1125
V O L U M E 21, N O . 9, S E P T E M B E R 1 9 4 9 Some advantage would accrue from the use of a logarithmic amplifier ( 6 ) , because the recorded wave heights would then be proportional to the concentration for those substances obeying Beer’s law. A modified form of apparatus would permit the measurement of light reflected from the sample to be recorded and the possibility of using the fluorescence of the material, although of restricted application, cannot be ignored any more than i t is in conventional chromatography. The extension to the ultraviolet, by either transmittance or reflectance, is even more important because then one is not restricted to colored substances. These, and other possibilities, are under investigation and will be reported later. The advantages of paper chromatography are not restricted to optical methods. I t has been found in this laboratory that conductance measurements are very useful in folloa~ingthe diffusion of ionic species through a paper strip and such measurements are even more easily recorded automatically. Although the use of paper as a chromatographic medium may be considered to have very limited use compared with other adsorbents, the possibility of impregnating i t lyith appropriate soluble or difficultly soluble reagents offers many opportunities to extend its usefulness (3). This instrument, and the several modifications that are under construction, are intended primarily for orientation studies on paper chromatography. Little or nothing lias been said here
about the type aiid tlilckness of the paper, its puiity a d structure. The instrument has proved very useful in evaluating these factors, the choice of eluting liquids, and almost every factor involved in a proposed separation. By a minor modification, in which the entire strip could be moved with uniform velocity across the scanning zone, one could obtain a conventional “densitome‘ter” record of a finished chromatogram. Whatex er advantages this mlght possess for quantitative work, it aould not provide as complete a picture of the actual dynamics of the process as the present arrangement. LITERATURE CITED
Consden, Gordon, Martin, and Synge, Biochem. J., 41, 690 (1947). Ibid.,41, 596 (1947). Flood, 2. anal. Chem., 120,327 (1940). Martin, Consden, and Gordon, Biochem. J . , 38, 224 (1944). hltlller. R. H., and Clegg, D. L.,ANAL.CHEM.,21, 192 (1949). Muller, R. H., and Kinney, G. F., J . Optical SOC.Am., 25, 342 (1936). Rheinboldt, H., in J. Houben’s “Die Methoden der organischen Chemie,” 3rd ed., 1‘01. I, p. 291, Leipzig. Gerge Thieme, 1926. Yagoda, H., IXD.EXQ.CHEM.,ANAL.ED., 9, 79 (1937). RECEIVEDDecember 29, 1948. A portion of 8 thesis submitted t o the Graduate School of New York University by Doris L. Clegg, in partial fulfillment of t h r requirrmrnts for the degree of doctor of philosophy.
Filter Paper Chromatography of Penicillin Broths M. L. KARNOVSKY AND RI. J. JOHNSON University of Wisconsin, Madison 6 , Wis. The c o n d i t i o n s for satisfactory paper-chromatographic separation of penicillins h a v e been e x a m i n e d critically, and a s y s t e m of o p e r a t i o n giving good r e s o l u t i o n is described. T w o assay methods a r e indicated, one rapid a n d of l i m i t e d applicat i o n based on m a x i m u m d i a m e t e r s of i n h i b i t i o n zones, a n d the o t h e r less s i m p l e but of greater accuracy and applicability. I n the l a t t e r the paper tape i s cut, a f t e r d e v e l o p m e n t , into u n i f o r m squares, and the penicillin c o n t e n t of e a c h is determined. The t y p e s of penicillin produced b y Penicillium rhrysopenum Q176 on a s y n t h e t i c m e d i u m , and t h e i r proportions. are discussed.
