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THE INVESTIGATION OF NEW COMPOUNDS
CHAPTER
38
THE INVESTIGATION OF NEW COMPOUNDS I. Smith and J. B. Jepson A F T E R the treatment of a one- or two-way chromatogram with a location reagent it is often found t h a t one or more "spots" appear which cannot immediately be identified. Reasons for such nonidentification fall into three main groups, viz.: the compound is a " n e w " one, i.e. it has not been previously recognized or reported by other workers; the compound has been previously reported by other workers b u t the present worker has none of the authentic material with which to compare chromatographic properties and colour reactions; the present worker is unaware of previous reports. I t must be realized, of course, t h a t the term " n e w " relates to the field of chromatography of naturally occurring compounds; for example, the compound later identified as pipecolinic acid (pipecolic or hexahydropyridine-2carboxylic acid), was referred to as a "new" compound because its R/ values and colour reactions with the usual location reagents were unknown and it had, therefore, to be identified from first principles, even though it was a h e a d y " k n o w n " as a synthetic compound (p. 540). I n the ensuing discussion it will be assumed t h a t the substance is a new compound in the sense defined above, t h a t it belongs to one of the families already discussed elsewhere in this book and t h a t it is first noticed on a two-way chromatogram in the case of those families normally examined in this way, e.g. nitrogenous compounds, or on a one-way chromatogram in the case of sugars and steroids. The first steps in the determination of structure will be the same whatever the nature of the compound but, obviously enough, the later steps will become more and more dependent on the type of compound being dealt with. Therefore, after a general discussion on methods we have taken a number of specific cases which illustrate the principles involved. Step 1. When it is decided to investigate a new spot on the chromato gram more chromatograms are run under the original conditions and some are located with the same location reagent to ensure t h a t the spot was genuine and not an artefact of one experiment. Having proved its authenticity, the other chromatograms should be treated with a variety of reagents, both singly and in multiple sequence, for two reasons; first, a great deal of knowledge about particular reactive groups present or absent in the molecule will be obtained and second, and perhaps more important here, should the new compound be the only one in the area to react with a particular reagent this will be of great use in its isolation. The mixture containing the unidentified component should also be chromatographed in a variety of solvents of other types, as this will give further information on its chemistry and may furnish a more suitable solvent for its separation. 968
T H E I N V E S T I G A T I O N OP N E W COMPOUNDS
969
Step 2. Recovery οϊ Pure Material (a) Milligram amounts. I n t h e preparation of chromatograms described in previous chapters, an amount of material is placed on t h e paper (Whatman Nos. 1, 2, or 4) so t h a t separation of the maximum number of compounds is obtained, with spots as discrete as possible. However, in isolation experiments neither discrete spots nor m a x i m u m separation of all the compounds is necessary a n d so thick papers (Nos. 31 or 3MM) are used a n d these are heavily overloaded with material in order t o isolate as much as possible with t h e minimum of labour a n d time. One-way chromatograms are prepared by placing a large number of spots along t h e origin line, although this m a y be done more simply by "streaking" the solution on to t h e paper by means of a pipette (Fig. 38.1A). Up to 0-2 ml. of solution m a y be apphed t o a thick paper along 9 in. of the 10 in. origin line and, after aUowing t o dry, this m a y be repeated m a n y times. The total volume of solution which m a y be apphed to the paper will depend on the concentration a n d n a t u r e of t h e substances present. If salts are present in large amounts only smaU volumes m a y be applied because of salting out effects, b u t if the solution is dilute, relatively pure a n d salt-free t h e n m a n y millilitres m a y be applied until a high enough concentration of material is present. Between five and twenty milligrams of solids can be applied t o one thick paper. Papers are then chromatographed in t h e usual way when t h e components separate in horizontal bands and, after drying, the new material is located using the most suitable reagent. Physical methods (radioactivity, ultra-violet fluorescence or adsorption, etc.) are preferred. Otherwise 1-2 cm. guide-strips are cut from each side of the paper in the direction of solvent flow, treated with the location reagent to find t h e position of t h e compound and t h e n realigned with the untreated paper. Thus if it was desired to obtain pure proline from a protein hydrolysate, the yellow colour obtained with ninhydrin would distinguish this aminoacid from t h e others, or t h e guide could be treated with isatin when t h e proline would produce a blue colour. Pencil hues are drawn on the untreated papers on either side of the band which is then cut out (Fig. 38.1B). This results in a very great primary purification, b u t the unknown is stiU usually contaminated with some of the other substances running close to it on t h e paper a n d a further chromatographic purification will be necessary. I t wiU be necessary to extract t h e material from t h e paper—a process known as elution—and this can be accomplished b y cutting the paper into small pieces and extracting with solvent. Alternatively, the excised strip m a y be treated as if it were a strip chromatogram and the material washed to the end of the paper by making use of a solvent in which it is very soluble and so travels with t h e front (e.g. water is used for sugars, and water or very dilute HCl for aminoacids, etc.). If the ascending method is used, the material is concentrated in a band about 2 cm. wide at t h e t o p of the pa,per and is t h e n extracted into solution as before. I t is more convenient, however, t o carry out this process using the descending technique and, if t h e lower end of the strip is cut to a point, the first few ml. of solvent which drip off into a
970
CHROMATOGRAPHIC T E C H N I Q U E S
small beaker held just below the tip will contain all of the material. I n both the above methods, the eluate may be applied to a new chrom atogram by streaking on as before. I n yet another method, known as the ''sewing in" technique, the substance is not eluted from the paper at all. A strip of paper, corresponding exactly in size to t h a t containing the substance, is cut out of a clean sheet. The old strip takes its place and is sewn into the new sheet by close stitching with a needle and cotton; this is then treated as if it were a single sheet of paper (Fig. 38.1c). On chromatographing again, in a solvent with different
y&mJt
strips
FIG. 3 8 . 1 . (A) A solution is "streaked" o n t o t h e c h r o m a t o g r a m which is t h e n run in s o l v e n t in t h e normal w a y . (B) T h e dried c h r o m a t o g r a m is t a k e n , guide strips are c u t from it a n d located w i t h a suitable reagent. T h e strips are realigned w i t h t h e u n t r e a t e d portion of the c h r o m a t o g r a m , t h e position of t h e n e w spot is m a r k e d w i t h pencil and t h e n c u t out. ( C ) A n identical section is cut from a clean sheet, t h e n e w - s p o t s e g m e n t is s e w n in a n d t h e paper is c h r o m a t o g r a p h e d again in a different s o l v e n t , Sg. A l t e r n a t i v e l y t h e n e w material is e l u t e d from t h e strip a n d its solution is again streaked o n to a clean c h r o m a t o g r a m and run in S j . ( S . F . = s o l v e n t front.)
properties, substances running close together in the first solvent will probably r u n separately in the second (thus if butanol-acetic acidwater Avas used first then butanol-pyridine-water might be used second). After this second run the material is again located and the untreated section of the paper containing the substance is cut out once more. Should the substance be completely separate from the other components of the original mixture it is eluted into solution for further chemical analysis, otherwise re-chromatography in a third solvent is necessary. The solvents used for these runs should be ones which can be com pletely dried off the paper in the cold without leaving a contaminating residue. The possibility of contamination by material unreactive to any of the location reagents always exists b u t the chance of such contamination is greatly reduced after each successive chrom atographic run, as the unreactive material is necessarily of a different chemical family and so wih have quite different running properties in the various solvents; this is a n important consideration in the determination of physical properties (infra-red or ultra-violet absorp tion, etc.) of materials purified in this way. Using five thick papers on
T H E I N V E S T I G A T I O N O F N E W COMPOUNDS
971
the Frame, the time necessary for the isolation of 5-25 mg. is less t h a n a week even if chromatography in three different solvents is necessary. Similar techniques are used for thin layers. The mixture is apphed as a single streak and chromatographed as usual. After drying, the main plate is covered with a second plate b u t lea\áng a margin of 0-5-1 cm. on both sides in the ascending direction; the margins m a y be separated from the main plate by drawing down a thin line with a spatula to avoid lateral movement of reagents. Subsequently, a reagent is sprayed on bcth margin strips to locate the separated compounds. Horizontal lines are drawn across the plate (as shown in Fig. 38.1) and the powder is removed by scraping off with a spatula onto a sheet of aluminium foil or by sucking u p with the vacuum cleaner device shown in Fig. 4.22. The substance is then eluted with a suitable solvent using a tube with a sintered disc as a micro-column. I t is possible to purify m a n y milli grams of material on the ordinary thin layers used for analytical work. (6) Gram Amounts, The techniques of preparative layer chromato graphy described in Chapter 34 are a remarkable advance a n d have replaced column preparative methods for m a n y types of compound. However ion exchange columns remain valuable for ionic materials and the two methods can be considered as complementary rather t h a n as alternatives.
