234
Anal. Chem. 1905, 57,234-237
(6) Karlsson, K.-E.; Novotny, M. HRC CC,J . High Resolut. Chromafogr. Chromatogr. Commun. 1983, 7 , 411-413. (7) Gluckman, J. C.; Hlrose, A.; McGuffin, V. L.; Novotny, M. Chromato-
(13) Novotny, M.; Alasandro, M.; Konishi, M. Anal. Chem. 1983, 55, 2375-2377.
oraahia 1983 f 7 ,. 303-309 - .- - - -
(8) ' A x k o i , k-sahlberg, B. L. Anal. Len. 1981, 14, 771-782. (9) Shackleton, C. H. L.; Whltney, J. 0. Clln. Chim. Acta 1980, 107, 231-243. (10) Setchell. K. D. R.; Alme, B.; Axelson, M.; Sjovall, J. J. Steroid Biochem. 1978, 7 , 615-629. (11) Lloyd, J. B. F. J . Chromatogr. 1979, 178, 249-258. (12) Voeker, W.; Huber, R.; Zech, K. J. Chromatogr. 1981, 277, 491-507.
RECEIVED for review January 24, 1984. Resubmitted and accepted September 4, 1984. This work was sutmorted bv Grant PHS kOl GM 24349-05 from the Institute-& General Medical Scientes, US. Department of Health and Human Services.
Liquid Chromatographic Separation of Diastereomers and Structural Isomers on Cyclodextrin-Bonded Phases Daniel W. Armstrong,*Wade DeMond, and Ala Alak Department of Chemistry, Texas Tech University, Lubbock, Texas 79409
Willie L. Hinze and Terrence E. Riehl Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109
Khanh H. Bui Advanced Separation Technologies, Inc., 37 Leslie Court, P.O. Box 297, Whippany, New Jersey 07981
Elghty compounds are separated from thelr Isomers by LC uslng cyclodextrln-bonded columns. A varlety of structural Isomers (lncludlng polycycllc aromatlc hydrocarbons and prostaglandlns), geometrlc Isomers, and sterold epimers are examined. Cyclodextrln-bonded packlngs appear to be more widely applicable than elther normal or reversed-phase packlngs for these types of separatlons. Indeed, compounds that cannot be well resolved on more tradltlonal columns are oflen easily separated on thls statlonary phase. The separation mechanism ls based on lncluslon complex formatlon and Is responsible for the unusual but oflen predlctable selectlvltles observed.
The separation of isomeric compounds by liquid chromatography is an interesting but unevenly characterized field. The separation of enantiomers, for example, has received a considerable amount of attention from a number of researchers employing a variety of techniques (1-3). By comparison, the LC separation of diastereomers and structural isomers has been somewhat neglected. Although one can find isolated examples of specific separations of pairs of isomers, there are few broadly applicable approaches and, generally, little evaluation of the principles involved. One of the few techniques that has been reasonably well studied is that of argentation chromatography. Silica gel impregnated with silver ions has been shown to be selective for the separation of certain geometrical isomers such as cisand trans-retinols, pheromones, pesticides, and unsaturated fatty acids (4-7).Thallium ion has been used in place of silver for analogous separations (8). Geometrical isomers are, of course, only one subclass of diastereomeric compounds. Argentation chromatography is generally less successful in separating structural isomers, other types of diastereomers, and saturated compounds which are not able to form charge-transfercomplexes. There is also the problem of a lack of reproducibility, high cost, and bleeding (due to the solubility of silver salts in many organic solvents).
