Development of Conditions for Rapid Thin-Layer

Chromatography of β-Lactam Antibiotics

Shalini Joshi*, Amrita Sharma, Mohan Singh Maniyari Rawat, and Charu Dhiman

Key Words
β-Lactam group
Ammonium chloride
Impregnated silica gel plates
UV detection

Summary HPTLC plates are commonly used for β-lactam antibi-

Penicillins and cephalosporins (subclasses of β-lactam antibiotics)
are widely used against Gram-positive and Gram-negative bacteria.
Use of HPLC, TLC–bioautography, and thin silica gel layers pre-
coated with fluorescent material has been reported in literature for
the analysis of these compounds. This manuscript deals with a
straightforward and sensitive method for rapid separation and
detection of selected β-lactams. Bulk impregnation of homemade sil-
ica gel G layers and impregnation of ready made silica gel 60 layers
with 0.2% ammonium chloride was carried out and various mobile
phases have been established for UV detection of the compounds.
Separation of penicillins (benzylpenicillin, ampicillin, and amoxi-
cillin) and cephalosporins (cephalexin, cefoperazone, ceftriaxone,
cefixime, and cefadroxil) was achieved by use of propanol–acetic
acid 4:1 (v/v) and butanol–acetic acid–water 4:1:2 (v/v) respectively,
as mobile phases.
1 Introduction
Penicillins and cephalosporins, subclasses of β-lactam antibi-
otics, act by inhibition of bacterial cell wall biosynthesis. The
free carboxyl group of thiazolidine is essential for its therapeu-
tic effectiveness. The stability of this class of compounds is
markedly affected by temperature, pH, and solvents, for exam-
ple methanol and acetonitrile, as studied by Tyczkowska et
al. [1]. Rapid and specific analytical methods are required to
detect the presence of structurally related compounds and degra-
dation products. HPLC, usually with C18 columns and UV detec-
tion, has been widely recommended for the purpose, as
reviewed elsewhere [2]. TLC, because of its rapid and sensitive
nature is still preferred in purity checking and in reaction moni-
toring of pharmaceuticals [3].
The literature reveals widespread use of reversed-phase layers,
because of the possibility of nucleophilic reactions of β-lactam
molecules after spotting on silica gel layers [4]. Various types of
otics [5–9]. Silica gel and silanized silica gel layers precoated
with fluorescent material have been studied for separation of
several of the compounds [10–12], although these methods are
time-consuming. A TLC–bioautography method using ammoni-
um chloride in the mobile phase has been established for analy-
sis of for β-lactam [13]. Kovács-Hadady et al. separated peni-
cillin G and V by over pressurized liquid chromatography
(OPLC) on silica layers impregnated with tricapryl methyl
ammonium chloride (TCMA, 0.1 M in 30% ethanol) [14].
Recently an ion-exchange material mixed with cellulose has
been used [15, 16] as stationary phase for β-lactams and sub-
sequently quantitative analysis of these compounds was
achieved by a method of Askal et al. [15] using 2,3-dichloro-5,6
dicyano-p-benzoquinone as detection reagent [16, 17].
In view of these results, it was considered worthwhile to develop
a straightforward and sensitive method for rapid separation and
detection of penicillins and cephalosporins in commercial sam-
ples. Impregnation of the stationary phase with ammonium salt
resulted in the appearance of fluorescent spots in the UV region.
2 Experimental
2.1 Chemicals and Materials
The penicillins and cephalosporins were obtained from various
manufactures, viz., Alembic, Solan, India (benzylpenicillin and
cephalexin); Ostrica Lifecare, New Delhi, India (cefadroxil, cef-
operazone, and ceftriaxone); Ind-Swift, Solan, India (amoxi-
cillin); Cadila Pharmaceutical, Ahmedabad, India (ampicillin),
and Intas Pharmaceuticals, Selaqui, India (cefixime). Ammoni-
um chloride and the solvents used were of analytical grade from
E. Merck, Mumbai (India) or Glaxo, Mumbai (India).

