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T H I N I A Y E R A H 0 GLASS P A P l R CHROMATOGRAPHY Cm 0. W I L L I A M S
Thin-layer --------. chromatography i s an a n a l y t i c a l method applying techniques of paper chromatography t o a t h i n l a y e r of adsorbents normally used in colwnn-chromatography. -This i s a r e l a t i v e l y new method of adsorption and p a r t i t i o n chrcjmatozraphy on a micro scale. Thin-layer chromtography i s an excellent supplenent t o t h e previously known methods of column, paper, ion-exchange and gas l i q u i d chrorcatography. Historically, the first attempts t o use a t h i n adsorbent l a y e r f o r chromatographic separations were described by Izmailov and Schraiber (8) over twenty years ago. These workers separated mixtures of organic compounds on layers of adsorbent powder, examining the rings produced under u l t r a v i o l e t l i g h t . After i t s introduction, very l i t t l e work was done with thin-layer chromatoGraphy u n t i l t h e period of 1949 t o 1951, A t t h i s time several vorkers (Meinherds and Ea11 (14); Kirchner, e t a l . (9) ) described the use of binding agents t o f i x t h e adsorbent
[email protected] t o t h e glass p l a t e , Actually the method remained i n obscurity u n t i l 1956 when Stahl described equipment and procedures f o r the preparation of chromatop l a t e s , and demonstrated the potential usefblness of thin-layer chromatography in t h e f r a c t i o n a t i o n of a wide variety of coupounds. Since equipment has become commercia1l.y available, thin-layer chroinatography has suddenly gained vide recognition. Very f e w laboratories, e6peciaU.y those engaged i n l i p i d research, a r e without t h i s valuable t o o l . I- I
Apparatus. The g l a s s p l a t e s a r e usually about 20 x 20 cm, b u t can be any s i z e depending on t h e dimensions of your applicator and developing tank. Thickness of the p l a t e i s very important with most types of applicators except the one based on the design of I h t t e r and Hof‘steSter (22)
The uniform coating of glass p l a t e s with a thin-layer of adsorbent requires t h e use of a s p e c i a l applicator. A t the present t h e r e a r e a t l e a s t three types of applicators on the marketa7D’C The equipment described by S t a h l i s probably the most widely used, but the applicator described by Xdtter and Hofstetter (22) i s advantageous i n a t l e a s t one l t h a t permits the respect. These l a t t e r workers have designed ~ lapparatus __ I^ .
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aCamg, A. G., Eomhurger S t r . 24, Nut’ienz, B. I,. Switzerland; , U.S. representative : Arthur H. Thomas Company, Philadelphia 5 , Pa. bC. Desaga h. b. H. Hauptstrasse 60, Heidelberg, Germany; U . S . representat i v e : C. A. BrinlaPann and Co., Inc., 115 CutterWll Road, Great Neck, Long Island, New York. CResearch Specialties Co., 200 South Garrard Blvd., Richmond, C a l i f ,
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regulation of t h e thickness of the adsorbent (about 250 microns desirable) regardless of t h e thickness of t h e g l a s s plates. Thus, inexpensive, good quality window glass may be used instead of p l a t e glass. Other applicators require the glass p l a t e s t o be of uniform thickness i f the adsorbent layer i s t o be t h e same on a l l plates. Preparative chromatograms may a l s o be obtained with t h i s applicator. A more v e r s a t i l e model of S t a h l ' s applicat o r has been produced, which permits one t o obtain uniform t h i n layers of any desired thickness between 250 U and ?.ma. This modified applicator is finding application in preparative work, but it also r e q a r e s t h e g l a s s p l a t e s t o be of uniform thickness, i f layers from p l a t e t o p l a t e are t o be uniform
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In addition t o t h e g l a s s p l a t e s and applicator, a chromatographic devgloping chamber, spotting template, desiccated storage cabinet and a 100 C drying w e n a r e e s s e n t i a l equipnent. Adsorbents used a r e about t h e same a s those used i n column chromatography, except in most cases a binder has been added. Adsorbents commercially a v a i k b l e contain 5 t o 15$ binder (usually celcinated calcium s u l f a t e ) , however, adsorbents can be o b t a b e d t h a t contain no binder. Work in our laboratory indicates t h a t a binder i s necessary i n the adsorbent only when t h e t u c k e r , preparative type, p l a t e s a r e being used. I * -
