C HROMATOG R A PHY Colin F. Poole and Salwa K. Poole
Department of Chemistry Wayne Slate University Detroit. MI 48202
Thin-layer chromatography (TLC) as practiced in the 1980s is very much an instrumental technology. TLC methods are most effective for low-cost analysis of simple mixtures when the sample workload is high, for rapid analysis of simple mixtures requiring minimum sample cleanup, for analysis of samples containing components that remain sorbed to the separation medium or contain suspended microparticles, and for analysis of substances with poor detection characteristics that require postchromatographic treatment for detection. In this REPORT we will introduce some of the important modern devel-
REPORT opmenta of TLC that will make the technique useful in the next decade and defme the scope and limitations of TLC aa an analytical tool. Rather than claim that TLC should replace HPLC aa a separation tool, we will attempt to show that the two techniques are complementary and that the experienced analyst should have both techniques at his or her disposal for problem solving and routine analysis. Modern TLC originated in the mid1970s with the commercial introduc0003-2700189/A361-1257/$01.50/0 @ 1989 American Chemical Society
tion of fine-particle layers optimized for fast and efficient separations with controlled sample application ( I ) . Table l compares these small-particle plates with conventional TLC plates. The tightness of the particle size distribution is just as important as the decrease in actual particle size in the performance of the new plates (2).In many cases, they were not well received when first introduced. Too often, they were used with methods considered more appropriate for conventional plates and were overloaded with sample and developed for too long a migration length, which resulted in longer separa-
Table 1.
tion times and poorer resolution than for conventional plates. Table I shows that these new layers require smaller sample sizes and shorter migration lengths to reveal their true separation potential and to deliver large savings in analysis time; better resolution; and, because spots are more compact and the optical properties of the layer more favorable for in situ detection, much better detection limits. Today, many types of fine-particle layers are available, including silica, bonded phases, and cellulose. In fact, just about all phases used for normal and reversed-phase HPLC are
Comparison of comrentionel and modem lLC
Plate size (an)
20 x 20
lox 10
Layer thickness @m) Particle SIZE (run) Average
100-250
10x20 200
Oistribuikm
sample volume bIL) Starting spot diameter (mm) oiameter of separated spots (mm)
Solvent migratim distance (cm) Time la development (mln)
20 10-60 1-5
3-6
Between5d15 "OW
0.1-0.2 1.0-1.5 2-6
515 10-15
3-6
30-200
3-20
1-5 50-100 10
0 1-0.5 5-10 18-36
Detection llmlts
Absorption (ne) Fluorescence (pg) Sample lanes per platea
ANALYTICAL CHEMISTRY, VOL. 61. NO. 22. NOVEMBW 15, 1989
12571
REPOR= available for TLC (3-5),and bonded chiral phases for the separation of enantiomers have been introduced recently (6-8). Theory Separations by TLC are usually achieved using the development mode with the migration velocity of the mobile phase controlled by capillary forces. For fme-particle layers it can be assumed that spot broadening in TLC is controlled entirely by molecular diffusion, resulting in a series of symmetrical, compact spots increasing uniformly in diameter with increasing migration distance (9, IO). For coarseparticle layers, contributions to the plate height from slow mass transfer can be significant and the spots can become distorted in the direction of migration. We will ignore this tendency and assume that the performance of both types of layers can be calculated assuming a Gaussian distribution of the sample within the spot according to
where n is the number of theoretical plates, Z. the distance migrated by the sample (spot) from its origin, W. the spot diameter, Rjis the retardation factor (ZJZj), and Z, is the distance moved by the mobile phase from the sample origii to the solvent front. Separated components migrate different distances through the layer; their zones are broadened to different extents and, therefore, the efficiency of the TLC plate is only constant for a specific migration distance. This differs from column chromatography using the elution mode and can he confusing when one compares the efficiency of TLC and column systems. By convention, the efficiency of a thin-layer plate is measured or calculated for a substance having an Rjvalue of 0.5,l.O. or some average value (IO). If the vapor phase in contact with the layer has attained equilibrium, the speed with which the mobile phase moves through the layer when governed by capillary forces will be described by
and the velocity constant K by
where KOis the permeability constant, dpis the average particle diameter, y is the surface tension of the mobile phase, q is the viscosity of the mobile phase, 8 is the contact angle, and t is the time 1258A