Step 3. Determination of Chromatographic Properties. I t is not
uncommon to find t h a t the R values of the pure compound are appreci ably different from those shown in the original mixture, possibly because of the mutual effect of compounds with close R values—a factor of increasing importance when the paper is overloaded. Hence these figures should be redetermined in the standard solvents used for com pounds which react with the particular location reagents; for example, compounds which react with ninhydrin are usuaUy r u n in butanolacetic acid-water, phenol-ammonia and butanol-pyridine-water (see Chapter 5). As it is possible t h a t t h e spot observed on t h e original chromatogram was composed of more t h a n one compound t h e pure material should again be treated with t h e various location reagents t o confirm t h a t all t h e original positive reactions are still obtained. At this stage it may be possible to say whether the material corresponds to one previously described b u t for which no authentic material is available for comparison and it is now necessary and worthwhile to synthesize a specimen for comparison of chromatographic and other properties. However, if the compound cannot be identified a t this stage a number of lines of attack present themselves. These are more conveniently discussed in relation to particular families of compounds although some of the methods are common to more t h a n one class.
Step 4. Further Investigation of Structure (a) AminO'Compounds. Amino-compounds m a y be subdivided as follows: amines, a-aminoacids, non-a-aminoacids, iminoacids, amides corresponding to one of the previous acid groups mentioned, peptides, multi-functional compounds such as an ammoacid containing an imidazole, indole, or guanidine nucleus or another amino, carboxyl, or amide group, and so on. All compounds in this group are located with
972
CHROMATOGRAPHIC T E C H N I Q U E S
ninhydrin and any which yield purple colours probably contain an NHg group attached to an aliphatic carbon, although the converse is not true. Acid hydrolysis for a period of 1 hour will yield the free acid derived from an amide and this will probably have a higher Rf in the butanol-acetic acid solvent t h a n the original; further more only one new spot will appear on the chromatogram. For a peptide it may be necessary to use hydrolysis periods up to 2 4 hours in order to split it completely into its constituent aminoacids but, as m a n y peptides contain amide groups, it is essential to carry out both t h e I and 24 hour hydrolyses in order to confirm t h a t the shorter period is not simply liberating a peptide free of amide groups. Most peptides of natural origin (e.g. glutathione, oxytocin, antibiotics, etc.) will yield, after hydrolysis, at least two spots on the chromatogram b u t other peptides are known which yield only one spot. For example, the peptides valyl-valine and leucyl-leucine are particularly resistant to hydrolysis and so may be found on the chromatograms of incompletely hydrolysed material; on hydrolysis of such peptides only one spot is obtained. Cases are also known where spots on chromatograms have been identified as standard aminoacids simply on the basis of position on the paper chromatogram when, in fact, they were actually peptides. Similarly the first naturally occurring sulphonium aminoacid, methyl methionine chloride, was originally "identified" as one of the basic aminoacids as it overlapped these compounds on chromatograms and yielded the same ninhydrin colour. I t should be noted here t h a t indoles are rapidly and completely destroyed by acid hydrolysis, t h a t iodo- and bromo-aminoacids are dehalogenated during 2 4 hour hydrolysis, and t h a t phosphorylated and sulphated compounds are split to form the parent hydroxy or guanidino compound after only a few minutes boiling with dilute acid. Having shown t h a t a compound is a sulphate or phosphate ester it is more rewarding, as well as being more simple, to endeavour to elucidate t h e structure of the parent compound first, as a great deal of chromato graphic data on amino-compounds is available whereas t h a t on such esters is scant. Alkaline hydrolysis is essential for the determination of the structure of indolic, iodine and bromine compounds, and for peptides containing these groups a combination of both acid and alkaline t r e a t m e n t is useful. Such alkaline hydrolysis is also useful in the study of guani dines as these are split to the parent amino-compound; t h u s Thoai and Robin^^^ could have used this to advantage in elucidating t h e structure of the recently reported guanidoethanol and guanidotaurine which yield the well known compounds ethanolamine and taurine. Furthermore, simple alkyl guanidines can be differentiated from the more complex guanidino analogues of aminoacids, as the former yield volatile amines which can be distilled off—a suitable apparatus is a modified Conway apparatus or manometric conical flask which can be partly immersed in a boiling water bath (see p. 293). If the compound is a peptide, it is probable t h a t t h e aminocompounds of which it is composed will be identifiable by further chromatography. If this is not so, the unidentifiable compounds
Plates I-I O are reproduced from "Paper & Th/n Layer Chromatography and Electro phoresis"—A Teaching Level Manual for use with the Unikit Apparatus. By kind permission of Shandon Scientific Company Ltd (see pió).
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Br,
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1 cm Plate I: PAPER CHROMATOGRAPHY, ascending. Separa tion of component dyes of writing inks. Solvent — butanol: ethanol: ammonia. Inks: Bk + Bri, Bk, Bri, ΒΓ2, Bl, Rd, Gn, Gn. Note that neither of the brown inks contains a brown pigment. The two are obviously distinguishable. Note also the reproducibility of the green. Paper 25x25 cm.
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1 cm P l a t e 3: PAPER CHROMATOGRAPHY, two-way. Separation of amino acids present in orange juice. Solvents — first: ethanol: water: ammonia; second: butanol: acetic acid: water. Note the yellow proline and the brown asparagine spots.
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Θ 1 cm Plate 5: CHROMATOGRAPHY-ELECTROPHORESIS, two-way. Separation on paper of pigments present in a mixture of brown and black inks. The first separa tion, vertically, was by chromatography; solvent — butanol: ethanol: ammonia in the 10 cm direction. The second separation, horizontally, was by electro phoresis; buffer — ammonia: ammonium acetate in the 30 cm direction. All pigments have migrated towards the anode, or have remair\ed stationary at the origin. Paper 10x30 cm.
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Θ 1 cm Plate 6: ELECTROPHORESIS-CHROMATOGRAPHY, two-way. Separation of amino acids present in fresh orange juice. The first separation, horizontally, was by electrophoresis: buffer — pyridine: acetic acid. The second separation, vertically, by chromatography: solvent — butanol: acetic acid: water.
