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Thin Layer Chromatography
JOSEPH SHERMA
Department of Chemistry, Lafayette College, Easton, PA, USA
I. INTRODUCTION
Thin layer chromatography (TLC) is a type of liquid
chromatography in which the stationary phase is in the
form of a layer on a glass, an aluminum, or a plastic
support. The term “planar chromatography” is often used
for both TLC and paper chromatography (PC) because
each employs a planar stationary phase rather than a
packed column. PC, which utilizes plain, modified, or
impregnated paper (cellulose) as the stationary phase,
involves many of the same basic techniques as TLC, but
it has not evolved into an efficient, sensitive, quantitative,
instrument-based analytical method and has many disad-
vantages relative to TLC. Consequently, PC will not be
covered in this chapter.
As originally developed in 1951 by J.G. Kirchner and
colleagues, later standardized by E. Stahl and colleagues,
and still widely practiced today (Fried and Sherma,
1999a; Sherma, 2002), classical, capillary-action TLC is
an inexpensive, easy technique that requires little instru-
mentation, which is used for separation of simple mixtures
and for qualitative identification or semiquantitative,
visual analysis of samples. In contrast, modern TLC
[usually termed as high performance thin layer chromato-
graphy (HPTLC)], which began around 1975 with the
introduction of high efficiency, commercially precoated
plates by Merck, is an instrumental technique carried out
on efficient, fine-particle layers. Instrumental HPTLC is
capable of producing fast, high-resolution separations
and qualitative and quantitative results that meet good
manufacturing practices (GMP) and good laboratory prac-
tices (GLP) standards. The accuracy and precision of data
obtained by HPTLC rival those of gas chromatography
(GC) and high performance column liquid chromato-
graphy (HPLC), and it has many advantages relative to
these methods.
TLC is an off-line process in which the various stages
this arrangement using an open, disposable layer com-
pared with an on-line column process such as HPLC
include the possibility of separating up to 70 samples
and standards simultaneously on a single plate, leading
to high throughput, low cost analyses and the ability to
construct calibration curves from standards chromato-
graphed under the same conditions as the samples; analyz-
ing samples with minimum sample preparation without
fear of irreversible contamination of the column or carry-
over of fractions from one sample to another as can occur
in HPLC with sequential injections; and analyzing a
sample by use of multiple separation steps and static post-
chromatographic detection procedures with various uni-
versal and specific visualization reagents, which is
possible because all sample components are “stored” on
the layer without chance of loss. TLC is highly selective
and flexible because of the great variety of layers that is
available commercially. It has proven to be as sensitive
995
(Fig. 1) are carried out independently. The advantages of
Copyright © 2005 by Marcel Dekker
as HPLC in many analyses, and solvent usage per sample
is extremely low.
TLC and HPTLC plates are usually developed by capil-
lary flow of the mobile phase without pressure in ascend-
ing or horizontal modes, but forced flow methods, in which
the mobile phase is driven through the layer by pumping
under pressure or by centrifugal force, are also used. In
capillary-flow TLC, the migration speed of the mobile
phase decreases with the square of the solvent migration
distance.
Limited separation efficiency is a disadvantage of TLC.
The maximum number of theoretical plates (N) for HPTLC
is ca. 5000 compared with ca. 15,000 for HPLC, while the
separation number (the number of spots that can be separated
over the distance of the run with a resolution of unity) is
ca. 15 in TLC compared with 200 in HPLC. This limited
efficiency is a result of the capillary-flow mechanism over
a restricted migration distance. Under forced flow conditions,
TLC separation efficiency is significantly improved. For a
discussion on the theory and mechanism of the various
An apparent disadvantage of TLC is that although all of
the individual steps have been automated and on-line
coupling with other chromatographic and spectrometric
techniques has been achieved, complete automation of
TLC has not been realized. However, changing the off-
line nature of TLC to an on-line closed system with com-
plete automation would eliminate many of the advantages
stated earlier. Several studies have shown that more
samples per day can be processed using stepwise-
automated TLC compared with a fully automated HPLC
system, and with lower cost (Abjean, 1993).
Analytical TLC differs from preparative layer chrom-
atography (PLC) in that larger weights and volumes of
samples are applied to thicker (0.5–2 mm) and sometimes
larger layers in the latter method, the purpose of which is
the isolation of 10–1000 mg of sample for further
analysis.
This chapter describes the TLC techniques and instru-
mentation with different levels of automation that can be
used in a contemporary analytical laboratory to produce
high-quality analytical results without sacrificing the
great flexibility of the method.
II. SAMPLE PREPARATION
Sample preparation (Fried and Sherma, 1999a, c; Sherma,
2003) procedures for TLC are similar to those for GC and
HPLC. The solution to be spotted must be sufficiently con-
centrated so that the analyte can be detected in the applied
volume, and pure enough so that it can be separated as a
discrete, compact spot or zone. The solvent in which the
sample is dissolved must be suitable in terms of viscosity,
volatility, ability to wet the layer, and potential for
unintended predevelopment during sample application.
Relatively pure samples or their concentrated extracts
can often be directly spotted for TLC analysis. If the
analyte is present in low concentration in a complex
sample, solvent extraction, cleanup (purification), and con-
centration procedures must precede TLC. Because layers
are not reused, it is often possible to apply cruder
samples than could be injected into a GC or HPLC
column, including samples with irreversibly sorbed impu-
rities. However, impurities that comigrate with the analyte,
adversely affect its detection, or distort its zone (i.e., cause
streaking or tailing), must be removed prior to TLC.
Carryover of material from one sample to another is
not a problem as it is in on-line column methods involv-
ing sequential injections. Therefore, sample preparation
is often simpler for TLC compared with other
chromatographic methods.
Common cleanup procedures include liquid–liquid
extraction, column chromatography, desalting, and depro-
teinization. Solid-phase extraction (SPE) using small,
Figure 1 Schematic diagram of the steps in a TLC analysis.
(Courtesy of Camag.)
996 Analytical Instrumentation Handbook
Copyright © 2005 by Marcel Dekker
modes of TLC, see Fried and Sherma (1999b), Kowalska
and Prus (2001), and Kowalska et al. (2003).
disposable columns and a manual vacuum manifold
(Fig. 2) or various semiautomated or automated instru-
ments has become widely used for isolation and cleanup
of samples prior to TLC analysis, such as pesticide resi-
dues in water (Hamada and Wintersteiger, 2002). Super-
critical fluid extraction has been used for off-line
extraction of analytes from samples (van Beek, 2002),
and has been directly coupled to TLC for analysis of
solid samples or solutions loaded on glass fiber filters
(Esser and Klockow, 1994). A derivative of the analyte
can be formed in solution prior to spotting, or in situ at
the origin by overspotting of a reagent, in order to
improve resolution or detection. Special plates with pread-
sorbent zone serve for sample cleanup by retaining some
interfering substances.
