above background as a function of micrograms of lead. The slope of the line, i.e., 50 cpm/bg, is used to convert all subsequent measurements. A distinct advantage of a radiochemical method over others lies in the fact that the sensitivity may be increased as the need arises merely by incorporating more of radioisotope *lOPbin the test anode, the amount added being limited only by the restrictions imposed by the By-product Materials License in force.
allurgy Research Center for his assistance and valuable suggestions.
ACKNOWLEDGMENT
RECEIVEDfor review November 7, 1975.Accepted January 22, 1976. Reference to specific equipment does not imply endorsement by the Bureau of Mines.
The authors are grateful to William L. Anderson of the Analytical Chemistry laboratory of the Salt Lake City Met-
LITERATURE CITED (1) W. D. Fairman and J. Sedlet. Anal. Chern., 40. 2004 (1968). (2) C. H. Wang and D. L. Willis. "Radiotracer Methodology in Biological Science", Prentice-Hall, Englewood Cliffs,N.J., 1965. 168. (3) I. Tsukahara and T. Yarnamoto. Anal. Chim. Acta, 61, 33 (1972).
Nematic Liquid Crystal for Gas-Liquid Chromatographic Separation of Steroid Epimers Walter L. Zielinski, Jr.,* Kevin Johnston, and Gary M. Muschik NCI Frederick Cancer Research Center, Frederick, Md. 2 170 1
Gas-liquid chromatography of underivatired steroid epimers has been successful in the nematic temperature region of N,N'-bis(pmethoxybenrylidene)-a,cr'-bi-ptoluidine. Application is made to the resolution of 5a/fl,3a/fl-ol solute combination pairs of the androstane, androstanone, and cholestane classes with separation factors exceeding those reported elsewhere in which polar phases and a variety of steroid derivatiration methods have been necessary. While 5 a and 5fl isomers readily yield large separation factors (2.3-3.8), factors of 1.1-1.2 are still attainable for difflcult separation of underivatized 5fl,3a/fl-ols in the temperature range of 185-250 'C. Separations on this nematogenic liquid crystal are greatly assisted by the difference in the molecular length-to-breadth ratio of the steroid epimers. The results of this study should be of direct relevance to programs in biochemical as well as in carcinogenesis research.
The significance of steroids in normal biological functions has been recognized for many years. Recently, specific aspects of steroid metabolism have also been of much interest to workers studying the abnormal case of malignancy. For example, while androsterone and its 5p epimer have been observed as the major steroids in urine and dehydroepiandrosterone in plasma, exceptions were 'noted in cancer of the adrenals and testes ( I ) . Androgen sensitivity and steroid binding has been studied in prostate cancer in rats ( 2 ) . Furthermore, a considerable level of research is currently being directed toward investigations of the involvement of steroids in human breast cancer (e.g., ( 3 ) ) ,as well as in 7,12-dimethylbenz[a]anthracene-inducedmammary tumors in experimental animals (4-10). In steroid biochemical studies, research has been substantially aided by the availability of useful analytical techniques for separation and measurement. While some studies have involved the use of radiolabeled steroids in which medium fractionation and scintillation counting are requisite, a substantial portion of such investigations has utilized non-labeled ma-
terials and one or more forms of chromatography-the most-widely used of which has been gas-liquid chromatography (GLC). While GLC has been successfully applied to the analysis of steroids in biological systems, the resolution of steroid epimers has not been possible without the use of polar liquid phases or the requirements for the preparation of a variety of volatile derivatives (11-19). The application of cholesteric mesophases for the separation of steroid epimers was reported several years ago (20),yet general utility of liquid crystals as stationary phases for such separations has been largely overlooked. We previously had reported ( 2 1 ) novel separations of 3-5 ring polynuclear aromatic hydrocarbons on a high-temperature nematic liquid crystal, N, N' - bis ( p - m e thox y benzylidene) - a ,a'- bi - p -toluidine (BMBT). We now report the use of this liquid phase for novel separations of steroid epimers of the androstane and cholestane series with separation factors greater than have been reported previously by GLC or any other form of chromatography.
