934
Anal. Chem. 1982, 5 4 , 934-938
solute with the stationary phase resulting in broader peaks as compared to the latter packing. Although the chromatographic performance of n-butylKel-F is presently inferior to present silica packings, some similarities such as surface area do exist between n-butyl-Kel-F and pellicular packings (24). Two major limitations in the column performance of n-butyl-Kel-F were undoubtedly the nonuniform particle distribution and the packing technique. Lately, use of a high-pressure pump has indicated derivatized 6061 Kel-F can be packed at 10000 psi. However, recent examination of derivatized Kel-F particles by light microscopy showed more fines than expected. Careful fractionation both before and after the derivatization reaction will apparently be required to achieve a narrower particle size distribution. It is expected that with the preparation of better packing materials n-butyl-Kel-F would be ideal for applications which now utilize pellicular packings such as purity verification, routine and/or rapid analyses, and initial screening of unknowns. In addition, chemically modifed Kel-F is relatively inexpensive and, therefore, would be useful in guard columns or for screening samples suspected of containing components which could irreversibly damage microparticle silica columns.
ACKNOWLEDGMENT The authors thank D. D. Anderson of the 3M Co. for the sample of 6300 Kel-F and W. Kuhn, Department of Material Science, University of Cincinnati, Cincinnati, OH, for the use of the Pilamec mill. LITERATURE CITED (1) Klrkland, J. J.; Snyder, L. R. "Introduction to Modern Llquld Chromatography", 2nd ed.; Wlley-Intersclence: New York, 1979; Chapters 5, 7. (2) Wehrll, A.; Hildenbrand, J. C.; Keller, H. P.; Stamfll. R.; Frel, R. W. J . Chromatogr. 1978, 149, 199-210. (3) Pearson, R. L.; Weiss, J. F.; Kelmers. A. D. Blochim. Biophys Acta 1971, 228, 770-774.
.
(4) Kelmers, A. D.; Heatherly, D. E. Anal. Biochem. 1971, 44, 486-495. (5) Roe, B.; Marcu, K.; Dudock, B. Biochim. Biophys. Acta 1973, 319, 25-36. (6) Slnghal, R. P. Biochim. Blophys Acta 1973, 3 79, 11-24. (7) Egan, B. 2. Biochim. Blophys Acta 1973, 299, 245-252. (8) Shum, B. W.; Crothers, D. M. Nucleic Acids Res. 1978, 5 , 2297-231 1. (9) Hardies, S. C.; Wells, R. D. R o c . Nafl. Acad. Sci. U.S.A. 1978, 73, 3117-3121. (10) Landy, A.; Foeller, C.; Reszelbach, R.; Dudock, B. Nucleic Acids Res. 1978, 3, 2575-2592. (11) Eshaghpour, H.; Crothers, D. M. Nucleic Acids Res. 1978, 5 , 13-21. (12) Blna, M.; Radonovich, M. F.; Roe, B. A. Anal. Blochem. 1981, 714, 105-1 11. (13) Usher, D. A. Nucleic Aclds Res. 1979, 6 , 2289-2306. (14) Zwier, T. A.; Burke, M. F. Anal. Chem. 1981, 53, 812-8113, (15) Plzak, 2.; Dousek, F. P.; Jansta, J. J . Chromatogr. 1978, 747, 137- 142. (16) Smolkova, E.; Zima, J.; Dousek, F. P.; Jansta, J.; Plzak, 2 . J. Chromatogr. 1980, 191, 61-69. (17) Danielson, N. D.; Taylor, R. T.; Huth, J. A,; Slerglej, R. W.; Galloway, J. G.; Paperman, J. B., Miami Universify, Oxford, OH,Sept 1981, unpublished work. (18) Scott, C. D. Anal. Biochem. 1988, 24, 292-298. (19) Wakeflekl, B. J. "The Chemistry of Organollthium Compounds"; Pergamon Press: Oxford, 1974; p 22. (20) Billmeyer, F. W., Jr. "Textbook of Polymer Science", 2nd ed.; WlleyInterscience: New York, 1971; Chapter 7. (21) Cagle, C. V. "Handbook of Adhesive Binding"; McGraw-Hill: New York, 1973; Chapter 19. (22) Deehan, D., 3M Co. Nov 1981, personal communication. (23) Saunders, K. J. "Organic Polymer Chemistry"; Chapman and Hall: London, 1973; Chapter 7. (24) Klrkland, J. J. J. Chromatogr. Sci. 1971, 9 , 206-214.