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T H E I N V E S T I G A T I O N O F N E W COMPOUNDS
973
can be examined as described for a simple compound. I t has often been assumed t h a t it is not necessary to isolate a compound in order to show t h a t it is a peptide and, if this is all t h a t is required, the original extract can be hydrolysed directly when further chromatography wiU show t h a t the new spot has now disappeared. With the discovery of more and more non-peptide substances unstable t o acid hydrolysis, this assumption can no longer be maintained, and before such arguments can be used, it is necessary to show t h a t t h e compound is, at least, not a phosphate, indole or iodine containing substance. Should the compound prove resistant to hydrolytic cleavage, the foUowing further experiments should be carried o u t : 1. Chromatography of the copper carbonate-treated material (see p . 126). A two-way chromatogram with solvent runs of about 6-8 in. is sufíicient as only a single substance is being investigated. 2. Chromatography after peroxide oxidation (see p . 126). Labile compounds are often oxidized under the conditions of acid or alkaline hydrolysis—a factor of importance in the previous discussion on pep tides. A change of will usually suggest the presence of sulphur in the molecule although the converse is not true because of the existence of some completely oxidized compounds such as taurine. 3. Charcoal adsorption (see p. 261). Aromatic compounds are selectively adsorbed on to the column and may be eluted after the nonaromatic materials have been washed through. 4. Electrolytic desalting of a fraction of the material (see p . 41). Strong acid conjugates will be lost via t h e anode solution, activated double bonds will be reduced, etc. 5. Nitrous acid deamination (see p . 258 for a suitable reagent). Nitrous acid rapidly deaminates aliphatic amino-compounds and use can be made of this in the following ways. A peptide is treated with the nitrous acid in acetic acid solution (mineral acid must be absent), hydrolysed and chromatographed when one aminoacid present in the original hydrolysate will now be found to be missing, i.e. the one a t the amino end of the chain; should none be missing then the peptide m a y weh be cyclic in nature. If a new compound has been shown to be a non-a-aminoacid (copper carbonate technique) then a further distinc tion may be dra\vn between those compounds containing a primary amino group and others such as secondary amino compounds (sarcosine), nitrogen-containing rings (piperidine, pyrrolidine, indole, and imidazole derivatives), etc. Thus on treating the mixture with nitrous acid primary amines are destroyed while secondary amines form N-nitroso' derivatives, m a n y of which are ether-soluble and can be regenerated to the parent compound by acid hydrolysis. This method is of value for isolating iminoacids from admixture with aminoacids. (b) Peptides and Proteins. Valuable methods are now available for structural studies of these compounds (see Chapters 6 and 7). (c) Sugars. Sugars m a y be divided into the following groups: aldoses, ketoses, reducing and non-reducing sugars, mono-, di- triand ohgo-saccharides, acids, etc. Reagents are available to determine into which group a monosaccharide falls and R^ determinations in three or four solvents will often give useful indications of whether the 63
974
CHROMATOGRAPHIC T E C H N I Q U E S
compound is a pentose, hexose, or ohgosaccharide. Acid hydrolysis (dilute HCl, I hour) will split all those sugars which travel from the origin during chromatography, i.e. up to heptaoses, into their consti t u e n t units and partial hydrolysis (chlute HCl, room temperature for varying periods) wiU yield intermediate breakdown products which may be investigated separately. Enzyme hydrolysis either in solution, or on paper, before or after chromatography, will indicate the type of bond present, e.g. a or β glucósido, etc. (d) Enzymic reactions. Aminoacid solutions have been treated with both D and L aminoacid oxidases, sugars have been treated with bond specific enzymes, and so on, and the solution then chromatographed in order to determine further properties of the compounds present in the original. I n m a n y cases it is possible to carry out the enzyme reaction on the paper chromatogram either at the origin and before chroma tography, or after chromatographic separation, by application of the enzyme to the whole of the paper after removing the solvent. (e) Reactions on the chromatogram. The above reactions have been carried out on the original extract or on the paper or layer prior to chromatography, b u t there is no reason why m a n y of them, or other reactions as well, should not be carried out after the chromatographic separation or even in between the first and second solvent runs in twoway chromatography. Thus sugars might be separated in one direction, the area of paper on which they now lie is sprayed with invertase and, after some 5-10 minutes, the paper is dried and placed in the second solvent for further separation. Steroids containing side chains have been run in one direction and, after drying, the side chains have been oxidized off and the steroids so formed have then been run in an appropriate solvent in the second direction.
Examples of Apphcation of Above Techniques 1. The Occurrence of Iodo-aminoacids in Certain AlgaeS^^ Two spots, A and B, were often observed on ninhydrin-treated chromatograms of alkaline hydrolysates of certain whole algae. Further tests on two-way chromatograms showed t h a t both spots contained iodine and spot Β was also positive to the nitrosonaphthol test. Ry of spot A : same as leucine in butanol-acetic acid-water, same as tyrosine in phenol-ammonia. Ry of spot Β : half way between A and tyrosine in same as tyrosine and A in PhAm.
BuA,
Known iodo-aminoacids include iodo-tyrosines and thyronines. Both spots disappeared after acid hydrolysis or copper carbonate treatment. Co-chromatography with authentic di-iodo-tyrosine showed enlargement of spot A, and with mono-iodo-tyrosine enlargement of spot B ; Electrolytic desalting resulted in the disappearance of A and Β and enlargement of tyrosine spot. Therefore the two new spots corresponded to the two iodo-tyrosines in running properties and colour reactions. These compounds were found to be present in aU red algae and some brown algae (laminariaceae) but not in other classes examined.
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P l a t e 8: T H I N LAYER CHROMATOGRAPHY. Separation of component dyes of writing inks. Solvent — butanol: ethanol: ammonia. Inks: Bk, Bri, Br2, Bl, Rd, Gn, R d + G n . Compare Plate I.
T H E I N V E S T I G A T I O N O F N E W COMPOUNDS
975
2. The Occurrence of PipecoKnic Acid, (a) Stewards proof.^'^ A new spot on chromatograms of extracts of the common bean yielded a bluepurple with ninhydrin, green-blue with isatin and travelled shghtly higher t h a n proline in the usual aminoacid solvents. F r o m colour reactions it A v a s thought probable t h a t the compound was related to pyrrohdine or piperidine compounds. The substance was stable t o acid and alkahne hydrolysis, and was shown to be a non-a-amino compound as it was unaffected hy copper carbonate treatment. The material was isolated as follows: passage down a cation exchange resin retained it, elution with dilute acid removed m a n y aminoacids b u t not the unknown, displacement with dilute ammonia yielded a n eluate containing the unknown plus basic aminoacids plus y-amino-butyric acid, and careful crystallization yielded pure unknown. Comparison of B,f and colour reactions with synthetic piperidine 2-, 3-, a n d 4-carboxylic acids showed t h a t the unknown corresponded t o the 2-acid. This was confirmed b y infra-red spectroscopy. B y a splendid modification of the copper carbonate technique (copper carbonate/alumina column) a large scale separation of pipecolinic acid yielded 15 gm. of material and the method was shown to be of great potential value for the isolation of non-a-amino acids. (6) Morrison's Proof.^^^ The unknown was independently discovered by identical reactions mentioned above. I t was isolated as follows: charcoal treatment removed aromatic compounds from t h e original extract b u t not the unknown; bulk separation on cation exchange resin yielded a mixture of unknown plus leucine, valine, prohne, a n d y-aminobutyric acid; fractionation of this on ceUulose yielded unknown plus vahne, and crystalhzation gave pure unknown. Comparison with piperidine acids foUowed b y X-ray powder photography identified the unknown as piperidine-2-acid. 3. Identification of urinary Indican as Indoxyl Sulphate. The treat ment with Ehrlich reagent of paper chromatograms from normal h u m a n urines invariably gives a n orange-brown spot, intensifying t o red on later application of ammonia (the fluorindal reaction, see p . 257). This chromogen is related in amount to the '^indican" level of t h e urine as shown b y the Obermeyer test, and disappears from t h e urine in a sub ject dosed with Chlortetracycline, a gut-sterUizing antibiotic. Tentative identification of the spot as *'indican" (acid conjugates of indoxyl formed from tryptophan by gut bacteria) was supported by (a) the dark blue colour (indigo) given by treating the area of t h e spot with acidic oxidizing agent; (b) the red colour (indirubin) given b y t h e spot on treating the paper with acidic isatin and heating; (c) the fact t h a t the chromogen was no longer present in electrolyticaUy-desalted urine, b u t could be recovered from the effluent acid. The indican was chromatographically identical (R^ and colour reactions) with synthetic indoxyl sulphate added to electrically desalted urine, though both were markedly displaced b y other urinary com ponents from the positions given b y indoxyl sulphate in aqueous solution. No other area of chromatograms from neat urine gave indigo blue on acidic oxidation. One-way chromatography of t h e urine, on