2.2 Sample Preparation

S. Joshi, A. Sharma, and C. Dhiman, Department of Chemistry, K.L.D.A.V. (P.G.)
College, Roorkee-247667, India; and M.S.M. Rawat, Department of Chemistry,
H.N.B. University Srinagar (Garhwal), Srinagar-246174, India.
The powdered contents of capsule and injection were extracted
E-mail: shajoshi@yahoo.com with methanol–water at pH 6 then left at room temperature in a

Journal of Planar Chromatography 22 (2009) 6, 435–437

0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest
DOI: 10.1556/JPC.22.2009.6.9
435
Rapid TLC of β-Lactam Antibiotics

Table 1 Table 3

Results obtained on 0.2% ammonium chloride-impregnated silica gel hRF values of cephalosporins on silica gel 60 plates impregnated with
G layers. 0.2% NH4Cl with butanol–acetic acid–water 4:1:2 (v/v) as mobile
phase.
Compound Mobile phase (v/v) Detection
limit Compound hRF

Benzylpenicillin Propanol–acetic acid 4:1 14 μg Cephalexin 43

Amoxicillin Propanol–acetic acid 4:1 9.1 μg Cefoperazone 50
Ampicillin Propanol–acetic acid 4:1 8.7 μg Ceftriaxone 22
Cephalexin Propanol–acetic acid 4:1 100 ng Cefixime 31
Diethyl ether–acetonitrile– Cefadroxil 38
ethyl acetate 2:1:2 5 μg
The development time was 30–35 min and the temperature 25 ± 2°C
Acetone–acetic acid 1:1 40 ng
Butanol–acetic acid–water 3:1:2 400 ng
Table 4
Chloroform–ethanol–acetic acid
Resolution of selected penicillins on silica gel G layers impregnated
5:2.5:0.3 400 ng
with 0.2% NH4Cl with propanol–acetic acid 4:1 (v/v) as mobile phase.
Acetonitrile–water 4:1 40 ng
Amoxicillin Ampicillin
Acetone–ethyl acetate 1:1 5 μg
Cefoperazone Chloroform–ethanol–acetic acid Benzylpenicillin 1.27 1.33
5:2.5:0.3 500 ng Ampicillin 2.6 –
Butanol–acetic acid–water 3:1:2 200 ng
Ceftriaxone Butanol–acetic acid–water 3:1:2 10 μg
Table 5
Chloroform–ethanol–acetic acid
5:2.5:0.3 10 μg Resolution of selected cephalosporins on silica gel 60 plates impreg-
nated with 0.2% NH4Cl with butanol–acetic acid–water 4:1:2 (v/v) as
Cefixime Butanol–acetic acid–water 3:1:2 5 μg mobile phase.
Propanol–water 4:1 10 μg
Sample Cefadroxil Cefixime Ceftriaxone Cefoperazone
Cefadroxil Butanol–acetic acid–water 3:1:2 100 ng
Cephalexin 1.00 2.57 4.57 1.14
Chloroform–ethanol–acetic acid
5:2.5:0.3 200 ng Cefoperazone 2.28 4.33 6.66 –
Ceftriaxone 3.42 2.33 – –
Cefixime 1.42 – – –
Table 2

hRF values of penicillins on silica gel G layers bulk impregnated with

0.2% NH4Cl with propanol–acetic acid 4:1 (v/v) as mobile phase. ammonium chloride solution (0.2%). These were dried
overnight at 60 ± 2°C.
Compound hRF
Solutions of the purified cephalosporins (1%) and penicillins
Benzylpenicillin 73 (10–3 M) were spotted on the plates with a Hamilton microliter
Ampicillin 80 syringe (10 μL). One or two drops of dilute HCl were added for
ampicillin and amoxicillin. Samples were applied 2.0–2.5 cm
Amoxicillin 65
from the bottom of the plate. For determination of detection
The development time was 30–35 min and the temperature 25 ± 2°C limit various dilutions of the sample solution were prepared and
applied to the plates.
Chromatograms were developed as mentioned elsewhere [18].
watch glass. The crystals obtained were checked for purity by Detection was performed in UV Chamber (long-wavelength UV
measurement of melting point and acquisition of IR spectra. 365 nm) and fluorescent spots appeared. These were marked and
hRF (RF × 100) values were determined.

