714
Anal. Chem. 1981, 53, 714-716
(14) Turiey, T. J.; Demas, J. N., unpublished results. (15) Peterson, s. H.; Demas, J. N.; Kennelly, T.; QfneY, H.; Novak, D. p. J . Phys. Chem. 1979, 83, 2991.
RECEIVED for review October 20,1980. Accepted January 12, 1981. We gratefully acknowledge support by the Air Force
Office of Scientific Research (78-3590)and the donors of the petroleum Research Fund, administered by the American Chemical Society. All calculations and plotting were carried out on the University of Virginia’s laser facility’s microcomputer which was purchased in part by the National Science Foundation Grant CHE 77-09296.
Controlled Migration in Thin-Layer Chromatography David Nurok Department of Chemistry, Indiana University- Purdue Universrly at Indlanapolis, Indianapolls, Indiana 46205
Thln-layer chromatography Is described in which the point of solvent application is moved during chromatographic development. For a given plate and solvent, the rate of solvent mlgration is dependent on the distance between the moving solvent applicator and the solvent front. This allows a plate of any length to be developed. This contrasts wlth conventional TLC In whlch plate length Is llmlted to about 20 cm. The technique is demonstrated wllh a simple dye mixture, whlch illustrates Its separatlng power.
Thin-layer chromatography (TLC) has classically been viewed as a useful, inexpensive and rapid technique for separating simple mixtures. Until recently, it has been considered a less useful technique than high-performance liquid chromatography (HPLC) for the following reasons: (1)Apparatus for quantitation was inferior to that available for HPLC. (2) Efficiencies obtainable on TLC plates were decidedly inferior to those found in a well-packed VPLC column. (3) The effective size of a TLC plate is limited because the rate of solvent migration is inversely proportional to the distance between solvent source and solvent front. This results in the rate of solvent migration becoming extremely slow after the solvent front has traveled W 2 0 cm. At this point, there is no benefit in further development as there is considerable loss in resolution due to diffusion. With the recent advent of high-performance TLC plates and high-quality spectrophotometric TLC scanners, the first two of the above points are no longer valid. In fact, for mgst simple mixtures consisting of less than about seven components, TLC is fully competitive with HPLC. Advantages that TLC have over HPLC include the fact that up to 76 samples can simultaneously be analyzed on a single TLC plate by use of suitable apparatus and that the optical properties of the development solvent can be ignored because solvent is removed before quantitation. This paper is addressed to overcoming the third limitation detailed above. The technique described below will allow more complex mixtures to be separated than is possible with classical TLC methods. It should enable TLC to take its place along side HPLC and capillary gas chromatography as a powerful and highly flexible modern analytical technique. EXPERIMENTAL SECTION Chromatography was performed on 20 X 10 cm Whatman HP-K HPTLC plates by use of toluene (Fisher Certified ACS) to separate the dye mixture (no. 646, Fotodyne, Inc., New Berlin,
WI). A Pyrex dish (10 in. X 6 in. X l3I4in.) was used as developing tank. The dish was lined with filter paper saturated in toluene and was covered with two glass plates. A 50-pL syringe (Hamilton No. 705) equipped with a 6 in. needle was used as sample applicator. The TLC plate was placed face up on the bottom of the tank and the syringe needle inserted in a in. gap between the cover plates so that the tip touched the plate surface. Conventional chromatography was performed by using the same TLC plates, solvent, and dye mixture. Development was in a closed TLC tank lined with filter paper.