.
.
RECEIVED for review November 30,1981. Accepted February 4,1982. This work was supported in part by grants from the Faculty Research Committee of Miami University, the Research Corporation, and the donors of the Petroleum Research Fund, administered by the American Chemical Society. J. A. Huth gratefully acknowledges support provided by a Dissertation Fellowship from Miami University. This work was partially presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 1981.
Steroid Structure and Solvent Composition in Thin-Layer Chromatography Slobodan M. Petrovlb," Ljlljana A. Kolarov, and Eva S. Traljld Institute of Mlcroblologlcal Processes and Applled Chemlstry, Faculty of Technology, University of Novi Sad, V. VlahoviEa 2, 2 1000 Novi Sad, Yugoslavia
Julljana A. Petrovlb Instltute of Chemistry, Unlverslty of Novl Sad, V. VlahoviEa 2, 21000 Novi Sad, Yugoslavia
To study the reiatlons between the chemical structure of solutes and their retention behavlor In thin-layer chromatography, we determlned the retention behavior of 15 mono-, di-, tri-, and tetrasubstltuted sterold derlvatives as a function of the composition of eight binary solvent systems. The slopes and Intercepts of the linear relations between the retention constant (RM) and the logarithm of the volume fraction of the polar solvent have been calculated and dlscussed In relation to the solute and solvent characteristics. The RM and seiectivlty parameter (ARM) of steroids largely depend on the retentlon behavior of the hydroxyl group.
The effects of solvent composition on retention behavior have great theoretical and practical significance since the 0003-2700/82/0354-0934$01.25/0
mobile phases employed in both partition and adsorption chromatography are usually mixed solvents. They have therefore been of interest to chromatographers since the beginnings of chromatographic techniques. Several treatments of this subject have been applied to liquid-solid chromatography (1-7). Some of them demonstrated a linear relationship between the retention and the binary solvent properties (1-6). The treatments of Soczewinski and co-workers (2-4) are based on the concept of a competition between solute and solvent molecules in the liquid phase for a place on the adsorbent surface. For adsorption from solution as a result of the competition between the solute and an electron donor solvent (S) for the active sites on the adsorbent surface, they derived the equation.
RM
= c - n log X s
0 1982 Amerlcan Chemical Soclety
(1)
ANAL TICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982
where Xsis the mole fraction of a polar solvent component in a binary solvent mixture and c and n are constants. When eq 1was tested in a number of experiments (4,8-26) with wide range of varilous classes of compounds, linear relationships were observed in most cases. Several experiments (14, 18,27-30) indicated that the mole fraction in eq 1 can be substituted by the volume fraction, thus
R M = c - n log us
n
m
x" w
(2)
where us denotes the volume fraction of a polar solvent in a binary solvent mixture. On the basis of the work of Snyder (31) it was generally concluded that n should increase with C.
