High Speed Separation of Catechins by UHPLC (UV detection)

August 24, 2021


The main components of catechins in green tea include epicatechin (EC), epigallocatechin (EGC, hydroxy derivative of EC), epicatechin gallate (ECg, gallic acid ester of EC), and epigallocatechin gallate (EGCg, gallic acid ester of EGC).

This article shows the applicability of UHPLC for ultra-high speed analysis of 7 components: the 4 catechins listed above, catechin(C), catechin gallate(Cg), and caffeine.


LC-4000 UHPLC system


Chromatographic conditions:
Column = X-PressPak AQ-C18W (2 mmID x 50 mmL, 2 µm),
Column temperature = 30°C
Mobile phase A = 0.2% phosphoric acid / acetonitrile (90/10)
Mobile phase B = 0.2% phosphoric acid / acetonitrile (50/50)
Gradient (A/B) = 0 min (100/0) –> 0.5 min (100/0) –> 4.0 min (70/30) –> 5.0 min (70/30) –> 5.05 min (100/0)
Flow late = 0.7 mL/min
Detection = UV detection (280 nm)
Injection volume = 1 µL


Figure 1 shows an UHPLC chromatogram of a standard mixture of 7 catechins. These components were clearly separated within 5 minutes by using a gradient elution method.

Figure 1. UHPLC chromatogram of standard mixture (Peaks: 1=Epigallocatechin (500 µg/mL), 2=Caffeine (10 µg/mL), 3=Catechin (100 µg/mL), 4=Epicatechin (100 µg/mL), 5=Epigallocatechin gallate (25 µg/mL), 6=Epicatechin gallate (20 µg/mL), 7=Catechin gallate (100 µg/mL))

Figure 2 shows an UHPLC chromatogram of the tea sample. A commercially-available tea beverage was filtered with 0.2 µm membrane filter. A portion of the filtrate was injected. Each component is well separated from unknown peaks.

Figure 2. UHPLC chromatogram of commercially-available tea beverage (Peaks: 1=Epigallocatechin, 2=Caffeine, 3=Catechin, 4=Epicatechin, 5=Epigallocatechin gallate, 6=Epicatechin gallate, 7=Catechin gallate)

About the Author

Chromatography Group