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Justus G. Kirchner
201
JUSTUS G. KIRCHNER
JUSTUS GEORGE KIRCHNER was born i n 1911, i n Cedar Rapids, Iowa. H e r e c e i v e d a B.S. e m Zaude i n c h e m i s t r y from C r e i g h t o n U n i v e r s i t y a t Omaha, Nebraska i n 1935 and h i s Ph.D. from Iowa S t a t e C o l l e g e , a t A m e s , Iowa, i n 1939. I n t h e f a l l of t h a t y e a r he took a postdoct o r a t e assistantship at the California Instt u t e of Technology. A y e a r l a t e r he became a Research Fellow a t t h e I n s t i t u t e . I n 1945 he accepted a p o s i t i o n w i t h t h e United S t a t e s Department of A g r i c u l t u r e F r u i t and Vegetable Laboratory i n Los Angeles and remained w i t h t h e l a b o r a t o r y when i t r e l o c a t e d i n Pasadena, C a l i f o r n i a . I n 1954 he j o i n e d Tenco, an Ins t a n t Coffee Company, and a f t e r o r g a n i z i n g a Research Department, became D i r e c t o r of Research i n 1955. I n 1956 he w a s named D i r e c t o r of Research and Development f o r t h i s company which became a D i v i s i o n of t h e CocaCola Company i n 1961. I n 1968 h e moved t o A t l a n t a as S e n i o r S c i e n t i s t f o r t h e C o r p o r a t e D i v i s i o n of t h e Coca-Cola Company. D r . Kirchner ret i r e d i n 1976. D r . Kirchner i s t h e a u t h o r and c o a u t h o r of o v e r 30 s c i e n t i f i c p a p e r s and t h e a u t h o r o f two books and t h r e e book c h a p t e r s . Thin-Layer Chromatography, an e x t e n s i v e t r e a t i s e of t h e s u b j e c t , w a s p u b l i s h e d i n 1967; t h e e x t e n s i v e l y r e v i s e d second e d i t i o n i s scheduled f o r p u b l i c a t i o n i n 1978. I n 1975 he was p l e n a r y l e c t u r e r on t h i n - l a y e r chromatography f o r t h e F e d e r a t i o n of A n a l y t i c a l Chemistry and S p e c t r o s c o p i c S o c i e t i e s ' meeting. D r . Kirchner founded t h e present-day system of t h i n - l a y e r chromatography, t h e r e s u l t s of which were p u b l i s h e d i n 1951-1954. I n t h e s e publ i c a t i o n s he i n t r o d u c e d f o r t h e f i r s t t i m e a number of i m p o r t a n t t e c h n i q u e s now u n i v e r s a l l y u t i l i z e d i n t h i n - l a y e r chromatography.
202 In 1939 I was hired as a research assistant to Dr. Zechmeister at the California Institute of Technology, but s nce his arrival in this country was delayed, I worked with Dr. A.J. Haagen-Smit for one year. On Dr. Zechmeister's arrival the following year I was offered the opportunity of remaining with Dr. Haagen-Smi h to work on the chemistry of the aroma and flavor principles in pineapple, or of transferring to Dr. Zechmeister's laboratories. Work on the chemistry of flavor and aroma was in its infancy at that time, and I decided to remain with it. It was while on this project that I first became acquainted with chromatography, and developed a column-chromatographic separation method for thep-phenylphenacylesters of acids ( 1 ) . The publication of the work on pineapple flavors (2, 3 ) then led to an offer to work on the chemistry of orange and grapefruit flavors at the Fruit and Vegetable Laboratory of the United States Department of Agriculture. This was indeed a challenge at that time because gas chromatography had not been developed and the minute amounts of flavoring and aroma materials required a micro method for isolating and identifying these constituents. Even though large quantities of juice were processed (3,000 gallons each of fresh, freshly canned, and stored canned orange juice, 2,760 gallons of fresh grapefruit juice, and 2,470 gallons each of freshly canned and stored canned grapefruit juice) the amount of these flavor materials was exceedingly small. This problem of quantity is exemplified by the sulfur-containing ester found in pineapple flavor ( 3 ) where the fruit was found to contain 112 to 250 mg of sulfur per kilogram