Instrumentation
vapor space over the plate is enclosed, usually by placing the TLC plate in a closed tank, the bottom of which is covered by the solvent. Given favorable conditions, the components of the mixture move in the direction of solvent advance and separate from each other as they move. After the solvent has substantially crossed the thin layer, the plate is removed from the enclosure and dried. Now deposited on the adsorbent as separate spots, the various components can be identified or assayed or recovered. Easily used and widely adaptable, thin-layer chromatography has grown phenomenally (7, 8).Each of the following characteristics of TLC was necessary for this growth; all must be present in any form of TLC that will be widely used: * Applicability to a chemically wide variety of materials Suitability for microanalysis (the genesis of TLC) Thin-layer plate (a major invention) Spot recoverability: of the spots that became separated, of the residue that did not move, and of the components that moved with the solvent front Solvent volatility Development without attention Adaptability to parallel processing, that is, to multiple, simultaneous, similar separations. John A. Perry Conventional TLC grew, despite in601 East 32nd Street, Chicago, 111. 60616 herent handicaps: TLC spot locations and areas reThomas H. Jupille and Louis J. Glum flect the respective locations and areas Regis Chemical Co., Morton Grove, 111. 60053 of the spot origins. Therefore, the resultant chromatograms depend unwontedly on operator attention and technique. A great deal of time must This paper is a review of and progbe and is spent applying spots or ress report on programmed multiple streaks of minimum spread along the development (PMD) (1-6), an apdirection of development. proach to thin-layer chromatography TLC spots spread. In conse(TLC) ( 7 , 8 ) . quence: sensitivity continually deIn conventional TLC a spot of a creases during development; one plate mixture to be separated is deposited can offer only a few separated spots or from solution near the edge of a thinstreaks; and a spot location soon canlayer plate. Usually, this plate is a thin not be precisely reported-the spot (0.25 mm) layer of silica gel supported has become too broad and too diffuse. on a piece of glass. After the spot has Methods for TLC dried a t what has become the spot origin, the plate edge below the spot is Various methods have been devised placed in a solvent. By capillary action that alleviate different aspects of the solvent moves into the thin layer; these handicaps. We review below this is the development of the thinmethods that are mechanical (the layer plate. During development the shaped bed), physical (the heated
Programmed Multiple Development
bed), chemical (solvent gradients), and repetitive (multiple development). After this, we discuss PMD, which has certain characteristics of its own. Shaped Bed. To increase sensitivity, Mazza et al. (9) shaped the thinlayer bed so that the solvent carried the spots into a narrow strip. Also, having isolated a given spot, they slowly removed the still-developing plate from the tank, so that the spot moved into the evaporating solvent front. The narrow bed countered lateral spot spreading, and the evaporating front reconcentrated the longitudinally spread-out spot. Heated Bed. In one heated-bed variation, Turina et al. (10, 11) raised the temperature of the thin-layer plate during development; and in another, provided a zone of sharp temperature increase across the plate. Raising the temperature of the plate during development increased both sensitivity and resolution. With a “high-resolution zone” across which the temperature of the developing solvent rose sharply in the direction of development, both resolution and sensitivity were highly improved within the zone (11 ). Solvent Gradients. The simplest solvent gradient depends on the solvent demixing (12, 13)of two solvents. The solvent demixing occurs when the thin layer retains the stronger of the two solvents while the weaker flows ahead. An internal solvent front then marks the advancing forward boundary of the stronger solvent. An adsorbed solute, not desorbed by the weaker solvent but strongly desorbed by the stronger, is found in that internal solvent front. The spot of such a solute can spread laterally within the front but is as sharply delineated along the direction of development as the front itself. When usable, such gradient techniques, more or less elaborate, counter spot spreading. They affect liquid-solid chromatography much as temperature programming affects gas-liquid chromatography, increasing the range of solute adsorptivities that can be handled within a given sample, but not the resolution of two similar solutes. Multiple Development. This technique does increase the resolution of similar solutes. Repeated development of the same plate by the same solvent in the same direction and for the same