2.3 TLC

TLC was performed on silica gel G (E. Merck, Mumbai, India)
containing calcium sulfate (13%), iron, and chloride (0.03%
each) and on ready made TLC silica gel 60 plates (Merck,
Darmstadt, Germany). Impregnated TLC plates were prepared
by spreading slurry of silica gel G (50 g) in 100 mL ammonium
chloride solution (0.2%) with a Stahl-type applicator and then
drying the plates overnight at 60 ± 2°C in an oven. Ready made
silica gel 60 plates were impregnated by development with
3 Results and Discussion
A variety of mobile phase reported for use on fluorescent
layers [3] were tried on ammonium chloride-impregnated silica
gel G layers. After several runs the concentration of ammonium
chloride was optimized as 0.2%. Suitable changes in mobile
phase were made and fluorescent spots of the samples were
observed in the UV chamber. Various mobile phases systemati-

436 Journal of Planar Chromatography 22 (2009) 6


Figure 1
Chromatogram showing separation of benzyl penicillin, ampicillin,
and amoxicillin.
cally established for separation of β-lactams are listed in
Table 1. The detection limit for each compound is also reported.
The detection limits for the β-lactams were quite low, i.e. the
method is very sensitive. hRF values of the penicillins and
cephalosporins and their resolution, R (= d/(w1 + w2/2), where d
is the distance between zone centers and w1 and w2 are the widths
of the zones) were calculated; the values are listed in
Tables 2–5. Pairs of compounds for which resolution was ≥1 are
regarded as separated. It is clear from the tables that
propanol–acetic acid 4:1 (v/v) enables successful separation of
benzylpenicillin, ampicillin, and amoxicillin on silica gel G
layers impregnated with 0.2% ammonium chloride, and that
butanol–acetic acid–water 4:1:2 (v/v) enables separation of the
five cephalosporins (cephalexin, cefoperazone, ceftriaxone,
cefixime and cefadroxil) on silica gel 60 plates impregnated
with 0.2% ammonium chloride. All of these cephalosporins
were not resolved on homemade impregnated plates, however,
because of the greater width of the spots. Typical chroma-
tograms are shown in Figures 1 and 2.
It was also observed that results were very sensitive to changes
in the pH of mobile phase, stationary phase, and sample solu-
tions, possibly because of degradation of the β-lactams. The
quantity of acetic acid in the mobile phase also seems to have an
important effect on the separation, and in furnishing compact
spots. An increase in concentration of impregnation reagent
resulted in a decrease of hRF values, indicating formation of
bulkier species between sample and impregnation reagent. The
surface of the TLC plate became uneven when the concentration
of impregnation reagent exceeded 0.25%.
It is possible the fluorescent spot appeared because interaction
between ammonium ion present in the stationary phase and β-
lactam compounds resulted in an electronic transition corre-
sponding to charge-transfer bands [19].
In conclusion, the normal-phase TLC method reported here is
rapid and truly economical. The method is sensitive and is thus
applicable in routine analytical quality control conditions and
can compete well with other, costlier, methods.
Acknowledgment
The authors acknowledge the help of Uttarakhand State Council
for Science and Technology, Dehradun for financial assistance
Journal of Planar Chromatography 22 (2009) 6
Rapid TLC of β-Lactam Antibiotics
Figure 2
Chromatogram showing separation of cephalexin, cefoperazone, cef-
triaxone, cefixime, and cefadroxil.
and the Department of Chemistry IIT Roorkee for providing
facilities.
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Ms received: December 20, 2008
Accepted: June 17, 2009
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