Infrared Analysis of Crystallhe Penicillins R. BOWLING BARNES, R. C. GORE, E. F. WILLIAMS, S. G. LINSLEY, AND E. M. PETERSEN Stamford Research Laboratories, American Cyanamid Company, Stamford, Conn. Qualitative infrared spectra in the solid phase of crystalline sodium penicillins G, F, X, K, and amyl are presented as a basis for quantitative analysis. Suitable analytical infrared absorption frequencies are listed. The details of an analysis in the solid phase, with an accuracy of *2.0$7& for crystalline sodium penicillin G are given. Spectroscopic
analytical interferences are discussed. The effects of crystal orientation or pleochroism are discussed. Methods for obviatiog the di83culties resulting from this phenomenon are given. Details are outlined sufficiently to enable the performance of the quantitative analysis of mixtures of the presently known five types of penicillin.
s
evaluated quickly from the standpoint of the spectra of its products. A series of samples of various penicillin content, as evi. denced by the biological assays, is shown in Figure 1. Close examination of such a series of spectra indicates clearly the progressive trend towards product purity. ' An early attempt was made to utilize this absorption band in the away of the amorphouf material. Although a positive correlation between the strength of the 1770 ern.-' absorption band and the biological assay was found, the variability of the latter in addition to effects which may now be attributable to the multiplicity of penicillins led to a concentration of effort in other directions. This correlation has been used throughout this work to check on biological assays. It has been established that the infrared spectrum of a given organic molecule may be used to characterize that particula~ compound. I t is one of the most truly typical of the physica! constants. Unlike many other physical properties, the spectrum of a mixture of several component molecules, which show no physi.
HORTLY after Fleming's discovery of penicillin it was evident that because of its instability and other chemical properties the purification, isolation, characterization, and analysis of the antibiotic would not be an easy task. As purification of the material progressed it was soon found that a multiplicity of types 3f penicillin was produced by the mold, each type possessing its own specific antibiotic activity and properties. Because of these iifferences between the several types of penicillin molecules it is of major importance to detect and estimate the quantities of each :ype present in a sample. In the early days, several methods of biological assay were lescribed. These were adequate for the problems then at hand. I s the multiplicity of types became evident modifications of the biological assay were required. Assay and identification through 9, bacterial spectrum or a simple activity ratio such as that obtained with bacillus subtzlis and staphylococcus aureus were introduced. These methods still embodied all the disadvantages and variabilities of biological techniques and as the purity of the various penicillin entities increased they often possessed too little resolution to indicate the presence of a few per cent of one or several types of penicillin in a sample of predominantly one type. I t was inevitable that chemical and physical methods of assay would be sought. The gravimetric precipitation of penicillin G by N-ethylpiperidine in suitable solvents has been described (6). Gravimetric procedures are subject to errors caused by changes in solubility produced by many possible solubilizing agents. The complex natural character of crude penicillins, as well as the effect upon the solubility of one type penicillin by another, are factors which must be carefully considered. A colorimetric method has been based upon the reaction product of pencillin and hydroxyl amine '4) which is subsequently coupled with an iron salt. ThiA method measures the total penicillin content of the sample without regard to type. The use of buffered silica gel columns (3) has also been proposed as an analytical procedure. Physical assay methods based upon solubility distribution ratios are open to the same objections as gravimetric precipitation procedures. Ultraviolet spectrometric techniques ( 4 ) ,also, have been described for the differential analysis of the penicillins. With the commercial advent of the temperature-stable crystalline sodium salt of penicillin G, attention has been sharply focused on the need for a rapid differential analysis of the penicillins indicating particularly the emount of crystalline sodium penicillin G in a sample. In none of the above-mentioned methods is the sample analyzed in the nrystalline state. Infrared spectrometric methods have been found useful in connection with many phases of penicillin research. The applicrbility of a given purification technique can be checked by examining the resulting products, since the intensity of the infrared %bsorption band near 1770 cm.