ANALYTICAL CHEMISTRY, VOL. 47. NO. 1, JANUARY 1975
65A
distance is called unidimensional multiple chromatography (UMC). First proposed by Jeanes et al. for column chromatography (14 1, UMC has been studied and described by Thoma (15, 16). His observations and conclusions are relevant to PMD. Observations on UMC Thoma ( 1 5 ) spoke of the “superb resolving power of UMC,” but commented that “the excessive time required (for its use in column chromatography) has pruhahly curtailed its u s e , , . . However,. . . (with) TLC, . . .it is now feasible to employ a larger number of solvent passes when attempting to resolve simple mixtures . . . . (A) combination of TLC and UMC should prove to he an ideal way to resolve mixtures. . . .” Thoma showed that
(1 - R f ) ” = 1 - R f , ”
(1)
where n is the number of multiple developments; Ri is the conventional single-development ratio of spot velocity to solvent velocity (the same Rf usually measured as the spot migration distance divided by the originto-solvent front distance); and Ri,,, is the apparent Ri after n multiple developments. Let the center-to-center separation of two similar solutes he divided by the total bed length used, and call this the reduced separation AS. (For instance, suppose the solvent level-tosolvent front distance is 10.0 cm, and two spots in this chromatogram show a center-to-center separation of 1.0 cm. Then the reduced separation A S of these spots is 1.0/10.0 or 0.1.) For a given solvent strength, the reduced separation A S first increases with an increasing number of multiple developments, then passes through a maximum, and ultimately decreases. However, for solvents of decreasing solvent strength-one curve per solvent in a family of plots of A S vs. n, the corresponding A S maxima not only increase but also correspond to higher and higher numbers of multiple developments. In each case, the reduced separation passes through a maximum “when the average distance migrated is (1 - e-l) or 0.632 of the length of the support. The number of irrigations (multiple developments) producing this separation is (-l)/ln (1 - Ri’), where Ri’is the average Ri of the two solutes” (15). We reiterate: a certain optimum number nopt of multiple developments will produce a maximum separation of two similar solutes when the two solute spots have on their average traversed 0.632 of the length of bed used. 66A
T h a t optimum number, if the Ri’s of the two solute spots are sniall but approximately equal, is
-1 no,, = In(1 - 1
(2)
~
In the two references cited (15,16), Thoma related the maximum reduced separation AS,., produced by nopt multiple developments to the average Ri; of two, nominal, similar solutes being separated. This separation increases with decreasing solvent strength and, thus, decreasing Ri’. Moreover, A S increases sharply once Ri’ decreases helow a certain level-for these two spots, 0.06. [From Figure 1 of ref. 15, one can see that a single development of these two solute spots, Ri’ = 0.10, produces a reduced separation of about 0,009. I t follows that in a 15-cm bed, the two superimposed spots, developed, would have a center-to-center separation of about (0,009) (15 cm) or 0.15 cm. Such a separation would he barely discernible in a composite spot of the usual 1-cm diameter.] Consider the three possible examples that follow for those two spots (the numbers come from Figures 1and 2 of ref. 15 and from Equation 2 of this paper): ~~~
Ri’ 0.06 0.02 0.007
AS,,,
n”Pt
16 50 133
0.2 0.3 0.4
The first example means that if the solvent strength is decreased until the (average) Ri’ is 0.06, then 16 multiple developments will cause the two spots of these two solutes to move the optimum fraction, 0.632, of the way across the bed. A t this point, the center-tocenter separation of the spots will be 0.2, or one-fifth of the bed length. (It is assumed that as the solvent strength is decreased, selectivity is neither increased nor decreased. Because a small change in solvent