EXPERIMENTAL Chemicals. The nematic liquid crystal used as the GLC stationary phase, N,N'-bis(p-methoxybenzylidene)-a,a'-bi-p-toluidine (BMBT), was obtained from Eastman Kodak Co. in a purity in excess of 99%, as determined by differential scanning calorimetry. Its nematic mesophase range (181-320 "C) and its heats of phase transition were reported previously (21). The steroids were purchased from Steraloids, Inc. (Pawling, N.Y.) and Sigma Chemical Co. (St. Louis, Mo.). Absolute ethanol was obtained from US.Industrial Chemicals Co. (New York, N.Y.), and all other solvents used were glass-distilled by Burdick & Jackson (Muskegon, Mich.). Apparatus and Procedure. A Hewlett-Packard 7610 gas chromatograph was employed containing a flame ionization detector and a 4-ft X 4- or 2-mm i.d. glass column run a t a,helium carrier gas flow rate of 70 or 40 ml/min, respectively. The column packing was 5 wt % of BMBT on 100/120 mesh H P Chromosorb W, prepared by the solvent slurry technique and fluidized drying with nitrogen. Prepared columns were conditioned a t 250 OC for 1-2 h. Chromatograms were generated on a I-mV f.s. strip chart recorder using an electrometer setting of 16 X lo2. Carrier gas flow was monitored by a calibrated Brooks 5840 Dual GC Mass Flow ConANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
* 907
Table I. List of Steroids Studied IUPAC nameo W v)
z
g Ya W a
0
a
0 K
5 10 TIME,MINUTES
0
Figure 1. Chromatogram of the 5alP-androstan-3alP-ols
Column: 4-ft X 2-mm i.d. glass. Packing 5 % (w/w). Conditions: Oven, 185 'C: injector, 200 OC: detector, 200 O C ; helium flow rate, 40 ml/min
Abbreviation used
5a-Androstan-3a-01 5,-Androstan-30-01 50-Androstan-3,-01 5/3-Androstan-SP-o1 3a-Hydroxy-5a-androstan-17-one 3P-Hydroxy-5P-androstan-17-one 3u-Hydroxy-5/3-androstan-17-one 3P-Hydroxy-5/3-androstan-17-one 3P-Hydroxy-5a-androstan-16-one 5a-Androstane-3/3,164-diol 5a-Androstane-3P,17/3-diol 5a-Cholestan-3a-01 5a-Cholestan-3P-01 56-Cholestan-3a-01 5p-Cholestan-3/3-01 58-Cholestan-3-one 511-Cholestane a
5,-A-30-01 5,-A-3p-01 5P-A-3a-01 5P-A-30-01 5a-A-3a-ol-17-one 5P-A-3P-ol-17-one 5P-A-3a-ol-17-one 5P-A-3P-ol-17-one 5a-A-3P-ol-16-one 5a-A-3/3-01-166-01 5a-A-3/3-01-17/3-01 5a-C-3a-01 5a-C-3P-01 5/3-c-3a-ol 5P-c-3P-01 5P-C-3-one 5B-C
Ref. (22).
W v)
2
g v) W
K K W
n K
0 0 W K
0
4
8
12
16
TIME, MINUTES
Figure 2. Chromatogram of the 3alP-hydroxy-5alP-androstan-17-
ones Column, packing, and conditions: same as Figure 1 except: Oven, 230 OC; injector, 190 OC; detector, 230 O C troller, while hydrogen and air flow rates (40 and 500 ml/min, respectively) were measured with a soap bubble flowmeter. Sample injection volumes were usually 1-2 pl using a Hamilton 501N 10-pl syringe.
