1094  Tavallali et al.: Journal of AOAC International Vol. 94, No.
4, 2011
DRUG FORMULATIONS AND CLINICAL METHODS
An Efficient and Simultaneous Analysis of Caffeine and
Paracetamol in Pharmaceutical Formulations Using TLC
with a Fluorescence Plate Reader
Hossein Tavallali, S. Faraneh Zareiyan J., and Maryam Naghian
Payame Noor University, Department of Chemistry, 19395-4697, Tehran, I.R. of Iran
A simple, rapid, and efficient method using
TLC with a fluorescence plate reader has been
described for simultaneous determination
of caffeine and paracetamol. Determination
was carried out using the fluorescence-
quenching action of caffeine and paracetamol
on a TLC plate with a fluorescent indicator at
λex = 254 nm in the linear ranges of 0.2–1.9
and 0.03–1.5 µg/L, respectively. Separation
of caffeine and paracetamol were performed
on the TLC plate, and the best results were
obtained using the optimized mobile phase
n-hexane–ethyl acetate–ethanol (2.5 + 1.5
+ 0.4, v/v). Some important parameters,
such as solvent type and ratio of the mobile
phase, the presence of other components,
and instrumental parameters, were studied.
Caffeine and paracetamol detection limits
were 0.025 and 0.032 µg/L, and RSD values for
0.6 µg/L caffeine and 0.06 µg/L paracetamol
(n = 5) were 1.93 and 2.06%, respectively.
Using this technique, some pharmaceuticals
containing caffeine and paracetamol were
analyzed with satisfactory results.
C
affeine (1, 3, 7-trimethylxanthine) is mainly
ingested by drinking coffee (1, 2). Paracetamol
(acetaminophen or N-acetyl-4-aminophenol) is a
popular antipyretic and analgesic agent. In several countries,
it is one of the most used alternatives to acetylsalicylic acid (3).
The use of a mixture of caffeine and paracetamol is
well established in pharmaceutical formulations (4, 5). In
order to achieve a better curative effect and lower toxicity,
it is very important to control the content of caffeine and
paracetamol in pharmaceutical tablets (6).
Hyphenated techniques combine chromatographic
and spectral methods to exploit the advantages of both.
Chromatography produces pure or nearly pure fractions
of chemical components in a mixture, and spectroscopy
Received May 15, 2010. Accepted by SW August 15, 2010.
Corresponding author’s e-mail: Tavallali@pnu.ac.ir, Tavallali@yahoo.com
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produces selective information for identification using
standards or library spectra (7).
TLC is one of the simplest, most useful, and most widely
applicable forms of chromatography for separation of
components yet devized. Analysis of separated components
is often carried out based on fluorescence or fluorescence
quenching (8–10).
A plate reader accessory can be connected to a
luminescence spectrophotometer and used for measuring
fluorescence on any flat surface. The reader enables an
operator to scan plates vertically or horizontally; it can also
be programmed to scan every part, or every single point,
or lines on plates at a definite scan rate and desirable data
interval, as seen in Figure 1 (11).
Solid-surface luminescence analysis is very sensitive
and selective for organic trace analysis. In addition, it is
simple, inexpensive, and rapid, and can be used selectively
for mixture analysis (12). Fluorescence-based techniques
after TLC separation are frequently applied to the detection
and quantitation of sample components (13).
For simultaneous determination of caffeine and
paracetamol in drug formulations, different methods
exploiting such techniques as zero-crossing second
derivative spectrophotometry (14), electrochemical methods
like voltammetric determination using a boron-doped
diamond electrode (15), solid-phase molecular fluorescence
and parallel factor analysis (16), a flow injection UV partial
least-squares multioptosensing device (17), RP capillary