T
111.: unalysis of mixtures of penicillins by paper chromatography was introduced by Goodall and Levi (,2, S), who dewribed an approximate determination of the different penicillins i n a mixture. A qualitative modification of this procedure, more rapid but possessing somewhat lower resolving power, has been reported by n’insten and Spark ( 7 ) . In the present work a number of factors involved in the paper-chromatographic separation of the penicillins have been studied, together with aspects of the assay of the different components. Procedures are outlined which, it is helieved, result in improved resolution, reproducihilitv, and accuracy. GENERAL PROCEDURE
A filter paper strip, impregnated with buffer solution aud then
dried, is suspended vertically over the lip of a trough which is later filled with ether. About 0.002 to 0.004 nil. of broth or other aqueous sample, containing 1 to 2 units of penicillin (optimal figures), is spotted at a point on the tape, below the lip, on the outside of the trough. Water-saturated ether, progressing down the tape, is the mobile phase. The stationary phase is water absorbed by the impregnated paper. The entire operation is carried out in a sealed ciiamber. The distance moved by a given penicillin is a function of the pH of the buffer on the tape, the water content of the system, the distribution coefficient of the penicillin component, and tlie volunie of the ether allowed to flow. After development, the positions and concentrations of the individual compoueuts are determined either by measurement of the zones of iiihihitio~iproduced when the entire strip is laid on a
large agar plate inoculated with Bocillus subtilis spores, or by cutting the strip into small uniform squares and measuring the circular inhibition zones produced on B. subtilis plates by the individual squares-Le., the equivalent of a filter-disk assay. Figure 1 illustrates results obtained hy the use of both these methods, and the general chromatographic procedure outlined above. It may ?.JP seen {,hat: The major penicillin coniponent,s are well separated. Plating out individual squares gives somewhat better reuolution, for there is 110 loss of resolution due to diffusion of one component into another-which does occur on plating the ent,ire t,ape. Rleasurement of such zones as that of the I< types is not easy when the entire strip is plated out. In addition, Some doubt was entertained as t,o the validity of the maximum diameter of a zone as a true criterion of the amount of penicillin present in that zone, irrespective of the shape. (This is the method of assay used by Goodall and Levi, 2 . ) In the “squares” method, a convenient measure of tlie amount of each penicillin is given by the a.rea under the relevant portior~of the curve.
A detailed desciiption of the chromatograpliic and analytical procedures of the method finally adopted is given below, as well aa several factors investigated before establishing this final method. The general procedures adopted during the examination of these factors were t.hose of the esperimental part, and only the particular conditions under invactigation in each section were varied.
ANALYTICAL CHEMISTRY
1126 CHRO3IATOGRAPIIIC CONDITIONS
Effect of pH of Impregnating Buffer Solution on Position of Penicillins on Tape. Figure 2 illustrates the results obtained when pure penicillin G was chromatographed on tapes a t different pII levels, and developed for 22 hours. It is clear that pH may be used as a top1 for controlling the rate of passage of the various penicillins doirn the tape, a greater rate of movement being possible a t lower pH. In addition, tapes a t higher pH would require longer development times in order to achieve results equal, in terms of resolution, to those a t lower pH. It emerges that when “20%” potassium phosphate solutions between pH 6.0 and 6.7 are used to impregnate the paper, no change in the distance traveled by penicillin G occurs. This is taken to Figure 1. Filter Paper Chromatogram of a Penicillin Broth mean that during the actual experiment, where the Paper tape was eut in half longitudinally after development. The inhibition zones obtained when one of the strips was placed, entire, on a seeded agar plate are shown small amount of water on the tape is saturated with in scale tracing A . The other strip was cut into squares. A determination of the penicillin content of each square gave the data plotted in curve B potassium phosphate, it is saturated with respect to . both species-i.e., mono- and dihydrogen phosphates-within the pH range of the impregnating buffer solution mentioned. p H 6.2, falling in the middle of the plateau, would be an extremely suitable choice for the 20% phosphate impregnating solution, for small variations on \ C rither side would have minimal effect. This is a satisfactory \ pH level under the chromatographic conditions specified \ 2 300 below, and for maximum stability of penicillin solutions. -0 The plateau effect is less marked with buffers of lower 0 concentration and is shifted to the acid side. In general, \ 2% Potassium phosphate buffer .? 