Moderní vysokoúčinná tenkovrstvá chromatografie (HPTLC) v praxi

Používání tenkovrstvé chromatografie (TLC) v různých oblastech chemie není žádnou novinkou, avšak automatizovaná HPTLC pro některé z Vás možná novinkou bude. Pojďme společně vyvrátit představy o pracnosti této metody a vyzdvihnout její výhody.

Přednáška je zaměřená na aplikační využití HPTLC v různých oblastech chemie (farmaceutický, potravinářský a kosmetický průmysl) a na moderní přístrojové vybavení švýcarského výrobce CAMAG.

CAMAG je světovým lídrem v planární chromatografii a na celosvětovém trhu působí již více než 60 let. Umožňuje přímou analýzu různých vzorků získaných ze složitých matric pomocí instrumentální HPTLC. Jejich portfolio obsahuje instrumentaci na zabezpečení každého nezbytného kroku této metody. Konkrétně nanášení na chromatografickou desku (Linomat 5, ATS 4), vyvíjení chromatogramu (ADC 2), derivatizace, semikvantitativní (Vizualizer) a denzitometrické vyhodnocení (Scanner 4) výsledků pomocí softwaru.

Why Thin Layer Chromatography?

• Clearly visual & documented

• Rapid

• Cost efficient

• Small application volumes

• Less sample preparation

• Parallel analysis for standards and samples under identical conditions

• No solvent limitations, no carry-over

• Ideal method for complex matrices (plant materials, lipids, high sugar content samples)

DONAU LAB: Why Thin Layer Chromatography

HPTLC Application fields

• Botanical
• Pharma
• Food & Feed
• Cosmetics
• Clinical
• Forensic
• Industrial
• Environmental

Application field:

Botanical & Pharma – Comparison of Curcuma samples

• One separation & four ways to analyse

DONAU LAB: Botanical & Pharma – Comparison of Curcuma samples

Botanical & Pharma – Falsification of Sage oil

DONAU LAB: Botanical & Pharma – Falsification of Sage oil

Food & Feed – Identification of fatty oils

USP general chapter <203> Identification of Fixed oils by Thin-Layer Chromatography.

DONAU LAB: Food & Feed – Identification of fatty oils

Botanical & Pharma, Cosmetics – Analysis of lipid-based formulations

HPTLC allows an easy, rapid and robust high-throughput testing of complex lipid-containing samples

The raw materials ( different liposomes ) can be checked using a mobile phase of Chlorophorm, Methanol, Water 65:25:4 (V/V) and Primuline reagent for derivaitzation.

DONAU LAB: Botanical & Pharma, Cosmetics – Analysis of lipid-based formulations

The liposome-based formulation (sample) contains RNA, liposomes (DODMA, DOPE, DOPS), and excipients (including high concentrations of sucrose).

DONAU LAB: Botanical & Pharma, Cosmetics – Analysis of lipid-based formulations

Food & Feed – Quantification of sugar in honey

DONAU LAB: Food & Feed – Quantification of sugar in honey

Food & Feed – Semi-Quantification of nicotine in e-liquid

DONAU LAB: Food & Feed – Semi-Quantification of nicotine in e-liquid

Forensics – Cannabinoids in different Cannabis samples

DONAU LAB: Cannabinoids in different Cannabis samples

CAMAG instruments & tools portfolio

DONAU LAB: CAMAG instruments & tools portfolio

TLC line – Basic kit

DONAU LAB: CAMAG TLC line - Basic Kit

HPTLC line – Sample Application: semi automatic

LINOMAT 5

DONAU LAB: CAMAG LINOMAT 5

• Applies samples by spray technique on 20x20, 20x10 & 10x10 cm plates & sheets

• Manual: Syringe cleaning. Syringe filling. Replacing the syringe

• Automatic sample spraying in homogeneous bands or spots and in exact preprogrammed positions

• Stand-alone or by visionCATS software operation

• Syringes: Standard 100 μL , optional 500 μL

AUTOMATIC TLC SAMPLER 4

DONAU LAB: CAMAG AUTOMATIC TLC SAMPLER 4

• Applies bands and spots by spray and by contact technique on 20x20, 20x10 & 10x10 cm plates & sheets

