High Performance Liquid Chromatography

Analyte: Pure Caffeine Samples with Theobromine

Internal Standard

CHEM 4211L-003

Blake Hopkins, Ethan Lehman

Abstract
Caffeine samples on an increasing concentration gradient (0ppm, 2ppm, 4ppm, 6ppm,
8ppm, 10ppm) were analyzed using high performance liquid chromatography (HPLC). These
samples contained an internal standard of theobromine that was at 1ppm concentration in every
sample. Ultraviolet-Visible spectroscopy was used on a pure caffeine solution of concentration
75ppm to determine the maximum absorbance wavelength of 289.4nm. The parameters of the
HPLC device were set according to the peak absorbance wavelength of 289.4nm and the
prepared samples were analyzed to create a calibration curve for the instrument.

Materials and Devices

 UV-Visible Spectrometer
 Glass or Plastic Cuvette
 50mL, 250mL, 500mL Volumetric Flasks
 0.2 micrometer syringe filter
 1mL syringe
 Disposable Pipettes
 25 micrometer blunt tip syringe (for sample injection)
 HPLC Instrument:
Liquid Chromatograph (LC-20AD)
Detector (SPD-20AV)
Communication Module (CBM-20A)

Sample Preparation

1. Prepare a stock caffeine solution of concentration 100 ppm by weighing 50 mg of pure

caffeine and quantitatively transferring it to a 500 mL volumetric flask and filling the
volumetric to the mark with DI water.
2. Prepare a stock theobromine solution of concentration 50 ppm for use as the internal
standard. Do this by weighing 25 mg of theobromine and quantitatively transferring it to
a 500 mL volumetric flask and diluting to the mark with DI water.
3. Make caffeine samples of 0 ppm (blank), 2 ppm, 4 ppm, 6 ppm, 8 ppm, and 10 ppm by
dilution of the caffeine stock with water including the addition of the internal standard at
1.0 ppm. Prepare a pure caffeine sample with concentration 5 ppm and no internal
standard as well (this will be used to find the caffeine retention time later to distinguish
sample from internal standard).
a. Label the 7 different 50 mL volumetric flasks with the appropriate concentrations.
 4. The standards must be filtered before they can be inserted in
the HPLC machine. This filtration will be performed using a 1
mL syringe and a 0.2 µm syringe filter as shown in Figure 1.
5. Label 7 microcentrifuge tubes with the concentrations of the
caffeine standards to hold the filtered samples.
6. Rinse the syringe and filter with the sample. Do this by
drawing up 1 mL of the sample, attaching the filter after the
syringe is full, and compressing the plunger emptying the
contents into a waste beaker at a rate of a few drops per
second. Repeat this rinse procedure 3 times.
7. After rinsing, draw up 1 mL of desired sample, attach the
filter, discard the first few drops into the waste beaker, and
filter the rest of the contents into the designated
microcentrifuge tube at a slow rate of a few drops per second.
8. Repeat steps 6 and 7 with the other samples, starting with the
lowest concentration of caffeine and advancing to the highest
concentration.
a. The order of filtration should be 0 ppm,
2 ppm, 4 ppm, 5 ppm, 6 ppm, 8 ppm, Figure 1. The image above depicts the 1
and 10 ppm caffeine samples. mL syringe and the 0.2 µm syringe filter
b. The same syringe and filter can be used used for sample filtration.
throughout the full filtration process. If
the filter clogs, use a new filter ensuring
to rinse through the new filter.

Finding Peak Absorbance Wavelength

The Ultraviolet-visible spectrophotometer (UV-Vis) is used to determine the maximum
absorbance wavelength of pure caffeine. The wavelength determined will be the wavelength
used for the HPLC device.

1. Prepare a caffeine sample of around 74.4 ppm by diluting 18.6 mg of pure caffeine with
DI water in a 250 mL volumetric flask. The concentration does not need to be exactly
74.4 ppm, but this concentration is known to produce optimal results.
a. If the concentration is too high the peak might exceed an absorbance reading of
1.000. Reduce the concentration and rerun the sample with the lower
concentration.
b. If the concentration is too low, the peak may not be evident enough to definitively
find a peak absorbance wavelength. Increase the concentration and rerun the
sample with the higher concentration.
2. Fill a glass or plastic cuvette with the caffeine solution prepared in the previous step.
Measure the absorbance of this solution using the ‘Spectrum’ setting on the UV-Vis
spectrometer, setting the wavelength range from 270 nm to 325 nm.
 3. After the sample has completed the spectrum, press the button labeled ‘Curve’. Move the
cursor over to the highest point of the peak and record the wavelength that corresponds to
the maximum absorbance value. The spectrum should look similar to Figure 2 shown
below.

