Caffeine as a Surrogate for Cocaine in an
HPLC Forensic Experiment
Ray A. Gross Jr, Indravadan Shah and Muhamed
Jasarevic
Department of Physical Sciences
Prince George’s Community College
Largo, MD 20774

A modern high performance liquid chromatography
(HPLC) method for the identification and quantitative determination of caffeine as a surrogate for cocaine in colas and other caffeine containing beverages is described. Our HPLC protocol resolves caffeine in three minutes with a
HP - 1100 HPLC system. A calibration curve is prepared from standard caffeine solutions, and commercially available beverages are assayed for caffeine. The procedure is suitable for use by sophomore- level students in a forensics oriented instrumental analysis course
.

HPLC chromatography involves the separation of mixtures into individual components by passing a fluid (liquid) through a stationary phase. It is a highly efficient method, which involves a stationary phase and a mobile phase.
The liquid samples to be analyzed by HPLC are injected directly into the column, where the solvent is pumped at high speeds and high pressures - up to 1500 lb/in 2 or more from glass or stainless steel reservoirs, each of which contains 500 mL or more solvent. The reservoirs are degassed to remove dissolved gases - usually oxygen and nitrogen - that interfere by forming bubbles in the detector system.

Molecular formula: C
8
H
10
N
4
O
2
Molecular weight: 194.19
Chemical name: 1,3,7-trimethylxanthine
Properties: soft, white crystal or powder. No smell. Test is slightly bitter.
Melting point: 235 - 238 ° C
Solubility: Soluble in chloroform and partially soluble in water and ethanol.
Chemicals: HPLC grade water, HPLC grade methanol,
HPLC grade acetonitrile, Caffeine for Reference
Standard preparation, Samples - beverages

CH
3
N O
H
3
C
N
O
N
CH
3

Instrument: HP-1100 HPLC
Column: 4 X 125 ODS Hypersil 5 µm (C18 column)
Mobile phase: A = HPLC Acetonitrile 40%, B = HPLC water and methanol (50:50) 60%
Flow rate: 1.0 mL/min
Elution: Isocratic
UV detector: 270/20 nm; Reference - 360/80 nm standard cell
Run time: 3 min
Injection volume: 1.0 µL
Reference Standard: Prepared as follows.
(1 ppm = 1mg/L = 1µg/mL)
100 µg/mL, 200 µg/mL, 400 µg/mL, 600 µg/mL

HPLC grade acetonitrile, methanol, and water solvents constitute the mobile phase in the reservoir.
Turn on the HPLC; Prime the pump using the 30 or 60 mL syringe; Adjust flow of solvent to 1.00mL/min. Let the
HPLC run approximately 15 minutes before injecting the sample. Make sure the waste is coming into the waste bottle; Open the Caffeine analysis method.
When ready mode is displayed, inject 1 µL of the reference standard and run the sample. HP chemstation automatically starts data acquisition (run each concentration twice for the reference standards).
Run the sample analysis for each soft drink.

Run
3
1
1
2
1
2
3
2
3
1
Calibration table and curve
Concentration
(µg/mL)
100.00
Area (mAU’s)
100.19
200.00
93.49
95.25
173.69
150.97
400.00
600.00
163.67
261.29
274.97
266.48
362.40
Average area
96.31
162.78
267.58
377.98
Standard deviation
%CV or RSD
3.47
3.60
11.39
7.00
6.91
2.58
14.54

Caffeine RS
400
350
300
250
200
150
100
50
0
0 y = 0.5535x + 46.123
R
2
= 0.9988
200 400 600
Concentration (PPM)
800

Beverages
Area, mAU’s
( y value)
Area x dilution factor (10)
Conc.mg/L
(x value)
Hansen’s energy 60.707
607.07
1013.45
AMP energy 51.633
SoBe adrenaline 76.890
KMX energy drink 40.439
High potency energy venom
Red Bull ( sugar free)
Red Bull
121.959
39.995
31.790
Coke 17.195
516.33
768.90
404.39
1219.59
399.95
317.90
171.95
849.52
1305.83
647.27
2119.92
639.25
491.02
227.33
Coca-cola
Pepsi
11.287
8.016
112.87
80.16
120.59
61.49

The experiment and its results serve as a basis for classroom discussion, including a general discussion of method, accuracy of results, limitations of method, and economical aspects. These results showed good comparison with other techniques. HPLC is an accurate, reliable method for determining the caffeine content of many popular beverages. Various colas may serve as cocaine surrogates in an instrumental analysis course emphasizing forensic techniques.

Glenda K. Ferguson . Quantitative HPLC Analysis of an Analgesic/Caffeine
Formulation: Determination of Caffeine J. Chem. Educ. 1998 75 467.
Glenda K. Ferguson
Wesleyan College, Department of Chemistry, 4760 Forsyth Road, Macon,
GA 312
National Soft Drink Association, Bunker & McWilliums, J Am Diet, 74: 28-
32, 1979 http://www.astro.umd.edu