1. Anhalt University of Applied Sciences
Welcome to my presentation.
Multidimensional Chromatography (HPLC-HPTLC) for Identification of Antifungal Substances in Rheum Root Extracts
M.Sc. Ing. ecotroph. Silvia Ballert
Bernburg, Germany
Anhalt University of Applied Sciences
HTPLC Symposium July 6th 2017
Institute of Bioanalytical Sciences

2. 1. Institute of Bioanalytical Sciences (IBAS)
I work at the IBAS at the applied university in BBG.
The core competence of the intitute are plant extracts and their various use in food, cosmetics or technical application.
One of the latest projects was the development of a biological plant protection agent which should be based on rhubarb root extracts.
We started with the harvest an extraction of Rheum leucorum cultivated on our own test field.
Core competence:
Production of plant extracts with defined activity spectrum for:
food and dietary supplements
cosmetics and drugs
technical applications
plant protection
Topic: Development of a biological plant protection agent based on rhubarb root extracts
Field with different Rheum genotypes
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

3. 2. Workflow Harvest and extraction of Rheum leucorum Rheum leucorum
I work at the IBAS at the applied university in BBG.
The core competence of the intitute are plant extracts and their various use in food, cosmetics or technical application.
One of the latest projects was the development of a biological plant protection agent which should be based on rhubarb root extracts.
We started with the harvest an extraction of Rheum leucorum cultivated on our own test field.
Followed by biological testing of the relative mycelium growth of plant pathogenes on extract spiked plates versus a control seen on the left.
Harvest and extraction of Rheum leucorum
Rheum leucorum
Rh. leucorum roots
Rh. leucorum dried extract
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

4. 2. Workflow Harvest and extraction of Rheum leucorum
I work at the IBAS at the applied university in BBG.
The core competence of the intitute are plant extracts and their various use in food, cosmetics or technical application.
One of the latest projects was the development of a biological plant protection agent which should be based on rhubarb root extracts.
We started with the harvest an extraction of Rheum leucorum cultivated on our own test field.
Followed by biological testing of the relative mycelium growth of plant pathogenes on extract spiked plates versus a control seen on the left.
Harvest and extraction of Rheum leucorum
Biological testing of the dried total extract
Control (left) vs. relative mycelium growth of P. capsici on plates with Rh. leucorum extract in different concentrations
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

5. 2. Workflow Harvest and extraction of Rheum leucorum
I work at the IBAS at the applied university in BBG.
The core competence of the intitute are plant extracts and their various use in food, cosmetics or technical application.
One of the latest projects was the development of a biological plant protection agent which should be based on rhubarb root extracts.
We started with the harvest an extraction of Rheum leucorum cultivated on our own test field.
Followed by biological testing of the relative mycelium growth of plant pathogenes on extract spiked plates versus a control seen on the left.
The active extract was then analyzed with 2d HPTLC revealing a multitude of substances, often even sharing a Rf-value, as seen in the scan.
Harvest and extraction of Rheum leucorum
Biological testing of the dried total extract
Analysis of the dried total extract
2D-HPTLC of total extract, toluene: ethyl acetate: formic acid, derivatization: vanillin reagent, white transmission light
1st Dimension: 1 : 8 : 1 (v/v/v)
2nd Dimension: 4 : 5 : 1 (v/v/v)
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

6. 2. Workflow Harvest and extraction of Rheum leucorum
I work at the IBAS at the applied university in BBG.
The core competence of the intitute are plant extracts and their various use in food, cosmetics or technical application.
One of the latest projects was the development of a biological plant protection agent which should be based on rhubarb root extracts.
We started with the harvest an extraction of Rheum leucorum cultivated on our own test field.
Followed by biological testing of the relative mycelium growth of plant pathogenes on extract spiked plates versus a control seen on the left.
The active extract was then analyzed with 2d HPTLC revealing a multitude of substances, often even sharing a Rf-value, as seen in the scan.
Harvest and extraction of Rheum leucorum
Biological testing of the dried total extract
Analysis of the dried total extract
1st Dimension: 1 : 8 : 1 (v/v/v)
2nd Dimension: 4 : 5 : 1 (v/v/v)
Phenolic constituents in Rheum spp.
Anthraquinone derivatives
Flavan-3-ols / Proanthocyanidins
Stilbenes
Naphthalenes
Phenylbutanones
Chromone
Phenolic acids
Hydroxycinnamic acids
Flavonols
Anthocyanins
Source: G. Nonaka, Y. Kashiwada and I. Nishioka several publications, Chem. Pharm. Bull
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

