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ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
(IO) D. J. Byrd, W. Kochen, D. Idzko, and E. Knorr, J . Chromatogr., 94,85 (1974). (11) G. H. Schenk. G. L. Sullivan, and P. A. Fryer, J . Chromatogr., 89,49 (1974). (12) 0.Hutzinger, J . Chromatogr., 40, 117 (1969). (13) 1. K. Genefke, Acta Pharmacol. Toxicol., 31, 554 (1972). (14) H. T. Gordon and M. J. Huraux, Anal. Chem., 31, 302 (1959). (15) J. M. Feldman, S. S. Butler, and B. A. Chapman, Clin. Chem. (Winston-Salem, N . C . ) , 20,607 (1974). (16) J. J. Vallon. A. Badinand, and C. Bichon, Ann. Biol. Clin. (Paris), 32, 359 (1974). (17) A. P. Graffeo and B. L. Karger, Clin. Chem. ( Winston-Salem, N.C.), 22, 184 (1976). (18) J. P. Sharma, E. G. Perkins, and R. F. Bevill, J . Chromatogr., 134,441 (1977). (19) F. J. Scandrett, Lancet, 270, 967 (1956). (20) J. Porath and N. Fornstedt, J . Chromatogr., 51, 479 (1970)
(21) R. Axdn, H. Drevin, and J. Carlsson, Acta Chem. Scand. Ser. B , 29, 471 (1975). (22) J. Porath and K. Dahlgren Caldwell, J , Chromatogr., 133, 180 (1977). (23) N. Fornstedt, Department of Clinical Chemisw, University Hospital, Uppsah, Sweden, unpublished work, 1977. (24) D. D. Chilcote and J. E. Mrochek, Clin. Chem. ( Winston-Saiem, N.C.), 18,778 (1972). (25) E. Grushka and E. J. Kikta Jr., J . Chromatogr., 143,51 (1977). (26) J. Jurand, in “High Pressure Liquid Chromatography in Clinical Chemistr”, P. F. Dixon, C. H. Gray, C. K.Lim, and M. S. Stoll, Ed., Academic Press, New York, N.Y., 1976,pp 125-130.
RECEIVED for review December 12, 1977. Accepted May 2 , 1978. This work was supported by a grant from the Swedish Natural Science Research Council (K-0220-045).
High Performance Liquid Chromatography with Metal-Solute Complexes Francis K. Chow and Eli Grushka” Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 74274
Cu( 11) was coordinated to two ligands, dithiocarbamate and diketone, which were chemically bonded to silica gel. Twenty-two aromatic amines were used as test solutes to study the chromatographic behavior of the copper-loaded packings. Both systems proved to be highly selective and stable. The presence of Cu( 11) was found essential for separation to occur. The dependence of capacity ratios on the concentration of methanol modifier in the mobile phase suggests that the modifier affects the retention by competing for the solutes, and by changing the amount of Cu on the column. Optimum concentration of methanol in the mobile phase allows the separation of 12 aromatic amines in less than 13 min. The effect of Cu(I1) concentration in the mobile phase on the capacity ratios was also studied.