-' is apparently dependent upon penicillin content-Le., the intensity of this absorption parallels ectivity. Likewise, the efficiency of any method designed spe-ifically for the separation of the various penicillin types can be
/
/
I
1400 I
I600 I
I
2000Cm-'
1800 I
I
/ 6 3 2 0.u
l
l
Figure 1. Correlation between Infrared Absorption near 1770 Crn.-' and the Biological Assays 620
62I
SEPTEMBER 1947
wnstitution of the acids rtsults in the use of larger Sam. plw, possible degradation 0' the molecules dependent UPOL their type, changes in com. position ratio and purity, and the introduction of many a t tendant uncertainties. The sodium salts exhibit sufficienl solubility only in polar sol. vents, such as water, which is in appreciable thickness c P opaque to infrared radiatior a in many spectral regions. I1 is obviously advantageow not to restrict the infrared ob servations to limited spectra 1330 intervals; accordingly, mosl of the observations herein re ported have been confined i r the solid phase. Early spectrograms wert often obtained by depositing 3400 3000 2600 I800 1600 1400 1200 1000 800 600 400 6lms on rock-salt plates fron F R E Q U E N C Y Cm" various solvents. After care. Vigure 2. Infrared 4bmrption Spectra of the Crystalline Sodium Salts of Variour ful evaporation of the solvent Penicillins apectra are obtained that show no absorption ban& other than those of the sample. Such films, however, are labor 4 or chemical intermtions, is simply the sum of the spectra of ioua to prepare and strongly scatter radiation. By far the mos1 the individual components with intensities proportional to their convenient method for the preparation of the sample has beer relative abundance in the mixture. As generally presented, an infrared spectrum is a plot of per cent transmission as ordinates found to be the mulling of a small portion (0.5 to 3.0 mg.) wit1 liquid petrolatum (Nujol) directly upon the rock-salt plates 0' versus the frequency in em.-', u = IO4) or the wave length the cell. The spectra obtained through such a technique exhibi P clean sharp absorption bands but, they do contain the charartpr in microns, p). The unique frequency positions furnish the data for qualitative analysis while the per cent Transmission valuas furnish the information rrecmary for quantitative analysis. The present paper presents the details of che application of the infrared method to the malysis of the sodium salt9 of the crystalb e penicillins. It is of particular value to dxamine the salts in the same phase (crystalline) in which they are sold because the dxtent of crystallinity bears a direct relation+hip to the purity and stability of the marerial. The infrared method possesses the followingfurther advantages:
E Ivv-' Y Y s z z m :
g r ? r "
(
-
No C
1. The detection of the presence of .the garious types of sodium salts of penicillin without subjecting the sample to chemical Cahange. 2. The quantitative estimation of the &mountsof these types present. 3. The use of small crystalline phase samd e s of 15- to 20-mg. weight. 2. The rapid performance of the analysis. i 4 relatively high analytical precision
_.-,-
No K
_,,_/,-
Ne F
QUALITATIVE ANALYSIS
before a quantitative infrared analysis is performed, it is highly advisable that a qualicative analysis of the sample be made. Accordingly, the qualitative spectra of each of the penicillin types were obtained with their future use in connection with quantitative malyses in mind. It is desirable to leave amples in the metallic salt condition and not to reconstitute the penicillin acids, although this latter terhnique haq hpen i i w l d . Anv re-
+
8
I
0
880
010
I
'
8Kl
I 850
a?! l 840
I
"1 l
830
/ 820
I
810
!
800
FRlOUENCY
Figure 3 .
74.
I
7%
t
1
720
110
1
1
700
I
ma ,
680
IN C M - '
Infrared Analytical Absorption Bands for Various Crystallin* Sndium Salts of Penidlin
V O L U M E 19, NO. 9
622
CRYSTALLINE BONDED NH
AMORPHOUS NH
z
0 a K
0
In
m
+
1
I
2200
I
I
1
I
I
2400 2600
I
I
I I I I I
3000
3500
FREQUENCY Cm-'
Figure 1.
