Journal of Chromatography B 1214 (2023) 123553

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Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

Planar chromatography – Current practice and future prospects

Ian D. Wilson a, *, Colin F. Poole b, *
a
Division of Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College, Burlington Danes Building, Du Cane Road, London W12 0NN,
UK
b
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Planar chromatography, in the form of thin-layer or high-performance thin-layer chromatography (TLC, HPTLC),
TLC continues to provide a robust and widely used separation technique. It is unrivaled as a simple and rapid
HPTLC qualitative method for mixture analysis, or for finding bioactive components in mixtures. The format of TLC/
TLC/MS
HPTLC also provides a unique method for preserving the separation, enabling further investigation of compo­
Automation
Effect-directed detection
nents of interest (including quantification/structure determination) separated in both time and space from the
Imaging original analysis. The current practice of planar chromatography and areas of development of the technology are
reviewed and promising future directions in the use of TLC/HPTLC are outlined.

1. Introduction
Thin-layer chromatography (planar) formats exist both as simple
laboratory tools, requiring little instrumentation, and as sophisticated
and fully instrumental techniques. In both cases, the stationary phase
consists of a sorbent bed containing homogeneously packed particles or
as a porous monolith [1,2]. When movement of the mobile phase
through the sorbent bed is controlled by capillary forces the separation
performance is suboptimal but requires little instrumentation affording
a convenient and flexible arrangement for simple separations at the
analytical or preparative scale. For faster separations, or separations
with a higher separation capacity, instrumentation such as over­
pressured layer chromatography (OPLC) and electroosmotically-driven
flow (or pressurized planar electrochromatography) can be employed
[3-6]. However, even though instrumentation for OPLC and pressurized
planar electrochromatography were once commercially available,
neither are in common use and TLC remains predominantly a capillary-
controlled flow technique. Conventional TLC still thrives in the labora­
tory environment as a quick, inexpensive, flexible, and portable quali­
tative method for mixture analysis. However, to obtain the best
performance, and perform accurate quantification, instrumentation is
required to enable the various steps in the analytical process to be
optimized. This instrumentalized methodology combined with the use of
fine particle layers has been termed high-performance thin-layer chro­
matography (HPTLC) to distinguish the technique from conventional
TLC [4,7,8]. The adoption of HPTLC however, which can trace its
development back to the 1970s [8], requires significant investment and
whilst the general advantages of utilizing HPLC conditions versus con­
ventional column chromatography are well-known the same argument
cannot be made so easily for HPTLC vs TLC. As a result, the general
migration of separations from conventional TLC practices to HPTLC has
not been universal and it remains the case that few laboratories are
currently equipped to perform HPTLC.
The general evolution of HPTLC during its first and second eras are
captured in a series of books, which if ordered chronologically, represent
the state-of-the-art at different times to the present day [8-16]. The main
features of the contemporary practice of HPTLC are the use of fine
particle layers for fast and efficient separations; sorbents with a wide
range of sorption properties to optimize selectivity; the use of instru­
mentation for semi-automated sample application, development,
derivatization and detection; the accurate and precise in situ recording
and quantification of chromatograms; biochemical and biological
detection to complement spectroscopic methods; and the development
of interfaces for mass spectrometric identification.
To understand why planar chromatography remains an important
mode of separation in the 21st century, when techniques such as U(H)
PLC/MS might have been confidently expected to have reduced it to
insignificance, it is perhaps worth restating its benefits. So, as things
currently stand, no one can deny that column liquid chromatography
(CLC) affords a better arrangement for operation at high pressures and
for variation of the separation conditions by control of external pa­
rameters. Hyphenation to spectroscopic techniques in CLC is generally

* Corresponding authors.
E-mail addresses: i.wilson@imperial.ac.uk (I.D. Wilson), cfp@chem.wayne.edu (C.F. Poole).

https://doi.org/10.1016/j.jchromb.2022.123553
Received 26 September 2022; Received in revised form 1 November 2022; Accepted 24 November 2022
Available online 28 November 2022
1570-0232/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
I.D. Wilson and C.F. Poole
simple and quantification is also easy. The thin-layer format on the other
hand, provides a better arrangement for high sample throughput, with
flexible detection strategies, and a greater tolerance of samples with a
high-matrix burden. The throughput advantage of TLC is a consequence
of its ability to separate multiple samples in parallel with each sample
occupying a single lane (or track) on the layer and several samples
assigned to different lanes for simultaneous separation. A single 20 cm
× 10 cm HPTLC plate can separate thirty-six samples and standards
simultaneously if developed in one direction and twice that number if
developed from two opposing edges to the center. In addition, many
plates can be developed simultaneously if rapid scale up is needed at the
trivial cost of adding more developing chambers.
CLC separations employ the elution mode in which all sample com­
ponents experience the same separation distance defined by the column
length, but because of the different nature of their interactions with the
stationary phase, are separated in time. In contrast, separations by TLC
are generally in the development mode, and the mobile phase moves
through the layer in a definite direction for a fixed distance with sample
components separated in space (have different migration distances (RF)
achieved with the same separation time. This has obvious consequences
for detection in TLC with sample components either detected in the
stationary phase, compared to the mobile phase in CLC or after extrac­
tion for “off plate” analysis. However, irrespective of the mode of post
chromatographic analysis, at the completion of the separation the
separated zones are stationary and can be interrogated free of time
constraints. This simplifies the use of e.g., MS or 1H NMR spectroscopic
methods of analysis and facilitates the use of chemical and biological
reagents for detection [17-20]. In addition, for TLC using the develop­
ment mode for separation the whole sample is contained in the sample
lane and is available for interrogation. This is important for determining
the integrity of a sample and contrasts with CLC where the only sample
components observed are those that are fully eluted from the column.
Typically, TLC plates are employed in single-use applications and are
then discarded. This contrasts with CLC where, because of their higher
cost, a single column is used for the separation of many samples, which
might result in carryover effects, and sample preparation is designed not
only to concentrate analytes but also to avoid damaging the column.
Whilst effective sample preparation can also improve the quality of in­
formation for target compounds in complex mixtures for TLC as well as
CLC a real advantage of the former is its ability to handle samples with a
heavy “matrix burden”. Thus, samples containing strongly retained
components or suspended particles which could be profoundly
damaging to CLC columns, rapidly degrading performance and leading
ultimately to column failure, are not a concern for TLC. Because reuse of
the TLC plate is not envisaged the methodology is ideal for e.g., initial
screening and the analysis of crude samples with minimal sample
preparation and is therefore the method of choice [4]. An alternative use
for TLC that exploits this relative insensitivity to the matrix is “planar
solid-phase extraction” where the layer is used for the partial purifica­
tion of the target analyte(s) now present in a matrix-reduced zone(s) and
available for subsequent analysis, often by mass spectrometry, as
described in section 6.
In our own studies we have preferred TLC for the screening step to
identify samples requiring a detailed analysis (target analyte possibly
present in a small number of samples), for samples with a heavy matrix
burden or unknown matrix properties, for effect-directed analysis
(target analytes unknown), for class fractionation (identification of
analytes by group membership rather than individual identity), and for
the standardization of plant materials by fingerprint analysis. The most
recent applications exploring these advantages are the subject of this
overview of the current state of the art and practice of HPTLC. For some
sample types, we generally prefer CLC, or use TLC for initial screening
and column chromatography for the analysis of the sub-set of samples
identified to contain target analytes (pyramid approach). Samples that
require a high peak capacity for their separation and identification are
usually better handled by U(H)PLC as are samples that require
2
Journal of Chromatography B 1214 (2023) 123553
techniques like affinity chromatography, size-exclusion chromatog­
raphy, or stereochemical selectivity, due to the limited number of
available stationary phases for TLC.
2. Automation
Whilst TLC can be performed in most locations, including the field,
using unsophisticated manual approaches many modern laboratories
performing HPTLC employ a high level of automation. Analysis by
HPTLC offers great flexibility in that the sample analysis workflow can
be separated into a series of discrete and largely independent steps.
Thus, early attempts at automation focused on the individual unit op­
erations which could be optimized independently rather than full
automation of the whole sample workflow [21]. Automated modules for
each unit operation have been available for some time, resulting in
improved performance through the removal of many error-prone
manual operations [7,8]. Such automation is particularly important
for the unit operations of sample application, plate development,
derivatization, and detection with manual intervention limited to
transporting the layer from module to module. Additional automation is
possible using robotic workstations, which provide a mechanism to
transfer the layer between individual instruments for specific unit op­
erations [22,23]. However, in practice HPTLC has generally remained
semi-automated but a fully automated system was recently launched by
CAMAG (Muttenz, Switzerland) using a series of linked modules
controlled through integrated software. Routine quality control labo­
ratories are considered the main market for this type of system although
individual modules can be operated independently. However, it remains
to be seen how widely such instrumentation will be deployed. A mini­
aturized 2LabsToGo system combining planar separations with effect-
directed detection in a single unit for field and laboratory use was
recently described [24]. This is not a commercial instrument, but lab­
oratories could construct their own instrument using additive
manufacturing practices with open-source software available. This
approach presents an alternative for the economic development of
automated TLC instrumentation.
