310 GLISTER AND GRAINGER MODIF1E:D RAPID TECHNIQUE FOR [Vol. 75 A Modified Rapid Technique for the Separation and Determination of Penicillin Types by Partition Chromatography on Paper BY G. A. GLISTER AND A. GRAINGER SYNOPSIS-A modified rapid-development technique for the micro-chromato- graphic assay of penicillin types is presented. Problems arising out of the quantitative interpretation are discussed, and it is indicated how, by using developed mixtures of pure penicillins as standards, the results are rendered more truly representative. THE method of Goodall and Levi,l using the principle of Consden and Martin,2 for the separation and estimation of penicillin types is well known andl requires no introduction. It is recognised as the method giving the most comprehensive picture of the constituents in a mixture of penicillins.The recently published methods of Winsten and Spark3 and KlueneI-4 describe rapid development at room temperature. In view of the advantages offered by this in routine practice, our procedure was modified and, by using a simplified developing apparatus and narrow paper strips, the basic principle of filter-paper impregnated with phosphate buffer as support for the stationary aqueous phase and diethyl ether as the mobile phase being retained, development time was cut from 24 hour!; at 4” C. to 4 hours at room temperature. Quantitative interpretation of chromatographic assays of this type has been the subject of much criticism and investigation, the issue being confused by the varying shapes of the inhibition zones that are produced and by the use of undeveloped standards prepared from only one entity.Maximum zone widths, lengths and areas have all been considered as measures of activity, and various corrective factors and formulae have been evolved, but so far none has proved satisfactory. The hydroxamic acid method of Baker, Dobson and Martin6 offers a useful alternative to the microbiological finish, but has the disadvantage that it is not so readily applicable to samples of low penicillin content, such as broth culture filtrates and the more dilute extracts therefrom. The quantitative interpretation of the microbiological chromatographic technique has therefore remained only comparatively accurate for any one technique. RAPID DEVELOPMENT- Experimental work finally led to the use of micro-strips of Whatman No.1 filter-paper (30 cm. x 1 cm.) impregnated with potassium phosphate, pH 6.2, and a decreased dose of penicillin of 5 units per strip instead of the original 30 units. Development of these strips with water-saturated diethyl ether as mobile phase for 3 to 4 hours at room temperature, and using a glass jar (14 inches x 8 inches diameter) and an evaporating basin as developing chamber and trough respectively, produced chromatograms which were reproducible and comparable with those produced by other methocis, especially by that of Goodall and Levi in which strips twice the size are used and developed at 4” C. for 24 hours. QUANTITATIVE INTERPRETATION- Goodall and Levi, using standard dilutions of penicillin G, found that the ratio of the biological assay “slopes” from the inhibition zones of developed to those from undeveloped, or stationary, spots was approximately 1.2.They therefore introduced this factor as a correction to be applied to plate slopes determined by using undeveloped standards. In our early experimental work we confirmed. the factor over the range 30 to 0-3 units of penicillin G per spot (a concentration ratio of 100/1), but found that it varied considerably outside these limits. Developed standards were then introduced as indicated below. The grade of filter-paper and the pH of the phosphate buffer used to impregnate the paper, as well as the concentration of penicillin per strip and vapour conditions within the developing chamber, are all known to be factors affecting chromatographic separation and EXPERIMENTALJune, 19501 SEPARATION AND DETERMINATION OF PENICILLIN TYPES 31 1 hence the shape of the final inhibition zones of the penicillins.Observation and measurement of numerous series of typical inhibition zones obtained from the paper chromatograms indicate that in descending order of development- $-Hydroxybenzyl penicillin (X) . . Produces a circular zone. Benzyl penicillin (G) .. . . 