Effect of Hydrogen Bonding upon the NH Absorption Band of Penicillin
istic absorption bands of Nujol. All of the spectroscopic work herein reported was done using a Perkin-Elmer spectrometer Model 12-B (I). It must be emphasized that many of the absorption bands recorded are associated with the crystalstructureof the material. Accordingly, the spectra of a given sample in the crystalline and the amorphous states may not coincide. Analyses can, however, be made with the sample in either form. Inasmuch as the cation of the salt affects the spectrum, it is necessary that the same cation be used throughout. In the present work all data were obtained upon sodium salts. The methods used, however, are general and may be applied to salts involving other cations. I t has been suggested ( 2 ) that the basic structure of sodium penicillin may be H
In Figure 2 are shown the spectra of crystalliae salts of these types. Although the samples from which these,spectra were obtained are the best the authors have been able to procure, several, in particular K and amyl, are known to be impure. [Penicillins K and dihydro F (n-amyl) hydrate readily and may carry variable amounts of water which interferes in both purification and analysis.] As purification methods are improved and as still other penicillins are isolated and characterized, it may become necessary to regard these standard samples as more impure than noTv suspected. Therefore, these standard qualitative curves, together with the quantitative results so far obtained, must be accepted at this time as tentative. There are many coincidences in the spectra of the five types. For example, all samples exhibit absorption in the nitrogen-hydrogen bond stretching vibration region near 3350 cm.-l The major absorption between 3000 and 2800 em.-' is that contributed by the carbon-hydrogen vibrations in Sujol used in mulling the samples to reduce loss of radiation by scattering a t the crystal interfaces. The three strong X=Y stretching vibration absorption bands between 1800 and 1600 are common to all types of penicillin, although slight shifts are observed. Absorption bands a t 1460 and 1375 cm.-' are again partially contributed by the angular bending vibrations of the methylene and methyl radicals in the Nujol. Although many of the remaining absorption bands are unassignable to definite vibrations in the respective molecules they serve to characterize or fingerprint each type. These are influenced greatly by the molecular environment such as is encountered in crystal structure or solution, and are very often found to be of value in quantitative analysis. It is readily possible to observe the presence of one or more other types of penicillin in a mixed sample predominantly of one kind. From the shape of the absorption bands, their purity and simplicity, it is possible to obtain a general over-all picture of the quality of the sample. Close inspection of the spectra of the five penicillin types reveals several possible analytical bands for each type. For convenience in observing these bands portions of the spectra of the five types are superposed in Figure 3. The analytical frequencies which are being used a t the present tinir are as follows: Sa-G 703 cIn.-l, 14.23 p sa-x 831 cm.-', 12.03 p (1220 crn.-', 8.2 p Na-F 971 cm.-l, 10.3 p --NaK 1330 cm.-l, 7.52 p 1166 ern.-', 8.57 p Naamyl 715 cm.-', 14.0 p
{
LOG 700
with R in the various types being:
G-
X-
F-
E-,.
=-E-,
H H H H H HC-C-C=C-C-, H H H
t benzyl
R =
4 (703~~') 1.
500
R 400
p-hydroxybenzyl
300 '
200 '
Alpentenyl
H H H H H H H H-C-C-C-C-C-C-C, n-heptyl H H H H H H H H H H H H HC-C-C-C-C--, n-amyl Amyl or dihydro FH H H H H
INTERNAL
100.
dl
STANDARD
ALANINE
K-
"0
IO
20
30
40
50
60
70
80
30
PERCENT CRYSTALLINE N o PENICILLIN G Figure 5. Working Curve for Determination of Crystalline Sodium Penicillin G ~
SEPTEMBER 1947 LOG
LOG
1,
623 1. The availability of pure standards typical of the components of the mixture (in order t o allow far the determination of accurate k
(103~6')
=
a.CONC=R
ALANINE
dI
Ip(851cm-') I A
No
G
703 Cm-'
W
values). 2. The presence in the spectrum of the mixture of absorption bands characteristic of the material or component being determined. 3. The precision of the determina tion of the radiation intensities I and Io. 4. A knowledge of the presence of interferine substances. 5. The acouraey of the measurement of the cell length, d.