of fruit. My first thoughts were to use paper chromatography which had been used with such excellent results with amino acids, but it was soon evident that paper was much too mild an adsorbent to accomplish the required task. Flood ( 4 ) and Hopf ( 5 ) had used alumina-impregnated paper for spot tests and this soon led us to the development of the first silica gel impregnated paper for use in chromatography (6). This was produced by soaking filter paper in sodium silicate solution and then precipitating silicic acid in the fibers by immersing in hydrochloric acid. This showed promise and was used to separate some 2,4-dinitrophenylhydrazones of aldehydes and ketones which could not be separated on unimpregnated paper. However, the impregnated paper was still not the answer to our needs, its capacity was still not great enough. During this time when Chemical Abstracts were much less expensive than they are today, I would clip out abstracts which were of interest to me and file them away for future use. On this particular day I had come across the abstract of Meinhard and Hall's work (7) on drop chromatography for the separation of inorganic ions. I clipped the abstract and laid it on the corner of my desk. Later in the day one of my assistants came in very much discouraged with his attempts at separating terpene constituents by paper chromatography. I picked up the abstract and said "Here, let's make layers of silicic acid on strips of glass and develop them in an ascending manner analogous to paper chromatography." This latter step proved to be the key to the development of the successful system, now
203
Fig. 22.1. Fractionating citrus oils at the U.S.D.A. Fruit and Vegetable Laboratory, in Pasadena, California, circa 1950.
better known as "thin-layer chromatography", existing today. The method was first published in 1951 (8) and was used successfuly by a large number of research workers prior to 1956 (9a) when Egon Stahl began publishing his first work. Drop thin-layer chromatography had first been demonstrated by Beyerinck in 1889 ( 2 0 ) which even predates Tswett's excellent work as well as the work of Reed in 1893 ( 1 1 ) . However, this drop method as demonstrated by Beyerinck and practiced by Izmailov and Shraiber ( 2 2 ) as well as others was too limited in its scope for our puropose. After we had proved to our satisfaction that the method would work, the next task was that of finding the best adsorbent and the optimum conditions for making thin-layer chromatography reliable and reproducible. Of the sixteen adsorbents tested, silicic acid was selected a5 the best for our purpose. This was combined with Amioca starch (National Starch Products Co.) to give a satisfactory layer. Later ( 1 3 ) this binder was replaced to advantage with Clinco 15 modified starch (Clinton Foods, Inc.) or by a 2:l mixture of cornstarch and Superior AA tapioca flour (Stein Hall and Co., Inc.), and at a still later date ( 2 4 ) we found that the starch binder could be reduced to advantage from 5% to 24% in the mixture.
204
Fig. 2 2 . 2 . Separation of p-cymene, pulegone and cinnamaldehyde on silicic acid thin-layers with 15% ethyl acetate in hexane, as shown under ultraviolet light on fluorescent chromatostrips. (8). (Reproduced with permission of the American Chemical Society). From top to bottom: p-cymene, pulegone, cinnamaldehyde. From left to right: l=blank (spot due to traces of impurity in solvent not removed by distillation); 2=p-cymene (143 pg), pulegone (3.3 ug) and cinnamaldehyde (3.3 pg); J=p-cymene (358 pg), pulegone ( 8 . 2 pg) and cinnamaldehyde (9.1 pg); 4= p-cymene (1.4 mg), pulegone (32.8 pg) and cinnamaldehyde (36.4 pg). One of the earliest discoveries we made and reported was that in order to obtain reproducible results, conditions had to be carefully controlled (standardized). To obtain these results, the silicic acid was screened in order to remove the coarser particles and the layers were dried and held under carefully controlled conditions.