2.1. Sample application
For stationary phases such as silica gel samples are applied, wherever
possible, in volatile solvents to HPTLC plates as spots or bands of a
minimum size, at an exact position, and with a homogeneous sample
distribution within the applied zone [25,26]. For reversed-phase (RP)
TLC aqueous samples can also be applied to the plates with some
advantage for samples such as urine in terms of limiting the band
spreading of analytes. Similarly, samples with a high matrix burden or
for preparative separations can be applied over a rectangular area and
focused into a narrow starting zone by pre-development with a strong
solvent. In such cases, the analytes are front migrated without separa­
tion to the upper edge of the rectangle and focused into a narrow band
often leaving matrix contaminants adsorbed over the total application
area. The sample solvent used for application should be a good solvent
for the sample, of low viscosity, volatile, must wet the stationary phase,
and of low eluotropic strength with respect to the separation mechanism
[25]. Water and other high viscosity/low volatility solvents are, in
general, not a good choice for sample application to silica gel. If un­
avoidable, aqueous solutions can be diluted with a miscible organic
solvent to facilitate their application using spray-on devices. Assuming
analyte stability is adequate heat may be used to increase the solvent
evaporation rate. Suspensions cannot be applied reproducibly to layers
and may block sample applicators. Filtration for particle removal is a
common sample pretreatment step prior to sample application.
The most popular devices for automated or semi-automated sample
application are based on either the contact transfer or the spray-on
technique. The latter, which provides compact starting zones by mini­
mizing sample diffusion during application is regarded as preferable to
I.D. Wilson and C.F. Poole
contact transfer. Spray-on devices employ a motor driven syringe as the
sample reservoir and a nitrogen (or compressed air) atomizer to spray
the sample in the form of an aerosol onto the layer as spots, bands, or
rectangles. The position of the atomizer is fixed, and the plate is moved
on a motorized stage beneath it to apply bands of any length between
(spots) and the maximum transit length of the atomizer for preparative
applications. Recommended default values for analytical separations
using bands are 8 mm for complex samples and those dissolved in
aqueous solution and 6 mm for relatively simple mixtures in volatile
organic solvents [26]. With a zigzag motion of the layer, samples can be
applied over a rectangular area, useful for preparative applications and
for samples dissolved in solvents of low volatility. The rate of sample
deposition is adjustable to accommodate sample solutions of different
viscosity and volatility. The advantages of band application for scanning
densitometry/in situ MALDI/DESI MS etc., are that the sample band can
be made longer than that required for visualizing the sample (e.g., the
slit length of the light source). This minimizes the chances of the sample
being missed during scanning and reduces quantification errors due to
positioning of the sample within the light beam/DESI sprayer etc. Over
spraying samples that have already been applied to the layer with a
solution of a standard facilitates quantification by the standard addition
method, and by over spraying with a reactive reagent pre-
chromatographic derivatization is also possible. Similarly, a MALDI
matrix for mass spectrometry can be applied in this way.
2.2. Plate development in TLC/HPTLC
Manual methods for TLC/HPTLC development have hardly changed
since the introduction of the techniques and simply involve placing the
plate into the solvent in an appropriately sized tank, that has been
preconditioned by allowing equilibration with the solvent, and
removing it when the solvent front has reached a predetermined dis­
tance. Such advances as have been made include the introduction of
overpressured layer chromatography (OPLC) and pressurized planar
electrochromatography (PPEC) [3-6] and in the automation of the
traditional plate developing process. Neither OPLC nor PPEC, however,
have gained widespread use. Automation of the development process in
the normal or sandwich chamber configuration on the other hand has
been more widely taken up in laboratories seeking to perform HPTLC.
Such automated plate development is possible with e.g., the CAMAG
automatic developing chamber (ADC 2) [7,8,14]. With the ADC pre­
conditioning, insertion, the mobile phase migration distance, removal of
the plate from the solvent, and post-chromatographic drying conditions
can be preselected and performed automatically without human inter­
vention. At the same time the repeatability of the separations is
improved due to the better control of the individual unit operations that
comprise the development process [27]. A universal standard mixture
has been recommended as a system suitability test to account for
experimental variables that effect RF values [28] with an interlaboratory
evaluation finding a confidence interval of 0.04 RF units. A more
advanced version of this system (the HPTLC PRO Module DEVELOP­
MENT combined with the HPTLC PRO Module PLATE STORAGE) can be
used to sequentially develop up to 5 plates and store the developed
plates for further treatment or evaluation.
As a means of increasing the coverage of components of widely
different polarity in complex mixture, the techniques of multiple
development (MD) using solvents of decreasing eluotopic strength to
provide what is effectively a solvent gradient, can have advantages.
Typically, each solvent development is 1–3 mm longer than the previous
one and employs a solvent of lower solvent strength. The first few de­
velopments employ a strong solvent to primarily focus the sample zones.
Subsequent development steps are longer employing weaker solvents for
the separation. The number of developments required for a separation
depends on the complexity of the mixture and the shape of the desired
gradient. Typically, 10–30 developments, requiring 1.5 to 4.5 h for
completion, are used (but by employing 0.1 mm-thick layers this can be
3
Journal of Chromatography B 1214 (2023) 123553
reduced by ca. 50%). However, to perform this type of separation
manually is clearly tedious, time consuming, and prone to irreproduc­
ibility. Automated multiple development chambers (i.e., the CAMAG
AMD 2) are available [7,8,29] and eliminate many of these disadvan­
tages. For mixtures of a wide polarity, bands remain focused and sharp
for all separation distances providing a higher zone capacity than
observed for non-gradient methods [4] and in many ways could be
considered an alternative to 2D-separations as a means of increasing
resolution.
2.3. Derivatization
2.3.1. Instrumentation
Early in the evolution of TLC numerous derivatization reactions were
employed for the visual detection of compounds lacking a suitable
chromophore or fluorophore. Simultaneously, chemical reactions
emerged that facilitated group-type and family-type detection
increasing the selectivity for target compound analysis. These early
studies have left a rich legacy of suitable reagents and conditions for
general use [8,13,15,30-32]. Derivatization reactions may be via pre-
chromatographic derivatization [30,32-34], either as part of sample
preparation or by over spraying the sample zones applied to the layer or,
more commonly, as post-chromatographic derivatization. Of course, in
addition to improving detection pre-chromatographic derivatization
alters the structure of the target compounds often assisting in their
separation and possibly enhancing their stability.
Post-chromatographic derivatization takes advantage of the open
nature of the layer, which affords easy access for the reagents to the
immobilized sample components, reduces chromatographic interference
from reaction by-products, and minimizes differences in reaction con­
ditions by derivatizing all samples and standards simultaneously. The
reagents and derivatives, however, must have different detection char­
acteristics as the background for detection is the reagent-impregnated
layer. Heating of the layer may be required to complete the reaction
in a reasonable time or to achieve complete reaction. Flat-bed, tem­
perature-controlled, plate heaters are commonly used for this purpose.
For post-chromatographic reactions, the reagents are applied to the
layer through the gas phase (volatile reagents only), or more commonly,
as a solution using an impregnation technique [8,15,30]. Spraying,
immersion, or contact with a transfer pad (overpressure derivatization)
are used for reagents in solution. Manual methods can be used for any or
all these steps but are time consuming and often provide poor precision
unless the operator is particularly skillful [35]. Manual spraying also
requires a ventilation device (spray chamber) or fume hood to minimize
exposure to hazardous vapors.
Many of the problems associated with post-chromatographic deriv­
atization have however, been solved using automated spray devices.
These typically allow the spray rate, reagent volume, and number of
cycles to be set and the x- and y- coordinates of the layer to be freely
chosen to allow lane, area, or whole plate reagent application [35,36].
Again, automated devices are commercially available such as e.g., the
“CAMAG Derivatizer” [37] where a sophisticated spray atomizer is used
to create an even distribution of the spray (and optimize factors such as
droplet size, spray rate etc.,) with excess reagent vapors removed by
suction at the end of the procedure.
However, automated immersion devices, that afford simpler opera­
tion and optimization compared with automatic spray systems, have
remained the most common devices for post-chromatographic deriva­
tization, and provide good quantification results, but consume a rela­
tively large amount of reagent [7,8,38]. They typically consist of a
motorized holder to maintain the plate in a vertical position and to lower
and retrieve it from an integrated dipping chamber containing the re­
agent solution. The fill volume for the dipping chamber is adjusted to
cover the selected area of the layer without displacing reagent from the
top. The rate of immersion and withdrawal (e.g., 2.5–4.5 cm/s) and
dwell time when the layer is stationary (e.g., 0 to 8 s) can be varied to
I.D. Wilson and C.F. Poole
obtain the desired layer impregnation. It is obviously critical that the
reagent solution wets the layer for rapid impregnation whilst not
washing the sample from the layer during the dipping cycle. Excessive
diffusion of sample zones following immersion is possible for reagents
which evaporate slowly during the drying step.
