EXERCISE NO.

6
HPLC ANALYSIS OF BENZOIC ACID AND CAFFEINE IN SOFTDRINK SAMPLE

REHANA V. LEBBE
CHEM 137.1 1L

DATE SUBMITTED: NOVEMBER 16, 2016

ABSTRACT
In the experiment, the chromatograms of caffeine and mixed standards
(caffeine and benzoic acid) were determined using UPLC. Optimization of
parameters including flow rate, sample load and solvent system were done
using chromatograms of mixed standards and caffeine. The parts of HPLC
were also explained throughout this paper. Information such as retention
factor, resolution and selectivity factor were calculated and their importance
in column efficiency were explained. Also, HETP and N values were
determined using the three known methods: tangent method, 5 method
and half-height method.

I.

INTRODUCTION:

High performance liquid chromatography (HPLC) is a chromatographic technique used to separate

mixture of compounds. This method is usually used in biochemistry and analytical chemistry to identify,
quantify and purify individual components of mixture. Generally, in liquid chromatography, sample mixture is
eluted through the packed column with a mobile phase supported by high pressure from the pump. The
components are separated from one another based on the interaction of these components with the
stationary phase and the mobile phase. These separated components are detected at the exit of the column
by a detector that can measure the amount of the components present. An output from this detector is known
as a chromatogram. (Harvey, 2000)

Figure 6.1 Typical HPLC unit.

In HPLC, narrow columns with internal diameters 2-80 mm are used. These columns are packed
with particles having an average diameter of less than 50 microns (50 x 10 -6m). Such columns require high
pressures (1000-6000 psi) to maintain a convenient flow of the eluting solvent, usually in the range 0.1-10
mL/min, but occasionally higher. Resolution is considerably superior to that achieved with an ordinary column,
in part because of the tight packing of the stationary phase, which reduces lateral diffusion and because of
the large surface area of the packing. In comparison to HPLC, classical column chromatography employs

columns that are gravity operated which can take hours or days before all components are eluted from the
column. Hence, HPLC has a better analysis time as well as resolution of chromatogram. (Harvey, 2000)
HPLC is especially suited to the analysis of compounds not readily analyzed by gas chromatography.
For example, thermally labile compounds can be analysed by HPLC at ambient temperatures as well as
highly polar or nonvolatile compounds can be analysed. Sample treatment is often minimal since aqueous
solutions can be used in HPLC. Since its inception in the late 1960's, HPLC has made significant practical
impact on the areas of pharmaceutical, clinical, forensic, environmental and industrial research and
development analysis. Preparative HPLC has also found an important use in the isolation and purification of
various compounds. (Harvey, 2000)
For most users of HPLC, analyzing the performance of a chromatographic column consists of
measuring the following four key performance parameters: the column efficiency (N or HETP), the retention
factor (k), and the selectivity factor () for important pairs of analytes. Also, other parameters of column such
as lifetime, chemical inertness, and sample load are also considered.
The objectives of the exercise were to optimize the following parameters: solvent system, best
working flow rate and establish the appropriate sample load. Also, amount of caffeine was quantitatively
determined.
II.

MATERIALS AND METHOD

A set of conditions for the Acquity Waters UPLC includes a C18 column with 17 m pore size, 21x50
mm size. The temperature was set at 45 at 15000 psi with wavelength detector at 210-400 nm.
The first run includes the injection of 10 L of mixed standard at a flow rate of 0.6 mL/min with two
different solvent system: 9:1 water: acetonitrile and 8:2 water: acetonitrile. Second run involves the injection
of 10 L of mixed standard using a solvent system 9:1 water: acetonitrile at two different flow rates: 0.3
mL/min and 0.60 mL/min. The third run involves the injection of 5 L and 10 L of mixed standard with a
flow rate of 0.6 mL/min using a solvent system 9:1 water: acetonitrile.
III.

RESULTS AND DISCUSSION

Liquid chromatography basically employs the idea of the partitioning of the analyte between the
sample-free phase known as mobile phase and another sample-free phase that remains fixed known as the
stationary phase. Once components are eluted along with the mobile phase, interaction of the components
with the stationary phase will determine its retention time on the column. This can be detected by a detector
to produce a chromatogram. (Harvey, 2000)
The sample used in the experiment was Mountain Dew softdrink. Using the UPLC, mixed standards
of benzoic acid and caffeine, caffeine and unknown sample was analyzed for their components. The first run
of involved the injection of 10 L of mixed standard at a flow rate of 0.6 mL/min with two different mobile
phase system: 9:1 water: acetonitrile and 8:2 water: acetonitrile. In this run, effect of changing the composition
of mobile phase is observed.