976
CHROMATOGRAPHIC T E C H N I Q U E S
thick paper in IPrAm, foUowed by elution of the appropriate strip, furnished a solution of the indican, chromatographically-free from other Ehrlich reactors, and shown colorimetrically to represent a good recovery of the total indican. Six solvents failed to show this eluted indican to be in any way different from synthetic indoxyl sulphate in aqueous solution. But there remained the possibility t h a t the urinary indican contained some other indoxyl conjugate, say the glucuronide, which was not separable under the conditions tried (a synthetic sample of indoxyl glucuronide is not avaUable for comparison). The eluted indican solution was, therefore, mixed with a n enzyme preparation from limpets, known to contain an aryl sulphatase of wide specificity, b u t showing no glucuronidase or phosphatase activity. After this treat ment, no indican of any type could be detected chromatographically, though insoluble indigo had been formed. Conversely, a limpet ßglucuronidase preparation free from sulphatase caused no detectable change in the indican eluate. Thus, subject only to the proviso t h a t the chromatographic process has not destroyed other indoxyl conjugates, the indican of normal urine, and of many abnormal urines, wpuld appear to be solely indoxyl sulphate. 4. Identification of a Urinary Glucuronide as Testosterone Glucuronide. When the 17-ketosteroid glucuronides were first separated from one another, a "new" steroid was detected, and this was also presumed to be a glucuronide. I t was far from pure, and in fact was always found in the tail of the aetiocholanolone glucuronide peak from the partition column. However, hydrolysis of this material with j5-glucuronidase and examination of the liberated steroid in the A system (p. 584) showed the *'new" steroid to have an R,(19) half t h a t of aetiocholanolone. I t s reactions on the paper were: (i) ultra violet light of about 240 ιημ, was absorbed; (ii) with m-dinitrobenzene and K O H (Zimmermann reaction) a blue colour was produced; (iii) an orange dinitrophenyl hydrazone was formed; all characteristic of Δ^-3-ketones. Reaction (ii) rules out a 17-ketosteroid which would give a purple colour, so it was concluded t h a t the usually oxygenated 17 position carried a hydroxyl group. From the Ry it could be assumed t h a t the ''new" steroid was only dioxygenated; trioxygenated compounds have R^ values less t h a n 10 in the A system. Testosterone is a steroid t h a t has aU these proper ties, and when an authentic sample of this compound was run alongside the "new" steroid in the A system they were found to have identical R/s. To check the identity, the "new" steroid and testosterone were oxidized by CrOg (1 per cent) in acetic acid solution at room temperature for 15 hours. After dUuting with water, the steroids were extracted into benzene, which was washed with N a O H solution and water to remove the acid. On running these in the A system in parallel with androst4-ene-3:17-dione, expected from the oxidation of testosterone, aU three compounds were found to have identical Ry.'s(54), as well as reactions (i) and (iii) above. Reaction (ii) produced a purple colour, characteristic of the 17-ketone group.
T H E INVESTIGATION O F N E W COMPOUNDS
977
The original compound, which was hj^drolysed b y the specific jS-glucuronidase preparation, was presumably a /S-glucuronide. The only position on the steroid available for the a t t a c h m e n t of this group is a t 17 and if this is so, the Δ^-3-ketone group must be intact and the compound should show absorption of 240 ηψ. light. This absorption was found to be present, whereas it was not shown by the other glucuronides from the same partition columns. The above argument is summarized diagrammaticahy below. W h a t would have happened if the " n e w " steroid had not been testosterone? Another possibility involves the oxidation of an 11-hydroxyl group to keto b u t this would not have brought about such a large change of R^, e.g. the oxidation of ll-hydroxy-setiocholanolone (R^ = 10) to ll-keto-setiocholanolone (Rf = 15). COOH
HO
The " n e w " steroid could have been the testosterone analogue without the unsaturation at the 4 position (17-hydroxy-5 (a or /9)-androstan-17one), in which the 240 m ^ . absorption would have been missing and it would have been oxidized to androstandione or setiocholandione [5 (a and ^)-androstan-3:17-diones], both of which have R^ values about 60, so they are ruled out. On examining the extensive steroid literature it is clear t h a t other positions for the hydroxyl group are unlikely to produce an original compound and an oxidation product with t h e R^'s found. Alternative chemical transformations are available, and these must also be considered before starting the investigation. Acetylation is technically easier t h a n oxidation, b u t the separation of C^g steroid acetates is very difiicult and cannot be effected in t h e A system as most of these move with the front. However, acetylation m a y be very valuable in the adrenocortical steroid series.
978
CHROMATOGRAPHIC T E C H N I Q U E S
Having, by the use of some 50 μg. of the "new" steroid, obtained a clear indication of its identity, the next step is the isolation of the compound on a larger scale, both as the original glucuronide and the free steroid, for the identification is not conclusive until the classical proofs of melting point, rotation and synthesis are obtained. A large degree of purification can be obtained by running broad bands of " n e w " steroid followed by elution as described earlier, b u t it is necessary, how ever, to avoid overloading the chromatogram as large amounts of con taminants are also washed out of the paper, and subjection of the paper to preliminary washing does not prevent this. Column chromatography with Bush systems supported on celite or kieselguhr yields good separa tions provided t h a t a "slower" system t h a n the corresponding paper solvent is used. This large scale work has not yet been possible on the testosterone compound described here, but it is hoped t h a t this dis cussion should provide a guide to the examination of " n e w " compounds which m a y be treated by the t3φe of chemical transformation and R^ identification described here.
Fingerprmt and Diagonal Techniques-^^^^ REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Partridge, S. M. Brit. Med. Bull., 1954, 10 (3), 2 4 1 . Partridge, S. M. Analyst, 1952, 77, 955. N g u y e n - v a n Thoai, a n d R o b i n , Y . Biochim. Biophys. Acta, 1954, 13, 533. D e n t , C. E . , a n d B o n e t t i , B . E . Biochem. J., 1954, 57, 77. E d w a r d s , R. W . H . (See chapter 23). S m i t h , I. (Unpubhshed.) Steward, F . C , Zacharius, R . M., a n d T h o m s o n , J . F . J. Amer. Chem. Soc, 1954, 76, 2908 a n d 2912. Morrison, R. I. Biochem. J., 1953, 53, 474. E d w a r d s , R. W . H . , a n d KeUie, A . E . Chem. and Ind., 1956, 250. W i t k o p , B . , and F o l t z . C. M. J . Amer. Chem. Soc, 1957, 79, 19. See cliapter 7 a n d vol. 2, chapter 8. General Reviews
and
Methods
Methods of Biochemical Analysis. Vol. I , 1954. Intersci., etc. Modern Methods of Plant Analysis. E d . P a e c h a n d Tracy, Springer. Vols. I - I V cover a wide range of m e t h o d s a n d chemical families. Progress in Chemistry of Fats. Vol. I, 1952. F a t t y acids a n d related s u b s t a n c e s . Methods in Carbohydrate Chemistry, Vol. I, 1960. Advances in Protein Chemistry, Vol. I , 1947. Separation of Biological Molecules. Brit. Med. Bull., 1966, 22(2), 103. Advances in Chromatography, Vol. I, 1965.