2.3.2. Saccharides, an illustrative example of derivatization in HPTLC
Saccharides are typical of compounds that lack suitable chromo­
phores for absorption or fluorescence detection and would be difficult to
analyze in complex mixtures by HPTLC without derivatization
[8,13,39]. As such, saccharides serve as a typical example of the general
capabilities of post-chromatographic derivatization for identification
and quantification in HPTLC. The aniline-diphenylamine-o-phosphoric
acid reagent (ADP) is the most widely used as it has broad scope and
sufficient sensitivity for typical applications [40-42]. It reacts with al­
doses, ketoses, galacturonic aid, and glucuronic acid forming variously
colored zones that assist in the identification of individual saccharides
by their relative migration distance and characteristic color. A repre­
sentative application is shown in Fig. 1 for the separation of the methyl
ethers and esters obtained by acid methanolysis of polysaccharides [43].
For other sample types, the limit of quantitation (LOQ) for mono- and
disaccharides in food matrices ranged from 60 to 900 ng/band (λ = 370
nm) [44]; for lactose in dairy products 6 ng/band (λ = 370 nm) [45]; for
mono- and disaccharides in plant extracts 40–230 ng/band (λ = 600 nm)
[46]; for glucose, fructose and sucrose in honey 65–100 ng/band (λ =
white light) [47]; and for glucose in hydrolyzed starch 10 ng/spot (λ =
520 nm) [48].
Alternatively, with saccharides 4-aminobenzoic acid (ABA) produces
reddish-brown bands on a colorless or pale brown background in the
absorption mode and turquoise bands on a blue-black background for
fluorescence (λex = 366 nm and λem > 400 nm) [30,49-51]. Detection
Fig. 1. Separation of methyl glycosides and methyl glycoside methyl esters on
silica gel with the mobile phase 2-propyl acetate-ethyl acetate-methanol-water
5:4:1:0.1 (v/v/v/v), development length 60 mm, and a controlled relative hu­
midity (saturated magnesium chloride). The lower plot is the track scan by
video densitometry after visualization using the ADP reagent. The composition
of mixture 1 and RF values and (color) are galactose 0.13 (gray), xylose 0.34
(blue), rhamnose 0.51 (gray), galacturonic acid 0.60 (green) and for mixture 2
glucose 0.15 (gray), mannose 0.20 (gray), arabinose 0.25 (blue), fucose 0.31
(gray), and glucuronic acid 0.67 (gray). (Reproduced from ref. [43] with
permission). (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
4
Journal of Chromatography B 1214 (2023) 123553
limits in the absorption mode are similar to the ADP reagent but lack the
possibility to confirm individual saccharides by their characteristic color
differences. The ABA reagent is typically used in the fluorescence mode
when higher selectivity or sensitivity is required compared with the ADP
reagent. For lactose in dairy products an LOQ of 3 ng/band was obtained
[45]; for mono- and disaccharides, glucuronic acid and galacturonic acid
in plant extracts about 35 ng/band (visual fluorescence) [49]; for
galactose, glucose, and fructose in fermentation processes 25 ng/band
[50]; and glucose, fructose, and sucrose in enzyme assays ≤ 60 ng/band
(absorption λ = 390 nm) [51].
As well as ADP and ABA, the 2-naphthol-sulfuric acid reagent can be
used for the detection of saccharides, producing dark brown bands on a
light-yellow background, and complements ADP for absorption mea­
surements [52-54]. For lactose in dairy products an LOQ of 3 ng/band (λ
= 500 nm) was obtained [45]; for sucralose in food extracts 5 ng/band
(λ = 500 nm) [53]; and for oligosaccharides in plant extracts 33–77 ng/
band (λ = 585 nm) [54].
2.4. Evaluation by slit-scanning and video densitometry
Clearly, one of the major advantages of TLC is that visual assessment
is a very easy means of obtaining a qualitative assessment of a sample.
However, where quantification is required then an instrumental method
such as scanning densitometry/imaging is required. The major com­
mercial systems for in situ quantification can be categorized as slit
scanning densitometers, diode-array densitometers, and image ana­
lyzers (cameras and flatbed scanners) [7,8,14,15,55,56]. These are
supplemented by special purpose devices for flame ionization detection,
radioisotope detection, and interfaces for spectroscopic detection and
identification. Consolidation in the manufacturing sector since 2000 has
reduced the number of companies competing in the marketplace while
the technical capabilities of instruments has continued to advance
Commercial instruments for slit-scanning densitometry share several
features in common, Table 1 [7,8,15,56]. In general, they support
measurements in the reflectance mode by absorbance or fluorescence.
Transmission measurements are possible with some densitometers, but
wavelength selection is limited by the transmission properties of the
layer support. Two light sources are required to cover the entire
UV–visible range from 190 to 800 nm. Electroluminescent lamps have
attractive features as general light sources with possible applications in
scanning densitometry [57]. A high-pressure mercury lamp is used to
excite fluorescence with a cutoff or band filter placed between the de­
tector and the layer to isolate the emission signal. A photomultiplier is
used to record the signal with a reference photomultiplier to correct for
short-term variation in the source output. A beam splitter located after
the monochromator is typically used to create the signal at the reference
photomultiplier. Due to the wide range of signal intensities encountered
some form of automatic voltage control is utilized to adjust the mea­
surement conditions for each plate and sample type. Lane scanning is
employed with selectable speeds up to 10 cm/s by mounting the layer on
a movable stage controlled by stepper motors. The only manual opera­
tion required is to position the layer on the scanning stage and to adjust
its position to scan the first track. The remaining tracks are then scanned
automatically under software control. The source beam is typically fixed
in position and shaped into a rectangular area on the plate surface by a
pair of adjustable slits through which the plate is transported in the
direction of development. Each scan, therefore, represents a lane whose
length is defined by the solvent front position and width by the slit di­
mensions. Most densitometers also provide for automatic recording of
UV/visible absorption spectra and dual- or multiple-wavelength scan­
ning. Multiple-wavelength scanning allows quantification at optimum
wavelengths for each sample zone. The main sources of uncertainty in
scanning densitometry have been determined as errors due to the optical
measurement, electronic amplification, and noise associated with the
recording device, resulting in a typical relative standard deviation of
0.2–0.7% [55]. The relative standard deviation from all errors including
I.D. Wilson and C.F. Poole
Table 1
Typical operating characteristics of slit-scanning densitometers.
Parameter Specification
Mode Reflectance for absorption or fluorescence
Transmission for some densitometers as an option
Source Deuterium lamp for absorption (190–450 nm)
Tungsten-halogen lamp for absorption (350–800 nm)
High-pressure mercury lamp for fluorescence (248, 254,
265, 280, 297,
302, 313, 366, 405, 436, 546, 577, 579 nm)
Monochromator Concave holographic grating 1200 lines / mm
Band width selectable 5 or 20 nm
5 nm for spectral recording and multiwavelength scanning
20 nm for maximum signal-to-noise
Wavelength setting ≤ 0.2 nm (accuracy ≤ 1 nm)
Slit images Slit height selectable from 0.2 to 12 mm
Slit width selectable from 0.02 to 2 mm
Motor driven filter Automated selection of filters to eliminate second order
wheel wavelengths 400 nm cut-off filter for fluorescence
Operation Single or multiple wavelengths for densitometry
Combination of measurements in absorption and
fluorescence mode
Full spectral recording (scan speed ≤ 100 nm / s)
Detection Two matched broadband photomultipliers. Measurement
beam divided by
a beam splitter with the second photomultipler providing
correction for
short term source fluctuations. Third photomultiplier
located below the
layer required for transmission measurements.
Acquisition Independent x- and y-direction control using stepper
motors. Position
reproducibility < 50 μm in the y-direction and < 100 μm in
the x-direction.
Image resolution 20–200 μm depending on scan settings.
Scan type Linear scan with various methods for track optimization.
Selectable
scanning speed with a maximum of 10 cm / s.
those associated with sample application and the chromatographic step
can be maintained below 2–3 % making instrumental HPTLC a reliable
quantitative tool [58].
Video densitometers (often referred to as image analyzers) use a
charged-coupled or smart phone digital camera or a flatbed office
scanner with appropriate software to capture a stationary image of the
layer. They are simpler and less expensive than slit-scanning densi­
tometers and can be assembled from components likely available in
many laboratories. They may perform basic functions adequately but
lack some of the versatility and advanced features provided by slit-
scanning densitometers.
