HIGH PERFORMANCE LIQUID CHROMATOGRAPHY:

SEPARATION AND QUANTIFICATION

OF COMPONENTS IN DIET SOFT DRINKS

REFERENCES:
1. Principles of Instrumental Analysis, 5th Edition, Douglas Skoog, F. James Holler,
Timothy Nieman, Saunders College Publishing, Philadelphia, 1998.

2. “The Analysis of Artificial Sweeteners and Additives in Beverages by HPLC,”

Journal of Chemical Education, vol. 68(8), August 1991, p A195-A200.

OBJECTIVES:

The purpose of this experiment is to quantify the caffeine content of a diet cola
sample using high performance liquid chromatography (HPLC). In order to quantify the
caffeine, it must be isolated from the other components in the mixture. In this
experiment, you will determine a set of HPLC conditions suitable for separating caffeine,
benzoic acid, and aspartame and then quantify the caffeine content of your cola sample
using a standard calibration curve. At the end of this experiment you should understand
the mechanisms by which components in a mixture are separated, identified, and
quantified using HPLC and understand how to vary experimental parameters to optimize
a separation.
 BACKGROUND:

The fundamentals of chromatographic separations and a detailed discussion of the

application of HPLC are covered in reference 1 (chapters 26 and 28). The following
discussion summarizes important concepts from these chapters, but the student is
encouraged to read the full text.

Introduction to HPLC and Instrument Components.

High performance liquid chromatography (HPLC) is an important analytical tool

for separating and quantifying components in complex liquid mixtures. By choosing the
appropriate equipment (i.e. column and detector), this method is applicable to samples
with components ranging from small organic and inorganic molecules and ions to
polymers and proteins with high molecular weights. The various types of HPLC and
their characteristics are summarized in the table below. In this experiment, we will use
reversed-phase partition chromatography.

Table 1. Various Types and Applications of HPLC

TYPE SAMPLE MOLECULAR STATIONARY MOBILE PHASE

POLARITY WEIGHT PHASE
RANGE
Adsorption non-polar to 100 - 104 silica or alumina non-polar to polar
somewhat polar
Partition non-polar to 100 - 104 non-polar liquid relatively polar
(reversed-phase) somewhat polar adsorbed or
chemically bonded to
the packing material
Partition (normal- somewhat polar to 100 - 104 highly polar liquid relatively non-polar
phase) highly polar adsorbed or
chemically bonded to
the packing material
Ion Exchange highly polar to ionic 100 - 104 ion-exchange resins aqueous buffers with
made of insoluble, added organic
high-molecular solvents to moderate
weight solids solvent strength
functionalized
typically with
sulfonic acid (cationic
exchange) or amine
(anionic exchange)
groups
Size-Exclusion non-polar to ionic 103 – 106 small, porous, silica polar to non-polar
or polymeric particles

HPLC
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 Figure 1. shows the components of our Hewlett-Packard Model 1100 HPLC. The
system consists of:

 reservoirs to hold the solvents used to make up the mobile phase

 a solvent degasser to prevent bubbles in the mobile phase
 a programmable quaternary pump that mixes the solvents in the prescribed ratios
and pumps them through the column and past the detector
 a column compartment that houses and thermostats the HPLC column
(in our case a ZORBAX, reversed-phase C18 column; dimensions 4.6mm x 15
cm)
 an autosampler that draws prescribed volumes from sample vials and injects them
onto the column
 a diode array detector that monitors the entire UV-vis spectrum of the column
effluent at regular intervals

Control of the above components and data acquisition and analysis are performed on a
personal computer.

Solvent Reservoirs

Autosampler

Column Compartment

Solvent Degasser

Column DAD
Bypass Valve
Quaternary Pump

Figure 1. HPLC 1100 Instrument Components

HPLC
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 Optimization of Resolution and Column Performance.

The goal of any HPLC experiment is to achieve the desired separation in the
shortest possible time. Time is critical because “time is money” and because as we’ll see,
the more time the sample spends on the column, the more the bands containing the
components spread, resulting in reduced resolution. Optimization of the experiment thus
usually involves manipulation of column and mobile phase parameters to alter the
relative migration rates of the components in the mixture and to reduce zone broadening.
These can generally be optimized fairly independently.