To further increase the reliability of results, it was established at that time that it was necessary to run standards to show that the layers were properly prepared and to serve as a reference for comparing RF values. Because most of the compounds with which we were working were colorless, it was necessary to develop methods for locating compounds. For ultraviolet absorbing compounds we adopted the technique of Sease (15) of incorporating inorganic fluorescent material with the adsorbent. Numerous spray reagents were then developed for other compounds, and in some cases these not only located the compound but also indicated the type of compound that was present. For example,the 2,4-dinitrophenylhydrazine spray disclosed the presence of carbonyl compounds and the dianisidine spray showed the presence of aldehyde groups. One of the more useful sprays was the 0 . 0 5 % fluorescein spray with subsequent exposure of the thin-layer to bromine vapor to detect unsaturated compounds or other compounds which would react with bromine vapor under the conditions used. There were some compounds, as for example, camphor, which could not be located by any of the methods thus far devised. I originated the idea of using plaster of Paris as a binder so the compounds could be sprayed with sulfuric acid containing an oxidizing agent. Thus on subsequent heating, the spot location was revealed by the charred areas. This was the origin of the so-called silica gel G, because when plaster of Paris sets, it takes up water of crystallization to form gypsum. Later this charring technique was developed by others into a quantitative analytical method. One of the techniques which wedevelopedand used for fractionating larger amounts was that of using thin-layer chromatography to monitor the fractions as they were eluted from a packed chromatographic column ( 1 6 ) . In this way by the time the column was completely eluted, it was possible to determine which fractions should be combined and which fractions were single compounds under the conditions of the separation used. Another column application was the use of thin-layer principles for the preparation of a self-supporting column unencumbered by a containing envelope ( 1 7 ) . This was designated as a "chromatobar", as it consisted of cylindrical or square bar of silicic acid bound with gypsum around a glass rod which added strength. This column was developed in an ascending manner and could be removed and checked for development by spraying with a visualizing agent on one side. The thin layer of sprayed material could then be scraped off and the column returned for further development. In addition to the use of narrow strips of adsorbent, we also introduced the use of square coated plates in our first publication (8); thus multiple samples could be run on a single plate or twodimensional chromatography could be carried out on an individual sample so as to give improved resolution. An important feature of our work included the introduction of the use of reactions on the thin-layer plate ( 1 8 ) . We used oxidations, reductions, dehydrations, hydrolysis reactions, and the preparation of derivatives. These could all be used to help in identifying a
206
Fig. 22.3 (left). Dr. Kirchner at the CAMAG First International Thin-Layer Chromatographic Symposium demonstrating preparative thin-layer chromatography without a backing plate to support the layer so that it is accessible from both sides for sample application and visualization. For details, see ref. 21. Fig. 22.4 (right). Separation of 100 mg each of six dyes on a 4 x 8 x 8 in. silica gel self-supporting layer with no backing plate. Top to bottom: Yellow OB, narrow line due to second solvent front, Sudan I , Sudan 111. Sudan 11, Methyl Red and Crystal Violet ( 2 1 ) . specific compound because the reaction products had different RF values than the original compounds. This technique is also useful in separating critical pairs of compounds that can not normally be separated by chromatography. This reaction technique proved to be very versatile as it may be applied to the initial spotted sample, or it may be applied after the chromatogram has been developed in one direction and prior to the development in a second dimension. Biphenyl is used by the citrus industry to prevent molding during shipping and storage of citrus fruit. Because there was a need for an accurate method for detecting and measuring the amount of biphenyl absorbed by the citrus fruit, we had the opportunity to apply the thin-layer technique to this problem and so introduced
