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Experiment 1

High Performance Liquid Chromatography

Alfred E. Neuman

Chemistry 436
September 15, 1999


In this experiment, high performance liquid chromatography (HPLC) was used to separate
a mixture of three similar alkaloids: caffeine, theobromine, and theophylline. The retention times
of the three compounds were measured, and a calibration model for caffeine was constructed
based on the chromatographic peak heights produced by a series of six caffeine standard
solutions. The derived calibration model was then employed in the prediction of caffeine content
in a commercial soft drink.


No deviations were made from the procedures described in the laboratory handout.


Retention Time Study

Retention Time (sec, + 0.1 sec)

Compound Trial 1 Trial 2 Trial 3

Caffeine 180.1 181.3 180.5

Theobromine 154.6 155.3 154.9
Theophylline 244.7 245.8 245.2

Caffeine Calibration Study

Aliquot Volume Peak Height (cm, + 0.05 cm)

(mL) Trial 1 Trial 2 Trial 3

5.0 2.10 1.25 1.50

10.0 3.82 3.20 3.80
15.0 5.02 5.09 5.60
20.0 8.40 8.35 7.70
30.0 11.90 10.25 11.05
40.0 14.55 15.34 13.80
Unknown 6.23 6.58 6.61


Retention Time Study

The retention times of caffeine, theobromine, and theophylline were analyzed to compute
the mean retention time, standard deviation of retention time, and % relative standard deviation.
Means and standard deviations were computed by use of functions resident in Microsoft Excel.
Compound Mean Retention Time Standard Deviation % RSD
(sec) (sec)

Caffeine 180.6 0.6 0.39

Theobromine 154.9 0.4 0.23
Theophylline 245.2 0.6 0.22

Caffeine Calibration Study

Calculation of Exact Concentrations of Standards

Cstock = (0.5000 g caffeine/1.0 L) x (1000 mg/g) = 500.0 mg/L = 500.0 ppm

For a given standard,

Cstd = (A)(Cstock)/100 mL
where A is an aliquot volume in mL.

Example: (Standard #1) Cstd = (5 mL)(500.0 ppm)/(100 mL) = 25.0 ppm

Table I
Exact Concentrations of Standards

Standard # Concentration (ppm)

1 25.0
2 50.0
3 75.0
4 100.0
5 150.0
6 200.0

Calculation of Mean Peak Heights and Confidence Limits

The means and standard deviations of the three replicate peak heights for each concentration
standard were computed by use of the built-in functions of Microsoft Excel. The 95% confidence
limits were computed as

Upper Limit = Mean + t*s / n½

Lower Limit = Mean - t*s / n½

where Mean is the computed mean peak height for a given standard, s is the corresponding
computed standard deviation, n = 3 replicates, and t = 4.303 for n - 1 = 2 degrees of freedom
(Microsoft Excel function “tinv”)
Table II
Mean Peak Heights, Standard Deviations, and Confidence Limits (CL)

Concentration Mean Peak Height S. Deviation Upper 95% CL Lower 95% CL

(ppm) (cm) (cm) (cm) (cm)
25.0 1.62 0.44 2.71 0.53
50.0 3.61 0.35 4.48 2.74
75.0 5.24 0.32 6.04 4.45
100.0 8.15 0.39 9.12 7.18
150.0 11.07 0.83 13.13 9.01
200.0 14.56 0.77 16.47 12.65

Calculation of Calibration Model

A least-squares calculation was used to compute the slope and intercept of the best calibration
model for the above data. The resident least-squares functions of Microsoft Excel were used for
this calculation.

Peak Height = -0.0486 + (0.074220)(Concentration)

To evaluate the quality of the calibration model, the correlation coefficient and the standard error
of estimate were computed. The built-in functions of Microsoft Excel were used for these

R2 = 0.9948 s = 1.14 cm

Plot of Calibration Model

A plot of the calibration model is included as Figure 1. This plot was generated with the
commercial software package, Axum (Version 3.0), operating on a Dell 466/L computer. The data
values from Table II are plotted along with the computed regression line.

Computation of Unknown Caffeine Concentration

The mean peak height of the unknown was calculated to be 6.47 cm. This value was
used with the computed slope and intercept to determine the concentration of caffeine in the
sample of "Pepsi".

Cunk = (6.47 + 0.0486)/0.07422 = 87.9 ppm

Figure 1: Mean peak height (cm) vs. caffeine concentration (ppm). Error bars are plotted as the
95% confidence intervals based on three replicate peak height measurements at each
concentration. The solid line identifies the computed regression line.


1. Two explanations for constituents not showing up in the chromatograms are (1) their possible
co-elution with other peaks in the chromatogram or (2) their being permanently retained on the
column. It is also possible that constituents may not absorb light at 254 nm, the wavelength used
by the detector employed here.

2. Enhanced qualitative structural information would best be obtained by use of an alternate

detector. Mass spectrometric detection would perhaps give the most structural information.


The calibration model produced an excellent fit to the experimental data, as evidenced by
the high value of the correlation coefficient. While no formal test of accuracy was performed, the
successful calibration model lends confidence to the determined caffeine value. The retention
time study indicated clearly that the three compounds could be separated using the C8-column.
This study also revealed that the retention times were quite reproducible (relative standard
deviations < 1%). No anomalies were encountered during the experimental procedure.