An examination of the recent literature seems to indicate that the separation of structural and geometric isomers is more easily accomplished by normal-phase LC than by reversedphase LC. For example, silica gel is by far the preferred packing in the separation of isomers of retinal, retinol, and retinyl esters (9, 10). The separation of 0-,m-, and p-nitroaniline has been achieved with near-base-line resolution on an alumina column but not by reversed-phase LC (11). One class of isomeric compounds (i.e., diastereomers) where reversed-phase LC seems to have had as much success as normal-phase LC is in the separation of steroid epimers (12,13). It has been noted that some steroid epimers are best separated by normal-phase LC and others by reversed-phase LC, and some are not well separated by either technique (12-15). Often recycling is needed to achieve these separations, particularly when the epimeric center is ?Czo of the steroid structure (12). It is apparent that in some cases the separation of a pair of isomers is facile (syn- and anti-azobenzene, for example) while in other cases very specific conditions are required. There are also several examples of intractable isomers for which no really effective LC technique currently exists. For example, the separation of all four epimers of estriol (i.e., estriol, 16-epiestriol, 17-epiestriol, and 16J7-epiestriol) has not been reported to our knowledge (13). Benzo[a]pyrene and benzo[e]pyrene (as well as some other polycyclic aromatic hydrocarbons) are known to be difficult to separate, particularly in complex mixtures (16-18). It is also apparent that there is no single packing or procedure that can be considered widely applicable for the LC separation of diastereomers and structural isomers. Certainly the development of an effective packing that can discriminate between a variety of similar compounds on the basis of their geometry or orientation and that would allow one to avoid using specially prepared packings, multiple columns, and recycling, would be useful in LC. It will be demonstrated in this report that the cyclodextrin column is particularly adept at separating both diastereomers and structural isomers. This is because the separation is largely due to inclusion complex formation which provides
0003-2700/85/0357-0234$01.50/00 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985 235
Table I. Isocratic LC Retention Data for a Series of Structural Isomeric Compounds no
0
6
12
RETENTION TIME, Min.
Figure 1. Isocratlc separation of benzo[e]pyrene from benzo[a]pyrene on a 10-cm 0-cyclodextrin column. The flow rate was 1.5 mL/min, and the mobile-phase composition was 50:50 (v/v) methanol/water.
mobile phaseb
7.9 6.2
I
50150
2
1,2:3,4-dibenzanthracene 1,2:5,6-dibenzanthracene
3.8
11‘
55/45
4.2
I
55/45
I
50150
I
50150
IIC
90110
I
40160
IIC
50150
IIC
50150
11‘
45/65
1.7 2.2
11‘
40160
0.1
I‘
55/45
IIC
40160
IIC
30170
Id
60140
IC
40160
14.0 5.0
11‘
60140
7.7 9.1 4.7
IC
30170
3.8 4.8 1.7
IIC
50150
3 4
5 6
EXPERIMENTAL SECTION
10
Table I gives the separation conditions and retention data for a variety of structural isomers. These include compounds which are isomers because of the translocation of an aromatic ring (numbers 1-3, Table I), translocation of a double bond (numbers 4-6, Table I), or a change in the location of a substituent on an aromatic nucleus (numbers 7-19, Table I). All isomers can be completely resolved (i.e., R, I 1.5) except for 3- and 5-methylindole and 0-and p-aminobenzoic acid which are partially resolved. Figures 1and 2 illustrate the isocratic separation of benzo[a]pyrene (b[a]p) from benzo[e]pyrene (b[e]p) as well as the separation of 0-,m-, and p-xylene. These particular isomeric compounds (as well as others such as 0-, m-,and p-cresol) are known to be difficult to separate by LC. As indicated in Figures 1and 2 and Table I, complete separations are easily achieved when using a (3-cyclodextrin-bonded packing and isocratic elution. It should be noted that there have been previous reports on successful gradient RPLC separations of b[a]p and b[e]p (16-18). These separations were shown to be very dependent on the nature of the bonded phase. Isomers of nitroaniline (Table I, number 16) are not
column”
benzo [a]pyrene benzo[e]pyrene
9
RESULTS AND DISCUSSION
k’
1
a physical basis for the resolution of isomeric compounds (19-23). Indeed, base-line resolution of several isomers will be demonstrated that cannot be achieved on any currently available normal or reversed-phase columns, even when using recycling techniques and heavy atom impregnation. Materials. Hydrolytically stable @-cyclodextrin-packedcolumns (10 x 0.46 cm and 25 X 0.46 cm) were obtained from Advanced Separation Technologies, Inc., Whippany, NJ. These differ from previously reported cyclodextrin packings (24,25)in that there is no nitrogen (i.e., amines and/or amides) in the linkage between the cyclodextrin and the silica gel. Consequently problems associated with these earlier packings (e.g., stability, selectivity,nitroxide formation, etc.) have been avoided (26). The average diameter of the packing material was 5 pm. HPLC grade methanol, acetonitrile, and water were obtained from Burdick & Jackson (Muskegon, MI).All steroid epimers, prostaglandins, and vitamin analogues were obtained from Sigma Chemical Co. were the (St. Louis MO). cis- and trans-benzo[a]pyren-7,8-diol generous gift of H. J. Issaq (Frederich Cancer Research Facility). All other compounds were obtained from Aldrich (Milwaukee, WI). Methods. All separations were done at room temperature (20 “C) using a Shimadzu LC-4A liquid chromatograph with a variable-wavelengthdetector containing a 13-pL flow cell. All solutes were detected at 254 nm except for the estrogen and steroid epimers containing unconjugated carbonyl groups, which were detected at 280 nm. All samples were dissolved in methanol prior to injection. Unless indicated otherwise, the flow rate was 1.5 mL/min. The void volume of the column was determined by injecting either 200 pL of neat methanol or water when pumping with a mixed mobile phase. The peak-trough combination caused by the change in refractive index was used as the marker.
