Thin layer chromatographic analysis was performed on various beta-lactam antibiotics. The methodology involved preparing TLC plates, applying antibiotic samples as spots, developing the plates in solvent systems, and detecting separated components. Iodine vapor, potassium iodide-iodine, and ninhydrin solutions after alkaline hydrolysis were used for detection. The results showed separation of some antibiotics like aminopenicillins and first generation cephalosporins was difficult. Detection was possible via fluorescence under UV light or by color reactions after spraying detection reagents. Thin layer chromatography is useful for preliminary screening and analysis of beta-lactam antibiotics.

1. Thin Layer Chromatographic Analysis of
Beta – Lactam Antibiotics
Submitted to:
Dr. Ranjit Mohapatra
M. Pharm, Ph. D
Lecture in Pharmachemistry
UDPS, Utkal University
Presented By:
Satyajit Ghosh
B. Pharm 7th Semester
Roll No.: - 16137 V 18 7034
UNIVERSITY DEPARTMENT OF PHARMACEUTICAL SCIENCES, UTKAL UNIVERSITY

2. Contents
 Aim and Objective
 Introduction
 Principle
 Drug Profile
 Mechanism of Action
 Methodology
 Preparation of TLC plates
 Application of sample
 Development of sample
 Detection
 Results
 Advantages of TLC
 Disadvantages of TLC

3. Aim and Objectives
The summery and primary objective of the proposed work is: -
 To determine the number of component in a mixture
 To identify the substances
 To monitor the progress of a reaction
 To determine the effectiveness of a purification
 To determine the appropriate conditions for a column chromatographic separation.

4. Introduction
 Among all the antibiotics, the group of beta-lactams ranks first regarding both the number
of compounds on the market and also in terms of their use in the treatment of infectious
disease. The most frequently used beta-lactams are the group of penicillins and
cephalosporins respectively [1].
 The most popular analytical methods for the determination of beta-lactams are the
chromatographic ones, including high performance liquid-chromatography (HPLC) and thin
layer chromatography (TLC) [2].
 TLC is a less expensive and less complicated chromatographic procedure, which can be
successfully used, in the preliminary screening of pharmaceutical substances.
 In modern analysis, TLC is usually used as a separation method, which establishes the
presence or absence of beta-lactam antibiotics above a defined level of concentration [3].
 Thin layer chromatography (TLC) is a quick, sensitive, and inexpensive technique used to
determine the number of components in a mixture, verify the identity and purity of a
compound, monitor the progress of a reaction, determine the solvent composition for
preparative separations, and analyze the fractions obtained from column chromatography
[3].
Continue…….

5. Introduction
 Thin-layer chromatography (TLC) is a chromatography technique used to separate non-
volatile mixtures. Thin-layer chromatography is performed on a sheet of an inert substrate
such as glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent
material, usually silica gel, aluminum oxide (alumina), or cellulose. This layer of adsorbent
is known as the stationary phase [2].
 After the sample has been applied on the plate, a solvent or solvent mixture (known as the
mobile phase) is drawn up the plate via capillary action.
 Because different analytes ascend the TLC plate at different rates, separation is achieved.
The mobile phase has different properties from the stationary phase. For example, with
silica gel, a very polar substance, non-polar mobile phases such as heptane are used.

6. Principle
 The principle of separation is adsorption. One or more compounds are spotted on a thin
layer of adsorbent coated on a chromatographic plate.
 The mobile phase solvent flows through because of capillary action (against gravitational
force). The components move according to their affinities towards the adsorbent.
 The component with more affinity towards the stationary phase travels slower. The
component with lesser affinity towards the stationary phase travels faster [2].
 Thus, the components are separated on a thin layer chromatographic plate based on the
affinity of the components towards the stationary phase.
 During development, molecules are continuously moving back and forth between the free
and adsorbed states.
 A balance of intermolecular forces determines the position of equilibrium and thus the
ability of the solvent to move the solute up the plate [5].
Continue…….