T h e use of metal ions in high pressure liquid chromatography for the enhancement of the resolution is drawing more and more attention. For example, silver ions have been used previously for the separation of unsaturated compounds (see ref. 1 and citations therein). Although in most cases the solid support was impregnated with silver ions, the use of Ag(1) in the mobile phase has also been reported (viz., 2, 3). Cations other than silver have been used in ligand-exchange chromatography to achieve selective separation (viz., 4 4 ) . Kunzru and Frei ( 7 ) have utilized silica impregnated with Cd(I1) for t h e separation of some aromatic amines. Yasuda (8, 9) has also separated aromatic amines on TLC plates loaded with metal ions. Sternsen and DeWitte ( 1 0 , I I ) have used Ni(I1) in t h e mobile phase t o facilitate t h e separation of amino phenols. Previous works have indicated the usefulness of metal ions in controlling solute retention behaviors and in improving the selectivity of chromatographic systems. T o capitalize on the advantage offered by such systems, Chow and Gruskha (12) have recently discussed the possibility of using metal ions which are bonded via a ligand to the support particles. Cooke e t al. (13)have also discussed such an approach. In addition, 0003-2700/78/0350-1346$01.OO/O
Cooke et al. have described the use of metal ions coordinated to an hydrophobic chelating agent in t h e mobile phases in conjunction with a reversed phase packing. In our previous paper (12),propylamine covalently bonded to silica gel was used to retain Cu(I1) as part of the stationary phase. The potential of such a chromatographic approach in achieving selective separations was clearly demonstrated. However, due to the fact that the alkylamine has only one site which can coordinate the metal ion, Cu(I1) can be leached from the support rather easily. To overcome this difficulty, a bidentate bonded ligand can be used t o ensure a stronger attachment of the metal ion. The present work describes two such bidentate chelating agents: (1) a dithiocarbamate and (2) a diketone. Both of these ligands have been studied extensively in the extraction of metal ions, and both can complex Cu(I1) quite strongly. Therefore these chelating agents, when covalently bonded to the support, should retain Cu(I1) and provide a stable and selective system for certain chromatographic separations. It might be anticipated t h a t the retention behavior would also be affected by the presence of the metal ion in the mobile phase, (Le,, over and above the metal ion in the stationary phase). One of the aims of this study was to determine t h e selectivity of the bonded-Cu(I1) columns with excess copper in the mobile phase. An additional benefit of such a study is in investigating the stability of t h e copper columns with and without copper ions in t h e mobile phase.
EXPERIMENTAL Apparatus. The liquid chromatograph consisted of an Altex Model 110 solvent metering pump (Berkeley, Calif.), and LDC (Riviera Beach, Fla.) UV detector model 1285 (254 nm) and a Rheodyne (Berkeley, Calif.) model 7120 injection valve. Columns were made of 316 stainless steel, 25 cm X 0.31 cm i.d. and 20 cm X 0.31 cm for the “Dithiocarbamate” and the “Diketone” columns, respectively. Reagents. Partisil-10 porous silica support was obtained from Whatman Inc., (Clifton, N.J.). y-Aminopropyltrimethoxysilane was purchased from Silar Labs (Scotia, N.Y.). Ethyl benzoylacetate, fluorenes, and fluorenones were bought from Aldrich Chemical Co. (Metuchen, N.J.). All other amines were obtained ‘C 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
Table I. Table of Solutes Tested compound 1. naphthalene 2. 1-aminonaphthalene 2-aminonaphthalene 3. 1-aminoanthracene 4. 2-aminoan thracene 5. 6. 2-amino-9-fluorenone 4-amino-9-fluorenone 7. 2-amino-9-hydroxyfluorene 8. 9-fluorenone 9. fluorene 10. 11. 2-aminofluorene o-chloroaniline 12. rn-chloroaniline 13. p-chloroaniline 14. o-nitroaniline 15. rn-nitroaniline 16. p-nitroaniline 17. o-Toluidine 18. rn-Toluidine 19. p-Toluidine 20. 0-Anisidine 21. p-Anisidine 22.