RESULTS AND DISCUSSION It was previously reported by Janini et al. (21) that excellent separations of 3-5 ring polynuclear aromatic hydrocarbons could be successfully obtained by the use of a hightemperature nematic liquid crystal (BMBT) as a novel GLC stationary liquid phase. These separations were based upon subtle differences in solute shape (molecular lengthto-breadth ratio). I t was felt that this unique liquid phase might selectively resolve steroid epimers differing in A/B ring configuration and in axial/equatorial alignment of the 3-hydroxy substituent. This paper describes the analytical results which validate this hypothesis for a series of androstane and cholestane alcohols and ketones (Table I). The separation of 5a/@-androstan-3a/@-olson BMBT is shown in Figure 1. The longer retention of the 5a derivatives on BMBT is due to much higher planarity of the 5aandrostane nucleus in which the A/B rings are trans-fused (23, 24). Furthermore, since the 3@-hydroxygroup is equatorial to the A ring in the 5a derivative, it possesses a greater length-to-breadth ratio than its 3a analogue and is re908
ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
0
2
4
TIME, MINUTES
Figure 3. Chromatogram of the trimethylsilyl ethers of the 5aIP-
androstan-3a/@-ols Column: 4-ft X 4-mm 1.d. glass. Packing: 5% (w/w). Conditions: Oven, 185 OC; injector, 200 O C ; detector, 235 OC: helium flow rate, 70 ml/min
tained longer by the nematic liquid phase. In the case of the 5P-androstanols, the 3P-hydroxy group lies more in the plane of the molecule than the 3a analogue, even though the former geometric substituent lies axial to the A ring. The separation factors for this epimer class are greater than reported elsewhere (15, 17). Larger separation factors are likewise observed here than reported by other workers for the TMS derivatives of these solutes (15, 17), for 3a/phydroxy-5a/P-androstan-17-ones (14, 16) or their TMS derivatives ( 5 , 17, 20), or for 5a/~-cholestan-3a/@-ols (12, 13). Shown in Figure 2 is the separation of 3alP-hydroxy5alP-androstan-17-ones. The explanations for the separation order observed here are the same as those given above for the androstanol epimers. Since the respective separation factors for each set of solutes shown in Figures 1 and 2 are fairly similar, it may be concluded that the presence of a keto group a t the 17 position (Figure 2) does not noticeably influence the elution pattern of 5alfl-androstan-3alP01s.
w v)
z
W
2
v)
z
:
Ya a
2a
a
a w a
Ya
8w
W D
a
0
K
4
TIME, MINUTES Figure 4. Chromatogram of the trimethylsilyl ethers of the 3 a / & hydroxy-5alP-androstan- 17-ones Column, packing. and conditions: same as Figure 2
TIME, MINUTES
Figure 6. Chromatogram of 5P-cholestanes having different substituents attached at the 3 position. Column, packing, and conditions: same as Figure 5
I
r: W
W
ul 2
2
ul
2 8 a
2 8 a a
a
P U 0
n
W
W
a
0
sa
YK
0
0
4
b
4
a
12
15
TIME, MINUTES
TIME, MINUTES Figure 5. Chromatogram of the 16p/17p-ols and the 16/17-ones of 50-androstan-3P-01 Column: 4-ft X 4-mm i.d. glass. Packing: 5 % (w/w). Conditions: Oven, 250 OC: injector, 225 OC; detector, 250 OC; helium carrier flow, 70 ml/min
Separation of the T M S derivatives of the 5alP-A-3alP-01 and 5tu/fl-A-3~~/6-17-one epimers on BMBT is shown in Figures 3 and 4, respectively. The decrease in solute retention is attributed to a decrease in solute length-to-breadth ratio caused by the introduction of the bulky trimethylsilyl group a t the 3 position as well as to an elevation in solute vapor pressure. I t is observed that the use of T M S derivatives results in a deterioration of epimer resolution. It should be noted, however, that the high degree of separation observed for the free epimers precludes the normal requirement for steroid derivatization on BMBT.
Figure 7. Chromatogram of the 5a/fi-cholestan-3cu/~-ols Column, packing, and conditions: same as Figure 5
Comparison of the retention behavior for keto and 6hydroxy substitution a t positions 16/17 in 5a-A-36-01 is shown in Figure 5. Keto substitution for a hydroxyl group enhances molecular planarity and thus contribures to solute retention on a liquid crystal phase. This is observed in Figure 5 for the greater retention of 16/15-ones over the 16/3/17/3-01s. This effect is also observed in Figure 6 for the retention of 56-cholestane derivatives having different functionalities. Figure 7 shows the separation of 5alP-cholestan-3a//I-ols a t 250 O C in the same elution sequence as noted previously for the 5a//3-androstan-3alP-ols (Figure 1).The temperature dependence of the separation factors for different 5a/@-C-Sa/P-o1epimer pairs (Figure 8 ) is quite negligible for the 5@-3a//3pair ( A a of 0.55) in the range of ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
909
(In.)
x
10'
150
100
n
d
R
in 1.8
26
32
36
,-ISOTHERMALA
185 190
. 1.85
200
210
220
230
240
250
COLUMN TEMPERATURE ( O C ) A
1.9
1.95
20
Figure 9. Chromatogram of a synthetic mixture of the 5 a l @ - A - 3 a l 0-ols, the 5a-A-3a, 16P(/17~)-diols,and the 5alP-C-3alfl-ols
2.05
Column: 4 4 X 4-mm i.d. glass. Packing: 5%(w/w). Conditions: Oven temperature program rate of 2 OC/min from 185-250 O C , then isothermal at 250 OC; injector 225 O C : detector, 250 O C ; helium flow rate, 70 ml/min
Table 11. Comparison of Literature Separation Factors with Those Obtained for Free Steroid Epimers on BMBT SeDaration factors Best literature value
Ref.
3.77
1.96a
(20)
2.45
1.54= 1.996 1.09b*
(201 (20) (20)
1.3lC
(13)
BMBT
1.74 1.14 3.51
17-one
5a//3-A-3a-o12.34 1.19d* (13) 17-one 5a-A-3P/a-011.73 1.47e* (15) 17-one 50-A-3PIa-011.15 1.21c* (13) 17-one 5a-A-30-011.12 1.131 (13) 16/17-one Cholestanols 5a/@-C-3P-ol 3.53 1.2SC (13) 5a/fl-C-3a-ol 2.57 1.09g (13) 5LY - c- 3/3/a-01 1.69 1.37k (13) 5/3-C-30/a-o] 1.23 1.14c* (13) On cholesteryl p-phenylbenzoate; on cholesteryl benzoate as the TMS derivatives; on QF-1; on JXR-CHMDS;e on XE-60 as the heptafluorohutyrates;f on SE-30 as the dimethylhydrazones; g on SE-30; on SE-30 as the heptafluorohutyrates. * Elution order reversed (reciprocal of separation factor is given). 220-265 "C.I t should be noted that the larger the difference in length-to-breadth ratio (5a-3p > 5a-3a > 5P-36 > 5@-3a),the larger is the value of a. Investigators who have employed GLC for the separation of steroids have observed the lack of resolution of steroid epimers on non-polar phases such as SE-30 (12-14, 17, 191, and were forced to resort to the preparation of a variety of derivatives [TMS ethers (5, 15, 17, 19, 20); alkyl (12) or perfluoroalkyl esters (12, 14); chloromethylsilyl ethers 910
24
TIME, MINUTES
1031~
Steroid pair Androstanols 5a/@-A-38-ol 5a/@-A-3a-o1 5a-A-3P/a-ol 5P-A3P/o(-ol Androstanones 5~dfi-A-3P-01-
20
16
12
50
ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
( 2 5 ) ;T M S ether-enol-TMS ethers ( 2 6 ) ;or others ( 1 4 ) ] or more polar liquid phases [QF-1; OV-17, -210, -220; polyesters EGSS-X and ECNSS-M; F-60; XE-60; NPGS]. The use of capillary columns (19) and cholesteric liquid crystal phases (20) have also been examined, as well as a computer-aided GC-MS method for the selective analysis of unresolved steroid epimers (18). However, the separation factors for underivatized epimeric steroids reported here on BMBT liquid crystal exceed all previous attempts for the GLC separation of these solutes, with or without prior derivatization (Table 11). The utility of this liquid phase for the programmed temperature separation of a number of solutes studied is shown in Figure 9. I t has been reported that column bleed of BMBT can occur during prolonged periods of operation a t elevated temperatures (21), yet routine operation a t temperatures between 182-250 O C a t liquid phase coatings below 5% w/w has been observed over a period of several weeks. Other high-temperature liquid crystals which exhibit substantially diminished column bleed rates have recently been synthesized (27). While it is recognized that other GLC systems may be required for the separation of the broad array