Flatbed scanners for office purposes are limited to detection in the
visible region (400–800 nm). They provide uniform illumination with
white light, high optical resolution, and produce an image of the plate in
a convenient form for storage or evaluation [7,54,56]. Free open source
or low-cost commercial software is available for converting the stored
image into chromatographic information [59]. The scan data is stored as
a set of RGB values (red 600–750 nm, green 520–565 nm, and blue
425–450 nm) in separate channels. Software evaluation then uses data
collected in any channel, or combination of channels, defined as a
greyscale. Flatbed scanners provide limited selectivity (RGB channels
only) and provide no spectral information for identification or peak
purity assessment. A few validated methods for samples at moderate to
high concentration have been described [60-62].
High quality commercial systems as well as laboratory-built systems
of varying sophistication are used for electronic scanning with a digital
camera [7,8,15,63-65]. Commercially available systems consist of an
illumination unit and a digital camera with an optimum design for TLC
purposes. The illumination compartment is sized to maximize the source
intensity and to obtain uniform illumination of layers of a standard size
with minimal interference from stray light. Tungsten light tubes are used
for measurements in the visible range by reflectance of polychromatic
5
Journal of Chromatography B 1214 (2023) 123553
light with a built-in optical glass filter or lenses coated with an organic
layer to eliminate interference from UV light. Broad band light tubes
with an emission envelope centered around 366 nm are used for the
measurement of fluorescence in the visible region. Mercury discharge
lamps are used for illumination at 254 nm for the detection of UV-
absorbing compounds on layers with a fluorescent indicator.
The Peltier-cooled CCD camera functions as a two-dimensional array
of unit detectors. The captured images are initialized, stored, and
transformed into chromatographic data. They are usually displayed as a
three-dimensional plot in which the x- and y-coordinates define the spot
or band position and the z direction the absorbance or fluorescence in­
tensity. Background subtraction, normalization, denoising, and thresh­
olding are common data transformation processes rarely detailed in the
system software [7,8,15,66-68]. In addition, automated peak detection,
RF or migration distance assignments, calibration for quantification, and
a comparison viewer for side-by-side display of tracks from the same or
different plates and/or different illumination modes are typical software
supported functions. The main performance limitation remains the dif­
ficulty of uniformly illuminating the layer.
Attractive features of image analysis for HPTLC are fast data acqui­
sition, simple instrument design, compatibility with two-dimensional
separations, and the storage, retrieval, and side-by-side comparison of
images. As we have noted above, from its inception, TLC has been
associated with visual data evaluation and image analyzers provide a
convenient means of continuing and extending this practice. On the
other hand, image analyzers do not generally compare favorably with
slit-scanning densitometers in terms of accuracy, precision, and wave­
length measuring range, and do not support spectral identification. They
have proven popular as a replacement for photographic documentation
of planar separations and have established a niche application in the
“fingerprint characterization” of herbal medicines and natural products.
Such fingerprints are chromatographic profiles or images that
represent the characteristic composition of a sample or sets of charac­
teristic chromatographic and/or spectroscopic signals, whose compari­
son allows the classification of samples. HPTLC-based chromatographic
fingerprinting provides an efficient, flexible, and cost-effective method
for the quality assessment of natural products [8,14,43,49,69-81].
Potentially, any separation technique can be used for fingerprinting, but
HPTLC offers several advantages. It is the only separation technique that
provides results in the form of images for multiple samples and reference
standards separated in parallel facilitating a straightforward comparison
of results. Classical fingerprinting is based on a visual assessment while
modern approaches allow instrumental assessment with chemometric
data analysis. Assessment may be based on a limited number of target
(marker) compounds or utilize all information provided in the finger­
print. Fingerprints can, and often are, obtained for several TLC systems
or visualization conditions, with each system allowing comparison of
selected compounds suitable for distinguishing different plant species or
to identify adulterated or counterfeit materials. The main disadvantage
of classical fingerprinting is its subjectivity when visual perception is the
only tool used for evaluation. Instrumental analysis, particularly when
combined with chemometric data analysis, provides a more robust
approach for sample classification [8,43,68,74-76]. Instrumental anal­
ysis is required when potentcy is to be determined simultaneouly with
sample classification. Standardized botanical reference materials play
an important role in classification studies as do suitable synthetic
marker compounds, but both approaches are limited by availability,
particularly for plant-based materials with limited commercial
potential.
There are numerous examples of the use of HPTLC for fingerprint
analysis of natural products, reviewed elsewhere to various extents
depending on date and focus [8,14,16,68-81]. Illustrative examples of
the flexibility of the HPTLC approach to fingerprint analysis is provided
by the classification of ginseng root powder [82] and tansy [83] extracts.
Authentic ginseng contains several ginsenosides (triterpenoid saponins)
that are suitable marker compounds to determine the authenticity,
I.D. Wilson and C.F. Poole
stability, consistency, and adulteration of ginseng root powder extracts.
Either visual comparison, or better densitometry, of the fluorescence
response excited at 366 nm allows distinction of white Panax ginseng
(WG), red Panax ginseng (RG), American ginseng (Panax quincefolius,
AG), and Panax notoginseng (NG) by comparison of the presence and
peak ratio of the ginsenoside reference compounds separated simulta­
neously, Fig. 2.
The complementary nature of chemical fingerprints obtained using
Fig. 2. Separation of ginsenosides on silica gel layers with a mobile phase of
chloroform-ethyl acetate-methanol-water 15:40:22:10 (v/v/v/v) and the dried
layers visualized by spraying with 10 % (v/v) sulfuric acid in ethanol. HPTLC
fingerprints obtained by scanning and video densitometry are shown for white
Panax ginseng (WG), red Panax ginseng (RG), American ginseng (AG), Panax
notoginseng (NG) and ginsenosides marker compounds. (Reproduced from ref.
[82] with permission). (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
6
Journal of Chromatography B 1214 (2023) 123553
different visualizing reagents for extracts of the aerial parts of the tansy
plant is illustrated in Fig. 3 [83]. The vanillin-sulfuric acid reagent is a
reasonably non-specific reagent that produces colored zones with
different compounds, the natural product reagent and aluminum chlo­
ride reagent are semi-specific for flavonoids producing characteristic
fluorescence, and the aniline-diphenylamine-o-phosphoric acid (colored
zones) and 4-aminobenzoic acid (fluorescence) reagents are reasonably
specific for saccharides and glycosides. The sequence of reagents and
their side-by-side comparison illustrate the flexibility of chemical fin­
gerprints to provide a variety of images (or scans) suitable for improving
the specificity of compound identification, in this case, as well as clas­
sification of related species. Biological detection can be used to expand
the identification/classification approach. The sequence of images in
Fig. 4 illustrates the varied fingerprints obtained using visualization at
selected wavelengths and after chemical derivatization with the
vanillin-sulfuric acid reagent (a to d), treatment with the 2,2-diphenyl-1-
picrylhydrazyl radical (antioxidant activity (i), antibacterial activity
(Bacillus subtillis, (e), Aliivibrio fischeri, (f), Xanthomonas euvesicatoria,
(g), and Pseudomonas syringae maculicola, (h), and enzymatic inhibition
(acetylcholinesterase, (j), and butylcholinesterase, (k). Any or several of
these chemical, biochemical, and biological fingerprints could be
adopted for specific identification/classification purposes and for addi­
tional flexibility, combined with different separation systems. Only
HPTLC provides this range of adaptability for crude extracts. Fig. 4 also
serves as an example of non-targeted screening for bioactive compounds
using effect-directed assays (section 5 below).
3. Planar solid-phase extraction
HPTLC has two useful attributes for the cleanup of complex samples
requiring high sample throughput. Matrix components comigrating with
target analytes can be observed and sample preparation and separation
steps actively optimized to minimize interferences. Secondly, parallel
sample processing affords higher sample throughput than serial column-
Fig. 3. Separation of extracts from the aerial parts of the tansy plant on silica
gel layers with a mobile phase of n-hexane-isopropyl acetate 9:1 (v/v). The
dried layers were treated with (a) vanillin-sulfuric acid reagent (white light),
(b) natural product reagent and poly(ethylene glycol) with fluorescence excited
at 365 nm, (c) aluminium chloride reagent with fluorescence excited at 365 nm,
(d) aniline-diphenylamine-o-phosphoric acid reagent (white light), (e) and 4-
aminobenzoic acid reagent with fluorescence excited at 365 nm. The combi­
nation of visualization techniques was used to provide insight for the pre­
liminary identification of the bioactive compounds indicated as TR1 to TR6.
(Reproduced from ref. [83] with permission).