Table 6.1. Data on the elution of caffeine using 9:1 water:acetonitrile solvent system.
Name
Caffeine
-

Retention Time
0.259
0.282
0.637
1.845

Area
19014
43110
234663
8

%Area
6.41
14.53
79.07
0

Table 6.2. Data on the elution of caffeine using 8:2 water:acetonitrile solvent system.
Name
Caffeine

Retention Time
0.482

Area
2924870

Height
2592665

As seen from the data gathered, four peaks were observed upon use of 9:1 ACN:water in comparison
to the one peak observed for the 8:2 ACN:water. Also, caffeine was retained far longer with use of less polar
mobile phase than use of more polar mobile phase. This shows that the best solvent system was 9:1
ACN:water.
Elution order of the analyte in HPLC is governed by polarity. In a normal-phase separation the least
polar solute spends proportionally less time in the polar stationary phase and is the first solute to elute from
the column. Retention times are controlled by selecting the mobile phase, with a less polar mobile phase
leading to longer retention times. (Harvey, 2000) Thus, upon use of 9:1 ACN:water, it is expected to have
better resolution due to longer retention times in comparison to 8:2 ACN:water. This is proven from the data
gathered from the experiment since components were more detected using the less polar mobile phase.
In the chromatography done in the experiment, the stationary phase is covalently bonded to silicate
particles. Bonded stationary phases are attached by reacting the silica particles with an organochlorosilane
of the general form Si(CH3)2RCl, where R is an alkyl or substituted alkyl group. The properties of a stationary
phase are determined by the nature of the organosilanes alkyl group. If R is a polar functional group, then
the stationary phase will be polar. Since the stationary phase is polar, the mobile phase used is a nonpolar
solvent. (Harvey, 2000)
Table 6.3. Data obtained for the mixed standards at 0.30 mL/min flow rate.
Name
Retention Time
Area
%Area
0.433
878
0.22
Caffeine
0.515
94652
24.04
0.55
69988
17.78
Benzoic acid
1.192
228173
57.96

%Height
201
38398
45158
121632

Table 6.4. Data obtained for the mixed standards at 0.60 mL/min flow rate.
Name
Retention Time
Area
%Area
0.259
17949
10.34
Caffeine
0.278
41383
23.84
0.412
44
0.03
Benzoic acid
0.64
113700
65.49
1.199
528
0.3

%Height
17269
45978
45
104783
312

The effect of different flow rates shows that longer flow rate results to better resolution of peaks in
comparison to shorter flow rates with less resolution of peaks. This is shown in data gathered in the
experiment. This is because longer flow rates allow the components of the analyte to interact with the mobile
and stationary phases longer. Once these components are retained, separation of the benzoic acid and
caffeine is distinctive from the chromatogram.
Table 6.5. Data obtained for the mixed standards with 10 L sample load.
Name
Retention Time
Area
%Area
0.259
17949
10.34
Caffeine
0.278
41383
23.84
0.412
44
0.03
Benzoic acid
0.64
113700
65.49
1.199
528
0.3

%Height
17269
45978
45
104783
312

Table 6.6. Data obtained for the mixed standards with 5 L sample load.
Name
Retention Time
Area
%Area
0.259
7976
9.2
Caffeine
0.267
22370
25.8
Benzoic Acid
0.626
56375
65.01

%Height
15345
27290
52795

On the other hand, effect of the amount of sample load shows that the sample is introduced using a
loop injector. Sampling loops are interchangeable, and available with volumes ranging from 0.5 mL to 2 mL.
A syringe with a capacity several times that of the sampling loop is used to place the sample in the loop. Any
extra sample beyond that needed to fill the sample loop exits through the waste line. After loading the sample,
the injector is turned to the inject position. In this position the mobile phase is directed through the sampling
loop, and the sample is swept onto the column. (Harvey, 2000) Hence, effect of amount of sample load should
not affect the peaks in the chromatogram. However, results showed a better resolution of peaks for the 10
L compared to sample load of 5 L.
The standard chromatogram eluted with mixed standard of equal amounts of benzoic acid and
caffeine. Theoretically, polar stationary phase should retained the more polar component. Comparing the
structure and polarity of benzoic acid and caffeine, it is shown that the benzoic acid is more polar compared
caffeine. Thus, elution order observed should be caffeine, first, and benzoic acid.