38
THE INVESTIGATION OF NEW COMPOUNDS I. Smith and J. B. Jepson A F T E R the treatment of a one- or two-way chromatogram with a location reagent it is often found t h a t one or more "spots" appear which cannot immediately be identified. Reasons for such nonidentification fall into three main groups, viz.: the compound is a " n e w " one, i.e. it has not been previously recognized or reported by other workers; the compound has been previously reported by other workers b u t the present worker has none of the authentic material with which to compare chromatographic properties and colour reactions; the present worker is unaware of previous reports. I t must be realized, of course, t h a t the term " n e w " relates to the field of chromatography of naturally occurring compounds; for example, the compound later identified as pipecolinic acid (pipecolic or hexahydropyridine-2carboxylic acid), was referred to as a "new" compound because its R/ values and colour reactions with the usual location reagents were unknown and it had, therefore, to be identified from first principles, even though it was a h e a d y " k n o w n " as a synthetic compound (p. 540). I n the ensuing discussion it will be assumed t h a t the substance is a new compound in the sense defined above, t h a t it belongs to one of the families already discussed elsewhere in this book and t h a t it is first noticed on a two-way chromatogram in the case of those families normally examined in this way, e.g. nitrogenous compounds, or on a one-way chromatogram in the case of sugars and steroids. The first steps in the determination of structure will be the same whatever the nature of the compound but, obviously enough, the later steps will become more and more dependent on the type of compound being dealt with. Therefore, after a general discussion on methods we have taken a number of specific cases which illustrate the principles involved. Step 1. When it is decided to investigate a new spot on the chromato gram more chromatograms are run under the original conditions and some are located with the same location reagent to ensure t h a t the spot was genuine and not an artefact of one experiment. Having proved its authenticity, the other chromatograms should be treated with a variety of reagents, both singly and in multiple sequence, for two reasons; first, a great deal of knowledge about particular reactive groups present or absent in the molecule will be obtained and second, and perhaps more important here, should the new compound be the only one in the area to react with a particular reagent this will be of great use in its isolation. The mixture containing the unidentified component should also be chromatographed in a variety of solvents of other types, as this will give further information on its chemistry and may furnish a more suitable solvent for its separation. 968
T H E I N V E S T I G A T I O N OP N E W COMPOUNDS
969
Step 2. Recovery οϊ Pure Material (a) Milligram amounts. I n t h e preparation of chromatograms described in previous chapters, an amount of material is placed on t h e paper (Whatman Nos. 1, 2, or 4) so t h a t separation of the maximum number of compounds is obtained, with spots as discrete as possible. However, in isolation experiments neither discrete spots nor m a x i m u m separation of all the compounds is necessary a n d so thick papers (Nos. 31 or 3MM) are used a n d these are heavily overloaded with material in order t o isolate as much as possible with t h e minimum of labour a n d time. One-way chromatograms are prepared by placing a large number of spots along t h e origin line, although this m a y be done more simply by "streaking" the solution on to t h e paper by means of a pipette (Fig. 38.1A). Up to 0-2 ml. of solution m a y be apphed t o a thick paper along 9 in. of the 10 in. origin line and, after aUowing t o dry, this m a y be repeated m a n y times. The total volume of solution which m a y be apphed to the paper will depend on the concentration a n d n a t u r e of t h e substances present. If salts are present in large amounts only smaU volumes m a y be applied because of salting out effects, b u t if the solution is dilute, relatively pure a n d salt-free t h e n m a n y millilitres m a y be applied until a high enough concentration of material is present. Between five and twenty milligrams of solids can be applied t o one thick paper. Papers are then chromatographed in t h e usual way when t h e components separate in horizontal bands and, after drying, the new material is located using the most suitable reagent. Physical methods (radioactivity, ultra-violet fluorescence or adsorption, etc.) are preferred. Otherwise 1-2 cm. guide-strips are cut from each side of the paper in the direction of solvent flow, treated with the location reagent to find t h e position of t h e compound and t h e n realigned with the untreated paper. Thus if it was desired to obtain pure proline from a protein hydrolysate, the yellow colour obtained with ninhydrin would distinguish this aminoacid from t h e others, or t h e guide could be treated with isatin when t h e proline would produce a blue colour. Pencil hues are drawn on the untreated papers on either side of the band which is then cut out (Fig. 38.1B). This results in a very great primary purification, b u t the unknown is stiU usually contaminated with some of the other substances running close to it on t h e paper a n d a further chromatographic purification will be necessary. I t wiU be necessary to extract t h e material from t h e paper—a process known as elution—and this can be accomplished b y cutting the paper into small pieces and extracting with solvent. Alternatively, the excised strip m a y be treated as if it were a strip chromatogram and the material washed to the end of the paper by making use of a solvent in which it is very soluble and so travels with t h e front (e.g. water is used for sugars, and water or very dilute HCl for aminoacids, etc.). If the ascending method is used, the material is concentrated in a band about 2 cm. wide at t h e t o p of the pa,per and is t h e n extracted into solution as before. I t is more convenient, however, t o carry out this process using the descending technique and, if t h e lower end of the strip is cut to a point, the first few ml. of solvent which drip off into a
970
CHROMATOGRAPHIC T E C H N I Q U E S
small beaker held just below the tip will contain all of the material. I n both the above methods, the eluate may be applied to a new chrom atogram by streaking on as before. I n yet another method, known as the ''sewing in" technique, the substance is not eluted from the paper at all. A strip of paper, corresponding exactly in size to t h a t containing the substance, is cut out of a clean sheet. The old strip takes its place and is sewn into the new sheet by close stitching with a needle and cotton; this is then treated as if it were a single sheet of paper (Fig. 38.1c). On chromatographing again, in a solvent with different
y&mJt
strips
FIG. 3 8 . 1 . (A) A solution is "streaked" o n t o t h e c h r o m a t o g r a m which is t h e n run in s o l v e n t in t h e normal w a y . (B) T h e dried c h r o m a t o g r a m is t a k e n , guide strips are c u t from it a n d located w i t h a suitable reagent. T h e strips are realigned w i t h t h e u n t r e a t e d portion of the c h r o m a t o g r a m , t h e position of t h e n e w spot is m a r k e d w i t h pencil and t h e n c u t out. ( C ) A n identical section is cut from a clean sheet, t h e n e w - s p o t s e g m e n t is s e w n in a n d t h e paper is c h r o m a t o g r a p h e d again in a different s o l v e n t , Sg. A l t e r n a t i v e l y t h e n e w material is e l u t e d from t h e strip a n d its solution is again streaked o n to a clean c h r o m a t o g r a m and run in S j . ( S . F . = s o l v e n t front.)
properties, substances running close together in the first solvent will probably r u n separately in the second (thus if butanol-acetic acidwater Avas used first then butanol-pyridine-water might be used second). After this second run the material is again located and the untreated section of the paper containing the substance is cut out once more. Should the substance be completely separate from the other components of the original mixture it is eluted into solution for further chemical analysis, otherwise re-chromatography in a third solvent is necessary. The solvents used for these runs should be ones which can be com pletely dried off the paper in the cold without leaving a contaminating residue. The possibility of contamination by material unreactive to any of the location reagents always exists b u t the chance of such contamination is greatly reduced after each successive chrom atographic run, as the unreactive material is necessarily of a different chemical family and so wih have quite different running properties in the various solvents; this is a n important consideration in the determination of physical properties (infra-red or ultra-violet absorp tion, etc.) of materials purified in this way. Using five thick papers on
T H E I N V E S T I G A T I O N O F N E W COMPOUNDS
971
the Frame, the time necessary for the isolation of 5-25 mg. is less t h a n a week even if chromatography in three different solvents is necessary. Similar techniques are used for thin layers. The mixture is apphed as a single streak and chromatographed as usual. After drying, the main plate is covered with a second plate b u t lea\áng a margin of 0-5-1 cm. on both sides in the ascending direction; the margins m a y be separated from the main plate by drawing down a thin line with a spatula to avoid lateral movement of reagents. Subsequently, a reagent is sprayed on bcth margin strips to locate the separated compounds. Horizontal lines are drawn across the plate (as shown in Fig. 38.1) and the powder is removed by scraping off with a spatula onto a sheet of aluminium foil or by sucking u p with the vacuum cleaner device shown in Fig. 4.22. The substance is then eluted with a suitable solvent using a tube with a sintered disc as a micro-column. I t is possible to purify m a n y milli grams of material on the ordinary thin layers used for analytical work. (6) Gram Amounts, The techniques of preparative layer chromato graphy described in Chapter 34 are a remarkable advance a n d have replaced column preparative methods for m a n y types of compound. However ion exchange columns remain valuable for ionic materials and the two methods can be considered as complementary rather t h a n as alternatives.