III. STATIONARY PHASES
TLC and HPTLC plates are commercially available in the
form of precoated layers supported on glass, plastic sheets,
or aluminum foil (Fried and Sherma, 1999d; Lepri and
Cincinelli, 2001; Rabel, 2003). HPTLC plates are smaller
(10 � 10 or 10 � 20 cm), have a thinner (0.1–0.2 mm)
more uniform layer composed of smaller diameter particles
(5–6 mm), and are developed over shorter distances
(ca. 3–7 cm) compared with classical 20 � 20 cm TLC
plates, which have a 0.25 mm thick layer of 12–20 mm
particle size and are developed for 10–12 cm. Optimal
development distances are the points beyond which
increased resolution of zones is offset by diffusion
effects. In comparison with TLC, HPTLC provides better
separation efficiency and lower detection limits.
The choice of the layer and mobile phase is made in
relation to the nature of the sample. Normal-phase (NP)
or straight-phase adsorption TLC on silica gel with a less
polar mobile phase, such as chloroform–methanol, is the
most widely used mode. Lipophilic C-18, C-8, and C-2
bonded silica gel phases with a polar aqueous mobile
phase, such as methanol–water, are used for reversed-
phase (RP) TLC. Other precoated layers include alumina,
magnesium silicate (Florisil), polyamide, cellulose, ion
exchangers, and chemically bonded phenyl, amino,
cyano, and diol layers. The latter three bonded phases
can function with multimodal mechanisms, depending on
the composition of the mobile phase. Silica gel can be
impregnated with various solvents, buffers, and selective
reagents to improve separations. Chiral plates composed
of a RP layer impregnated with copper acetate and a
chiral selector, (2S,4R,20RS)-4-hydroxy-1-(2-hydroxydo-
decyl)proline, can be used to separate enantiomers
through a ligand-exchange mechanism. Preparative
layers are thicker than analytical layers to provide higher
sample capacity.
Ultra-thin silica layers (Hauck et al., 2002) are the
newest type available commercially. Unlike all other
TLC and HPTLC layers, these do not consist of particulate
material but are characterized by a monolithic silica struc-
ture. They are manufactured without a binder, which is
usually needed to stabilize the sorbent particles on the
support, and have a significantly thinner layer (10 mm),
leading to short migration distances, fast development
times, and very low solvent consumption.
Important manufacturers of TLC plates include Merck,
Whatman, Analtech, and Macherey-Nagel, and literature
from these companies should be consulted for details of
availability, properties, usage, and applications.
IV. MOBILE PHASES
Unlike GC, in which the mobile phase (carrier gas) is not
a factor in the selectivity of the chromatographic system,
the mobile phase in liquid chromatography, including
TLC, exerts a decisive influence on the separation.
In HPLC, the analyte passes through the on-line detector
in the presence of the mobile phase. Therefore, solvents
must be chosen not only to provide the required resolution,
but also to not absorb at the ultraviolet (UV) detection
wavelength. Because in TLC the mobile phase is
removed (evaporated) before the zones are detected, a
wider variety of solvents can be used to prepare mobile
phases compared with HPLC.
Figure 2 J.T. Baker vacuum processor for 12 or 24 BAKER-
BOND SPE or Speedisk columns. (Courtesy of Mallinckrodt
Baker Inc., Phillipsburg, NJ.)
Thin Layer Chromatography 997
Copyright © 2005 by Marcel Dekker
The mobile phase (Fried and Sherma, 1999e) is usually a
mixture of two to five different solvents selected empirically
using trial and error guided by prior personal experience and
literature reports of similar separations. In addition, various
systematic mobile phase optimization approaches
(Cimpoiu, 2003; Prus and Kowalska, 2001) involving
solvent classification (selectivity) and the elutropic series
(strength) patterned after HPLC have been described, most
notably, the PRISMA model based on Snyder’s solvent
classification system as developed by Nyiredy. For
NP-TLC (silica gel), the following 10 solvents from
Snyder’s eight selectivity groups are used for exploratory
TLC of the mixture: diethyl ether (group I), isopropanol
and ethanol (group II), tetrahydrofuran (group III), acetic
acid (group IV), dichloromethane (group V), ethyl acetate
and dioxane (group VI), toluene (group VII), and chloroform
(group VIII). Hexane (solvent strength ¼ 0) is used to adjust
the Rf values within the optimum range (0.2–0.8), if necess-
ary. Between two and five solvents are then selected for
construction of the PRISMA model that leads to identifi-
cation of the optimized mobile phase. A similar procedure
is followed for RP-TLC (e.g., C-18 bonded phase layer)
using mixtures of methanol, acetonitrile, and/or tetrahydro-
furan with water (solvent strength ¼ 0).
TLC is usually carried out with a single mobile phase,
rather than a mobile phase gradient as is often used in
HPLC. Equilibration between the mobile phase and the
layer occurs gradually during TLC development, and the
mobile phase composition can change because different
constituents migrate through the layer at different rates
(solvent demixing). This leads to solvent gradients along
the layer during “isocratic” TLC, the formation process
of which is very different than the intentional, well-
controlled gradients in a fully equilibrated HPLC column.
Mobile phase selection for automated multiple develop-
ment (AMD) is described in Section VI,A,4.
V. APPLICATION OF SAMPLES
Application of small, exactly positioned initial zones of
sample and standard solutions (Fried and Sherma, 1999f)
having accurate and precise volumes, without damaging
the layer surface, is critical for achieving maximum resol-
ution and reliable qualitative and quantitative analysis. The
volumes applied and the method of application depend on
the type of analysis to be performed (qualitative or quanti-
tative), the layer (TLC or HPTLC), and the detection limit.
For the greatest separation efficiency, the solvent in which
the sample is dissolved should have high volatility and be
as low in solvent strength as possible (nonpolar for NP
systems and polar for RP systems) to retard the possibility
of “prechromatography” during application.
Application of round spots allows the maximum
number of samples to be applied onto a given plate. For
TLC, 0.5–5 mL volumes are usually applied manually
with a micropipet to produce initial zones with diameters
in the 2–4 mm range. For TLC or HPTLC, initial zones
in the form of spots can be applied from a disposable
0.5, 1, 2, or 5 mL fixed-volume, selfloading glass capillary
pipet, which is held in a rocker-type spotting device (the
Nanomat 4, Camag Scientific Inc., Wilmington, NC) that
mechanically controls its positioning and brings the capil-
lary tip in gentle and uniform contact with the layer to
discharge the solution without damage to the layer. The
Nanomat and other instruments for sample application
and the later steps in the TLC process have been described
by Reich (2003).
The TLS100 (Baron, Reichenau, Germany) is an auto-
mated apparatus for applying spots of up to 30 samples and
four standards using a 1, 10, or 100 mL motor driven
syringe. Locations and volumes can be chosen with a
keypad for application onto as many as six HPTLC plates.
Sample application in the form of bands is advan-
tageous for high-resolution separations of complex
samples, improved detection limits, and for precise [1%
relative standard deviation (RSD)] quantitative scanning
densitometry using the aliquot technique (scanning with
a slit of one-half to two-thirds the length of the applied
band). Narrow, homogeneous sample bands of controlled
length [from 1 (spot) to 195 mm] can be applied by use
of a spray-on device (the Camag Linomat 5, Fig. 3), in
which the plate is mechanically moved right to left in
the X-direction beneath a fixed syringe from which
Figure 3 Linomat 5. (Courtesy of Camag.)
