High Performance Thin-Layer Chromatography The suggestion that thin-layer chromatography can provide quantitative results comparable or even superior to those obtained with column techniques would appear quite implausible to most practitioners of chromatographic analysis. Not that TLC is without merit as a separation technique, but its established utility has been as a simple, bench-top procedure for quick, qualitative examinations, or even as a clean-up operation used in preparing samples for other chromatographic determinations. Perhaps it is this very simplicity that has led many to contend that quantitative TLC is a contradiction in terms, particularly when the separated components are measured in situ. After all, there would seem to be a certain element of imprecision associated with determining the amount of material in a volume as undefined as a spot on a thinlayer plate. That TLC is limited to, at most, semiquantitative determinations is nevertheless a misconception, for recently TLC has begun to attract attention as a highly sensitive, precise, and extremely rapid technique with potential application in nearly every area of analytical interest. This has not been the result of any specific breakthrough in instrumentation or materials, but is the culmination of improvements in practically all of the operations of which TLC is comprised. The manifest increase in performance over that obtained in the usual practice of TLC has led to the adoption of "HPTLC" to describe this system, paralleling similar usage in liquid column chromatography to distinguish HPLC from its less instrument-oriented predecessor. Comparisons of HPTLC and HPLC are inevitable because there are many areas where either of these systems has application. The strengths of column chromatography are well-recognized, as witnessed by its enormous popularity, but its weaknesses are often tolerated in the belief that no better alternatives exist, or that the shortcomings will eventually yield to refinement of instrumentation. HPTLC, however, repre2 5 2 A · ANALYTICAL CHEMISTRY, VOL. 5 3 , NO. 2, FEBRUARY
sents a totally different approach, and as such, avoids many of the problems inherent in column chromatography. The principal difference between column and thin-layer chromatography, as described by many authors, is simply that one is a "closed" system and the other is "open," and this obvious distinction is the basis of the respective advantages of either chromatographic process. Gas chromatography must by the very nature of the mobile phase operate within a closed system ; consequently, there is little point in including it in further discussion. But where the mobile phase is liquid, the chromatographic process can function in either a "closed" column or on an "open" thin-layer plate. It is understandable that there has been more activity in applying column liquid chromatography to quantitative analysis because of the availability of technology previously established in gas chromatography. The operations of sample introduction, chromatographic separation, and detection are performed in exactly the same way in HPLC as in GC except for those modifications necessary to accommodate a liquid rather than a gas as the mobile phase. That is, the sampling is sequential, the separated components are eluted with the mobile phase, and detection is a dynamic timedependent process. With thin-layer chromatography these operations are totally different (Table I) : Many samples may be introduced and run simultaneously, the separation is by development (the components remain in the chromatographic bed), and the detection is static (independent of time) . If HPTLC is to be preferred in any given analytical situation over established column techniques, these differences in operation could be clearly advantageous. While this report is not intended as a polemic as 1981
0003-2700/81/0351-252A$01.00/0 © 1981 A m e r i c a n Chemical Society
Report David C. Fenimore Clarke Analytical Systems P.O. Box 744 Sierra Madre, Calif. 91024
Chester M. Davis Texas Research Institute of Mental Sciences 1300 Moursund Ave. Houston, Tex. 77030
to whether H PLC or HPTLC is the superior system, a discussion of benefits that derive from open chromatography is in order. HPTLC Operations Simultaneous Sample Development. A matter of some controversy, which may not be resolved to everyone's complete satisfaction for quite some time, is the relative efficiency of HPTLC compared to HPLC. A column system can operate under rather extreme conditions of pressure differential across the chromatographic bed, and consequently the length of that bed is not as restricted as it is with HPTLC, where mobile phase movement is determined solely by capillary action. HPLC is therefore capable of generating more total theoretical plates than HPTLC and may even produce a given separation in less time than HPTLC. However, on a sample-for-sample basis, HPTLC is clearly superior to HPLC in rapidity of analysis. This derives simply from the capability an open chromatographic bed has for permitting the separation of many samples at the same time. Although this feature of TLC is well-known, the exent to which simultaneous development in HPTLC can contribute to separation speed is perhaps less well appreciated. For example, it is often sufficient to carry the development on an HPTLC plate no more than 3 or 4 cm in order to achieve satisfactory separation, and this can be accomplished in 5 min, more or less, depending on the development solvent. A 10-by-20-cm HPTLC plate will readily accommodate 30 samples along each 20-cm edge, and by using a developing chamber in which the solvent travels concurrently from opposite edges toward the center, 60 samples can be developed simultaneously. This amounts to only 5 s per sample ! These few seconds per sample do not include the time required for sample application or quantitation of resolved components,
