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 three 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. The selectivity factor, capacity factor and
column efficiency were calculated using the mixed standards.
II.

MATERIALS AND METHOD

A set of conditions for the Acquity Waters UPLC includes a C18 column with 17 m pore size,
2.1x50 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)
Using the UPLC, mixed standards of benzoic acid and caffeine, caffeine and unknown sample was
analyzed for their components. The first run 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 was observed.

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

Retention Time
0.637

Area
234663

%Area
79.07

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 water:acetonitrile in
comparison to the one peak observed for the 8:2 water:acetonitrile. Also, caffeine was retained far longer
with use of more polar mobile phase in comparison to the less polar mobile phase. This shows that the best
solvent system was 9:1 water:acetonitrile because better separation of peaks can be observed for this
solvent system.
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. In reverse-phase chromatography, on the oher hand the stationary phase
is nonpolar and the mobile phase is polar. The most common stationary phases used are
organochlorosilane for which R group is an n-octyl (C8) or n-octyldecyl (C18) hydrocarbon chain. The
mobile phase of RPLC are usually using a buffered aqueous solution. The pH of the mobile phase must be
less than 7.5 since the silica substrate is subject to hydrolysis in basic solution. (Harvey, 2000)
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 of mobile phase 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)
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 C18. The properties of a stationary phase are determined by
the nature of the organosilanes alkyl group. Since the R group of the stationary phase is nonpolar, the
mobile phase used is highly polar. (Harvey, 2000)
Table 6.3. Data obtained for the mixed standards at 0.30 mL/min flow rate.
Name
Retention Time
Area
%Area
Benzoic Acid
0.515
94652
24.04
Caffeine
1.192
228173
57.96

%Height
38398
121632

Table 6.4. Data obtained for the mixed standards at 0.60 mL/min flow rate.
Name
Retention Time
Area
%Area
Benzoic Acid
0.278
41383
23.84
Caffeine
0.64
113700
65.49

%Height
45978
104783

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. 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. This is shown in the data
gathered in the experiment. Comparing the data obtained in Table 6.3 and Table 6.4., it shows that
components were retained longer in 0.30 mL/min flow rate than in 0.60 mL/min flow rate.
Table 6.5. Data obtained for the mixed standards with 10 L sample load.
Name
Retention Time
Area
%Area
Benzoic Acid
0.278
41383
23.84
Caffeine
0.640
113700
65.49

%Height
45978
104783

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

%Height
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) The effect of amount of
sample load should not affect the peaks or resolution in the chromatogram. Results showed almost the
same values of retention time of the 5 and 10 L sample load.
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
to caffeine. Using the reverse-phase LC, the less polar compound is likely to be retained longer than the
more polar compound. Thus, benzoic acid is expected to elute first than caffeine.

Figure 6.2. Structure of benzoic acid and 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 was 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)
L
N=
H
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
t
N=16 ( R )
W
b. 5 method
tR
W 4.4

N=25
c. Half-height method
tR
W 1/ 2

N=5.54
Table 6.7. Data calculated for N and HETP using three different methods.
Parameters
N
1154.187
Tangent Method
899.1841
5 method
Half-height method

1245.37

HETP
0.004015
0.005561
0.004332

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[t 'R , A t 'R , B ]

Rs =
w A+ w B
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.
t t
k= R M
tM
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:
'

k A t R , A t M
a= ' =
k B t R ,B t M
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 t R , A is greater than t R , B . (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.
For the quantitative analysis of the unknown, single-point calibration was used to determine the
amount of caffeine in diluted Mountain Dew.
Table 6.8. Data on the analysis of unknown in UPLC.
Name
Retention Time
Area
Benzoic acid
0.279
40780
Caffeine
0.641
70217

%Area
31.3
53.78

%Height
46649
64297

The calculations are shown in the Sample Calculations part of the paper and results showed that the
amount of caffeine present in Mountain Dew is 4.10597 mg/L.
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 water:acetonitrile, 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 these three methods were
used to calculate for HETP. The following are the N and HETP obtained: 1154.1841, 899.1841, 1245.37 for
N and 0.004015, 0.005561, 0.004332 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

Calculation of void volume:

Void Volume=

0.17d 2L
4
2

0.172.1 50
Void Volume=
=0.1177626006 mL
4
Calculation of dead time:
Dead time=

void volume
flow rate

Dead time=

0.1177626006 mL
=0.196271001min
0.6 mL /min

Using caffeine with 0.60 mL/min flow rate:

Calculate number of theoretical plates, N:
a. Tangent method
N=16 (
N=16 (
N=1154.187

0.637 2
)
0.3 cm

tR
)
W

b. 5 method
tR
W 4.4

N=25
0.637
0.05 cm

N=5.54

c. Half-height method
tR
W 1/ 2

N=5.54
0.637
0.1cm

N=5.54
N=1245.37
Calculation for the HETP of 5 cm length column:
L
H=
N
H=

5 cm
=0.004015
1245.37

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

'

k
t t
a= 'A = R , A M
k B t R ,B t M
a=

0.6400.2
=5.641026
0.2780.2

Calculation for capacity factor:

Caffeine:
t t
k= R M
tM
k=

0.2780.2
=0.39
0.2

Benzoic Acid:
0.6400.2
k=
=2.2
0.2

Calculation for resolution:

2[t 'R , A t 'R , B ]
Rs =
w A+ w B
Rs =

2[2.20.39]
=12.0666
0.1+ 0.2

Quantitative analysis of caffeine in diluted Mountain Dew:

S=kC
S
k=
C
k=

Using the data of caffeine in Table 6.5. (mixed standard):

65.49
=13.098 L/mg
5 mg/ L

S
C=
k
C=

Using data of unknown in Table 6.8:

53.78
=4.15097 mg/L
13.098 L/mg

VI.

REFERENCES

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

Skoog, D. A., West, D. M., Holler, J. F., & Crouch, S. R. (2014). Fundamentals of
Analytical Chemistry. Belmont, USA.