(11, 12). Experimental values for KO tend to vary more widely than reported values for columns, for which a value of 1X 10-3 is generally considered to be a good approximation. Values for TLC tend to be somewhat higher, around 8 X 10-3; thus, the packing density may be higher in columns than that observed for layers (13).Assuming a narrow particle-size distribution, the velocity constant should increase linearly with the average particle size and thus the solvent front velocity is greater for coarse-particle layers. The velocity constant also depends linearly on the ratio of the surface tension of the splvent to its viscosity, and solvents that maximize this ratio (and not iust outimize one of the parametersj are preferred for TLC (13). Althoughfor silicagellayers the coutact angle for all common solvents is close to zero, for reversed-phase layers containing bonded, long-chain alkyl groups the contact angle of the solvent increases rapidly with increasing water content of the mohile phase and, a t about 3040% (v/v) water, cos 8 becomes smaller than 0.2-0.3 (4, ll).The mobile phase is virtually unable to ascend the thin-layer plate, and chromatography becomes impossible. To improve solvent compatibility, modern reversed-phase TLC plates are prepared from larger size particles (10-13 pm) than other high-performance plates or from sorbents with a defined degree of modification that is lower than that of exhaustively silanized sorbents. Plates with lower degrees of modification can be used without any solvent composition restrictions, and pure water may also he used. Solvent compatibility is much less of a problem for polar, bonded sorbents such as 3aminopropylsilanized and 3-cyanopropylsilanized sorbents, which are wetted by all solvents including pure water. If the mobile-phase velocity is controlled extemally, as in forced-flow TLC, then the restrictions imposed by using capillary action for solvent migration are removed. The mobile-phase velocity can be controlled and optimized independently of the solvent migration distance and is no longer dependent on the contact angle. Two approaches to forced-flow development have been used (14). In rotational planar chromatography, centrifugal force generated by spinning the sorbent layer about a central axis is used to drive the solvent through the layer (15).The rate of solvent migration is a function of the rotation speed and the rate a t which the mobile phase is supplied to the layer. Because the layer is not enclosed, the ultimate velocity of the solvent front is limited by the amount of
ANALYTICAL CHEMISTRY, VOL. 61. NO. 22, NOVEMBER 15. 1989
solvent that can be kept within the layer without floating over the surface. At high rotation speeds the migration of the solvent front as a function of time becomes approximately constant in the linear development mode. If the sorbent layer is sealed (using a flexible membrane or an optically flat rigid surface under hydraulic pressure) and the mohile phase is delivered to the layer by a mechanical pump, the mohilephase velocity can he controlled and optimized by adjusting the output from the pump feeding the mobile phase to the layer. For a pump with a constant volume delivery using linear development mode, the migration of the solvent front will be a linear function of time. Figure 1 demonstrates the importance of using an optimal and constant mobile-phase velocity. Under capillary flow-controlled conditions with fineparticle layers, the average plate height first passes through a minimum and then increases sharply for longer migration distances. For coarse-particle layers the plate height is less dependent on the migration distance and eventually the two curves cross, indicating that a greater number of theoretical plates can be obtained by using coarse-particle layers and long migration distances. This contrary finding is easily explained by the relative permeability of the layers. The mobile-phase velocity for the fine-particle layer declines rapidly with the migration distance until it becomes so slow that diffusion causes the spots to broaden at a rate faster than the spot centers are migrating through the layer. The coarse-particle layer is more permeable than the fineparticle layer, and both the solvent velocity and the efficiency are higher a t longer plate lengths. For fine-particle layers with a development length of 5-7 cm it should be possible to obtain up to about 5000 theoretical plates, but it is nearly impossible to exceed this number using capillary flow-controlled development. For coarse-particle layers ( d , 15pm), a development length of about 15 cm is required to obtain around 5ooo theoretical plates and, although higher numbers are possible, they will lead to long separation times. Fine-particle layers are preferred for capillary flow-controlled conditions because they provide faster separations; separations that can be achieved only with more than 40005000 theoretical plates are too difficult to be worthwhile. In forced-flowTLC the average plate height is largely independent of the migration distance and is more favorable for fine-particle layers than coarse ones
-
Conventional TLC plate, normal development
Figure 2. Change in resolution of two closely migrating spots as a function of the R,value of the faster moving spot.
\
Conventional TLC plate, forced-flowdevelopment
(Adapted with p~rmls6ion (mm RefwnDB 2.)
HPTLC plate, forced-flow development
Flgm 1. Variation of the efficiency(average plate height) of fine- and coarseparticle layers as a function of migration distance and development technique. (Adapted with permiMbn bun RBlwence 2.)
