Dec., 19501 BAKER, DOBSON AND MARTIN 661 Analysis of Penicillin Mixtures by Paper Chromatography of the Hydroxamic Acid Derivatives BY P. B. BAKER, F. DOBSON AND A. J. P. MARTIN* (Read at the meeting of the Society on Wednesday, AfiriL 6th, 1949) SYNOPSIS-A rapid method that is independent of biological assays and suitable as a routine procedure is described for estimating one or more species of penicillin in a mixture. This method requires less than 8 hours for completion; it is based on the fact that the relatively stable hydroxamic acid derivatives of the various penicillins show different partition coefficients between isopropyl ether - isopropyl alcohol and phthalate buffer, at a given pH, and can therefore be separated by paper chromatography. A novel apparatus for use with volatile solvents and heavily buffered papers, is described.A direct, qualitative result is obtained by developing the chromatograms with dilute ferric chloride solution. A quantitative result is attained by extracting the iron complexes of the various hydroxamic acids with butyl alcohol, measuring the degree of extinction in a colorirneter and reading the penicillin concentration from standard curves. GOODALL and Lev9 have described a method of analysis of penicillin based on partition chromatography on buffered paper strips and using bacteria-sown plates to detect and estimate the various penicillins. When the present work was carried out that was the only published method capable of routine application to give a reasonably complete analysis of a mixture of several different penicillins.The accuracy of the method is essentially that of the biological assay, which is sufficient for most purposes when the content of one species of penicillin is high, but barely adequate when more than one penicillin is present in considerable amount. A further disadvantage of the method is the length of time, three days, that it takes. Although we have had but little experience of the Goodall and Levi method, we believe that considerable experience is required before results comparable with those obtained by its originators can be obtained. For these reasons it seemed worth while to reinvestigate the possibilities of other methods that use paper chromatography. Little progress was made, however, until the colorimetric method of Ford2 was adapted to our requirements.Ford made hydroxamic acids by mixing penicillin and hydroxylamine- S CH, S CH, / \ / -i- NH,OH -+ R.CO.NH.CH/:H C / \/ R.CO.NH. CH-CH I I PCHs CO-Y __- CH.COONa ~ H O H and showed that the Fe"' complexes with the hydroxamic acids could be used for colori- metric estimations. These hydroxamic acids are relatively stable substances that can be separated on paper chromatograms at room temperature without serious loss. After separation the paper is sprayed with ferric chloride to give reddish-brown spots on a buff background at the positions of the individual hydroxamic acids. These spots can be extracted from the paper and the various penicillins estimated colorimetrically in the extracts. It is suitable for crude or pure salts of penicillin, but not for culture fluids without a preliminary extraction.A The colorimetric method is far less sensitive than the biological. * Present address. National Institute for Medical Research, Mill Hill, London.652 BAKER, DOBSOh' AND MARTIN: ANALYSIS OF PENICILLIN MIXTURES BY [VOl. 75 strongly coloured spot is given by 100 pg. of penicillin. A convenient amount for quantitative estimation is 1 mg. With buffer-loaded paper chromatograms it is difficult to keep control of the humidity of the paper during development, and this difficulty is accentuated when a volatile solvent is used. Goodall and Levi, by rigorous attention to the routine of handling the paper, succeeded in reproducing their conditions, even though their paper was loaded with strong phosphate solution and their chamber had walls covered with cloth that was wet with water.It is evident that no eaquilibrium can be attained in such a system, and it is to be expected that the chromatographic behaviour will vary with every change in the previous history of the paper, with the shape and size of the vessels and with the temperature and time of running. An attempt was made to arrange conditions so that the chromatograms could run under equilibrium conditions. The air within the chamber was kept saturated with respect to both phases on the paper. To do this was found to be difficult, and not until both phases were pumped continuously over the walls of the chamber were reproducible results obtained. No method of simply stirring the air or the liquid, or both, was adequate.With the apparatus described below, however, it was found that the paper came to substantial equilibrium in half an hour and that the temperature was comparatively un- important, a variation of as much as 10" C. during the run being without any deleterious effect. It is believed that this type of apparatus will be of general utility when volatile