200 bufier solutions of about 20% concentration have been found 0 more suitable for impregnation purposes than those of lower g concentration. \ Optimal Time of Flow of Mobile Phase. -4close correla\ 20% Potassium 2 100 z tion exists between the amount of mobile phase that is permitted to flow down the tape and the pH conditions employed, if a given penicillin is to be brought to a particular I I I I I I 0 point on the tape. pH of buffer It may be shown that the region of maximum resolution in a chromatogram lies a t the middle of its length from the Figure 2. Variation of Movement of Penicillin G with point of application of the substance under investigation to pH of Buffer Solution Used to Impregnate Paper the solvent front. Because penicillin K is the component Mid-point between leading and m a r edges of zones plotted least likely to be a single entity, and is the most rapidly moving common component reported, it was considered deGoodall and Levi ( b ) , has proved perfectly satisfactory in this sirable to bring it to the middle of the chromatogram’s total laboratory, it wm felt that local conditions elsewhere might cause length. In order, also, to use the full length of the paper tape, difficulty, because the latter workers have noted the critical nait was desired to bring the K zone to a point just short of the end ture of this factor. For example, tapes stored in a room of high of the tape, and to make this point about the middle of a theohumidity and then humidified 15 minutes in a saturated atmosretical tape twice the length of the actual tape used. phere would conceivably contain more water than those stored in The time required for the ether front to reach the end of such a a relatively dry place and then humidified. The present authors theoretical tape may be determined by the use of a dye such as have encountered no difficulty over a period of 10 months, coverSudan 111, which, being soluble in the ether only, clearly demaring a wide range of humidity conditions in the laboratory, but it cates the front. The inclusion of one calibrated tape in each run, was decided to attempt to define absolute humidity conditions, from which the progress of the ether front may be followed, allows duplicable anywhere. a time to be set for the development of a particular set of tapes. In addition, the systems devised by Goodall and Levi, and Figure 3 illustrates the type of curve which may be drawn for such adopted as routine practice in the present work, are not coma tape, and the extrapolation t o double its length, from which the pletely in equilibrium. The ether used is saturated with water, time of development is determined. A constant rate of drip from and the atmosphere with water vapor, whereas the paper is still the end of the tape is assumed. Slight variations in the time recapable of absorbing more water. Changes in the conditions obquired for development of different sets of tapes do occur, due to taining on the tape therefore occur as the water-saturated ether temperature changes, use of tapes from different batches of paper, moves down, and a state of equilibrium is attained eventually etc. However, the period is usually between 15 and 20 hours for only. An attempt was made to introduce conditions such that the paper of the type of Whatman No. 1. process of humidification of the paper would be absolutely deIt has been found that, with a time of flow determined as above, fined, so that the amount of water on the paper and in the ether, and a tape impregnated with 20% potassium phosphate buffer, and the humidity of the atmosphere in the apparatus would be pH 6.2, penicillin K does indeed reach a point just short of the end constant and in equilibrium throughout the process of developof the actual tape. ment. The system envisaged w ~ t s89 follows: Standardization of Humidity Conditions throughout Chromatographic Operation. Although the method of humidifying A hygroscopic salt, A (with the same action as that of the impregnating buffer solution), was to be included in the buffer soluthe tapes for 15 minutes before chromatographing, adopted from i
-
.-
L
V O L U M E 21, NO. 9, S E P T E M B E R 1 9 4 9
117.1
analog does not. This m&ht bell di&h on the tape,
Figure 3. Movement of Ether Front, as Followed with Sudan 111 W h a t m s n No. 1 paper
~ ,~ ~ ~ ~ , . , ties~higher than those of a saturated solution of A. By this means, a series of tapes was to be obtained containing varvine ~
~~~~~
1
~
,
~~
D~~
water. EverGthing was to be stored"inAthed d room: at a con-