symbol N 1N 2N 1A 2A 2,9 4,9 2AOH 9F F 2AF OCA mCA PCA ONA mNA PNA OT mT PT OA PA
from various other sources. The solutes used are shown in Table I. Methanol was distilled before use; all other solvents were reagent grade and used without further purification. Procedure. (I) Preparation of the alkylamine bonded phase: 30 mL of 10% y-aminopropyltrimethoxysilanein dry toluene was added to 5.0 g of Partisil-10 which had been vacuum dried at 150 O C for 6 h. The mixture was then refluxed under anhydrous conditions for 6 h. It was then washed with dry toluene, isopropanol, acetone, methanol, and acetone again, and vacuum dried a t 100 “C for 1 h. CHN analysis showed 2.9 pmol/m2 surface coverage as calculated by the method suggested by Unger (8). N analysis was used for the calculation. (11) Preparation of the “Dithiocarbamate” System: 2.5 g of the alkylamine-bonded Partisil-10 prepared previously were added to 10 mL dry toluene, 2 mL isopropanol, 2 mL carbon disulfide, and 1 mL of 10% methanolic solution of ammonium hydroxide. The mixture was stirred for 20 min a t room temperature as suggested by Leyden and Luttrel (14). The reaction is as follows:
The presence of carbon disulfide in the bonded phase was tested qualitatively by adding concentrated hydrochloric acid to some of the packing (15). (111)Preparation of the “Diketone” System: 2.5 g of previously bonded-amine Partisil-10 were added to 30 mL of 10% ethyl benzoylacetate solution in dry toluene. The mixture was refluxed for 6 h. It was expected that the reaction will yield the 0-diketone, compound 1. However NMR studies of the reaction between an alkyl amine and ethyl benzoylacetate indicated that most likely compound 2 is also present on the silica gel:
~
i s 0 s ICH,),y
1
,
0
o,u~~”2~ocA
($ =Pclenyl
CHN analysis showed that about 50% of the amine sites have reacted with the reagents. Both 1 and 2 can coordinate Cu(I1). Although the reactions between ethyl benzoylacetate and the bonded propylamine did not yield just the expected diketone (see
1347
reaction scheme), that column will be referred to as the “diketone” column. (IV) Chromatographic Studies: h’vdue was collected before loading the columns with Cu(I1). Then a 5.0 x M CuS04 solution in methanol was passed through the columns in order to load them with copper. h’value was collected again with the same batches of mobile phases. The mobile phases used are: (1) 20% v/v cyclohexane in methanol (mobile phase A) without and with Cu2+, and (2) a mixture of hexane:methylene chloride: methanol having the ratio 7:1:0.5 (mobile phase B). In one study, the relative ratios of hexane to methanol were changed. Columns were washed with HC1 solution (pH -1.5) and the concentration of Cu(I1) was determined by titration as discussed by Belcher and Nutten (16). Mobile phases were tested in an increasing order of polarity. Hold-up times were measured by injecting 10 pL of a solvent having a slightly different composition than that of the mobile phases. The flow rate used throughout, the whole study was 1.0 mL/min.
RESULTS AND DISCUSSIONS T h e concentration of complexed Cu(I1) on the “dithiocarbamate” and “diketone” columns was found t o be 0.35 pmol/m2 and 0.42 pmol/m2, respectively. These values were obtained after the columns were equilibrated with methanol, and in essence they represent the lower limit of copper loading. Our studies show, as will be discussed later, that mobile phases lean in methanol allowed larger amounts of Cu(I1) to be retained on the column. The amount of Cu(I1) bonded to the support in the present study is about 1.5 to 2 times larger than t h a t reported by us previously. Still, this amount of Cu(I1) is small compared to t h e total number of sites available for bonding. Several solutions differing in the concentration of Cu(I1) were used to bond copper to t h e support; yet the capacity ratios of the solutes studied here remained constant when the CuS04 concentration in the loading solution was above about 5 X M. Table I1 shows the k’values of 22 solutes on the “diketone” and t h e dithiocarbamate columns with and without Cu(II), using mobile phases A and B. With t h e dithiocarbamateCu(I1) column, mobile phase A contained CuS04 (2.2 X lo4
MI. The capacity ratios of all the solutes on both columns were very small when Cu(I1) was not present and mobile phase A was used. The presence of copper on the stationary phase causes longer retention for all but a few solutes. Those solutes which do not have an amine group, naphthalene, 9-fluorenone and fluorene, eluted faster on t h e bonded copper columns when mobile phase A was used. The complexed Cu(I1) seems to prevent the interaction between the above three solutes and the stationary phases. The increase in the capacity ratio values of the less basic aniline isomers (e.g., o-chloroaniline) in the presence of Cu(I1) was rather small. Using mobile phase A, separations not possible on t h e diketone or t h e dithiocarbamate columns alone can be achieved when Cu(I1) is added to the support. As a n example, Table 111 shows some selectivity factors, a , using mobile phase A on both columns with and without Cu(I1). T h e retention behavior is different when a mobile phase “lean” in methanol is used: see Table 11, mobile phase B. With such a mobile phase, the separations of some of t h e aromatic amines are possible even without t h e presence of Cu(I1); (e.g., nitroanilines, or aminofluorenones). Still, many solutes are not separated; e.g., the aminonaphthalenes or the toluidines. With the copper bonded phase the capacity ratios and the selectivity factors, a , increase, as illustrated in Table 111. Even though the capacity ratios are much larger when mobile phase B is used, in general t’he CY values do not differ greatly between the mobile phases. However, because of the very rapid elution of solutes with mobile phase A, errors in measuring k’ values, and hence, CY, can be significant.