compound
7
8
11 12 13
14
15 16 17
18 19
phenanthrene anthracene prostaglandin A, prostaglandin B1 prostaglandin A, prostaglandin B2 vitamin Dz lumisterol previtamin D a-naphthol @-naphthol a-naphthoflavone @-naphthoflavone a-ethylphenethyl alcohol @-ethylphenethylalcohol 1,2-naphthoquinone 1,4-naphthoquinone quinoline isoquinoline o,o’-biphenyl p,p ’-biphenyl o-xylene
m-xylene p-xylene o-cresol m-cresol p-cresol o-nitrophenol m-nitrophenol p-nitrophenol o-nitroaniline m-nitroaniline p-nitroaniline o-bromobenzoic acid m-bromobenzoic acid p-bromobenzoic acid o-aminobenzoic acid m-aminobenzoic acid p-aminobenzoic acid 1-methylindole 2-methylindole 3-methylindole 5-methylindole 7-methylindole
2.6 3.4 3.8 2.6 4.6 2.0 0.4 0.7 1.2
9.0 11.5 3.4 5.3 1.7
2.0 0.7
0.9
2.2
3.1 2.5 3.8 2.3 1.9
3.5 1.3 0.6 2.4
5.9 5.4
1.4 1.1
1.6 0.8
“Column “I” is 10 cm in length and contains @-cyclodextrin packing. Column “11” is 25 cm in length and contains @cycledextrin packing. Numbers represent the volume ratio of methanollwater. CFlowrate was 1.0 mL/min (see Experimental Section). dFlow rate was 0.5 mL/min (see ExDerimental Section). only easily separated but are also very useful as an indication of cyclodextrin loading on the stationary phase (26). The greater the amount of (3-cyclodextrin bonded to the silica gel, the greater the separation between the ortho and para isomers, with the retention of p-nitroaniline increasing dramatically. When no cyclodextrin is present, the para isomer elutes first rather than last. A more complete discussion of this is given elsewhere (26). An additional advantage of the cyclodextrin column is that there is a consistency in the elution pattern of isomeric compounds which enhances the possibility of using LC to predict or verify molecular structure. For example, in naphthalene-based a- and @-substitutedcompounds, the p isomer is eluted last (Table I). This is because an a substituent sterically limits complete penetration of the cyclodextrin cavity. For disubstituted benzene compounds, the elution order is generally meta, ortho, and para (Table I). Smaller
236
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985 0
Table 11. Isocratic LC Retention Data for a Series of Diastereomeric Compounds no.
compound
k'
column" mobile phaseb
Geometric Isomers
m 1 cis-clomiphene 2
3 P
4 5
trans-clomiphene cis-stilbene trans-stilbene cis-benzo[a]pyrene7,8-diol trans-benzo[a]pyrene7.8-diol cis-3-hexen-1-01 trans-3-hexen-1-01 sy n-azobenzene anti-azobenzene
5.4 I1 65/35 3.6 7.3 I 55/45 4.5 (18) (19) (11) (111) (35165) (30170) (20) (24) (11) (111) I1
50/50
I
55/45
I
60140
I
80/20
I
65/35
I
55/45
I
55/45
I
65/35
I
65/35
5.2 4.9
I
40160
2.0
I
75/25
I
75/25
0.3 0.5 2.6 4.0 Epimers
8
0
16
RETENTION TIME, Min.