of steroid structures, the separations reported here should enhance the capabilities of workers in the clinical, biochemical, and carcinogenesis research fields. ACKNOWLEDGMENT The authors express their appreciation to George M. Janini for his previous work with this unique liquid phase (21), in showing its high temperature application for novel GLC separations. LITERATURE CITED (1) G. I. Fujimoto and R. W. Ledeen, "Comprehensive Biochemistry", M. Florkin and E. H. Stotz, Ed., Vol. 10, Elsevier, New York, 1963, p 32. (2) W. Voight, M. Feldman. and W. F. Dunning, Cancer Res., 35, 1840 (1975). (3) K. E. Horwitz and W. L. McGuire. Steroids,25, 497 (1975). (4) A. G. Jabara and M. S. Maritz, Br. J. Cancer, 28, 161 (1973). (5) W. R. Miller, A. P. M. Forrest, and T. Hamilton, Steroids, 23, 379 (1974). (6)C. Lee and R. Oyasu. J. Natl. Cancer lnst., 52, 283 (1974). (7) P-C. Chan and L. A . Cohen. J. Nat. Cancer lnst., 52, 25 (1974). (8) L. Terenius, Eur. J. Cancer, 7 , 65 (1971). (9) S. K. Quadri, G. S. Kledzik, and J. Meites, Cancer Res., 34, 499 (1974). (10) H. Nagawasaand R. Yanai, J. Nat. Cancer Inst., 52, 1219 (1974). (11) R. J. Hamilton, W. J. A. VandenHauvel, and E. C. Horning, Biocbim. Biophys. Acta, 70, 679 (1963). (12) W. J. A. VandenHeuvel and E. C. Horning, "Biochemical Applications of Gas Chromatography", H. A. Szymanski, Ed., Plenum Press, New York, 1964, p 89.
(13) E. C. Horning and W. J. A. VandenHeuvel. Adv. Chromatogr., 1, 161 (1965). (14) E. C. Horning, "Gas Phase Chromatography of Steroids", K. E. Eik-Nes and E. C. Horning, Ed., Springer-Verlag,New York. 1968, p 1. (15) A. Vermeuien. Clin. Chim. Acta, 34, 223 (1971). (16) A. Hiscoe, D. W. Mathieson, and R. H. Perrett, J. Chromatogr., 81, 144 (1973). (17) A. Kuksis, Fette, Seifen, Anstrichm., 75, 420 (1973). (18) J. D. Baty and A. P. Wade, Anal. Biochem., 57, 27 (1974). (19) E. Bailey, M. Fenoughty and J. R. Chapman, J. Chromatogr., 96, 33
(23) D. Kritchevsky, "Comprehensive Biochemistry", M. Florkin and E. H. Stotz, Ed., Vol. 10, Eisevier, New York, 1963, p 1. (24) L. F. Fieser and M. Fieser. "Steroids", Reinhold. New York, 1959, Chap. 1. (25) J. R. Chapman and E. Bailey, Anal. Chem., 45, 1636 (1973). (26) E. M. Chambaz, G. Defaye, and C. Madani, Anal. Chem.. 45, 1090 (1973). (27) G. M. Janini. G. M. Muschik, and W. L. Zielinski, Jr., personal mrnmunication, 1975.
(1 974).
(20) D. N. Kirk and P. M. Shaw, J. Chem. SOC.C, 1971, 3979. (21) G. M. Janini, K. Johnston, and W. L. Zielinski, Jr.. Anal. Chem., 47, 670 (1975). (22) IUPAC Commission on the Nomenclature of Organic Chemistry and IUPAC-IUB Commission of Biochemical Nomenclature, J. Steroid Bio-
chem., 1, 143 (1970).
RECEIVEDfor review September 29, 1975. Accepted January 5, 1976. This study was sponsored by the National Cancer Institute under Contract No. N01-CO-25423 with' Litton Bionetics, Inc.
Determination of Trace Amounts of Diethylene Glycol in Nitroglycerin by Infrared Spectrometry A. S. Tompa Research and Technology Department, Naval Surface Weapons Center, White Oak Laboratory, Silver Spring, Md. 209 10
Diethylene glycol concentrations of 0.01 to 0.20% in nitroglycerin were determined to within 0.005 YO by infrared spectrometry using the 1122 cm-' C-0 absorption band.