Z
In cases where the sample is measured as a gas, the accurate determination of d presents no problem, d l ALANINE AND A since the cells used are, in general, S A M P L E O F PENICILLIN, of the order of several centimeters in MULLED WITH N U J D L length. Where the samples are liquids, solutions, or continuous solids, the I , , I I values of d are small. In such cases 7DD 730 850 900 the standard practice has been to avoid thedeterminatiouof thisquantity FREQUENCY IN CM4 entirely bv measurine: the knowns and _r -_.. "." _the u n L i w n s in fixed-length cells. In Crystalline Sodium Penicillin G the analyses herein reported. however. it was notpossible to follow this practice, inasmuch as no suitable solvent was available. As indicated A comparison of the spectrum of a given sample with that of above, the salt nature of the penicillin samples limits the number its prototype reveals the extent to which nonpenicillin materials of Dossible solvents to those of an extremely polar nature. Many occur. The D X S ~ ~ofCsmall ~ amounts of material which hydro.. gen bond to the penicillin molecule msy be detected by observing suih scIlvents, including water and deuterium oxide, mere investhe presence of lower frequency absorption on the side of the tigated and found to be impractical. Even if it were not for the nitrogen-hydrogen stretehiug frequency near 3400 cm.-'as shown strong hindered rotation band of water near 510 cm.-', the use of .. wouia preciuae, m,. e empioymem, or~m,e conven. aqueous SOIULLO~S in Figure 4. Both the nitrogen-hydrogen stretching vibration and the amide carbonyl ne= 1700 cm.-'are especially sensitive tional rock salt or potassium bromide cell windows, leaving only to the presence of hydrogen-bondable materials. When crystalthe less convenient and more expensive materials such as fluorite and silver chloride as possibilities. A further reason for not line penidlir I i y h r r s w is plwrJ in aqueous solution these t w o analyzing penicillin in solution is the desire to avoid the marked stretrliirig frr.iurncies + i i r to II 1 ~ ~ rvsliie . r indicarivr of ilir important role of their structures in the hydration of the molecule. Y
K N D W N MIXTUUE OF
_____-
ll_l
i_
I
1
QUANTITATIVE ANALYSIS
All quantitative analyses per-
. ' p C_i
R'
-
\;}
.. .
%
$.
formed by spectrometric means depend upon the fact that the absorption of eleetromgnetic energy a t a given frequency by a partially transparent substance is a function of the specific absorption of the material, and the total number of molecules of the sample through which the energy passes. This relationship may be written in the form of Beer's law as follows:
I
= I, e--bd
where Ia and I are functions, respectively, of the radiant energy falling upon and passing through the sample, k is an absorption c o e f f i c i e n t c h a r a c t e r i s t i c of the sample, e is a concentration, and d is the length of path through the sample, or the cell length. The success of any particular quantitative spectrometric analysis depends upon the following factors:
Figure 7. Crystal Orientation Effeots A. Platelike habit which orients in the mull B. Lath-shaped -stale,
nonorienfing
V O L U M E 19, N O . 9
624