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
Table 11. k' Values with and without Cu(I1) Bonded t o t h e Support
1.
2. 3. 4. 5. 6. 7. 8.
9.
solutes N IN 2N 1A 2A 29 49 2AOH 9F
dithiocarbamate column no Cu(I1) Cu(I1) bonded mobile mobile mobile mobile phase AC phase BC phase A' phase B 0.40 0.40 0.38 0.38 0.44 0.44 0.21
1.60 1.60 1.78 1.86 2.71 3.48 6.73
0.36 0.20 0.36 0.04 0.40 1.35 12. OCA 0.28 0.73 13. mCA 0.28 1.40 14. PCA 0.28 1.40 15. ONA 0.34 2.13 16. mNA 0.35 3.53 17. PNA 0.35 7.53 18. OT 0.20 0.67 19. mT 0.20 0.87 20. PT 0.20 0.87 21. OA 0.20 0.77 22. PA 0.20 1.48 'Mobile phase A contained 2.2 X MCuSO,. Hexane :methylenechloride :methanol) (7 :1:0.5). 10. 11.
F 2AF
diketone column n o Cu(I1) Cu(11) bonded mobile mobile mobile mobile phase A phase B phase A phase B 0.89 0.7 0.11 0.56 0.90 2.78 0.36 0.89 0.17 4.16 1.94 7.36 0.73 0.89 0.17 9.58 1.11 1.06 2.39 0.18 0.46 5.08 1.84 0.18 3.39 9.32 1.32 0.79 0.97 0.45 0.18 1.60 6.24 7.46 0.39 0.25 0.18 1.72 3.78 4.34 2.68 b 38.7 0.82 0.086 2.42 0.16 0.03 0.11 0.18 0.56 0.36 0.16 0.00 0.11 0.18 0.14 0.22 8.06 2.74 13.6 1.31 0.18 0.92 0.26 0.11 1.40 0.85 0.17 0.58 0.71 1.00 3.70 0.17 0.25 4.76 1.29 1.00 0.44 7.88 6.85 0.17 0.31 0.21 2.84 2.58 1.53 0.11 6.67 0.53 0.25 6.68 2.28 0.14 11.8 10.7 0.14 0.42 0.21 9.88 0.84 2.06 3.12 0.34 0.53 0.027 1.61 5.70 7.56 0.71 0.53 0.027 9.94 12.36 1.04 2.29 0.53 0.027 2.63 4.44 0.45 0.50 0.070 0.87 b 33.6 2.07 0.84 3.45 0.070 Retention too long. A = 20% v/v cyclohexane in methanol. B =
Table 111. Comparison of Selectivity Factors, a , of Some Solutes with and without Cu(I1) Bonded to Stationary Phases mobile phase B __ k ' 2N
Column dithiocarbamate column "diketone" column
k ' IN 1.00 1.00
dithiocarbamate-Cu(I1) column 2.65 "diketone"-Cu(I1) column 2.30 ' The mobile phase contained 2.2 x lo-, M CuSO,.
k'2.9
k'PCA
k'2N -
k'2,9 -
k ' PCA
k'4,9
ktmCA
k'lN
k'4,9
k'mCA
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.85 1.65
2.16' 2.03
2.4ga 1.80
1.82' 1.76
1.28 (k'a
mobile phase A
Jk'2,9)
1.0s (k'4,Jk'2,9)
1.65 1.72
A and B same as Table 11.