but improved methods of sample spotting and rapid densitometer scanning add very little to the analysis time of an individual sample. Development Chromatography. Although it is possible to utilize development chromatography in a column system, it is of little practical importance because of the inaccessibility of the chromatographic bed. On the other hand, elution chromatography can be used with thin-layer plates but would serve little useful purpose because development chromatography as an open system carries with it several important advantages. By far the greatest number of chromatographic analyses are concerned with the measurement of only one or two components of a sample mixture (together with appropriate internal standards). With development chromatography the conditions are optimized for resolution of only the components of interest, while the remainder of the sample material is left at the origin or is moved away from the region of maximum resolution. This results in a considerable saving of time compared to elution chromatography, where every component of the sample must traverse the length of the chromatographic bed in order to be measured. The selection of a solvent system to accomplish the required separation may involve a number of trials, but the choice of solvent is quite unrestricted by considerations
Table 1. Compari son of Chromatographic Systems Closed
Open
Sample introduction
Single, sequential
Multiple, concurrent
Chromatography
Elution
Development
Detection
Dynamic
Static
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981 · 253 A
of interference with detector response or possible deteriorating effect on the stationary phase. Indeed the purity of the solvents employed in HPTLC is not nearly as critical as in HPLC for these same reasons. From the preceding discussion it should not be inferred that develop ment chromatography is confined to determination of only a few compo nents within any given mixture. An open system permits multiple devel opment with any number of solvent systems, hence fractions of widely varying polarity may be moved away from the origin into the region of mea surement by successive development in solvent mixtures of increasing strength. For example, this technique has been employed quite effectively in the separation of mycotoxins (2 ) and the determination of anticonvulsant drugs in blood plasma (2). In addition, such successive developments need not be carried out in the same direc tion, for, unlike column chromatogra phy, TLC permits true two-dimen sional development with separations that rival and even surpass those ob tained in high-efficiency liquid col umns. Static Detection. Elution chroma tography as performed by most present-day HPLC instruments is a totally dynamic process with detection an integral and inseparable compo nent of the total system. As such, the chromatographic fractions passing through the detection device cannot be observed any faster or slower than the rate at which they are being eluted from the column. In other words, dy namic detection must operate within the time constraints established by the chromatographic conditions. Detection in development chroma tography is completely separate from the chromatographic operation and, by contrast, may be considered a stat ic process. Any given chromatographic fraction can be examined for as long as necessary to extract the maximum amount of information, or for as short a period as the time constant of the
detection device and its associated electronics will allow. This freedom from time constraints is potentially the most important aspect of TLC, be cause it permits utilization of any of a variety of techniques to enhance the sensitivity of detection, such as reac tions to increase light absorbance or fluorescent emission and wavelength selection for optimum response of each component to be measured. In addition in situ spectra can be ob tained for component identification. Not the least of these advantages is the possibility of exploiting computer techniques for optimization of the de tected signal by signal averaging, digi tal filtering, background subtraction, or cross-correlation operations that are either impossible or extremely dif ficult when the detector has but one opportunity to view a component as it is eluted from a chromatographic column. It is worth remembering that, un like HPLC, the operations that make up the HPTLC system are not "on line" and this lends considerable flexi bility in the scheduling of the separate steps of sample application, develop ment, and detection. Thus, samples may be processed "in parallel," requir ing no more than a change of devel oping solvent or scanning wavelength to move rapidly from one group of samples to another having totally dif ferent characteristics. HPTLC Methods Quite satisfactory qualitative re sults have been achieved in conven tional TLC with no more than a dis posable pipet for sample application, a glass tank for development, and a se lection of suitable spray reagents for visual detection. The procedures used in HPTLC are basically the same, but beyond that rudimentary similarity the techniques differ considerably, much as in the case of HPLC when compared to conventional liquid col umn chromatography. In order to achieve the resolution, sensitivity, and
Table II. Comparison Of TLC and HPTLC* HPTLC
TLC
20 X 20 c m
10 X 10 c m
Sample volume (capillary application)
1 - 5μΙ_
0.1-0.2 μ ι
Diameter of spots
3 - 6 mm
1.0 mm
Diameter of separated spots
6 - 1 5 mm
2 - 5 mm
Solvent migration
10-15 m m
3 - 6 mm
Detection limits Absorption Fluorescence
~ 5 ng ~ 0 . 1 ng
~ 0 . 5 ng ~ . 0 1 ng
Plate size
• From: Hezel. U.B. (3)
254 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