RESULTS AND DISCUSSION The implicit assumption has always been made that the point of solvent application should remain constant throughout the development of a TLC plate. Normally this is achieved by having one edge of a TLC plate contact a pool of the solvent. Other techniques such as circular or anticircular chromatography also have a fixed solvent application point or area. All these methods result in the slowing of the solvent front which, as discussed above, results in very poor efficiencies when plates longer than 10-20 cm are used. These techniques are also very time-consuming due to the slow solvent advancement during the latter part of a TLC development. All of these disadvantages can be overcome if the application point (or area) is moved during development by having a movable solvent applicator. Then for a given TLC plate and solvent, the solvent front will move at a constant rate dependent only on the distance between the solvent applicator and the solvent front. The closer the applicator is to the solvent front, the more rapidly will the front move. With this technique, a TLC plate of a n y length can be developed. If it is considered that a HPTLC plate has the same efficiency per unit length as a HPLC column which generally is limited to about 1 m due to pressure considerations, it is clear that more theoretical plates can be generated by TLC than by HPLC even though elution time will generally be longer by TLC. In fact, if it is considered that HPTLC plates have about 20-30 times the efficiency per unit length than a capillary column for gas chromatography, it becomes clear that this technique may prove to be the most powerful of all chromatographic methods. It is expected that TLC plates of suitable configuration (see below) will have an extremely high number of theoretical plates. Controlled migration TLC (CMTLC) is suggested as a name for this technique. It will be necessary to dry that section of the plate that the solvent front has already traversed to obviate spot spreading due to diffusion. The suggested name for the intersection of solvent and dry plate behind the applicator is the solvent intersection line. Solvent will flow from the applicator in a
0003-2700/81/0353-0714$01.25/00 1981 American Chemical Society
715
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981
forward (desirable) direction toward the solvent front and in a backward (undesirable) direction toward the solvent intersection line. The forward to backward migration ratio can be maximized by keeping the distance from applicator to solvent front very much smaller than the distance from applicator to the solvent intersection line. 'rhus, if the former distance is 0.4 cm and latter distance is 3 cm, the ratio of migration rates will be approximately 1/(0.4):1/ (3); Le., the forward migration rate will be about 7.5 times greater than the backward rate which can be effectively ignored. Both the solvent intersection line and the solvent front can be defined by using two screens. The first screen would follow the applicator at a fixed distance to define the solvent intersection line, the second would lead the applicator by a short distance to define the solvent front. A stream of warm inert gas would be blown onto the plate behind the first applicator and in front of the second applicator to evaporate solvent. For achievement of maximum efficiency on a long plate there should be a series of applicators each generating a solvent front and followed by a solvent intersection line. These applicators could in principle be controlled by a computer using information provided by a spectrophotometric TLC scanner. A 1-m plate could have as many as 25 applicators working simultaneously if the distance from solvent front to solvent intersection line was about 4 cm. This will significantly decrease the separation time and will confer the advantage of many repeated developments. It is well established that on repeated development, spots are sharpened and resolution is in general improved. When a plate is redeveloped, the trailing edge of a spot contacts the solvent and moves before the leading edge moves. This results in a decrease of spot size. The most sophisticated technique of performing multiple developments in conventional TLC is called programmed multiple developments (1). In this technique the solvent path length is increased with each successive development. With sufficient developments, very good resolution between spots can be obtained, even when R, values are similar. However, as is obvious, the solvent path length can only be increased until it corresponds to the length of the TLC plate. As discussed in the introduction to this paper, the maximum plate length used in conventional TLC varies between 10 and 20 cm. After a sufficient number of developments over the same section of plate, spot resolution decreases, and eventually all spots merge into the solvent front at the end of the plate. In contrast, there is no theoretical limit to the length of plate used in CMTLC. All successive CMTLC developments lead to improved spot resolution provided that a sufficiently long plate is used. Thus, a higher resolution can be obtained with CMTLC than with any conventional form of multiple developments. CMTLC will also be a faster technique as can be demonstrated by the following argument. The relationship between the distance traveled by the solvent front and time is well approximated by the equation (2) 2'
= Kt
where z is the distance in mm between the solvent origin and the solvent front, t is the time in seconds, and K is the velocity constant in square millimeters per second. Consider the situation where two spots are partially separated after a certain number of multiple developments on a 10-cm plate. Let the trailing edge of the slower spot be 5 cm from the solvent origin and let the velocity constant for the system be 10 mmz/s. If the next development is performed by conventional TLC, the time taken for the solvent to reach the spot will be, t = 502/10 = 250 s = 4.2 min. This is essentially wasted time as no separation occurs during this period of the development. The total time for the development of the 10-cni plate is t = 1002/10 = 1000 s = 16.7 min.