998 Analytical Instrumentation Handbook
Copyright © 2005 by Marcel Dekker
0.1–2000 mL of sample is sprayed by an atomizer operat-
ing with a controlled nitrogen gas pressure for analytical
and preparative applications. The user selects the sample
volumes and Y-position via a keypad or by downloading
a method from a personal computer, and the instrument
exactly positions the initial zones, which facilitates auto-
mated scanning after chromatogram development and
overspraying samples with a reagent for in situ prechroma-
tographic derivatization or with spiking solutions for vali-
dation of quantitative analysis by the standard-addition
method. With the correct choice of application parameters,
less volatile and higher strength sample solvents can be
tolerated without forming broadened initial zones. The
ability to apply larger volumes to an HPTLC plate
without loss of resolution lowers the determination limits
with respect to the concentration of the solution, which
aids in trace analysis. Complex, impure samples can
often be successfully quantified only if bands are applied
rather than spots. The Linomat facilitates quantitative
analysis by allowing different volumes of the same stan-
dard solution to be applied to produce the densitometric
calibration curve, rather than the same volume of a
series of standards when spots are applied.
The AS30 (Desaga, Weisloch, Germany) is a software
controlled, fully automated band or spot applicator that
also works according to a spray-on technique, in which a
stream of gas carries the sample from the cannula tip
onto the plate. The syringe does not have to be manually
filled by the user, as with the Linomat. During the filling
process, the dosing syringe is positioned over the tray,
which collects rinsing and flushing solvent and excess
sample. The sample is injected into the body of the
syringe through a lateral opening. After the syringe has
been filled, a stepping motor moves the piston downwards
to dose the fillport. A second stepping motor moves the
tower sideways across the plate. The microprocessor con-
trols both motors and the gas valve for accurate and precise
application in the form of spots or bands. All parameters
for application of up to 30 samples are entered via the key-
board. The user is guided through the clearly structured
menu by the two-line LCD display.
Figure 4 shows the Camag Automatic TLC Sampler
IV (ATS 4), which is an advanced, fully automated,
computer-controlled device for sequential application of
up to 66 samples from a rack of vials or 96 samples
from well plates through a steel capillary as spots by
contact transfer or as bands by the spray-on technique.
The speed, volume, and X- and Y-position pattern of appli-
cation are controllable, and a programmable rinse cycle
can eliminate cross-contamination. Low concentration
samples can be applied as rectangles, which are focused
into narrow bands by predevelopment with a strong
mobile phase. An optional heated spray nozzle allows
increased application speed, which is important for
aqueous solutions. Analyses performed with this
applicator combined with densitometric chromatogram
evaluation controlled by the same computer conform to
GMP/GLP standards.
VI. CHROMATOGRAM DEVELOPMENT
TLC is almost always carried out in the elution mode.
Methods and applications of displacement TLC have been
described (Bariska et al., 2000), but this method is not yet
widely used and will not be covered in this chapter.
Electro-osmotically driven TLC (Nurok et al., 2002) is a
quite new method that has not yet been shown to have a
significant number of important practical applications, and
it also will not be covered.
Linear development of TLC plates is used almost exclu-
sively today. Therefore, circular (radial) and anticircular
development will not be discussed here. TLC development
times are typically in the range of 3–60 min, depending on
the layer, mobile phase, and development method chosen.
However, the development time does not significantly influ-
ence the overall analysis time per sample, because many
samples and standards can be chromatographed simul-
taneously. Development modes and chambers have been
described in detail elsewhere (Fried and Sherma, 1999g).
A. Capillary-Flow TLC
1. Ascending Development
The results of TLC are strongly dependent upon the
environmental conditions during development, such as
small changes in mobile phase composition, temperature,
Figure 4 Automatic TLC Sampler 4 (ATS 4). (Courtesy of
Camag.)
Thin Layer Chromatography 999
Copyright © 2005 by Marcel Dekker
humidity, and the size and type of the chamber and its
solvent vapor saturation conditions.
In the classical method of linear, ascending develop-
ment TLC and HPTLC, the developing solvent is con-
tained in a large volume, covered glass tank (N-tank).
The spotted plate is inclined against an inside wall of the
tank with its lower edge immersed in the developing
solvent below the starting line, and the solvent begins to
rise immediately through the initial zones due to capillary
flow. As the mobile phase ascends, the layer interacts with
the vapor phase as well as the mobile phase and the
mixture components. The space inside the tank is more
or less equilibrated with solvent vapors, depending on
the presence or absence of a mobile phase-soaked paper
liner, and the period of time the tank is allowed to stand
before the plate is inserted. Reproducible results are
obtained only if all development conditions are maintained
as constant as possible. Solvent consumption is high with
classical chambers.
A sandwich chamber (S-chamber) consists of the TLC
plate, a spacer of 3 mm thickness, and a cover- or counter-
plate that is either blank glass or a solvent-soaked TLC
plate. These parts are clamped together so that the
bottom 2 cm of the layer is uncovered and are placed in
a trough containing the mobile phase. Interaction between
the layer, dry or wetted, and the gas phase is largely
suppressed in an S-chamber, and reproducibility of the
separation is improved. Both ascending and horizontal
chambers can be operated as S-chambers (Gocan, 2001a).
The twin-trough chamber is an N-chamber modified
with an inverted V-shaped ridge on the bottom dividing
the tank into two sections, which allow development
with only 5–20 mL of solvent, depending on the plate
size, on one side, and easy pre-equilibration of the layer
with vapors of the mobile phase or another conditioning
liquid (e.g., a sulfuric acid–water mixture to control
humidity) or volatile reagent on the other side.
2. Horizontal Development
The horizontal developing chamber (Fig. 5) permits
simultaneous development from opposite edges to the
middle of 72 sample spots on a 20 � 10 cm HPTLC plate,
or 36 samples from one end to the other. The developing
solvent, held in narrow troughs, is carried to the layer
through capillary slits formed between the trough walls
and the glass slides. The chamber is covered with a glass
plate during pre-equilibration and development and can be
operated in N-type or S-type configurations, including
humidity control by placing an appropriate sulfuric acid–
water mixture in a conditioning tray. Use of the horizontal
developing chamber allows separation conditions to be effi-
ciently standardized, and only low amounts of mobile phase
are required.
The Camag HPTLC Vario System is a horizontal
chamber that facilitates development and optimization of
separation parameters. Simultaneous development can be
tested with up to six different mobile phases, sandwich
or tank configurations, and pre-equilibration conditions
in any combination.
3. Continuous Development
The short bed continuous development chamber (Regis,
Morton Grove, IL) is used for continuous development,
which leads to improved resolution of zones with low
migration rates because of the larger effective separation
distance. Four glass ridges and the back wall of the
chamber support the plate at five different angles of incli-
nation, each of which allows increasing lengths of the
layer to protrude out of the top of the chamber. Solvent
continually evaporates from the external layer at the top
and is replaced by additional solvent drawn from the
chamber at the bottom.
4. Gradient Elution TLC Combined with AMD
AMD generally involves 10–30 individual linear ascend-
ing developments of an HPTLC plate (usually silica gel)
an N-type chamber equipped with connections for feeding
and releasing mobile phase and pumping a gas phase in
and out, storage bottles for pure solvents and waste, a gra-
dient mixer, syringes for measuring solvent volumes, and a
charge coupled device (CCD) detector for monitoring
migration distances. The developments are performed in
the same direction with a stepwise mobile phase gradient
Figure 5 Horizontal development chamber. 1, HPTLC plate with layer facing down; 2, glass plate for sandwich configuration; 3, reser-
voir for mobile phase; 4, glass strip; 5, cover plate; 6, conditioning tray. (Courtesy of Camag.)