strength can make a great change in
Ri, the assumption is probably accurate enough.) In the third example (we will skip describing the second example), if the solvent streneth is decreased further u n w m e average ai IS U.UUI, men i a a multiple developments will cause the spots to be separated by two-fifths of the bed length. For a 15-cm length of bed used, the center-to-center separation would be 6 cm (vs. 0.15 cm for one development). As the solvent strength and Ri’ decrease still further, the fraction of the total bed length separating the two all-hut-identical spots increases even more sharply. Thus, increasingly large numbers of multiple developments by suitably weak solvents yield rapidly increasing separations. Very few separations require such numbers, hut theory indicates such numbers are useful if needed and so does experiment “. . .since the maximum extent of separation increases as Ri falls, difficultly resolvable components should be repeatedly irrigated (multiply developed) with solvents which produce low Ri values. This recommendation is in accord with experimental observations” (16). Programmed Multiple Development In PMD the frequency (as, for example, 10 or 20 in the first hour) and numbers of programmed multiple developments are large compared to conventional practice. However, although the PMD prototype could handle 999 multiple developments and the present commercial model can still handle 99, the genesis of PMD had nothing to do with multiple development. PMD was a bv-product of a deliberate attempt io exploit the resolution potential of TLC. The plate efficiency of the adsorptive thin-layer plate, and thus its resolution potential, far exceeds that usually achieved. Because adsorption-desorption is very fast compared to dif-
P b
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I
PMD
* ANALYTICAL CHEMISTRY, VOL. 47, NO. 1, JANUARY
1975
I
fusion through a liquid ( 1 7 ) ,TLC plate efficiency is limited by solvent diffusivity (18).Solvent diffusivity, however, varies positively and exponentially with temperature (19). Therefore, TLC plate efficiency always increases with temperature for a given solvent (20). Our early experiments exploring higher temperatures with solvents of high diffusivities involved strongly heating TLC plates during developments with such solvents, which are volatile. These, of course, quickly evaporated. Multiple developments with successively more extended solvent excursions ensued as a convenient way of allowing the development phenomena to evolve and to be observed the while. The unusual character of the multiply developed spots soon became apparent and led to PMD. During the development and design of PMD instrumentation, we kept two goals present and equal: To conserve intact each of the highly useful, operational characteristics of conventional TLC-those that were set forth earlier in this paper T o produce a highly usable instrument that would als? embody an open-ended experimental format. This format would foster the widest possible subsequent growth of the PMD concept. It would allow PMD practice to evolve, rather than lock it in. Because we have not yet fully characterized PMD, nor even become aware of all relevant theory (both goals recede!), this paper is still only a progress report. The several effects of PMD that have become clear will be better understood if PMD itself is first described in somewhat more detail. Description. Programmed multiple development consists of a succession of initially brief but increasingly extended solvent advances. Each solvent advance is followed by a solvent removal that returns the solvent front to the spot origin. Solvent is removed by an inert gas sweep, or radiant heat, or both. In the same way, development is halted on termination of the PMD. The whole process is automatically controlled. Throughout, the thin-layer plate remains in contact with the solvent, requires no attention, and does not move (Figure 1). Each programmed multiple development is carried out by a programmer controlling one or more developers (for a given program, one programmer can control any number of developers) (Figure 2). The conditions for a given programmed multiple development are set into the programmer by the operator. Programmer switch labels include
.