decrease in spectral absorption intensity which acoompanies the trsasition from the crystal to the solution 3tate, and which therefore necessitates the use of a much larger sample. The use of a discontinuous solid rample, such as crystalline penicillin, renders the determination of a true d u e ford extremely difficult. In such u s e s the addition to the sample of s solid phase iuternal stmdard in a known concentration eliminates the necessity for malring a direct determination of the cell length. A good internal standard should possess the following characteristics: 1. No absorption interfering with the analytical bands. 2. A strong l~bnorptionband convenient to the spectral regions of hterest. 3. Easilv weiehable and mullable with the sample. 4. Easily procurable in &' reproiucible state of purity. From among *vera possible internal standard materials dl-alanine was chosen for !he analysis of the penicilljhs. I
Figure 8. 4ppearanoe of Final Mull, Penicillin plus dl-Alanine plus Nujd After the quantitative mixing of the 4. hodentation incomplete R. Deotisnfed Intern1 standard and the peniciUin the w c t r a are observed and wed in the versus the per cent of G as abscissas. From a consideration 01 sstablishment of a working curve relating absorption ohsraoterBeer's law it can be seen that the use of this ratio eliminstee lstics and amount of penicillin, the amount of penicillin being varied bhe necedtv for measurine the samule thickness. A differeni by diluting the stmdard sample with a noninterfering and n o m e but equally Gseful calibration curve may he.ohtained by measur ing an I and Io deflection employing the cell in-cell out technique kcting solid such as magnesium oxide, sodium chloride, or another type of penidin. The establishmentof accurate analytical workIng curves for all the combinations of the five penicillins has not been possible because of the scarcity of standard specimens. The working CUNC now In use for the estimation of cryarsl.Ine rodiiiui penicillin-(; in an unknown, t'iwrc 8, was obtained rhrouch tha fdlowinz orocctlure: 1.- Weiah out 1; &R. of the penicillin sample (thorouphlx mound, seeFigure 11). 2. Weigh out 7.5 mg. of internal standard and add to above. 3. Dry-mix the above using a small mortar and pestle. 4. Place a drop of Nujol on one of the polished rock-sall phtes to be assembled as the Apsorption cell. 5. To the drop of Nujol add about 10 mg. of the dry mixture. 6. Using t h e second rocksalt plate of the absorption cell null the sample thoroughly. Upon being held up to a llght the mull should be uniformly translucent over an area. large enough to cover tbS"s1it of the spectrometer. The presence of large c v t d fragments, resulting in a grainy appearance of the mull, d l lead to erroneous values. 7. Adjust the cell thickness so 88 to give about 50% transmission at 851 om.-' 8. Set the spectrometer at 925 om.-' with the cell in the beam, and adiust the slits and amplification factor to give a fullwale deflection. Run the spectnun from 925 to 775 om.?, recording the zero at the beginning and the end of the run: For this purpose a &utter suitable for minimizing scattered hght should be used. 9. Set the spectrometer at 760 cm.-', adjust for full-scale lcflection, and reciml from 760 10 695 ern.-' IO. Sotc the slit acttin@ and amplification facum used, in 3 and 9. The slit sftrines musc be duiilicatcd on all S I I U C P S ~ ~ V ~ ~~
'739 740 742 747 737 750 741 758 726
100.1 100. 2 100.6 101.2
99.9
733 722 720 760 747 763 746 743 737 730 736 725 730 726 720
737 724 732 738 752 740 AT. 738
f0.2 f0.5 +1.2
-0.1
f1.6 f0.2
C2.6 -1.8 f1.3 -0.6
748
I
fO.1
-2.2
-2.6
+2.e
C1.2 f3.1 t l . 1 .W,"
99.9 99.0 89.8
98.3
99.0 98.3
97.4 99.Q
CO.0 -0.1 -1.0
-0.2 -1.7 -1.0 -1.7 -2.8
98.a
-0.1 -1.8
99.4 100.0 1a.8
f1.8
100.2
-0.6 0.0
+o.a
1.x
~
71118.
11. Draw in the referonce lines of Fiwre 6 in ordor to allow tor the empirical i ~ ~ e a ~ u r r moil e n tnnd l a . 12. Rrucat on suvctssivelv diluted SBmnlPR and cstablish n
ralihration'curveby plotting
R=
.
log In (703 om.-') I pen.