Nitroglycerin (NG) is a powerful and very shock-sensitive explosive plasticizer which is used with nitrocellulose in double-base propellants. Accordingly, the shipment of NG is required by Interstate Commerce Commission regulations to be desensitized by the addition of 25%diethylene glycol (DEG). However, before NG can be used in propellant formulations, the DEG must be removed because it has deleterious effects on propellant properties. NG is therefore water-washed three times in order t o lower the DEG concentration to below 0.02%. The determination of DEG in water-washed NG has been attempted by dichromate oxidation ( 1 ) and near-infrared spectrometry (2). The oxidation method is time consuming (requires several extractions and a two-hour digestion period) and is nonselective as any organic impurities present in the extract are also oxidized. The near-infrared procedure is a pass or fail method based on a weak shoulder band a t 2.92 Mm. Any residual water must be corrected for by a Karl Fischer titration. The correction factor is usually of a greater magnitude than the DEG concentration and, hence a serious source of error. Therefore, alternate methods of analysis which are quick, safe, and accurate are highly desirable. Two promising methods ( 3 ) developed in this laboratory are gas chromatography and infrared spectrometry. The gas chromatography method is based on Trowell's ( 4 ) method for analysis of aged double-base propellants.
EXPERIMENTAL Apparatus. The infrared spectra were recorded on a PerkinElmer Model 521 spectrophotometer. The spectra were obtained from 1170 to 1090 cm-' using a 2X slit program and a scanning speed of 30 cm-'/min. The mechanical slit opening was 541 Fm a t 1122 cm-'. The infrared cells had pathlenghs of 1.0 and 0.10 mm. Reagents. Reagent grade DEG and chloroform were used. T h e water-washed NG was at least 99.7% pure and was either a clear solution or slightly cloudy because of the presence of trace amounts of water.
Procedure. Three procedures were investigated. The first method used neat NG as the solvent. The second was a modification of the first by using the method of additions. The third used a solution of NG in chloroform. In the first method, 0.50 to 15.0 mg of DEG was added directly to 1 to 4.000 g of NG to give 0.01 to 1.20% solutions of DEG in NG. T h e vials containing the solutions were gently swirled to ensure thorough mixing. The solutions were then run in a 0.1-mm cell with neat NG in a 0.1-mm reference cell. The cell thicknesses should be sufficiently matched so that neat NG in both the sample and reference cells would give a straight line in the 1170 t o 1090 cm-' region. If the sample contains 0.01% or less of DEG in NG, then 5X scale expansion should be used t o increase the precision of the measurement. The second method involves adding a known amount of DEG to the sample and running the original and spiked samples in the same infrared cell. Generally, the spiked samples would contain 0.01 t o 0.05% DEG in NG. In the third method, solutions of 0.001 to 0.02% DEG in chloroform containing 8.00 to 10.00% NG were prepared. Two to 10 mg of DEG were added to the 10-ml volumetric flasks and filled to the mark with chloroform. Aliquots of 1 t o 2 ml of these solutions were added to 10-ml volumetric flasks with plastic stoppers. Chloroform was added to approximately half-fill the flask, the flask weighed, and then 0.8 to 1.000 g of NG was added. The flask was reweighed and then diluted to the mark with chloroform. A standard reference solution of 10.00% NG in chloroform was prepared. Aliquots of this solution were diluted with chloroform t o match the NG concentration in the sample solution. The chloroform solutions are run in 1.0-mm cells. Chloroform in a 1.0-mm cell has a strong absorption band in the 1100 to 1000 cm-' region. It was observed that the absorption band a t 1122 cm-' was more intense and symmetrical when the mechanical slit width was doubled. The 10 baseline was taken as the horizontal portion of the spectrum between 1170 to 1140 cm-'. I t was found using chloroform solutions in order to get good quality spectra suitable for quantitative analysis, the product of the NG concentration and cell thickness in the reference cell must be a t least 95% of the product of the NG concentration and cell thickness in the sample cell. On some occasions, the nitroglycerin sample will contain 0.1% of 2-nitrodiphenylamine which acts as a stabilizer. I t imparts a characteristic red color to the sample. The stabilizer has a sharp band a t 1150 cm-' of medium intensity in a 0.1-mm cell a t the 0.1% level. It does not interfere with the DEG determination; however, better precision is obtained if the method of addition is used.
RESULTS AND DISCUSSION The infrared spectra of DEG and NG in the 4000 to 400 cm-' region showed that the 1122 cm-l absorption band of ANALYTICAL CHEMISTRY, VOL. 48,
NO. 6,
MAY 1976
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