7 All produce elliptical zones of similar shape, h2-Pentenyl penicillin (F) . . e.g., the ratios of major to minor axis are n-Amy1 penicillin (dihydro F) . J constant. ut-Heptyl penicillin (K) . . . . Produces an elliptical zone which is elon- gated out of all proportion to the others. From consideration of the zone shapes it may be seen that if penicillin G standards (developed under identical conditions as the test penicillins) are used and the maximum zone width is taken as the criterion against log units then, as there is a slight decrease in zone width for an increase in zone length, there results an over-estimation of penicillin X and an under-estimation of penicillin K.Therefore a standard should be strictly only applicable to penicillins producing zones of identical shape. Fortunately for practical purposes, penicillin X is rarely present in commercial penicillin in quantities exceeding 0-5 per cent. (of activity) and may be neglected. One or two closely associated unknown antibiotics are often present in small amounts and, as they are almost inseparable from penicillin K, they may contribute slightly to the zone length, although in practice the effect is negligible.While working with mixtures of pure penicillins G and K, a procedure was evolved by which this under-estimation of K could be overcome. An equal number of units of penicillins G and K were mixed together in the same solution, and a series of dilutions, such as those shown in Table I, was prepared, each containing the TABLE I A DEVELOPED “STANDARD CURVE,’ , , ) Penicillin K, however, has to be considered. Standard solution, containing penicillins G and K in equal amounts, subtilis units per ml. 10,000 7000 4000 2000 1000 700 400 200 100 70 40 20 Maximum zone width G zone, K zone, mm. mm. 37 35 36 34 34 33 33 31 31 30 28 21 27 26 25 24 22 20 20 20 18 19 16 18 A r \ number of units of G and K per ml.stated. Each sample was chromatographed in replicates of six strips and plated out as usual on assay plates, using B. subtiZis as test organism. Maximum widths of the inhibition zones obtained were measured and graphs plotted against log units. The results showed that in each case the graph of maximum zone width plotted against log units was linear, but that the slope for developed K was less than that for developed G, as shown in Fig. 1. The graph clearly demonstrates how penicillin K may be under-estimated by using a G slope for zone width of K. In mixtures where the proportion qf K is very small, the reverse holds, an over-estimation being obtained below the intersection. EXPERIMENTAL CONCLUSIONS- From the experimental findings it was concluded that the inclusion of penicillin K with G as developed standard would greatly improve the accuracy of the quantitative procedure, G, F and dihydro F being calculated as usual with the G standards, and K being calculated separately with a standard of similar type.Mixtures containing appreciable amounts of penicillin X should be accompanied by standards which include X.312 GLISTER AND GRAINGER : MODIFIXD RAPID TECHNIQUE FOR MODIFIED METHOD APPARATUS AND REAGENTS- [Vol. 75 Developing chamber and trough-A wide-necked cylindrical glass jar, 14 inches high and 8 inches in diam. fitted with a gas-tight lid, is suitable. The trough consists of a 3-inch porcelain evaporating basin resting on the top of an 11 to 12-inch cylinder which stands vertically in the centre of the jar.Buflered paper strips-Sheets of Whatman No. 1 filter-paper are soaked in 20 per cent. w/v potassium phosphate buffer solution, pH 6.2, blotted between blotting paper, and air- dried. The sheets are then cut by means of a razor and a steel. straight-edge into strips 30 cm. x 1 cm. in size. The cylinder is bandaged with absorbent lint. Fig. 1. Difference of slope for G and K n! :cro-pipettes-These are made from 1-mjm. bore heavy-walled glass tu,,,ig drawn out to give a long capillary tip. A calibration mark is made on the capillary at a point from which delivery of water on to No. 1 filter-paper produces a 2 to 3-mm. diameter spot. This volume is approximately 2 p1. These pipettes give relative or comparative measure- ments required by the technique.Accuracy must be maintained in the primary preparations of the standard and test solutions. Assay plates-These are prepared from sheets of plate glass 19 inches x 14 inches. Half-inch wide strips may be cemented to form walls by means of cements such as Reuter’s &chromate gelatin mixture. Standard solutions-As the diameter - log units relationship is linear over a wide range, it is only necessary to prepare two standard solutions. (a) High standard-containing 5000 B. subt& units each of sodium penicillin G and K per ml. (b) Low standard-containing 50 B. subtilis units of sodium penicillin G and K per ml. The solutions are made in 1 per cent. phosphate buffer, pH 6.5, and usually the low standard is prepared from the high one by dilution. Nutrient agar for assay plates-The medium contains: 10 g.of Peptone, 3 g. of