TLC

30

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


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


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.)



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.)



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.)


carried out in an AMD instrument (Fig. 6), which includes


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.)


scan shown in Fig. 7 illustrates a high-resolution chroma-

Section VI.B)


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.]



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.)


(Fig. 11)


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.)


Fig. 13) includes visible and UV lighting, CCD camera

when different detection methods are used to give multiple


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.)



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.)



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.]



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


spectra at multiple wavelengths (Fig. 7). These spectra can

scanners

shown in Fig. 13. Integration of analog curves can be per-


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


linking an OPLC 50 instrument (Fig. 8) and a Micromass


(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.)


analyzer is the RITA (Raytest, Wilmington, NC; Fig. 19),


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.)



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.

REFERENCES

Abjean, J. P. (1993). Screening of drug residues in food of animal

origin by planar chromatography: application to sulfona-

mides. J. Planar Chromatogr.-Mod. TLC 6(2):147–148.

Bariska, J., Csermely, T., Furst, S., Kalasz, H., Bathori,

M. (2000). Displacement thin layer chromatography. J. Liq.

Chromatogr. Relat. Technol. 23(4):531–549.

van Beek, T. A. (2002). Chemical analysis of Ginko biloba

leaves and extracts. J. Chromatogr. A 967(1):21–55.

Busch, K. L. (2003). Thin layer chromatography coupled with

mass spectrometry. In: Sherma, J., Fried, B., eds. Handbook

of Thin Layer Chromatography. 3rd ed. New York: Marcel

Dekker, Inc., pp. 239–275.

Cazes, J., Ed. (2001). Encyclopedia of Chromatography.

New York: Marcel Dekker, Inc.

Chai, W. G., Leteux, C., Lawson, A. M., Stoll, M. S. (2003).

On-line overpressure thin layer chromatographic separation

and electrospray mass spectrometric detection of glycolipids.

Anal. Chem. 75(1):118–125.

Cimpoiu, C. (2003). Optimization. In: Sherma, J., Fried, B., Eds.

Handbook of Thin Layer Chromatography. 3rd ed. New York:

Marcel Dekker, Inc., pp. 81–98.

Esser, G., Klockow, D. (1994). Detection of hydroperoxides in

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-

sis. J. AOAC Int. 84(4):1265–1276.

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.



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