7. 2. Workflow Harvest and extraction of Rheum leucorum
I work at the IBAS at the applied university in BBG.
The core competence of the intitute are plant extracts and their various use in food, cosmetics or technical application.
One of the latest projects was the development of a biological plant protection agent which should be based on rhubarb root extracts.
We started with the harvest an extraction of Rheum leucorum cultivated on our own test field.
Followed by biological testing of the relative mycelium growth of plant pathogenes on extract spiked plates versus a control seen on the left.
The active extract was then analyzed with 2d HPTLC revealing a multitude of substances, often even sharing a Rf-value, as seen in the scan.
Harvest and extraction of Rheum leucorum
Biological testing of the dried total extract
Analysis of the dried total extract
1st Dimension: 1 : 8 : 1 (v/v/v)
2nd Dimension: 4 : 5 : 1 (v/v/v)
Phenolic constituents in Rheum spp.
Anthraquinone derivatives
Flavan-3-ols / Proanthocyanidins
Stilbenes
Naphthalenes
Phenylbutanones
Chromone
Phenolic acids
Hydroxycinnamic acids
Flavonols
Anthocyanins
Source: G. Nonaka, Y. Kashiwada and I. Nishioka several publications, Chem. Pharm. Bull
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

8. 2. Workflow Harvest and extraction of Rheum leucorum
I work at the IBAS at the applied university in BBG.
The core competence of the intitute are plant extracts and their various use in food, cosmetics or technical application.
One of the latest projects was the development of a biological plant protection agent which should be based on rhubarb root extracts.
We started with the harvest an extraction of Rheum leucorum cultivated on our own test field.
Followed by biological testing of the relative mycelium growth of plant pathogenes on extract spiked plates versus a control seen on the left.
The active extract was then analyzed with 2d HPTLC revealing a multitude of substances, often even sharing a Rf-value, as seen in the scan.
To reduce the variety of compounds per tested extract, a fractionation with the preparative HPLC was executed.
Harvest and extraction of Rheum leucorum
Biological testing of the dried total extract
Analysis of the dried total extract
Fractionation using preparative HPLC
Chromatogram of total extract, column Zorbax Eclipse XDB-C18 30 x 250mm, 5µm, Agilent Technologies; water and 0,5 % acetic acid : methanol, gradient from 10 % to 100 % methanol, detection 280nm
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

9. 2. Workflow
I work at the IBAS at the applied university in BBG.
The core competence of the institute are plant extracts and their various use in food, cosmetics or technical application.
One of the latest projects was the development of a biological plant protection agent which should be based on rhubarb root extracts.
We started with the harvest an extraction of Rheum leucorum cultivated on our own test field.
Followed by biological testing of the relative mycelium growth of plant pathogens on extract spiked plates versus a control seen on the left.
The active extract was then analyzed with 2d HPTLC revealing a multitude of substances, often even sharing a Rf-value, as seen in the scan.
To reduce the variety of compounds per tested extract, a fractionation with the preparative HPLC was executed.
Obtained fractions were then again biologically tested and analyzed with HPTLC-MS to determine the relation between a substance structure and it´s antifungal effect.
Harvest and extraction of Rheum leucorum
Biological testing of the dried total extract
Analysis of the dried total extract
Fractionation using preparative HPLC
Biological testing of obtained fractions
Analysis of obtained fractions using HPTLC-MS
Determination of the relation between substance structure and antifungal effect
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

10. 3.1 HPTLC - Materials Stationary phases
For the development we use three different normal phases silica gel plates to compare efficiency. The first one I will refer to as „basic“, then we have Lichrosphere and AMD called „extra thin“.
The mobile phases consist of toluene, ethyl acetate and formic acid in different ratios.
For derivatization vanillin reagent with sulfuric acid, natural product reagent and polyethylene glycol are applied.
Stationary phases
HPTLC Silica gel 60 F254 , Merck (basic)
LiChrospher HPTLC Silica gel 60 F254S, Merck
HPTLC Silica gel 60 F254 AMD, extra thin, Merck
Mobile phases
Toluene : ethyl acetate : formic acid
1 : 8 : 1 (v/v/v)
4 : 5 : 1 (v/v/v)
Gradient: 100 % ethyl acetate : formic acid (9 :1 (v/v)) to 100 % toluene in 11 steps of 10 % (AMD 2)
Derivatizations
Vanillin reagent with sulfuric acid (VR)
Natural product reagent (NPR)
Polyethylene glycol solution (PEG)
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