In our preliminary investigation of the retention behavior of some steroids, chromatographed on thin layers of silica with a binary ciolvent mixture of various component proportions, the data obtained indicated that slope n in eq 2 is simply the sum of particular contriibutions of steroid functional groups and skeleton and that a linear relation exists between constant c and slope n. The aim of this paper is to provide more complete experimental confirmation of these observations. Therefore, the retention characteristics of 15 steroid samples on a silica gel layers were examined as a function of the compositiion of several binary solvent systems containing the same diluting component. EXPERIMENTAL SECTION The steroids listed in Table I were chromatographed on Silica Gel 60 G (E. Merck A. G., G.F.R.) thin layers. Thirty grams of silica gel was suspended in 60 mL of distilled water, and the suspension was coated on to glass plates (20 X 20 cm) with Desaga equipment (C. Desaga GmbH, Heidelberg, G.F.R.) in a thickness of about 0.25 mm. The layers were dried in air (relativehumidity 2630%) at room temperature (about 21 "C) for about 24 h before use. After the sample solutions were spotted (0.5% of each steroid in chloroform), the layew were conditioned for 2 h in the chromatographic chamber above the solvent mixture. The chromatograms were developed by the ascending technique and the steroids were detected lay spraying with 30% sulfuric acid in methanol and heating in an oven for 10-15 min at 100--110"C. RESULTS! AND DISCUSSION The solvents used in this work were selected according to the classiification of Snyder (32): class N, benzene (BZ), chloroform (CHI,); class P, diethyl ether (DEE), ethyl acetate (EtAc), methyl acetate (MeAc), methyl ethyl ketone (MEK), acetone (An),dioxane (Dx); class AB,1-propanol (PrOH). In all binary systems benzene was used as the diluent. The solutes were mono-, di-, tri-, and tetraaubstituted steroids containing moderately polar functions, such as acyloxy, methoxy, lactone, ether, ester, keto, and hydroxyl groups. The structures of the solutes are shown in Figure 1. For reproducible experimental conditions providing reliable values for the retention characteristics, the following parameters were kept constant: preparation of layers, humidity in storage, distance of starting line from the bottom, quantity of solute, the solvent volume in the tank, conditioning time for layers, developing temperature, and the Rf range (0.1-0.9) when it was possible. For each solute and solvent combination at least five chromatograms were prepared and the Rf values were averaged. The standard deviation for a determination of Rf was 0.015. When 1 2 was ~ plotted against the logarithm of the reciprocal value of the volume fraction (l/us) of the more polar solvent component, a linear relationship with a positive slope was obtaiined for all compounds studied. The numerical data for slopes (n)and constant (c) in eq 2 for each compound and polar solvent are presented in Table I. Correlation between the Slope (n) a n d Molecular S t r u c t u r e of Steroids. From Table I it is evident that n varies with the type and number of substituents in the steroid
v
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982
936
b
0
HO
HO
6
I
11
P
0 HO
Me0
RM
7
12
1
Ac 0
HO
&
--..---
I
Me0
8
8 L
9
IL CBH!,
AcO
5
&
Me0
IO
C HOL A N A T E DERIVATIVES
15
ANDROSTENE D E R I VAT I V E S
ESTRONE D E R I VAT I V E S
Figure 1. Structural formulas of the steroids examined. For identification, see Table I.
Table 11. bn Increment of Steroid Skeleton and Substituent skeleton
An
substituent aliphatic side chain
0
-OH
1.0
> c=o
0.6
>O -0IVk
0.5 0.4
-0Ac
0.3
An
n
H
-0.3
log&
Figure 2. Plots of R , vs. logarithm of the reciprocal value of volume fraction (l/vs)of methyl ethyl ketone (a) and 1-propanol (b) in a binary solvent mixture with benzene as the diluent. Designation of samples is as in Table I.
Me0
OH
O
iog 1 VS
13
COOMe
HO
I
05
0
molecule. In the binary systems containing polar solvent of class N or P introduction of each hydroxyl group into the methyl cholanate molecule increases n by 1; the position of the additional hydroxyl group has no influence on the slope. The 3 @-hydroxylgroup of cholesterol gives a slope equal to 1 and the aliphatic side chain has no influence on the slope. In general, each polar substituent in the solutes studied influences the slope. Taking the slope of androstene and methylcholanate equal to 0, the introduction of certain functional groups in the molecules increases the slope by a value An (Table 11). In the case of estrogens, the presence of the aromatic A ring in the molecule decreases the slope by about