I.D. Wilson and C.F. Poole Journal of Chromatography B 1214 (2023) 123553

Fig. 4. Separation of tansy root extract using the same conditions as Fig. 3. Images of chemical, biological and biochemical fingerprints obtained at (a) absorption
254 nm, (b) absorption 365 nm, (c) after derivatization with vanillin-sulfuric acid reagent (white light), (d) as for (c) at 365 nm, (e) after treatment with B. subtillis, (f)
after treatment with A. fischeri, (g) after treatment with X. euvesicatoria, (h) after treatment with P. maculicola, (i) after visualization with 2,2-diphenyl-1- pic­
rylhydrazyl radical, (j) after treatment with acetylcholinesterase, and (k) after treatment with butylcholinesterase. (Reproduced from ref. [83] with permission).

based methods. Coeluting matrix interferences can be a major limitation
for column-based methods employing mass spectrometric detection for
routine analysis often creating the need for multi-step and time-
consuming sample preparation procedures.
In planar solid-phase extraction (pSPE) the goal is the separation of
target compounds as one or a few zones free of matrix components that
might interfere in their subsequent analysis [84]. Thus, the separation
conditions are optimized to minimize within group separation of target
compounds in favor of focusing these compounds into a minimum
number of zones adequately separated from sample matrix. The pSPE
method typically employs two development steps in which the second
development occurs in the reverse direction to the first. In the first
development, the target compounds are front end eluted and separated
from most matrix components with a higher affinity for the stationary
phase. In the second development, the target compounds are front end
eluted in the reverse direction with a stronger solvent and focused into a
narrow zone at some location in the migration region with matrix
components occupying the remaining positions of the migration region.
The efficient cleanup of pesticide residues in extracts from fruits and
vegetables [84-86] and antibiotic residues in foods of animal origin [87]
was demonstrated using pSPE. Typically, crude extracts isolated by a
modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe)
method or solvent extraction were applied as bands to layers of different
selectivity and developed as explained above. The sharp pesticide- or
antibiotic-containing zones were in situ solvent extracted using the TLC-
MS interface for analysis by LC-MS or flow-injection analysis coupled
with high-resolution mass spectrometry. The chromatograms obtained
were virtually free of matrix interferences. This allowed calibration with
a solvent standard instead of matrix-matched standards generally rec­
ommended for the analysis of contaminants at residue levels. An
enhancement to the automation of pSPE was achieved using an auto­
mated TLC-MS interface for the serial extraction of target compound
zones [87,88] and validated for the screening of 66 multi-class antibi­
otics residues in meat, eggs, and milk for residues in the low μg/kg
range.
For some products employed as technical mixtures their complexity
may confound the possibility of identifying all individual components in
environmental samples, and in any case, may not be essential for an
initial estimate of their hazard potential. These compounds may be
monitored as a sum value. In this case, it is often advantageous to adopt
a method that provides isolation of the target compounds as a group
with limited separation within the group, relatively free of contamina­
tion from other compounds, and a detection method that is relatively
unselective for the individual mixture components. For example, chlo­
rinated paraffins are complex mixtures of straight-chain, C-10 to C-30,
chlorinated hydrocarbons with a variable chlorine content (30–70 % w/
w) providing mixtures potentially consisting of thousands of congeners
and isomers. Planar solid-phase extraction allows their separation from
matrix components in oil-based foods as a single zone [89]. The chlo­
rinated paraffins are isolated by solvent extraction and applied as bands
to a silica gel layer. The sample is initially developed to a length of 80
mm with the mobile phase cyclohexane-toluene (94:6 v/v) providing a
good separation of nonpolar lipids from the chlorinated paraffins, which
are dispersed over a broad migration region from 0 to 0.55 RF units. A
second development in the same direction of 45 mm with acetone
focusses the chlorinated paraffins into a compact zone with an apparent
RF of 0.60. The chlorinated paraffins were visualized as grey zones on an
orange background after dipping into a solution of o-toluidine in acetone
after exposure of the dried layer to UV light. The chlorinated paraffins
were subsequently quantified by absorption (λ = 645 nm) using a

7
I.D. Wilson and C.F. Poole
technical mixture of chlorinated paraffins as a calibration standard. The
LOD for the chlorinated paraffins was 0.2 mg/kg of vegetable oil with
recoveries near 100%. Further examples using the above approach
include the determination of ergot alkaloids in rye flour using fluores­
cence detection [90], nonylphenols in extracts of wastewater using the
pYES bioassay for detection [91], and mineral oil hydrocarbons in
vegetable oils with selective chemical derivatization [92].
A variation of planar solid-phase extraction is known as solvent front
position extraction utilizing a multi-step development in the same di­
rection in which the final location of the target compounds is at the
solvent front for the last development [93,94]. This has the advantage
that the target compounds are concentrated into a narrow zone with an
easily identifiable location for subsequent solvent extraction and anal­
ysis but comes at the expense of a longer time for sample processing.
Both approaches to pSPE have become more attractive with the devel­
opment of the semi-automated TLC-MS interface for in situ solvent
extraction with in-vial collection of extracts.
4. Multidimensional chromatography
Multidimensional separations take advantage of the higher separa­
tion power of coupled systems. In practice, only two-dimensional sys­
tems are of practical importance for both CLC and TLC. Two general
operations are considered multidimensional separations. These are
heart-cut methods, in which a part of the separation obtained in the first
chromatographic system is passed to the second system to improve the
separation of the components in the heart-cut fraction. This type of
separation is commonly used for target compound analysis in which a
few compounds are designated important and need to be separated from
each other and from the sample matrix and is more widely used for
column methods. The alternative method of multidimensional chroma­
tography is known as comprehensive chromatography, in which the
complete separation from the first dimension is passed to the second
dimension for separation with preservation of the separation obtained in
the first dimension, and is the type generally associated with planar
chromatography. These “comprehensive” separations provide high peak
capacities and are used for fingerprint-type analyses and to characterize
complex mixtures, where the object is to separate as many compounds as
possible for either classification or identification.
The introduction of specialized TLC-MS interfaces (see section 6
below) has led to several methods to increase peak capacity by on– or
off-line coupling of HPTLC to column chromatography [7,20]. The most
recent iteration of the TLC-MS interface is a second-generation design
for the solvent extraction of individual zones from HPTLC plates in what
is, effectively, 2D pSPE. Here, instead of direct connection to the ion
source of a MS, the outlet capillary of the extraction device is connected
to the injection valve for column chromatography. Alternatively, the
eluate from the target zone can simply be collected into a vial for further
off-line coupling to spectroscopic or chromatographic techniques, free of
time constraints, and with the possibility of solvent exchange and
sample concentration [8,95-98]. This sort of approach offers a
straightforward approach to heart-cut multidimensional chromatog­
raphy employing HPTLC for sample fractionation and any form of
chromatography (e.g., TLC, HPLC, GC or SFC) for the final separation.
The sensitivity of in-vial collection is typically lower compared with on-
line transfer, as only an aliquot is subsequently taken for chromato­
graphic analysis unless the eluted zone is concentrated prior to HPLC
[87,88].
On-line coupling and automation are less straightforward, but
possible, for HPTLC-HPLC using a valve interface in the outlet capillary
to transfer eluted samples to a column with the possibility of analyte
trapping on a sorbent cartridge to remove interfering background ma­
terials for structural elucidation by mass spectrometry
[20,87,88,99,100]. It is typically employed for non-target compound
analysis using normal-phase HPTLC with effect-directed detection for
screening and reversed-phase HPLC with mass spectrometric detection
8
Journal of Chromatography B 1214 (2023) 123553
for the identification of bioactive compounds in complex matrices. It is
not a settled development and further advances can be anticipated,
which hopefully will lead to a commercially available fully automated
interface.
In comprehensive separations significant differences and challenges
exist for both columns and layers [101,102]. The goals are the same, but
the processes are quite different. Comprehensive separations in CLC
occur in time and for TLC in space. In both cases a high peak capacity is
achieved when the retention mechanism in each dimension is dissimilar.
It also presupposes that the sample components are varied in properties,
since highly similar compounds will occupy a narrow range of the
selectivity space and their peak-to-peak separation is only weakly
coupled to the dimensions of the selectivity space.
For maximum performance in comprehensive two-dimensional
planar chromatography, it is assumed that layers can be operated as
flat columns with optimal fixed mobile phase velocity (forced-flow
conditions) [97]. Two-dimensional HPTLC with capillary-controlled
flow produces lower performance and slower separations than pre­
dicted for forced-flow methods but requires little in the way of instru­
mentation and is simple to implement [104-106]. If a single stationary
phase is employed for the separation, then the separation mechanisms
differ only in the choice of mobile phase. Two-dimensional HPTLC
typically employs normal-phase separation conditions effectively
limiting the orthogonality of the system due to site-specific interactions
of polar compounds and their influence on solvent strength. Often a
better arrangement is the use of chemically bonded layers for both
normal-phase and reversed-phase separations. Two-dimensional HPTLC
separations employing reversed-phase conditions on polar chemically
bonded phases are comparatively easy to optimize and currently are an
underutilized option [107]. Compared with forced-flow development,
capillary-controlled flow results in additional band broadening in both
separation directions due to the inadequate and variable mobile phase
velocity, a limited migration distance for the solvent front due to the
weak capillary forces driving the mobile phase, and low resolution for
compounds close to the origin or solvent front. The zone capacity in two-
dimensional capillary-controlled flow HPTLC is difficult to model
because of the varying mobile phase velocity but it has been estimated,
and in reasonable agreement with practice, that it should be relatively
easy to generate between 100 and 250 separated spots in about 30 min
with little prospect of exceeding 500, at least for a single development in
both orthogonal directions [102]. This is sufficient to handle mixtures of
moderate complexity. Video densitometry has solved the problem of
recording separations, which was never straightforward using lane
scanning densitometers, but calibration for quantitative analysis re­
mains an issue. Two-dimensional HPTLC also loses the high-throughput
advantage of unidimensional HPTLC as it is limited to a single sample
per separation (or layer).