Migration Rates
The length of time it takes for a given component/solute to travel through the
column and be detected is determined by the flow rate of the mobile phase, , and the
partitioning of the solute between the mobile and stationary phases. Since the solute
molecules can only travel when they are dissolved in the mobile phase, the greater their
concentration in the mobile phase, the faster they will elute. The partition coefficient, K,
is defined in equation 1

CS
K (1)
CM

where CS is the concentration of the solute dissolved in or adsorbed to the stationary

phase, and CM is the concentration of the solute in the mobile phase.
The quantities CS and CM, however, are rarely determined in chromatographic
experiments. Instead, a quantity called the retention factor, k’, is determined. The
retention factor for a component A is defined as

t t
k 'A  R M (2)
tM

where tR is the retention time of component A and tM is the retention time of an unretained
species (also called the dead time). The average rate of linear migration of component A
is related to both the flow rate of the mobile phase and the retention factor.

1
v  
1  k 'A  (3)

HPLC
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 The retention factors should normally lie in a range of 2-5, but for complex
mixtures a larger range may be required to separate all the components. The value of the
retention factor for a given component depends on the chemical identity of the
component and the following experimental variables:

 mobile phase flow rate

 mobile phase composition
 column temperature
 column composition

Zone Broadening
The extent to which the component bands spread as they travel down the column
affects the efficiency of the separation. The theoretical plate height, H, is defined in
equation 4 and is based on a Gaussian analysis of the peak width, , as it exits the column
at point L.

2
H (4)
L

where

LW
 (5)
4t R

and W is the width of the peak at the base. The data analysis program on our HPLC
actually reports the width at half maximum, W1/2, for each peak rather than the width at
the base. Assuming a Gaussian peak shape,

W  1.6994 W1 / 2 (6)

so,

L W1 / 2  2
H (7)
5.540 t R  2

The number of theoretical plates in the column, N, is

L
N (8)
H

Efficient columns have small H and large N for a given component. The theoretical plate
height is affected by the following experimental parameters:

HPLC
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  mobile phase flow rate
 diffusion coefficient of the solute in the mobile phase
 diffusion coefficient in the stationary phase (depends on temperature and
viscosity)
 retention factor
 diameter of the particles packing the column
 thickness of the liquid coating on the stationary phase

Resolution
The resolution of two adjacent peaks, RS, is determined by their separation and
their widths.

2 ( t R )B  ( t R )A  2 ( t R )B  ( t R )A 
RS  
WA  WB 
1.6994  W1 / 2  A   W1 / 2  B  (9)

In other words, RS depends on both migration rates and zone broadening. A resolution of
1.5 means that the overlap of the peaks is about 0.3%, so conditions should be optimized
to achieve at least this resolution if possible.

In this experiment, you will adjust only the composition of the mobile phase to
optimize the retention factors and resolution. We will not attempt to optimize the zone
broadening independently by changing the column or the flow rate.

HPLC
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 Components in Diet Soft Drinks

The ingredient list for most diet soft-drinks includes caffeine, benzoic acid, and
aspartame (Nutrasweet®). The structures of these compounds are shown below along
with their UV-vis spectra.

CH3

O N N

N
H3C N

CH3
O
200 250 300 350 nm

Caffeine
C8H10N4O2
MW = 194.19
pKa = 10.4

O OH

200 250 300 350 nm

Benzoic Acid
C7H6O2
MW = 122.12
pKa = 4.2

HPLC
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 NH2 O

NH
OH

O
O O

HO
200 250 300 350 nm

Aspartame
(L-Aspartyl-L-phenylalanine methyl ester)
C14H18N2O5
MW = 294.31

HPLC
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 EXPERIMENTAL PROCEDURE:

Solutions:

Provided by instructor:
mobile phase - HPLC-grade methanol
20 mM phosphate buffer, pH 3
standard samples - mixture containing caffeine, aspartame, benzoic
acid and uracil
- individual samples of each of the above components

Student prepared:
diet cola sample - degassed for 20 minutes with air stream followed by
filtration with 0.22m syringe filter
caffeine standards - 100% = 0.045g/250 mL water
85% dilution
70%
65%
50%
(and additional dilutions as necessary to bracket
cola sample)

Equipment:

