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Determination of Caffeine in Coffee using High Performance Thin Layer Chromatography


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, see Figure 1. A four-level calibration curve was constructed and used for quantitation purposes, see Figure 2. Each sample was analyzed in triplicate performed in parallel, a benefit with planar chromatography over both GC and HPLC.

Chromatographic data

Track Compound hRf [Caffeine]
Application volume 
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

Developed plate at 254 nm. Chromatographic data

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

Graph from the polynomial regression mode

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


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

Application data

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

Ordering Information

Description Product No.
HPTLC Si60 F254, 20x10 cm 1.05642
Acetonitrile gradient grade for liquid chromatography LiChrosolv® 1.00030
n-Heptane for liquid chromatography LiChrosolv® 1.04390
Methanol gradient grade for liquid chromatography LiChrosolv® 1.06018
Millex®-FH filter, 0.45 μm hydrophobic PTFE, 25 mm, non-sterile SLFH025