(Figure 1). The quadratic decrease in linear velocity with time is defeated, and an optimum constant linear velocity-similar to that for column chromatography-leads to the optimum plate height (IO, 16). Compared with capillary flow-controlled systems, zone broadening by diffusion is now minor even for long migration distances becaw the optimum mobile-phase veloci@ is always higher than that observed for ascending development by capillary flow. Commercially available TLC layers and equipment make it possible to achieve an upper limit of about 31,000 plates (Zf = 25 cm),but it will be difficult to exceed this value. However, this limit is not theoretical; the ultimate efficiency of the forcedflowsystem is limited only by the particle size and homogeneity of the bed, the
available bed length, and the pressure required to maintain the optimum mobile-phase velocity. The object of any chromatographic analysis is to rapidly obtain a certain resolution between individual components of a mixture. To control resolution we must know how it varies with experimental parameters such as the layer efficiency,the ratio of the equilibrium constants governing the separation process, and the position of the zones within the chromatogram (17). This variation can he expressed approximately by Equation 4 for a single unidimensional development under capillary flow-controlledconditions:
- 4[(k,/kJ\InrR, - 4 [ 1 - RR]
R -
ANALYTICAL
(4)
where Rs is the peak-to-peak resolution, nf is the number of theoretical plates for a zone migrating with the solvent front, and k is the capacity factor [(l - R,)/Rfj. The subscripts 1and 2 refer to the individual zones numbered such that the larger number corresponds to the zone with the highest R, value. The variation of resolution with R, is not a simple function (see Figure 2). The resolution increases with the square root of the layer efficiency, which depends linearly on the R, value. The influence of the position of the zones in the chromatogram on resolution shows the opposite behavior to that of the layer quality. At greater values of Rp,the term 1- R p will decrease and the resolution will become zero at a value of R p = 1. Differentiation of Eguation 4 indicates that the optimum resolution of two closely migrating zones will occur a t an Rf value of about 0.3. Figure 2 indicates that the resolution does not change significantly for R, values between 0.2 and 0.5; within this range, the resolution is greater than 9% of the maximum value. This Rf region is thus the target zone for solvent selection to achieve the optimum resolution of the most difficult solute pair in a mixture. TLC separations generally a n be optimized by increasing the selectivily of the system because relatively small changes in selectivity make it much easier to obtain a given separation. The total number of theoretical plates that can be made available for a separation cannot be increased sufficiently t o make kinetic optimization the most worthwhile approach to improving resolution. In ,planar chromatography, separations are easy when (Rfi R p ) is greater than 0.1 and very difficult or impossible when ( R p Rfi) is less than or equal to 0.05 in the region of the
-
-
CHEMISTRY, VOL. 61, NO. 22. NOVEMBER 15, 1989
125DA
REPORT
Migi Figure S. Separation of a mixhlre of PTHgmino acid derivativt concentration mechanism used to ccmt~olzone broadening.
optimum R, value for the separation. Window diagrams, simplex methods, statistical design, and the prisma method have emerged in the past few years as powerful solvent optimization strategies for TLC (18-20). These methodological guides and computational techniques minimize the numher of trial-and-error experiments required to identify the most valuable solvent systems for a separation. Under forced-flow conditions there is no maximum obtained for resolution as a function of migration distance (16).In this case, the resolution continues to increase with increasing migration distance; the upper limit is estahliihed by practical constraints such as the available sorbent length, acceptable separation time, and the inlet presswe required to maintain the optimum mobile-phase velocity. A popular approach for improving resolution under capillary flow-controlled conditions is to use multiple development (21-23).Either unidimensional or two-dimensional separations are poasible in planar chromatography. In unidimensional multiple development, the TLC plate is developed for some selected distance or time (continuous development), the plate is withdrawn from the developing chamber 12001
rnidimensior
and the adsorbed solvent evaporated, and the plate is returned to the developing chamber and the development process repeated. This versatile strategy for separating complex mixtures all o w variation among the primary experimental components: plate length (or time), composition of the mobile phase for each development, and the number of developments. Another unique feature of the multiple development process is the wne reconcentration mechanism, illustrated in Figure 3, which enhances the layer efficiency by counteracting the zone diffusion broadening mechanism. Each time the solvent front traverses the stationary phase, it compressesthe spot in the direction of development. Initially the spot will he round, but gradually it will hecome more oval shaped until-if a sufficiently large numher of developments are used-it w i l l be compressed to a thin hand. The compression occura hecause the mobile phase first contacts the bottom edge of the spot where the sample molecules start to move forward before those moledes that are still ahead of the solvent front. Once the solvent front has reached beyond the spot, the reconcentrated spot migrates and is broadened hy diffusion in the usual way. It is thus possible to
ANALYTICAL CHEMISTRY. VOL. 61, NO. 22. NOVEMBER 15, 1989
listancl
levelopment, Illustrating the spot re
