an introduction to hptlc and hptlc-ms · 2018-10-26 · tlc vs. hptlc. tlc analysis of asian...
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![Page 1: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/1.jpg)
Make new connections: TLC-MS Workshop - a collaboration of Waters, Camag and Merck
An introduction to HPTLC and HPTLC-MS Michaela Oberle, Merck KGaA Eschborn 24th Oktober 2018
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
![Page 2: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/2.jpg)
CAMAG – more than half a century dedicated to Chromatography CAMAG was founded 1958 in Muttenz, Switzerland
Started with the production of Aluminum Oxide for chromatography (still sold)
Since 1961 CAMAG is dedicated to Thin-Layer Chromatography (TLC)
From 1975 on CAMAG concentrated its activities on the development of instruments & automated solutions for TLC
Since the mid 80’s CAMAG provides software (currently visionCATS) to control the automation of TLC steps and to evaluate the results
Currently, CAMAG is designing the next generation of analytical instruments for the High-Performance Thin-Layer Chromatography (HPTLC)
In addition, CAMAG provides an automated high-throughput elution instrument (DBS-MS 500) to the clinical diagnostics industry
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
![Page 3: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/3.jpg)
Instrumentation for HPTLC
Sample Application
Chromatogram Development Derivatization Detection
Evaluation Documentation Hyphenation of TLC
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
![Page 4: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/4.jpg)
TLC vs. HPTLC
TLC analysis of Asian Ginseng
HPTLC analysis of 4 Ginseng species
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
![Page 5: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/5.jpg)
Advantages of planar chromatography
Nearly unlimited choice of stationary and mobile phases
Multiple samples can be analyzed in parallel
Single use of stationary phase: no clean-up
„Archive“ of separated substances on each track
Multiple detection on the same plate
Quantitation
Chemical and biological detection
Directed, cost-effective MS VISUAL REPRODUCIBLE
COST-EFFECTIVE
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
![Page 6: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/6.jpg)
Identity - HPTLC Fingerprint (Cimicifuga)
C. racemosa C. foetida
C. racemosa
adulterated
with C. foetida
C. dahurica
C. heracleifolia
C. racemosa
adulterated with
C. dahurica
C. heracleifolia
C. pachypoda
C. americana
Comparison of tracks from different plates and analysis files
Useful tool for identification and detection of adulteration in the quality control of raw materials
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
![Page 7: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/7.jpg)
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
TLC – The cost-effective way to precise results…
Sreening in front of HPLC • search for one target compound
• complex matrix
• high number of samples
Simple Qualitative Identification
• quick identification
Cost-Effective Quantification • fast results
• high throughput
• simple sample preparation
![Page 8: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/8.jpg)
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
…in particular for matrix rich samples…
![Page 9: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/9.jpg)
Simplify Analysis!
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
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Simple workflow for complex samples
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
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ATS 4 – Application
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ADC 2 – Development Chromatogram Development
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
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CAMAG TLC-MS Interface 2 with Waters QDa
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
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Principle of the TLC-MS coupling
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
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Elution
TLC/HPTLC plate with substance zones
Pressure controlled piston is lowered
HPLC pump delivers the solvent
Mass spectrometer analyzes the sample
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
![Page 16: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/16.jpg)
TLC-MS Interface 2: Round / oval elution head
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
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Principle of TLC-MS Interface 2
Positioning of the plate with the laser on the
specific spot
Piston lowers with 4 bar and the elution starts
Spot before & after elution
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
![Page 18: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/18.jpg)
TLC-MS Interface 2
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
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Elution-based TLC – MS
Acetonitrile / Water 95/5 + 0.1% formic acid Acetonitrile / Water 50/50 + 0.1% formic acid Methanol / Water 80/20 + 0.1% formic acid Methanol / Water 50/50 + 0.1% formic acid Solvent grade: hypergrade for LC – MS possible adducts: [M+H]+, [M+Na]+, [M+K]+, [M+MeOH+H]+, [M+ACN+H]+
Acetonitrile / Water 95/5 + 10 mM ammonium formate / acetate buffer
Acetonitrile / Water 50/50 + 10 mM ammonium formate / acetate buffer
Methanol / Water 80/20 + 10 mM ammonium formate / acetate buffer
Methanol / Water 50/50 + 10 mM ammonium formate /acetate buffer
Solvent grade: hypergrade for LC – MS
possible adducts: [M-H]-, [M-2H+Na]-, [M+HCO2]-
Eluents for positive ionization: Eluents for negative ionization:
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
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Elution-based TLC – MS
Step Time Description
1 5 min Rinsing: Measurement of system
2 1 - 2 min First plate blank
3 2 min Rinsing: Measurement of system
4 1 - 2 min Second plate blank
5 2 min Rinsing: Measurement of system
6 1 – 2 min Substance measurement
7 2 min Rinsing: Measurement of system
8 Result Substance measurement subtracted by plate blank
Measurement procedure including background evaluation
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
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Elution-based TLC – MS
21
Step Description Spectrum
1 Measurement of system
4 Second plate blank
6 Substance measurement
8 Substance measurement subtracted by plate blank
[M+H]+
Caffeine measurement with Methanol / Water 1/1 + 0.1% formic acid
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
![Page 22: An introduction to HPTLC and HPTLC-MS · 2018-10-26 · TLC vs. HPTLC. TLC analysis of Asian Ginseng . HPTLC analysis of 4 Ginseng species. Make new connections: TLC-MS workshop │Hohenheim](https://reader035.vdocument.in/reader035/viewer/2022062219/5e94d115faec561b7935a450/html5/thumbnails/22.jpg)
HPTLC-MS: In-process control chemical synthesis (CAMAG A-99.1)
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :
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https://www.sigmaaldrich.com/analytical-chromatography/thin-layer-chromatography.html
https://www.sigmaaldrich.com/technical-documents/articles/analytical-applications/tlc.html
Thank you for your attention!
Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :