Topics in..
.
Chemical Instrumentation Fdiled by GALEN W. W I N G , Seton Hall University, So. Orange, N. J. 07079
These articles are intended to serve the reoders o f ~ ~JOURNAL rs by calling attention lo new developments in the theory, design, or availability of chemical laboralory inslrumentation, or by presenting useful insights and ezplanalions of topics that are of practical importance to those who use, or teach the use of, modern instrumatatiun and instrumental techniques. The editor invites correspondence from prospective contributors.
LVIII. lnstrumentation for Thin-Layer Chromatography PETER F. LOTT, Chemistry Department, University of Missouri-Kansas City, Kansas City, Missouri 641 10, and ROBERT J. HURTUBISE. Chos Pfizer & Co., Inc., Terre Hade, Indiana
Thin-Layer Chromatography Virtually all methods of separation require the distrihobion of & component between two different phases. Thin-layer chromatography is in general one of the simplest, fastest, and most widely applicable chromxtographic techniques. I n the uwal thin-layer chromatographic process, very small drops of a s d ~ i t i o n are applied to a thin layer of an adsorhent surface that ha5 been coated on a solid support oalled a plate. Generally the chromatophte (plate coated with solid adsorhent) has been activated a t 100250°C before the sample is applied ( 1 ) . After the solvent is $lowed to dry, the spotted chromatoplate is placed in a closed container whose atmosphere has been saturated previously with vapor from a small amount of the eluting solvent, and enough of the eluting solvent is left in the container to wet the hottom of the plate. By capillary action, the fiolvent travels up the chromatoplate. When the solvent reaches the sample, separation begins. As the solvent and components travel up the chrom~ltoplate, the individual components then continuously undergo a combination of adsorption, ion-exchange, and partition chromatography with one type of chromatography predominating. The process is terminated by removing the chromatoplate from the container aiter the solvent has migrated a sufficient distance, perhaps 10 em. Accordingly, a separation has taken place if a selective retardation has prevailed. This retardation not only depends upon the particular characteristics of the substance being separated, but d s a on its interaction with the adsorptive surface and its distribution in the eluting solvent plus other factors (8). The separated components are distributed on the chromatoplate as a series of spots.
Rr Values The ratio of the distance of migration of the component to the distance the solvent has travelled in the same time is known as the Rr (rate of flow) value. Rr values range from zero to unity and serve a5 a semiqualitative means far the identification of substances. Because Rr values can vary through factors that cannot he controlled, i t is good practice to run a known substance with the unknown mixture on the same chromstoplste. In this way the known and unknown substances are submitted t o the same experimental conditions and relative Rr values can he calculated. An unknown compound may also be identified by spotting the unknown and a genuine sample on the same area of the plate. If after developing with several different solvents a single spot is obtained each time, the compounds are probably identical. The reproducibility of Rr values depends on many factors such as quality of the layer material, humidity, layer thickness, and development distance. For the separation of substances with similar Rr values, the use of wedge-strips, V-shaped wedge-strips, and two-dimensional chromatography has been very useful (3). Tailing of spots is sometimes a problem and reduces the efficiency of the separation. Tailing can he reduced' by usingsmaller quantities of sample; also, the eluting- solvent and adsorbent can be altered. Tailing very often occurs if the spot migrates too rapidly L ~ the I plate.
Applications of TLC Numerous new TLC applications have appeared recently. For example, a new TLC method for the qualitative detection and quantitative determination of microgram amounts of the commercial food antioxidant BHA (2- and 3-terl-butyl-4-
Dr.Peter P. Lott obtained his B.R. and M.S. degrees from St. Lawrence Tlniver3ity (1'349.19501,and his 1'11.D. from the Universitv of Conneotieut l195Di. He ?as been iesenrch chemist with dupont tnd with the Pure Carbon Company. 3 e has taught s t The University of \fissouri in Rolls, and at St. John's Jniversity. Jamaica. N. Y. He was nfluential in establishing a doctoral pro:ram in analytical ohemistry at the Uni,ersity of Missouri-Kansas City, where ie presently teaches. His research inwests are in the fields of analytical and 8hysiod ohemistry. He has published %any papers, including several eonributions to "Topics in Chemical Instru"entation!'
Dr. Robert J. Hurtibise rereivril his 3.S. (1004) imd h l . S. (I!)Iili)i n clie~n~i~try
rom Xwier Ilniversity. Cinr.innnti. Ihio, and his P h D . (19G9) in iu~alytical hemistry from Ohio TJniversity. He ,reviously was assistant professol. of hemistry at Roekhurst College in Kansas Xty, Mo. Presently he is employed by :has. Pfizer & Co., h e . , at Terre Hmte. nd. His research interests include umineseence analysis, separation methds, environmentd pollution, and kinetic lethods of analysis.
(Continued on page A458)
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Chemical Instrumentation
Table 1.
Suppliers of Adsorbents a n d Precoated Plates Preeoated layers on-
Powders--.,dllass-,-Plastichydroxyanisole) in breakfast cereals has been developed (4). TLC has been used to isolite eleven crystalline wmponents of expressed lime oil (5). The Rr values of isolated components were one means used for identification. Many other methods have been developed (6). Thin-layer chromatography permits the separation of samples from the microgram to the gram range. The procedure is both selective and sensitive; inorganic as well as organic compounds from low molecular weight to high polymers o m be separated. A separation is accomplished usually if a solvent bath dissolves the sample and selectively retards the components in the sample. The solvent and adsorbent can be altered for improved separation; however, varying the solvent is generally favored before altering the adsorbent. Surface Coating of t h e Chromotoplote Mast critical to the TLC process is the surface coating of the chromstoplate. Generally in most TLC separations, adsorption and partition processes occur simultaneously. Conditions can be adjusted to favor one process over the other; most commonly, the sepnratian~are performed that favor the adsorption technique with the adsorbent serving = the stationary phase and the organic solvent as the mobile phase of the two-phase system. Silica gel, the most commonly nsed hnorhent, is employed to separate acidic or nentral compounds, and alumina genernlly for the separation of hasio compo~unds (7). Some componnd~ that have been separated are amino .acids, amines, sao dyes, alcohols, alkaloids, fatty acids, carotenes, food dyes, narcotia, steroids, sogars, tranquilizers, and vitnmins. Other commonly used adsorbent,~ are kieselgnhr and cellulose powder. Kieselguhr ha5 been found valuable for sep,zrations of compounds such as sugars, while rellulose powder ha? been employed for the separation of water soluble materials. With the latter adsorbent,, t.he sepavations tend to he slower and the procedure is more of n true partition process paralleling tho conditions of paper chromatography. The chromatoplate is the predominant factor in n soccessfnl chromatographic separation. Even with the same type of adsol.bent, conditions for t,he sepavation can be varied tremendorrsly. Silien gel, for oxnmple, can he plcpared at diBerent pH values and ooatod on plntes with different layer thickness. Pel.ticle sise can have a pronounced influence on sepamtioa. A chromat,oplnte prepared with a larger particle sire adsorbent generally develops more rapidly and may show less resolution. Keeping the particle sise of the fine adsalhent uniform is very much of a problem. Chromatographeir; who coat their own plates sometimes notice this nonuniformity (the, presence of undesirahk larger sise partxles) in the adnorbent when the seme amount of water added to a fresh hatch
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Analabs Analtech Applied Sci. Lahs: Baker, J. T.a Bio-Rad Labs. CAMAG Chemapol* Cheng-Chin Corning DPI (Eastman: GallardSchlesinger Gelman Instruments' Mache Nagel ~allin%rodtb Mann Research Merck Dmmstadta Pharmacia Quantum Reanal Reeve Angel Riedel de Haen Schleicher & Schuell Supelco VEB Farhenfabriken Wako Woelma
' Also various precoated
layers on metal. Also provides impregnated silica. or alumina.
of adsorbent produces a much more fluid slurry. Binders such as starch, calcium sulfate and polymeric materials such as polyamides, are employed so that the adso~.hentwill adhere readily to the plate. The amount and type of hinder added to the aqueous slurry of the adsorbent can affect greatly the separation as the binder may he preferentially adsorbed and deactivate the surface. Also, pretreatment conditions for the coated plate, such as the temperature for drying, may he an important factor. Consequently, it is a matter of debate whether or not to e m.~ l . o v.rec coated olates or if it is better for the experimedter to coat his awn plates. Strong arguments can be offered for both viewpoints. Many precosted plates are coated on a flexible hacking such as a thin aluminum or a plastic sheet which allows them to he cut into smaller strips for testing a method and for ease in storing the developed chromatograms for later reference. The Gelman "Instant TLC" material consists of the adsorbent impregnated into a. glass fiber support. The precoated plates possibly tend to be more uniform in coating thickness and more reproducible from hatch to hatch. Furthermore, it is unnecessary to purchase a plate coater for TLC work. On the other hand, the experimenter making his own plates can vary the type of hinder, choose adsorhents from many manufacturers, and prepare, if he wishes, any type of adsorbent mixture. The adsorbent is usually cdated an glass plates.
Preparative thin-layer work, where larger quantities of materials are separated, requires chromatoplatefi that are prepared nearly binder-free and, because of the fragility of the surface coating, need to be prepared in the laboratory. The worker making his own chromatoplates needs not only s. plate caater but also the patience to wash the plates scrupulously clean prior to coating. Users of large numbers of chramatoplstes may also find it cheaper to make their own chromstoplstes. In Table 1 are listed sources of adsorhents and precoated plates. Plate Cooters For those who desire to prepare their own chromntoplates, the sophistication of plate coaters varies considerably. The simplest vemions, such as introduced in the Mallinckrodt kit, utilise a. piece of trvpe dong two side8 of the plate for height adjustment and a. glass rod to spread the slurry. Such units are quite satisfactory for producing an occasional chromataplate. The more common practical plate coaters consist primarily of two types: The Desaga type, distrjbuted by Brinkman Instruments, and the type distributed by CAMAG, Inc. In the Desaga unit, the hopper containing the adsorbent is pulled over a row of glass plates (Fig. 1). Variations of this type of plate coater are the "Quickfit" which employs s. lever arrangement to affix all the glass plates in the row against a flat surface, and the (Cvntinued on page A440)
Chemical Instrumentation
is evaporated. For the above three methods, it is doubtful whether a very uniform thickness of the adsorbent layer can be obtainrid.
Activation Once a chromatoplate of silica gel or alumina is ready for use it is usually heated between 110"-2.50" for about 1 hr. This procedure is called activation. Plates coated with cellulose need no activation became partition chromatography predominates, and they may be dried s t room temperature. The Rr values vary according to the degree of activation with silica. eel and alumina because adsorption chromatography predominates.
-
Figure 1. plicator.
Derago (Brinkmann) Adjustable Ap-
Reeve Angel model that. employs an inflatable hag to press the glass plates against a guide row over which the hopper slides. The CAMAG type plate coator, in contrast, uses a fixed hopper that is raised in a. vertical direction. The plates are slid one-by-one under the hopper. The unit is offered both in 8. manual and automatic model (Fig. 2). A large number of firms distribute plate coaters, as shown in Table 2. The efficiency of a plate coster can be e d y judged by a prospective user. The coater should make plates that are uniform in thickness ovkr the whole surface and should not have a leading or trailing edge. Provisions should alsa be made in the coater so that plates of different layer thickness may be prepared.
Application of Sample When a solution of a mixture is applied to the chromatoplate, the solvent is allowed to dry between successive applications. In some cases, drying may be hastened by using a hair dryer or a stream of filtered sir. Generally a few milligrams of a mixture can be chromatographed on a layer of silica gel 250 pm thick, with alumina layers about a tenth of a milligram, and kieselguhr about 25 to 60 pg. On cellulose layers the quantity that can be applied is relatively small.
Figure 3.
Figure 2.
CAMAG mutomatis TLC mater.
O t h e ~methods of preparing chromw toplates are pouring, spraying, and dipping. "Poured" plates are not as reproducible as "spread" plates and it is doubtful whether the pouring method will become papular. The spraying method is useful far coating a large number of small plates (microscope slides). With the dipping method a pair of plates is held together and dipped into a slurry of the adsorbent and then withdrawn. The plates are separated and the solvent Table 2.
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CAMAG Chromat~shor~er.
For qoditative work the sample can be applied with micropipettes or melting point capillaries. For quantitative work a micrometer pipette can be used, far example, the "Agls" micrometer syringe (Burroughs Wellcome and Co., London, England). Hamilton syringes may alsa be used and are very popular (Hamilton Co., Ine.); they are available from a number of scientific supply houses. For preparative thin-layer chromatography, the sample is usually applied as a wide band. Wide b,and applicators, as indicated in Figure 3, are available for this purpose. Devices for simultaneous multiple spot application are avdable. The Morgan applicator can be obtained from A. H. Thomas Co. and a multinle
Commercially Available Plate Cooters
Stationary Hopper Baird and Tatlook (London), Ltd. CAMAG, Inc. Labor Muszeripari Murek Pleuger Toueart et Matignon Movable Hopper Applied Science Laboratories, Inc. Brinkmann Instruments, Inc. Chemetron Milauo Kensington Scientific Carp. Quickfit Inc. Shandon Scientific Co.
Figure 4.
CAMAG sample-spotting guide.
spot applicator is avail.ilable through the Kensington Scientific Corporation. A number of spotting guides can be purchased thst allow samples to be applied in a straight line, reference lines to be drawn, and length measurements to be made. A number of manufacturers have avsilahle transparent plastic spatting guides. The CAMAG guide is illustrated in Figure 4.
Eluting Solvent Once the sample is spotted the chrome toplate is placed in a. developing container thst has been presatursted with vapor from the eluting solvent. Presaturation shortens developing time and helps eliminate the "edge effect." The choice of eluting solvent is aided by referring to an elutropic series of solvents. The series lists solvents in order of increasing polarity which is usually in the same order as increasing elutive power. Generally with a. given compound the more polar the solvent the greater the migration of the compound. By spotting the unknown on 8. series of small chromatoplates and developing the chromatoplates in a series of eight or so solvents, the choice of eluting solvent can easily be made by noting the distance the unknown migrates. Also, a guideline the chromatographer can use is "like dissolves like." For example, if an unknown is soluble in hexme but insoluble in ethanol, then the choice of elue ing solvents is narrowed. A typical eluotropic series in order of increasing eluting power follows (8). Approximate Dielectric Constant n-Hexme Cyclohexane Carbon betrachloride Bensene Toluene Trichloroethylene Diethylether Chloroform Ethyl acetate 1-Butanol 1-Proprtnol
-
Ardnne . . ... ...
1.9 2.0
2.2 2.3 2.4 3.4 4.3 4.8 6.0
17 20 21
Ethanol Methanol Water
Development U~uallychromatoplates m e developed by the ascending technique, in circular or rectangular chamhers. One type of rectangular vessel is illustrated in Figure 5. To assure a. fully saturated stmosphere, the walls of the chamber can be lined with fdter paper and the chamber shaken with developing solvent in the chamber. The chamber can then be allowed to stand one or more hours before the chromstaplates are inserted. The sandwich-type developing chamber is illustrated in Figure 6. This type of chamber requires less developing solvent and the limited space means good vapor s a t u r e tion. Suitable chambers can he obtained from any firm that upp plies thin-layer chromatography equipment. (Cmztinued on page A44B) Volume
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Chemical instrumentation
absorb ultraviolet light may give off visible fluorescence and can be readily deteoted. If the adsorbent layer is
ultraviolet iight usnaliy csn be detected. The compounds appear as dark spots against a fluorescent background because they quench the fluorescence of the indicator. Plstes aoated with aluminum oxide, cellulose, kieselguhr, polyamide, magnesium silicate and silica gel can he obtained with a. fluorescent indicator from eomvanies such ss J. T. Baker. Brink-
no color, do not absorb in the ultraviolet, or show fluorescence, by carrying out chemical reactions on the chromatoplate. The reagents are applied by means of a sprayer like one illustrated in Figure 7. Figwe 5.