1000 Analytical Instrumentation Handbook
carried out in an AMD instrument (Fig. 6), which includes
Copyright © 2005 by Marcel Dekker
that becomes progressively weaker (i.e., less polar) over
distances that increase by 1–5 mm for each stage. The
solvent is completely removed from the chamber, and
the layer is dried under vacuum applied by a pump for a
preselected time after each development. The layer is
then preconditioned with the vapor phase of the next
batch of fresh solvent, which is fed into the chamber
before the following incremental run.
The solvent strength may be changed for each develop-
ment, or several stages may be carried out with the same
solvent before changing its strength. The repeated move-
ment of the solvent front through the chromatographic
zones causes them to become compressed into narrow
bands during AMD, leading to peak capacities of more
than 50 over a separation distance of 80 mm. Typical
“universal gradients” for AMD are produced from metha-
nol or acetonitrile (polar); dichloromethane, di-isopropyl
ether, or t-butylmethyl ether (medium polarity); and
hexane (nonpolar). The central or base solvent and the
nonpolar solvent have the greatest effect on selectivity.
By superimposing the densitogram of a chromatogram
with a matched-scale diagram of the gradient, required
modifications of the solvent system can be predicted.
Gradient development in TLC has been reviewed
(Golkiewicz, 2003).
Complex mixtures containing compounds with widely
different polarities can be separated by AMD on one chro-
matogram, in which sharply focused zones migrate differ-
ent distances according to their polarities. Zone widths are
independent of migration distance and size of the starting
zone, leading to high resolution and the ability to resolve
relatively large samples for trace analysis. Migration dis-
tances of individual components are largely independent
of the sample matrix, and detection limits are improved
because of the highly concentrated zones (typically
1 mm peak width) that are produced. The densitometer
togram that can be produced by gradient AMD.
5. Two-Dimensional Development
Two-dimensional (2D) TLC involves spotting the sample
in one corner of the layer, developing (ascending or hori-
zontal) with the first mobile phase, drying the plate, and
developing at a 908 angle with a second mobile phase
having a diverse, complementary separation mechanism
(selectivity). Computer simulation has been used to opti-
mize 2D separations based on one-dimensional (1D)
data. Resolution (spot capacity) in 2D TLC is greatly
improved compared with 1D TLC because sample com-
ponents are resolved over the entire area of the layer,
and is usually superior to that obtained by HPLC.
Resolution by 2D forced flow planar chromatography
(FFPC) (see exceeds 2D capillary-flow
TLC because spot diffusion is smaller. Disadvantages of
2D TLC include a limit of one sample per plate and
the time required for two developments and intermediate
drying. In addition, quantitative calibration or qualitative
identification standards cannot be developed in parallel
on the same plate at the same time. Improved video densito-
meters may allow more reliable quantitative 2D TLC in
the future than is possible today with the commonly
used slit-scanning densitometers. 2D TLC and other
multidimensional methods have been reviewed (Gocan,
2001b).
B. Forced Flow Planar Chromatography
The mobile phase can migrate through the layer by capillary
action, as is the case in the procedures described earlier, or
under the influence of forced flow. FFPC has theoretical
advantages relative to capillary flow, including independent
optimization of mobile phase velocity, higher efficiency,
lower separation time, and use of solvents that do not wet
the layer, but it requires specialized, complex commercial
instrumentation. Forced flow is produced by mechanically
pumping solvent through a sealed layer [overpressured layer
chromatography or optimum performance laminar chrom-
atography (OPLC) (Mincsovics et al., 2003; Rozylo,
Figure 6 AMD 2 automated multiple development instrument.
(Courtesy of Camag Scientific Inc.)
Thin Layer Chromatography 1001
scan shown in Fig. 7 illustrates a high-resolution chroma-
Section VI.B)
Copyright © 2005 by Marcel Dekker
2001)] or by spinning a glass rotor covered with sorbent
around a central axis to drive the solvent from the center
to the periphery of the layer by centrifugal force [rotation
planar chromatography (RPC)]. Separations can be
accomplished with a dry layer (off-line FFPC), but the
closed system arrangement also allows the separation to
be started after the layer is equilibrated with the mobile
phase, similar to the situation in HPLC (on-line FFPC).
In RPC, samples are applied to the rotating stationary
phase near the center, and centrifugal force along with
capillary action drives the mobile phase through the
sorbent from the center to the periphery of the plate. Up
to 72 samples can be applied for analytical separations,
and in situ quantification is possible. One circular
sample is applied for micropreparative and preparative
separations, which can be carried out off- and on-line.
Various chambers are used for RPC, which differ mainly
in the volume of the vapor space. The major commercial
instruments for RPC are the Chromatotron 7924 (Harrison
Research, Palo Alto, CA), CLC-5 (Hitachi, Tokyo, Japan),
Extrachrom (RIMP, Budakalasz, Hungary), and Rota-
chrom Model P (Petazon, Zug, Switzerland). Although
analytical applications have been proposed, RPC appears
to be most useful for preparative applications.
OPLC combines many advantages of classical TLC and
HPLC. A special glass- or aluminum-backed layer, sealed
at the edges, is covered by a polyethylene or Teflon foil
and pressurized by water. Development modes include
linear unidirectional, linear bidirectional, circular, on-
line, off-line, parallel coupled multilayer, serial coupled
multilayer, isocratic, and gradient. The fully automatic,
computer controlled personal OPLC BS-50 instrument
(OPLC-NIT, Budapest, Hungary) is shown in Fig. 8
(Mincsovics et al., 1999). It consists, in general, of a
separation chamber and a liquid delivery system with a
Figure 7 Multiwavelength densitogram of 16 pesticides separated by AMD. (Courtesy of Camag.)
Figure 8 Automated personal OPLC BS-50 system. 1, Liquid
delivery system; 2, separation chamber; 3, cassette; 4, mobile
phase inlet; 5, mobile phase outlet; 6, mobile phase switching
valve; 7, mobile phase reservoirs; 8, liquid crystal display. [Repro-
duced from Mincsovics et al. (1999) with permission.]
1002 Analytical Instrumentation Handbook
Copyright © 2005 by Marcel Dekker
two-in-one hydraulic pump and mobile phase delivery
pump. The chamber contains a holding unit, hydraulic
unit, tray-like layer cassette, and drain valve. The separ-
ation chamber has two mobile phase connections and a
5 MPa maximum external pressure.
VII. ZONE DETECTION
After development with the mobile phase, the plate is dried
in a fumehood (with or without heat) to completely evap-
orate the mobile phase. Separated compounds are detected
(or visualized) on the layer by their natural color, natural
fluorescence, quenching of fluorescence, or as colored,
UV-absorbing, or fluorescent zones after reaction with
an appropriate reagent (post-chromatographic derivatiza-
tion) (Fried and Sherma, 1999h; Sherma, 2001a).
