Analysis

of
Coldeze
Super
pharmaceutical cold and flu
remedy solution.
Introduction
Medications intended to treat the common cold often contain a number of active
ingredients which may be separated by various means and analysed. Modern
separation techniques may yield a tranche of both quantitative and qualitative
data regarding the composition of a sample.
An umbrella term for modern separation techniques is chromatography, which
covers a wide range of separatory methods, some of the more widely used
including thin layer chromatography (TLC); gas chromatography (GC), and
reverse phase high performance liquid chromatography (RP - HPLC). Each
approach varies in terms of speed, expense, performance parameters, and so on;
from the more basic information provided by TLC (key data : the Rf = distance
spot
travels
/
distance
solvent
travels)
to
the
multitude
of
quantitative/qualitative/performance parameters which it is possible to
determine via RP-HPLC, for example.
In this procedure, the composition of an oral solution for cold and flu - Coldeze
Super, was investigated and an attempt was made to analyse the exact
proportions of its active ingredients. The stated proportions of the active
substances were 250 mg/5ml paracetamol, 20 mg/ml caffeine and 10 v/v %
ethanol.

Methods and materials

Thin Layer Chromatography for chemical analysis of analgesics
solution
Three TLC plates were spotted with standard solutions for caffeine, paracetamol,
aspirin and ibuprofen, and Coldeze Super, an oral pharmaceutical solution of
unknown content. Each plate was developed with a different mobile phase and R f
values recorded. Solvents used:
1. 95: 5 % ethyl acetate : acetic acid;
2. 100 % ethyl acetate,
3. 50 % ethyl acetate : 50 % hexane
The process initially gave no useful result and so was repeated with a freshly
acquired set of TLC plates, which did then yield useful data.

Analysis of ethanol by GLC

In order to determine the ethanol content of the unknown Coldeze Super
solution, an analytical GLC apparatus containing a packed column at a fixed
temperature with a nitrogen flow rate of approx. 50 cm 3.min-1 and flame ion

detector was utilized, with the method of internal standards for calibration.
(Waters Alliance 2695 System 28, Column: Zebron Waxplus, Column Length
15 metres, Carrier gas hydrogen 5.5psi Flow Rate - ~3ml/min, Injection Temp
100C Injection Temp 230 C Detector 1: 150).

Solutions, as shown in Table 1, were made up in 25cm3 volumetric flasks to the

proportions shown, topped up with distilled water, and mixed thoroughly. From
each flask, approximately 1l was injected into the apparatus.

Table 1. GLC calibration standard preparation

No.

Ethanol
/cm3

1
2
3
4
5
6
7
8
9
10

0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50

Gives %
(v/v)
Ethanol
1
2
3
4
5
6
7
8
9
10

Propan1-ol
/
cm3
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50

Gives %
(v/v)
Propanol
6
6
6
6
6
6
6
6
6
6

A malfunction with the GLC apparatus arose approximately halfway through the
laboratory session, which prevented any further data from being collected. As an
exercise in gaining familiarisation with the techniques involved, data from a
previous session were used in order to plot absolute peak area and peak area
ratios for ethanol and propan-1-ol. (Table 2)

Table 2: Data from a previous session

Tables 3 & 4. Determination of mean and SD

EtOH
23489.3
area 1
4
(V.s)
EtOH
area 2 27901
(V.s)

Peak
area
ratio
Peak
area
ratio 2

0.65
0.67627
9

EtOH
27270.5
area 3
3
(V.s)
EtOH
area
mean

26220.2
9

EtOH
area SD

2385.98
8

Peak
area
ratio 3
Peak
area
ratio
mean
Peak
area
ratio SD

0.65302
5
0.66

0.01372
7

Analysis of paracetamol and caffeine by HPLC

Instrumental Conditions
Mobile phase: 50:50 v/v MeOH:H2O
Injection loop: 20 microlitres
Column: 25 cm, 4mm i.d .containing 5 m silica coated with octadecylsilane (5micrometres C18)
Detector: 273nm, ~0.1 AUFS
Flow rate: ~1 cm3 min-1

Premarked solutions of paracetamol and caffeine were provided of varying

concentrations as shown in Table 5 below. Each was injected in turn and
chromatographs were obtained for all.

