© CAMAG 2020
HPTLC
HIGH-PERFORMANCE
THIN-LAYER CHROMATOGRAPHY
Ing. Aneta Hartmanová
DONAU LAB, s.r.o.
▪ 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)
Why Thin Layer Chromatography ?
HPTLC Application fields
Botanical
Pharma
Food & Feed
Cosmetics
Clinical
Forensic
Industrial
Environmental
One separation & four ways to analyse
Application field:
Botanical & Pharma
– Comparison of Curcuma samples
GC
Application field:
Botanical & Pharma
– Falsification of Sage oil
USP general chapter <203>
Identification of Fixed oils by Thin-
Layer Chromatography.
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
4
2 µL
Cacao oil
13
2 µL
Nigella oil
5
2 µL
Henbane oil
14
2 µL
Rape seed oil
6
2 µL
Hypericum oil
15
2 µL
Shea butter
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:
Food & Feed
– Identification of fatty oils
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
Calibration curve of DOPE
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.
Chromatograms at 366 nm after
derivatization with primuline reagent
HPTLC densitogram at 366>/400 nm after
derivatization with primuline reagent
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(A-138.1)
The liposome-based formulation (sample) contains RNA, liposomes (DODMA, DOPE, DOPS), and
excipients (including high concentrations of sucrose)
Primuline is preferred over copper II
sulfate for the formulations
(samples), because with Primuline,
sucrose (one of the excipients) is not
derivatized ( not showed in the
chromatogram ) and Primuline
allows a detection in fluorescence
mode (better sensitivity, good S/N
ratio and linear working range)
primuline
Cu (II
) sulfate
liposomes
Formulations
DOPS
DOPE
DODMA
sucrose and mRNA are not migrating (stay at the application line)
and no degradation products can be seen in the sample
Application field:
Food & Feed
– Quantification of sugar in honey
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 )
Calibration curve of sucrose
CBS 126
Suc
ros
e
Maltos
e
Gluc
os
e
F
ruc
tos
e
(A-106.1)
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
Track no.5 is the 20 mg/ml limit
which is the maximum
concentration allowed in Europe
(A-98.1)
Application field:
Forensics
– Cannabinoids in different Cannabis samples
CAMAG instruments & tools portfolio
CAMAG instruments & tools portfolio
TLC line
– Basic kit
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: semi 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
– Sample Application: automatic
▪ 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
Automatic Developing Chamber ADC2
Humidity effect on RF (test dye)
17% RH.
47% RH.
75% RH.
saturated salt
solution
% relative
humidity
NaCl
75
KSCN
47
MgCl2
33
KCOOH
20
(Molecular sieve)
0-5
CAMAG instruments & tools portfolio
HPTLC line
– Plate Development
▪ 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
AUTOMATED MULTIPLE DEVELOPMENT AMD 2
CAMAG instruments & tools portfolio
HPTLC line
– Plate Development
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
CAMAG instruments & tools portfolio
HPTLC line
– Derivatization
▪ 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
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
– Derivatization
▪
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
TLC SCANNER 4
CAMAG instruments & tools portfolio
HPTLC line
– Detection
White light
UV 254
UV 366
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
▪ Professional industrial digital camera
▪ Suitable for plates & sheets up to 20x20 cm
▪ Fully controlled by visionCATS software
TLC Visualizer 2 with VisionCATS - Data view options
CAMAG instruments & tools portfolio
HPTLC line
– Documentation
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
▪ 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
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)
Cross-hair laser
External filter in a ferrule:
Easy cleaning, easy changing
Sledge, for
head cleaning
Scaled Table
Magnetic plate
positioner (ruler)
Elution Head
CAMAG instruments & tools portfolio
HPTLC line
– Hyphenation (MS coupling)
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Stánek č. E 13