quantitative reliability expected of a high-performance system, refinement of materials and methods is necessary. A brief description of HPTLC meth ods follows. For a more detailed dis cussion of methods and materials the reader is referred to two recent vol umes on the practice of HPTLC (3, 4). The HPTLC Plate. HPTLC plates available today from commercial sources are actually fairly similar to standard TLC plates. Most are glass plates coated, for the most part, with silica gel held together with various proprietary binders. The difference is primarily that the average size of the silica gel particles is smaller and the size distribution is tighter. The layer itself is usually somewhat thinner and the surface more uniform than that of standard TLC plates. The increased efficiency realized by the smaller particle size chromato graphic bed results in less band broad ening and, hence, improved resolution and greater sensitivity of detection of the separated fraction. In addition, al though the small particle size reduces the velocity of mobile phase flow, the length of the chromatographic bed re quired is markedly less than that en countered in conventional TLC. A comparison of HPTLC and TLC pa rameters is shown in Table II. Sample Application. Experience has shown most practitioners of HPTLC that the operation most influ ential on the quality of separation and quantitation is sample introduction. In order to attain optimum resolution and sensitivity, the size of the applied sample spot must be small—no great er than 1.0 mm in diameter for most commercial HPTLC plates. In addi tion the sample spots should be as uniform as possible in both shape and size for precise estimation of sepa rated components. Clearly, this elimi nates the usual fire-drawn glass pipet as a means of sample delivery. Assuming an even distribution of solute in a sample solution deposited on the adsorbent layer, a volume of approximately 0.1 μL will produce a 1-mm spot on most HPTLC plates. If the solute has a low Rf (ratio of sample migration to solvent travel) in the sample solvent, larger volumes may be used; but often those solvents have low polarity or solvent strength and are not the best for quantitative trans fer of the solution from the spotting device. One of the more convenient means for manipulating such small volumes is a micropipet constructed from platinum-iridium capillary tubing fused into the end of a length of glass tub ing. These pipets are usually mounted in a fixture that permits close control of the pressure exerted by the tip on the absorbent layer and also precisely
positions the pipet for repetitive delivery of sample, an important consideration if greater amounts of sample must be applied than can be accommodated by the small volume of the pipet. Very reproducible sample delivery can be achieved with such devices, providing the solution flows easily in and out of the capillary tubing. Unfortunately, many sample solutions, particularly extracts of biological materials, become quite viscous when concentrated to low volumes. Therefore, successive applications of a dilute solution must be made to the same spot if sensitivity is to be preserved. An alternative method recently reported (5) removes most of the solvent from the sample solution prior to application to the HPTLC plate. With this procedure, termed "contact spotting" (Figure 1), relatively large volumes of sample solution are evaporated on a treated fluoropolymer film positioned over a number of depressions in a metal plate. After the solvent is evaporated by gentle heat and nitrogen flow, the thin-layer plate is positioned adsorbent side down over the film, and the sample residues are transferred by bringing the film into contact with the adsorbent. Thus, many samples can be applied simultaneously, and spot diameters a fraction of a millimeter can be obtained with starting volumes as large as 0.1 mL. Plate Development. The open, two-dimensional chromatographic bed of a thin-layer plate allows more choice in the direction of mobile phase flow than do closed systems, and three different systems are presently in use with HPTLC: linear, circular, and anticircular. Of these, linear development (Figure 2a) is the most familiar
Figure 1. Sample introduction by contact spotting. A specially treated fluoropolymer film is pulled into a series of depressions in a metal plate by application of vacuum (a and b). Sample solution is delivered by pipet (c), and after evaporation a residue remains (d), which is transferred to the HPTLC plate by replacing the vacuum with slight pressure (e)
to users of TLC, and in its simplest form requires no more than contact of one edge of the plate with the developing solvent—usually accomplished by standing the plate vertically in a
(a)
few milliliters of solvent in a suitable container. Better performance can be achieved if the plate is horizontal during development (solvent travel is unimpeded by gravity) and if the free volume surrounding the plate is minimal (more rapid and uniform equilibration of liquid and vapor phases). In such an arrangement, the solvent is usually fed to the plate by capillary action, and with the plate horizontal there is no difficulty in supplying solvent from opposite edges at the same time, as mentioned previously. An example of this type of linear development is illustrated in Figure 2b. As mentioned previously, TLC permits two-dimensional development, particularly useful for examining many components of a complex mixture. By utilizing the entire surface of the plate for separation of a single sample the resolving power is nearly the square of that attainable in a onedimensional development. The usual two-dimensional development (Figure 3a) accommodates only a single sample applied to one corner of the plate; but four samples can be chromatographed simultaneously using convergent solvent flow (Figure 3b). In circular chromatography the solvent is introduced by a wick or pumped through capillary tubing to the adsorbent layer and allowed to move the samples radially from a central point. This mode of development (Figure 4a) appears to improve resolution of components at low Rf and is faster than linear HPTLC. Anticircular chromatography supplies the solvent through an annular capillary space to the periphery of a circle, and the solvent then moves inward to converge at the center, as shown in Figure