0.3 with respect to the corresponding steroids with an alicyclic A ring. Thus, n = An,k + Can,,, where the subscripts sk and sb stand for skeleton and substituent, respectively. Thus, on the basis of our results, we can conclude: (a) the slope (n)of particular steroids studied depends on the type and the number of substituents in the molecule and on the skeletal structure; it is the sum of the particular An values; (b) the contribution of a hydroxyl group to the slope is equal to unity; (c) the position of the substituent in the molecule of methyl cholanates has no influence on the slope; (d) binary solvent systems containing a solvent of class N or P produce the same n values for particular solutes, with deviations that fall within the experimental error. On the other hand, the binary systems containing 1propanol (class AB), as the polar component, give smaller values of n than those of class P and N (for comparison see Table I and Figure 2). The slope values in systems with 1-propanol increase in the same order as in systems with other solvents, but the contribution of substituents to the slope is not proportional to the contribution of the hydroxyl group; therefore the solute n values are not the sums of particular An values. The general conclusions of Hara and co-workers (24-26), deduced from data obtained by HPLC, are similar to those which can be drawn on the basis of our results, e.g., the slope and, of course, retention for any solvent system increases in the order: acyloxy < keto < hydroxyl group. We can supplement this sequence: acyloxy < methoxy < lactone = ether < keto < hydroxyl, but our numerical slope values are not in agreement with their (25,26)values, especially for the same binary systems and solutes. Their conclusion that n values for difunctional steroids are higher than those of monofunctional compounds but lower than the sums of the values for monofunctional compounds that contain the corresponding substiuents is not borne out by our results, except for systems with 1-propanol. Correlation between Retention and Molecular Structure of Steroids. Plotting the values of constant c for steroids examined against the corresponding slope (n) values, the
) Slopes in Figure 3 for Table 111. Retention Constants of Hydroxyl Group (AR'MoH) and Skeleton ( R o M s kand Methyl Cholanates and Other Steroids in Pure Solvents of Class N and P
methyl cholanates
CHL
DEE
EtAc
MeAc
MEK
An
0.75 0.78 -1.35 0.73
0.60
1.46 -1.63
0.95 0.97 -1.35 0.93 -1.75
-1.41 0.61
-1.81
-1.82
0.62 0.63 -1.48 0.57 -1.83
0.35 0.35 -1.39 0.31 -1.73
AR"Mo~
slope
other steroids
ROMSk
slope
RoMsk
0.62
Dx
mean value
0.20 0.20
-1.38
-1.39
0.24
-1.75
-1.76
-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982
I n METHYL ACETATE
METHYLETHYLKETONE
r---
937
Equations 3 and 4 approximately apply to any steroid studied. Of course, exceptions exist, as seen from graphs in Figure 3 and from Table I, e.g., for cholesterol, whose retention is greater than calculated from eq 3. As mentioned earlier, the position of the second hydroxyl group in methyl cholanttte does not influence the slope n (the slopes of dihydroxy clerivatives are equal), but it affects the retention. The 3a,6ladihydroxy derivative has the greatest retention due to the proximity of those functional groups causing polarization of the whole molecule. The increased distance between two hydroxyl groups in 3a,7a and 3a,12a derivatives gradually decreases the molecule polarization and, therefore, those derivatives are weakly adsorbed. The presence of the aromatic A ring in the estrogen molecule skeleton has also a negative effect on the chromatographic retention. In all polar solvents used, RM values for estrone are lower than those for dehydroepiandrosterone. This is a consequence, probably, of both the planarity and the better solubility in the diluent of the aromatic ring. Experimental results reveal that the AROMOH values decrease with increasing solvent polarity (Table 111). Certainly, the R o M = c values decrease in the same way. With respect to a particular solute, the eluotropic activity or polarity of solvents increases in the following order for any diluent/strcng solvent proportion: CHL < DEE < EtAc < MeAc = MECK < An < Dx < PrOH. In general, experimental results showed that the solute retention parallels the solute polarity for all binary mixtuires and solvents in the following order: 5 > 4 > 3 > 2 > 6 7 2 13 > 1 > 9 > 11 2 14 > 8 1 12 > 15 > 10. Some parts of this order are unexpected, e.g., in all solvent systems cholesterol was more strongly retained than estrone, although estrone is more polar; thus, the slope n of estrone is higher than of