Figure 2. PMD instrumentation. Programmer (left);developer (right)
the terms cycle, segment, advance, removal, scheduled, fixed, and mode. Most of these explain themselves. Each development cycle has two segments, a solvent advance segment and a solvent removal segment. The duration of each solvent advance segment conforms to the development mode schedule, set out in the next paragraph. The duration of each solvent removal segment may be either fixed or scheduled: if fixed, it is invariant; if scheduled, it too conforms to the development mode schedule. Development mode governs segment duration and maximum cycle number. The segment duration T varies with the current cycle number n and the unit t i m e t according to the development mode: Segment duration Max cycles Mode T available 1 T=nt 99 2 T = [n(n+ 1 ) / 2 ] t 28 3 T = n2t 20 Thus, the operator selects among three development modes. Mode 3 most closely approximates conventional TLC, yields the fewest cycles per overall program time, produces ~~
"c
the maximum center-to-center separation of spots (but yields the widest spots), and best resolves the spots of higher Rp On the other hand, Mode 1 yields the most cycles per overall program time, produces the least centerto-center separation (but yields the narrowest spots), and best resolves spots of lower Rp (Also, after 10 or 15 cycles, Mode 1closely resembles UMC-and the PMD could begin on, say, the 11th cycle.) Mode 2 is intermediate. Figure 3 shows diagrammatically how the successive positions of the solvent front are related to mode and cycle number. In practice, then, the operator selects individual unit times-0-100 sec in 10-sec steps-for solvent advance and solvent removal, chooses to have solvent removals fixed or scheduled, and decides how many multiple devel opments-cycles-are to be carried out. To determine bow many minutes his projected PMD will take, he press es an EVALUATE button. The required number of minutes then appears on a three-digit display. (After a program has been started, the display shows the number of cycles com-
a,
Figure 3. Comparison of
m .-ul c
5 cycles in Mode 3 with 10 cycles in Mode 1 for
0
same unit solvent ad-
vance time in each
mode Horizontal lines: distances of
I Time Overall
successive solvent front excursions in each mode. (Note distance-coincidence of second Mode 3 cycle with fourth Mode 1 and of third Mode 3 cycle with ninth Mode 1)
ANALYTICAL CHEMISTRY, VOL. 47, NO. 1, JANUARY 1975
67A
pleted, unless the EVALUATE button is pushed. In that case, it shows the number of minutes already elapsed in the current program.) The operator also selects among 11 power levels (0-10, inclusive) for the radiant heat level to be applied during both solvent advance and solvent removal. For maximum plate efficiency, as we have pointed out, the radiant heat levels should be as high as possible (while still permitting the necessary, successively more extended solvent advance into the thin layer). Apparently or possibly thermally labile sample components may make above-ambient temperatures intolerable. Therefore, solvent removal can be carried out solely by a sweep of gas. Each developer carries a socket that is activated by the house current (Le., in the U S . , for example, 110 V ac) a t all times except during solvent advance segments. This can be used to control a flow of nitrogen across the plate to effect solvent removal. If the radiant power level is set a t 0, the temperature within the thin-layer bed drops below ambient during solvent removal. The operator selects a preprogram time from 0 to 100 sec, in 10-sec steps, to have the first solvent advance begin under the same conditions as each successive solvent advance. This is more important for the shorter threeand four-cycle programs than for the higher-cycle programs. Finally, the operator selects the conditions he wishes to obtain once the PMD has been automatically terminated, and further development permanently halted. The plate can then be kept free of solvent by nitrogen flow, infrared radiation a t a separately chosen power level (one of ll), or both. These same conditions can be invoked in midrun by pressing a PAUSE button, which interrupts the program without aborting or terminating it. Having completed his instructions to the programmer, the operator prepares and installs the thin-layer plate (or plates) in the developer (or developers) (Figure 4 ) . The developer accommodates plates 10 or 20 cm high and up to 20 cm wide. Spot Shape and Location Until very recently, the effect of PMD on spot shape and location has preoccupied us. We have hardly touched the effect of a large number of multiple developments on the centerto-center separation of two difficultly separable spots, as described by Thoma (15, 1 6 ) ,though what little we have done (we shall be doing much more) agrees with his predictions. Most of what we have to report concerns spot reconcentration, which quickly renders spot shape and spot 68A
Figure 4. Thin-layer plate arrangement for PMD (A) Scored and spotted thinnonporous spalayer plate, (6) cer, (C)facing plate: clamped and held in solvent trough during PMD