compiete iquilib&m wodd obtain. After a considerable number of trials i t was found that equilibrium with a saturated solution of a salt of humidity greater than 95% would be required, and because saturated solutions of potassium phosphate yield humidities somewhat below this when in an enclosed space, no additional ingredient A would he necessary in the impregnating buffer. Thus, a system equilibrated against saturated calcium sulfate solution (about 98% humidity) was tried. The tapes were stored over this solution for several days a t 4" C. and the ether and apparatus were equilibrated against it, On chromatagrqhing, good separation of penicillins was obtained, and the positions of the components matched those specified below, mhioh were obtained by the ordinary method involving 8. 15-minute humidification period for the tapes. A system fully equilibrated against saturated calcium sulfate solution a t 4" to 5" C. may be regarded &s a standard condition. For ordinary routine work, the conditions for humidifying the tapes described in the experimental section have been found to give very satisfactory results in this laboratory. During the course of the investigation of the above factors, several points of oonsiderable interest mere noted:
Positions of Individual Penicillin Components on Tape. It would be useful if, undcr defined conditions, figures analogous to the R, values of Cansdon et al. ( 1 ) could he assigned to penicillin components. It was thought convenient to express the position of any given penicillin (established by the position of the peak of its inhibition zone) as a prrcentage of tho movement of the fastest moving common erystallinc penicillin-Le., penicillin K. However, in the application of this system to certain broths-.g., Figures 1 and 5-it was found that several "K type" penicillins m y occur, and that the K zone had no real point of maximum diameter, and was cxtrcmcly long and flat. As a eonsequenoe the use of penicillin Ii as a standard is inconvenient in these cases. Positional data have therefore been based on penicillin dihydro F as st,andard, its laeation being arbitrarily designated aa 100. Under the conditions of operation, it was found possible to reproduce with reammhle osaetncss the ohsraoteristic positions oceupiod by definite components. Results of the determination of these oharacteristic eonstank for the various oommon crystalhe penicillins are given in Table I, column 2. These data were obtained by plating the entire tape, but similar results m v he calculated from- the positions of t h e peaks when t h e "squares" m e t h o d is used. As a result of the a p p l i c a t i o n of the chromatographic f a o tors discussed above, and laid down in the h a 1 met.hod, resolutions of the type exemplified in Figures 4 and 5 have been consistently ohtdned. DETERMINATION OF PROPORTIONS OF DlFFERENT COMPONENTS
(a)When the ether used was shaken with saturated solutions of salts with humidities up t o G67& and all other conditions as deIined in the final method, no movement of the uenicillins was ob-
limit being, in t6e secmd case, a uoint halfwav down the t a m . Resolutioi'was extremely poor: (a) Sodium phosphate buffer was found totally unsuitable for impregnating the paper. At pH 6.2 the penicillins moved comoletelv off the taue. and in order to retain them bhe ~ €had 1 to be Faised to about 8:3.' The Bones were poorly defined and there was considerable irregularity in the shape of the zones. ( c ) Tests with sodium and potassium citrate buffers gave moderately good results, which were identical. The anomaly of
the humidity ;onditi&
Figure 4. Chrnmatographic Separation of a Mixture of Crystalline Pcnicillins Three tapes shown hare equd volumes of n given penicillin aolntiun ut thccc dilutions (1, I/,, and I / % from loft te right). Tho major pcnioillin. from thc top of the tape to the bottom are: X, G , , F , dihydm F, and K . O n the t r p o with t l m greatest amount of penicillin fhc prosEllre of^' ofpOnisininK) may be noted. Paper tapes were removed hefore printine
"Entire P l a t i n g " Method. Some d o u b t was e n t e r t a i n e d as t o t h e validity of the maximum diameter of an inhibition zone, irrespective of its shape, as a measure of its penioillin c o n t e n t . Goodall and Levi ( 8 ) have reported that p e n i c i l l i n G, when moved various distances down t h e tape, showed an increase in maximum diameter with movement a t % level of 30 units, and a decrease a t a level of 0.1 unit. At an intermediate level, 3 units, no effect
ANALYTICAL CHEMISTRY
1128 Table 1. Positions of C o m m o n Crystalline Penicillins, and Those Produced h y Penicillium ehrysogenum Q176 on a S y n t h e t i c Medium" cryeta.11ine Preparation. AT. diveraenae
Synthetic Broths
No. of Timer Enoountered
... 8.0 ...
-0:9
..
12
?d
28.0
-i;7
?d
.is:o
3 .. 12 11 12 12 8
Taw .. 38 .. .. 6 .. 38 28 38 13
..
..
Daaienetion Polar tYPe*b
6 X ?' ?'
G F FHI K'$ K K"
Position
...
77.0 1001 136 159
...
-i:7 *1.7 -418 -8.2
...
Runs
..
.. ..
38
Position 4.3
.. . . .11.0 1Y.5
...
82.7 32.7
50.9. 77.5 1001 146 168 188
A". divergence * 1.0 6'n c.4
* 1.7 *' i . 4 * 1.6 * 1.8 *' 7 . 6 r12
-11
No. of Times Enaauntered Runs Tapes 7 14
...
i
2 2
3
...
s
5 7
14 16 16
7 7
lo
5
16 9
7 5
C o m ~ a n e n t ?found in two BoUroegwhoae poeitions overlap when average divergence figure ix taken into BOOOUnt are plsoed in same category of designation. Cf. Sa and 8%( 1 ) . broths these, when present in very small amoynts, were probably pait of tail of pear-shaped ''pols? G In type'' zone (Figure 5 ) . d cf.sa(7). a This component ia not definitely penicillin G : ita position is the only criterion SO fsr considered. I BY definition.