5. Effect-directed detection - HPTLC-bioautography
Target compound analysis generally fails to provide information for
non-target compounds resulting in an incomplete assessment of bioac­
tivity and the provision of information for risk assessment. To detect
contaminants or native compounds having biological activity in survey
or routine monitoring campaigns, it is essential for the chosen method to
be as universal as possible and focused on specific biological effects. The
selectivity of the method for individual compounds is not as import as its
ability to detect as many compounds as possible in the sample that
contribute to a particular biological response. Typically, this requires
the detection of both anticipated compounds and unknown or unex­
pected compounds in a complex matrix at low concentrations. Complete
chemical analysis is generally not feasible in this case and would be too
costly for routine use. The preferred approach is to focus on the iden­
tification of compounds with a demonstrated biological response, which
is the basis of effect-directed analysis [18-20,102,103].
There is rapid growth of interest in effect-directed detection in TLC
I.D. Wilson and C.F. Poole
employing various bioassays based on the inhibition/stimulation of
growth or activity of an organism, such as bacteria, yeast cells, mold
spores, cell organelles, enzymes, etc., for detection (see e.g., recent re­
views on its use for detecting enzyme inhibitors [110], antimicrobial
activity in plants [111], and applications for foodstuffs [112]). After
incubation of the layer with the test organism and possibly application
of additional reagents, the zones with biological activity are typically
identified and sometimes quantified using a charged-coupled device
[7,8,71,80,87,113,114].
To preserve the separating power of column separation methods
using bioassay detection the eluent must be fractionated into many small
volume increments, each of which must be assayed separately. This is a
tedious process and may require solvent exchange to maintain
compatibility with the bioassay. A significant advantage of HPTLC is
that the bioassay can be performed directly on the surface of the layer, in
a compatible medium since the mobile phase is completely evaporated
after the separation. The whole bioactivity of the sample can be
observed and not just the fraction that elutes from the column. Spray-on
sample application techniques, common for HPTLC, support the use of a
wider range of extract volumes to increase method flexibility. The par­
allel processing of samples by HPTLC results in an increase in sample
throughput and lower costs per sample. On the other hand, currently
there are only limited automation possibilities for bioassay protocols,
and some bioassays require relatively long incubation times, causing
significant zone broadening of the separated zones if the cells or mi­
croorganisms used for detection require an aqueous medium for
viability. Suitable approaches to mitigate this problem, however, have
emerged for specific bioassays [36,37,115-119]. An interesting, perhaps
general, approach using zone fixation by application of a poly(isobutyl
methacrylate) coating was demonstrated to be suitable for integrating
multiple bioassays on the same plate simplifying the workflow for multi-
detection and multidimensional separations [119].
Bioactivity tests employing the luminescent bacteria Aliivibrio fischeri
are among the oldest, most applied, and fully supported bioassays for the
detection of compounds that exhibit non-specific toxicity
[8,17,71,80,100,108-114,120-122]. A suitable apparatus for this
bioassay consists of a tray for holding the thin-layer plate, a device to
humidify the coated layer to stabilize the microorganism, a light-tight
housing, and a CCD camera for detection. After a short incubation
period bioactive substances are indicated as zones with a different
response to the plate background coated with the microorganism.
Compounds that interfere in the bacterium’s metabolism by various
complex mechanisms are detected by changes in the intensity of the
bioluminescence (see examples in Fig. 4). Toxic effects can be caused by
compounds that interact with cell surface receptors, that alter cell
membrane functions, undergo chemical reactions with cellular compo­
nents, or inhibit the bacterium enzyme system [120]. Inhibition zones
are observed as dark bands in areas of reduced fluorescence against a
uniform light background. The intensity of inhibition zones is non-linear
with respect to concentration, and for comparison the bioluminescence
intensities are transformed into a linear scale by a logarithmic function
that incorporates the relative intensity of the inhibition zones and of
reference zones at either side of the inhibition zones [109,120]. The
activity of individual compounds is typically expressed as a reciprocal
iso-inhibition volume equal to the calculated sample volume responsible
for 50 % inhibition of the bioluminescence signal.
Neurotoxic compounds, which include organophosphorous and
carbamate pesticides, and compounds with a possible connection to
dementia and its treatment, can be detected after separation on HPTLC
layers by inhibition of cholinesterases applied as a post-
chromatographic visualization reagent [19,72,81,86,95,108,109,114,
123-128]. This approach is suitable for the selective detection of target
compounds as well as for non-target analysis of neurotoxins. Acetyl­
cholinesterase and butylcholinesterase are the most widely used en­
zymes [19] slowly giving way to multienzyme preparations such as
rabbit liver esterase, cutinases, and horse serum esterases [124,128].
9
Journal of Chromatography B 1214 (2023) 123553
Multienzyme preparations provide wider compound coverage, simplify
the analytical protocol, and afford marginally better detection limits.
Without removal of the enzyme, the layer is dipped into a substrate
solution, for example, naphthyl acetate and fast blue salt B or indoxyl
acetate, and following a short reaction time, the enzyme is deactivated
by briefly heating at 50 ◦ C. If naphthyl acetate is used as a substrate, the
enzyme releases naphthol in regions corresponding to low inhibition,
which reacts with fast blue salt B to give a violet background with
colorless inhibition zones due to limited substrate conversion. Quanti­
fication is by absorbance at λ = 530–550 nm with LOQs at the low pg/
band range for organophosphorus pesticides (strong inhibitors) and low
ng/band range for carbamate pesticides.
Acetylcholinesterase was used with the metabolic activator S9 frac­
tion of rat liver in a streamlined process for toxicological evaluation of
pesticide residues at threshold levels required for regulatory compliance
[128,129]. In this case, the S9 mixture is over sprayed on each sample
band and incubated in a humidity chamber prior to development. For
general screening of pesticides, separations were performed on silica gel
layers with a mobile phase of hexane–ethyl acetate-dichloromethane
65:20:15 (v/v/v) [125]. Three further mobile phases adjusted to the
polarity of the pesticides were used for confirmation.
For the detection of estrogenic compounds, the yeast estrogen screen
(YES) has been used for effect-directed analysis for many years and can
be combined with HPTLC as the planar YES bioassay (pYES)
[8,18,36,91,101,109,112,130-133]. YES is a reporter gene assay using
genetically modified yeast cells housing the human estrogen receptor.
Activation of the receptor induces the reporter gene lacZ to encode the
synthesis of the enzyme β-galactosidase. The substrate, typically 4-
methylumbellifery-β-D-galactopyranoside (MUG), is applied to the layer
separately after incubation of the yeast cells with the separated sample.
The activity of the expressed reporter enzyme is detected by the for­
mation of the blue fluorescent 4-methylumbelliferone released by the
enzyme. The pYES bioassay offers high sensitivity with LODs for 17β-
estradiol (E2) and 17α-ethenylestradiol (EE2) at the low pg/band or less
depending on the protocol used. Using resorufin-β-D-galactopyranoside
(RGP) as substrate an LOQ < 10 pg/band for E2 and EE2 was obtained
[130]. To expand the scope of the pYES bioassay genetically modified
yeast cells engineered to contain other endocrine receptors have been
developed [131] and to simplify the general workflow, yeast cells that
release easily detected fluorescent proteins [90,132]. These yeast
endocrine assays do not require a separate substrate and further incu­
bation and can be used as mixtures for the simultaneous detection of
more than one endocrine endpoint, so long as the reporter proteins can
be detected without spectral overlap. Several bioassays for hormones
related to the pYES bioassays have been developed, for example, the
pYAS assay for androgens, pYDS for dioxin, pYTS for compounds
interacting with the thyroid receptor, pYVS for compounds interacting
with the vitamin D receptor, pYRas for compounds interacting with the
retinoic acid receptor, and pYMEES a multi-endocrine screen for com­
pounds interacting with the estrogen, androgen, or progesterone re­
ceptors [131,132]. Three androgenic substances in the influent and two
additional substances in the effluent samples from a wastewater treat­
ment plant were detected using the pYAS bioassay [132]. The LODs for
testosterone and 5α-androstan-17β-ol-3-one were 0.37 ng/L and 0.59
ng/L, respectively. The wastewater treatment plant removed > 99% of
the androgenic activity.
