Direct Estimation of Penicillin G in Small Broth Samples J. A. THORN AND MARVIN J. JOHNSON College of Agriculture, University of Wisconsin, Madison, Wis.
A method is presented for the estimation of penicillin G in broths by chromatographic adsorption. The method is specific for penicillin G, samples require no preliminary purification, and only 100 to 200 units of penicillin are required for an analysis. The application of aqueous chromatography to resolution of mixtures of the other known penicillins is described.
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mixture of penicillins containing penicillin G is chromatographed, the latter is adsorbed more strongly than the other penicillins and is eluted as a separate elution band, Penicillin F is the only known penicillin which is sometimes not completely separated from penicillin G by this procedure (Figure 2). The extent of overlapping of the bands of the two penicillins is rarely greater than that illustrated, and the leading edge of the penicillin G elution curve may be extrapolated easily to the abscissa to facilitate calculation of recovery. Although penicillin G could be separated from other penicillins in a “synthetic” mixture, it was necessary to determine how the separation Tvould be affected bv extraneous materials normally found in broths before the method could be applied to broth analysis. To this end, the penicillin in steep liquor broth was inactivated bv adjusting to pH 2 and heating in a steam bath for an hour. The broth 1%-as then cooled, adjusted to pH 4.6 with alkali, and added to known mixtures of penicillins to the extent of 2 5 7 , by volume. These samples were then chromatographed in the normal manner, and the inactivated broth lvas found to have no effect on the resolution. hlthough the inactivated broth was undoubtedly not identical to untreated broth, the absence of effect indicated that broth components would not noticeably de-
NICILLIK, as produced by mold growth, consists of a mixture of several antibiotics (10). h t present, five antibiotics of the penicillin class are known: A”pentenv1penicillin (F), n-amylpenicillin (dihydro F), n-heptylpenicillin (I(),benzylpenicillin (G), and p-hydroxybenzylpenicillin (S). Each has its own specific biological activity and properties. The penicillin being produced at the present time is largely penicillin G. I n order to increase yields of this penicillin, precursors, such as phenylacetic acid and p-phenylethvlamine, have been added to media to increase the amount of benzylpenicillin produced. I t is obvious that any analytical method allowing the easy and rapid analysis of broth samples for penicillin G would be of value in studying the effects of such precursors. The methods for the determination of penicillin G so far described in the literature are not particularly applicable to the analysis of broth samples. The gravimetric determination (9), based on the insolubility of the S-ethylpiperidine salt of the penicillin in certain solvents, requires reasonably pure penicillin G in milligram quantities. The recently descr-ibed polariscopic-ultraviolet absorption method is applicable to crystalline and amorphous penicillin preparations ( 5 ) ,whereas the method employing infrared analysis requires crystalline samples (1). Although the microbiological differential assay (6) can be used to analyze broths, it is limited by the assumption that only three known penicillins are present in any sample. The value of the countercurrent distribution method ( 2 , 3 )in analyses for penicillin G depends greatly upon the composition of the samples analyzed. The method is of limited value for samples containing appreciable proportions of penicillins F and dihydro F. The method described in this paper has evolved from a study of the adsorption chromatography of penicillins in aqueous solutions. I t is based on the fact that, on a column of Super Filtrol (an acid-treated bentonite), penicillin G is more strongly adsorbed under given conditions than any other known penicillin and may be eluted as a separate fraction. The method is particularly applicable to fermentation broths and is not affected by the number or types of penicillins occurring in the sample to be analyzed. The method is not intended for use on purified samples, for which the more accurate physical and chemical methods are available.
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PENICILLIN X (26 UNITS)
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PENICILLIN DIHYDRO (34 UNITS)
F
F
PENICILLIN
(58 UNITS)
EXPERIMENTAL
The chromatograms obtained when single penicillins are adsorbed and eluted on columns of Super Filtrol are shown in Figure 1. The columns were prepared in the manner described under procedure. It is seen that the sequence in which the penicillins are eluted is: X and dihydro F (first eluted), F, then G (last eluted). A tendency for dihydropenicillin F to be adsorbed more strongly than penicillin X is indicated by the longer “tail” of the elution band of the former penicillin. Penicillin K appears to be largely, if not entirely, inactivated upon adsorption. When a
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I20 EFFLUENT IN ML.
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Figure 1. Chromatograms of Single Penicillins on Super Filtrol Columns at pH 4.6 614
V O L U M E 20, NO. 7, J U L Y 1 9 4 8
615
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per Filtrol. Figure 4 shows the resolution obtained with a column containing 4 grams of one lot, that obtained with 4 grams of a second lot, and that obtained with an increased amount (6.3 grams) of the second lot.
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REAGENTS AND MATERIALS
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:I 50.5
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80 120 E F F L U E N T IN ML.
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Figure 2. Chromatogram of a .Mixture of Penicillins X , F, K, and G on a Super Filtrol Column Column of pH 4.6 contained 4 grams of Super Filtrol and 4 grams of Celite 545. Penicillins adsorbed: X, 23 units; F, 27 units; K , 37 units; G, 84 units
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I PROCEDURE
1.5-
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Preparation of Samples. Column samples should be 1 ml. or less in volume and should contain between 50 and 200 units of penicillin. A standard solution of sodium penicillin G may be prepared with pH 6.1 buffer, providing the latter is not of sufficient
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Super Filtrol, obtained from the Filtrol Corporation, Los Angeles, Calif. Celite 545, obtained from Johns-Manville Corporation, Kew York, K . I'. PhosDhoric Acid. 5070. Phosphate Buffer, 1 "&I, pH 6.1, containing 114.4 grams of potassium monobasic phosphate and 28.6 grams of potassium dibasic phosphate per liter. Potassium Monobasic Phosphate. Solution, 0.05 (DH about 4.6). Penicillins. Pure sodium penicillin G is required as a standard. The penicillin G used in these experiments was identical in potency with a standard sample of crystalline penicillin G from the U.Y. Food and Drug Administration. Corn steep liquor broth containing a small proportion of penicillin G, or broth from a synthetic medium (JThich contains no penicillin G), may be used as a second standard. Chromatogram Tubes. Sections of Pyrex tubing, 14 mm. in inner diamet,er and about 22 cni. in length, are used. A constriction at one end gives an opening of approximately 2 mm., and glass wool is tamped above the constriction as a base for the adsorbent. A4pressure liquid-feed system is used to control the rate of flow of eluent.