(b)
Figure 2. Linear HPTLC development with (a) solvent flow in one direction, and (b) simultaneous flow from opposite directions 256 A • ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
(b)
(a)
Figure 3. Two-dimensional development, (a) Solvents A and Β used successively at right angles, (b) Solvent flow from opposite edges permits simultaneous four-sample, two-dimensional development
4b. Resolution at high Rf values is re ported to be favored by this approach, and, again, the speed of development is much greater than with linear flow. While each of the various systems may offer advantages in certain ana lytical applications, linear develop ment does not require much in the way of sophisticated equipment, and in practice provides the resolution and reproducibility required for most quantitative determinations. Scanning Densitometry. Quanti tation of the components separated by HPTLC is usually performed in situ with photometric measurement of ab sorbed light or emitted fluorescence. In absorption densitometry the spots on the HPTLC plate are scanned by a beam of monochromatic light formed into a slit image with the length of the slit selected according to the diameter of the largest spot. The diffusely re flected—or scattered—light may then be detected with a photomultiplier. Those areas of the plate that are free of light-absorbing materials will yield a maximum signal, and the separated spots will cause a diminution of re flected light that is concentration de pendent. At this time there are three optical systems that are employed predomi nantly in scanning densitometers. The first, shown in Figure 5a, is a simple single beam arrangement with the light emerging from the monochromator perpendicular to the surface of the thin-layer plate. Diffusely reflected light is detected by a photomultiplier. The plate is mounted on a movable stage, usually motor driven in the di rection perpendicular to the slit length, and either manually operated or motor driven in the orthogonal di
rection. This arrangement is quite ca pable of producing excellent quantita tive results, but baseline drift may be troublesome due to extraneous ad sorbed material in the thin-layer, which can move during chromato graphic development. This problem may be overcome by using a double-beam scanner (Figure 5b) with a reference beam scanning the intervening space between sample lanes. The difference signal thus elim inates the contribution of general plate background. Small irregularities in the plate surface as well as undesired background contributed by the sample itself may still pose a problem. Consequently, the dual-wavelength system (Figure 5c) is designed to mini mize these effects. Two monochromators alternately furnish to the same sample lane a reference wavelength with minimal sample absorbance and a sample wavelength chosen for maxi mum absorbance. The reference wave length cancels out background and corrects for plate irregularities. These various densitometers provide for a choice of light sources and optics, per mitting a range of wavelengths from below 200 nm through the visible spectrum. An example of an absorb-
Figure 4. Circular chromatography (a) with development solvent introduced at a central point to a ring of initial sample spots. Anticircular chromatography (b) requires solvent delivery from an annu lar capillary space to the periphery of the circle enclosing the sample spots. A zone of adsorbent layer is removed to prevent solvent migration in the oppo site direction, and the solvent con verges at the center of the circle
258 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
(a)
(b)
Figure 5. (a) Simple, single beam scanning densitometer. L, lamp; MC, monochromator; PM, photomultiplier; P, thin-layer plate, (b) Dual beam arrangement with beam splitter. Reference beam corrects for plate background by scanning between sample lanes, (c) Single beam, dual wavelength scanner. A reference wavelength chosen for minimal sample absorption corrects for plate irregularities as well as background in the sample lane. CH, chopper
Figure 6. Absorption-reflectance scan of imipramine (I) and desipramine (D) extracted from a 1.0 mL blood plasma sample, at concentration of 100 ng/mL. Internal standard (IS) is butaperazine
ance scan of the tricyclic antidepressant drug imipramine and its metabolic product desipramine extracted from a blood plasma sample is shown in Figure 6. The response of such reflectanceabsorbance scans is nonlinear with concentration and, as would be expected, subject to perturbations not encountered in solution densitometry. This is not a serious problem because the multiple sample capability of HPTLC permits the inclusion of calibration standards with each sample run. Consequently, all samples, both standards and unknowns, are subjected to exactly the same chromatographic conditions, and systematic errors remain very much at a minimum. The relative standard deviation of determinations such as that shown in Figure 6 is usually less than 4%, and may even fall below 2%. Determination of pure standards often produces errors less than 1%. Despite the excellent results ob-
Figure 7. Fluorescent scan of Prolixin (fluphenazine) from blood plasma (upper trace) at a concentration of 0.6 ng/mL. Internal standard is triflupromazine. Lower trace is blank blood plasma 262 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
Chlorpromazine Imipramine
Now determine As, Ser and Hg with a light heart and an IL AVA.