position independent of origin shape and position (Figures 5-7). Spot Reconcentration. This occurs twice per PMD cycle by interaction with the solvent front, first during solvent advance, and then during solvent removal (Figure 5 ) . During solvent advance, the solvent first reaches and moves upward the molecules that are lower on the thinlayer plate. I t reaches and moves these before it reaches similar molecules that happen to be higher. So, each solvent advance brings similar molecules closer toget her. [In his 1963 paper (151, Thoma mentioned this briefly, once, but that paper described almost exclusively the variation of center-to-center separation with the number of multiple developments, rather than the variation of resolution. Thoma considered spot shape as part of a later paper ( 1 6 )but not as a function of multiple developments.] During solvent removal (Figure 51, the solvent front is caused to recede, preferably slowly. Because in PMD the thin-layer plate remains in contact with the solvent reservoir, solvent moves constantly toward the solvent front. Some solvent, vaporizing a t the front, moves through it. Spot molecules, however, cannot be carried through the front but are deposited in it. During solvent removal, higher molecules are deposited in the front while similar lower molecules continue to move upward. So, each solvent removal also brings similar molecules together. Jupille has related ( 4 , 6 ) the final separation distance X f of two same R f molecules to the original separation distance X,of these molecules. The distances are taken along the line of chromatographic development, and each molecule represents a statistical aggregate in its behavior. After n solvent advances,
ANALYTICAL CHEMISTRY, VOL. 47, NO. 1, JANUARY 1975
This equation refers only to the effects of solvent advance under isothermal conditions and neglects diffusional spreading, which depends on the causative concentration gradient (21 ). Equation 3 helps to explain why the relatively short, earlier developments of any PMD are so effective. The effects of spot reconcentration are exponentially proportional only to the n u m b e r of multiple developments. (Also, because each PMD cycle lasts longer than its predecessor, the frequency of the twice-per-cycle spot reconcentration is highest a t the start of any PMD. And because capillary flow continually decreases in velocity with increase in capillary length, the distance advanced per unit time by the solvent is also highest a t the start. Therefore, the effects of spot reconcentration are attained in a relatively short time and over a relatively extended distance.j A numerical evaluation from Equation 3 is instructive. Consider two molecules of R f = 0.5 a t the top and bottom of a just-deposited spot 1 cm in diameter. The equation predicts that within four cycles, the molecules would be within 1 mm of each other (four PMD cycles usually take 10 to 15 min). This prediction neglects diffusional spreading and the effects of solvent removal. The efficiency of spot reconcentration by solvent removal is less simply described than that of solvent advance. The solvent front should recede slowly relative to the spot forward motion ( I ). Solvent Removal by Heating. Recently, we have been able to isolate experimentally the two types of spot reconcentration that are connected with solvent removal by heating. One is due only to solvent advance that follows
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During front traversals lower molecules move toward \ upper. nonmovinq ones
\\
SOLVENT FRONT M O V E M E N T
solvent advance
SOLVENT FRONT
solvent moves at all times into front
solvent removal
Figure 5. Spot reconcentration
solvent removal by heating. The other is due only to solvent removal by heating. The two are complementary, one increasingly effective as the other weakens. The more severe the heating, the more effective the solvent advance mechanism and the less effective the solvent removal mechanism. As the heating becomes less and less severe and therefore necessarily more prolonged, the solvent removal mechanism becomes increasingly effective while the solvent advance mechanism decreases in efficiehcy, approaching as a minimum the efficiency described in Equation 3. In the near future, we hope to publish a study showing these effects. We may now consider again the Turina-heated bed (IO, 11 ). When the solvent in PMD is being removed by infrared radiation, simply touching the plate reveals that the plate is cool where wet, warm or hot where dry. Following solvent removal, the solvent again begins to advance into the moreor-less hot bed. At the solvent front the solvent cools the bed and the plate by evaporating. Because the solvent evaporates, the solvent front advances less rapidly than it would under isothermal conditions. This overall set of conditions corresponds closely to Turina’s “high-resolution zone” ( I 1 ), except that here the zone is moving, sweeping the chromatogram twice per cycle. Channeling. We can now also consider again the shaped-bed techniques of Mazza (9). Channeling prevented lateral spreading, and a slow removal of the still-developing plate concentrated the longitudinally spread-out spot. We have shown (3) that normal PMD spot reconcentration brings about its effects throughout the whole chromatogram without requiring special plate movement. Also, because the original spot may he spread out over several centimeters longitudinally, the channels themselves may be unusually narrow. Deposited in such narrow 70A
* ANALYTICAL CHEMISTRY, VOL.
