1. Quantitative effect-directed analysis based on TLC/HPTLC-direct bioautography
Irena Choma, UMCS Lublin, Poland
HPTLC Berlin, 2017

2. Effect –directed analysis
Extracts
extraction
fractionation
BIOLOGICAL DETECTION
Complex mixture
Fractions
refractionation/
purification
Pure constituents
Chemical identification
LC-MS, GS-MS, IR, NMR
Toxicological studies
I.M. Choma, W. Jesionek, Instrumental TLC, ed. C.F. Pool, chapter 11

3. Frequently used TLC-bioassays
Estrogenic activity (Yes test).
Enzymatic tests (acetylocholinesterase inhibition test).
Antioxidant and radical scavenging activity tests (DPPH, ABTS).
Antimicrobial tests.
Spira D. et al., J. Planar Chromatogr.
26 (2013) 395
Zheng X. et al., J. Planar Chromatogr, 24 (2011) 470
Jesionek, W. et al., J. Liq. Chromatogr. & Rel. Tech., 38 (2015)
Jesionek, W. et al.,
JPC., 26 (2013)

4. contact bioautography immersion/overlay bioautography
direct bioautography
immersion in agar
overlay with agar
dipping in broth
spraying with broth
TLC plate
Petri dish
(mirror image)

5. Visualization in TLC-DB
Bacillus subtilis
Aliivibrio fischeri

6. TLC-DPPH Lihua Gu et al., J. Planar Chromatogr. 27 (2014) 5-10
TLC chromatogram, scanning profile of Magnoliae officinalis Cortex samples from different locations in China
Linear calibration curves of magnolol and honokiol, based on their antioxidant effect
Lihua Gu et al., J. Planar Chromatogr. 27 (2014) 5-10

7. TLC-DB: Estrogenic compounds
S. Buchinger, D. Spira, Anal. Chem., 85 (2013) 7248−7256

8. TLC-DB: Estrogenic compounds
M. B. Muller, C. Weins, Chromatography, 60 (2004)

9. TLC-DB: Estrogenic compounds
HPTLC-pYES dose-response curves (n = 4) for six EDCs
I. Klingelhöfer, G. E. Morlock, J. Chromatogr. A., 1360 (2014) 288–295

10. TLC-DB: Estrogenic compounds
D. Schick, W. Schwack, J. Chromatogr.A (2017)

11. CALIBRATION IN TLC-DB
Calibration curves: rhombus – enrofloxacin, square – ciprofloxacin
I.M. Choma et al., J. Liquid Chromatogr. & Rel. Technol., 27 (2004) 2071

12. CALIBRATION IN TLC-DB
Exponential calibration curves: rhombus – enrofloxacin, square – ciprofloxacin. The data from four bioautograms.
I.M. Choma et al., J. Liquid Chromatogr. & Rel. Technol., 27 (2004) 2071

13. NP TLC
RP HPLC
I.M. Choma, J. Planar Chromatogr., 19 (2006)

14. Cefacetril in milk
Excretion of cefacetril after intramammary application at 2, 3, 6, 8, 10, 24, 36 and 48 hours. Lower trial - 5 μl, upper trial 10 μl.
I.M. Choma et al., J. Liquid Chromatogr. & Rel. Technol., 31 (2008) 1903

15. Cefacetril in milk HPLC TLC - DB
I.M. Choma et al., J. Liquid Chromatogr. & Rel. Technol., 31 (2008) 1903

16. Cefacetril in milk
Concentration of cefacetril (ppm) in milk as a function of time after administration of the drug to a cow. Circle – HPLC data, rhombus – TLC - DB data.
I.M. Choma et al., J. Liquid Chromatogr. & Rel. Technol., 31 (2008) 1903

17. A. Ramirez et al. J. Chromatogr. B 784 (2003) 315–322

18. Chlorogenic acid in coffee TLC-DB Bacillus subtilis DPPH
NP-PEG

19. Chlorogenic acid in coffee
HPLC 254nm
TLC-densitometer 254
TLC-VideoScan 254 nm
Coffee
C CQA [mg/mL]
SD n = 5
RSD %
SD n = 4
RSD %
SD n = 9
Brazil
2,34
0,10
4,42
0,21
8,87
1,66
0,65
39,21
Salvador
2,22
0,11
4,96
2,14
9,93
1,82
0,71
39,05
Colom-bia
2,79
0,23
8,30
2,39
0,14
5,90
2,06
0,73
35,27
Papua
3,10
0,17
5,53
3,05
0,45
14,85
2,41
0,95
39,38
Papua Black
0,19
0,02
7,90
7,03
0,32
0,03
8,59
Thank you for your kind attention

20. Chlorogenic acid in coffee [mg/ml]
HPLC 325
TLC-densit. 325
HPLC 254
TLC-densit. 254
TLC-Video Scan 254
TLC-Video Scan 366
TLC-DB
NP-PEG 366
B.subtilis VIS
DPPH VIS
Brazil green
2,44
2,93
2,18
2,34
1,66
3,56
4,87
2,05
1,70
Salvador green
2,59
2,71
2,22
2,14
1,82
2,75
5,44
1,94
1,61
Colombia green
3,09
3,24
2,79
2,39
2,06
3,31
6,17
2,50
1,73
Papua green
3,49
4,20
3,10
3,05
2,41
4,27
8,58
3,77
2,62
Papua
black
0,18
0,17
0,19
0,21
0,32
0,27
0,58
0,37
0,26

21. Total amount of polyphenols in coffee
TLC ,,dot-blot’’-NP/PEG
Spectrophotometry UV-VIS
Folin-Ciocalteu
Brazil green
4,31
4,53
Salvador green
4,15
4,25
Colombia green
4,34
2,90
Papua green
4,55
5,76
Papua black
1,11
1,99
CQA 20 mg/mL, MeOH, μL μL
CQA 0,1 mg/mL, ,5 mg/mL, mg/mL, mg/mL, mg/mL mg/mL,
Brazili G Salvador G Colombia G Papua G Papua B.

22. Thank you for your
kind attention