P a r t i c l e size and uniformity of the adsorbent powder a r e much more c r i t i c a l in thin-layer chromatography than in standard column chromatography. A standardized 200 mesh adsorbent i s preferable, The most widely used adsorbent is s i l i c a , s i l i c a g e l or s i l i c i c acid. These terms a r e often used synonymously i n the l i t e r a t u r e . They r e f e r t o powdered s o l i d s of t h e general formula Si02. X 0 which consists of porous, t h r e e dimensional siloxane ( t e t r a h e d r a l Si.0. i structures with surface silanol (Si-0H)group. There i s usually a mono o r multimolecular layer of adsorbed water on the adsorbent p a r t i c l e s . During t h e a c t i v a t i o n process, which is merely heating t h e chranatoplates a t about 105°-1200C, t h i s adsorbed "free water" i s reverslbly removed, S i l i c i c acid p l a t e s should not be exposed t o temperatures above 170%', because a t t h i s temperature t h e silanol groups Will begin condensing, l i b e r a t i n g t h e "fixed water", The degree of thermal degradation of the s i l a n o l groups increases with r i s i n g temperatures until, a t about llOO°C only SiOz remains. This thermal degradation i s r a t h e r i r r e v e r s i b l e , because it destroys pore s t r u c t u r e and permanently impairs adsorbent properties by reducing t h e number of active sites available for hydrogen bonding (26),
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S i l i c i c acid i s most frequently used f o r the separation of n e u t r a l and acidic lipids. The s i l i c i c acid is s l u r r i e d with d i s t i l l e d water in a r a t i o of 1:2 W/V and applied t o t h e p l a t e s in a uniform layer. S t a h l (21) prepared a c i d i c s i l i c i c acid chromatoplates by using aqueous 0.5 N oxalic acid solution instead of water i n making t h e slurry. This same worker a l s o prepared alkallne chromatoplates in a similar manner u t i l i z i n g aqueous 0.5 N KoE, Mangold and Kamereck (13) used chromatoplates of s i l i c i c acid containing about lo$ ammonium s u l f a t e f o r t h e separation of phospholipids and strongly a c i d i c f a t t y acid derivatives. These layers prepared in basic OT a c i d i c solutions do not adhere w e l l t o t h e glass plates. It i s recommended that these p l a t e s be dried a t room temperature f o r several hours before they a r e placed in t h e drying oven.
151. Diatomaceous e a r t h or Kieselguhr i s especially suited for p a r t i t i o n separations of substances which a r e strongly hydrophilic or Amphoteric. After impregnation, i’k can a l s o be used a s an i n e r t c a r r i e r f o r the separation of hydrophobic substances. Like s i l i c i c acid diatomaceous e a r t h i s applied t o t h e p l a t e s a s a s l u r r y (1:2 W/Vj made with d i s t i l l e d water. Weill and Hanbe (24) used t h i s adsorbent f o r the fractionation of Malto-oligosaccharides. A l u m i n u m oxide (alumina) i s r a r e l y used for the chromatography of complex mixtures because it causes hydrolysis of e s t e r linkages and isomerizations of double bonds. However, alumina i s superior t o any other adsorbent for t h e chromatographic fractionation of t h e Vitamin A group, hydrocarbons and various classes of l i p i d s , especially the basic ones. It i s also often used for t h e chromatography of s t e r o l s (16).
Other adsorbents such as magnesium oxide, magnesium carbonate, calcium hydroxide, calcium carbonate, dicalcium phosphate, f l o r i s i l cellulose and many others have been described i n t h e l i t e r a t u r e (l2j Almost any adsorbent used i n column chromatography may be adapted t o t h i n layer chromatography
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Reversed-phase p a r t i t i o n TIC has been used i n resolving a l i p i d c l a s s i n t o i t s individual constitueats. T h i s i s done by impregnating t h i n layers of adsorbent with s i l i c o n e (11)or sane other hydrophobic substance.
Ion exchange TIC has not been used a s extensive a s adsorption
TLC, however, I can see no reason whyr the present ion exchangers could not
Ecteola cellulose bound t o glass p l a t e s by collodion was used by Randerath (20) t o separate various purine and pyrimidine nucleotides.
be adapted t o this technique.
Selection of the solvent i s based upon the w e l l known principles of adsorption chromatography. Polar solvents cause a greater migration than sa%le-spo”ctraveg ) of a non-polar solvents or t h e Rf value Distance solveni; front t r a v e l s substance increases with increasing p o l a r i t y of the solvent. Frequently, it i s convenient t o adjust t h e p o l a r i t y of t h e solvent by adding small amounts of a polar solvent t o a non-polar solvent. Generally this i s the case, b u t it i s recommended t h a t these solvent mixtures be kept as simple a s possible f o r b e t t e r reproducability (12) P I -
The solvent required f o r good separation depends on t h e type and number of functional groups i n t h e compounds being separated. Hydrocarbons a r e adsorbed very l i g h t l y and require a low p o l a r i t y solvent for t h e i r separation. Ir“ functional groups a r e introduced i n t o a hydrocarbon, t h e adsorption a f f i n i t y increases i n t h e following sequence: R cH?, R-O-alkyl, R-C = 0, R NHz, R OH, R COOH. Within each of t h e above c asses, saturated compounds a r e eluted e a s i e r than unsaturated compounds. Polyunsaturated compounds with i s o l a t e d double bonds a r e more e a s i l y eluted than those containing a conjugated system of double bonds. Cis-isomers a r e more e a s i l y eluted than t h e i r t r a n s counterparts.
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The a c t u a l selection of a solvent system t o nee% a p a r t i c u l a r need may be accomplished by using a simple spot test and a thorough knowledge of
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the order of solvents. The spot test is carried out by placing a drop of sample on t h e layer and a f t e r drying, slowly adding solvent fran a micropipette. Tbe movement of t h e sample i s then observed. In the case of an unknown mixture, the spot test is s t a r t e d by using a solvent of medium polarity. If t h e sample remeins in the v i c i n i t y o f t h e s t a r t i n g spot, then a stronger solvent must be used. E migration is t o o f a s t then a weaker solvent mixture must be employed. By t h i s method one can s e l e c t the proper solvent mixture with a minimum of extra work.
xisualization and I d E t i f i c a t f a n . Many chromagenic spray reagents, In commollly employed i n paper chrwt-phy, can a l s o be applied t o TIL!. addition, a number of corrosive sprays such a s concentrated which cannot be used on paper, can be u t i l i z e d on these A f t e r spraying with these oxidizing acids, the chromatoplate i s heated and the coanpounds present can be see0 as dark spots OD a l i g h t background. Spray reagents such a s 1, 4 dichlorofluorescein or Rhodamine 6 G may be used i n conjunction with an u l t r a v i o l e t lamp. Fluorescent chemicals and oxidizing acids may be incorporated i n t h e adsorbent layer f o r visualizing spots, and this eliminates t h e necessity of spraying. Autoradiographs a r e readily obtained when chrmatoplates cont a i n i n g radioactive labeled substances a r e exposed t o No-Screen Medical X-Ray Safety Film.
Documentation of Chrcaaatopams. Many people consider documentat i o n t o be a major disadvantage with this technique. We have not found t h i s t o be true. We-record chromatograms by t r a c i n g them onto acetate paper or tracing them d i r e c t l y i n t o a laboratory manual. This gives a permanent record Of' ell chromatograms. Other methods of documentation t h a t a r e being used include photograpby, radiograpby and dipping the chrctlretagrams i n t o a solution of label glaze. After dipping a chromatogram in l a b e l glaze the adsorbent Layer i s pulled off the glass p l a t e as a film.
Thin Layer Chranatogr-.
Present methods f o r of thin-leyer chromatograms leave much t o be desired. Few of t h e methods a r e exact and others a r e impractical.