Although dependent upon the particular analyte and the
detection method chosen, sensitivity values are generally
in the low microgram to nanogram range for absorbance
and picogram range for fluorescence.
Layers are frequently heated after applying the detec-
tion reagent in order to accelerate the reaction upon
which detection is based. Heating is carried out with a
hair drier in a fume hood, in an oven, or with a TLC
plate heater. The plate heater (Fig. 9), which contains a
20 � 20 cm flat, even heating area, a grid to facilitate
proper positioning of TLC and HPTLC plates, program-
able temperature between 258C and 2008C, and digital
display of the actual temperature, provides the most
consistent heating conditions.
Compounds that are naturally colored are viewed
directly on the layer in daylight, whereas compounds
with native fluorescence are viewed as bright zones on a
dark background under UV light. Viewing cabinets
incorporating shortwave (254 nm) and longwave (366 nm)
UV lamps are available for inspecting chromatograms in
an undarkened room.
Compounds that absorb around 254 nm, particularly
those with aromatic rings and conjugated double bonds,
can be detected on an “F-layer” containing a phosphor or
fluorescent indicator (often zinc silicate). When irradiated
with 254 nm UV light, absorbing compounds diminish
(quench) the uniform layer fluorescence and are detected
as dark violet spots on a bright (usually green) background.
Universal or selective chromogenic and fluorogenic
liquid detection reagents are applied by spraying or
dipping the layer. Various types of aerosol sprayers are
available for manual operation, including the Desaga
Sprayer SG 1 with a quiet, built-in pump and PTFE
spray head (Fig. 10). The ChromaJet DS 20 (Desaga)
is a spraying instrument that reproducibly
applies selectable, accurate amounts of reagent to individ-
ual plate tracks under computer control. For safety
purposes, spraying is carried out inside a laboratory fume-
hood or commercial TLC spray cabinet with a blower (fan)
and exhaust hose. The most uniform dip application of
reagents can be achieved by use of a battery operated chro-
matogram immersion device (Camag), which provides
selectable, consistent vertical immersion and withdrawal
speeds between 30 and 50 mm/s and immersion times
between 1 and 8 s for plates with 10 or 20 cm heights.
This mechanized dipping device can also be used for pre-
washing TLC plates prior to initial zone application,
impregnation of layers with reagents that improve resol-
ution or detection prior to initial zone application and
Figure 9 TLC plate heater. (Courtesy of Camag.) Figure 10 Sprayer SG 1. (Courtesy of Desaga.)
Thin Layer Chromatography 1003
(Fig. 11)
Copyright © 2005 by Marcel Dekker
development, and for postdevelopment impregnation of
chromatograms containing fluorescent zones with a fluor-
escence enhancer and stabilizer such as paraffin. A few
detection reagents (HCl, sulfuryl chloride, iodine) can be
transferred uniformly to the layer as vapors in a closed
chamber. The preparation, procedure for use, and results
for many hundreds of TLC detection reagents have been
described (Jork, et al., 1990, 1994; Zweig and Sherma,
1972).
A variety of biological detection methods are available
for compound detection. As an example, immunostaining
of thin layer chromatograms provides very sensitive detec-
tion of glycolipids (Putalan et al., 2001).
VIII. DOCUMENTATION OFCHROMATOGRAMS
TLC plates contain complete chromatograms that provide
a great amount of information for sample identification,
visual semiquantification, and comparison, especially
images of the same sample.
TLC separations are documented by photography,
video recording, or scanning (Fried and Sherma, 1999i;
Morlock and Kovar, 2003). Commercial systems for
photographic documentation contain a conventional,
instant, or digital (Fig. 12) camera and associated lighting
accessories for photography of colored, fluorescent, and
fluorescence-quenched zones on TLC plates in shortwave,
midrange, and longwave UV light and in visible light.
Special software is needed for reproducible, GMP/GLP-
compliant use of a digital camera.
The Camag video documentation system (VideoStore,
with zoom lens, monitor, and video color printer. The
VideoStore software is GMP/GLP-compliant for
capture, editing, annotation, documenting, and archiving
of images. Desaga offers a similar color video documen-
tation system (VD 40) with eightfold motor zoom and
autofocus CCD camera, pentium powered computer, and
ProViDoc GLP-conforming software.
Documentation can also be carried out by scanning the
spots on the plate with a flatbed office scanner (Rozylo
Figure 11 ChromaJet DS 20 automatic spray apparatus. (Courtesy of Desaga.)
Figure 12 Reprostar 3 lighting unit and camera stand with
cabinet cover and mounted digital camera. (Courtesy of Camag.)
1004 Analytical Instrumentation Handbook
Fig. 13) includes visible and UV lighting, CCD camera
when different detection methods are used to give multiple
Copyright © 2005 by Marcel Dekker
et al., 1997). The equipment required includes a computer,
scanner, and monochrome or color printer. Computer
scanning can be used only for visible spots, but not those
that are fluorescent or quench fluorescence unless the
flatbed scanner is modified.
IX. ZONE IDENTIFICATION
The identity of TLC zones (Fried and Sherma, 1999i;
Morlock and Kovar, 2003) is obtained in the first instance
by comparison of Rf values between samples and reference
standards chromatographed on the same plate, where Rf
equals the migration distance from the origin to the
center of the zone divided by the migration distance of
the mobile phase front. Identity is more certain if a selec-
tive chromogenic reagent yields the same characteristic
color for sample and standard zones, or if an Rf match
between samples and standards is obtained in at least
two TLC systems with diverse mechanisms, for
example, silica gel NP and C-18 bonded silica gel RP.
Comparison of standard and sample in situ UV-visible
absorption or fluorescence emission spectra, obtained by
using the spectral mode of a slit-scanning or video densi-
tometer, can also aid identification, but these spectra may
contain inadequate structural information for complex
mixtures.
The TIDAS TLC 2010 scanner (Flowspek AG, Basel,
Switzerland) (Fig. 14) allows rapid scanning of TLC
plates with simultaneous acquisition of a complete spec-
trum for all substances on a layer. Fiber optics technology
is combined with diode array detection in this instrument,
which has the following specifications: 190–1000 nm
wavelength range, 0.8 nm pixel resolution, deuterium
and tungsten light sources, ,160 mm optical resolution
Figure 13 Camag VideoStore/VideoScan including Repros-
tar 3 with cabinet cover, camera bellows, camera support, and
3-CCD camera with zoom objective. (Courtesy of Camag.)
Figure 14 TLC 2010 diode array scanner. (Courtesy of Flowspek.)
Thin Layer Chromatography 1005
Copyright © 2005 by Marcel Dekker
on the layer, 5 mm/s scanning speed, 0.1 mm positioning
accuracy, and software with parameters including peak
purity, resolution, and identification via spectral library
matching. Figures 15 and 16 show the identification and
confirmation of codeine in urine with the TLC 2010
diode array scanner.
Zone identity can also be confirmed by the application
of combined TLC-spectrometry methods described in
Section XI.
X. QUANTITATIVE ANALYSIS
A. Nondensitometric Methods
Quantification of thin layer chromatograms (Fried and
Sherma, 1999j; Prosek and Vovk, 2003) can be performed
after manually scraping off the separated zones of samples
and standards and elution of the substances from the layer
material with a strong, volatile solvent. The eluates are
concentrated and analyzed by use of a spectrometry, GC,
HPLC, or some other sensitive microanalytical method.