207
the use of this versatile technique in the quantitative field. By using a spectrophotometric method on the eluted spots we were able to obtain results of +2.8% on amounts ranging from 0.1 to 600 ppm. This helped to establish the reliability of our thin-layer technique at that time. Another technique that we introduced because of our work with thin layers, was the chromatographic preparation of terpeneless oils ( 1 9 ) . I had noticed in running thin-layer chromatograms in hexane on silicic acid that this solvent moved the hydrocarbons, but not the oxygenated components which remained behind at the origin. A check of numerous compounds confirmed this observation and it was then a fairly simple matter to apply the technique using packed columns. On entering into industrial work in 1954, the opportunity was not available to continue research in chromatography although I managed to keep somewhat active in the field by producing a text on thin-layer chromatography ( 9 ) , which has just recently been revised (20). However, there was also an opportunity to develop a preparative thick-layer method ( 2 1 ) . Up to this time the thin-layer methods consisted in running numerous thin-layer chromatograms and combining the separated components until a sufficient quantity of material had been collected. The quantity that could be separated on an individual plate was limited by the thickness of the layer which ranged from 1 to 5 mm thick. The thicker the layer, the more difficult is is to prepare a layer without developing cracks on drying. For the 5 mm layers this resulted in only 50% of the layers being usable. It occurred to me that a thick-layer plate could perhaps be prepared based on the chromatobar technique. The first attempt at the preparation of a 1/4 in. layer without a backing plate was successful; however, the layers were rather fragile, especially when dry, with a tendency for the corners to break. This then led to the use of a stainless steel frame with 0.025 in. stainless steel wires stretched across the frame and imbedded in the layer to help support the latter. These wires in no way interfered with the chromatographic development. The layers were cast in a mold that had been carefully machined so that the layer thickness was extremely uniform. The main problem to be overcome was the tendency of the wet layer to stick to the top and bottom of the mold. Various materials and techniques were tried to solve this difficulty, and this was finally accomplished by covering the top and bottom of the mold with thin sheets of plastic so that the stainless steel plates could be easily removed; then the plastic was rolled back to expose the undisturbed layer. Layers ranging in thickness from 1/8 to 1/2 in. thick were successfully prepared and used. REFERENCES 1 J.G. K,irchner,A.N. Prater and A.J. Haagen-Smith, Ind. E n g . Chem. 18 (1946). 2 A.J. Haagen-Smith, J.G. Kirchner, A.N. Prater and C.L. Deasy, J . Amer. Chem. SOC. 67 (1945) 1646.
208 3 A.J.
Haagen-Smith, J . G .
Kirchner, C . L .
J . h e r . Chem. SOC. 67 (1945) 1651. 4 H. F l o o d , Anal. Chem. 120 (1940) 327. 5 P.P. Hopf, J . Chem. Soc. (1946) 785.
Deasy and A . N .
Prater,
6 J . G . K i r c h n e r and G . J . Keller, J . Amer. Chem. Soc. 7 2 (1950) 1867. 7 J . E . Meinhard and N.F. H a l l , Anal. Chem. 21 (1949) 185. 8 J . G . K i r c h n e r , J . M . Miller and G . J . Keller, Anal. Chem. 23 (1951) 420. 9 J.G. K i r c h n e r , Thin-Layer Chromatography, I n t e r s c i e n c e P u b l i s h e r s , N e w York, 1967; a, p. 5. 10 M.W. B e y e r i n c k , 2. Phys. Chern. 3 (1889) 110. 11 L. Reed, Proc. Chem. Soc. 9 (1893) 123. 12 N . A . Izmailov and M.S. S h r a i b e r , Farmatsiya ( S o f i a ) 3 (1938) 1. 13 J . G . K i r c h n e r , J . M . Miller .and R.G. R i c e , A g r . Food. Chem. 2 (1954) 1031. 14 J . G . K i r c h n e r and V.P. F l a n a g a n , Gordon Research Conference, Colby J u n i o r C o l l e g e , N e w London, N. H., August 1962. 15 J . W . S e a s e , J. Amer. Chem. Soc. 69 (1947) 2242. 16 J . M . Miller and J . G . K i r c h n e r , Anal. Chem. 24 (1952) 1480. 17 J . M . Miller and J . G . K i r c h n e r , Anal. Chem. 2 3 (1951) 428. 18 J . M . Miller and J . G . K i r c h n e r , Anal. Chem. 25 (1953) 1107. 19 J . G . K i r c h n e r and J . M . M i l l e r , Ind. Eng. Chem. 44 (1952) 318. 20 J . G . K i r c h n e r , Thin-Layer Chromatography, 2nd e d . , W i l e y - I n t e r s c i e n c e , N e w York, 1978. 21 J . G . K i r c h n e r , J. Chromatogr. 63 (1971) 45.
