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 :

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 :

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 :

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 :

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 :

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 :

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

Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :

…in particular for matrix rich samples…

Simplify Analysis!

Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :

Simple workflow for complex samples

Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :

ATS 4 – Application

Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :

ADC 2 – Development Chromatogram Development

Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :

CAMAG TLC-MS Interface 2 with Waters QDa

Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :

Principle of the TLC-MS coupling

Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :

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 :

TLC-MS Interface 2: Round / oval elution head

Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :

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 :

TLC-MS Interface 2

Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :

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 :

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 :

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 :

HPTLC-MS: In-process control chemical synthesis (CAMAG A-99.1)

Make new connections: TLC-MS workshop │Hohenheim 11th October 2018│A collaboration between :

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 :