7. Schematic representation of ascending development chamber
for conventional TLC

8. Principle
 This balance depends on: -
 The polarity of the TLC coating material,
 The polarity of the development solvent,
 The polarity of the sample molecule(s).
 The information provided by the chromatogram includes
the migration behavior of the separated substances, given
by the Rf value: -
𝐑𝐟 𝐯𝐚𝐥𝐮𝐞 =
𝐃𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐭𝐫𝐚𝐯𝐞𝐥𝐥𝐞𝐝 𝐛𝐲 𝐬𝐨𝐥𝐮𝐭𝐞 (𝐒𝐭𝐚𝐫𝐭𝐢𝐧𝐠 𝐳𝐨𝐧𝐞 − 𝐒𝐮𝐛𝐬𝐭𝐚𝐧𝐜𝐞 𝐙𝐨𝐧𝐞)
𝐃𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐭𝐫𝐚𝐯𝐞𝐥𝐥𝐞𝐝 𝐛𝐲 𝐬𝐨𝐥𝐯𝐞𝐧𝐭(𝐒𝐭𝐚𝐫𝐭𝐢𝐧𝐠 𝐳𝐨𝐧𝐞 − 𝐒𝐨𝐥𝐯𝐞𝐧𝐭 𝐟𝐫𝐨𝐧𝐭)

9. Drug Profile
 β-lactam antibiotics (beta-lactam antibiotics)
are antibiotics that contain a beta-lactam ring in
their molecular structure.
 This includes penicillin derivatives (penams),
cephalosporins and cephamycins (cephems),
monobactams, carbapenems and carbacephems
[6].
 Most β-lactam antibiotics work by inhibiting cell
wall biosynthesis in the bacterial organism and
are the most widely used group of antibiotics [1].
 The first β-lactam antibiotic discovered,
penicillin, was isolated from a rare variant of
Penicillium notatum [5].
 Bacteria often develop resistance to β-lactam
antibiotics by synthesizing a β-lactamase, an
enzyme that attacks the β-lactam ring. To
overcome this resistance, β-lactam antibiotics
can be given with β-lactamase inhibitors such as
clavulanic acid.

10. Mechanism of Action
 β-lactam antibiotics are bactericidal, and act by inhibiting the
synthesis of the peptidoglycan layer of bacterial cell walls.
 The final transpeptidation step in the synthesis of the
peptidoglycan is facilitated by DD-transpeptidases, also known
as penicillin binding proteins (PBPs).
 PBPs vary in their affinity for penicillin and other β-lactam
antibiotics. The number of PBPs varies between bacterial
species.
 β-lactam antibiotics are analogues of d-alanyl-d-alanine—the
terminal amino acid residues on the precursor NAM/NAG-
peptide subunits of the nascent peptidoglycan layer.
 The structural similarity between β-lactam antibiotics and d-
alanyl-d-alanine facilitates their binding to the active site of
PBPs.
 The β-lactam nucleus of the molecule irreversibly binds to
(acylates) the Ser403 residue of the PBP active site. This
irreversible inhibition of the PBPs prevents the final
crosslinking (transpeptidation) of the nascent peptidoglycan
layer, disrupting cell wall synthesis.
Continue…….

11. Mechanism of Action
 β-lactam antibiotics block not only the division of bacteria,
including cyanobacteria, but also the division of cyanelles, the
photosynthetic organelles of the glaucophytes, and the division of
chloroplasts of bryophytes.
 In contrast, they have no effect on the plastids of the highly
developed vascular plants. This is supporting the endosymbiotic
theory and indicates an evolution of plastid division in land
plants.
 Under normal circumstances, peptidoglycan precursors signal a
reorganization of the bacterial cell wall and, as a consequence,
trigger the activation of autolytic cell wall hydrolases.
 Inhibition of cross-linkage by β-lactams causes a build-up of
peptidoglycan precursors, which triggers the digestion of existing
peptidoglycan by autolytic hydrolases without the production of
new peptidoglycan. As a result, the bactericidal action of β-lactam
antibiotics is further enhanced.

