• Application Note

Analysis of Herbal Medicine on an XBridge HPLC Amide Column

Analysis of Herbal Medicine on an XBridge HPLC Amide Column
  • Waters Corporation
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Healing roots and herbs in wooden bowls

This is an Application Brief and does not contain a detailed Experimental section.

Abstract

This application brief demonstrates analysis of herbal medicine on an XBridge HPLC amide column.

Introduction

Compound

Compound

Experimental

HPLC Conditions

Column:

XBridge Amide, 3.5 μm, 4.6 x 250 mm

Part Number:

186004870

Mobile Phase:

80:20 MeCN:H2O

Isocratic Flow Rate:

0.8 mL/min

Column Temp.:

60 °C

Sample Temp.:

10 °C

Injection Vol.:

20 μL

Needle Wash:

95:5 MeCN:H2O

Seal Wash:

10:90 MeOH:H2O

UV:

203 nm

Sampling Rate:

20 Hz

Filter Time Constant:

0.1 sec

Total Run Time:

18 min

Instrument:

Alliance 2695 and 2998 PDA

Pretreatment

  1. Weigh 2 g of herbal medicine powder into a centrifuge tube.
  2. Add 30 mL of 60% MeOH/40% H2O.
  3. Shake for 15 min.
  4. Centrifuge at 4,000 rpm for 10 min.
  5. Obtain the supernatant.
  6. Repeat steps 2-5 with the residue using 15 mL of 60% MeOH/40% H2O.
  7. Combine the supernatant, and make exactly 50 mL by adding 60% MeOH/40% H2O.
  8. Take 10 mL of this solution and add 3 mL of NaOH test solution (1 mol/L).
  9. Let stand for 30 min.
  10. Add 3 mL of HCl test solution (1 mol/L).
  11. Add 60% MeOH/40% H2O to make exactly 20 mL.

Solid-Phase Extraction

SPE Device:

Sep-Pak Plus C18 cartridge 360 mg (55-105 μm)

Part Number:

WAT020515
  1. Condition with 2 mL MeOH.
  2. Equilibrate with 2 mL of 30% MeOH/70% H2O just before loading.
  3. Load 5 mL of the solution from step 11 in the pretreatment stage.
  4. Wash with 2 mL of 30% MeOH/70% H2O.
  5. Wash with 1 mL of Na2CO3 test solution (1 mol/L).
  6. Wash with 10 mL of 30% MeOH/70% H2O.
  7. Elute with 5 mL MeOH (this is the injection solution).

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