteins, enzymes, peptide hormones, and other
physiologically active compounds .
Analysis of amino acids in medicine, biochemistry,
molecular genetics, etc. is problem of current unterest.
Modern procedures for analysis of amino acid deriv-
atives in albumin hydrolyzates, peptides, and bio-
medical samples are based on high-performance liquid
chromatography. However, data on application of
high-performance quantitative thin-layer chromatog-
raphy (HPTLC) to separation of multicomponent mix-
tures of free amino acids (including 20 natural acids)
In this study we developed chromatographic proce-
dures for separation and quantitative determination of
free amino acids in culture broths (CBs) of certain
producer cultures. The main analytical method was
HPTLC, and high-performance liquid chromatography
(HPLC) was used for comparison . In some spe-
cific cases, an amino acid analyzer (AA) was used.
It should be noted that the requirements to the re-
liability and adequacy of chromatographic procedures
in biochemistry and biotechnology are essentially
different. For example, procedures providing exper-
imental error of no more than 15% and reproducibility
of 90% are acceptable in biochemistry .
Similar requirements are imposed upon analytical
chromatographic procedures in biotechnology at the
stage of development of the producer cultures of
amino acids. However, at the stage of optimization of
the fermentation procedure the analysis error should
not exceed 5%. The same requirement is imposed
upon the chromatographic analysis in recovery and
purification of the desired amino acid, because the
error of 10315% may cause either production of the
preparation of insufficient purity or a significant loss
unacceptable in the large-scale production.
In our study we used densitometers (Shimadzu,
Japan), a High Speed TLC Scanner CS-920 (Shima-
dzu, Japan), a TLC-Scanner-II (Camag, Switzerland),
a DenSkan-01 video system (Lenkhrom, Russia),
a DenSkan-04 video system (Lenkhrom, Russia), and
a Delta Prep 3000 liquid chromatograph equipped
with a Lambda Max 481 spectrophotometric detector
and a 4.6 0 250-mm stainless steel column packed
with m-Bondapak C18 sorbent (5 mm, Waters, USA).
The eluent was 10
M solution of copper(II) sulfate
in 20% aqueous acetonitrile (0.5 ml min
l 235 nm).
The concentrations of some amino acids in CBs
were measured using a Biocal amino acid analyzer
The tests were carried out using the following chro-
matographic plates: Sorbfil PTSKh-P-V (Institute of
Macromolecular Compounds, Russian Academy of
Sciences, Sorbpolymer Company, St. Petersburg, Rus-
sia) and Kieselgel 60 (Merck, Germany).
Mixtures of 1-propanol, 2-propanol, ethanol, ethyl
acetate, chloroform, acetone, acetic acid, 25% aqueous
ammonia, and water were taken as mobile phases.