Zingiber officinale – rhizome

1. Scope
This method identifies dried rhizomes scraped, partially scraped, or unscraped of Zingiber
officinale Roscoe by HPTLC fingerprint and discriminates the herbal drugs described under
results.
2. Authors and adoptions
HPTLC Association
Adopted in USP41-N36 S1 (change to SST requirements).
3. Procedure
Test solution Mix 1.00 g of powdered sample with 10.0 mL of methanol and
sonicate for 10 minutes, then centrifuge the mixture Use the
supernatant as test solution.
Reference solutions Standard solution A: 2 mg/mL of USP Ginger Constituent Mixture
RS in methanol*.
Standard solution B: 100 mg/mL of USP Powdered Ginger RS in
methanol. Sonicate for 10 min, centrifuge, and use the
supernatant as reference solution.
*Deviation from monograph with higher concentration of
Standard solution A
Stationary phase HPTLC Si 60 F254 (Merck)
Application 15 tracks, band length 8 mm, track distance 11.4 mm, distance
from left edge 20 mm, distance from lower edge 8 mm, application
volume 6.0 µL of Standard solution A, 2.0 µL each of Standard
solution B and test solution.
Developing solvent Toluene, ethyl acetate 3:1 (v/v)
Developing distance 70 mm from lower edge
Saturation time 20 min, with a saturation pad
Relative humidity 33%, saturated MgCl2
Temperature 22 ± 5°C
Derivatization reagent Anisaldehyde reagent
Preparation: Slowly and carefully mix 170 mL of ice-cooled
methanol with 20 mL of acetic acid and 10 mL of sulfuric acid.
Allow the mixture to cool to room temperature, then add 1 mL of
anisaldehyde (p-methoxy benzaldehyde).
Use: Derivatize (Immersion device: time 0, speed 5; Derivatizer:
3 mL, blue nozzle, spraying level 3), heat at 100°C for 3 min.
Detection A) Underivatized, UV 254 nm*
B) Underivatized, UV 366 nm*
C) Underivatized, white light RT*
D) Derivatized, UV 366 nm
E) Derivatized, white light RT
*Deviation from monograph with additional detection modes

International Association for the Advancement of High Performance Thin Layer Chromatography
A non-profit organization dedicated to the promotion of HPTLC in plant analysis and other analytical fields • www.hptlc-association.org
Page 1 of 3
4. Results
Note: These chromatographic fingerprints are representative of the samples used in this
analysis. Fingerprints obtained may vary from sample to sample. Analysts must validate the
most appropriate fingerprint for their identity standard.
System suitability test (under white light, after derivatization)

 6-gingerol: brown zone at RF ~ 0.24

 6-shogaol: brown zone at RF ~ 0.53

Figure 1: HPTLC fingerprints UV 254 nm (A), UV 366 nm (B) and white light (C) prior to derivatization, and UV 366
nm after derivatization (D), and white light after derivatization (E).

Track Sample Origin

1 USP Ginger Constituent Mixture RS (6-gingerol and 6-shogaol with increasing RF) --
2 USP Powdered Ginger RS --
3-8 Zingiber officinale – powdered rhizome Unknown
9-10 Zingiber zerumbet – powdered rhizome (same preparation as test solution) Unknown
11-12 Alpinia galanga – powdered rhizome (same preparation as test solution) Unknown

International Association for the Advancement of High Performance Thin Layer Chromatography
A non-profit organization dedicated to the promotion of HPTLC in plant analysis and other analytical fields • www.hptlc-association.org
Page 2 of 3
Identification
Compare the results with the reference images. The fingerprint of the test solution prepared
from the sample is similar to those obtained from the corresponding botanical reference
materials. Additional weak zones may be present.
Under white light, after derivatization, the chromatogram of Standard solution A shows two
prominent zones, the lower due to 6-gingerol, the upper due to 6-shogaol. The chromatogram
of Standard solution B shows a succession of dark-violet zones between the origin and the
intense dark-brown zone corresponding to that of the 6-gingerol in Standard solution A;
immediately above the 6-gingerol zone in the Standard solution B chromatogram, less intense
zones due to 8-gingerol and 10-gingerol are observed. A variable number of low-intensity dark-
gray zones appear between 10-gingerol and the second prominent zone corresponding to 6-
shogaol in Standard solution A. In the upper third part of the chromatogram, a dark-purple
somewhat diffuse zone is observed. Under UV 366 nm after derivatization, the chromatograms
of the Standard solutions exhibit patterns similar to those observed under white light. The
zones due to all gingerols and shogaols are bright orange; the zones between the origin and
the 6-gingerol zone are dark-red to brown, somewhat less prominent than when observed in
white light. The zones between 10-gingerol and 6-shogaol are variably colored; frequently, a
light-gray zone appears halfway within these zones with a light-purple diffuse zone between it
and the orange zone due to 6-shogaol. The diffuse zone in the upper-third part assumes a
purple-pink hue.
The chromatogram of the test solution under white light and UV 366 nm exhibits the zone
pattern similar to that observed with Standard solution B. The zone in the upper-third part of
the chromatogram, however, has no diagnostic significance. Its color may range from purple-
pink to muddy yellow, or may be absent.

Test for other species

Under white light after derivatization, the chromatogram of Zingiber zerumbet rhizome lacks
the zones due to 6-gingerol and 6-shogaol and instead shows an olive green zone in the upper
third section. Alpinia galangal rhizome does not show any of the zones detected in standard
solution B.

Version Revision history Released by

1 Created by: EC/18 Nov 2009 ER/12 Dec 2012
2 Harmonized with USP40 monograph, images updated: VW/05 MS/01 Dec 2018
Oct 2018
3 Harmonized with the new template: DF/05 Aug 2019 M Sharaf/25 Mar 2020

International Association for the Advancement of High Performance Thin Layer Chromatography
A non-profit organization dedicated to the promotion of HPTLC in plant analysis and other analytical fields • www.hptlc-association.org
Page 3 of 3