12. METHODOLOGY
(Preparation of TLC plates)
 After preparing the slurry, which is a mixture of stationary phase and water, the TLC plates can
be prepared by using any one of the following techniques pouring, dipping, spraying and
spreading [5].
 In pouring technique, the slurry is prepared and poured on to glass plate which is maintained on
a levelled surface. The slurry is spread uniformly on the surface of the glass plate. After setting,
the plates are dried in an oven. The disadvantage is that uniformity in thickness can be ensured.
 In dipping technique, two plates (either of standard dimensions or microscopic slides) are
dipped in to the slurry and are separated after removing from slurry and later dried. The
disadvantage is that a larger quantity of slurry is required even for preparing fewer plates [4].
 Spraying technique resembles that of using a perfume spray on a cloth. The suspension of
adsorbent or slurry is sprayed on a glass plate using a sprayer. The disadvantage is that the layer
thickness cannot be maintained uniformly all over the plate.
 Spreading is the best technique where a TLC spreader is used. The glass plates of specific
dimensions (20cm × 20cm/10cm/5cm) are stacked on a base plate. The slurry after preparation
is poured inside the reservoir of TLC spreader. The thickness of the adsorbent layer is adjusted
by using a knob in the spreader [6].
Continue…….

13. METHODOLOGY
(Preparation of TLC plates)
 Normally a thickness of 0,25mm (150-250 μm) is used for analytical purpose and 2mm
thickness for preparative purpose. Then the spreader is rolled only once on the plates. The plates
are allowed for setting (air drying) at 80-90℃ for about 30 min. This is done to avoid cracks on
the surface of adsorbent.
 After setting, the plates are activated by keeping in an oven at 100℃ to 120℃ for 1 hour.
Activation of TLC plates is nothing but removing water / moisture and other adsorbed
substances from the surface of any adsorbent, by heating at high temperature so that adsorbent
activity is retained. The activated plates can be stored in thermostatically controlled oven or in
desiccator and can be used whenever required [2].

14. METHODOLOGY
(Application of sample)
 The most common technique for TLC sample application is to use a glass capillary spotter. The
spotting capillaries are extremely small and easy to make from glass Pasteur pipets.
 Heat the middle part of the Pasteur pipet over a Bunsen burner blue flame. Hold both ends
carefully, as they will get hot.
 When the middle part is hot enough, it will become pliable; pull both ends apart quickly and
smoothly. This will draw the middle part into a thin string-like tube.
 After cooling for a few minutes, gently break the middle string-like part into 2- in. sections to
provide good TLC capillary spotters. The end should be as flat as possible, as a jagged edge will
lead to unsymmetrical spots or no spots on the TLC plate.
Continue…….

15. METHODOLOGY
(Application of sample)
 Fill the prepared capillary spotter by quickly dipping it into the organic sample solution. With
aqueous solutions, filling will be much slower—an Eppendorf pipettor with 10-μl tip is thus
recommended for applying aqueous sample solutions [4].
 Place the capillary spotter at the starting line (labeled in pencil) on the coated side of the plate
vertically and carefully, to allow capillary action to draw the solution onto the plate. If more than
one sample is running at the same time, make sure to properly label the plate with a pencil and
use a different capillary for each sample to avoid contamination [6].
 Blow with cold or hot air to facilitate solvent evaporation of the applied sample.

16. Methodology
(Development of sample and development tank)
 In most cases, ascending TLC is applied in a TLC
chamber as for development once with a single
solvent system (single development). Commercial
TLC chambers of different sizes are readily
available, but usually a small wide-mouth flat
bottom glass jar/bottle with a lid large enough to
fit the TLC plate works just as well [11].
 First, fill the chamber with development solvent to
a depth no greater than 0.5 cm.
 Use tweezers to place the TLC plate in the prepared
development chamber with its back layer leaning
against the chamber’s inside wall, and immediately
cover the chamber with the lid [4].
Continue…….