JUSTUS G. KIRCHNER
JUSTUS GEORGE KIRCHNER was born i n 1911, i n Cedar Rapids, Iowa. H e r e c e i v e d a B.S. e m Zaude i n c h e m i s t r y from C r e i g h t o n U n i v e r s i t y a t Omaha, Nebraska i n 1935 and h i s Ph.D. from Iowa S t a t e C o l l e g e , a t A m e s , Iowa, i n 1939. I n t h e f a l l of t h a t y e a r he took a postdoct o r a t e assistantship at the California Instt u t e of Technology. A y e a r l a t e r he became a Research Fellow a t t h e I n s t i t u t e . I n 1945 he accepted a p o s i t i o n w i t h t h e United S t a t e s Department of A g r i c u l t u r e F r u i t and Vegetable Laboratory i n Los Angeles and remained w i t h t h e l a b o r a t o r y when i t r e l o c a t e d i n Pasadena, C a l i f o r n i a . I n 1954 he j o i n e d Tenco, an Ins t a n t Coffee Company, and a f t e r o r g a n i z i n g a Research Department, became D i r e c t o r of Research i n 1955. I n 1956 he w a s named D i r e c t o r of Research and Development f o r t h i s company which became a D i v i s i o n of t h e CocaCola Company i n 1961. I n 1968 h e moved t o A t l a n t a as S e n i o r S c i e n t i s t f o r t h e C o r p o r a t e D i v i s i o n of t h e Coca-Cola Company. D r . Kirchner ret i r e d i n 1976. D r . Kirchner i s t h e a u t h o r and c o a u t h o r of o v e r 30 s c i e n t i f i c p a p e r s and t h e a u t h o r o f two books and t h r e e book c h a p t e r s . Thin-Layer Chromatography, an e x t e n s i v e t r e a t i s e of t h e s u b j e c t , w a s p u b l i s h e d i n 1967; t h e e x t e n s i v e l y r e v i s e d second e d i t i o n i s scheduled f o r p u b l i c a t i o n i n 1978. I n 1975 he was p l e n a r y l e c t u r e r on t h i n - l a y e r chromatography f o r t h e F e d e r a t i o n of A n a l y t i c a l Chemistry and S p e c t r o s c o p i c S o c i e t i e s ' meeting. D r . Kirchner founded t h e present-day system of t h i n - l a y e r chromatography, t h e r e s u l t s of which were p u b l i s h e d i n 1951-1954. I n t h e s e publ i c a t i o n s he i n t r o d u c e d f o r t h e f i r s t t i m e a number of i m p o r t a n t t e c h n i q u e s now u n i v e r s a l l y u t i l i z e d i n t h i n - l a y e r chromatography.