40
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I20 EFFLUENT IN ML.
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COLUMN
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200
Figure 3. Chromatogram of a Steep Liquor Broth Sample
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Column of pH 4.6 contained 4 grams of Super Filtrol and 4 grams of Celite 545. Penicillin adsorbed was 73 units from steep liquor broth, 28Vo of which was found t o be penicillin G
COLUMN 2
crease the efficiency of the adsorbent. This is, in fact, found t o be the case, provided that a new column is used for each analysis. Repeated use of a column for broth analyses, however, leads to decreased resolution, probably due to saturation of the adsorbent \T ith nonpenicillin broth components. Figure 3 shows a typical chromatogram obtained with a sample of steep liquor broth Containing no penicillin G precursor. -11though the total penicillin applied to the column vias only 7 3 units, a clear-cut analysis was possible. The first band consisted of a mixture of penicillins other than G, and penicillin G a-aq recovered as an almost completely separated band. The adsorptive capacity of Super Filtrol has been found to vanfrom batch to batch. This means that each batch of Super Filtrol to be used in the estimation of penicillin G must be tested to determine the amount of adsorbent required for satisfactory resolution. This m a r easily be done b> the method of trial and error. For example, for four different lots of adsorbent, amounts ranging from 4 to 6.5 grams gave satisfactory separation of penicillin G, equal amounts of Celite 545 being used in each case. Figure 4 gives a comparison of chromatograms for a mixture of penicillins X, F, K, and G obtained mith columns of two different lots of Su-
Figure 4. Comparison of Chromatograms of a Mixture of Penicillins X, F, K, and G Adsorbed on Columns of Two Different Lots of Super Filtrol Column 1. 4 grams of Super Filtrol A and 4 grams of Celite 545 Column 2. 4 grams of Super Filtrol B and 4 grams of Celite 545 Column 3. 6.3 grams of Super Filtrol B and 6.3 grams of Celite 545 Penicillin mixture consisted of 28 units X, 20 units F, 21 units K , and 83.5 units G
ANALYTICAL CHEMISTRY
616 strength to affect the pH of the column. FOIeaainple, 1 5 (0.07 M ) phosphate buffer, pH 6.1, may be employed, as 1 ml. of this solution does not have a noticeable effect on the column pH. Such a solution of known penicillin G concentration may be kept for some time and makes unnecessary the preparation of a new solution for each test. Broth samples are prepared as follows: Tht, mycelium is strained from the liquid by drawing the latter into a pipet, the tip of which is covered with a small amount of cotton. As soon as the column (described below) is ready for use, the broth liquid is adjusted to pH 4.6 with 50% phosphoric acid anti diluted, if necessary, with0.05 M potassium monobasic phosphatc so as to contain between 50 and 200 units of penicillin per ml. One milliliter of this broth liquid is then applied to the column and an aliquot of the same solution is inimediatley diluted with appropriate buffer for awav hp the Staphylococczrs mirei(s (*up plat? method (4, 8).
7 4.0
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Figure 5 .
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50 V,
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70 IN ML.
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Relationship between WS arid 1;
Column contained 4 grnrnu of adsorhant m d 4
gram*
must b e taken a t the initial and final stages of elution of penicillili G to ensure separation of penicillin G from other adsorbed penicillins and elution of all the pmicillin G. The fractions are atljusted as collected to about pII 6 by the addition of 1 ml. of 1 -I/ phosphate buffer and are thrn ready for assay, without t’urthet dilution, by the cup plate method. When the adju*ttd effluent fractions cannot be assayed immediately, they may be kept in th:. refrigerator for at least 48 hours without, noticeahle loss in ptxiiicillin content. Standardization and Calculations. Sat penicillin G from the other penicillins is n the retention volume of penicillin G (the volume of ef€luent>collected before penicillin G is first observed in the efflurnt) is 70 I I I 80 ml. If 4 grams of Super Filtrol are not sufficient to cause satirfactory separation, more adsorbent must he used. The Supt’i Filtrol-Celite 545 ratio is maintained at unity t o :ilIn~vt’asy f l o 01’ ~ eluent t,hrough the columns.
i*F, the ret,ention volume of penicillin C; for a coluiiii~containilly 4 granis of a given lot of Super Filtrol and 4 grams of Celite 545. was determined for each of a number of Super Filtrol lots. Also determined was TI’*, the amount of each lot of adsorbent rcquird for satisfactory resolution. I n Figure 5, II’, is plotted against era1 Super Filtrol lots. Such a curve is of value hecausr. O I I C Y I; is determined for a batch of Super Filtrol, TVa mag he i ~ . a t directly l from the curve. Ttits average recovery of adsorbed pcnicillin U varies gericrall! iruiii 7 5 t o depending upon the particular lot of Super E’iltroI hviny tlmployed for adsorption. The value used in calculating thc amount of penicillin G in unknown samples must be based 0 1 1 csperiment and is obtained by adsorbing known amounts of penicdliri G on several columns. This figure should be che sionally by rontrol runs. Assuming a recovery of 79 cillin G, for example, the penicillin G content of a broth sample is