This method of quantification is laborious and time con-
suming, and the difficulty in recovering samples and stan-
dards uniformly is a major source of error. Although its
importance has declined relative to densitometry, the
indirect scraping and elution quantification method is
still being rather widely used, for example, for some
drug assays according to the US Pharmacopoeia.
Direct TLC semiquantitative analysis can be performed
by visual comparison of sample spot intensities with the
intensities of reference spots developed simultaneously
on the same layer. For this comparison, the bracketing
method is used in which standard spots with concen-
trations equal to, greater than, and less than the expected
sample concentration are placed on either side of duplicate
sample spots. The concentrations of samples and standards
should lie within the linear response range of the detection
method. The use of TLC for compliance screening of drug
products is an important example of this approach.
Figure 15 Contour plot, densitogram, and codeine spectrum on one screen shot, with codeine appearing at 13.6 mm. (Courtesy of
Flowspek.)
Figure 16 Codeine spectrum (top) and library spectrum
(bottom) with a match of 98%. (Courtesy of Flowspek.)
1006 Analytical Instrumentation Handbook
Copyright © 2005 by Marcel Dekker
B. Densitometric Evaluation
Most modern HPTLC quantitative analyses are performed
by in situ measurement of the absorbance or fluorescence
of the separated zones in the chromatogram tracks using an
optical densitometic scanner (Reich, 2003; Sherma,
2001b) operated with a fixed sample light beam in the
form of a rectangular slit. The length and width of the
slit is selectable for optimized scanning of spot or band-
shaped zones with different dimensions. The densitometer
measures the difference between the optical signal from a
zone-free background area of the plate and that from the
calibration standards and sample zones. With automated
zone application, precision ranging from 1% to 3% RSD
is typical for densitometric analyses.
The plate is mounted on a moveable stage controlled in
the X- and Y-directions by a stepping motor, which allows
each chromatogram to be scanned, usually in the direction
of development. The single beam, single wavelength scan-
ning mode most often used gives excellent results with
high quality plates and a mobile phase that result in
compact, well separated zones. A schematic diagram of
the light path of a densitometer is shown in Fig. 17. A
tungsten-halogen lamp is used as the source for scanning
colored zones in the 400–800 nm range (visible absorp-
tion) and a deuterium continuum lamp for scanning the
absorption of UV light by zones on layers with or
without fluorescence indicator in the 190–400 nm range.
The monochromator used with these continuous wave-
length sources can be a quartz prism or, more often, a
grating. The detector is a photomultiplier or a photodiode.
For fluorescence scanning, a high intensity xenon or
mercury vapor lamp is used as the source, the optimum
excitation wavelength is selected by the monochromator,
and a cutoff filter is placed between the plate and detector
to block the exciting UV radiation and transmit the visible
emitted fluorescence. The light beam strikes the plate at a
908 angle, and the photomultiplier for reflectance scanning
is at a 30o angle to normal. Part of the light beam is
directed to a reference photomultiplier by a beam splitter
to compensate for lamp variations and short-term fluctu-
ations. A detector mounted below the stage is used when
scanning in the transmission mode (TLC plates or electro-
phoresis gels). Zig-zag (or meander) scanning with a small
spot of light is possible with scanners having two
independent stepping motors to move the plate in the
x- and y-axes. Computer algorithms integrate the maxi-
mum absorbance measurements from each swing, which
Figure 17 Light path diagram of the TLC Scanner 3. 1, Lamp selector; 2, entrance lens slit; 3, monochromator entry slit; 4, grating
monochromator; 5, mirror; 6, slit aperture disk; 7, lens system; 8, mirror; 9, beam splitter; 10, reflectance monochromator; 11, object to be
scanned; 12, measuring photomultiplier; 13, photodiode (transmission). [Reproduced from Reich (2003) with permission.]
Thin Layer Chromatography 1007
Copyright © 2005 by Marcel Dekker
corresponds to the length of the slit, to produce a distri-
bution profile of zones having any shape. Disadvantages
of scanning with a moving light spot include problems
with data processing, lower spatial resolution for
HPTLC, and unfavorable error propagation upon aver-
aging of readings from different points within the zone.
Most modern have a computer controlled
motor driven monochromator that allows automatic
recording of in situ absorption and fluorescence excitation
aid compound identification by comparison with cochro-
matographed standards or stored standard spectra, test
for identity by superimposition of spectra from different
zones on a plate, and check zone purity by superimposition
of spectra from different areas of a single zone. The spec-
tral maximum determined from the in situ spectrum is
usually the optimal wavelength for scanning standard
and sample areas for quantitative analysis.
The scanner is connected to a recorder, an integrator, or a
computer. A personal computer with software designed
specifically for TLC is most common for data processing
and automated control of the scanning process in modern
instruments. With a fully automated system (e.g., winCATS
from Camag), the computer can carry out the following
functions: selectable scanning speed up to 100 mm/s;
evaluation of 36 tracks with up to 100 substances in
sequence; integration with automatic or manual baseline
correction; single or multilevel calibration with linear or
nonlinear regression using internal or external standards,
and statistics such as RSD or confidence interval with full
error propagation; subcomponent evaluation to relate uni-
dentified fractions to the main component, as required by
various pharmacopoeias; dual-wavelength scan to eliminate
matrix effects or quantify incompletely resolved peaks;
multiwavelength scan (up to 31 different wavelengths) to
obtain the optimum wavelength for quantification of each
fraction and achieve maximum analytical selectivity;
scanner qualification (automatic tests of mechanical,
optical, and electronic functions); track optimization
(repeated scanning of each track with small lateral offsets
in order to optimize measurements of distorted chromato-
grams); and spectrum library. For GMP compliance, all
conditions and data are automatically recorded and held
in a secure format.
Because of light scattering from the sorbent particles, a
simple, well-defined mathematical relationship between
amount of analyte and the light signal has not been
found. Plots relating absorption signal (peak height or
area) and concentration or weight of standards on the
layer are usually nonlinear, especially at higher concen-
trations, and do not pass through the origin. Modern
integrators and computer software programs can routinely
perform linear or polynomial regression of the calibration
data, depending upon which is most suitable. Fluorescence
calibration curves are generally linear and pass through the
origin, and analyses based on fluorescence are more
specific and 10–1000 times more sensitive than those
employing absorbance. Because of these advantages, com-
pounds that are not naturally fluorescent are often deriva-
tized pre- or postchromatography to allow them to be
scanned in the fluorescence mode if an appropriate rea-
gent is available. However, absorbance in the 190–300 nm
UV range has been most used for densitometric
analyses.
Validation procedures (Fried and Sherma, 1999k) for
quantitative analysis are in some aspects very similar to
those for HPLC and GC, with additional considerations
related to procedural aspects specific to TLC. Protocols
for validation of TLC results (Ferenczi-Fodor et al.,
2001), especially for pharmaceutical analysis, are available
in the literature. Some densitometers include automatic
instrument validation programs in their software.
The Camag TLC Scanner 3, Shimadzu (Columbia, MD
USA) CS 9000, and Desaga Densitometer CD 60 are
examples of modern computer-controlled slit-scanning
densitometers.