CAMAG development tank.
Visualization
If the compouents separated on a chromatoplate all have their own color, then there may be no problem locating the components. However, this situation usually does not occu~.. If the components are colol.less, then some means must m w t be employed to visualize them. Many organic compounds become visible under ultraviolet light. Either shorb wave ultraviolet light (254 nm) or longwave ultraviolet light (366 nm) can he used. For example, compounds that
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Journal o f Chemical Edvcotion
Figure 6. Sandwich development chomber, Dirtillation Products Industrier, Inc.
Care must be taken not to spray an excess of reagent because the spots may be washed off.
Figure 7.
CAMAG Loborotory reagent rprmy.
Iodine vapors are widely used to visualize organic compounds. Ciystals of iodine are placed in the bottom of a container; then the chromatoplate is placed in the container. The rate of iodine vapor evolution can he increased by placing the container and contents in a hot water bath or using heat from an infrared lamp. The compounds usually appear as brown spots. In many cases, the iodine sublimes from the chromatoplate after it is removed from the container, leaving the compounds unaltered. For compounds that absorb ultraviolet light a direct ~hotoprintcan be made. The chromatoplate is placed on copy paper and exposed to ultraviolet light. The photocopy is developed and the compounds are indicated by spots. Permanent records of the chromatogram can he made by tracing the spots on semitransparent paper, making a photoprint, or treating the plate with paraffin. A plastic dispersion that is sprayed on the chromatoplate tend then allowed to set is also rwdahle from Merck. Afterwards, the whole layer can he removed from the glass plate and stored.
Quantitative Thin-layer Measurements The best method of quantitatively determining the concentrt~tions of the substances separated by TLC is by a direct measurement of concentration on for such work. Beelluse the measurements are made either through or more generdly by reflection from the chromatoplate, the reproducibility of the measurements by such scanners is not as precise as the conventional spectrophotometric or fluorometric measurements. Scanners can operate in both the visible and ultraviolet region. With fluarometric scanners, ultraviolet light from a fluorometer is employed to excite the sample, and the fluorescent light emitted by the sample is then measured. The CAMAG scanner that is adaptable to the Turner Model 111 fluorometer is shown in Figure 9 and the optical diagram in Figure 10. As can be seen in the optical diagram, a small optical port restricts the measurements to only one segment of the chromatoplate. The excitation and emission light selections are made with conventional filters. The chromatoplate is t,raversed horisontally past this segment for a complete scanning and the emitted fluorescent light is gathered by a "light pipe" far detection by the fluorometer. After the chromatoplate has heen moved completely in a horizontal direction past the port, the chromatoplate is then moved vertically by hand, and another horizontal scan is taken. The fluorescent intensities of the spots are recorded; standard suhstrtnces are employed for a quantitative correlation between the recorded intensities and concentration. In a general sense all scanners operate somewhat on this same principle whereby the chromatoplate is measured one por-
Removal of Zones Removal of sones from the chromatoplate can he accomplished by scraping with an object such ss a raror hlade, or when flexible plates are used the spots can he cut out with scissoe. "Vacuum cleaner" type collectors, as indicated in Figure 8, are avdahle from a number of companies. The separated sones can he removed from the chromatoplate with eluting solvent, transferred to a spectrophotometer or spectrophotofluorometer cell, and characterized qualitat',rve1y or quantitatively by appropriate measurements.
Figure 9.