202 In 1939 I was hired as a research assistant to Dr. Zechmeister at the California Institute of Technology, but s nce his arrival in this country was delayed, I worked with Dr. A.J. Haagen-Smit for one year. On Dr. Zechmeister's arrival the following year I was offered the opportunity of remaining with Dr. Haagen-Smi h to work on the chemistry of the aroma and flavor principles in pineapple, or of transferring to Dr. Zechmeister's laboratories. Work on the chemistry of flavor and aroma was in its infancy at that time, and I decided to remain with it. It was while on this project that I first became acquainted with chromatography, and developed a column-chromatographic separation method for thep-phenylphenacylesters of acids ( 1 ) . The publication of the work on pineapple flavors (2, 3 ) then led to an offer to work on the chemistry of orange and grapefruit flavors at the Fruit and Vegetable Laboratory of the United States Department of Agriculture. This was indeed a challenge at that time because gas chromatography had not been developed and the minute amounts of flavoring and aroma materials required a micro method for isolating and identifying these constituents. Even though large quantities of juice were processed (3,000 gallons each of fresh, freshly canned, and stored canned orange juice, 2,760 gallons of fresh grapefruit juice, and 2,470 gallons each of freshly canned and stored canned grapefruit juice) the amount of these flavor materials was exceedingly small. This problem of quantity is exemplified by the sulfur-containing ester found in pineapple flavor ( 3 ) where the fruit was found to contain 112 to 250 mg of sulfur per kilogram of fruit. My first thoughts were to use paper chromatography which had been used with such excellent results with amino acids, but it was soon evident that paper was much too mild an adsorbent to accomplish the required task. Flood ( 4 ) and Hopf ( 5 ) had used alumina-impregnated paper for spot tests and this soon led us to the development of the first silica gel impregnated paper for use in chromatography (6). This was produced by soaking filter paper in sodium silicate solution and then precipitating silicic acid in the fibers by immersing in hydrochloric acid. This showed promise and was used to separate some 2,4-dinitrophenylhydrazones of aldehydes and ketones which could not be separated on unimpregnated paper. However, the impregnated paper was still not the answer to our needs, its capacity was still not great enough. During this time when Chemical Abstracts were much less expensive than they are today, I would clip out abstracts which were of interest to me and file them away for future use. On this particular day I had come across the abstract of Meinhard and Hall's work (7) on drop chromatography for the separation of inorganic ions. I clipped the abstract and laid it on the corner of my desk. Later in the day one of my assistants came in very much discouraged with his attempts at separating terpene constituents by paper chromatography. I picked up the abstract and said "Here, let's make layers of silicic acid on strips of glass and develop them in an ascending manner analogous to paper chromatography." This latter step proved to be the key to the development of the successful system, now
203
Fig. 22.1. Fractionating citrus oils at the U.S.D.A. Fruit and Vegetable Laboratory, in Pasadena, California, circa 1950.
better known as "thin-layer chromatography", existing today. The method was first published in 1951 (8) and was used successfuly by a large number of research workers prior to 1956 (9a) when Egon Stahl began publishing his first work. Drop thin-layer chromatography had first been demonstrated by Beyerinck in 1889 ( 2 0 ) which even predates Tswett's excellent work as well as the work of Reed in 1893 ( 1 1 ) . However, this drop method as demonstrated by Beyerinck and practiced by Izmailov and Shraiber ( 2 2 ) as well as others was too limited in its scope for our puropose. After we had proved to our satisfaction that the method would work, the next task was that of finding the best adsorbent and the optimum conditions for making thin-layer chromatography reliable and reproducible. Of the sixteen adsorbents tested, silicic acid was selected a5 the best for our purpose. This was combined with Amioca starch (National Starch Products Co.) to give a satisfactory layer. Later ( 1 3 ) this binder was replaced to advantage with Clinco 15 modified starch (Clinton Foods, Inc.) or by a 2:l mixture of cornstarch and Superior AA tapioca flour (Stein Hall and Co., Inc.), and at a still later date ( 2 4 ) we found that the starch binder could be reduced to advantage from 5% to 24% in the mixture.
204
Fig. 2 2 . 2 . Separation of p-cymene, pulegone and cinnamaldehyde on silicic acid thin-layers with 15% ethyl acetate in hexane, as shown under ultraviolet light on fluorescent chromatostrips. (8). (Reproduced with permission of the American Chemical Society). From top to bottom: p-cymene, pulegone, cinnamaldehyde. From left to right: l=blank (spot due to traces of impurity in solvent not removed by distillation); 2=p-cymene (143 pg), pulegone (3.3 ug) and cinnamaldehyde (3.3 pg); J=p-cymene (358 pg), pulegone ( 8 . 2 pg) and cinnamaldehyde (9.1 pg); 4= p-cymene (1.4 mg), pulegone (32.8 pg) and cinnamaldehyde (36.4 pg). One of the earliest discoveries we made and reported was that in order to obtain reproducible results, conditions had to be carefully controlled (standardized). To obtain these results, the silicic acid was screened in order to remove the coarser particles and the layers were dried and held under carefully controlled conditions.