11. 3.1 HPTLC - Materials Stationary phases
For the development we use three different normal phases silica gel plates to compare efficiency. The first one I will refer to as „basic“, then we have Lichrosphere and AMD called „extra thin“.
The mobile phases consist of toluene, ethyl acetate and formic acid in different ratios.
For derivatization vanillin reagent with sulfuric acid, natural product reagent and polyethylene glycol are applied.
Stationary phases
HPTLC Silica gel 60 F254 , Merck (basic)
LiChrospher HPTLC Silica gel 60 F254S, Merck
HPTLC Silica gel 60 F254 AMD, extra thin, Merck
Mobile phases
Toluene : ethyl acetate : formic acid
1 : 8 : 1 (v/v/v)
4 : 5 : 1 (v/v/v)
Gradient: 100 % ethyl acetate : formic acid (9 :1 (v/v)) to 100 % toluene in 11 steps of 10 % (AMD 2)
Derivatizations
Vanillin reagent with sulfuric acid (VR)
Natural product reagent (NPR)
Polyethylene glycol solution (PEG)
Gradient AMD 2
Ethyl acetate : formic acid (9:1(v/v)) [%]
Toluene [%]
Distance [mm]
100 20 90 10 30 80 37 70 42 60 40 47 50 52 57 62 67 72 75
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

12. 3.2 HPTLC - Development F1, F2:
As a result of the preparative HPLC run, which you can see at the top, 32 fractions were obtained. 16 fractions contained substances of high purity, marked blue in the chromatogram of the HPLC.
F1, F2:
Toluene : ethyl acetate : formic acid (1 : 8 : 1 (v/v/v)), NPR + PEG, 366nm
F4, F9, F14, F15:
Toluene : ethyl acetate : formic acid (1 : 8 : 1 (v/v/v)), VR, white transmission mode
F7: Gradient AMD,
VR, white transmission mode
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

13. 3.2 HPTLC - Development F16, F17, F30, F31, F32:
As a result of the preparative HPLC run, which you can see at the top, 32 fractions were obtained. 16 fractions contained substances of high purity, marked blue in the chromatogram of the HPLC.
F16, F17, F30, F31, F32:
Toluene : ethyl acetate : formic acid (1 : 8 : 1 (v/v/v)), underivatized, 366nm
F20:
Toluene : ethyl acetate : formic acid (1 : 8 : 1 (v/v/v)), NPR, 366nm
F18, F27: Toluene : ethyl acetate : formic acid (1 : 8 : 1 (v/v/v)), VR, white transmission mode
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

14. 3.3 HPTLC - Development F3, F5, F6, F8, F10, F11:
However the 16 other fractions, marked green in the HPLC run above, contained several substances, which required further method development for the separation with HPTLC to reach the optimized separations as shown here.
F3, F5, F6, F8, F10, F11:
Toluene : ethyl acetate : formic acid
(1 : 8 : 1 (v/v/v)),
VR, white transmission mode
F12:
Gradient AMD,
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

15. 3.3 HPTLC - Development F19, F21:
However the 16 other fractions, marked green in the HPLC run above, contained several substances, which required further method development for the separation with HPTLC to reach the optimized separations as shown here.
F19, F21:
Toluene : ethyl acetate : formic acid
(1 : 8 : 1 (v/v/v)), NPR + PEG, 366nm
F22, F23:
(1 : 8 : 1 (v/v/v)), NPR, 366nm
F24, 25, 26, 29:
(1 : 8 : 1 (v/v/v)), underivatized, 366nm
F28: Gradient AMD, NPR, 366nm
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