Figure 2. Isocratic separation of 0 - , m-, and p-xylene on a 25-cm /3-cyclodextrin column. The flow rate was 1.0 mL/min, and the mobile-phase composition was 40:60 (v/v) methanoVwater.
0
7
14
RETENTION TIME, Min.
Figure 3. Isocratic separation of the four epimers of estriol on a
6 estriol 16-epiestriol 17-epiestriol 16,17-espiestriol 7 testosterone 7-epitestosterone 8 17a-estradiol 17P-estradiol 9 1la-hydroxyprogesterone 116-hydroxyprogesterone 10 20a-hydroxy-4-pregnen-3-one 20P-hydroxy-4-pregnen-3-one 11 5a-androstan-3,17dione 5P-androstan-3,17dione 12 50-androstan-3P-01-17one 50-androstan-3a-01-17one 13 d-aldosterone 17-isoaldosterone 14 5P-androstan-3P-01-17one 5a-androstan-3P-01-17one 15 5a-androstan-3a-ol-17one 50-androstan-3a-01-17one
3.8 11.5 5.8 2.8
9.0 3.5 1.9 5.0 2.3 3.7 8.5 10.7 8.9 4.4 3.1 2.2
10.1 1.8
6.4
lOcm, Pcyclodextrin column. The peaks represent (from left to right) 16,17-epiestrioi, estriol, 17-epiestrioi, and 16-epiestriol. The flow rate was 1.5 mL/mln and the moblie-phase composition was 60:40 (v/v) methanoVwater.
" Column I is 10 cm in length and contains P-cyclodextrin packing. Column I1 is 25 cm in length and contains p-cyclodextrin packing. Column I11 is 10 cm in length and contains y-cyclodextrin packing. Numbers represent the volume ratio of methanolfwater.
diameter a-cyclodextrin-bondedphases produce elution orders of ortho, meta, and para (26). The stereochemical rationale for this has been discussed (20,22,23). Table I1 lists the separation conditions and retention data for several diastereomeric compounds. These include geometrical isomers (numbers 1-5, Table 11) and epimers (numbers 6-15, Table 11). All isomers can be completely resolved (i.e., R, 2 1.5) except for cis- and trans-benzo[a]pyrene-7,8diol. This compound is nearly resolved when using a 6-cyclodextrin column (R,= L4), and it is completely resolved when using the larger y-cyclodextrin column (Table 11). While some diastereomers are reasonably well separated on standard normal and reversed-phase columns (e.g., syn- and anti-azobenzene or 17a- and 176-estradiol) others are more difficult. For example, Figure 3 shows the base-line resolution of all four epimeric estriols on a 10-cm 6-cyclodextrin column. To our
knowledge this is the first time this has been accomplished although others have been able to separate two or three out of the four epimers (13). It is apparent that the P-cyclodextrin column is applicable for the separation of a greater variety of diastereomers than either normal or reversed-phase packings. Whether or not it is more useful than all the current techniques combined cannot be said at this point. Certainly the strengths and shortcomings of cyclodextrin packings will become more apparent upon further study. The ease with which cyclodextrin packings discriminate between solutes that differ only in geometry or spatial orientation is obviously due to its ability to form intimate inclusion complexes with these compounds (19-23). Different-shaped solutes form inclusion complexes of widely dif-