To further increase the reliability of results, it was established at that time that it was necessary to run standards to show that the layers were properly prepared and to serve as a reference for comparing RF values. Because most of the compounds with which we were working were colorless, it was necessary to develop methods for locating compounds. For ultraviolet absorbing compounds we adopted the technique of Sease (15) of incorporating inorganic fluorescent material with the adsorbent. Numerous spray reagents were then developed for other compounds, and in some cases these not only located the compound but also indicated the type of compound that was present. For example,the 2,4-dinitrophenylhydrazine spray disclosed the presence of carbonyl compounds and the dianisidine spray showed the presence of aldehyde groups. One of the more useful sprays was the 0 . 0 5 % fluorescein spray with subsequent exposure of the thin-layer to bromine vapor to detect unsaturated compounds or other compounds which would react with bromine vapor under the conditions used. There were some compounds, as for example, camphor, which could not be located by any of the methods thus far devised. I originated the idea of using plaster of Paris as a binder so the compounds could be sprayed with sulfuric acid containing an oxidizing agent. Thus on subsequent heating, the spot location was revealed by the charred areas. This was the origin of the so-called silica gel G, because when plaster of Paris sets, it takes up water of crystallization to form gypsum. Later this charring technique was developed by others into a quantitative analytical method. One of the techniques which wedevelopedand used for fractionating larger amounts was that of using thin-layer chromatography to monitor the fractions as they were eluted from a packed chromatographic column ( 1 6 ) . In this way by the time the column was completely eluted, it was possible to determine which fractions should be combined and which fractions were single compounds under the conditions of the separation used. Another column application was the use of thin-layer principles for the preparation of a self-supporting column unencumbered by a containing envelope ( 1 7 ) . This was designated as a "chromatobar", as it consisted of cylindrical or square bar of silicic acid bound with gypsum around a glass rod which added strength. This column was developed in an ascending manner and could be removed and checked for development by spraying with a visualizing agent on one side. The thin layer of sprayed material could then be scraped off and the column returned for further development. In addition to the use of narrow strips of adsorbent, we also introduced the use of square coated plates in our first publication (8); thus multiple samples could be run on a single plate or twodimensional chromatography could be carried out on an individual sample so as to give improved resolution. An important feature of our work included the introduction of the use of reactions on the thin-layer plate ( 1 8 ) . We used oxidations, reductions, dehydrations, hydrolysis reactions, and the preparation of derivatives. These could all be used to help in identifying a
206
Fig. 22.3 (left). Dr. Kirchner at the CAMAG First International Thin-Layer Chromatographic Symposium demonstrating preparative thin-layer chromatography without a backing plate to support the layer so that it is accessible from both sides for sample application and visualization. For details, see ref. 21. Fig. 22.4 (right). Separation of 100 mg each of six dyes on a 4 x 8 x 8 in. silica gel self-supporting layer with no backing plate. Top to bottom: Yellow OB, narrow line due to second solvent front, Sudan I , Sudan 111. Sudan 11, Methyl Red and Crystal Violet ( 2 1 ) . specific compound because the reaction products had different RF values than the original compounds. This technique is also useful in separating critical pairs of compounds that can not normally be separated by chromatography. This reaction technique proved to be very versatile as it may be applied to the initial spotted sample, or it may be applied after the chromatogram has been developed in one direction and prior to the development in a second dimension. Biphenyl is used by the citrus industry to prevent molding during shipping and storage of citrus fruit. Because there was a need for an accurate method for detecting and measuring the amount of biphenyl absorbed by the citrus fruit, we had the opportunity to apply the thin-layer technique to this problem and so introduced