cholesterol. The exceptions were only systems with chloroform, which afforded greater retention of estrone than cholesterol. A negative effect of the aromatic A ring in the estrogen skeleton on the chromatographic retention is unexpectedly high in systems containing solvents of class P and AB. In systems with chloroform, the solubility of androskne and cholestane derivatives in the mobile phase is higher than of estrone derivatives; the retention of dehydroepiandrosterone (7) is lower than that of 3-methoxy-l7-oxa-~-homoestra1,3,5(1O)-trien-16-01(13) only in systems with chloroform. In all pure solvents 3-methoxy-17-oxa-~-homoestra-1,3,5(10)trien-16-01 (13) was more strongly adsorbed than 3-methoxy-17-oxa-D-homoestra-1,3,5(10)-triene-16-one (14), except in pure diethyl ether. In order to provide an insight into HPLC on the basis of TLC data, Hara (24) directly compared the TLC R, values and HPLC mobilities (R) of some steroids by applying the same binary solvents as mobile phases, and obtained the following relation: .Rf(TLC) X 1.5 = R(HPLC) = l/(k'+ l), where k' is capacity factor, with N + N and N + P class binary solvents. We compared our and Hara and co-workers (25, 26) c values for the same (cholesteryl acetate and cholesterol) and similar (3~-acetoxy-5a-cholestane, 5a-cholestan-36-01, dehydroepiandrosterone, dehydroepiandrosterone acetate, 3/3-hydroxy-5a-androstan-17-one, 3@-acetoxy-5a-androstan17-one) steroids and same solvents (DEE, EtAc, An, Dx) and found that R(HPLC) = 0.95R,(TLC) = R,(TLC), which is in agreement with the calculations of Soczewinski and Golkiewics (33). One of the essential differences between TLC and column techniques is the fact that TLC is an open three-phase system (solid-liquid-gaseous). During conditioning of silica layers probably similar conditions as in HPLC are attainled. Therefore a solute retention in both techniques is similar. On the other hand, silica used for HPLC in Hara experiments (24) was previously deactivated by equilibration with ambient b:
L-A
oL---J 1
n
2
Figure 3. Relationship between R o M= c and slope n values (Table
I) of methyl cholanates (upper line) and androstenes and estrones (lower line). Designation of samples is as in Table I.
graphs presented in Figure 3 were obtained. The slopes of straight lines in Figure 3 for particular solvents of class P are actually equal to the retention constants of the hydroxyl group, AROMOH, calculated as mean values of constant c differences between methyl litocholate, methyl chemodeoxycholate, and methyl cholate (Table 111). Because the constant c is equal to the retention constant of a solute in pure solvent, ROM,the linear relation between c and n can be expressed, for solvent systems containing benzene as diluent, by R o M = n m o M O H "t RoMvIsk
(3) where RolMsk is the retention constant in pure solvent of methyl cholanato (upper line) and of androstene and 1,3,5(10)-estratriene (lower line), elqual to the corresponding axis intercepts in Figure 3 (see Table 111). Introducing eq 3 into eq 2 we obtain
RM = n(&'"~oa
log Us) + R o M s k
(4)
938
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982
LITERATURE CITED
Table IV. Experimental and Calculated (Eq 5 ) A R M Valuesa converted functional group
ARM
An
-OH
sol- ref ventb 41
I
0.88
I1
1.09 0.36 0.22
I
=O
Oe6
I1
= O -+ -OH
I
0.15
Oo4
I1
=O + -OH
0'4
I1
0.37 0.37 0.40 0.37
,
T\TT
I I
our data
calcd data
0.91 1.09
0.90 1.05 0.54 0.63 0.36 0.42 0.36 0.42 0.63
0.49 0.43 0.34 0.44 0.34 0.44 0.60
I 0.08 0.28 0.54 Oa6 I1 0.27 0.40 0.63 a AR"MoH(EtAc) = 0.75, AR"MoH(An) = 0.35. Solvent: (I)benzene-ethyl acetate (3:7);(11)benzeneacetone (4:l). moisture. It is well-known that deactivation of silica decreases the retention; therefore Rf(TLC) X 1.5 x R(HPLC). The HPLC selectivity parameter CY = k,'/k,' (34) corresponds in TLC to A R M = log (k,'/k,'). Several authors (35-41) have used this value in correlations with steroid structures, mainly for the evaluation of the contribution of a functional group and the selectivity of developing solvents. From eq 4, it is evident that
where 1 and 2 denote compounds which differ in molecular structure. In the derivation of the above equation we assumed the RoWk value to be constant. Although RoMsk values for esters of bile acids are different from those of other steroids studied (Table 111),when they are based on R, values the difference is about 0.02,a value very close to the error in a determination of Rp Comparison of our experimental TLC A R M data with those of Hara and Mibe (41) for the same solvent systems and with those calculated from eq 5 (Table IV) shows a good agreement between calculated and experimental A R M values.