Anal. Chem. 1985, 57. 237-242
fering stabilities. Since traditional normal- and reversed-phase packings separate solutes by entirely different mechanisms (i.e., adsorption or partition), they cannot be expected to achieve selectivities analogous to those of cyclodextrin-bonded packings. An evaluation of the quantitative effects of solvent composition and temperature on k' and efficiency for this system will be discussed in a subsequent publication. Registry No. 0-Cyclodextrin, 7585-39-9; benzo[a]pyrene, 50-32-8; benzo[e]pyrene, 192-97-2; 1,2:3,4-dibenzanthracene, 215-58-7; 1,25,6-dibenzanthracene, 53-70-3; phenanthrene, 85-01-8; anthracene, 120-12-7;prostaglandin Al, 14152-28-4;prostaglandin B1, 13345-51-2;prostaglandin A2, 13345-50-1;prostaglandin B,, 13367-85-6;vitamin D,, 50-14-6; lumisterol, 474-69-1; previtamin D2,21307-05-1; a-naphthol, 90-15-3; @-naphthol,135-19-3;a-naphthoflavone, 604-59-1; 0-naphthoflavone, 6051-87-2; a-ethylphenethyl alcohol, 701-70-2; @-ethylphenethylalcohol, 2035-94-1; 1,2-naphthoquinone, 524-42-5; 1,4-naphthoquinone, 130-15-4; quinoline, 91-22-5; isoquinoline, 119-65-3; o-xylene, 95-47-6; m-xylene, 108-38-3;p-xylene, 106-42-3;0-cresol, 95-48-7; m-cresol, 108-39-4;p-cresol, 106-44-5;o-nitrophenol, 88-75-5; m-nitrophenol, 554-84-7; p-nitrophenol, 100-02-7; o-nitroaniline, 88-74-4; mnitroaniline, 99-09-2;p-nitroaniline,100-01-6;o-bromobenzoic acid, 88-65-3; m-bromobenzoic acid, 585-76-2; p-bromobenzoic acid, 586-76-5; o-aminobenzoic acid, 118-92-3;m-aminobenzoic acid, 99-05-8; p-aminobenzoic acid, 150-13-0;1-methylindole,603-76-9; 2-methylindole, 95-20-5; 3-methylindole, 83-34-1; 5-methylindole, 614-96-0; 7-methylindole, 933-67-5; cis-clomiphene, 15690-55-8; trans-clomiphene, 15690-57-0;cis-stilbene,645-49-8; trans-stilbene, 60657-25-2; 103-30-0; cis-7,8-dihydrobenzo[a]pyrene-7,8-diol, trans-7,8-dihydrobenzo[a]pyrene-7,8-diol, 57404-88-3;cis-3-hexsyn-azobenzene, en-l-ol,928-96-1; trans-3-hexen-l-ol,928-97-2; 1080-16-6;anti-azobenzene, 17082-12-1;estriol, 50-27-1; 16-epiestriol, 547-81-9; 17-epiestriol,1228-72-4;16,17-epiestriol,793-89-5; testosterone, 58-22-0; 17-epitestosterone,481-30-1; 17a-estradiol, 57-91-0; 17@-estradiol,50-28-2; Ha-hydroxyprogesterone,80-75-1; ll@-hydroxyprogesterone,600-57-7;20a-hydroxy-4-pregnen-3-one, 145-14-2;20@-hydroxy-4-pregnen-3-one, 145-15-3;5a-androstan3,17-dione, 846-46-8; 5@-androstan-3,17-dione,1229-12-5; 50-
237
androstan-3@-01-17-one,571-31-3; 5@-androstan-3a-ol-17-one, 53-42-9; &aldosterone, 52-39-1; 17-isoaldosterone, 13479-36-2; 5@-androstan-3@-01-17-one,571-31-3; 5a-androstan-3@-01-17-one, 481-29-8; 5a-androstan-3a-ol-17-one, 53-41-8;5O-androstan-3aol-17-one, 53-42-9; 2,2'-biphenol, 1806-29-7;4,4'-biphenol, 92-88-6.