CAMAG-Turner TLC scanner.
t,ion at a time. Either the seannine head chromatoplate is moved under the scanning head. Quite commonly optical methods are employed unless the chromatographed substance is a. naturdly radioactive substance or s. labeled compound. The optical scanning detectors measure tho vifiihle reflectance from (or transmission through) the chromatoplate, ultraviolet excited fluorescence (as described in the CAMAG-Turner scanner), or fluorescence quenching. For the visible reflectance scanning measurements, the separated spot must first be visualized, if it is not colored. The methods of
Figure 8
Brinkrnann *pot collector,
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Chemical Instrumentation visualization such as spraying with reagents, acid charring, or iodine color developing have been previously described. The chromatoplate must be uniformly treated with the developing agent, as otherwise an uneven scan will he produced. This is particularly important when transmittance measurements through the chromatoplate are attempted with a scanning densitometer; here again the necessity of a uniform plate coating is readily ondemtood. Very commonly to cnt down such errors, the measurements are made on areas closely bordering the spot,, rather than over the chromatoplate ns s. whale. Reflectance type measurements are not as critical to t,he uniformity of the layer t,hirkness since the measnrements are made from the surface layer, rather than through the chromatoplate.
Ultraviolet scanners t,end to give good quantitative results, and they are capable of detecting quantities down to the nanogram range. The baselines are generally straight, as meitsurements are done usually in a reflectance manner so that layer thickness is not 8. critical factor. However, by their great sensitivity, they are more susceptible to "random noise" in the adsorbent layer. Such noise can be mused hy impurities in the chromatoplate or the developing solvent. The latter effect can he part.icdarly had if the impurities or the solvent fluoresce under the same excitation and emission conditions as the measured srthstance. Fluorer cent measurements also are susceptible to an additional error if the substance shows photochemical decomposition upon excitation. Flnorescence quenching me* sorements may also be performed. In this case, one uses x chromatoplate containing s. fluorescent indicator in the
Photomultiplier U.V.Source
Plate hol
binder (as previously described) and measures the decrease in fluorescence, the degree of quenching, caused by the separated suhstance. Snch measurements are very commonly employed. They again are very sensitive, but subject to the errors previously discussed. I n m y of the methods the find qosnlitatian of the substance is nsnslly made hy comparison against a calihrstion curve prepared with standards of known concentration run under identical conditions. The different methods have recently been reviewed (9). References (1) DEAN. J. A., "Chernioal Separation ~ e t h ode," Van Nostrand Reinhold Company, NewYork. N . Y . , 1969, pp. 182-199. (2) R * m e n * ~ ~ , K., "Thin-Layer Chroma. tography" (2nd ed.), Academic Press, New York. N. Y., 1966, pp. 7-11. (3) STAHL. E.. %n "Thin-Layer Chromatogmphy" (2nd ed.). Stahl, E., ed.. Springer Verlhg Ino., New York, N. Y., 1969, pp. 88-94. ~ , J., AND LATE, H. W.. J . Agl. (4) H n n ~ u s l s R. Food Chem., 18,377 (1970). ( 5 ) Lnm, H. W., AND MADBEN,B. C., A n d . Chem., 41, 1180 (1969). (6) GUNTER,Z., MOORE.R. B., AND SXERMI, J.. ANAL.CREU.,42, 352R (1970). (7) B o a a r ~ ~J., J., SCHWARTINO, A. E., AND Gnm~nR , . J.. "Introduction to Chrome togra~hy,'' Reinhold Book Corporation, New York, N. Y., 1968, pp. 39-83. (8) Trsoxes. T. N.. A N D BAITSHOLTB. A. D., American Laboiofory, May. 1970, p. 69. (9) LEFAn, M. 8.. AND LEWIB. A. D., Anal. Cham.. 42, No. 3, 79A (1970).
Plate Figure 10.
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Opticd diagram for the CAMAG-Turner scanner.
Journal of Chemicol Education
(To be e a c l u d e d in the August issue.)