Thiothixine
Clozapine
I 220
240
-i_
_i_
260
280
_i_
300 320
Wavelength ( n m )
220
240
260
280
300 320
Wavelength ( n m )
Figure 8. In situ U V s p e c t r a of four p s y c h o p h a r m a c o l o g i c a g e n t s o b t a i n e d b y s u c cessive scans from 220 to 3 2 0 nm
tained with reflectance-absorbance densitometry, fluorescence measurements are usually preferred if the substance in question is or can be made fluorescent under appropriate excitation. The limit of detectability is considerably enhanced—it is frequently in the low picogram range—and the linearity of response and selectivity of detection are much improved. A single beam mode of operation of the densitometer is usually quite sufficient for
Chlorpromazine
fluorescence photometry, with the simple addition of a cutoff filter ahead of the photomultiplier to block the excitation wavelength. Mercury or xenon lamps are available with most instruments to provide excitation. An example of a scan in this mode is shown in Figure 7. This, again, is a drug extracted from blood plasma and is illustrative of the sensitivity that can be attained with in situ fluorescent photometry. It is of interest that this particular compound has a relatively modest fluorescent yield compared to such strongly fluorescent compounds as the porphyrins or the aflatoxins, which are detectable in amounts below 10 pg on the thin-layer plate (1, 3). Future Trends
Imipramine Clozapine Thiothixine
Figure 9 . C r o s s - c o r r e l a t i o n of a c h r o m a t o g r a m against t h e U V s p e c t r u m of a single c o m p o n e n t ( c h l o r p r o m a z i n e ) of a drug m i x t u r e , s h o w i n g peak a n d b a c k ground suppression
Although TLC is one of the oldest forms of chromatographic separation, HPTLC is still very much in its infancy. Refinement accompanies use, which has certainly been the case with GC and HPLC, and there remain many intriguing possibilities for improvements of HPTLC methodology. The HPTLC plates currently available from commercial sources, while very much better than TLC plates, cannot be regarded as the ultimate by any means. Market demands will very likely stimulate development of plates with much smaller particle size distributions, more uniform adsorbent thicknesses, and even smaller average particle sizes. The minimum spot diameter that can be achieved on a developed plate is a function of plate ef-
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ficiency which, in turn, is a reflection of these aforementioned factors. Bear ing in mind that on a surface such as a TLC plate, concentration increases by the inverse square of its spot diame ter, improvements of efficiency can greatly extend the sensitivity of detec tion as well as enhance resolution. Smaller developed spots also place greater demands on the optical sys tems of scanning densitometers, so it follows that improved instrumenta tion must surely be in the offing. With only one or two exceptions the scan ners now in use were designed with macro TLC plates in mind and have merely been adapted to HPTLC. Thus, densitometers will most certain ly be developed to answer the unique requirements of HPTLC from the standpoint of both refined optical sys tems and digitized control and data acquisition. The application of com puter techniques to HPTLC scanners will also simplify the optical systems, because rapid scanning with data stor age and processing is a very effective alternative to optical correction of baseline drift. An even more promising use of com puter techniques is in the manipula tion of the acquired data to suppress response of some peaks while preserv ing that of others. For example, the ul traviolet spectra obtained directly from scans of an HPTLC plate are shown for four drugs in Figure 8. By scanning a chromatogram at various selected wavelengths and then per forming a cross-correlation operation against the previously determined UV spectrum for one of these compounds, only the peak which corresponds to that spectrum remains undiminished
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