Step 3. Determination of Chromatographic Properties. I t is not
uncommon to find t h a t the R values of the pure compound are appreci ably different from those shown in the original mixture, possibly because of the mutual effect of compounds with close R values—a factor of increasing importance when the paper is overloaded. Hence these figures should be redetermined in the standard solvents used for com pounds which react with the particular location reagents; for example, compounds which react with ninhydrin are usuaUy r u n in butanolacetic acid-water, phenol-ammonia and butanol-pyridine-water (see Chapter 5). As it is possible t h a t t h e spot observed on t h e original chromatogram was composed of more t h a n one compound t h e pure material should again be treated with t h e various location reagents t o confirm t h a t all t h e original positive reactions are still obtained. At this stage it may be possible to say whether the material corresponds to one previously described b u t for which no authentic material is available for comparison and it is now necessary and worthwhile to synthesize a specimen for comparison of chromatographic and other properties. However, if the compound cannot be identified a t this stage a number of lines of attack present themselves. These are more conveniently discussed in relation to particular families of compounds although some of the methods are common to more t h a n one class.
Step 4. Further Investigation of Structure (a) AminO'Compounds. Amino-compounds m a y be subdivided as follows: amines, a-aminoacids, non-a-aminoacids, iminoacids, amides corresponding to one of the previous acid groups mentioned, peptides, multi-functional compounds such as an ammoacid containing an imidazole, indole, or guanidine nucleus or another amino, carboxyl, or amide group, and so on. All compounds in this group are located with
972
CHROMATOGRAPHIC T E C H N I Q U E S
ninhydrin and any which yield purple colours probably contain an NHg group attached to an aliphatic carbon, although the converse is not true. Acid hydrolysis for a period of 1 hour will yield the free acid derived from an amide and this will probably have a higher Rf in the butanol-acetic acid solvent t h a n the original; further more only one new spot will appear on the chromatogram. For a peptide it may be necessary to use hydrolysis periods up to 2 4 hours in order to split it completely into its constituent aminoacids but, as m a n y peptides contain amide groups, it is essential to carry out both t h e I and 24 hour hydrolyses in order to confirm t h a t the shorter period is not simply liberating a peptide free of amide groups. Most peptides of natural origin (e.g. glutathione, oxytocin, antibiotics, etc.) will yield, after hydrolysis, at least two spots on the chromatogram b u t other peptides are known which yield only one spot. For example, the peptides valyl-valine and leucyl-leucine are particularly resistant to hydrolysis and so may be found on the chromatograms of incompletely hydrolysed material; on hydrolysis of such peptides only one spot is obtained. Cases are also known where spots on chromatograms have been identified as standard aminoacids simply on the basis of position on the paper chromatogram when, in fact, they were actually peptides. Similarly the first naturally occurring sulphonium aminoacid, methyl methionine chloride, was originally "identified" as one of the basic aminoacids as it overlapped these compounds on chromatograms and yielded the same ninhydrin colour. I t should be noted here t h a t indoles are rapidly and completely destroyed by acid hydrolysis, t h a t iodo- and bromo-aminoacids are dehalogenated during 2 4 hour hydrolysis, and t h a t phosphorylated and sulphated compounds are split to form the parent hydroxy or guanidino compound after only a few minutes boiling with dilute acid. Having shown t h a t a compound is a sulphate or phosphate ester it is more rewarding, as well as being more simple, to endeavour to elucidate t h e structure of the parent compound first, as a great deal of chromato graphic data on amino-compounds is available whereas t h a t on such esters is scant. Alkaline hydrolysis is essential for the determination of the structure of indolic, iodine and bromine compounds, and for peptides containing these groups a combination of both acid and alkaline t r e a t m e n t is useful. Such alkaline hydrolysis is also useful in the study of guani dines as these are split to the parent amino-compound; t h u s Thoai and Robin^^^ could have used this to advantage in elucidating t h e structure of the recently reported guanidoethanol and guanidotaurine which yield the well known compounds ethanolamine and taurine. Furthermore, simple alkyl guanidines can be differentiated from the more complex guanidino analogues of aminoacids, as the former yield volatile amines which can be distilled off—a suitable apparatus is a modified Conway apparatus or manometric conical flask which can be partly immersed in a boiling water bath (see p. 293). If the compound is a peptide, it is probable t h a t t h e aminocompounds of which it is composed will be identifiable by further chromatography. If this is not so, the unidentifiable compounds
Plates I-I O are reproduced from "Paper & Th/n Layer Chromatography and Electro phoresis"—A Teaching Level Manual for use with the Unikit Apparatus. By kind permission of Shandon Scientific Company Ltd (see pió).
SF
Bk + Br,
Bk
Br,
Br,
Bl
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1 cm Plate I: PAPER CHROMATOGRAPHY, ascending. Separa tion of component dyes of writing inks. Solvent — butanol: ethanol: ammonia. Inks: Bk + Bri, Bk, Bri, ΒΓ2, Bl, Rd, Gn, Gn. Note that neither of the brown inks contains a brown pigment. The two are obviously distinguishable. Note also the reproducibility of the green. Paper 25x25 cm.
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1 cm P l a t e 3: PAPER CHROMATOGRAPHY, two-way. Separation of amino acids present in orange juice. Solvents — first: ethanol: water: ammonia; second: butanol: acetic acid: water. Note the yellow proline and the brown asparagine spots.
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Θ 1 cm Plate 5: CHROMATOGRAPHY-ELECTROPHORESIS, two-way. Separation on paper of pigments present in a mixture of brown and black inks. The first separa tion, vertically, was by chromatography; solvent — butanol: ethanol: ammonia in the 10 cm direction. The second separation, horizontally, was by electro phoresis; buffer — ammonia: ammonium acetate in the 30 cm direction. All pigments have migrated towards the anode, or have remair\ed stationary at the origin. Paper 10x30 cm.
Θ
Θ 1 cm Plate 6: ELECTROPHORESIS-CHROMATOGRAPHY, two-way. Separation of amino acids present in fresh orange juice. The first separation, horizontally, was by electrophoresis: buffer — pyridine: acetic acid. The second separation, vertically, by chromatography: solvent — butanol: acetic acid: water.