channels, normal liquid sample volumes do spread out to an extent that would ruin normal resolution. However, spot reconcentration obliterates the original spread of the spot and yields normal PMD resolution, while the very narrow channel enhances normal PMD sensitivity, itself well above the conventional level ( 3 ) . In general, because of spot reconcentration, neither the original extent of a spot nor the amount of solution originally applied affects the size or shape or location of the final PMD spot. The initial narrowing of a spot reflects primarily the quick decrease from whatever hounds t h e deposited component may have had. Later narrowing reflects primarily the course of the particular PMD program being used. Spot Width. We close our general comments on spot reconcentration by considering top-to-hottom spot
Figure 6. TLC plate showing that final location of given molecular type does not reflect original location
“width” in PMD. That width depends mainly on three variables: component amount, program characteristics, and adsorbent particle size. Spot size varies directly with component amount, but the minimum spot size does not become zero ( 8 ) .We wish to determine the PMD minimum spot top-to-hottom “width.” Perhaps 20 particle diameters, side-by-side, could “sample” the concentration distribution across a minimum spot “width.” The particle diameters on the commercial precoated plates we have generally used range from 20 to 40 pm, somewhat larger than the 5-25-pm diameter particles Trnter mentioned as commercially available, and considerably larger than the 1-5-pm diameter particles he called “ideal” (8). Side-by-side,-twenty 20-40-pm diameter particles would cover 0.4-0.8 mm. On plates having such particles, the usual minimum PMD spot widths range from 0.9 to 1.1mm. We conclude, for now, that the PMD minimum spot width equals 25-50 diameters of the adsorbent particles. For a given plate and thus a given particle size range, PMD spot widths depend on the program and the amount of component per spot. For a given program, the spot width shows the balance between the repeated reconcentrating of the spot and the diffusional gradients that result. However, for the usual program of 20 cy-
Figure ?. Chromatograms showing that PMD spots quickly become characteristically tight and mutually aligned without regard to location or spread of origin Sudan Black. benzene, Silica Gel 0: Mode 2; 38 min overall: 7 cycles: “nil ~ d v e nadvance t time, 20 sec: solvent removal time, fixed, 90 sec
b
47, NO. 1, JANUARY 1975
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I
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cles or less, the spots are essentially uniform in width throughout the chromatogram and usually less than perhaps 2.5 mm in top-to-bottom width. Also, the position of the chromatogram itself depends on the program hut not on the distance of the origin from the edge. For these reasons, PMD chromatograms differ markedly from conventional TLC chromatograms. The following discussion illustrates various uses of these differences. Advantages Because PMD concentrates all molecules of a given type into one tight spot, regardless of how spread out the original spot was, trace sensitivity is enhanced, large volumes of dilute solutions can be used in just one application of the original spot, greater overloading without trace obliteration is tolerable, and the location of the center of the original spot is not particularly important. These results are illustrated in Figures 8 and 9. Figure 10 shows how a high sensitivity for quinine can he had from the deposition of 50 pl of raw (quinine-spiked) urine directly onto the thin-layer plate: the resultant quinine spots are perhaps at most one-tenth as wide, top to bottom, as the original spot. (The quinine is visible down to 0.625 pglpl using PMD, but only to 10 wglfil in a matching conventional development.)