17. Methodology
(Development of sample and development tank)
 Make sure that the starting line is above the solvent level. Saturation of the chamber atmosphere
with solvent vapor is important for TLC.
 Development starts once the TLC plate is immersed; when the solvent front has reached an
appropriate level (usually within 0.7 cm of the top of the plate), quickly remove the lid, take out
the plate with tweezers, and mark the solvent front with a pencil.
 Allow the plate to dry in a fume hood or with a heat gun (heat gently) before proceeding to the
visualization step.

18. Methodology
(Detection)
 Three detection procedures were used; first with iodine vapors and then using in situ plate color
reactions with iodine and ninhydrin, after an alkaline hydrolysis.
 A few iodine crystals were placed on the base of tightly sealed chromatographic chamber, stored
in a fume cupboard. After a few hours during which violet iodine vaporizes and distributes itself
homogenously throughout the interior of the chamber, the chromatographic plates were
introduced in the chamber. After 30 minutes the plates were sprayed with a 1% starch solution
[4].
 Chromatograms were first sprayed with a 1N sodium hydroxide solution, in order to hydrolyze
the beta-lactam ring, and after 15 minutes with a solution containing 0.2 g potassium iodine, 0.4
g iodine dissolved in 20 ml ethanol and 5 ml 10% hydrochloric acid.
 Chromatoplates were first sprayed with a 1N sodium hydroxide solution, in order to hydrolyze
the beta-lactam ring, and after 15 minutes with a 0.1% ninhydrin solution in ethanol, and heated
in an oven at 120 °C for 10 minutes [12].

19. Results
 All beta-lactams can be detected in UV light at 254 nm (green fluorescence) and 366 nm (blue
fluorescence).
 Applying reagents such as ninhydrin or exposing the chromatoplate to iodine vapor can diminish the
detection limit.
 Difficulties appear at the separation of the two aminopenicillins (AMP, AMO) and of the three first
generation oral cephalosporins (CEL, CFD, CFC).
 When detection takes place by spraying the chromatoplate with an iodine-potassium iodide solution,
the iodine contained in the reagent reacts chemically with the penicilloic acid, as the beta-lactam ring
is initially opened by alkaline hydrolysis.
 The subsequent treatment with starch solution employed after the iodine treatment for the
stabilization and enhancement of the “iodine” chromatogram zones cannot be employed here since the
layers - even after evaporation of the excess iodine - still contain so much iodine that the whole
background is colored blue.
 Aminopenicillins (AMP, AMO) proved to be more sensitive to ninhydrin reaction after an alkaline
hydrolysis than PEN or OXA, which were scarcely visible.

20. Chromatogram obtained at the separation of penicillins
using mobile phase III (ethyl acetate – water – acetic acid
60:20:20), detection in UV light at 254 nm(A) and 366
nm(B) (1 - AMP, 2 - AMO, 3 – PEN, 4 - OXA)
Chromatogram obtained at the separation of cephalosporins using
mobile phase III (ethyl acetate – water – acetic acid 60:20:20),
detection in UV light at 254 nm (A) and 366 nm (B) (1 - CFD, 2 - CEL, 3
– CFC, 4 – CFR, 5 – CZI, 6 - CTR)