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T H E I N V E S T I G A T I O N O F N E W COMPOUNDS
973
can be examined as described for a simple compound. I t has often been assumed t h a t it is not necessary to isolate a compound in order to show t h a t it is a peptide and, if this is all t h a t is required, the original extract can be hydrolysed directly when further chromatography wiU show t h a t the new spot has now disappeared. With the discovery of more and more non-peptide substances unstable t o acid hydrolysis, this assumption can no longer be maintained, and before such arguments can be used, it is necessary to show t h a t t h e compound is, at least, not a phosphate, indole or iodine containing substance. Should the compound prove resistant to hydrolytic cleavage, the foUowing further experiments should be carried o u t : 1. Chromatography of the copper carbonate-treated material (see p . 126). A two-way chromatogram with solvent runs of about 6-8 in. is sufíicient as only a single substance is being investigated. 2. Chromatography after peroxide oxidation (see p . 126). Labile compounds are often oxidized under the conditions of acid or alkaline hydrolysis—a factor of importance in the previous discussion on pep tides. A change of will usually suggest the presence of sulphur in the molecule although the converse is not true because of the existence of some completely oxidized compounds such as taurine. 3. Charcoal adsorption (see p. 261). Aromatic compounds are selectively adsorbed on to the column and may be eluted after the nonaromatic materials have been washed through. 4. Electrolytic desalting of a fraction of the material (see p . 41). Strong acid conjugates will be lost via t h e anode solution, activated double bonds will be reduced, etc. 5. Nitrous acid deamination (see p . 258 for a suitable reagent). Nitrous acid rapidly deaminates aliphatic amino-compounds and use can be made of this in the following ways. A peptide is treated with the nitrous acid in acetic acid solution (mineral acid must be absent), hydrolysed and chromatographed when one aminoacid present in the original hydrolysate will now be found to be missing, i.e. the one a t the amino end of the chain; should none be missing then the peptide m a y weh be cyclic in nature. If a new compound has been shown to be a non-a-aminoacid (copper carbonate technique) then a further distinc tion may be dra\vn between those compounds containing a primary amino group and others such as secondary amino compounds (sarcosine), nitrogen-containing rings (piperidine, pyrrolidine, indole, and imidazole derivatives), etc. Thus on treating the mixture with nitrous acid primary amines are destroyed while secondary amines form N-nitroso' derivatives, m a n y of which are ether-soluble and can be regenerated to the parent compound by acid hydrolysis. This method is of value for isolating iminoacids from admixture with aminoacids. (b) Peptides and Proteins. Valuable methods are now available for structural studies of these compounds (see Chapters 6 and 7). (c) Sugars. Sugars m a y be divided into the following groups: aldoses, ketoses, reducing and non-reducing sugars, mono-, di- triand ohgo-saccharides, acids, etc. Reagents are available to determine into which group a monosaccharide falls and R^ determinations in three or four solvents will often give useful indications of whether the 63
974
CHROMATOGRAPHIC T E C H N I Q U E S
compound is a pentose, hexose, or ohgosaccharide. Acid hydrolysis (dilute HCl, I hour) will split all those sugars which travel from the origin during chromatography, i.e. up to heptaoses, into their consti t u e n t units and partial hydrolysis (chlute HCl, room temperature for varying periods) wiU yield intermediate breakdown products which may be investigated separately. Enzyme hydrolysis either in solution, or on paper, before or after chromatography, will indicate the type of bond present, e.g. a or β glucósido, etc. (d) Enzymic reactions. Aminoacid solutions have been treated with both D and L aminoacid oxidases, sugars have been treated with bond specific enzymes, and so on, and the solution then chromatographed in order to determine further properties of the compounds present in the original. I n m a n y cases it is possible to carry out the enzyme reaction on the paper chromatogram either at the origin and before chroma tography, or after chromatographic separation, by application of the enzyme to the whole of the paper after removing the solvent. (e) Reactions on the chromatogram. The above reactions have been carried out on the original extract or on the paper or layer prior to chromatography, b u t there is no reason why m a n y of them, or other reactions as well, should not be carried out after the chromatographic separation or even in between the first and second solvent runs in twoway chromatography. Thus sugars might be separated in one direction, the area of paper on which they now lie is sprayed with invertase and, after some 5-10 minutes, the paper is dried and placed in the second solvent for further separation. Steroids containing side chains have been run in one direction and, after drying, the side chains have been oxidized off and the steroids so formed have then been run in an appropriate solvent in the second direction.
Examples of Apphcation of Above Techniques 1. The Occurrence of Iodo-aminoacids in Certain AlgaeS^^ Two spots, A and B, were often observed on ninhydrin-treated chromatograms of alkaline hydrolysates of certain whole algae. Further tests on two-way chromatograms showed t h a t both spots contained iodine and spot Β was also positive to the nitrosonaphthol test. Ry of spot A : same as leucine in butanol-acetic acid-water, same as tyrosine in phenol-ammonia. Ry of spot Β : half way between A and tyrosine in same as tyrosine and A in PhAm.
BuA,
Known iodo-aminoacids include iodo-tyrosines and thyronines. Both spots disappeared after acid hydrolysis or copper carbonate treatment. Co-chromatography with authentic di-iodo-tyrosine showed enlargement of spot A, and with mono-iodo-tyrosine enlargement of spot B ; Electrolytic desalting resulted in the disappearance of A and Β and enlargement of tyrosine spot. Therefore the two new spots corresponded to the two iodo-tyrosines in running properties and colour reactions. These compounds were found to be present in aU red algae and some brown algae (laminariaceae) but not in other classes examined.
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P l a t e 8: T H I N LAYER CHROMATOGRAPHY. Separation of component dyes of writing inks. Solvent — butanol: ethanol: ammonia. Inks: Bk, Bri, Br2, Bl, Rd, Gn, R d + G n . Compare Plate I.
T H E I N V E S T I G A T I O N O F N E W COMPOUNDS
975
2. The Occurrence of PipecoKnic Acid, (a) Stewards proof.^'^ A new spot on chromatograms of extracts of the common bean yielded a bluepurple with ninhydrin, green-blue with isatin and travelled shghtly higher t h a n proline in the usual aminoacid solvents. F r o m colour reactions it A v a s thought probable t h a t the compound was related to pyrrohdine or piperidine compounds. The substance was stable t o acid and alkahne hydrolysis, and was shown to be a non-a-amino compound as it was unaffected hy copper carbonate treatment. The material was isolated as follows: passage down a cation exchange resin retained it, elution with dilute acid removed m a n y aminoacids b u t not the unknown, displacement with dilute ammonia yielded a n eluate containing the unknown plus basic aminoacids plus y-amino-butyric acid, and careful crystallization yielded pure unknown. Comparison of B,f and colour reactions with synthetic piperidine 2-, 3-, a n d 4-carboxylic acids showed t h a t the unknown corresponded t o the 2-acid. This was confirmed b y infra-red spectroscopy. B y a splendid modification of the copper carbonate technique (copper carbonate/alumina column) a large scale separation of pipecolinic acid yielded 15 gm. of material and the method was shown to be of great potential value for the isolation of non-a-amino acids. (6) Morrison's Proof.^^^ The unknown was independently discovered by identical reactions mentioned above. I t was isolated as follows: charcoal treatment removed aromatic compounds from t h e original extract b u t not the unknown; bulk separation on cation exchange resin yielded a mixture of unknown plus leucine, valine, prohne, a n d y-aminobutyric acid; fractionation of this on ceUulose yielded unknown plus vahne, and crystalhzation gave pure unknown. Comparison with piperidine acids foUowed b y X-ray powder photography identified the unknown as piperidine-2-acid. 3. Identification of urinary Indican as Indoxyl Sulphate. The treat ment with Ehrlich reagent of paper chromatograms from normal h u m a n urines invariably gives a n orange-brown spot, intensifying t o red on later application of ammonia (the fluorindal reaction, see p . 257). This chromogen is related in amount to the '^indican" level of t h e urine as shown b y the Obermeyer test, and disappears from t h e urine in a sub ject dosed with Chlortetracycline, a gut-sterUizing antibiotic. Tentative identification of the spot as *'indican" (acid conjugates of indoxyl formed from tryptophan by gut bacteria) was supported by (a) the dark blue colour (indigo) given by treating the area of t h e spot with acidic oxidizing agent; (b) the red colour (indirubin) given b y t h e spot on treating the paper with acidic isatin and heating; (c) the fact t h a t the chromogen was no longer present in electrolyticaUy-desalted urine, b u t could be recovered from the effluent acid. The indican was chromatographically identical (R^ and colour reactions) with synthetic indoxyl sulphate added to electrically desalted urine, though both were markedly displaced b y other urinary com ponents from the positions given b y indoxyl sulphate in aqueous solution. No other area of chromatograms from neat urine gave indigo blue on acidic oxidation. One-way chromatography of t h e urine, on