migrate a spot a substantial distance without significantlychanging the wne dimensions in the direction of migration. One disadvantage of multiple development is that for samples requiring a largenumber of theoretical plates (long migration length) a lot of time is wasted while the solvent level reaches the level of the lowest spot on the plate. For capillary controlled-flow conditions, the mobile-phase velocity may no longer he adequate to maintain optimum separation conditions. A solution to this problem is to position the solvent entry point higher on the plate at each development step (24, W), thereby increasing efficiencyabout eightfold over that of conventional development. This method can be accomplished within a similar separation time by using 10 development steps. A theoretical framework for multiple development would be very complex and has only been solved for repetitive development without change of solvent or migration distance (17). kltidimensiara~ and muftlmodalnc In two-dimensional TLC the sample is spotted at the comer of the layer and developed along one edge of the plate. The solvent is then evaporated and the
plate rotated 90° and redeveloped in the orthogonal direction. If the same solvent is used for both developments, only a slight increase in resolution can be anticipated (a factor of $2). This increase will correspond to the increased migration distance for the sample. More efficient separation requires the resolved sample to be dmtributed over the entire plate surface, necessitating complementary separation mechanisms for the orthogonal development that may be challenging to achieve. Difficulties in scanning two-dimensional chromatograms with slit-scanning densitometers and in the construction of a two-dimensional forcedflow instrument have prevented twodimensional TLC from reaching its true potential as a separation tool. The potential of a chromatographic system to provide a certain separation can be estimated from its separation number, referred to as the spot capacity in TLC (i.e., the number of spots resolved with a resolution of unity that can he placed between the sample spot at the origin and the spot of an unretained compound) (17,26). An exact spot capacity value is more difficult to calculate than the equivalent value for a column system because it deuends on , many experimental variables-that are ’ not well understood. For a single, unidimensional development using capillary flow-controlled conditions, it is not difficult to achieve a spot capacity between 10 and 20, but it is very difficult to reach 25 and practically impossible to exceed 30. To obtain separation numbers greater than 25, very long plates and prohibitively long separation times would be needed. (In HPLC, separation numbers around 150 can be achieved; in exceptional cases, a value of 500 can be obtained.) In theory, the spot capacity of forcedflow TLC should he identical to that of column chromatography. In practice, however, shorter bed lengths and limited inlet pressures compared with thwe used in column chromatography set a practical limit of about 80 with commercially available equipment (with a plate length of 30 cm, a particle size of 5 pm, and a pressure drop of 36 atm). For two-dimensional TLC under capillary controlled-flow conditions i t should be easy to achieve a spot capacity of 1W250, but it will be difficult to reach 400 and nearly impossible to exceed 500 except in very favorable circumstances. If forced-flow development is used in the first direction and elution in the second, a spot capacity of a few thousand is achievable. This represents an order of magnitude increase in separation potential over column systems, but the experimental difficul- I
ties in implementing such a separation system are formidable (27). The confident analysis of moderateto-complex mixtures requires a large separation capacity that is most readily obtained by using multidimensional and multimodal separation techniques (21,23). In multimodal separations two different separation techniques are combined via an interface that ideally allows independent and optimized use of the two separation techniques. Examples of multimodal techniques include GCA’LC, HPLCmLC, and SFCI TLC. Spectroscopic techniques such as IR, Raman, and mass spectrometry can be used in alternative multimodal approaches in which partially separated sample zones are further resolved based on differences in their spectroscopic properties (23, 28). The TLC plate can function as a storage medium, enabling spectroscopic evaluation that is free of time constraints and timeconsuming techniques such as signal averaging to improve sensitivity. Interface requirements for coupling microcolumn HPLC or SFC to TLC are comparatively simple. With microbore HPLC columns that operate a t flow rates of 5 1 0 0 pLlmin, the complete
column effluent can be deposited on the TLC plate using a modified sprayjet band applicator (29,30). For example, Jaenchen used this approach to separate a mixture of 56 pesticides by a combination of reversed-phase microbore HPLC and normal-phase automated multiple development (31). More than a decade ago, Stahl(32) described an apparatus for supercritical fluid extraction (SFE) and deposition onto a TLC plate, which used a very fme heated capillary (2550 pm) as the interface. Decompression of the supercritical fluid occurs with rapid cooling that favors the deposition process without inhibiting the evaporation of the decompressed fluid. The current strong interest in SFE should encourage reevaluation of Stahl’s early work, particularly if spectroscopic evaluation for structural identification is required. The TLC plate in this case is a much more convenient fluid elimination interface for spectroscopy than some of the on-line approaches currently under development. Certain spectroscopic techniques such as lowtemperature fluorescence line-narrowing spectroscopy, surface-enhanced Raman spectroscopy, and liquid sur-
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