solvents (e.g., ethyl ether) and heavily buffered papers must be used, If the stationary phase is an aqueous solution of a non-volatile substance, it is of course not necessary that it should be circulated round the vessel, but it can be replaced by anj. other solution with the same vapour pressure, and this may be advantageous when expensive or corrosive solutions are concerned. The role of the buffer with which the payer is loaded is worthy of some discussion.Satisfactory chromatograms are obtained with either citrate or phthalate buffers, but not with oxalate or phosphate of the same pH value. With oxalate or phosphate the spots of the hydroxamic acid complex are elongated and the RF values are small. With citrate or phthalate the spots are compact and the RI: values much greater. This may be explained by the assumption that the hydroxamic acids exist as dimers in the mobile phase (a mixture of isopropyl ether and isopropyl alcohol) when phosphate or oxalate buffers, which are insoluble in the mobile phase, are used. When citrate or phthalate buffers are used, citric or phthalic acid dissolves in the mobile phase to an extent that is large compared with the amount of hydroxamic acid present and chelation between the hydroxamic acid and the buffer acid occurs.Since the buffer is in excess, the amount of chelated product is pro- portional to the concentration of hydroxamic acid and not, as with the dimer, to the square of the concentration. As a result the partition coefficient is more in favour of the mobile phase and is comparatively insensitive to the concentration of the hydroxamic acid. Hence the spots on the chromatograms are faster running and more compact with phthdate or citrate than with oxalate or phosphate buffers. METHOD Paper-Whatman No. 4 paper is used. It is dipped in 0.10M potassium hydrogen yhthalate solution and air-dried. The paper comes finally into equilibrium with 0.50 M buffer solution and should be dry enough to gain rather than lose water when it is placed in the chromatogram box.To retain heptyl penicillin hydroxamiq acid on a reasonable length of paper while the other penicillins are adequately developed, the lower third of the paper should be dipped in phthalate buffer of pH 6.2. Whatman No. 1 paper will give satisfactory chromatograms but a much longer time of development is then required. Preparation of hydroxamic acids-In 1 ml. of a mixture of equal volumes of 4 N hydroxyl- amine hydrochloride and 3 N sodium hydroxide are dissolved 10 to 40 mg. of penicillin salt. Ten p1. of this solution are applied as a spot to the chromatogram and air-dried. Ten sqch spots can be accommodated on a sheet 15 cm. wide. A single spot suffices for a qualitative analysis, ten for a quantitative analysis.Mobile phase-The mobile phase is isopropyl ether containing 15 per cent. v/v of isopropyl alcohol. To each 100ml. of the mixture are added 2.4ml. of O.lOIW potassium hydrogen phthalate to give approximate saturation with respect to the stationary phase. The mixtureDec., 19501 PAPER CHROMATOGRAPHY OF HYDROXAMIC ACID DERIVATIVES c G 1 I L A B C D Scale Fig. 1. Diagram of chromatogram 110s for usc ith .i-olatile solvents ,I. Front clevation (trough absent) B. Side elevation (trough present) C . Plan 2). Isometric view of lid. The box is constructed of 3/16-inch Perspex. (a) Flanged, gabled lid, with stoppered opening for filling trough; ( b ) rods for supporting cloth lining; (c) perforated tube delivering pumped liqnid to cloth lining; (d) outlet (with gauze filter) for return of liquid to pump: (e) support for trough; ( 1 ) stainless stcel trough (end view alone shown); (6) wooden base 653654 BAKER, DOBSON AND MARTIN: ANALYSIS OF PENICILLIN MIXTURES BY [VOl.75 should be free from aldehydes or peroxides: distillation of the solvents from saturated bisulphite or 5 N sodium hydroxide ensures this. Apparatus-The apparatus used, Fig. 1, consists of a Perspex box 15 x 25 x 60 cm. with a gabled lid, so that liquid condensing on the top does not drip on to the chromatograms. The walls of the box are lined with cotton cloth, over which both phases are pumped con- tinuously by a diaphragm pump delivering 1500ml. per minute. The liquids are drawn from the bottom of the box, and, in setting up the apparatus, special attention is paid to ensuring that both phases are circulated.The papers, already spotted with hydroxarnic acids and hanging from an cmpty trongh, are placed in the chromatogram box and the pump is started. After thirty minutes the trough is filled with 50 ml. of the mobile phase. Six hours later the paper is removed, air-dried and sprayed with 2 per cent. ferric chloride solution in 0.01 N hydrochloric acid. Inspection of the sheet now permits a qualitative analysis. K group "MiXTURE" ''INTS OF ORIGIN Benzyl Pent ctlli n ~- Benzyl Penicillin Fig. 2. Chromatograni showing various penicillin types, 100 pg. per "spot" Extraction techniqwe-The chromatograms are cut across into strips, each containing the 10 spots from