21. Results
 Cephalosporins couldn’t be detected clearly with ninhydrin, as ninhydrin reacts actually with a
degradation product of penicillins in alkaline environment, D-penicillamine.
 In general, amino - acid related compounds could be visualized using the ninhydrin reagent. Penicillins
can be also detected by spraying the plates with sulphuric acid – formaldehyde reagent (37%
formaldehyde solution in conc. sulphuric acid 1:10), dark yellow spots were detected for the two
aminopenicillins (AMP, AMO), PEN gave a brown spot while OXA a yellow one.
 Dragendroff’s reagent (solution of potassium bismuth iodide) was also applied for detection, but beta-
lactams proved to be less sensitive to this commonly used chromatographic reagent, as all the
penicillins gave a very pale orange spot.
 Although the stability of beta-lactam antibiotics in solid state is generally satisfying, beta-lactams
dissolved in water or other solvents gradually convert to different degradation products through
hydrolysis.
 After dissolution of each antibiotic the sample solutions were analyzed using mobile phase III, several
times over duration of a week. The extent of the hydrolysis of beta-lactams is highly dependent on the
time and temperature.
 Some degradation products detectable by TLC were found in the case of PEN after 48 hours storage.
Possible degradation was noticeable on the chromatoplate for OXA, CEL, CFD and CFC after samples
were stored in a refrigerator for a week, as they produced more than one spot on the TLC plate.

22. Rf values of the studied beta-lactams in the six-development system
Beta lactam
derivatives
Rf values
I II III IV V VI
AMP 0.43 0.45 0.48 0.36 0.40 0.45
AMO 0.38 0.41 0.42 0.34 0.38 0.39
PEN 0.79 0.82 0.87 0.66 0.75 0.80
OXA 0.84 0.85 0.92 0.71 0.77 0.84
CFD 0.15 0.17 0.19 0.14 0.15 0.18
CEL 0.20 0.20 0.22 0.18 0.21 0.24

23. Colours and fluorescence of beta-lactams developed by the six TLC systems
Beta lactam derivatives Detection in (with)
UV 254 nm UV 366 nm Iodine vapors Ninhydrin reaction
AMP
Brown Fluorescent blue Yellow – Brown Reddish
AMO
Brown Fluorescent blue Yellow – Brown Reddish
PEN
Brown Fluorescent green Yellow Pale Red
OXA
Brown Fluorescent green Yellow Pale Red
CFD
Brown Fluorescent blue Yellow – Orange Pale Red
CEL
Brown Fluorescent blue Yellow – Orange Pale Red

24. ADVANTAGES OF TLC
 Simple method and cost of the equipment is low.
 Rapid technique and not time consuming like column chromatography.
 Separation of ug of the substances can be achieved.
 Any type of compound can be analyzed.
 Efficiency of separation: Very small particle size can be used which increases the efficiency of
separation. Flow rate is not altered because of the particle size since it is not a closed column. It
is a planar type having thin layer of adsorbent.
 Detection is easy and not tedious.
 Capacity of the thin layer can be altered. Hence analytical and preparative separations can be
made.
 Corrosive spray reagents can be used without damaging the plates.
 Needs less solvent, stationary phase and time for every separation when compared to column
chromatography.

25. DISADVANTAGES OF TLC
 The results obtained from the experiment are difficult to reproduce.
 Applicable for soluble mixture components only.
 Qualitative analysis, not quantitative analysis.
 Not an automatic process.
 The humidity and temperature can affect the results as Thin layer chromatography works in an
open system.
 Separation process takes place only to a certain length as plate length is limited.

26. Reference
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 Wall P. Thin-layer chromatography – A modern practical approach, RSC Chromatography Monographs.
London: Royal Society of Chemistry; 2005.
 European Pharmacopoeia. 7th ed. Strasbourg: Council of Europe; 2010.
 Hendrickx S, Roets E, Hoogmartens J, Vanderhaeghe H. Identification of penicillins by thin-layer
chromatography. J Chromatogr A 1984; 291:211-8.
 Quintens I, Eykens J, Roets E, Hoogmartens J. Identification of cephalosporins by thin layer
chromatography and color reactions. J Plan Chromatogr 1993; 6:181-6.
 Nabi SA, Laiq E, Islam A. Selective separation and determination of cephalosporins by TLC on stannic
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 Choma IM. TLC Separation of cephalosporins: searching for better selectivity. J Liq Chromatogr Relat
Technol 2007;30(15):2231-40.
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11th ed. Philadelphia: Lippincott Williams and Wilkins; 2004.