Video densitometers (image processors) are an alter-
native to the optical/mechanical slit scanners. Video
densitometry is based on electronic point-scanning of a
stationary plate using an instrument composed of UV
and visible light sources, a CCD camera with zoom capa-
bilities, and a computer with imaging and evaluation soft-
ware. Video scanners have advantages including rapid
data collection and storage, simple design with virtually
no moving parts, easy operation, and the ability to quan-
tify 2D chromatograms, but they have not yet been shown
to have the required capabilities to replace slit-scanning
densitometers. Current video scanners can function only
in the visible range to measure colored, fluorescence-
quenched, or fluorescent spots. They lack the spectral
selectivity and accuracy based on the ability to scan
with monochromatic light of selectable wavelength
throughout the visible and UV range (190–800 nm) that
are inherent in classical densitometry, and they cannot
record the in situ spectra. The VideoScan software
program (Camag) allows quantitative evaluation (video-
densitometry) of images at any time after they are
captured with the VideoStore documentation system
formed automatically, quantification performed via peak
areas or heights, and single or multilevel calibrations
with linear or polynomial regression. The ProResult soft-
ware package is available for quantification with
Desaga’s video documentation system.
Special software packages have been used for quantifi-
cation of zones on chromatogram images produced with a
flatbed scanner. Both colored (visible) (Johnson, 2000)
and fluorescent (Stroka et al., 2001) zones have been
1008 Analytical Instrumentation Handbook
spectra at multiple wavelengths (Fig. 7). These spectra can
scanners
shown in Fig. 13. Integration of analog curves can be per-
Copyright © 2005 by Marcel Dekker
measured for the analyses, the latter after modification of
the scanner by adding a black light tube.
XI. TLC COMBINED WITH SPECTROMETRICMETHODS
A. Mass Spectrometry
The identity of TLC zones can be confirmed by mass spec-
trometry (MS) analysis (Busch, 2003; Rozylo, 2001). The
most used ionization modes for TLC/MS include electro-
spray ionization (ESI), and matrix- and surface-assisted
laser desorption ionization (MALDI and SALDI).
ESI is used with solvent extracts of zones scraped from
the TLC plate. The liquid is sprayed through a charged
needle as an aerosol mist at atmospheric pressure, and
the ions created from desolvation and charge distribution
processes in the droplets are extracted through skimmer
cones into the mass analyzer of the mass spectrometer.
MALDI and SALDI involve ionization directly from
the surface layer held under vacuum after addition of an
energy-buffering matrix. The ionization occurs as a
result of surface irradiation by a laser beam, with mass
analysis usually carried with a time of flight (TOF) mass
analyzer.
On-line TLC/ESI–MS has been carried out by directly
(Beverly, MA USA) Q-TOF mass spectrometer (Chai
et al., 2003). Aluminum-backed silica gel layers with a
perimeter seal were used. After initial layer precondition-
ing to reduce background noise, the limit of detection of
glycolipids was in the 5–20 pmol range.
Quadrupole, ion trap, and Fourier Transform (FT) mass
spectrometers and fast atom bombardment and liquid sec-
ondary ion mass spectrometry ionization techniques have
also been used in various applications of TLC/MS.
B. Infrared Spectrometry
Zones can also be confirmed by combining TLC with
infrared (IR) spectrometry (Morlock and Kovar, 2003).
Indirect TLC–IR coupling is carried out by transfer of
the sample from the layer to an IR-transparent pellet or
powder such as KBr or in situ measurement of scraped
TLC zones by the diffuse reflectance infrared Fourier
transform spectrometry (DRIFT) technique.
TLC has been directly coupled with DRIFT, in which
case difficulties occur because conventional stationary
phases, for example, silica gel, absorb strongly in the IR
region, and the influence of the refractive index on the
spectra must be considered. Intense interference bands
between 1350 and 1000 cm21 and above 3550 cm21 are
superimposed on the DRIFT spectra of the analytes and
restrict the available wavenumber range. In addition,
the large active surface area of silica gel, with its hydroxyl
groups, makes adequate compensation of sample and
reference spectra more difficult. It has been found that
HPTLC plates with 10 mm particles, small particle size
distribution, 0.2 mm layer thickness, and glass backing
are best for direct TLC–DRIFT. A Brucker IFS 48 FTIR
spectrometer with a special mirror arrangement and
MCT (mercury, cadmium, telluride) small band detector
has been constructed to enable DRIFT measurement
despite the self-absorbance of TLC sorbents (Glauninger
et al., 1990).
Although mainly useful for qualitative identification
and confirmation of zones, quantification by TLC–IR
has been carried out in some applications by evaluation
of Kubelka–Munk spectra with integration of their
strongest bands, especially for substances lacking chro-
mophores that absorb in the UV-visible range. However,
the limit of determination is about 10 times higher than
that of densitometry, and precision is poorer.
C. Raman Spectrometry
Surface-enhanced Raman scattering (SERS) spectrometry
(Morlock and Kovar, 2003; Somsen et al., 1995) has sen-
sitivity in the nanogram or the picogram range using a
Raman spectrometer with argon ion, HeNe, or YAG
monochromatic light source and CCD detector. The
method is most useful for identification of compounds
with groups of atoms that are IR-inactive; quantitative
analysis has not been successfully carried out.
For in situ analysis, the HPTLC plate, after develop-
ment and drying, is dipped into or sprayed with a colloidal
silver suspension prepared by reduction of silver nitrate
with sodium citrate. Alternatively, the silver molecules
can be evaporated onto the layer in order to eliminate
the zone diffusion and lowering of enhancement caused
by dipping or spraying.
Highly Raman-active substances (dyes, optical bright-
eners) can be determined at low nanogram levels without
surface-enhanced scattering on specially modified silica
gel plates. EMD chemicals Inc. (Gibbstown, NJ, an
affiliate of Merck KGaA) sells silica gel 60 plates with a
0.1 mm layer of 3–5 mm particles on an aluminum
support. The spherical silica gel produces a 10-fold
increase in Raman spectrometry signal intensity compared
with a similar layer made with irregular silica gel particles.
XII. PREPARATIVE LAYERCHROMATOGRAPHY
PLC for isolation of larger amounts of material than nor-
mally separated by TLC can be carried out by classical
PLC (Fried and Sherma, 1999l) by use of thicker layers
Thin Layer Chromatography 1009
linking an OPLC 50 instrument (Fig. 8) and a Micromass
Copyright © 2005 by Marcel Dekker
(usually 0.5–2 mm) developed in the ascending direction
in a large volume chamber with detection of zones by a
nondestructive method such as iodine vapor or fluor-
escence quenching. Fractions are scraped from the layer,
and the purified analytes recovered by elution with a
solvent for use in other laboratory work or further analysis.
The construction and use of the commercial instru-
ments available for forced flow PLC have been described
by Nyiredy (Nyiredy, 2003). These include the older
Chrompres 10 and 25 chambers (LABOR Instrument
Works, Budapest, Hungary) and Personal OPLC 50
system (Section VI.B, capable of semipreparative separ-
ations only) for OPLC and the Chromatotron CLC-5,
Rotachrom, and Extachrom for RPC.
