HPTLC
HIGH-PERFORMANCE
THIN-LAYER CHROMATOGRAPHY
Ing. Aneta Hartmanová
DONAU LAB, s.r.o.
© CAMAG 2020
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)
Botanical Pharma Food & Feed Cosmetics
HPTLC Application fields
Clinical Forensic Industrial Environmental
Application field:
Botanical & Pharma – Comparison of Curcuma samples
One separation & four ways to analyse
Application field:
Botanical & Pharma – Falsification of Sage oil
GC
Application field:
Food & Feed – Identification of fatty oils
Track Volume Sample Track Volume Sample
1 2 µL Corn oil (SST) 10 2 µL Walnut oil
2 2 µL Olive oil –USP (SST) 11 2 µL Castor oil
3 2 µL Avocado oil 12 2 µL Laurel oil
2 µL 2 µL
USP general chapter <203>
4 Cacao oil 13 Nigella oil
5 2 µL Henbane oil 14 2 µL Rape seed oil
Identification of Fixed oils by Thin-
6 2 µL Hypericum oil 15 2 µL Shea butter Layer Chromatography.
7 2 µL Jojoba oil 16 2 µL Grape seed oil
8 2 µL Weat germ oil 17 2 µL Linseed oil
9 2 µL Argan oil
Application field:
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
liposome-based liposomes
formulation ) can
(sample) be checked
contains RNA, using a mobile
liposomes phase DOPE,
(DODMA, of DOPS), and
excipients (including
Chlorophorm, high concentrations
Methanol, Water 65:25:4of sucrose)
(V/V) and Primuline reagent for derivaitzation.
DOTMA
DODMA
DOPS
DOTMA
lyso-DOPE
DOPE
Cholesterol
DODMA
Cholesterol
lyso-DOPE
DOTAP
DOPE
DOTAP
Oleic acid
DODMA
Oleic acid
primuline Cu (II) sulfate
DOPS
DOPE
Primuline is preferred over copper II
sulfate for the formulations
(samples), because with Primuline,
sucrose (one of the excipients) is not DOPS
derivatized ( not showed in the
Formulations
chromatogram ) and
Chromatograms Primuline
at 366 nm after HPTLC densitogram at 366>/400 nm after
allows a detection in fluorescence Calibration curve of DOPE
derivatization with primuline reagent derivatization with primuline reagent liposomes
mode (better sensitivity, good S/N
ratio and linear working range) sucrose and mRNA are not migrating (stay at the application line)
and no degradation products can be seen in the sample
(A-138.1)
Application field:
Food & Feed – Quantification of sugar in honey
Fructose
Maltose
Sucrose
Glucose
Calibration curve of sucrose
Mobile phase: 1-butanol, 2-propanol, aqueous boric acid (5 mg/mL) 30:50:10 (v/v/v) in ADC 2
Derivatization with ADPA reagent ( aniline-diphenylamine-phosphoric acid )
CBS 126
Application field:
Food & Feed – Semi-Quantification of nicotine in e-liquid
Tracks 1–5: reference solutions
Tracks 6–7: samples without flavor
Tracks 8–9: samples with flavor
(A-106.1)
Track no.5 is the 20 mg/ml limit
which is the maximum
concentration allowed in Europe
Application field:
Forensics – Cannabinoids in different Cannabis samples
(A-98.1)
CAMAG instruments & tools portfolio
CAMAG instruments & tools portfolio
TLC line – Basic kit
CAMAG instruments & tools portfolio
HPTLC line – Sample Application: semi automatic
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 : Standard100 L , optional 500 L
CAMAG instruments & tools portfolio
HPTLC line – Sample Application: automatic
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 L, optional 10 L &100 L
CAMAG instruments & tools portfolio
HPTLC line – Plate Development
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
CAMAG instruments & tools portfolio
HPTLC line – Plate Development
Humidity effect on RF (test dye)
17% RH.
47% RH.
saturated salt % relative
solution humidity
NaCl 75
KSCN 47
MgCl2 33
75% RH. KCOOH 20
(Molecular sieve) 0-5
CAMAG instruments & tools portfolio
HPTLC line – Plate Development
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
CAMAG instruments & tools portfolio
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
CAMAG instruments & tools portfolio
HPTLC line – Derivatization
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
CAMAG instruments & tools portfolio
HPTLC line – Derivatization
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
CAMAG instruments & tools portfolio
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
CAMAG instruments & tools portfolio
HPTLC line – Detection
CAMAG instruments & tools portfolio
HPTLC line – Documentation
TLC VISUALIZER 2 ▪ Completely dark compartment with large sliding door
and small viewing window
▪ Three types of illumination
White light UV 254 UV 366
▪ Professional industrial digital camera
▪ Suitable for plates & sheets up to 20x20 cm
▪ Fully controlled by visionCATS software
CAMAG instruments & tools portfolio
HPTLC line – Documentation
TLC Visualizer 2 with VisionCATS - Data view options
CAMAG instruments & tools portfolio
HPTLC line – Documentation
TLC Visualizer 2 with VisionCATS – Images comparison
CAMAG instruments & tools portfolio
HPTLC line – Quantitative Evaluation
CAMAG instruments & tools portfolio
HPTLC line – Software
visionCATS
▪ 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
CAMAG instruments & tools portfolio
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
CAMAG instruments & tools portfolio
HPTLC line – Hyphenation (MS coupling)
External filter in a ferrule: Elution Head
Easy cleaning, easy changing
Cross-hair laser
Sledge, for
head cleaning
Magnetic plate
positioner (ruler)
Scaled Table
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