Some retention reversal should be noted. Without copper, 2-amino-9-fluorenone elutes before the 4-amino isomer. When Cu(I1) is bonded to either ligand, t h e 2-amino isomer is retained longer than t h e 4-amino isomers. T h e interaction between the Cu(1I) and the 2-amino isomer is stronger than the interaction with the 4-amino isomer. It might be possible t h a t the 2-amino isomer can form a bidentate complex with t h e bonded copper whereas the 4-amino isomer cannot. The retention order of the nitroanilines with mobile phase A as compared to B is also of interest. The meta nitroaniline isomer is t h e most basic of t h e three, and with the Cu(I1) columns a n d mobile phase A, this isomer elutes last among t h e three. With mobile phase B, the retention order, which depends most likely on the solubility, is ortho < meta < para, irrespective whether or not Cu(I1) is present in the stationary phase. The basicity influence on the retention is still evident, however, and the capacity ratio of rn-nitroaniline shows largest change when t h e Cu(I1) is introduced to t h e support. Figure 1 shows t h e separation of several solutes on the diketone column without copper using mobile Phase B. Figure 2 is a chromatogram of the same solutes under the same conditions, with t h e exception t h a t Cu(I1) is bonded t o the diketone. The solute 2-amino-9-hydroxyfluorene,not shown in t h e figure, elutes after about 30 min. With the Cu(I1) column, 2-aminofluorene elutes after 2-amino-9-fluorenonone and 4-amino-9-fluorenone, and as mentioned before, t h e
Table IV. Comparison of Plate Height (mm) of Two Solutes for Columns with and without Cu(I1) in the Stationary Phase. Mobile-Phase ' B solutes
a
column dithiocarbamate column "diketone" column dithiocarbamate column-Cu(I1) " diketone" colu mn-Cu( I1 ) Conditions same as Table I.
1N 0.15 0.21 0.31 0.28
2,9 0.17 0.23 0.58 0.31
elution of the 2-amino and the 4-amino is the reverse of that shown in Figure 1. Apparently, the ketone group of the phenone delocalizes t h e electrons in t h e aromatic rings. Consequently the aminofluorene is a stronger base than t h e two aminofluorenones, and forms a stronger complex with the bonded copper. The aminohydroxyfluorene can form a strong bidentate complex with the copper and it is therefore retained t h e most. Although t h e selectivity is higher on t h e Cu(I1)loaded column, the efficiency, especially as measured from the more retained solutes, is decreased. This decrease presumably is due to t h e slow dissociation of the solute-Cu(I1) complex (12, 13). Table IV shows the plate heights for the columns with and without copper, measured with l-aminonaphthalene and 2-amino-9-fluorenone. Future research
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
Figure 1. Separation of some fluorenes and fluorenones. No Cu(I1) in stationary phase. Mobile phase: Hexane/methylenechlorkIe/mehnol (7/1/0.5). Flow rate: 1.0 mL/min. Diketone column. (1) fluorene, (2) 9-fluorenone, (3) 4-amino-9-fluorenone, (4) 2-amino-9-fluorenone, (5) 2-arninofiuorene, (6) 2-amino-9-hydroxylfluorene
2 1
4
n 5
Figure 2. Separation of some fluorenes and fluorenones. Cu(I1) bonded to support. Mobile phase: Hexane/methylene chloride/methanol (7/1/0.5). Flow rate: 1.0 mL/min. Diketone column. Solutes are the same as in Figure 1
should be directed a t improving the plate height of the bonded-metal system. Figure 3 shows a chromatogram of the aminonaphthalenes on a copper column. Although the efficiency is rather poor for present day LC technology, the selectivity is so large that the column can be overloaded and used as a preparative one for the separation of these isomers. The same can be said about the separation of various substituted anilines. Some band tailing was observed in the Cu(I1)-loaded column. This tailing might be due to the slow rate of dissociation and association of the Cu(I1)-amine complexes. Effect of Cu(I1) Ions in Mobile Phase. Metal cations in the mobile phase, in addition to those in the stationary phase, can have a pronounced effect on the retention behavior and on the separation. Table V shows that the capacity ratios, in general, increase with the concentration of Cu(I1) in the mobile phase. Cooke et al. (13) found that with Zn(I1) in the mobile phase above a certain concentration, the capacity ratios decrease with further increase of the metal ion concentration. Unfortunately in the present study, higher concentration of
(MiN.)