An additional commercial instrument for rotation or
centrifugal preparative planar chromatography is the
CycloGraph II from Analtech (Newark, DE; Fig. 18). Sep-
arations typically occur within 20 min without need to
scrape off the separated zones. The sample solution is
applied using a solvent pump or hand-held syringe inside
the adsorbent ring of a precast rotor. The eluent (mobile
phase) is pumped through the rotating layer (variable
speed control from 100 to 1400 rpm), and as each separ-
ated ring reaches the outer rim of the rotor it is spun off
of the edge of the glass into a circular trough. The adjusta-
ble angle of the trough allows the eluent to settle at the
bottom and drip out of the collection port into individual
tubes for different fractions. High performance, reusable
silica gel rotors (9.5 in diameter) with 1000–8000 mm
thickness are available.
XIII. THIN LAYER RADIOCHROMATOGRAPHY
Location and quantification of separated radioisotope-
labeled substances on a thin layer requires the use of
contact autoradiography, zonal analysis, or direct scanning
with a radiation detector. These thin layer radiochromato-
graphy (TLRC) methods (Fried and Sherma, 1999m;
Hazai and Klebovich, 2003) are especially important in
drug and pesticide metabolism studies in plants, animals,
and humans and in studies of the fate of labeled chemicals
in the environment.
Contact autoradiography involves exposure of X-ray or
photographic film to emissions from radioisotope zones to
produce an image on the film. After exposure and develop-
ment of the film, the radioisotopes are visible as dark spots,
which can be compared with standards for qualitative
identification or quantified by measurement of their
optical densities with a scanning densitometer.
Zonal analysis involves scraping radioactive zones
from the plate, placing the sorbent in counting vials,
adding scintillation fluid or cocktail to elute the radio-
active components, and liquid scintillation counting.
Direct measurement of radioactive zones on a layer is
most often carried out today by use of a linear analyzer,
digital autoradiograph (DAR), or an imaging analyzer.
Linear analyzers incorporate a position-sensitive window-
less gas-flow proportional counter as the detector, which
measures all radioactive zones in a chromatogram (track)
simultaneously and then is moved under computer
control for measurements at other selected positions
across the layer. The fill gas (e.g., argon–methane) is
ionized when radioactive emissions from the TLC zones
enter the detector, producing electrons. The electron
pulses are detected electronically and stored in computer
memory to provide a digital image of the distribution of
radioactivity on the layer. An example of a TLC linear
which has a gold plated detector with an active length of
200 mm and an active width selectable from 20 to 1 mm
by choice of the diaphragm.
Multiwire proportional counters (DAR or microchannel
array detector) are 2D detectors based on a measuring
principle similar to the linear analyzer, but they can
detect all areas of radiation from a 20 � 20 cm layer
simultaneously without moving a detector head. Many
research studies have been reported on applications of
HPTLC and OPLC coupled with an LB 287 Berthold
DAR (EG&G Berthold, Wilbad, Germany) for analysis
of tritiated or 14C-labeled metabolites in a variety of
Figure 18 CycloGraph II centrifugal preparative planar
chromatography instrument with built-in UV lamp and hinged
lid. (Courtesy of Analtech.)
1010 Analytical Instrumentation Handbook
analyzer is the RITA (Raytest, Wilmington, NC; Fig. 19),
Copyright © 2005 by Marcel Dekker
biological matrixes. This instrument has a 20 � 20 cm
sensitive area that contains a 600 � 600 wire grid.
Measurements are made using argon–methane (9:1) as
counting gas bubbled through methylal at 2.88C and a
flow rate of 5 mL/min. The positive high voltage potential
used for 3H- and 14C-labeled compounds is 2040 and
1200 V, respectively, and signal analysis is achieved by
measuring 5 � 360,000 detector cells/s.
Bioimaging/phosphor imaging analyzers represent the
newest and most advantageous technology for measuring
radioactive TLC zones. The layer is exposed to a phosphor
imaging plate (IP) that accumulates and stores irradiating
radioactive energy from the zones. The plate is then
inserted into an image-reading unit and scanned with a
fine laser beam. Luminescence is emitted in proportion
to the intensity of the recorded radiation, collected by a
photomultiplier tube, and converted to electrical energy
to produce the instrumental readout. Resolution, linear
range, and sensitivity are equal to, or better than, those
of the other detection methods. The BAS 5000 (Raytest)
(Fig. 20) is an example of a modern phosphor IP TLC
scanner. Specifications include 20 � 25 cm IP size, 25/50 mm pixel size, 5 min (50 mm) reading time, detection
limit 0.9 dpm/mm2/h (14C), and four to five orders of
magnitude (16 bits) dynamic range.
XIV. APPLICATIONS OF TLC
TLC can provide rapid, low cost qualitative analyses and
screening in order to obtain information such as sample
stability, purity, and uniformity and to follow the course
of a reaction, whereas instrumental HPTLC can provide
accurate and reproducible (1–3% RSD) quantitative
results. Samples that are difficult to prepare can be ana-
lyzed readily, and detection is especially flexible in the
Figure 20 BAS 5000 phosphor IP scanner. (Courtesy of Raytest.)
Figure 19 RITA radioactivity intelligent thin layer analyzer. (Courtesy of Raytest.)
Thin Layer Chromatography 1011
Copyright © 2005 by Marcel Dekker
absence of the mobile phase and with a variety of
parameters.
TLC has been applied virtually in all areas of analysis,
including chemistry, biochemistry, biology, industrial, agri-
cultural, environmental, food, pharmaceutical, clinical,
natural products, toxicology, forensics, plant science, bac-
teriology, parasitology, and entomology. Table 1 lists the
compound classes that are covered in three books that
contain detailed information on specific applications
of TLC analysis, discussion of which is beyond the scope
of this chapter. The applications of TLC, as well as
theory, techniques, and instrumentation, is regularly
updated in a biennial review of planar chromatography
(Sherma, 2004) and the Camag Bibliography Service,
which is published every March and September and is avail-
able in paper and CD-ROM format free of charge.
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combustion aerosols by supercritical fluid extraction
coupled to thin layer chromatography. Mikrochim. Acta
113(3–6):373–379.
Ferenczi-Fodor, K., Vegh, Z., Nagy-Turak, A., Renger, B.,
Zeller, M. (2001). Validation and quality assurance of
planar chromatographic procedures in pharmaceutical analy-
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Table 1 Sources of Information on the TLC Analysis of Different Compound Classes
Fried and Sherma
(1999n)
Sherma and Fried
(2003) Cazes (2001)
Amino acids � � �
Antibiotics � �
Carbohydrates � � �
Carboxylic acids �
Ceramides �
Coumarins �
Dyes � � �
Enantiomers � �
Indoles �
Inorganics �
Lipids � � �
Nucleic acid derivatives � �
Organometallics �
Peptides and proteins �
Pesticides � �
Pharmaceuticals and drugs � �
Phenols � �
Pigments � � �
Plant extracts �
Steroids � � �
Taxoids �
Terpenoids �
Toxins � �
Vitamins � � �
Note: � indicates the book contains a chapter on the compound class.
1012 Analytical Instrumentation Handbook
Copyright © 2005 by Marcel Dekker
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1014 Analytical Instrumentation Handbook
Copyright © 2005 by Marcel Dekker
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