electrochromatography (18), and chemometric methods (19,
20) have been proposed.
The advantages of the proposed method, TLC with
a fluorescence plate reader (FLPR), over the available
analysis methods for caffeine and paracetamol listed
above are simplicity, rapidity, and ease of operation.
While some techniques need expensive and time-
consuming pretreatment or mathematical calculations,
TLC-FLPR is a low-cost method without the complexity
needed in chemometric and electrochemical techniques;
it is suggested for QC testing and measuring various
materials in medical laboratories.
 Tavallali et al.: Journal of AOAC International Vol. 94, No. 4, 2011  1095
Figure  1.  FLWINLAB software for TLC scans.
Experimental
Apparatus and Software
Fluorescence intensity was monitored with a
PerkinElmer (Waltham, MA) LS 50 luminescence
spectrophotometer with a 150 W xenon arc lamp.
Instrumental parameters and processing data were
controlled by the Fluorescence Data Manager software
(FLWINLAB). A plate reader accessory from PerkinElmer
connected to the luminescence spectrophotometer was
applied. Silica gel 60 FTLC aluminum-backed plates
(20 × 20 cm; Part No. 4AJ-9130050; Merck, Darmstadt,
Germany) were also used. A syringe (100 μL, Hamilton
Co.; Bonaduz, Switzerland) was used for precise and
accurate sample spotting on TLC plates.
Figure  2.  Schematic diagram of plate reader and
luminescence spectrophotometer.
Materials and Reagents
All experiments were carried out with analytical
reagent grade chemicals from Merck, and deionized
water was used.
Caffeine and paracetamol stock solutions (0.1 M) were
prepared separately by dissolving 4.8552 and 3.7792 g
analytical grade caffeine and paracetamol, respectively,
in ethyl acetate in 250 mL volumetric flasks. Working
solutions were prepared by appropriate dilution of the
stock solutions.
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Ethyl acetate, n-hexane, and ethanol were used as
mobile phase solvents.
For the interferences study, phenobarbital, phenytoin,
codeine, and aspirin were prepared in the same
concentration with caffeine and paracetamol.
Commercial capsule and tablet products
acetaminophen (paracetamol), caffeine, and aspirin
(ACA; Alborz Ind., Tehran, Iran; Batch No. 08027);
adult cold (Kharazmi Pharmaceutical Co., Karaj, Iran;
Batch No. 119); children cold (Pharmashimi, Karaj, Iran;
Batch No. 130); acetaminophen plus codeine (Kharazmi
Pharmaceutical Co.; Batch No. 24); acetaminophen
(Soha, Tehran, Iran; Batch No. 130); and Novafen
(Alhavi, Karaj, Iran; Batch No. 0390887) were prepared
by dissolving each tablet in ethyl acetate in a 250 mL
volumetric flask.
Procedure
The selected mobile phase mixture, including
n-hexane, ethyl acetate, and ethanol, was optimized for
separation of caffeine and paracetamol to give the best Rf
values. One-microliter spots of caffeine and paracetamol
were applied to the TLC plate according to caffeine and
paracetamol standards using a 100 μL syringe. The plate
was developed with the mobile phase n-hexane–ethyl
acetate–ethanol. Spots were placed at a 10 mm distance
from the bottom of the TLC plate, and then the plate
was left for about 180 s in the developing tank until
the mobile phase reached up to 25 mm. The quenching
effects of caffeine and paracetamol on the TLC plate
were measured by TLC-FLPR at λex = 254 nm and λem =
270  nm (fluorescent indicator wavelengths) in different
concentrations for calculating linear ranges.
Various excipients and active ingredients available in
tablets were also spotted and their interference effects
studied.
At the end of the method development experiments,
various types of pharmaceuticals containing caffeine
and paracetamol, such as ACA, adult cold, children’s
cold, acetaminophen with codeine, acetaminophen, and
Novafen, were analyzed by the same method.
1096  Tavallali et al.: Journal of AOAC International Vol. 94, No. 4, 2011