'
was noted. These results were obtained by allllotving varying amounts of the mobile phase to flow. In the present work a single oomponent of a mixture of penicillins was usually less than 0.4 unit, and diminution of the maximum diameter of its zone of inhibition would he expoot,ed, from the above, with inrreare in sone length. In general, diminution o? the diameter of the inhihitian none with increasing elliptical nature a? the zone would he expected from the cireumstance t h a t tho maximum penicillin c o n c e n t r a t i o n at any point becomes less as the zone lengthens. In this investiga, tion p e n i c i l l i n G w&s moved varying distances by vmying the pH of the buffer used to impregnate the tape, m a i n t a i n i n g the amount of flow of t h e s o l v e n t eonetant. From 0.2 to 0.8 unit of penicillin was employed, and comparisons w e r e made with relevant undeveloped standards on paper of the samepH. Inmost cases no distinguishable effect could be detected. Figure 5. Chromatographic SepaIt was, however, ration of Penicillins Produced b y not possible t o Penicillium ehrysogenum Q176 on move the penicillin a Synthetic Medium G as far dawn the Rsoulie for three sepnmfe bmfhs am ahown. The multiple nature of the K tape &8 was desired m n e i s evident. Zones designated s, 6. without using unand e nrr d-rrihed in t e ~ t
toward conditions of pH or unduly long times of development, which might result in destruction of the penicillin. The use of penicillin K would have allowed a greatmrangeof move ment, but was undesirable because of the greater instability of that penicillin. As a result of the above expericnce a rapid approximstx method ?or the determination of the oomponents of a mixture has been used, based on the maximum diameter criterion of Goodall and Levi (3). The s o l u t i o n under investigation vas ehromatogmphed at two and sometimes three dilutions
h i d out on an agar plato oi uniform depth, seeded with B . subtilis, and then incubated (Figure 4). No standards were necessary, and the assay slope for each plate was readily obtained from tho diameters of the components a t the different levels. (Assay slopc may be defined as the increase of diameter, in millimeters, of an inhibition aono occurring when the concentration of the penicillin is doubled.) The dope used in oalculations xias the average of the slopes of all components. The composition of the mixture could he obtained in duplicate or in triplicate (though these were not truly independent determinations) by constructing a curve having the relevant assay slope on semilog pape-i.e., diameter us. lag relative concentration. The amount of each component could t,hen be read off in arbitrary units from the m x i m u m diameters of the zones, and the composition of the mixture calculated. A typical calculation is exemplifiedin Table 11. Table I11 shom the results obtained by the entire plating method on some mixtures of crystalline penicillins. The result@ me ressonzbly good. It is in connection with the use of such a method on cwtain penicillin broths, where extremely elongated zones may he encountered, that difficulties occur. This aspect is discussed in the section on penicillins produced by Penicillium chrysogenun Q176 on synthetic medium. Severs.1 additional points of interest mere noted: For components of comparable order of magnitude, the asmy dope value tended t o inorease from the top of the tape to the middle, and then to decrease again to the end-Le., penicillins X and K tended t o have lower assay slope values than G, F. and dihydro F. The assay slope was not eonst,ant, hut decreased st very high concentrations of penioillin. The itssay slope of daveloped penicillins proved, as indicated by Goodall and Levi ( 8 ) , t.o be great.er t,han that for undeveloped
Table 11. Caloulation of c o m p o n e n t s of a Penicillin Mixture by E n t i r e P l a t i n g Method No Dilution Zone
X G F
FH.
K
Penicillin. Diameter, arbitrary mm. units 35.9 62 38.1 100 21.0 2.4 36.0 63 36.1 64
Fourfold Dilution Penioilli".
Diarnetor, arbitrary mm. units 30.0 17 31.8 25, Diluted O u t 30.0 17 30.9 21 A". Percentam Com~oeitionof Mixture
Penicillin < Peroenthge Diln. 1 21.3 34 Diln.L/d 21.3 31 * Minor components may be
x
slope 3.0 3.2 3.0 2.6 3.0
I129
V O L U M E 2 1 , NO. 9, S E P T E M B E R 1 9 4 9 -.