Figure 2. The above image shows the spectrophotometer screen after running a caffeine sample to determine the
peak absorbance wavelength measurement.

4. The recorded maximum absorbance wavelength determined from the peak absorbance of
the spectrum will be the wavelength used for the HPLC machine. (In this instance the
HPLC will be run at 289 nm)
HPLC Instrument Operations
Startup
1. Turn on individual components in specific order: Ultraviolet (UV) Detector, Liquid
Chromatograph (LC) Pump, and Communication connector (i.e., middle component, top
component, bottom component).
2. Ensure the solvent, water, and waste containers are at adequate levels.
3. Open Lab Solutions (LS) software on the PC and turn on the pump at the main screen.
4. At the menu for the UV Detector, set to zero.
5. Check physical LC component for bubbles in the tube:
a. To purge: turn the pump off, turn the valve to open, press purge and wait for
dialogue on LS, turn valve to close, and turn the pump back on.
6. Allow 15 minutes for startup, the pump is conditioned with a mixture of containers A and
B (water and methanol, respectively).

Before Running Samples

1. Select New Method in LS
2. Edit instrument parameters: select data acquisition time. Change end time to five minutes.
Change LC Stop Time to 0.01 minutes.
3. In pump menu, select low pressure gradient, change pump flow to 1.0 mL / min, change
solvent B concentration to 40%, and set maximum PSI to 3000
4. In Detector menu, use the maximum λ from data acquired from the UV-Vis
Spectrophotometer.
5. Select “download and close.”
6. Save method file to a safe location for easy access.
7. When changing any parameters, navigate to the “Method” tab and perform a baseline
check. Wait for system dialogue and then press the “Baseline Check” button located on
the second row of icons at the top of the screen.

Running Samples
1. Use a 25 µL syringe and gather your sample of lowest concentration. Rinse the syringe
twice with the same solution before obtaining the sample you wish to inject.
2. In LS, select “Single Run.”
3. Set injection volume to 20 µL, and create a unique sample name, save name, and save
location for the data.
4. Allow the dialogue to appear with the start button, insert the syringe into the column,
push the plunger, switch the handle to inject, remove the syringe after the start dialogue
has disappeared, and switch the handle back to load.
5. Repeat for each sample in triplicate, increasing the concentration after each triplicate, and
rinsing the syringe in between each trial.

Exporting Data
1. Select Data Analysis.
2. Select the data you wish to export by double clicking.
3. Select file, export as ASCII.
 4. Name your data and add “.csv” to your file name.
5. Select “chromatogram” in the drop-down menu.
6. For your data delimiter, select “comma.”

Data Analysis and Calculations

The following data and calculations are based on our trials conducted in lab. To begin, samples
were created by following the procedure described above and actual concentration values were
noted versus the theoretical values.

The caffeine stock solution used to create the individual samples was 100.8 ppm or 100.8 mg
caffeine / L water. Variable concentrations were created using the dilution equation (1) shown
below.

M 1 V 1=M 2 V 2
Equation 1. Dilution Equation

Where M 1 is the concentration of the stock, V 1 is the volume of the stock, M 2 is the desired
concentration of the dilution, and V 2 is the desired volume of the dilution.

The table below shows concentration values in a final volume of 50 mL.

Concentration Volume of Volume of

Standard of Caffeine in Caffeine Stock Theobromine Internal
Sample Samples (ppm) Solution (mL) Standard (mL)

Blank 0.000 0.0 1.0

1 2.016 1.0 1.0

2 4.032 2.0 1.0

3 6.048 3.0 1.0

4 8.064 4.0 1.0

5 10.080 5.0 1.0

6* 5 2.5 0.0

Table 1. Sample Preparation using Caffeine and Theobromine Stock Solutions.

*Sample is for determining caffeine retention time in HPLC to easily find peak difference between caffeine
and Theobromine. Actual concentration of caffeine does not have to be precise, just quantitatively similar
to the median of the other samples.
Peak values were measured in intensity and after average calculations produced a graph in order
to visualize the data from over 20 trials. The peak height graph is shown below in Figure 3.