• Fully automatic sample application and syringe cleaning

• Standard rack of 66 positions

• Automatic sample spraying in homogeneous bands or spots and in exact preprogrammed positions

• Fully controlled by visionCATS software

• Syringes : Standard 25 μL, optional 10 μL &100 μL

HPTLC line – Plate Development

Automatic Developing Chamber ADC2

DONAU LAB: Automatic Developing Chamber ADC2

• Fully automatic development of only HPTLC plates 20x10 cm
• Saturation, preconditioning & humidity are controlled which leads to unsurpassed reproducible RFs
• Stand-alone or via visionCATS software operation

DONAU LAB: Humidity effect on RF (test dye)

saturated salt solution / % relative humidity

• NaCl / 75
• KSCN / 47
• MgCl₂/ 33
• KCOOH / 20
• (Molecular sieve) / 0-5

AUTOMATED MULTIPLE DEVELOPMENT AMD 2

• Multiple development using a solvent gradient of only HPTLC plates 20x10 cm
• Between runs, the solvent is completely removed from the developing chamber and the layer is dried under vacuum.
• Each following run uses a solvent of lower elution strength than that of the one used before. Separation power improved - over regular HPTLC development
• Fully controlled by visionCATS software

DONAU LAB: CAMAG AUTOMATED MULTIPLE DEVELOPMENT - AMD 2

HPTLC line – Derivatization

Chromatogram Immersion Device III

• Derivatization by dipping
• Selectable immersion time and vertical speed
• Uniform reagent transfer
• Dip tanks for 20x20 & 20x10 cm plates & sheets
• It takes 200 ml to fill the glass dip tank

DONAU LAB: Chromatogram Immersion Device III

DERIVATIZER

• Derivatization by spraying
• Reproducible & homogeneous reagent distribution, thanks to Micro droplet spraying technology
• Low reagent consumption ( 2 - 4 ml )
• Standalone instrument with small footprint
• Suitable for plates & sheets up to 20x20 cm
• Environment & user friendly thanks to the closed hood and the wash bottle aspiration system

DONAU LAB CAMAG Derivatizer

TLC PLATE HEATER III

• Derivatization by thermal activation
• Heating the plate after spaying or after dipping in chemical reagent
• Temperature of heating surface is programmable from ambient to 200° Celsius and continuously displayed
• Suitable for plates & sheets up to 20x20 cm

DONAU LAB: TLC PLATE HEATER III

HPTLC line – Detection

TLC SCANNER 4

• Detection by scanning plates & sheets up to 20x20 cm under specific wavelength in the UV or visible range
• Works in absorption, fluorescence or spectrum mode
• Spectral range from 190 to 900 nm
• Fully controlled by visionCATS software

DONAU LAB: CAMAG TLC Scanner 4

DONAU LAB: CAMAG HPTLC Line - Detection

HPTLC line – Documentation

TLC VISUALIZER 2

• Completely dark compartment with large sliding door and small viewing window
• Three types of illumination

DONAU LAB: CAMAG TLC VISUALIZER 2 - Three types of illumination

• Professional industrial digital camera
• Suitable for plates & sheets up to 20x20 cm
• Fully controlled by visionCATS software

DONAU LAB: TLC Visualizer 2 with VisionCATS - Data view options

DONAU LAB: TLC Visualizer 2 with VisionCATS – Images comparison

DONAU LAB: TLC Visualizer 2 with VisionCATS

HPTLC line – Quantitative Evaluation

HPTLC line – Software

• CAMAG software to control CAMAG instruments, create and store TLC/HPTLC methods and analysis, process TLC/HPTLC data and print analysis reports.
• Microsoft® SQL Server® 2017 Express 64 bit database
• Has server/client architecture
• 21 CFR Part 11 compatible visionCATS

HPTLC line – Hyphenation (MS coupling)

TLC-MS Interface 2

• Hyphenating TLC and mass spectrometry opens new application areas for both techniques
• The classical procedure is : scratch, dilute, filter - Discontinuous and prone to contamination
• TLC-MS Interface 2 is a stand-alone, flexible, fast and easy to use alternative
• Suitable for plates & sheets up to 20x20 cm

DONAU LAB: Hyphenation (MS coupling)