penicillin from the two halfstrips has not been identical, it has been found that the (Entire plating method) proportions of the different Penicillin components have not been F + FHsb K, AIixt w r X, G, c* affected No. % "a '70 5% % 17.0 17.0 27.0 I . Theory" A considerable advantage 39.0 18.1 26.2 4.8 (26.2) 21.4 29.5 Poiind offered by the squares method 16.5 2'7.6 2 9 (20.7) 17.8 34.9 13.8 27.6 (16.8) 16.8 10.0 over the entire plating method 14.3 27. I 2.6 (17.3) 14.7 24.3 14.8 26.2 . . (12.8) 12.8 47.6 is the ease with which the re12.5 29.0 3.6 (18.1) 14. R 41.0 covery of penicillin, in absolute 10.4 23.2 2.4 (16,s) 14.4 48.0 14.6 36.0 12.7 2. Theor> 36.8 units, may be determined in 11.4 38.1 2.0 (10.1) 8.1 40.6 Found 10.0 36.2 the former. Because the true ... ( 7.5) 7.5 46.5 1 4 . 7 36.6 --14,4 34.1 3. 'Thtmry amount of penicillin on each 25.6 26 1 26.1 18.8 3.4 (22.2) Foiind 19.7 28.6 0.8 (153) 14.5 36.3 square is determined, the total 24.3 24.3 .., (13.6) 13.6 37.2 20.3 26.4 amount recovered on the tape 31.8 4.9 ( 1.6) 16.7 21.5 26.4 '2.6 (23.7) 21.1 2s.3 after development may be com26.5 22.7 (18.4) 18.4 31.8 28.7 26.7 .. (18.3) 18.3 26.7 puted, and referred t o the Average grosa difference from t,heoryc, 8.3'70 original volume of solution apAverage absolute percentage crrord, 25% plied and its potency. The Maximum proas difference ob>.erred, 14.7% potency may be detcrminrd Composition of known rniuturs crystalline peniciiliri. Penicillins F aud dihydro F were calciiiated together, bocaiiie dihydro F uspd contained sbo,it 15% F. and with the same test organism penicillin G used 1.972 F. Penicillin F was not separately added t o mixture. 0 Theory 2 5 % ; found 3052; gross diferrnce 5'7,. and the filter-disk method of d Theory 2 5 % ; found 30%: absolnte pprccntaqe error 20%. assay with squares, as described, Data on the recovery of the various penicillins have Tahle IV. Analysis of hIixtures of Crystalline Penicillins by hIethod of Plating Square-a been assembled through the PtxnlclllL use of mixtures of solutionq of __---___ F FH2 different crystalline penicillins \IlKtlll 't $, Q __ K, YO. C s % 5 R of kno-xn potency. These are 21,8----.12.8 30.4 35.2 1. Theory reported in Table V. Over-all 31.6 42.5 :n.3 Found .5 '1 (16.5) 10.8 ,17.4 25.4 43.6 12.9 2. Theory recovery is satiafsctory, and 20.7 33 1 12. I 3.; (18.1) 14.4 Found -17.0 -. 28.6 penicillin IC ir the most sus3. Theory i4.3 40.3 33.0 15.3 33.7 Found 1.7 (15.9) 14.2 ceptible t o loss, so that slightly 15.2 .2*.6 27.5 29.8 4. T!iciiry Z n , $1 33.1 27.1 F~lirid (19.7) lli . 7 Ion, results might be expected 28 8 25.0 -.1!3 m.2) lS.0 25.G 35.1 for this conlponmt. 2 1 .A 2 0 (19.4) 17 1 24,2 31.4 20.9 1 8 (17.0) lli.1 29.1 In the application of the 27.6 17,7--Theory (check) 2 4 . Y 20.6 ,0.0 -31.2 5 . ' Theory 34.3 33.!) method to corn steep liquor 32.6 33. ' Found ... 0.0 ... 33.7 broths, the few cases studied 38.1 36.3 25.4 0.0 ... 6 . Theory 26.8 _____-. o- -, n 30 L' 43.; have shown over-all recoveries 53 ,.a 27.5 20 . o Found .. 0.0 ... 29.4 7. Theory 42,7 -0.0 27.9 never 1es.jthan 7OP$,and rang26.3 ... 0.0 ... Found 43.8 31 n 0.0 26.0 ing to 1 0 0 ~ cand 8. Theory 13.2 , are regarded 31 0 :v,n 31.8 34.4 Found . . 0 0 .. as satisfactory. In a study .4verage gross difference from theory, 3.3'4" of the penicillins produced by Average absolute percentage error, 15Yo Maxiinurn gross difference observed, 10.2% Penicillium rhrysogenum Q176 See footnotes to Table 111. on the synthetic medium of * hfixtures 5 to 8 early results; no penirillin F (contaminant of G ) detected. Jarvis and Johnson (4),however, the situation is disappointing. As may be seen from penicillins. The conversion factor (undeveloped assay Slope to Table VI, recoveries have been somewhat variable. In any one assay 'lope) Of quoted by these authors for peniset of tapes, or "run," the recovery tended to be rather uniform cillin G is reasonable. Table 111. Analysis of Mixtures of Crystalline Penicillins by Measurement of Maximtun Diameters of Inhibition Zones
.