IO
8
6
4
2
1349
0
Figure 3. Separation of isomeric aminonaphthalenes on the diketone column with bonded Cu(I1). Mobile phase: Hexane/methylene chloride/methanol (7/1/0.5). Flow rate: 1.O mL/min. (1) naphthalene, (2) 1-aminonaphthalene, (3) 2-aminonaphthalene
Table V. Effect of Concentration of Cu(I1) in Mobile Phase A. Diketone-Cu(1i) Column (Flow Rate, 1.0 mL/min) 5.5 x 2.2 x 10-4 M 10-4 M CUZ+ no. Cu(I1) CuZ+ solutes 0.14 N 0.11 0.3.0 0.83 0.36 0.61 1N 2.14 0.73 1.51 2N 0.97 1A 0.46 0.75 2.09 0.79 1.49 2A 1.02 0.45 0.78 29 0.30 0.25 0.27 49 2.11 0.82 1.63 2A04 0.083 0.11 0.10 9F 0.083 F 0.11 0.1.0 3.05 1.31 2.43 2AF 0.23 0.11 0.13 OCA 0.77 0.25 0.64 mCA 1.49 PCA 0.44 1.01 0.26 ONA 0.21 0.10 0.43 0.25 0.27 mNA 0.26 PNA 0.21 0.10 0.89 0.34 0.158 OT 1.97 mT 0.71 1.43 2.89 1.04 2.19 PT 1.14 0.45 0.81 OA 4.71 2.07 4.31 PA Cu(I1) could not be used because of strong UV absorbance at 254 nm. The increase in the retention time probably is due to the fact that Cu(I1) from the mobile phase is being taken up by the support, resulting in a greater probability of forming solute-bonded copper complex. Complexation with the bonded Cu is more favorable (or more probable) than with the metal ion in the mobile phase. Figure 4 shows that effect of Cu(I1) in the mobile phase on the retention times and the separation of the aminonaphthalenes. In general, k’values increased with concentration of Cu(I1) a t least in the concentration range we studied. Effect of Methanol Concentration. Table I1 shows that the concentration of methanol in the mobile phase affects
1350
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978 I
Table VI. Effect of Amount of Methanol in Mobile Phase" on 12' Values h' values dike tone column-Cu(I1)
dithiocarbamate column-Cu(I1)
B B, B, B3 B, B B, B, B3 B4 B5 0.25 0.27 0.11 0.56 0.17 1N 4.16 2.07 1.56 1.40 0.50 2.78 1.93 1.54 1.30 0.70 0.33 9.58 5.42 3.39 3.40 2N 1.22 7.36 4.30 3.88 3.31 1.60 0.79 1A 5.08 2.61 1.60 .67 2.39 1.34 1.14 0.79 0.45 9.32 5.36 3.27 1.22 3.39 1.78 1.57 1.49 0.80 2A 2.24 1.83 0.58 6.24 2.89 2.37 1.70 0.81 0.47 29 7.46 3.48 1.30 0.86 0.27 3.78 1.62 1.41 0.93 0.41 0.24 4.34 1.50 49 6.16 5.40 1.19 14.80 11.03 6.81 2.29 1.06 2AOH 38.6 11.40 9F 0.56 0.38 0.45 0.27 0.083 0.03 0.32 0.072 0.30 0.16 0.076 F 0.30 0.27 0.083 0.00 0.14 0.072 0.30 0.16 0.076 0.22 0.23 2AF 13.6 8.60 4.98 5.07 2.17 8.06 5.76 5.20 4.70 2.23 1.30 0.82 0.47 0.083 0.85 0.74 0.67 0.54 0.27 0.11 OCA 1.40 0.65 4.76 2.13 1.60 1.33 mCA 0.44 3.70 2.35 1.93 1.53 0.71 0.68 2.51 2.43 0.78 6.85 4.00 3.31 2.73 1.25 1.01 PC A 7.88 3.94 0.88 0.63 0.17 2.58 1.32 1.17 0.81 0.33 0.17 2.84 1.03 ON A 6.68 2.39 1.67 1.21 0.22 6.67 2.80 2.28 1.62 0.61 0.29 mNA PN A 1.67 0.97 0.22 11.8 3.54 3.01 1.62 0.61 0.29 8.84 2.39 OT 3.12 1.75 1.33 1.17 0.37 2.06 1.46 1.36 1.14 0.59 0.25 3.05 2.85 0.94 5.70 3.58 3.35 2.79 1.47 0.74 mT 7.56 4.44 4.88 4.67 1.41 9 94 6.25 5.61 4.69 2.40 1 24 12.36 6.78 PT 0.22 1.60 0.57 2.63 1.64 1.41 1.23 0.70 0.29 OA 4.44 2.10 1.02 11.27 2.77 18.86 16.04 12.40 5.07 2.37 PA 33.6 " Mobile phase: hexanelmethylene chloride/methanol: 700/100/50, B; 600/100/150, B , ; 550/100/200, B,; 450/100/ 300, B,; 200/100/550, B,; 50/100/700, B,.