16. 3.3 HPTLC - Development
Therefore the twin trough chamber was replaced by the automatic development chamber, to increase reproducibility and greater control of the development conditions, resulting in a better separation. The different stationary phases showed a comparable separation between the basic and extra thin material for F3 and F5, while the Lichrospher had a poorer resolution. With the AMD chamber the goal was to achieve a better resolution and further separation of the critical pair of spots in F3. Although the two spots were now more distinct, their distance had even reduced, which meant this application could not be used for the TLC-MS due to the size of the elution head of the interface.
TTC and ADC: Toluene : ethyl acetate : formic acid (1 : 8 : 1 (v/v/v)), VR, white transmission mode
AMD: Gradient, VR, white transmission mode
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

17. 3.3 HPTLC - Development
Therefore the twin trough chamber was replaced by the automatic development chamber, to increase reproducibility and greater control of the development conditions, resulting in a better separation. The different stationary phases showed a comparable separation between the basic and extra thin material for F3 and F5, while the Lichrospher had a poorer resolution. With the AMD chamber the goal was to achieve a better resolution and further separation of the critical pair of spots in F3. Although the two spots were now more distinct, their distance had even reduced, which meant this application could not be used for the TLC-MS due to the size of the elution head of the interface.
TTC and ADC: Toluene : ethyl acetate : formic acid (1 : 8 : 1 (v/v/v)), VR, white transmission mode
AMD: Gradient, VR, white transmission mode
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

18. 3.3 HPTLC - Development
Therefore the twin trough chamber was replaced by the automatic development chamber, to increase reproducibility and greater control of the development conditions, resulting in a better separation. The different stationary phases showed a comparable separation between the basic and extra thin material for F3 and F5, while the Lichrospher had a poorer resolution. With the AMD chamber the goal was to achieve a better resolution and further separation of the critical pair of spots in F3. Although the two spots were now more distinct, their distance had even reduced, which meant this application could not be used for the TLC-MS due to the size of the elution head of the interface.
TTC and ADC: Toluene : ethyl acetate : formic acid (1 : 8 : 1 (v/v/v)), VR, white transmission mode
AMD: Gradient, VR, white transmission mode
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

19. 3.3 HPTLC - Development
Therefore the twin trough chamber was replaced by the automatic development chamber, to increase reproducibility and greater control of the development conditions, resulting in a better separation. The different stationary phases showed a comparable separation between the basic and extra thin material for F3 and F5, while the Lichrospher had a poorer resolution. With the AMD chamber the goal was to achieve a better resolution and further separation of the critical pair of spots in F3. Although the two spots were now more distinct, their distance had even reduced, which meant this application could not be used for the TLC-MS due to the size of the elution head of the interface.
TTC and ADC: Toluene : ethyl acetate : formic acid (1 : 8 : 1 (v/v/v)), VR, white transmission mode
AMD: Gradient, VR, white transmission mode
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

20. 3.4 HPTLC - Derivatization
After the optimization of the development the derivatization was optimized as well. Here we have a comparison between the dipping and spraying technic performed with the immersion device and the new derivatizer from camag. As you can see the coloring is equal, if not even better with the spraying device while requiring only 2-3ml of solvent instead of ml for a 10 by 20 cm plate.
Source:
Derivatizer
Immersion Device
Spraying
Dipping
Spraying
Dipping
F5, F15, F21 (f.l.t.r.): : Toluene : ethyl acetate : formic acid (1 : 8 : 1 (v/v/v)), VR, white transmission mode
Total extract, F5, F15, F21 (f.l.t.r.): : Toluene : ethyl acetate : formic acid (1 : 8 : 1 (v/v/v)), NPR, 366nm
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

21. 3.4 HPTLC - Derivatization
A further advantage is the possibility to use partial derivatization. This can be necessary if a sample contains substances which require different derivatizations as shown on the right, this way you can apply the sample several times on one plate and compare the different derivatizations.
This technic has also special importance for the HPTLC-MS, as you often need to derivatize compounds to make them visible, however for the MS the tracks should not be derivatized. By partially covering the plate with an old plate as seen on the left, the stationary phase underneath stays untouched, which is proven by the picture at 254nm with the derivatized area in the middle. The edges are so clear, that a 2mm distance between the tracks suffices.
Partial derivatization with the Derivatizer (CAMAG)
Different substances in one sample require different derivatizations
Different samples on one plate
HPTLC-MS (mass spectrometry)
Two dimensional HPTLC
Partially covered plate
Partially derivatized plate, 254nm
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