Lab. Lemco, 2 g. of sodium chloride, 20 g. of agar and 1 litre of distilled water. Dissolve the ingredients by boiling, and adjust the pH to 7.5. While still molten, .dispense the medium into 410-ml. lots and sterillise in an autoclave. Developing solven&Anaesthetic diethyl ether saturated with distilled water at room temperature.June, 19501 SEPARATION AND DETERMINATION OF PENICILLIN TYPES 313 PROCEDURE- For ordinary work nine are used, three for each standard and three for the test. Fold each strip 2 inches from one end and make a pencil-mark a half-inch from the fold in the centre of the strip on the long end of the fold. This serves as a point for the spot. The identification mark of the sample may be marked on the strip in pencil.By means of the same micro-pipette, spot identical volumes of the standards on to their respective strips. In the case of the test sample one should aim at spotting a similar amount of penicillin. With concentrated samples this may be achieved by preliminary dilution with 1 per cent. phosphate buffer to approximately 5000 B. subtilis units per ml. When the test sample is of low activity, e.g., as with culture filtrates, it is necessary to superimpose several spots, drying between applications, in order to keep a small concentrated spot. Solutions in organic solvents may be applied direct in the same manner. When the spots are dry, hang the strips in the developing chamber which should be previously prepared by soaking the cylinder bandage with water and placing a l-inch layer of diethyl ether in the bottom of the jar.Place the short ends of the strips in the basin, with the long ends hanging down. Then fill the basin to within half an inch of the top with the wet ether and place a small glass block in the basin to hold the strips firmly. Cover the jar with the lid, and allow development to proceed for 3Q to 4 hours. In the meantime assay plates should be poured with agar seeded with B. subtilis spores and stored in a cold chamber. When development is complete, remove the strips and lay them on the surface of the B. subtiZis-seeded agar. Allow diffusion to proceed for half anqhour and then incubate the plates at 27” C. overnight. Measure the maximum zone widths with a transparent plastic rule graduated in millimetres and calculate the averages.Take the required number of buffered strips. Thus the required units per spot may be obtained. CALCULATION (PENICILLIN x PRACTICALLY ABSENT)- Mean max. zone width high G standard - Mean max. zone width low G standard = G slope log conc. ratio (e.g., 100/1) Similarly for the K slope. The mean maximum zone widths of G, F and dihydro F of the test divided by the G slope give values the antilogs of which are proportional to the activities (in B. subtilis units) of these types. Similarly, the mean maximum width of the K zone of the test divided by the K slope gives a value whose antilog is proportional ,to its activity. The individual proportionate activities expressed as percentage of the sum of these activities. E percentage activity (B.subtilis units) for each type. The percentage activity (S. aureus units) may be obtained by using this organism in place of B. subtilis in the technique. Alternatively, a series of standards could form a “developed standard curve” and a graph be plotted. Test readings could be made on the graph direct. Typical results are shown in Tables I1 to IV. TABLE I1 ANALYSIS OF ARTIFICIALLY PREPARED MIXTURES OF PENICILLINS Results expressed as activity (B. subtilis units), per cent. Known penicillin K content, 5 10 20 50 67 % Found A f 5 Goodall and Levi method, Modified method, % % 3.3 5.1 6.8 9.8 10.8 19.6 32.0 61.4 40.2 64.8314 GRAY: CRITICAL FACTORS I N THE RESORCINOL REACTION TABLE 111 COMPARISON OF RESULTS FROM CULTURE FILTRATES Wol. 75 Goodall and Levi method Modified method L A I 1 r- \ X G F,and F, K x G F,andF, K (dih ydro) (dihydro) % % Yo Yo % Yo trace 94 3 3 trace 87 5 8 trace 97 1 2 trace 95 1 4 trace 92 4 4 trace 88 6 7 trace 80 6 14 trace 75 7 18 TABLE 1:V REPLICATE ANALYSES OF THE SAME SAMPLE G F, and F, (djhydro) K 92.4 3.1 4.5 93.8 2.2 4-0 91.0 2.7 6.3 We wish to thank the Directors of the Distillers Company (Biochemicals) Limited for their kind permission to publish this paper. REFERENCES 1. 2. 3. 4. 5. Goodall, R. R., and Levi, A. A., Analyst, 1947, 72, 277. Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J . , 1944, 38, 224. Winsten, W. A., and Spark, A. H., Science, 1947, 106, 192. Kluener, R. G., J . Bact., 1949, 57, 101. Baker, P. B., Dobson, F., and Martin, A. J . P., Private communication. THE DISTILLERS COMPANY (BIOCHEMICALS) LTD. SPEKE, LIVERPOOL November, 1949