Rough estimations a r e obtained by e l u t i n g and weighing. This method i s p r a c t i c a l only i f individual spots contain from 20 t o 50 mg. of material. Spectrophotcmetry and c o l o r b e t r y may also be used f o r quantitative estiolstims a f t e r t h e sam@e is eluted from the adsorbent. When u s i n g these techniques, care must be taken t o assure removal of a l l t h e adsorbed substance. As t h e p o l a r i t y of a canpound increases elution becmes more d i f f i c u l t .
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Photodensitmetry a f t e r charring a s applied by P r i v e t t 9. (18) (19) gives good results in a range 5 t o 35 ug o f material, The spots a r e measured in a Photovolt Pbotodensltcmeter with a stage attached f o r semiautanatic p l o t t i n g of curves. The quantitative estimation is made by measuring t h e area under t h e curve. Individual standard curves must be prepared f o r a l l campounds t h a t do not give spots of the same d e n s i t m e t r i c a l l y determined response (18)
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Measuring spot areas may be useful in supplementing t h e photodensitometric method, as it can be applied t o amounts of 30 t o 100 ug with an accuracy of f 576. Applications, Although it has been stated t h a t t h e greatest usefulness of TIC i s f o r t h e separation of l i p i d s and e s s e n t i z l o i l s , a b r i e f review of the l i t e r a t u r e reveals that it has extremely wide scope.
In t h e separation of l i p i d s , T U has been investigated quite extensively. Good resolutions have been obtained in the separation of s t e r o i d s (7) (lo), f a t soluble vitamins (Z), phospholipids (23) and crude preparations of various f a t s , o i l s and waxes. Basic fractionations between t h e various classes (aldehydes, ketones, acids etc.) of compounds have been obtained, These classes may be f u r t h e r separated according t o degree of unsaturation, chain length, and isomeric forms. Thin-layer chromatography has a l s o been employed i n t h e f r a c tionation of carbohydrates, amino acids, and various proteins. This technique has been applied t o the stu&j of various sulfur-and nitrogencontaining compounds such a s sulfa drugs (25) and nucleotides (20). Thin-layer chromatography i s a l s o being employed In the detection of food additives such a s antioxidants and coloring agents. Other f i e l d s such a s toxicology and pharmacology a r e using this technique f o r t h e rapid detection of impurities and poisons. S m a r y . Thin-layer chrcmatography i s achieved by f i r s t making a s l u r r y of t h e adsorbent, The slurry i s usually made with two p a r t s of d i s t u e d water and one p a r t adsorbent, but use could be made of various other solvents. T h i s slurry i s then applied uniformly t o glass p l a t e s with t h e a i d of a standard applicator. After the adsorbent has been applied, t h e p l a t e s a r e allowed t o dry a t room temperature, usually t h i s requires 5 t o 15 minutes. The p l a t e s a r e then placed i n a drying oven (usually 105120°C) f o r activation. Degree of activation depends on the length of time t h e p l a t e s a r e allowed t o remain i n the oven and t h e teuperature of t h e oven. The activated p l a t e s a r e now stored i n a desiccated container, u n t i l they a r e used. If t o o much moisture i s adsorbed, the p l a t e s may be r e a c t i vated. The materials t o be chromatographed a r e applied t o the adsorbent with a micropipette or a microsyringe. The spotted p l a t e s a r e then placed i n an equilibrated developing chamber containing t h e solvent. The solvent ascends in the adsorbent layer and separates t h e compound mixture a s it moves ,
The advantages of t h i s method a r e quite evident. Development i s much f a s t e r f o r thin-layer chromatography than f o r column or paper chromatography, The separating time, depending on solvent used and layer thickness, ranges from 20 t o 40 minutes f o r most applications. Only i n r a r e cases, such a s f o r amino acids, a r e longer developing times required. Simplicity i s another major advantage of t h i s technique. With a minimum of orientation good r e s u l t s can be obtained by ine,xperienced personnel, S e n s i t i v i t y and capacity a r e a l s o very important a t t r i b u t e s of t h i s technique. A m i n i m u m of 0.5 ug and up t o 500 ug, can be applied t o one spot,
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Among the r e l a t i v e l y few disadvantages i s t h e f a c t t h a t Rf values a r e not reproducible, which suggests the running of standards alongside the sample for comparison purposes. The quantitative removal and extraction of individual spots finom the p l a t e may a t times be more tedious than elution from paper chromatograms, Glass-Paper Chromatography T h i s i s a technique t h a t combines paper chromatography and thinlayer chromatography i n t o one technique, It makes use of a glass fiber f i l t e r paper irnprepated with an i n e r t phase such a s s i l i c i c acid. Development time i s much f a s t e r than paper chramatography and Rp values a r e more reproduc3bl.e than those of TU.