22. 4. Two dimensional HPTLC F22
As a preliminary step for the TLC-MS a 2d development of the fractions is done to ensure spot purity. Here you can see that a single development with the solvent system of dimension 2 only shows one peak with a shoulder in the scan, while the solvent of the first dimension separates F22 in 4 spots or peaks.
F22
2nd Dimension: 4 : 5 : 1 (v/v/v)
1st Dimension: 1 : 8 : 1 (v/v/v)
Scan (366 nm) of reference development of 2nd dimension: Toluene : ethyl acetate : formic acid 4 : 5 : 1 (v/v/v)
Scan (366 nm) of reference development of 1st dimension: Toluene : ethyl acetate : formic acid 1 : 8 : 1 (v/v/v)
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

23. 4. Two dimensional HPTLC F22 Spot 1 1st Dimension: 1 : 8 : 1 (v/v/v)
The scan of the 2d development shows that the single peak of the 2. dimension solvent would not have been pure.
2nd Dimension: 4 : 5 : 1 (v/v/v)
1st Dimension: 1 : 8 : 1 (v/v/v)
Scan (366 nm) of 2nd dimension in 2D-development: Toluene : ethyl acetate : formic acid 4 : 5 : 1 (v/v/v)
F22
Spot 1
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

24. 4. Two dimensional HPTLC F22 Spot 3 Spot 2 Spot 1
While the scans of the 1. dimension in the 2d development are all pure. Therefore the solvent of the first dimension is used for the TLC-MS analysis.
F22
1st Dimension: 1 : 8 : 1 (v/v/v)
2nd Dimension: 4 : 5 : 1 (v/v/v)
Scans (366 nm) of 1st dimension in 2D-development: Toluene : ethyl acetate : formic acid 1 : 8 : 1 (v/v/v)
Spot 3
Spot 2
Spot 1
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

25. 5. HPTLC – MS Devices TLC-MS-Interface 2 (CAMAG)
For the TLC-MS analyzis the Interface from Camag coupled with a triple quadrupole with linear ion trap is used. This way not only a full scan but also an enhanced product ion scan can be performed.
Devices
TLC-MS-Interface 2 (CAMAG)
4000 Qtrap (Sciex), Triple Quadrupol with Linear Ion Trap (LIT)
Mobile phase for elution
Methanol 0,1 ml/min
Mode MS
Full Scan 100 – 1300 Da
Product Ion Scan (MS/MS)
Enhanced Product Ion Scan (EPI)
Source:
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

26. 5. HPTLC – MS F22 Spot 1 Chrysophanol-glucopyranoside
The identification of the substances in the spot is done by the motherion (blue) and it´s fragments.
F22 Spot 1
Chrysophanol-glucopyranoside
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

27. 5. HPTLC – MS F22 Spot 2 Torachrysone-glucopyranoside
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

28. 5. HPTLC – MS F22 Spot 3 Torachrysone-acetylglucopyranoside
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

29. 5. HPTLC – MS F22 Spot 4 Torachrysone derivative
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

30. 6. Conclusion
Concluding it can be said, that the separation and based on that the identification with HPTLC-MS was successful. Rheum leucorum mainly contains flavan-3-ols and torachrysone derivatives as well as many anthraquinons. Unidentified substances will be determined with NMR soon. Those fractions with highest antifungal activity mainly contained procyanidins and torachryson derivatives. (naphtholglucosides)
Blumeria graminis f.sp. hordei
Rh. leucorum contains flavan-3-ols, naphthol-glucosides and anthraquinones
NMR for structural determination
Biological testing against a barley pathogen showed high antifungal activity in fractions with procyanidins and torachrysone derivatives
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

31. Thank you for your attention.
Welcome to my presentation.
Thank you for your attention.
Co-authors: M.Sc. Marit Gillmeister
Dr. Kathrin Kabrodt
Prof. Dr. Ingo Schellenberg
Anhalt University of Applied Sciences
HTPLC Symposium July 6th 2017
Institute of Bioanalytical Sciences

32. Relative infestation of barley leafs with Blumeria graminis f. sp
Relative infestation of barley leafs with Blumeria graminis f.sp. hordei
PB1
P + T T
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences

33. Relative infestation of barley leafs with Blumeria graminis f. sp
Relative infestation of barley leafs with Blumeria graminis f.sp. hordei
Control
Treated leafes
Anhalt University of Applied Sciences
Institute of Bioanalytical Sciences