Model 1100 Hewlett-Packard HPLC system

ZORBAX reversed-phase bonded C18 HPLC column (4.6 mm x 15 cm)
250mL volumetric flask
40-200 L automatic pipet
200-1000 L automatic pipet
1mL HPLC vials with caps
vial crimper
analytical balance

HPLC
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 Notes on Use of HPLC:

 Your instructor will show you how to use the equipment and the software. A
primer guide/refresher is also found in the appendix.
 At the beginning of the day, first open the valve with the black knob on the front
of the pump manifold. This sends the mobile phase to waste instead of the
column. Allow the pump to run for about 10 minutes at 5 mL/min to purge the
lines of any air bubbles.
 At the end of the day, run an 80% water/20% methanol mixture through the
column at 1 mL/min for 10 minutes followed by 100% methanol for 10 minutes.
This should flush the column of any potential salt forming materials and stores the
column in a compatible solvent.

Sequence of Analysis:

1. Determine optimum conditions for separation of caffeine, benzoic acid and

aspartame using the standard sample provided. Start with a 75% buffer/25%
methanol mobile phase mixture at 1 mL/min, 1L injection volumes, and a 20
minute run time. Uracil has been added to the standard mixture to provide a
dead time marker (i.e. uracil is unretained). The diode array detector can
monitor several wavelengths at once as well as record the entire spectrum at
specified intervals. Use the UV-vis spectra of the components shown above to
choose appropriate wavelength(s) to monitor the chromatograms. Vary the
ratio of buffer to methanol to achieve an acceptable separation and finally
adjust the run time to end after the last component exits the column. Use
these data to calculate the retention factors for each component, the resolution
between each pair of adjacent peaks, and the values for H and N for caffeine
under these conditions.

2. Using the conditions determined above and the pure samples of each
component provided, determine the retention time of each component (i.e.
identify the peaks).

3. Using the column conditions determined above and 5L injection volumes,
run three samples of your most concentrated caffeine standard to determine
the precision of the injections. Then run the remaining caffeine standards and
the diet cola sample to determine the concentration of caffeine in your sample
using a calibration curve. Check the full spectrum of each peak against
spectra of the pure components to verify that the peaks represent isolated
components.

HPLC
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 APPENDIX: SETTING HPLC PARAMETERS
The chemstation control software is accessed via the start menu, programs, HP
Chem Stations, and then instrument 1 online. All of the components are controlled via
the run control window shown below by accessing the instrument sub-menus for each of
the components. Notice also that the status of each component is indicated on the run
control screen.
The parameters are set in the various “set” menus. The “more” menus contain control sub-
menus that allow you to actually turn these components on.

You can either run individual samples one at a time using the Run Method command
under the Run Control menu, or run a sequence of samples using the Sequence menu and it’s
various sub-menus. If you choose the run method option, use the Sample Info menu under the
Run Control menu to tell the computer where to store your files and what to name each run. If
you use the sequence mode, you can enter this information in the sequence parameters sub-menu.

Once the data has been collected, use the View menu to see the report containing the
chromatograms at each monitored wavelength and the integrated peak areas etc. From here you
can print the report as well as view and print full spectra at any peak.

HPLC
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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY:
SEPARATION AND QUANTIFICATION
OF COMPONENTS IN DIET SOFT DRINKS

NAME ________________________________ DATE ______________

Solutions:

Mass caffeine _____________

Dilution volumes: standard / water

A: _______ / _________
B: _______ / _________
C: _______ / _________
D: _______ / _________
E: _______ / _________

Optimization of Conditions:

% buffer ____________
% methanol ____________
flow rate ____________
column pressure ____________

Retention Time Retention Factor W1/2

(min) (k’)
Uracil
Caffeine
Aspartame
Benzoic Acid

Rcaffeine,aspartame _______________
Hcaffeine ___________________
Ncaffeine ___________________
Quantitation of Caffeine:

wavelength chosen for quantitation __________________

Sample [caffeine] (mg/L) Peak Area

A

B
C
D
E
diet cola

avg. peak area for A ____________________ 95% CIm __________________

slope ___________________
std. dev. slope _________________ 95%CI slope ____________________
intercept ___________________
std. dev. intercept _______________ 95%CI intercept __________________
R2 ________________
covariance ___________________
[caffeine]diet cola _____________________ 95%CI ____________________