Figure 8. PMD chromatograms showing same amount of solute deposited from 1, IO,and 100 pi Sudan Black. benzene. Silica Gel G 10 multiple developments, 54 min overall. Mode 1: Unit solvent advance time, 40 sec: solvent removal time. fixed. 100 sec CouITesy of the Jwrnslofthe Assaclation ofOlf/cbl anslwlcal Chemlsfs (3) . .,
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ANALYTICAL CHEMISTRY, VOL. 47. NO. 1. JANUARY 1975
Figure 9. (A) Gross overloading causes increasingly gross spreading in conventional TLC development. (B) Gross overloading in PMD brings out traces more clearly Volumes in A and 0 , left to right: 1, 5, and 10 &I. Other conditions Same as in Figure 8 Courtesy of the Journal of the Aszociatian of OfficdlAnalflical Chemists (3)
pmd" PROGRAMMED MULTIPLE DEVELOPMENT ..is gaining recognition as a new, significant approach to thin layer chromatography. Among the reasons for this are: I. At comparable development time, PMD offers greater resolution than conventional TLC with still greater improvement for longer developments. 2. For equal resolution, PMD is faster than conventional TLC. 3. Because PMD developed spots are much narrower, PMD offers sensitivity that is 5 to 10 times greater than conventional TLC for major components, and even more for normally undetectable trace components and longer developments. The PMD story, complete with photographs and diagrams, is presented in a new booklet entitled "HOW TO GET BETTER TLC RESULTS". The booklet is free upon request. Also, each month Regis issues "PMD NEWS," a newsletter devoted exclusively to PMD applications and instrumentation. For a copy of the booklet "HOW TO GET BETTER TLC RESULTS'' and/or to receive the free monthly newsletter, "PMD NEWS," write or phone the Regis Professional Service Department.
.
The same concentrating of the spot benefits qualitative and quantitative analysis. Figure 11 shows an improved chromatogram for the separation of four drug components. As already noted, Figures 6 and 7 demonstrate the independence of the final spot location from the location of the deposited spot. How marked or valuable these advantages are depends on the case at band, However, the advantages of PMD over conventional TLC tend to increase with increasing program length, hecause the spots are reconcentrated twice per cycle as they separate. Limitations There are, of course, various troubles and limitations encountered in PMD. An unfortunate one frequently encountered is sample thermal lahility, either real or potential. Solvent removal by gas and without heat is then required. This entails a special plate holder and gas. Much more important from the physical point of view, the lower resultant temperatures exponentially decrease solvent diffusivity. Either time or plate efficiency must then in some degree be sacrificed. Usually plate efficiency is given up rather than time. The final PMD spots, though now considerably broader than they might he, are still useful-
ly more concentrated and better separated than conventional TLC spots. However practical this outcome, it nevertheless remains painful to the physical chemist. Another trouhle intermittently encountered is curved solvent fronts. These afflict conventional TLC also but are more irritating in PMD because of spot alignment. The same mechanisms that reconcentrate spots also align them, freeing them from dependence on origin position-see, for example, Figures 6 and 7. The trouble is less serious than it might he, because the frequent use of standards is merely good practice, but the trouble does surely perplex. Among the limitations of PMD we must list newness, cost, and complexity. Acceptably illustrative documentation of PMD accumulates only very slowly, partly because the most striking PMD results are often proprietary. Also, the very advantages and characteristics of PMD that we have been discussing would obviously, to be used, require a change in approach and in routine; such change might well be upsetting.