976
CHROMATOGRAPHIC T E C H N I Q U E S
thick paper in IPrAm, foUowed by elution of the appropriate strip, furnished a solution of the indican, chromatographically-free from other Ehrlich reactors, and shown colorimetrically to represent a good recovery of the total indican. Six solvents failed to show this eluted indican to be in any way different from synthetic indoxyl sulphate in aqueous solution. But there remained the possibility t h a t the urinary indican contained some other indoxyl conjugate, say the glucuronide, which was not separable under the conditions tried (a synthetic sample of indoxyl glucuronide is not avaUable for comparison). The eluted indican solution was, therefore, mixed with a n enzyme preparation from limpets, known to contain an aryl sulphatase of wide specificity, b u t showing no glucuronidase or phosphatase activity. After this treat ment, no indican of any type could be detected chromatographically, though insoluble indigo had been formed. Conversely, a limpet ßglucuronidase preparation free from sulphatase caused no detectable change in the indican eluate. Thus, subject only to the proviso t h a t the chromatographic process has not destroyed other indoxyl conjugates, the indican of normal urine, and of many abnormal urines, wpuld appear to be solely indoxyl sulphate. 4. Identification of a Urinary Glucuronide as Testosterone Glucuronide. When the 17-ketosteroid glucuronides were first separated from one another, a "new" steroid was detected, and this was also presumed to be a glucuronide. I t was far from pure, and in fact was always found in the tail of the aetiocholanolone glucuronide peak from the partition column. However, hydrolysis of this material with j5-glucuronidase and examination of the liberated steroid in the A system (p. 584) showed the *'new" steroid to have an R,(19) half t h a t of aetiocholanolone. I t s reactions on the paper were: (i) ultra violet light of about 240 ιημ, was absorbed; (ii) with m-dinitrobenzene and K O H (Zimmermann reaction) a blue colour was produced; (iii) an orange dinitrophenyl hydrazone was formed; all characteristic of Δ^-3-ketones. Reaction (ii) rules out a 17-ketosteroid which would give a purple colour, so it was concluded t h a t the usually oxygenated 17 position carried a hydroxyl group. From the Ry it could be assumed t h a t the ''new" steroid was only dioxygenated; trioxygenated compounds have R^ values less t h a n 10 in the A system. Testosterone is a steroid t h a t has aU these proper ties, and when an authentic sample of this compound was run alongside the "new" steroid in the A system they were found to have identical R/s. To check the identity, the "new" steroid and testosterone were oxidized by CrOg (1 per cent) in acetic acid solution at room temperature for 15 hours. After dUuting with water, the steroids were extracted into benzene, which was washed with N a O H solution and water to remove the acid. On running these in the A system in parallel with androst4-ene-3:17-dione, expected from the oxidation of testosterone, aU three compounds were found to have identical Ry.'s(54), as well as reactions (i) and (iii) above. Reaction (ii) produced a purple colour, characteristic of the 17-ketone group.
T H E INVESTIGATION O F N E W COMPOUNDS
977
The original compound, which was hj^drolysed b y the specific jS-glucuronidase preparation, was presumably a /S-glucuronide. The only position on the steroid available for the a t t a c h m e n t of this group is a t 17 and if this is so, the Δ^-3-ketone group must be intact and the compound should show absorption of 240 ηψ. light. This absorption was found to be present, whereas it was not shown by the other glucuronides from the same partition columns. The above argument is summarized diagrammaticahy below. W h a t would have happened if the " n e w " steroid had not been testosterone? Another possibility involves the oxidation of an 11-hydroxyl group to keto b u t this would not have brought about such a large change of R^, e.g. the oxidation of ll-hydroxy-setiocholanolone (R^ = 10) to ll-keto-setiocholanolone (Rf = 15). COOH
HO
The " n e w " steroid could have been the testosterone analogue without the unsaturation at the 4 position (17-hydroxy-5 (a or /9)-androstan-17one), in which the 240 m ^ . absorption would have been missing and it would have been oxidized to androstandione or setiocholandione [5 (a and ^)-androstan-3:17-diones], both of which have R^ values about 60, so they are ruled out. On examining the extensive steroid literature it is clear t h a t other positions for the hydroxyl group are unlikely to produce an original compound and an oxidation product with t h e R^'s found. Alternative chemical transformations are available, and these must also be considered before starting the investigation. Acetylation is technically easier t h a n oxidation, b u t the separation of C^g steroid acetates is very difiicult and cannot be effected in t h e A system as most of these move with the front. However, acetylation m a y be very valuable in the adrenocortical steroid series.
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CHROMATOGRAPHIC T E C H N I Q U E S
Having, by the use of some 50 μg. of the "new" steroid, obtained a clear indication of its identity, the next step is the isolation of the compound on a larger scale, both as the original glucuronide and the free steroid, for the identification is not conclusive until the classical proofs of melting point, rotation and synthesis are obtained. A large degree of purification can be obtained by running broad bands of " n e w " steroid followed by elution as described earlier, b u t it is necessary, how ever, to avoid overloading the chromatogram as large amounts of con taminants are also washed out of the paper, and subjection of the paper to preliminary washing does not prevent this. Column chromatography with Bush systems supported on celite or kieselguhr yields good separa tions provided t h a t a "slower" system t h a n the corresponding paper solvent is used. This large scale work has not yet been possible on the testosterone compound described here, but it is hoped t h a t this dis cussion should provide a guide to the examination of " n e w " compounds which m a y be treated by the t3φe of chemical transformation and R^ identification described here.
Fingerprmt and Diagonal Techniques-^^^^ REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Partridge, S. M. Brit. Med. Bull., 1954, 10 (3), 2 4 1 . Partridge, S. M. Analyst, 1952, 77, 955. N g u y e n - v a n Thoai, a n d R o b i n , Y . Biochim. Biophys. Acta, 1954, 13, 533. D e n t , C. E . , a n d B o n e t t i , B . E . Biochem. J., 1954, 57, 77. E d w a r d s , R. W . H . (See chapter 23). S m i t h , I. (Unpubhshed.) Steward, F . C , Zacharius, R . M., a n d T h o m s o n , J . F . J. Amer. Chem. Soc, 1954, 76, 2908 a n d 2912. Morrison, R. I. Biochem. J., 1953, 53, 474. E d w a r d s , R. W . H . , a n d KeUie, A . E . Chem. and Ind., 1956, 250. W i t k o p , B . , and F o l t z . C. M. J . Amer. Chem. Soc, 1957, 79, 19. See cliapter 7 a n d vol. 2, chapter 8. General Reviews
and
Methods
Methods of Biochemical Analysis. Vol. I , 1954. Intersci., etc. Modern Methods of Plant Analysis. E d . P a e c h a n d Tracy, Springer. Vols. I - I V cover a wide range of m e t h o d s a n d chemical families. Progress in Chemistry of Fats. Vol. I, 1952. F a t t y acids a n d related s u b s t a n c e s . Methods in Carbohydrate Chemistry, Vol. I, 1960. Advances in Protein Chemistry, Vol. I , 1947. Separation of Biological Molecules. Brit. Med. Bull., 1966, 22(2), 103. Advances in Chromatography, Vol. I, 1965.