High Speed Analysis of Piperine in Pepper using UHPLC

May 17, 2021


Piperine is a particular spicy constituent that is contained in Piperaceae plants and is effective in improving energy metabolism, circulation of the blood and excessive sensitivity to cold.

It is also known that piperine has an antibacterial action, an antiseptic action and an insecticidal action, and it is contained more in black pepper. In this application, piperine in pepper was analyzed using Ultra High-performance Liquid Chromatography with Photodiode Array Detection.


LC-4000 UHPLC system


Chromatographic conditions
Column:   ZORBAX Eclipse Plus C18 (3.0 mmID x 50 mmL, 1.8 µm)
Eluent A:   0.1% Formic acid
Eluent B:  0.1% Formic acid in Acetonitrile
Gradient condition (A/B): 0 min (70/30) -> 7 min (10/90) -> 7.5 min (10/90) -> 7.55 min (70/30)  1 cycle; 10 min
Flow rate:   0.8 mL/min
Column temp.:   40ºC
Wavelength:   200 – 500 nm
Injection volume:  1 µL
Standard sample:  Piperine 0.1 mg/mL in Water/Acetonitrile (90/10)


UHPLC, Pepper, Piperine, 1.8 mm, C18 Column, PDA detector


Figure 1 shows the chromatogram and the contour plot of the piperine standard sample. A longer method analysis time was used than typical for UHPLC taking into consideration that the actual sample may require separation of other components.

Figure 1. Chromatogram and contour plot of a piperine standard sample (1: Piperine)

Figure 2 shows a spectrum of piperine.

Figure 2. Spectrum of piperine sample

Figure 3 shows the chromatogram and the contour plot of the extract of coarsely ground pepper after extraction using a Supercritical Fluid Extraction system.

Figure 3. Chromatogram and contour plot of coarsely ground pepper (1: Piperine)

Sample Preparation
Coarsely ground pepper was extracted using a Supercritical Fluid Extraction system and its extract was dissolved in 1 mL of Acetonitrile and diluted with Acetonitrile to 1/100 concentration. Then it was filtrated using 0.2 mm membrane filter.

Super Critical Fluid extraction condition
Sample :  Coarsely ground pepper  1.0 g
Extraction vessel :   10 mL ( Glass wool has been mounted in both IN and OUT
Temperature :         40 ºC.
Back Pressure :      20 MPa
CO2 Flow Rate :  3.0 mL/min

About the Author

Chromatography Group