-_.-
c
m 0
-0
l
0
i 1
l
t
2
l 3
i
l 4
~ 5
I
i 6
~
0_ _ -_
00
0
l
r 7
l 8
-~
i
I 9
i
I
10
i
t
11
Figure 1. CMTLC development of a dye mixture with seven applicator passes.
0
a.000 Q
0
0 c m l ~ l i l i l i il 1 1 1 i I i 1 i I 1 I i I i 0
1
2
3
4
5
6
7
8
9
10
11
Figure 2. Conventional development of a dye mixture: (a) one development: (b) two developments: (c) three developments.
Only 12.5 min of this time is used for the actual separation process. Now contrast the situation where the development of the same system is performed by CMTLC. If the applicator is placed 5 mm behind the trailing spot, the solvent will reach it after 52/10 = 2.5 s = 0.04 min. If the applicator was not moved from this initial position, the development would be complete after 552/10 = 302.5 s = 5.0 min. In actual practice the applicator would follow the spots which would result in an even shorter developmenttime. In this situation, CMTLC would be at least 3 times faster than conventional TLC. The actual degree to which CMTLC is faster than conventional TLC will of course depend on the position of the spots on the plate after the preceding development. Example of CMTLC Separation. A test of the CMTLC technique was performed by using a simple dye mixture. The TLC plate was scored into a small channel to restrict the flow of solvent to one direction. The applicator used was a hand-held 50-pL syringe. Successive passes were made on alternative sides of the channel. This has the effect of moving the spots toward the center of the channel. It is expected that a fully automatic system with a properly controlled rate of solvent flow and applicator advancement would give better results than those described here. Nevertheless a hand-held syringe is satisfactory for demonstrating the technique as is shown in Figure 1. This shows the separation of the dye mixture after seven CMTLC passes which together with drying takes a total of about 40 min. The time can probably be significantly reduced by improving the control of solvent flow and by optimizing the distance between the applicator and the solvent front. The separation could also have been improved by changing the solvent strength between successive developments. This and other aspects of the technique are currently being investigated and will be reported shortly. It is interesting to contrast the separation by CMTLC with that obtained with conventional TLC. Figure 2 shows the same separation system developed by conventional TLC. Figure 2a-c corresponds to one, two, and three replicate developments with toluene. It is seen that three developments completely separate all the spots in a total development time of 55 min which is considerably longer than the time required for the equivalent or better separation shown in Figure 1for CMTLC. The CMTLC spots are sharper than the conven-
~
i
~
~
~
716
-
Anal. Chem. 1881, 53,716-718
._
0
~0 _0 _
~
0
0
0
~ ~ l 1 1 ~ 1 1 1 1 1 1 1 1 1 1 1 , 1 1 1 , l , l , l , l , l , l , l , l ~ l , l , ~ 0 I 2 S 4 8 I 7 I 9 10 II 12 13 I4 15 11 11 11 19 20
Figure 3. CMTLC development of a dye mixture.