Sheets of glass f i b e r paper* of a size --C_hroma_to&rain fieparation. suitable f o r available equipment i s freed of organic impurities by heating i n an oven a t 600°C f o r one hour. Dieckert e t a l e (3) accomplished t h i s by placing the paper i n a borosilicate glass container with a loose f i t t i n g lid, This container was then placed i n an annealing oven and heated a t 6OO0C f o r one hour. I -
The impre@ating solution i s a potassium s i l i c a t e solution, which is prepared by the addition of concentrated KOH t o a slurry of s i l i c i c acid (6) have successfully used sodium s i l i c a t e for and water, Muldrey et the separation of some phosphorus containing l i p i d s , and these workers omitted the Hcl treatment, The Hcl treatment - t r i l l be explained l a t e r . After the s i l i c i c acid has dissolved, t h e solution i s f i l t e r e d .L;hrough a sintered glass filter t o remove insoluble debris, The f i l t e r e d solution i s then diluted t o about 0.4% (15) potassium s i l i c a t e . This solution has a If CO i s useful l i f e of about one week, because it readily absorbs CO absorbed, it i s held u n t i l the Hcl treatment of the chromato&arn and & t h i s time i s released a s a g a s . T h i s gas w i l l form undesirable pockets In t h e chromatogram (5)
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The sheets of glass paper free of organic impurities a r e then dipped i n the 0.49 potassium s i l i c a t e solution and allowed t o dry i n an oven. Some workers stroke the dipped glass paper with a glass rod t o remove the excess impregnating solution, After t h e imgregnated strips a r e thoroughly dry, they a r e treated Hitb 4 N Hcl for 5 minutes. The acid treatment precipitates t h e potassium s i l i c a t e a s s i l i c i c acid. The impregnated chromatogram is washed with d i s t i l l e d water u n t i l a l l acid is removed. The chromatogram i s then dried in 8 100°C oven and stored in a closed container u n t i l used (3),
The basic principles of t h i s technique a r e quite similar t o those discussed e a r l i e r f o r TLC, Selection of a solvent i s about the same as t h a t for T U or paper chrmatograplqy, Visualization of the developed chromatogram i s a l s o very similar t o T U , I ~ a ~ e v e rit , has the d e f i n i t e sdvantage over paper chromatography i n t h a t the use of corrosive sprays i s possible,
*E, Reene Angel and Cos,
52 Duene St., New York, M.Y.
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Applications, --Most l i t e r a h r e concerning the use of glass-paper chromatography deals with the separation of l i p i d s , however, Dieckert e t a l . (3)(4) have successfully separated sugars a s well a s l i p i d s with t h i s technique. This same group of workers have reported t h e separation of glycerides, cholesterol and cholesteryl e s t e r s by glass-paper chromatography (5)(4). Ory (17) has a l s o reported the separation of glycerides of mixed f a t t y acid composition by glass paper chromatography. Dieckert et (6), and Muldrey e t al . (15) have used t h i s technique foi: the fractionation of phosphorus containing l i p i d s .
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Very l i t t l e work has been reported on the glass-paper chromatographic separation of proteins. Arimura and Dingman (1)have reported quite good success with the technique when it i s used a s an assay for vasopressin and oxytocin.
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MR. PEARSON: This morning our discussion so far has centered upon chranatographic techniques. 3basauch as we want to break up the morning somewhat more evenly, we would l i k e t o go ahead with the next paper which is quite a d i f f e r e n t tqic--and probably not so new--in that it bas been used for a great marly years. It deals with Spectrophotometric Methcds. This paper i s prepared by Dr. Damel E, GoU. and Dr, Harry E. Snyder of Iowa S t a t e University, and I am got o ask Dr. OOU- t o give the paper.
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