Figure 6.1. Structure of benzoic acid

Figure 6.2. Structure of caffeine

If there is no standard chromatogram present, other detectors could be used such as mass
spectroscopy, diffractional refractometer or UV-vis spectroscopy. These detectors can result to other
properties of the components of the analyte that can lead to the determination of their identity.
As the chromatographic run is observed, the peak widths of each component of the analyte
continually increases. This is called as band broadening. Column efficiency is used to measure the extent of
band broadening. (Harvey, 2000)

As you see from the equation above, column efficiency increases upon the increase in the length of
the column and decrease in the height of theoretical plate. The column efficiency calculated for the three
different methods are the following:
a. Tangent method

= 16( )
0.637 2
= 16(
)
0.3
= 1154.187

b. 5 method
= 25(
= 5.54(

c. Half-height method

2
)
4.4

0.637 2
) = 899.1841
0.05

 2
)
1/2
0.637 2
= 5.54(
)
0.1
= 1245.37
= 5.54(

Using the value obtained from the half-height method, the HETP calculated was 0.004015.
The main of goal of chromatography is to separate the components into chromatographic peaks.
Resolution is then used to measure the degree of separation of two peaks in a chromatogram. (Harvey, 2000)

2[,
,
]
=
+
Using the mixed standards with the optimized parameters of 0.60 mL/min and sample load of 10 L, the
resolution calculated for the two peaks of benzoic acid and caffeine is 12.0666. A solutes capacity factor
(k) can also be determined from a chromatogram by measuring the columns void time, tM, and the solutes
retention time, tR.

=

The value of capacity factor indicates the solutes distribution into the mobile phase and stationary phase.
The columns void time was 0.2. Using the mixed standard chromatogram with optimized parameters, the
capacity factor of caffeine and benzoic acid are 0.39 and 2.2, respectively. The relative selectivity of a
chromatographic column for a pair of solutes, on the other hand, is given by the selectivity factor, a, which is
defined as:

,
= =
,
The identities of the solutes are defined such that solute B always has the smaller retention time. Accordingly,
the selectivity factor is equal to 1 when the solutes elute with identical retention times, and is greater than 1
when , is greater than , . (Harvey, 2000)The calculated selectivity factor for the same chromatogram of
mixed standard is 5.641026. These parameters are important in indicating the column efficiency of the
instrument in separating the components of an analyte.
To effect a better separation between two solutes it is often necessary to improve the selectivity
factor, a. One approach is the adjustment of pH for ionic analytes. When a solute is a weak acid or a weak
base, adjusting the pH of the aqueous mobile phase can lead to significant changes in the solutes retention
time. At more acidic pH levels, both weak acids are present as neutral molecules. They partition then
favorably into the stationary phase resulting to fairly long retention times for the solutes. When the pH is more
basic, the solutes present as their conjugate weak base anions are less soluble in the stationary phase and
elute more quickly. (Harvey, 2000)

IV.

SUMMARY AND CONCLUSION

The experiment involves the application of high-performance liquid chromatography specifically UPLC to
mixed standards of caffeine and benzoic acid as well as caffeine alone. The instrument was first optimized
for the following parameters: best solvent system, flow rate and sample load. Results showed that the
following are the optimized parameters: 9:1 acetonitrile:water, 0.60 mL/min and 10 L sample load.
Using the chromatogram of mixed standard with these optimum parameters, the capacity factor,
selectivity factor and column resolution were calculated. These parameters are very important in determining
whether the separation of the components of mixed standard was good and efficient. Results showed that
there was good separation of benzoic acid and caffeine. Also, elution order of the two were determined based
on the polarity of these components. Since benzoic acid is more polar with low molecular weight, caffeine
was eluted first and detected in the chromatogram followed by benzoic acid.
The chromatogram of caffeine was also used to calculate for the column efficiency, N, using the three
known methods: tangent, 5 and half-height method. The value obtained from this was used to calculate for
HETP.
The use of HPLC/UPLC is very essential since it can be used for analysis of many substances.
Substances that cannot be analysed using gas chromatography can be analysed using HPLC. This is very
useful in isolation and determination of identities of components of analyte.
V.

SAMPLE CALCULATIONS

Using caffeine with 0.60 mL/min flow rate:

Calculate number of theoretical plates, N:
d. Tangent method

= 16( )
0.637 2
= 16(
)
0.3
= 1154.187

e. 5 method
= 25(
= 5.54(

f.

Half-height method

2
)
4.4

0.637 2
) = 899.1841
0.05

2
)
1/2
0.637 2
= 5.54(
)
0.1
= 5.54(

 = 1245.37
Calculation for the HETP of 5 cm length column:
=
=

5
= 0.004015
1245.37

Using mixed standard with 0.60 mL/min flow rate and 10 L sample injection:
Calculation for selectivity factor:

,
= =
,
0.640 0.2
=
= 5.641026
0.278 0.2
Calculation for capacity factor:
Caffeine:

0.278 0.2
=
= 0.39
0.2
=

Benzoic Acid:

Calculation for resolution:

0.640 0.2
= 2.2
0.2

2[,
,
]
+
2[2.2 0.39]
=
= 12.0666
0.1 + 0.2

VI.

REFERENCES

Harvey, D. (2000). Modern Analytical Chemistry. McGraw Hill.