tional TLC spots due to the greater number of multiple developments involved. The power of the technique is well illustrated in Figure 3 which shows a chromatogram which took about 1 h to complete; i.e., the running time was approximately the same as for the conventional development shown in Figure 2c. In the latter stages of the development shown in Figure 3, only one spot was moved a t a time. This is achieved by starting each development with the applicator behind a spot to be moved. Care is taken to terminate each development before solvent touches the next spot of lower Rp The separation shown in Figure 3 would be achieved in a shorter time if several applicators were used in series together with controlled drying as discussed earlier. The spots in Figure 3 are between 2 and 3 mm in diameter. This would in principle allow between 60 and 100 spots to be separated on a 20-cm plate which is the longest HPTLC plate currently available commercially. Smaller spots could be achieved by having a large number of short developments for each spot. The above refers to separation performed in a saturated atmosphere with a single-componentsolvent. Similar results are obtained with a two-componentsolvent system consisting of 75% toluene and 25% heptane in either a saturated or unsaturated atmosphere. Other Considerations. An advantage of CMTLC is that optimum solvent strength may in principle be chosen for each component of a mixture. Consider a mixture in which several components have a Rf of 0 and others have a Rf of 1 when developed with a solvent of moderate solvent strength. An increase in solvent strength will move and separate the com-
ponents of low Rf but will cause more components to have an Rf of 1; Le., to travel with the solvent front. An inverse effect occurs when a solvent of decreased solvent strength is used. High Rf solutes no longer travel with the solvent front but more solutes of low mobility remain at the origin. This difficult problem for classical TLC can easily be solved by having a solvent gradient by CMTLC such that solvent strength decrease from very high to very low as the applicator traverse the plate; i.e., the solvent delivered to a particular section of a plate could be tailored to the separation of the compounds present in that section. The technique could be further refined to allow, for instance, a chiral resolving solvent to be delivered when the applicator passes a racemic mixture of compounds. A reactive solvent delivered to a specific section of the plate could even be used to derivatize certain compounds, to enhance either their separation or their detection. CMTLC will not be limited to development in a linear mode. If a circular channel was used, the effective path length could be of any length desired, as the same spot could move around the channel many times. Very long TLC channels could be scribed on the outside of a drum as with Edison’s original phonograph record or in a spiral as with the modern phonograph record. ACKNOWLEDGMENT The author thanks Jeff De Wester for valuable assistance in running the chromatograms shown in Figures 1-3. LITERATURE CITED (1) Perry, J. A,; Jupllle, H. H.; Glunz, L. J. A n d . Chem. 1975, 47, MA74A.
(2) Gulochon, G.; Bressolle, F.; Slouffl, A. J. Chromatogr. Scl., 1979, 77,
368-386.
RECEIVED for review July 7, 1980. Accepted December 12, 1980.
CORRESPONDENCE Oxygen-Induced Responses with a Fixed-Frequency Pulsed Electron Capture Detector for Gas Chromatography Sir: During the past few years we have reported several studies of the effects on the electron capture detector (ECD) for gas chromatography (GC) of oxygen intentionally added to the carrier gas (1-4). With relatively large amounts of oxygen added to the carrier gas, many normally poorly responding compounds give increased responses due to what is thought to be assistance provided by the negative ion, Of, in the capture of negative charge by the sample. Others (5-7) have also reported analysis procedures whereby improvements in sensitivity of the ECD are made by using 02-doped carrier gas. With one exception (5),the ECDs used in these studies were of the constant-current (CC) or frequency-modulated types (8,9),where the frequency of pulses applied to the cell is continuously varied to maintain a constant, preselected level of current. The frequency of pulses, rather than a current measurement, is then taken as the analytical signal. A detailed model of the 02-doped ECD and its response to samples has been described for the constant-current ECD (3). While the constant current approach is rapidly becoming
,
the ECD mode of choice due to its extended linear dynamic range, many instruments in use today are of the fixed-frequency (FF) type for which the response to sample is taken simply as a reduction in the measured current. One might expect that the oxygen-doping technique would be applicable to this ECD, also, but until now no comparative study of its operation with 02-doped carrier has been reported. In this correspondence we describe the application of a fixed-frequency ECD to the oxygen-enhanced response of methyl chloride, a compound which has been extensively studied with the constant current ECD (2,3). It will be shown that with the fixed-frequency ECD, much smaller oxygen-induced response enhancements are observed than with the constantcurrent ECD. Furthermore, it will be shown that this result for the fixed-frequencyECD is consistent with the model for the O2 enhancement process previously proposed (3). EXPERIMENTAL SECTION Two ECD systems have been used. The CC-ECD accompanies
0003-2700/81/0353-0716$01.25/00 1981 American Chemical Soclety