solutes N
,
3 r2
i 2
3
h
i j ;
. OL.-
0
~
2
3
4
5
6
7
8
9
20
LOGiMeOH]
min
Flgure 4. Separation of isomeric aminonaphthalenes on diketone-Cu(I1) column with various Cu(I1) concentrations in mobile phase A. Flow rate: 1.O mL/min. Chromatogram A: 20 % cyclohexane/methanol M Cu(I1). Chromatogram 6: 20% cyclohexane/ with 5 . 5 X M Cu(I1). Chromatogram C: 20% cymethanol with 2.2 X clohexane/methanol with no Cu(I1). (1) naphthalene, (2) l-aminonaphthalene, (3) 2-aminonaphthalene
Figure 5. Correlation of log [methanol] to log k'for some fluorenes and fluorenones. Diketone column loaded with Cu(I1). (A) 4-amino9-fluorenone, (B) 2-amin@9-fluorenone,(C) 2-amino-9-hydroxyfluorene. (D) 2-aminofluorene
Bl
12.
greatly the retention times of the solutes. T o better understand the effect of methanol, a series of mobile phases made of various ratios of hexane:methylene ch1oride:methanol were prepared. Table VI shows the capacity ratios of the solutes on t h e two Cu(I1)-loaded columns with various amounts of methanol. As expected, the h'values decreased as the amount of methanol is increased. This decrease, however, is not linear. Figures 5 and 6 show plots of log h'vs. log of the concentration of MeOH. For the sake of clarity, the behavior of only four solutes is shown in the figures. Increasing the methanol content of the mobile phase changes t h e capacity ratios via two mechanisms: (1) better solvation of the solutes and (2) leaching out some of the bonded copper (This effect will be discussed shortly). T h e change in the retention of the amino-9-fluorenones and the amino-9-hydroxyfluorene seems to
a1
c!
~2
3
4
5
0
7
8
9
-~~ 0
1
-3
-35 'Vecy'
Figure 6. Correlation of log [methanol] to log k'for some fluorenes and fluorenones Dithiocarbamatecolumn loaded with Cu(I1) Solutes are the same as in Figure 5
~
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
1351
1
,& Figure 7.