-
7-
7
7
f-
-
,
7
-
---
Method of Squares. In this method, the tape was cut into uniform squares after the development, and these were serially formed and a plated. From the diameters of $he circular standard curve made with identical squares and penicillin solutions of known concentration, the absolute amounts of the various Components could be determined (Figure 1). Table IV gives the results of several analyses carried out' by this method on mixtures of crystalline penicillins. The results obtained in the squares assay were somewhat better than those from the entire plating assay. Because only half the tapc is used in each determination (the other may be used for qualitative work, or as a duplicate assay) it was of interest to determine how uniformly the penicillin n-as distributed across the tape. Figure 6 illustrates a typical tape split down the middle, the two halves being assayed separately. Agreement was good for the larger components, and someTvhat less satisfactory for miall ones. Even when total recovery of
-
Table V.
Recovery of Penicillins on Analysis of Crystalline Mixtures by Squares >lethod
Total Recoveryn,
%
93 90 80
100
in7 77 86 77 94 90 9n
95
a
Recovery of Individual Peni~illin,, % 0 F FHzb K in6 .. 71 127 107 .. 70 78 89 ... 72 75 !3 8 ... 1no 80 101 75 121 85 in8 98 83 in4 112 '31 8fi 93 72 78 113 89 119 103 I n? 94 99 84 73 112 79 84 74 107 109 83 81 95 103 88 84
X 110
Of oriyirral total activity of solution. See footnote t o Table 111.
+
1130
ANALYTICAL CHEMISTRY Right side
Left side
nated as K’,K, and K ” ; the middle one is apparently authentic penicillin K (cf. 6).
9 s might be expected, methods for the determination of the individual components depending on “maximurn diameters” of the zones ( 2 ) are not t 1 X 10.8% y 9.4% satisfactory when applied to mixtures of this type, LO inasmuch as low values are obtained for the I< penicillins, on account of the extreme length of the zone, which has no true point of maximum diameter. The method of squares on the other hand gives higher and, it is believed, more correct results for the K type penicillins in such broths. F Comparative data on the application of both methods to synthetic broths (some of which contained various precursors) are given in Table 1711. FH2 Positions of Various Components. Table I shows the positions of the components shown in Figure 5 , and which are typical of those produced by Penicillium chrysogenum Q176 on a synthetic medium without precursor. The penicillins in the K region have, as might be expected, less satisfactorily reproducible positions than that of crysI I I I I I I I talline penicillin K, owing to the flat nature of 150 100 50 0 50 100 150 the zone. Milli-units penicillin Average Relative Amounts of Different PenicilFigure 6. Comparison of Two Halves of a Chromatographic Tape lins. The following indicates the average amounts Squares assay method. Of the total recovered penicillin, 52% was on the left side of the tape, and 48% on the right side. Percentage composition figures calculated of the different components produced by Penicilfrom each side are entered under each component lium chryaogenum Q176, on a synthetic medium without precursor. Variations with the time of fermentation, etc., probably do occur, but the general composiTable VI. Recovery of Penicillin from Synthetic Broths tion indicated appear? norinal in this laboratory: Analyzed by Squares iMethod Polar types of ,(a) 6.0%: types of ( b ) and ( c ) 3.0%; perilR u n h-0. Recovery. % KO.of Tapes in Run cilhn F 45.0%; dihydro F 13.0’%0; K type penicillin 33.0%. 1 51 4 0 -
Point of application.
