Determination of Caffeine in Coffee using High Performance Thin Layer Chromatography

Introduction

High Performance Thin Layer Chromatography (HPTLC) is a very useful analytical technique and requires low sample preparation, here illustrated through the analysis of caffeine in coffee. Only extraction and filtration, through a 0.45 μm PTFE syringe filter, is needed for boiled coffee samples prior analysis. The HPTLC plate was pre-conditioned with the mobile phase, and a TLC-scanner was a used for the quantitation after the chromatographic separation was completed.

Results and Discussion

Caffeine was detected under UV light at 254 nm, hRf = 56, (Figure 1). A four-level calibration curve was constructed and used for quantitation purposes, (Figure 2). Each sample was analyzed in triplicate performed in parallel, a benefit with planar chromatography over both GC and HPLC.

Table 1 Chromatographic data

Track	Compound	hRf	[Caffeine] (mg/mL)	Application volume (µL)	[Caffeine] (mg/g)
1, 2, 3, 4, 9, 10, 11, 12 17, 18, 19, 20	Caffeine standard	56	1.0	0.1, 0.5, 1.0, 2.0	-
5, 13, 21	Filter Coffee 1	56		1	27.5
6, 14, 22	Filter Coffee 2	56		1	15.2
7, 15, 23	Pad Coffee	56		1	15.5
8, 16, 24	Chai Latte	56		1	4.9

Figure 1. Developed HPTLC plate at 254 nm, chromatographic data shown in the table above.

Figure 2. Calibration graph following the polynomial regression mode (r = 0.9970).

Conclusion

Caffeine can easily be quantitated in different coffee types with minimum sample preparation when using High Performance Thin Layer Chromatography.

Table 2 Application Data (Chromatography)

Chromatography
Plate	HPTLC Silica gel 60 F254, 20x10cm
Sample preparation	Caffeine standard diluted in Methanol; Coffee boiled with water and filtered with a 0.45 μm syringe filter.
Sample application	ATS4 (Camag)
Application volume	0.1 to 2.0 μL
Pre-conditioning and mobile phase	Acetonitrile / Methanol / n-Heptane 6/4/1
Pre-conditioning distance migration distance	7 cm 5 cm
Migration time	9 min

Table 3 Application Data (Detection)

Detection
Wavelength	UV light at 254 nm and 366 nm
Staining	None
MS equipment	expression CMS (Advion)
MS detection	ESI (+) mode (m/z 150 - 400)