IO log I internal standard
(851 om.-])
x
10'
88
ordinntss
The spread of the values was from +3.1 to -2.6%. Unknowns. Disinterested persons were asked to make up t e s ~ ~ ~ m p l etos be submitted for analysis. The samples were made by diluting sodium penicillin G, whose spectra established the sample as being 100% sodium G (referred t o the standard). with magnesium oxide. Included in the results will he, there fore, the errors introduced by several weighing8 and the diffi-
625
SEPTEMBER 1947
results of other assay methods. Microscopic examina. DEGREES FROM tion revealed that this Sam. PLATES- PREFERENTIAL ple had been crystallized u OR1 EN TAT1 0 N such a manner as to produw 22 flakes with some evidence oi 34 overgrowths (Figure 7). All 49 other samplea of crystalline 56 d t s used were found to ex. hibit a less flaky structure Microscopic examination of the respective Nujol mulls re. vealed that the flaky struc9 0 0 Cm-1 ture of the unusual sample had prevented the completf random comminution of tht crystals over the preparatioE $rea as shown in Figure S [t was suspected that the platelets contained dipoles oriented in such a fashion a+ to weaken the interactior with the electric vector of the Figure 9. Infrared Spectra of Two Forms of Crystalline Sodium Penicillin G as AiTected incident radiation, therebj by Sample Orientation Lowering the absorption in. tensity. Such a lack of rand t i e s in thoroughly mixing two solid materials. The analytical domness, as compared with that of the usual sample, could readil) results for these samples are rn follows: account for the low absorption a t 703 em.-' and the subsequently low infrared assay of this sample. An extended pre. Jarnple %G % G 70 grinding of this sample resulted in assay values approarhing 100% Denstion No. by Weight by Infrared As a check on the effect of the orientation of this sample in the 1 22.5 24.2 f1.7 spectrometer beam the following experiments were performed 2 50.0 47.8 -2.2 DEGREES FROM NORMAL 0 13
3
4
50.0 26.5 26.6 26.5 26.5 26.5 26.5 43.2
48.6 26.5 25.8 27.1 26.3 27.4 26.4 t0.4
-1.4 0.0
-0.7
tO.6 -0.2
4-0.9 -0.1 -2.6 Av. 1.06
[nterfefing Substances. Any molecule exhibiting absorption aear the 703 cm.-l sodium G analytical band will cause inter-
This sample was distributed by shaking it onto an oversizt rock-salt plate and the distribution was controlled by tapping the edge of the plate. The whole plate was then wet with Nujol and the upper rock-salt plate was carefully placed on top of the preparation in order to avoid fracturing the flat crystals. The whole cell was clamped together, resulting in a large area preparation having a great number of platelike crystals with their larger areas parallel to the faces of the rock-salt plates. This prepara. tion was then placed in the spectrometer in the usual position with the area of the plates normal to the incident radiation and the spectrum was recorded from 925 to 750 cm.-l It was the: rotated from the normal position to successive positions of 13 22O, 34", 49", and 56" off of normal and the spectrum recorded
ference with the analysis. It is to be expected, however, that during the crystallization of the penicillin most of the impuritiep will be removed. The b r e a k d o k pmducts and congeneric impurities of penicillin G which might interfere include such monosubstituted phenylFIO PLAT E S -NO 0 RlENTATlON bearing molecules as the sodium salt of phenyl wetic acid. Interference from sodium phenyl wetate may be detected by additional absorption bands at 682 and 728 cm.-I I n general, if any apn n n v W preciable amount of interfering material is present 0 5 Cni in a given sample, absorption bands characterisI .o tic of the impurity should be seen in the qualitative malysis of the sample which always precedes all quantitative analyses. Most samples of crystalline R sodium penicillin G will show only small amounts af impurities, usually other penicillin types, and little interference will be found at 703 em.-' If $odium penicillin X is present to any appreciable extent, a n unlikely possibility because of the great differences in solubility, it may be necessary to z:orrect for interference a t 703 em.-' This should not be a difficult operation, inasmuch as the amount of X may be readily estimated from its analytical absorption bands, as is shown in Figure 13. I I I I 0 15 30 45 60 Crystal Orientation Effects. During the course of this work a certain sample of penicillin of reputed DEGREES R O T A T I O N FROM NORMAL P O S I T I O N high potency was examined many times with Figure 10. Effect of Sample Orientation upon Infrared Absorption of a result that was inconsistent flow) with the Crystalline Sodium Penicillin G at 805 Cm.-'