LITERATURE CITED (1) Davankov, V. A. Adv. Chromatogr. 1080, 18, 139-195. (2) Davankov, V. A.; Kurganov, A. A; Bochkov, A. S. Adv. Chromafogr. 1080, 2 2 , 71-116. (3) Armstrong, D. W. J . Llq. Chromatogr. 1084, 7 , Suppl. 2, 353-367. (4) Houx, N. W. H.; Voerman, S. J . Chromafogr. 1078, 129, 456-459. (5) Heath, R. R.; Tumlinson, J. H.; Doolittle, R. E.; Duncan, J. H. J . Chromafogr. Scl. 1077, 15, 10-13. (6) Lam, S.; Grushka, E. J . Chromatogr. Scl. 1077, 15, 234-238. (7) Heath, R. R.; Sonnet, P. E. J . Llq. Chromatogr. 1880, 3 , 1129-1135. (8) Siouffi, A. M.; Traynard, J.-C.; Guiochon, G. J . Chromatogr. Scl. 1077, 15, 469-474. (9) Tlyoshi, T.; Kodama, M; Ito, M.; Kawamota, M.; Takahashi, K. J . Nuh. Scl. Vlfamlnol. 1077, 2 3 , 263-204. (10) Paanakker, J. E.; Groenendijk, G. W. T. J . Chromafogr. 1070, 168, 125-132. (11) E. Merck Technical Bulietln. No. 74-11, Darmstadt, Germany. (12) Redel, J. J.; Capilllon. J. "Steroid Analysis by HPLC", Kautsky, M. N., Ed.; Marcel Dekker: New York, 1981; Vol. 16, pp 343-358. (13) Lln, J.-T.; Heftmann, E. J . Chromatogr. 1081, 212, 239-244. (14) Lin, J.-T.; Heftmann. E. J . Chromatogr. 1082, 237, 215-224. (15) Lin, J.-T. Llq. Chromafogr. Mag. 1084, 2 , 135-138. (18) Ogan, K.; Katz, E. J . Chromatogr. 1080, 188, 115-127. (17) May, W. E.; Wise, S. A. Anal. Chem. 1084, 5 6 , 225-232. (18) Sander, L. C.; Wise, S. A. Anal. Chem. 1984, 56, 504-510. (19) Bender, M. L.; Komlyama, M. "Cyclodextrin Chemistry"; Springer-Verlag: Berlin, 1978. (20) Hlnze, W. L. Sep. furif. Methods 1981, 10, 159-237. (2 1) Szejtli, J. "Cyclodextrins and Their Inclusion Complexes"; Akademlai Kiado; Budapest, 1982. (22) Hinze, W. L.; Armstrong, D. W. Anal. Left. 1080, 73, 1093-1104. (23) Burkert. W. G.: Owensbv, C. N.; Hlnze, W. L. J . Lla. Chromatow. 1981, 4 , 1085-1085. (24) Fujimura, K.; Veda, T.; Ando, T. Anal. Chem. 1083, 5 5 , 446-450. (25) Kawaguchl, Y.; Tanaka, M.; Nakae, M.; Funazo, K.; Sheno, T. Anal. Chem. 1083, 55, 1852-1857. (26) Armstrong, D. W.; DeMond, W. J . Chromafogr. Sci. 1084, 2 2 , 411-415.
RECEIVED for review July 30,1984. Accepted October 9,1984.
Liquid Chromatographic Separation of Enantiomers Using a Chiral ,8-Cyclodextrin-Bonded Stationary Phase and Conventional Aqueous-Organic Mobile Phases Willie L. Hinze* and Terrence E. Riehl Department of Chemistry, Wake Forest University, Box 7486, Winston-Salem, North Carolina 27109
Daniel W. Armstrong,* Wade DeMond, Ala Alak, and Tim Ward Department of Chemistry, Texas Tech University, Lubbock, Texas 79409 A chlral statlonary phase composed of chemically bonded @-cyclodextrln (@-CD)molecules was used to separate enantiomers of dansylsulfonamlde, pnaphthamlde, or @-naphthyl ester derlvatlves of amlno aclds, barbiturates, substituted phenylacetic aclds, and dloxolanes. The separatlons are reasonably ratlonallred In terms of the lncluslon process between the enantlomers and @-cyclodextrlnand consideration of a three-polnt attachment model. The effects of mobllephase composltlon, temperature, and flow rate upon the observed enantiomeric selectivity and resolution were crltlcally assessed. Lastly, a brlef prospectus on the usefulness of cyclodextrln chlral stationary phases In high-performance liquid chromatographlc enantiomeric separations Is presented.
The liquid chromatographic separation of enantiomers is
an important and challenging task. The enantioselectivity of biological systems is well-known to most scientists and is often of paramount importance to many pharmacologists and biochemists. The determination of enantiomeric purity and the routine separation of these isomers are also of great use to many synthetic organic chemists, kineticists, and researchers interested in geochronology, etc. Traditionally, the resolution of enantiomers was a time-consuming often inefficient process that involved the use of naturally occurring optically active compounds to cocrystallize with the desired isomer. In some cases, enzymatic systems were useful as separation tools as a result of their stereospecific influence on reactivity. These more traditional techniques (despite their successes) are often considered tedious and/or not generally applicable. As a result there has been a considerable impetus toward the development of chromatography as a tool for en-
0003-2700/85/0357-0237$01.50/0 0 1984 American Chemlcal Soclety