one species of penicillin, which will have an average area of 75 sq.cm. Each strip is macerated with 1 ml. of 20 per cent. ferric chloride in 0.1 N hydrochloric acid, 10 ml. of n-butanol, 2 g. of anhydrous sodium sulphate and 0.6 g. of sodium chloride. The tubes are held in a water-bath at 20" C. until measured. The paper and salt can be packed at the bottom of the tube with a glass rod so that 7 to 8 ml. of the butanol can be poured off for the colorimetric measurement. The measurements were made in a photo-electric colori- meter (Evans Electroselenium Ltd.) with a "tricolour" green filter, No. 404. A more elaborate instrument would no doubt give greater accuracy.Dec., 19503 PAPER CHROMATOGRAPHY OF HYDROXAMIC ACID DERIVATIVES 655 The extinction of the butanol solution prepared in this way increases with temperature and the extraction and measurement should be made at a constant temperature.The colour appears to be stable for many hours and the solution obeys Beer’s law within the error of measurement. Satisfactory extraction of the hydroxamic acid from the paper required a somewhat polar solvent, which unfortunately also dissolved enough ferric phthalate to make a large and variable blank. To displace the hydroxamic acid from an aqueous solution into, say, butanol, without multiple extractions The reagent mixture was arrived at in the following way. 0.5 I .o I .5 2.0 1.5 AMMONIUM PENlClLLtN, mg. PER 10 ml. OF BUTANOL Fig. 3. Standard curves from pure penicillins Curve G, benzyl penicillin; curve F, pent-2-enyl penicillin; curve D, B-amyl penicillin; curve K, n-heptyl penicillin, etc.it was necessary to “salt out” with sodium sulphate or some other salt. The depth of colour of a given butanol solution of hydroxamic acid is dependent upon the amount of ferric chloride in it. Since the amount of ferric chloride sprayed on the paper is liable to variation, a large excess of ferric chloride was added to the extraction mixture. In the presence of saturated sodium sulphate, the ferric chloride is almost completely converted to ferric sulphate, which is not extracted by the butanol; hence no colour is obtained. By saturating the solution with sodium chloride also, the ferric chloride activity is raised and good colour development of the hydroxamic acid is obtained with a small and constant ferric chloride blank.The high concentration of chloride and sulphate in the aqueous phase, by competing with the relatively small amount of phthalate, so reduces the amount of ferric phthalate dissolved in the butanol layer that the blank remains independent of the area of paper taken. By this procedure no dilution or making up to known volume is required and practically the whole of the hydroxamic acid is dissolved in the 10 ml. of butanol, of which a large proportion is available for use in the colorirneter. RESULTS A typical chromatogram is shown in Fig. 2. The RF values3 of the hydroxamic acids Standard curves from pure penicillins are given in Fig. 3, showing the relation between If the gradient for penicillin G Pure penicillin from different penicillins are shown in Table I.log current and amounts of penicillins of different kinds. is taken as 100, the gradients of the other curves are F 95.5, D 86.3, K 75.8.656 BAKER, DOBSOS ,4ND MARTIN [Vol. 75 X has not been available and it is uncertain whether the K penicillin is wholly 92-heptyl. Other K varieties have not been available. TABLE I RF VALUES OF HYDROXAMIC ACIDS FROM T*rlHIOUS PENICILLINS Parent penicillin 13, value $2-Heptyl (K) . . . . . . . . . . 0.57 Pent-2-enyl (1;) . . . . . . . . 0.20 Benzyl (G) . . . . . . . . . . 0.13 w-Amy1 (D) . . . . . . . . 0.27 p-Hydroxy benzpl (X) . . . . . . 0.0i Tables I1 and I11 show analyses of control mixtures of pure penicillins, Table IV analyses of an unknown mixture. T-SBLIS 11 ,!XALYSIS OF A CONTROL MIXTURE CONTAISING 75 PER CENT. OF BENZYL PEKICILLIN AND 25 PER CENT. OF n-mYJ; PENICILLIN 73.2 7 2 4 76.3 76.6 75.2 26.8 37.6 23.i 23.4 24.8 TABLE 111 ANALYSIS OF CONTROL MIXTURE CONTAINING 50 PEK CENT. OF BENZYL, 8 PER CENT. OF PENT-2-ENYL, 22 PER CENT. OF n--kMYL AND 20 PER CENT. OF PE-HEPTYL PENICILLINS Benzy 1, Pent-2-eny1, +z-Xniyl, ?a-Heptyl, 48.8 9.3 14.7 27.2 49.1 7-7 19.6 23.6 46.8 7.7 22.1 23-2 47.9 7.2 23.0 91-7 % % Y O % TABLE IV SAMPLE 316 AMMONIUM SALT Benzyl, % 43.7 44.8 46.0 47.0 41.0 44-0 43.8 44.0 47.6 44.1 Pent-2-eny1, 0 1 / O 14.6 14.0 17.0 16.0 15.5 17.8 16.3 16.4 13.5 l5.8 I<-group, % 17.3 16.3 9.7 12.0 18.8 13.1 14.9 14.3 13.5 13.8 The authors wish to thank Sir Jack Drummond, F.R.S., Director of Research, Boots Pure Drug Co., for his encouragement and for permission to pubIish this work. REFERENCES 1. 3. Ford, J. H., Anal, Chew., 1947, 19, 1004. 3. Goodall, 13. R., and Levi, A. A,, ..Imdyst, 1947, 72, 277. Consden, R., Gordon, A. H., and '1\Iartin, -4. J. P., 13ioclieiJf. J . , 1944, 38, 224. RESEARCH DEPARTMENT BIOCHEMISTRY DIVISION HOOTS PURE DRUG COMPANY LIMITED First submitted, May, 1949 NOTTINGHAM Amended, J u ~ , 1050