EXPERIMENT 9

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

SEPARATION OF COMPONENTS OF SOFT DRINKS
2
A. PURPOSE
The principal purpose of this experiment is to introduce you to procedures and
instrumentation used for high performance liquid chromatography (HPLC).
B. OVERVIEW
In this experiment you will separate mixtures of components frequently found in
soft drinks, namely caffeine, benzoate, and aspartame. You will be given seven solutions
containing known amounts of the three species and record the chromatograms.
C. INSTRUMENTATION
1. Liquid chromatograph
A commercially available liquid chromatograph (PM-80 pump and CC-5 Injector,
Bioanalytical Systems, West Lafayette, IN) is used in this experiment where separation
is performed on a reversed phase column (#MF 8954).
Chromatograph and column settings are as follows:
Flow rate: 0.5 mL/min
Minimum pressure setting: 250 PSI
Maximum pressure setting: 4000 PSI
Column dimensions: 3(i.d.) 100 mm
Column has silica packing (3m particle size) and is functionalized with ODS
(C18). pH range is 2-7.
2. Detector
Detection is by absorption spectroscopy. Although the detector (Model UVVIS
200, Linear) is capable of measuring several wavelengths, all the components of interest
absorb at 254 nm. Accordingly, only one wavelength, 254 nm, will be used in this study.
Detector settings are as follows:
Rise time: 0.3 s
Wavelength: 254 nm
Range: 0.2 AUFS (absorbance units full scale)
3. Data acquisition
Data will be recorded on line using a digital voltmeter and a digital computer. The
following settings should be used:
Time per point: 0.1 s
Time per chromatogram: 10 min initially
4. Saving data
Data will be saved on floppy disks. Each group should bring at least two
floppy disks with full storage capability free of other files.
D. SOLUTIONS
1. Solutions provided
a. Mobile phase (Eluent). The mobile phase (eluent) is prepared by mixing 400 mL
of acetonitrile with 2.3 mL of glacial acetic acid and diluting the solution to 2 L
with water. The resulting solution is then adjusted to pH = 4.2 by addition of
sodium hydroxide. The solution is then filtered through a 0.2 m Nylon 66
membrane filter under vacuum to degas the solution and to remove solids that

could plug the chromatographic column. The mobile-phase solution is very

labor intensive to prepare; please use no more than necessary!
b. Stock standard solutions. Three standard solutions, each containing one of the
analytes, will be provided. These solutions will contain a) caffeine (0.4 mg/mL), b)
aspartame (6 mg/mL), and c) benzoate (0.7 mg/mL). You should record the
exact concentration of each analyte.
2. Standard Solutions
These solutions have been prepared for you in seven numbered vials in accord
with Table 1.
Table 1. Volumes of stock standards used to prepare 50 mL of working standards.
Standard
Volume (mL) of each standard to be added
Number
Caffeine
Benzoate
Aspartame
1
4
0
0
2
0
4
0
3
0
0
4
4
1
1
1
5
2
2
2
6
3
3
3
7
5
5
5
[The following instructions apply only if you need to prepare the seven solutions
yourself. Rinse seven 50-mL volumetric flasks first with distilled water and then with
three 5-mL volumes of the mobile-phase solution. Mark the flasks 1-7.
Clean three 25-mL burets (provided) thoroughly by washing with detergent and
rinsing with distilled water. Rinse each buret with several small volumes of one of the
stock standard solutions and fill the buret just above the 0.00-mL mark with the stock
standard with which it was rinsed. Adjust the buret to the 0.00-mL mark and add the
volumes given in Table 1 to appropriately marked 50-mL volumetric flasks. Then, dilute
each flask to volume with the filtered mobile phase solution and mix thoroughly.
Note: It is very difficult to mix solutions in small volumetric flasks. Each solution
should be inverted at least 25 times allowing the air bubble to go all the way between the
top and bottom of the flask during each inversion.
After the working standards are mixed thoroughly, label seven vials Std 1, Std 2,
Std 3 ...... Std 7 and transfer small portions of the standards to appropriately marked
vials.]
E. PROCEDURE
Confirm that all instrumental settings are as described under the Instrumentation
section (Section III) and that the waste line is in a waste container and not in the
reservoir for the mobile phase. Rinse a syringe with several volumes of one of the
working standards containing all three components (e.g. Std 6) and fill the syringe with
that solution. With the INJECTOR handle in the LOAD position, inject 0.10 mL of solution
through the septum. DO NOT REMOVE THE SYRINGE at this point. Move the
INJECTOR handle to the INJECT position to inject the sample onto the column and
Click Start on the computer screen immediately. The three peaks will appear on the
screen in sequence. Click Stop on the computer screen at about 60 s after the third
peak returns to baseline. Note the time, convert it to minutes, and set the time per
chromatogram (Part III-3) to this time.

Save these data under a suitable file name (e.g. Std_1, Std_2, etc.) on your
floppy disk.
Remove the syringe from the septum and repeat the above process for each of
the working standards using the same time per chromatogram determined from the first
run.
Save your data on a floppy disk. Be careful to identify each file (e.g. Std_1,
Std_2, ....Std_7) so that it can be correlated with the correct working standard. Though
not required, you are free to run repetitions of the standards if you wish.
F. WASTE DISPOSAL
Consult with your TA regarding waste disposal.
G. DATA PROCESSING
Your principal responsibilities for this part of the experiment are to show plots of
chromatograms and to prepare calibration plots of peak height vs. concentration.
1. Organizing data. Given that time axes are the same for all chromatograms, it is
recommended that you consolidate data for all chromatograms into one file using
Column A for the time data and Columns B-G for peak data.
2. Chromatograms
You will have obtained seven chromatograms. It is suggested that you plot the three
chromatograms for the single-component standards (Std. 1-Std. 3) on one graph.
Include this plot as Figure 1 in your report. These chromatograms can be used to
identify the three components in the mixture chromatograms.
Plot the four mixture chromatograms on a single graph using different line symbols
(solid, dash, dot, dot/dash etc.) to differentiate among the separate plots. Include this
plot as Figure 2 in your report.
3. Calibration plots, peak height
If Origin is used to plot data, the Screen reader tool can be used to read peak
heights directly from the screen with reasonable reliability. Normalize the concentrations
for each species to the highest concentration of that species by dividing each
concentration by the highest concentration. Using these normalized concentrations,
prepare a single graph to include peak height vs. the normalized concentration for each
species. Include the normalization factors in the legend of the calibration plot and include
this plot as Figure 3 in your report.
H. REPORT
Your report should contain the usual information.
I.

NOTE TO TAS
Before leaving the laboratory, move the waste line from the waste container
to the reservoir for the mobile phase!!!