Table 5. Concentrations of paracetamol and caffeine

Solutio
n

Paraceta
mol
concentra
tion
(mg.dm-3)
500

100

50

400

300

200

Paraceta Caffeine
mol area concentra
(V.s)
tion
(mg.dm-3)

Caffeine
area (V.s)

9622455.
1
122868.6
9
1693137.
09
690865.4
9
3937017.
1
2183863.
11
1544197.
36

500

2762202.4

100

526923.5

400

1740623.09

50

3606631.71

200

1185123.68

300

1295327.94

A rough calibration plot was plotted from the data. Due to time constraints it was
not possible to run a duplicate calibration chromatogram.

Next, 2.5cm3 of the unknown oral solution was diluted to 50 cm 3 with methanol,
labelled, then from this, 5 cm3 was taken and diluted to 50cm -3 with methanol.
This was injected and a chromatogram was obtained.

Results and discussion

Thin layer chromatography
As a simple, inexpensive method for basic chemical separation, thin layer
chromatography was an ideal first line procedure.
A first set of chromatograms failed to develop at all, indicating a problem with
the TLC plate such as prior adsorbtion of water. From the second set, similarities
in Rf values were found in two out of the three TLC solvent systems used,
strongly indicating the presence of paracetamol and caffeine in the unknown
sample.
In the acid containing system, no solution movement at all was detected, which
could again indicate a problem with the TLC plate.
The most efficient system in terms of solute separation distance was found to be
the 50 : 50 hexane : ethyl acetate which was in line with expectations since the
compounds in question contain both polar and non-polar moieties. Identical Rf
values were recorded for the unknown and the standard paracetamol sample,
and fairly close values for the caffeine. A mixture of other proportions of solvent
in order to achieve a further variance/ spread of polarities may separate with
further accuracy.
Table 6. TLC data
Mobil
e
phas
e

Solve
nt
front

95:5
100
50:5
0

5
5
5

Parac
etam
ol
front
(cm)
2.8
0.7

Parace
tamol
Rf
value

Caffeine
front
(cm)

Caffein
e
Rf
value

Ibuprofen
front (cm)

Ibuprofe
n
Rf
value

Unkno
wn
front 1

Unknow
n f. 1
value

Unkn
own
front
2

U2 Rf value

1.79
7.14

0.85
0.3

5.88
16.6

4.4
3.8

1.14
1.32

0.9
0.25

5.6
20

2.7
0.7

1.85
7.14

Analysis of ethanol by GLC

Since the volumes of liquid used in GC at the microlitre scale, errors are often
introduced during calibration. In order to mitigate this, an indirect method of
standardisation is used; the method of internal standards. However, with this

method, great care must be taken to ensure accuracy. The data used in this
investigation were of dubious accuracy.

EtOH in unknown =
23489.34; 29275.53, 23489.34
Mean area EtOH in unknown = 24751.40
SD = 2185.95

Calculation of unknown: y = mx + c

y = 4267.6x 5593.2
(y+5593.2) / 4267.6 = x
x = 7.11

The concentration of EtOH in the unknown was found to be 7.11 % v/v

Analysis of paracetamol and caffeine by HPLC

Errors appear
to have been
introduced
during
the
procedure of
calibration.
This
was
noted during
the laboratory
session
but
time
constraints
restricted the
availability of
the
HPLC
apparatus. In future the researchers will need to plan more proactively rather
than relying on scheduled as/when availability of equipment.

Fig 1 Sample chromatograph

References
Higson. Seamus. Analytical Chemistry