Table  1.  Mobile phase selection of ethanol reduced tailing of spots. It was concluded
that a mixture of a nonpolar solvent (n-hexane) and a
Rf
Mobile phase moderately polar solvent (ethyl acetate) with ethanol
System and solvent ratioa Caffeine Paracetamol addition ensured the optimum separation between the
S1 Methanol plus one drop NH3 0.20 0.22 tested drugs.
S2 Chloroform–ether–formic acid 33
0.24 0.34
By testing different mobile phase solvent ratios,
(7 + 2 + 1) the best Rf values were obtained with n-hexane–ethyl
S3 Chloroform–methanol–acetic 0.22 0.26
acetate–ethanol (2.5 + 1.5 + 0.4, v/v/v). When the solvent
acid 33 migrated to 25 mm, the first zone with the lowest Rf
(17 + 3 + 1 drop) (0.48) was caffeine, and the second was paracetamol
S4 n-Hexane–ethyl acetate 0.20 0.22 (0.73), which were located at distances of about 12 and
18.4 mm, respectively.

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(1 + 1)
S5 n-Hexane–ethyl acetate 0.48 0.56–0.70
(2.5 + 1.5) Fluorescence Quenching Effect of Caffeine and
S6 n-Hexane–ethyl acetate–ethanol 0.48 0.73 Paracetamol on the Fluorescent Indicator
(2.5 + 1.5 + 0.4)
a Zinc silicate activated by manganese coated on
  All mixtures of solvents are expressed as volume ratios.
TLC plates contains various types of fluorescent
centers that differ in wavelength of emitted radiation,
probability of fluorescence transition, and vibrational
Results and Discussion interaction with the surroundings. The centers are
assumed to be manganese ions with different numbers
of other manganese ions at neighboring sites (clusters).
Scanning Parameters Quenching of fluorescence is due to radiationless
Various slit widths for the excitation and emission processes starting from the excited state of the centers.
beams were examined; the best S/N was obtained by If particular conditions are fulfilled, quenching may
selecting a large slit width, 10 mm for both excitation and also occur from (higher) excited states of a different
emission. According to the number of spots placed on type (34). In this case, caffeine and paracetamol
TLC plates and for the best responses, the scan rates and act as quenchers by binding to manganese centers
data intervals were set at appropriate values, e.g., 300 or and producing complexes. The dependence of the
100 mm/s for scan rates and 0.1, 0.4, or 0.5 mm for data fluorescence intensity upon quencher concentration
intervals. Vertical or horizontal scanning and the number is easily derived by consideration of the association
of scans were carried out based on the number of spots constant for complex formation. This constant (Ka) is
(Figure 2). given by (35):

Mobile Phase Selection

TLC is a simple, rather inexpensive, and robust

separation and identification method for the components
of pharmaceutical mixtures (21, 22). Because silica gel is
a polar substance, a nonpolar mobile phase is necessary
for separation of drugs in NP chromatography (23). The
separation results were expressed as Rf values, which are
the migration distance of the compound from the origin
divided by the migration distance of the mobile phase
front from the origin (24–32).
Table 1 indicates different solvent mixtures in different
ratios used as mobile phases, such as methanol with
one drop of NH3, S1; chloroform–ether–formic acid
(7 + 2 + 1, v/v), S2 (33); chloroform–methanol–acetic
acid (17 + 3 + 1 drop), S3 (33); n-hexane–ethyl acetate
(1 + 1, v/v), S4; and chloroform–n-hexane–ethyl acetate
where [F]0 and [F]f are the fluorescence intensities in the
(2.5 + 1.5, v/v), S5, tested for the separation of caffeine
absence and presence of quenchers, respectively, [FQ]
and paracetamol. The increase in the developing solvent
is the concentration of complex, and [Q] and [F] are the
polarity by adding ethyl acetate to n-hexane enhanced
quencher and fluorophore concentration, respectively.
the migration distance for paracetamol, and addition
 Tavallali et al.: Journal of AOAC International Vol. 94, No. 4, 2011  1097

Table  2.  Analytical figures of merit for caffeine

and paracetamol
Component Linear Correlation
name range, µg/L LOD, µg/L RSD (n = 5) coefficient
Caffeine 0.2–1.9 0.025 (0.6 µg/L) 0.9999
1.93%
Paracetamol 0.03–1.5 0.032 (0.06 µg/L) 0.9999
2.06%