HPLC
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 SamDle PreDarat ion
The extracts of tamarind fruit pulp was prepared
as follows: 1 gram of the pulp was dispersed in 5 ml
of water, stirred for 15 minutes and then centrifuged.
Solutions of the internal standards were prepared by
dissolving 50 mg of ascorbic acid in 1 ml of water, 50
mg of caffeine in 1 ml of water and 120 mg of rhamnose
in 1 ml of water.
HPW: Analvsis
HPLC was performed using RI detector, Differential
Refractometer R 401 (Waters Associate, Inc., Milford,
MA), a programmable solvent delivery system, Series 3B
(Perkin-Elmer Corp., Norwalk, Conn.); a manual
injection valve, with 50 ul loop (Valco Instruments
Co., Houston, TX) and a chart recorder (Laboratory Data
Control, Riviera Beach, FL).
Water containing 0.01 M KH2PO4 was used as a
mobile phase to analyze various components from
tamarind fruit pulp solution on Whatman Partisphere-5
NH2 column. Ascorbic acid served as an internal
standard. A mixture of water and acetonitrile (90:10,
v/v) was used as a mobile phase to resolve components
such as alanine from serine, tartaric acid from
phenylalanine and proline from leucine on Partisphere-5
WCX column. Caffeine was used as an internal standard
in these cases (Fig. 2, 3 and 6). Resolution of oxalic
acid from glucose and fructose mixture was achieved on
Partisphere-5 PAC column by using aqueous solution of
0.01 M KH2PO4 (Fig. 4) as a mobile phase. Weight
against area calibration curves was used to quantify
oxalic acid in the mixture. Water and acetonitrile
Downloaded by [Michigan State University] at 01:36 09 February 2015
422 KHURANA AND HO
mixture (20:80, v/v) was used as a mobile phase to
resolve glucose from fructose on Partisphere-5 PAC
column (Fig. 5). Fthamnose served as an internal
standard to quantify glucose and fructose. Authentic
samples of glucose, fructose, alanineserine, leucine,
proline, phenylalanine, pipcolinic acid, oxalic acid,
citric acid, succinic acid and tartaric acid were used
to analyze these components on various columns.
Result, discussion
High performance liquid chromatography has been
used to analyze various nonvolatile components from
tamarind fruit pulp solution. A partial resolution of
sugars, amino acids and organic acids was achieved on
Partisphere-5 NH2 column (Figure 1). The components
were quantified by using ascorbic acid as an internal
standard. Glucose, fructose and oxalic acid coeluted
on this column. Similarly, the amino acids like
alanine, phenylalanine and leucine did not resolve from
serine, tartaric acid and proline respectively. The
unresolved components were resolved, identified and
quantified by using Partisphere-5 PAC and Partisphere-5
WCX columns (Figures 2-6). The unresolved mixture of

HPLC
Appendix
alanine and serine was collected from Partisphere-5 NH2
column and resolved on Partisphere-5 WCX by using
caffeine as an internal standard (Figure 2). Similarly
tartaric acid-phenylalanine and leucine-proline
mixtures were collected from Partisphere-5 NH2 column
and resolved on Partisphere-5 WCX column. The
components were quantified by using caffeine as an
internal standard (Figures 3 and 6). Partisphere-5 PAC
column was used to resolve oxalic acid from sugars in
the mixture as collected from Partisphere-5 NH2 column
(Figure 4). The sugar mixture containing glucose and
fructose was collected from Partisphere-5 PAC column
Downloaded by [Michigan State University] at 01:36 09 February 2015
FLAVOR COMPONENTS IN TAMARIND 423
II

HPLC
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Discussion Questions:

Discuss how the choice of monitored wavelength affects the sensitivity of the analysis.
Would you choose the same wavelength to quantify benzoic acid or aspartame?

Based on the pKa’s of the components in our samples, why do you think a mobile phase
buffer of pH 3 was chosen?

If significant zone broadening had resulted in unsatisfactory resolution in our experiment,

what might we have changed to reduce its affect? Comment on the feasibility of this.

Compare the precision obtained for the three injections of the same sample to the
precision of your calibration curve. Which limits the precision of your unknown
concentration? Would using an internal standard be justified?

Show sample calculations and attach all relevant chromatograms and spreadsheet
printouts.

HPLC
Appendix