207
the use of this versatile technique in the quantitative field. By using a spectrophotometric method on the eluted spots we were able to obtain results of +2.8% on amounts ranging from 0.1 to 600 ppm. This helped to establish the reliability of our thin-layer technique at that time. Another technique that we introduced because of our work with thin layers, was the chromatographic preparation of terpeneless oils ( 1 9 ) . I had noticed in running thin-layer chromatograms in hexane on silicic acid that this solvent moved the hydrocarbons, but not the oxygenated components which remained behind at the origin. A check of numerous compounds confirmed this observation and it was then a fairly simple matter to apply the technique using packed columns. On entering into industrial work in 1954, the opportunity was not available to continue research in chromatography although I managed to keep somewhat active in the field by producing a text on thin-layer chromatography ( 9 ) , which has just recently been revised (20). However, there was also an opportunity to develop a preparative thick-layer method ( 2 1 ) . Up to this time the thin-layer methods consisted in running numerous thin-layer chromatograms and combining the separated components until a sufficient quantity of material had been collected. The quantity that could be separated on an individual plate was limited by the thickness of the layer which ranged from 1 to 5 mm thick. The thicker the layer, the more difficult is is to prepare a layer without developing cracks on drying. For the 5 mm layers this resulted in only 50% of the layers being usable. It occurred to me that a thick-layer plate could perhaps be prepared based on the chromatobar technique. The first attempt at the preparation of a 1/4 in. layer without a backing plate was successful; however, the layers were rather fragile, especially when dry, with a tendency for the corners to break. This then led to the use of a stainless steel frame with 0.025 in. stainless steel wires stretched across the frame and imbedded in the layer to help support the latter. These wires in no way interfered with the chromatographic development. The layers were cast in a mold that had been carefully machined so that the layer thickness was extremely uniform. The main problem to be overcome was the tendency of the wet layer to stick to the top and bottom of the mold. Various materials and techniques were tried to solve this difficulty, and this was finally accomplished by covering the top and bottom of the mold with thin sheets of plastic so that the stainless steel plates could be easily removed; then the plastic was rolled back to expose the undisturbed layer. Layers ranging in thickness from 1/8 to 1/2 in. thick were successfully prepared and used. REFERENCES 1 J.G. K,irchner,A.N. Prater and A.J. Haagen-Smith, Ind. E n g . Chem. 18 (1946). 2 A.J. Haagen-Smith, J.G. Kirchner, A.N. Prater and C.L. Deasy, J . Amer. Chem. SOC. 67 (1945) 1646.
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6 J . G . K i r c h n e r and G . J . Keller, J . Amer. Chem. Soc. 7 2 (1950) 1867. 7 J . E . Meinhard and N.F. H a l l , Anal. Chem. 21 (1949) 185. 8 J . G . K i r c h n e r , J . M . Miller and G . J . Keller, Anal. Chem. 23 (1951) 420. 9 J.G. K i r c h n e r , Thin-Layer Chromatography, I n t e r s c i e n c e P u b l i s h e r s , N e w York, 1967; a, p. 5. 10 M.W. B e y e r i n c k , 2. Phys. Chern. 3 (1889) 110. 11 L. Reed, Proc. Chem. Soc. 9 (1893) 123. 12 N . A . Izmailov and M.S. S h r a i b e r , Farmatsiya ( S o f i a ) 3 (1938) 1. 13 J . G . K i r c h n e r , J . M . Miller .and R.G. R i c e , A g r . Food. Chem. 2 (1954) 1031. 14 J . G . K i r c h n e r and V.P. F l a n a g a n , Gordon Research Conference, Colby J u n i o r C o l l e g e , N e w London, N. H., August 1962. 15 J . W . S e a s e , J. Amer. Chem. Soc. 69 (1947) 2242. 16 J . M . Miller and J . G . K i r c h n e r , Anal. Chem. 24 (1952) 1480. 17 J . M . Miller and J . G . K i r c h n e r , Anal. Chem. 2 3 (1951) 428. 18 J . M . Miller and J . G . K i r c h n e r , Anal. Chem. 25 (1953) 1107. 19 J . G . K i r c h n e r and J . M . M i l l e r , Ind. Eng. Chem. 44 (1952) 318. 20 J . G . K i r c h n e r , Thin-Layer Chromatography, 2nd e d . , W i l e y - I n t e r s c i e n c e , N e w York, 1978. 21 J . G . K i r c h n e r , J. Chromatogr. 63 (1971) 45.