effects on the fluorescence indicator intensity. So the most

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Figure  3.  Quenching effects of caffeine and
paracetamol on the fluorescence intensity of a TLC
plate.
According to the above equations, the quencher
concentration will increase .
Wavelength Selection
As the experiment is carried out according to the
quenching effects of caffeine and paracetamol on TLC
plates purchased from Merck, λex, which is the reported
excitation wavelength of the fluorescent indicator coated
on these plates (manganese-activated zinc silicate), would
be equal to 254 nm. After wavelength scans, λem was set
at 270 nm as the emission wavelength. Fluorescence
intensity of TLC plates at λex = 254 and λem = 270 nm was
about 112 (arbitrary unit).
Effect of Caffeine and Paracetamol Concentration
on the Quenching Intensity of the TLC
Fluorescent Indicator
The most concentrated substances have more quenching
quenching effect can be expected for 1.9 µg/L, while
that of 0.038 µg/L would be the least. The same results
were expected for the paracetamol quenching effect.
The fluorescent quenching value is the only difference
between caffeine and paracetamol, which is less for the
latter (Figure 3). It can be explained by the number of
rings in the caffeine and paracetamol structures.
Because of the linear dependence, quenching data
are usually presented as plots of F0/F versus [Q]. This
plot, which is known as a Stern-Volmer plot, shows
the mechanism of quenching. This plot should yield
an intercept of unity on the y-axis and a slope equal to
KSV, that is, the Stern-Volmer quenching constant. It is
useful to note that 1/KSV is the quencher concentration
at which F0/F = 2, or 50% of the intensity is quenched.
A linear Stern-Volmer plot is generally indicative of a
single class of fluorophores that are all equally accessible
to the quencher. If two fluorophore populations are
present, and one class is not accessible to the quencher,
the Stern-Volmer plots deviate from linearity toward
the x-axis (downward; 36). As can be seen in Figure 4,
fluorescence intensity of the fluorescent indicator coated
on TLC plates showed a linear relationship in the presence
of the quenchers caffeine and paracetamol.
pH Effect
Manganese-activated zinc silicate with maximum
absorption at 254 nm is often used as the fluorescent
indicator on 20 × 20 cm silica gel 60 F254 plates. It is
relatively susceptible to acids, and its fluorescence is
completely quenched by acidic solvents; therefore, all
tests were carried out at relatively neutral pH (37).

Table  3.  Pharmaceuticals investigated as

interferences

Interferences, Caffeine, Paracetamol, Rf for optimized

0.6 µg/L µg/L µg/L mobile phase
Phenobarbital 0.6 ± 2.0 0.4 ± 2.5 0.78
Phenytoin 0.5 ± 2.5 0.5 ± 1.5 0.80
Codeine 0.7 ± 1.5 0.6 ± 1.0 0.75
Figure  4.  Stern-Volmer plots for caffeine and Aspirin 0.5 ± 2.0 0.7 ± 2.5 0.15
paracetamol.
1098  Tavallali et al.: Journal of AOAC International Vol. 94, No. 4, 2011

Table  4.  Caffeine and paracetamol quantitation results for pharmaceutical samples
Taken, mg Found, mg Recovery, %
Tablet weight, Caffeine Paracetamol Caffeine Paracetamol
Tablet name mg content content content content Caffeine Paracetamol
ACA 600.0 32.5 165.5 31.8 163.8 98 99
Novafen 586.0 40.0 325.0 39.6 321.7 99 99
Children’s cold 321.0 — 80.0 — 79.6 — 99.5
Acetaminophen-codeine 374.6 — 300.0 — 294.0 — 98
Acetaminophen 383.0 — 325.0 — 323.3 — 99.5
Adult cold 360.0 — 325.0 — 321.7 — 99

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Analytical Figures of Merit for Caffeine and
Paracetamol
As can be seen in Table 2, this method showed relatively
excellent reproducibility. Using this technique, according
to LOD values and the linear range of calibration curves,
it would be possible to easily measure very small amounts
of caffeine and paracetamol in a short time.
it was found that due to complete separation of these
components from caffeine and paracetamol they had no
effect on the TLC analyses. The experiment showed that
recovery of 0.6 µg/L caffeine and paracetamol did not
significantly change in the presence of these substances
(Table 3).
Applications

Interference Study To evaluate the applicability to real samples and to test

In many pharmaceuticals, substances such as
phenobarbital, phenytoin, codeine, and aspirin are mixed
with caffeine and paracetamol for the best medical effect.
Interference effects of these drugs were studied, and
its accuracy and precision, the method was applied to
simultaneous determination of caffeine and paracetamol in
commercially available pharmaceuticals. The quantitative
results and the curves of this analysis are shown in Table 4
and Figure 5. The good agreement between these results

Figure  5.  Real sample identification: (a) Novafen, (b) acetaminophen tablet, (c) ACA tablet, and (d) adult cold
tablet.
 Tavallali et al.: Journal of AOAC International Vol. 94, No. 4, 2011  1099
and known values indicated the successful applicability
of TLC-FLPR for simultaneous determination of caffeine
and paracetamol in pharmaceutical preparations.
Conclusions
It is concluded that this technique yields good results
for the separation and identification of caffeine and
paracetamol. Using this technique, separation of caffeine
and paracetamol is carried out with excellent precision;
therefore, it can be used in medical and pharmaceutical
laboratories.
Acknowledgments
We gratefully acknowledge the support of this work by
the Shiraz Payame Noor University Research Council.
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