The number of bioassays continues to grow, as exemplified by a
recent report of the use of HPTLC-bioautography to screen plants ex­
tracts for anti-Leishmania compounds [134] and human cancer cells for
the detection of cytotoxic compounds [135], and those indicated above
provide only a flavor of the research on bioassays that is currently un­
derway. For better targeting of specific activities, the increasing interest
in genetically modified microorganisms should be highlighted [136].
Bioassays can be used individually or in batches by splitting extracts
between several HPTLC layers, each employing a different bioassay, and
in some cases by using mixed bioassays [20,80,83,91,111,131,132].
I.D. Wilson and C.F. Poole
Identification of unknown compounds is typically by in situ extraction
using the TLC-MS interface coupled to electrospray ionization mass
spectrometry. After bioassay the layer is impregnated by numerous
organic and inorganic compounds from the sample matrix, the support
media for the bioassay system, and typical layer contaminants not
removed during cleaning of the layer prior to sample application. These
can cause problems in the extraction step and interference in the mass
spectra. This problem can be handled by developing two plates simul­
taneously, performing the bioassay on one plate, and using the co­
ordinates for detected zones from the first plate to reference locations on
the second plate for mass spectral identification. A more elegant solution
using heart-cut multidimensional chromatography, employing a filter
and short reversed-phase/cation-exchange column installed between
the solvent extraction interface and mass spectrometer, shows promise
for an automated solution for the identification of bioactive compou
nds from bioassay-impregnated layers by mass spectrometry
[87,88,99,100].
6. Mass spectrometric detection
The coupling of mass spectrometry for in situ detection in HPTLC has
evolved at a slower pace than for column-based methods, with instru­
mental HPTLC/MS (as opposed to manual “scrape and elute” methods)
only really becoming accessible in the last decade, while quickly
becoming the primary tool for identification and increasingly for
quantification. Early approaches to coupling TLC with mass spectrom­
etry (MS) are summarized in [137-141] and more recent developments
in [8,142-144]. Apart from the perfectly serviceable manual scrape and
elute procedures, that require patience rather than instrumentation, and
are only suitable for low throughput “structure determination work”,
there are two main approaches to TLC/MS. The first of these involves a
direct interface via a liquid junction that makes a seal with the plate and
then delivers a solvent to a selected spot eluting the analyte into the ion
source of a mass spectrometer. The second approach involves the use of
atmospheric pressure (or ambient) ionization sources which have pro­
vided a breakthrough in the direct coupling of HPTLC to MS systems
[145].
6.1. Liquid-microjunction surface analysis probes
The liquid-microjunction surface sampling probe now incorporated
in the liquid extraction surface analysis (LESA) interface uses solvent
extraction to isolate material from the surface of the layer and transports
it in solution to the ion source of a mass spectrometer [146,147]. The
sampling head consists of a coaxial arrangement of two capillary tubes,
with the annular space between the inner and outer tubes used for sol­
vent delivery and the inner tube to draw liquid from the surface and
transfer it to the ion source. The sampling probe is held at a short dis­
tance above the layer and forms a stable liquid film, or junction, between
the surface and the extraction head, with the rate of solvent infused to
the layer balanced by the solvent extracted from the film. A primary
requirement is that the extraction solvent does not penetrate the layer
surface, and to date, this approach has been demonstrated for reversed-
phase layers with aqueous extraction solvents only. Solvents that wet the
layer induce radial development of zones with loss of resolution and low
signal intensity. In principle, sample is extracted from material located
at the surface in contact with the extraction solvent only. The thin-layer
plate is placed on a motorized sample stage that can be moved in the x-
and y-direction and the gap between the probe head and layer is
monitored optically and adjusted to automatically maintain a constant
distance.
6.2. Elution head interfaces
Elution head interfaces were designed for the solvent extraction of
samples from a thin-layer plate [141]. It differs from the liquid-
10
Journal of Chromatography B 1214 (2023) 123553
microjunction surface sampling probe in that the whole sample zone,
or that portion contained by the probe head, is eluted from the layer.
Lane scanning is not supported by commercially available interfaces as
the plate needs to be manually positioned under the elution head for
each zone of interest for elution. However, it has proved to be a popular
interface for TLC/MS with three commercial versions available (with
some operational differences) [7,8,95,111,148]. Broadly they function
as follows: an oval or circular stainless-steel elution head with a cutting
edge is manually positioned over the target zone and driven by pneu­
matic pressure through the layer to the backing support sealing and
isolating the zone. The inlet capillary of the elution head is connected to
a pump to deliver the extraction solvent to the sealed sample zone
(organic solvent or aqueous buffer-organic solvent mixture at a flow rate
of about 0.1–0.2 mL/min). The outlet capillary is connected via an inline
particle filter to the ion source of the mass spectrometer or to a collection
vial. By operation of a valve the extraction solvent is switched from the
ion source (standby mode) and routed through the sample zone and back
to the ion source (extraction mode). Between extraction steps, the probe
head is moved to a docking station, where the sampling surface is
cleaned of accumulated particle material by compressed air. To mini­
mize carryover between samples the extraction head can be cleaned by
solvent. Matching the size and shape of the extraction head to the sample
zone enhances sample detectability and minimizes the contribution
from the layer background [149,150]. For separations with an acidic
eluent predevelopment of the layer with a mobile phase containing the
same acid was shown to reduce ion suppression when using the elution
head interface with electrospray ionization [151].
The current versions of the elution head interfaces are best consid­
ered semi-automatic devices with automation of the lifting, lowering,
and cleaning of the elution head. Positioning of the elution head over the
selected sample zone is still performed manually by moving the plate.
However, non-commercial upgrades to the CAMAG TLC-MS interface 2
have been described for full-automation using click-on image zone se­
lection, software-controlled plate positioning and an improved elution
and cleaning process [87,88,152], and to provide fully automated
normal-phase HPTLC-reversed-phase liquid chromatography-mass
spectrometry [87,99,100,153] (and also for automated sample
cleanup by planar solid-phase extraction using solvent front elution of
target compounds [94]). Clearly elution-based methods are designed for
targeted analysis and are not suitable for scanning. However, via semi-
automation, sequential analysis of target zones within a separation is
possible and vial collection-based elution methods facilitate the exam­
ination of the extracted zones by a wide variety of spectroscopic tech­
niques in addition to mass spectrometry [96-98].
6.3. Laser desorption/ionization and matrix-assisted laser desorption/
ionization (MALDI)
Laser desorption/ionization has been a popular and widely used
strategy in surface analysis and one of its best-known forms is matrix-
assisted laser desorption/ionization (MALDI). MALDI is a soft ioniza­
tion technique that employs a laser striking a matrix applied to a surface
to ionize analytes and propel them into the gas phase with minimal
fragmentation and has seen numerous applications in TLC with early
applications reviewed in [140] and more recent progress in [154,155].
Specific applications of TLC/MALDI/MS for lipid analysis have also
recently been reviewed [156].
MALDI provides high spatial resolution but, for good sensitivity and
signal persistence, the application of organic matrices (by post-
chromatographic dipping or spraying with a solution of the matrix)
such as 2,5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxy
cinnamic acid (CHCA), 3-beta-indoleacrylic acid (IAA), dithranol (AT)
and glycerol is required, thereby introducing an additional post-
chromatographic sample preparation step into the analysis. MALDI
mass spectra can then be obtained in either the positive or negative ion
mode and are generally dominated by a few adduct ions showing limited
I.D. Wilson and C.F. Poole
fragmentation and multiply charged ions in low abundance for the an­
alyte. The choice of matrix is often the critical step in optimizing the
mass spectrometer response and for avoiding overlap of sample ions
with those of the matrix. Any mass spectrometer equipped for MALDI
imaging can, in principle, perform this for TLC plates [155,157]. Un­
surprisingly, given its use for e.g., tissue imaging etc., spatially resolved
imaging of an entire TLC plate is also possible using MALDI.
TLC/MALDI is generally performed using UV lasers but there have
also been applications of infrared lasers for direct IR-MADLI/TLC (see
for example [155] and references therein) as well as innovations in the
matrices employed. One reason for using IR rather than UV-lasers for
MALDI is that the former is thought to offer a “softer” method for
desorption/ionization [158]. This, and the deeper ablation provided by
IR-lasers can have a benefit when the analysis of small amounts of un­
stable analytes is required, for example, for the analysis of lipids [155].