-
h
I
-
4
I
V O L U M E 19, NO. 9
626
The other sample exhibited the platelike type of crystal easily oriented on the rockLOG (703 Cm-l) salt plates. The ratios of the Roptical densities a t 703 ern.-' LOG ' . (851 Cm-l ) and 851 cm.-l, the positions I N O PLATES-NO O R I E N T A T I O N of the analytical frequency for sodium G and the absorption of the dl-alanine, are plotted versus the grinding time in minutes. The differences in the spectral behavior with deorientation are clearly shovin. It is also possible to estimate the amount of grinding necessary to deorient the platelike crystals. It must be emphasized that in solid phase analyses isomorphism, polymorphism, pleochroism, or crystal oricntation effects must be considered. Although these effects, if not properly recognized and controlled, may alter the reGRINDING T I M E - M I N U T E S sults of the infrared analysis Figure 11. Deorientation of Crystalline Penicillin Produced by Grinding of crystalline sodium penicillin G, the present results demonstrate that thev need through the above region for each position. From a previous not cause any serious difficulties. .As vet no conclusive evidence study it was noticed that the absorption band near 900 cm.-l was has been obtained for the occurrence of either isomorphism or not affected by orientation, so its intensity was used as a referpolymorphism. ence band for consideration of the orientation changes occurring in an absorption band near 805 em.-* I
ANALYSES INVOLVING OTHER PEYICILLIY TYPES
The spectra of this sample a t the various orientation angles is shown in Figure 9. It may be seen that the 805 cm.-l absorption band, which is all but absent a t normal incidence, increases in intensity with degrees rotation from the normal position. For comparison purposes, the spectra of a sample containing no plates are shown. In this case the 805 em.-' absorption is fully developed and independent of the angle of incidence. A plot of the ratio log
R =
.
log
As implied above, techniques similar to that outlined for the establishment of the analytical working curve for sodium G, may be used to establish a quantitative method for any of the components of a mixture of penicillins. Figure 12 shows such a curve for determining the amount of sodium penicillin F contained in a sample predominately sodium G. In arriving a t this curve the values of I and 10 a t 971 and 831 em.-' were obtained by direct measurement (cell in-cell out), rather than by the graphic use of
(SO5 cm.-l) I IO
(900 cm.-l)
hiI
97' versus the rotation angle is shown in Figure 10 along with the values of R obtained with the deoriented sample a t various angles. (The ratio of these two absorption bands has been used in this case in order to eliminate the effect of the sample rotation upon the effective thickness.) This deorientation was effected by a thorough grinding of the sample in order to break up the platelike crystals. A study of the grinding necessarv to produce complete s p e c t r o s c o p i c a l randomness was made. The results of this study are shown in Figure 11. Two samples were ground with dlalanine in a mullite mortar for varying periods of time under approximately similar conditions. One sample possessed the usual nonplatelike crystals of crystalline penicillin and is designated as the nonoriented sample.
3401 0
, 5
, IO
, I5
Figure 12. Determination of Sodium Penicillin F Left. Infrared spactra of known mixtures of crystalline sodium penicillin G and F Right. Analytical working ourve for determining modium F
627
S E P T E M B E R 1947
Figure 13 shows a similar analysis for sodium penicillin X contained in a sample of sodium penicillin G.
d I-A LA N I N E
ACKNOWLEDGMENT
I
3001
t R
I
1
0
I
290
The authors wish to acknowledge the assistance of various members of the laboratory staff who have helped in this work. In addition, they wish to express their appreciation to the Food and Drug Administration, hntibiotics Study Section of the Sational Institute of Health, and to numerous commercial producers of penicillin for submitting some of the samples studied.
