Identification and separation of iridoids

– Developing solvent 2 –
1. Scope
This method is suitable for identification and separation of iridoids by HPTLC.

2. Authors and adoptions

Tiên Do, HPTLC Association.
Part of the HPTLC Association Substance database.

3. Procedure
Reference solutions Universal HPTLC mix (UHM) – ready to use solution
(SST)
Test solutions 1.0 mg/mL in methanol
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 2.0 µL of reference solution and test
solutions.
Developing solvent Ethanol, dichloromethane, water 45:70:6.5 (V/V)
Developing distance 70 mm from lower edge of the plate
Saturation time 20 min, with a saturation pad
Relative humidity 33%, saturated MgCl2
Temperature 22 ± 5°C
Derivatization reagent 1 Anisaldehyde sulfuric acid (AS) 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: Spray 3.0 mL of reagent (Derivatizer: blue nozzle, spraying
level 3), heat at 100°C for 3 min
Derivatization reagent 2 Vanillin reagent
Preparation: Dissolve 1.0 g of vanillin in 100.0 mL of ethanol
96% and carefully add, dropwise, 2.0 mL of concentrated
sulfuric acid. Use within 48h.
Use: Spray (Derivatizer: yellow nozzle; spraying level 4), or dip,
heat at 100°C for 3 min, let cool to room temperature.
Detections A) Underivatized, shortwave UV (254 nm)
B) Underivatized, longwave UV (350 nm broadband)
C) Underivatized, white light RT
D) Derivatized, longwave UV (350 nm broadband)
E) Derivatized, white light RT

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4. Results
System suitability test – Detection A)
 Quenching zone at RF ~ 0.249 ± 0.016*
 Quenching zone at RF ~ 0.374 ± 0.037*
 Quenching zone at RF ~ 0.684 ± 0.037*
 Quenching zone at RF ~ 0.816 ± 0.030*
* standard deviation STDEV (n=2)

Prior to derivatization

Figure 1: HPTLC in shortwave UV (A), in longwave UV (B) and in white light RT (C) prior to derivatization.

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 Mean RF (n = 2)
Track Sample Origin
± STDEV
1 UHM (ready to use solution) Sigma-Aldrich (ref 91816) -
2 Loganic acid (1.0 mg/mL) Extrasynthese; 0612/0 #01 0.169 ± 0.001
3 Harpagide (1.0 mg/mL) Extrasynthese; 1121/0 #01 0.261 ± 0.006
4 Catalpol (1.0 mg/mL) Extrasynthese; 1012/0 #01 0.304 ± 0.004
5 Aucubin (1.0 mg/mL) Extrasynthese; 0423/0 #01 0.334 ± 0.008
6 Ajugol (1.0 mg/mL) Extrasynthese; 0423/0 #01 0.342 ± 0.009
7 Hastatoside (1.0 mg/mL) Extrasynthese; 0907/0 #01 0.471 ± 0.003
8 Nuzhenide (1.0 mg/mL) Extrasynthese; 0302/0 #01 0.467 ± 0.008
9 Loganin (1.0 mg/mL) Extrasynthese; 1204/0 #01 0.525 ± 0.002
10 Swertiamarine (1.0 mg/mL) Extrasynthese; 1102/0 #01 0.541 ± 0.005
11 Geniposide (1.0 mg/mL) Extrasynthese; 1102/0 #01 0.548 ± 0.001
12 Sweroside (1.0 mg/mL) Extrasynthese; 1102/0 #01 0.556 ± 0.000
13 Gentiopicroside (1.0 mg/mL) Extrasynthese; 1102/0 #01 0.577 ± 0.000
14 Harpagoside (1.0 mg/mL) Extrasynthese; 0907/0 #01 0.587 ± 0.004
15 Agnuside (1.0 mg/mL) Extrasynthese; 1003/0 #01 0.595 ± 0.001
16 Verbenalin (1.0 mg/mL) Extrasynthese; 1102/0 #01 0.627 ± 0.004
17 Oleuropein (1.0 mg/mL) Extrasynthese; 0303/0 #01 0.657 ± 0.002
18 Amarogentin (1.0 mg/mL) Extrasynthese; 0212/0 #01 0.817 ± 0.002
19 Genipin (1.0 mg/mL) Extrasynthese; 1102/0 #01 0.862 ± 0.005
20 Valtrate (1.0 mg/mL) Extrasynthese; 0114/0 #01 0.957 ± 0.002

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After derivatization with anisaldehyde sulfuric acid reagent

Figure 2: HPTLC in longwave UV (D) and in white light RT (E) after derivatization.

Track Sample
1 UHM (ready to use solution)
2 Loganic acid (1.0 mg/mL)
3 Harpagide (1.0 mg/mL)
4 Catalpol (1.0 mg/mL)
5 Aucubin (1.0 mg/mL)
6 Ajugol (1.0 mg/mL)
7 Hastatoside (1.0 mg/mL)
8 Nuzhenide (1.0 mg/mL)
9 Loganin (1.0 mg/mL)
10 Swertiamarine (1.0 mg/mL)
11 Geniposide (1.0 mg/mL)
12 Sweroside (1.0 mg/mL)
13 Gentiopicroside (1.0 mg/mL)
14 Harpagoside (1.0 mg/mL)
15 Agnuside (1.0 mg/mL)
16 Verbenalin (1.0 mg/mL)
17 Oleuropein (1.0 mg/mL)
18 Amarogentin (1.0 mg/mL)
19 Genipin (1.0 mg/mL)
20 Valtrate (1.0 mg/mL)

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After derivatization with vanillin reagent

Figure 3: HPTLC in longwave UV (D) and in white light RT (E) after derivatization.

Track Sample
1 UHM (ready to use solution)
2 Loganic acid (1.0 mg/mL)
3 Harpagide (1.0 mg/mL)
4 Catalpol (1.0 mg/mL)
5 Aucubin (1.0 mg/mL)
6 Ajugol (1.0 mg/mL)
7 Hastatoside (1.0 mg/mL)
8 Nuzhenide (1.0 mg/mL)
9 Loganin (1.0 mg/mL)
10 Swertiamarine (1.0 mg/mL)
11 Geniposide (1.0 mg/mL)
12 Sweroside (1.0 mg/mL)
13 Gentiopicroside (1.0 mg/mL)
14 Harpagoside (1.0 mg/mL)
15 Agnuside (1.0 mg/mL)
16 Verbenalin (1.0 mg/mL)
17 Oleuropein (1.0 mg/mL)
18 Amarogentin (1.0 mg/mL)
19 Genipin (1.0 mg/mL)
20 Valtrate (1.0 mg/mL)

Version Revision history Released by

1 Created by: TD/27 Aug 2021 ER/27 Aug 2021

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