1
2
28
sn
5 2
analysis of a number of synthetic broths without precursor has given similar results for percentage composition over several runs in which the recoveries were different and it is therefore probable that no great dislocation of the relative amounts of the different components occurs. PENICILLINS PRODUCED BY PENICILLIUM CHRYSOGENVM 4176 ON SYNTHETIC MEDIUM
A study has been made of the application of the methods described above to the determination of the individual penicillins produced by Penicillzurn chrysogenum Q176 on a synthetic medium (4)without the addition of any precursors. Entire plab ing of tapes after development has shown results exemplified by Figure 5 . There occur: ( a ) A pear-shaped zone around the point of application, tailing downward. More than one component is probably present, and in some cases definite indications of the multiple nature of this zone vere obtained, there being apparently three zones overlapping. From the positional figure for this zone of polar compounds, it is clear that at least one large component more polar than penicillin S is present (cf. SI and Szof Winsten and Spark, 7 ) . ( b ) A second, rather small zone. ( c ) A zone agreeing almost identically in position with penicillin G. ( d ) Zones that are clearly penicillins F and dihydro F, and whose identity has been established by the inclusion of crystalline penicillin F and dihydro F i n the broth. ( e ) A series of X type penicillins, which form an elongated zone, often showing evidence of at least three components, desig-
EXPERIMENTAL
Materials. PAPER. Whatman Yo. 1, available in large sheets, was extremely satisfactory. This was impregnated with buffer and then cut into 1.25-em. (0.5-inch) ribbons. Whatman No. 4, though affording a more rapid rate of flow, proved inferior to No. 1 in uniformity and resolution obtained. Eaton-Dikeman No. 613 paper, available in rolls of 1000 feet, 0.5 inch wide, was similar in behavior to Whatman No. 1. ETHER.A good commercial grade of ether was thoroughly washed to remove any alcohol and treated with alkaline pyrogallol several times. Finally it was washed exhaustively with water, and stored in a dark glass bottle at 4” to 5” C. BUFFERSOLUTIONS.The potassium phosphate buffer referred t u throughout as (‘20% buffer” was prepared as folloivs. Potassium monohydrogen phosphate was made up at 20% (w/v) and the pH of the solution adjusted to 6.2 (glass electrode) by the addition of 85% phosphoric acid. Phosphate buffers of different pH, where used, were similarly prepared. CRYSTALLINE PENICILLINS. The following were available: penicillin G, containing 1.9% F, and a trace of a component found between penicillins X and G on the chromatogram; penicillin X, chromatographically pure; penicillin dihydro F, containing about 15% F; and penicillin K, containing a trace of a component referred to in this work as K’. A sample which was mainly penicillin F, with various amounts of other components, TTas used qualitatively. AGAR; \ ~ E D I L M . The nutrient medium used in the assays consisted of 1 gram of glucose, 1.5 grams of meat extract, 6 grams of Bacto-peptone, 3 grams of yeast extract, and 15 granls of agar per liter of water. B. srthtilis SPORESUSPENSION. A nutrient medium, consisting of 3 grams of peptone and 3 grams of meat extract per liter, was inoculated with B. subtilis, Slarburg type, arid incubated for 6 days on a shaker at 30” C. The suspension was then pasteurized a t 80” C. for 10 minutes, and stored in the refrigerator until required. The titer of any spore suspension-i.e., the amount t o be added to 1 liter of nutrient agar-was determined by carrying out tests at various levels under the conditions outlined for penicillin standards below, and should be the least amount that, yields satisfactory zones. Apparatus. CHRO.UATOGRAPHIC CHAMBER.Figure 7 is a diagram of the essential apparatus required for the chromatographic
V O L U M E 2 1 , NO. 9, S E P T E M B E R 1 9 4 9
1131
it. Care was taken to touch the tapes as little as possible with the fingers. As far as was possible forceps were used, but FHz, K‘, U Typesn, where hundling was unavoid% % 75 able the tapes were touched 11.7 ... 26.6 0.8 3.1 51.3 1 Entire 6.6 lightly by the edges only. 10.0 ... 28.9 4.16 49.4 Squaws i.9 SPOTT~NQ THE PAPER.The 12.0 0.9 14.8 0.6 3.0 64.0 2 . Entire 1.1 1.1 11.2 14.6 1.2 4.0 59.6 tapes were clipped a t each end, 7.6 1.7 10. I 27.4 Squares 3.5 60.8 and placed in position in the 1.4 11.8 28.4 4.3 3.9 44.8 trough, as illustrated. The 3 Entire 14.9 ... 18.1 1.3 3.8 54.7 7.3 0.8 10.0 12.9 0.5 3.1 68.0 penicillin solutions, varying in 4 . ? Squares 1 .o 11.6 34.8 1.7 3.7 41.1 i .J volume with their potency, were then applied a t the marked * Where K’ was well enough separated, it is reported separately, and K types then refers t o the rest of this group. Where resolution was not good enough t o allow a separate figure for I
