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HomeThin Layer ChromatographyDetermination of Caffeine in Coffee Using High Performance Thin Layer Chromatography

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.

TrackCompoundhRf[Caffeine]
(mg/mL)
Application volume 
(µL)
[Caffeine]
(mg/g)
1, 2, 3, 4, 9, 10, 11, 12 17, 18, 19, 20Caffeine standard561.00.1, 0.5, 1.0, 2.0-
5, 13, 21Filter Coffee 156 127.5
6, 14, 22Filter Coffee 256 115.2
7, 15, 23Pad Coffee56 115.5
8, 16, 24Chai Latte56 14.9
Table 1Chromatographic data
A developed High-Performance Thin-Layer Chromatography (HPTLC) plate under ultraviolet light at 254 nm. The plate displays a series of numbered bands from 1 to 24, indicating different sample spots. A dashed rectangle highlights an area with the text ‘hRf = 56’ below it, suggesting the measurement of a particular spot’s retention factor.

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

A graph displaying a series of data points with a polynomial regression line fitted through them, indicating a strong correlation with an r-value of 0.9970. The data points are scattered along the line, suggesting a high degree of accuracy in the calibration process.

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.

Chromatography
PlateHPTLC Silica gel 60 F254, 20x10cm
Sample preparationCaffeine standard diluted in Methanol; Coffee boiled with water and filtered with a 0.45 μm syringe filter.
Sample applicationATS4 (Camag)
Application volume0.1 to 2.0 μL
Pre-conditioning and mobile phaseAcetonitrile / Methanol / n-Heptane 6/4/1
Pre-conditioning distance
migration distance
7 cm
5 cm
Migration time9 min
Table 2Application Data (Chromatography)
Detection
WavelengthUV light at 254 nm and 366 nm
StainingNone
MS equipmentexpression CMS (Advion)
MS detectionESI (+) mode (m/z 150 - 400)
Table 3Application Data (Detection)
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