(1) Snyder, L. R. "Principles of Adsorption Chromatography"; Marcel Dekker: New York. 1968. (2) Soczewinski, E. Anal. Chem. 1969,41, 179. (3) Soczewinski, E.; Golkiewicz, W. Chromatographia 1971,4 , 501. (4) Soczewinski, E.; Golkiewicz, W.; Szumilo, H. J . Chromatogr. 1969, 45, 1. (5) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1975, 172,425. (6) Scott, R. P. W. J . Chromatogr. 1976, 722.35. (7) Oscik, J.; Rozylo, J. K. Chromatographla 1971,4 , 516. ( 8 ) Gaiik, A. Anal. Chlm. Acta 1971,5 7 , 399. (9) Soczewinski, E.; Golkiewlcz, W. Chromatographla 1972,5, 431. (IO) Goklewicz, W,; Soczewinski, E. Chromatographia 1972,5, 594. (11) Soczewlnski, E.; Szumilo, H. J. Chromatogr. 1973,81, 99. (12) Soczewinski, E.; Golklewlcz, W. Chromatographla 1973,6 , 269. (13) Gallk, A. Anal. Chlm. Acta 1973,6 7 , 357. (14) Jandera, P.; ChuraEek, J. J . Chromatogr. 1974,93, 17. (15) Szumilo, H.; Soczewinski, E. J. Chromatogr. 1974,9 4 , 219. (16) Soczewinski, E.; Szumilo, H. J. Chromatogr. 1974,94, 229. (17) Soczewinskl, E.; Golkiewicz, W.; Markowski, W. Chromatographia 1975,8,13. (18) Jandera, P.; Janderova, M.; ChuraEek, J. J. Chromatogr. 1975, 115, 9. (19) Goikiewicz, W. Chromatographla 1976,9 , 113. (20) Soczewinski, E.; Dzldo, T.; Golkiewicz, W. Chromatographb 1977, 10, 298. (21) Gazda, K. J. Chromatogr. 1977, 131, 408. (22) Soczewinski, E.; Golkiewicz, W.; Dzido, T. Chromatographb 1977, 70, 221. (23) Wawrzynowicz, T.; Dzido, T. Chromatographla 1978, 1 1 , 335. (24) Hara, S. J. Chromatogr. 1977, 137, 41. (25) Hara, S.;Fuji, Y.; Hirasawa, M.: Miyamoto, S. J. Chromatogr. 1978, 749, 143. (26) Hara, S.; Ohlsawa, A. J. Chromatogr. 1980,200,85. (27) SoczewinskivE. J. Chromatogr. 1977, 130, 23. (28) Jandera, P.; Janderova, M.; ChuraEek, J. J. J. Chromatogr. 1978, 748, 79. (29) Soczewinski, E.; Kuczmierczyk, J. J. Chromatogr. 1978, 150, 53. (30) Soczewlnskl, E. Chromatographla 1978, 11 , 534. (31) Synder, L. R. I n "Chromatography", 2nd ed.; Heftmann, E., Ed.; Reinhold: New York, 1967; p 43. (32) Snyder, L. R.; Poppe, H. J. Chromatogr. 1960, 784, 363. (33) Soczewinskl, E.; Goiklewicz, W. J. Chromatogr. 1976, 118, 91. (34) Karger, 6. I n "Modern Practice of Liquid Chromatography"; Kirkland, J. J., Ed.; Wiley: New York, 1971. (35) Bush, I. E. "The Chromatography of Steroids"; Pergamon Press: Oxford, 1961. (36) Neher, R. "Steroid Chromatography"; Elsevier: Amsterdam, 1964. (37) Cathro, D. M.; Cameron, J.; Birchall, K. J. Chromatogr. 1965, 17, 362. (38) Lisboa, 6 . P. J. Chromatogr. 1964, 13, 391. (39) Lisboa, 6. P. Steroids 1965,6 , 605. (40) Lisbora, 6. P. J. Chromatogr. 1965, 19, 81. (41) Hara. S.; Mibe, K. Chem. Pharm. Bull. 1975, 23, 2850.
RECEIVED for review October 16,1981.Accepted January 19, 1982.