This may indeed be important as the thickness of the stationary phase
has been linked to the quality of the mass spectra of lipids obtained by
MALDI [159]. Advances in matrices include the development of “inor­
ganic” matrices such as magnetic nanoparticles functionalized with 2,5-
dihydroxybenzoic acid (DHB), a widely used MALDI matrix. Such
nanoparticles have been applied to TLC/MALDI/MS profiling of sugars
on TLC plates and saccharides and oligosaccharides in human milk
analyzed by HPTLC. The dihydroxybenzoic acid-functionalized mag­
netic nanoparticles (DHB@MNPs) were applied to the plates dispersed
in an ionic liquid via “spin coating” [160]. Another example of the
application of an inorganic matrix is for the analysis of anthocyanins in
red wine, apple juice and extracts of roses [161]. Core-shell silica-coated
iron oxide magnetic nanoparticles were used as a MALDI matrix, applied
as an ethanolic dispersion directly to the bands of interest, for the
spectrometric identification and dereplication of analytes. Using this
methodology, the detection of anthocyanins using positive ionization
and dihydrochalcones, flavanols, flavanols and phenolic acids (after
derivatization) in the negative ion mode was possible. Where DHB was
found to be wanting, improvements were obtained for certain com­
pounds (e.g., erectile dysfunction drugs) via the use of MALDI cocktails
made from glycerol, graphite and either DHB, CHCA, IAA or AT [161].
In an interesting approach, that removes the need for a MALDI matrix
completely, TLC plates have been prepared as monolithic layers formed
via the copolymerization of ethylene dimethacrylate (EDMA) and gly­
cidyl methacrylate (GDA), with the latter proving essential for successful
MALDI [157]. When investigated as a TLC medium, using several dyes,
copolymers containing a high ratio of glycidyl methacrylate (e.g., GDA/
EDMA, 3:17) provided separations of lower efficiency than those made
using higher proportions of ethylene dimethacrylate (e.g., GDA/
EDMA,1:27), but both were considered acceptable. However, the best
MALDI/MS spectral data were obtained with mixtures containing from
19 to 29% GDA.
6.4. Atmospheric pressure (or ambient) ionization sources
Depending on the type used these “ambient” approaches require
neither vacuum nor direct connection with the stationary phase of the
HPTLC plate and often with minimal pretreatment can be used to
interrogate individual spots, scan lanes or fully image plates. Ambient
ionization techniques can also more easily facilitate the manipulation of
relatively large objects, like thin-layer plates compared to conventional
vacuum ion sources. Desorption-based methods usually allow recording
of a mass chromatogram using the total ion current (TIC) or an extracted
ion chromatogram (EIC) for greater selectivity.
6.4.1. Direct analysis in real time (DART)
Direct analysis in real time (DART) uses a thermal desorption-based
atmospheric pressure ion source employing an electrical discharge in a
heated helium gas stream to create energetic neutral metastable helium
species [123,162-168]. The resulting narrow beam of these metastable
helium species interacts with the air to produce predominantly charged
11
Journal of Chromatography B 1214 (2023) 123553
water clusters (for positive ion spectra) and oxygen ions (for negative
ion spectra), as well as other species. The collision of the hot gas plasma
with compounds on the layer surface is responsible for ionization by
proton or charge transfer and their introduction into the gas phase by
thermal desorption. The ions originating from the surface are introduced
into the mass spectrometer via a ceramic probe located close to the
sampling position on the layer and mass analyzed. DART ionization
typically results in the formation of singularly charged molecular and
molecular adduct ions with some fragmentation as well as the appear­
ance of oxidation products in some cases (e.g., see [123]). Moving the
layer, typically a 1–2 cm-wide strip, on a motorized platform beneath
the focused gas beam facilitates spot sampling and lane scanning. Res­
olution is limited by the cone diameter of the source beam with opti­
mized configurations providing ca. 3 mm. Sensitivity is limited by only
sampling material from the zone surface but LODs in the low ng/band
can still be achieved, and quantification with reasonable repeatability is
possible [165,167]. An example of the utility of DART for the analysis of
difficult samples is provided by the determination of the phytoestrogen
equol in an ethanol extract of cattle manure using HPTLC-DART-TOF-
MS with concentrations estimated to be ca. 70 µg/g [168]
6.4.2. Desorption electrospray ionization (DESI)
Desorption electrospray ionization (DESI) is a liquid surface sam­
pling ambient ionization technique based on the spraying of charged
solvent droplets onto a surface, such as a TLC plate, to desorb analytes
and transfer them, via a vacuum transfer line, into the ESI source of a
mass spectrometer. It’s ease of operation has meant that it has become a
widely used tool for both surface analysis and imaging that is easily
adapted for TLC. Early examples of the use of DESI with TLC include its
use for the analysis of alkaloids from goldenseal [169], pharmaceutical
formulations (using a combination of ion mobility mass spectrometry
and MS (TLC/IMS/MS)) [170], the analysis of lipids following 2D TLC
[171] and the determination of salvinorium in extracts of the leaves of
Salvia divinorum [172]. Until recently such studies were usually per­
formed on lab-made DESI systems, and this continues to be a means of
performing such work (e.g., [169]), but the availability of commercial
systems that can be used generally for DESI imaging means that there is
no barrier to its use in TLC. Such systems can be targeted at individual
spot analysis or lane scanning and plate imaging, provided that the layer
fits onto the target stage. These devices are essentially agnostic with
regard to the surface being analyzed. As a result, there are an increasing
number of examples of its use e.g., [146,173-175] all related to phyto­
chemicals including phytoecdysteroids [173,175], anthraquinones
[174] and bioactive compounds in extracts of Lepidium peruvianum
[146]. In Fig. 5, a 2D image of a reversed-phase HPTLC plate showing
the result of the analysis of an extract of the plant Silene otites by DESI/
MS is shown, together with a representative spectrum of one of the
major constituents, 20-hydroxyecdysone [175]. Whilst TLC/DESI/MS is
relatively easy to implement, various factors need to be optimized to
obtain good results including e.g., the position of the sprayer, the
transfer line temperature, sampling rate, the composition and flow rates
of solvent and nebulizer gas, etc. DESI solvent choice and composition
are key parameters controlling ionization efficiency and interestingly, a
recent study suggested that the type of stationary phase could also affect
the mass spectra obtained [175].
6.5. Trends and opportunities in HPTLC/MS
Irrespective of the MS techniques used for HPTLC/MS the significant
number of applications that include HPTLC/MS of one sort or another,
especially in combination with bioautography to enable the detection
and characterization of bioactive compounds, appears to be an area of
significant interest in planar chromatography and can only be expected
to increase. And, whilst the methods described above currently repre­
sent the major techniques in use for HPTLC/MS innovations in this area
continue to appear such as e.g., low-temperature plasma probe MS
I.D. Wilson and C.F. Poole
[176], electrostatic field induced spray ionization-MS [177,178] and
cluster-induced desorption/ionization [179]. Another innovation that
can be expected to make a significant impression on the field is ion
mobility (IM)-based methods. The advantages of incorporating ion
mobility are twofold. Firstly, IM provides a second, orthogonal,
dimension of separation which can separate co-migrating compounds
present in a complex mixture that are unresolved by HPTLC. This sep­
aration can result in much cleaner spectra as a result of resolving
comigrating compounds. Secondly, the collision cross sections that can
be obtained via IMS provide another feature which, together with the
MS data, can help to further characterize and identify compounds (e.g.,
see [173]).
7. Conclusions
Even in an age where high resolution HPLC and GC dominate the
field of chromatographic separations the unique properties of HPTLC
have ensured that it continues to provide fit-for-purpose solutions to
many problems in analysis. Perhaps the biggest advantage of HPTLC is
that the separation is preserved at the end of the analysis and can be used
to store it prior to further evaluation. This ability to store the physical
separation enables the investigator to undertake qualitative, quantita­
tive, and structural or effect-directed analysis as required. Indeed, with
care all these analyses could be performed sequentially on the same
separation, and in different locations. This flexibility, which derives from
the open format of HPTLC itself, is simply not shared by other separation
techniques. TLC also has the potential to scale up analysis very simply
and inexpensively to provide a high throughput methodology, where
each step can be automated if needed. The addition of relatively simple
direct MS-based analysis via MALDI or DESI (or other approaches),
possibly with IMS, represents a real advance and one that will, in the
opinion of the authors, continue to keep planar chromatography as
12
Journal of Chromatography B 1214 (2023) 123553
Fig. 5. A DESI MS Image of the 2D HPTLC
separation of a crude extract of S. otites
following separation with aqueous acetone-
based solvent in the 1st (vertical axis) and
an ethanol-based solvent for the 2nd (hori­
zontal) dimensions respectively. The image
was acquired using a heated solvent transfer
line set at 450 ◦ C and MS spectra were ac­
quired for the various circled regions of in­
terest (ROI). The mass spectrum shown is for
20-hydroxyecdysone (ROI 9) (structure inset
to Figure). [175]. Composite image adapted
with permission of the copyright owners.
relevant for analysts in the 21st century as it was in the 20 th.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
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