Conclusions Conventional TLC can be inexpensive indeed and can yield immediate and excellent results even to the inexperienced, let alone the experienced and skilled professional. Although
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73A
TOTAL
TIC Figure 10. Chromatogram of quininespiked urine Silica Gel G, acetone, 5O-pl samples, PMD Mode 2, 7 cycles. Arrow marks quinine spots a: 5 pg/pl b: 2.5 p g l p l
c: 1.25 pg/pl d: 0.625 pg/pl e: Urine blank
(3) J. A. Perry and L. J. Glunz, J . Ass. Offic. Anal. Chem., 57, 832 (1974). (4) T. H. Jupille and J. A. Perry, J . Chromatogr., 99,231 (1974). (5) T. H . Jupille and H . M. McNair, Amer. Lab. (Sept. 1974). (6) T . H . Jupille and J. A. Perry, J . Chromatogr. Sci., in press. (7) E. Stahl, Ed., “Thin-Layer Chromatography: A Laboratory Handbook,” 2nd ed., Academic Press, New York, N.Y., 1972. (8) E. V.Truter, in “Advances in Chromatography,” J. C. Giddings and R. A. Keller, Eds., Vol 1, p p 113-52, Marcel Dekker, New York, N.Y., 1965. (9) L. Mazza, L. Sardo, and R. Frache, Ann. Chim. Rome, 57,1177 (1967). (IO) S. Turina, Z . Soljic, and V. Marjanovic, J . Chromatogr., 39, 81 (1969). (11) S. Turina and V. Jamnicki, Anal. Chem., 44, 1892 (1972). (12) M. Brenner, A. Niederwieser, G. Pataki, and R. Weber, in “Thin-Layer Chromatorranhv: A Laboratorv Handbook;” E. Stahl, Ed., 1st id., p p 75-133, Academic Press, New York, N.Y., 1965. (13) A. Niederwieser and C. C. Honegger, in “Advances in Chromatography,” J. C. Giddings and R. A. Keller, Eds., Vol 2, un 123-67. Marcel Dekker. New York. N.Y., 1966. (14) A. Jeanes, C. S. Wise, and R. J. Dimler, Anal. Chem., 23,415 (1951). (15) J. A. Thoma, ibid., 35, 214 (1963). (16) J. A. Thoma, in “Advances in Chromatography,’’J. C. Giddings and R A. Keller, Eds., Vol6, p p 61-117, Marcel Dekker, New York, Ii.Y., 1968. (17) J. C. Giddings, “Dynamics of Chromatography,” pp 254-55, Marcel Dekker, New York, K.Y., 1965. 118) J . C. Giddings, J . Chromatogr., 13. 301 (1964). 119) E . A. Moelwvn-Hughes. “Phvsical Chemistry,” 2 i d ed., p’ 729, Pergamon, 1961. New York, N.Y., (20) L. R. Snyder, “Principles of Adsorption Chromatography,” p 108, Marcel Dekker, New York. N.Y., 1968. (21) E. A. Moelwyn-Hughes, “Physical Chemistrv.” 2nd ed.. n 15. Perramon. New York, N.Y., 196f.
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PMD is not expensive compared to the average instrument-a densitometer, a GC-MS, or HPLC-its cost is fantastic compared to a coated microscope slide. Also, it requires, as the slide does not require, training and considerable insight. No instrument does well if used poorly, and PMD is no exception. Nor does it help that we do not know all the answers a priori. References (1) J. A. Perry, K. W. Haag, and L. J .
Glunz, J . Chromatogr. Sei., 11,447 (1973). (2) J. A. Perry, T . H . Jupille, and L. J . Glunz, Ind. Res., 16, 55 (1974).
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Figure 11. Improved resolution with PMD suggested by densitometric traces Slit width should not be more than 0.1 spot width. Conventional solvent: 1:l CHC13:ethylacetate. PMD solvent: 1O:l CHCI3:ethyl acetate (7 cycles, Mode 1 ) Traces reproduced courtesy of American Laboratory
permit savings over 30% and convenience. A 20 x 20 cm plate easily snaps to multiples of 5 cm (e.g., two 10 x 20 cm or four 5 x 20 cm plates).
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