28 0 LITERATURE CITED
800 I
I
I
I
Figure 13.
851 Cm-'
P
% S O D I U M X I N SODIUM G
(1)
270.
I
I
I
0
5
IO
Determination of Sodium Penicillin X
Left. Infrared spectra of known mixtures of crystallihe sodium penicillin G and X Right. Analytical working curve for determining sodium penicillin X
I
15
Barnes, R. B., McDonald, R. E.. Williams, 5'. Z., and Kinnaird, K . F., J . A p p l i e d Phys., 16, 77-86 (1945).
(2) Committee on Medical Research,
O.S.R.D., W a s h i n g t o n , anld Medical Research Council, London,
Science, 102, 627-9 (1945). (3) Fishback, H., Mundell, M., and Eble, T. E., Ihid., 104, 84-5 (1946). (4) Food and Drug Administration. com-
munication.
recorded spectra. In the figure the spectra shown are those corresponding to samples of sodium penicillin G to a-hich 0, 5, 10, 15,-33, and 50% sodium penicillin-F have been added. As may is be seen, a rapid estimation Of the F content of such a possible by visual examinat,ion.
( 5 ) Sheehan, J. C., hfader, SOC.,68, 2407 (1946).
W. J., and Cram, D. J., J . Am.
Chem.
PREBEXTED in part before the Conference on Antibiotic Research held a t Washington, D . c., on j a n . 31 a n d F e b . 1, 1947, under the auspices of t h e Antibiotics S t u d y Section of the S a t i o n a l Institute of Health.
Analysis of Thermite-Type Incendiary and Pyrotechnic Mixtures CHARLES E. DANKER AND JEROnlE GOLDENSON Chemical Corps Technical Command, Edgewood Arsenal, M d . Procedures are gi7en for the rapid estimation of composition and partiele size of thermite-type incendiary and pyrotechnic mixtures. The ingredients of the mixtures are separated with a magnet or by se1ectiT.e solvents and weighed, followed by particle size determination as required.
T
HE composition of U. S. Army thermite-type incendiary mixtures is given in the appropriate Chemical Corps Pro-
curenient Specification such as Chemical Corps Tentative Specification S o . 196-131-1228. Since these specifications provide for certification of composition by the manufacturer in addition to performance tests, there has been no need to include chemical method. for determining composition in the specifications. However, chemical methods have been frequently needed for determining the composition of incendiary mixtures in connection nith tests required by experimental work and for controlling production. There has also been a need for a quick qualitative procedure to identify unknon-n incendiary mixtures found in captured munitions. A consideration of the main analytical methods available indicated that a procedure utilizing the principle of separation of ingredients of a mixture by the action of selective solvents (2-5) was best suited, chiefly because it is simple and rapid; ingredients such as iron oxide scale, hammer scale, or iron ore, which are miatures of the various oxides of iron, can be best estimated by
weighing them in their original condition rather than by a procedure which requires putting them into solution; and mesh size analyses of specification ingredients cannot be made if the entire sample is put into solution. PROCEDURES
These methods were developed over a period of three years at Edgevood Arsenal in connection 71-ith experimental work and plant development on known incendiary fillings, and analyses of unknown foreign incendiary mixtures. Procedures are given for estimation of the composition and mesh size of mixtures whose components are known; a quick qualitative scheme is also given to determine the components in an unknown mixture. Plain or Commercial Thermite. This incendiary mixture,, which is used alone as a filling in nonmagnesium cont'ainers and and as an ingredient of other incendiary formulas, is a mixture of magnetic iron oxide scale and aluminum.
PROCEDURE I F ; 1 L n n N u x Is OF USKSOWX OR LOWPURITT. A'lix the entire sample thoroughly on glazed paper, as segregation