Average Peak Height vs Caff. Concentration

Average Peak Height (Intensity)

20000
f(x) = 1868.4380952381 x + 138.36507936508
15000 R² = 0.999145788292311

10000

5000

0
0 2 4 6 8 10 12
Caffeine Concentration (ppm)

Figure 3. Intensity of response from caffeine given by HPLC

Additionally, calculations must be done for limit of detection and limit of quantification of the
HPLC. These are the lowest concentration value that can be detected reliably and the value
where precision is reliable at a specified range.
3.3 σ
LOD=
m
Equation 2. Limit of Detection

Where LOD is the limit of detection, σ is the standard deviation of the replicate low
concentration response values, and m is the slope of the calibration curve.
10 σ
LOQ=
m
Equation 3. Limit of Quantification

Where LOQ is the limit of quantification, σ is the standard deviation of response, and m is the
slope of the calibration curve.

The calculated values for above limits are shown in Figure 4 below. LOD = 0.52 ppm and LOQ
= 3.49ppm based on the standard deviation of the 0 ppm and 10 ppm caffeine concentrations,
respectively.
 20000

Average Peak Height (Intensity)

18000 f(x) = 1868.4380952381 x + 138.36507936508
16000 R² = 0.999145788292311
14000
12000
10000
8000
6000
4000
2000
0
0 2 4 6 8 10 12
Caffeine Concentration (ppm)

Limit of Detection Limit of Quantification

Figure 4. Consistent with Figure 3. But including measurements for LOD and LOQ.

The integration data was provided via the data exported from LS and used to create a
concentration curve in excel with the AVERAGE function. The mean peak intensity integration
area for each trial done in triplicate was calculated and put against our caffeine concentration
shown in Table 1.
For our final calibration curve, the purpose of this procedure, we plotted the mean area of the
caffeine peaks given via HPLC against the caffeine concentration values below.

Peak Integral Values vs. Concentration

200000
Integral Value (intensity avg.)

f(x) = 18266.1753590325 x + 1773.36507936504

150000 R² = 0.999412295597017

100000

50000

0
0 2 4 6 8 10 12
Caffeine Concentration (ppm)

Figure 5. Calibration Curve. Measured peak integration values averaged and plotted against samples with increasing
caffeine concentration and constant theobromine concentration (1.5 ppm).

By using the slope of our calibration curve, we were able to calculate the actual concentrations of
the samples. The difference between these values is expressed in percent difference in the table
below.

Caffeine Measured Caffeine %

Concentration (ppm) Concentration (ppm) Difference
0 0.07
2.016 1.94 3.66
4.032 4.11 1.85
6.048 5.93 1.87
8.064 8.01 0.71
10.08 10.12 0.98

Table 2. Difference in Caffeine concentrations based on HPLC Results.

Lastly in our data analysis, it is important to mention that every trial conducted with HPLC
produced average peak values for theobromine and caffeine at their respective retention times.
While these peaks showed a definitive height, there were also “tails” trailing the peak that gave a
peak after with a fraction of the intensity. The figure below shows an example of this tail peak.

Figure 6. HPLC Response of trial 3 of 10 ppm caffeine and 1.5 ppm theobromine.

Figure 7. LS Interface of 5.0 ppm caffeine used to find caffeine retention time.

Note the peak tails at the end of the main peaks for both caffeine and theobromine. These peaks
are possibly due to issues with the solvent filter or high pressure on the system. While it doesn’t
directly interfere with the data we acquired, the possibility of instrumentation issues should be
considered in replication of the procedure.

References
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theophylline in coffee by near infrared spectroscopy (NIRS) compared to high-performance
liquid chromatography (HPLC) coupled to mass spectrometry. Analytica Chimica Acta, 538(1–
2), 195–203. https://doi.org/10.1016/j.aca.2005.01.064
Ferguson, G. K. (1998). Quantitative HPLC Analysis of an Analgesic/Caffeine Formulation:
Determination of Caffeine. Journal of Chemical Education, 75(4), 467.
https://doi.org/10.1021/ed075p467

Potard, G. (1999). Quantitative HPLC analysis of sunscreens and caffeine during in vitro
percutaneous penetration studies. International Journal of Pharmaceutics, 189(2), 249–260.
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Douglas A. Skoog, F. James Holler, S. R. Crouch, "Principles of Instrumental Analysis", 7th

Edition, Boston: Cengage Learning, 2018.