Separation of some fluorenes and fluorenones in a methanol-rich mobile phase. Mobile phase: Hexane/methylene chloride/methanol (21115.5). Flow rate: 1.O mL/min. Diketone-Cu(1I) column. Solutes are the same as in Figure 1
be the same, on each column, as the concentration of methanol is increased from -0.2 M to about 0.9 M. The change in k ' of the aminofluorene is less than that of the other solutes, as judged by the slope of the plots of Figures 5 and 6. Aminofluorene has only one site which can hydrogen bond with methanol, and perhaps the major cause for the decrease in the retention of that solute is due to the loss of copper from the column as the alcohol content is increased. The other aromatic amines can form H-bonds with the methanol, and solvation by the polar modifier might be the major contribution to the decrease in the k'values. Once the amount of methanol in the mobile phase exceeds a certain limit, the change in the k ' values is more pronounced, especially with the amino-9-hydroxyfluorene.As can be seen in Figures 5 and 6 and in Table VI, reversal in some retention order occurs. T h e increase in the rate of change of the capacity factors is probably a result of the greater extent of Cu(I1) stripping from the column by the higher concentration of methanol in the mobile phase. Comparison of chromatograms shown in Figure 2 and 7 presents a graphical illustration of the effect of the methanol. The mixture and the column are the same in either case. However, in the case of Figure 7 , the mobile phase is richer in methanol. Under these conditions, the amino-9-hydroxyfluorene elutes before the aminofluorene. Using plots such as those shown in Figures 5 and 6, an optimum mobile phase can be found for the separation of a large number of aromatic amines. Figure 8 shows the separation of 12 solutes on the diketone-Cu(I1) column in less than 13 min. T h e use of capacity ratio plots vs. the concentration of the mobile phase to obtain an optimum resolution is similar to the utilization of stationary phase composition as developed by Purnell and Laub in gas chromatography (viz. 17 and references therein). Figure 9 shows a separation of eight substituted anilines on the dithiocarbamate-Cu(I1) column. Column Equilibration and Stability. After the columns were loaded with Cu(I1) (by passing 5 x IO3 M CuSO, in MeOH through the columns), the mobile phase was introduced. A t that time, monitoring of the volume of mobile phase
Figure 8. Separation of a mixture of 12 aromatics and aromatic amines. Mobile phase: Hexene/methylene chloride/methanol (6/1/1.5). Flow rate: 1.O mL/min. Diketone-Cu(I1) column. (1) fluorene, (2) 9fluorenones, (3) o-chloroaniline, (4) o-nitroaniline, (5) 4-amino-9fluorenone, (6) 1-amiflonaphthalene, (7) rn-nitroaniline, (8) 2-amino9-fluorenone, (9) p-chloroaniline, (1 0) 2-aminonaphthalene, (11) 2aminofluorene, (12) 2-amino-9-hydroxyfluorene
Flgure 9. Separation of a mixture of eight anilines on the Dithiocarbamate-Cu(I1) column. Mobile phase: Hexanelmethylene chloride/methanol (5.5/1/2). Flow rate: 1.0 mL/min. (1) benzene, (2) o-chloroaniline, (3) o-nitroaniline, (4) o-anisidine, (5) rn-chloroaniline, (6) rn-nitroaniline, (7) p-nitroaniline, (8) p-toluidine
passing through the column was started. Injections of several solutes were made as soon as enough TJV light was transmitted M Cuz+the absorbance was much through the cell (at 5 x too high). Figure 10 shows typical behavior of the k'values of these solutes as a function of time on the diketone-Cu(I1) column. When mobile phase A was used, it took about 600 rnin (flow rate 1 mL/min) before the capacity ratio of the amino-9-hydroxyfluorene stabilized a t 0.84. On the other hand, with the solutes which are less sensitive to small variations in the amount of Cu(II), equilibration is much
1352
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
3.
B
c D
L
-
1 -
~~~ ~~~
--
L I i ’ = ; E-~ -
0
0
ZOC
400
VOLUVE
500
BOC
IWQ
(d)
Figure 10. Column equilibration curves for the Diketone column after bonding Cu(I1). Mobile phase flow rate, 1 mL/min. Curves C, D, and F are for 2-amino-9-hydroxyfluorene, 2-amino-9-fluorene and 9fluorenone, respectively using mobile phase A. Curves A, B, and E are for the same solutes with mobile phase A containing 5.5 X lo-, M CuSO,
Table VII. Reproducibility Test for Diketone column-Cu( I1)” - h’ values original return after solute run several days F 0.10 0.12 9F 0.10 0.12 29 0.78 0.81 49 0.27 0.30 2A F 2.43 2.65 2AOH 1.63 1.76 a Mobile phase: 20% cyclohexane/methanol 2.2 x M Cu(II), flow rate = 1.0 mL/min. Table VIII. Reproducibility Test for Diketone Column-Cu(I1k-h’ Valuesa values after adding 5.0 mL of lo-*M Cu(I1) in return MEOH after using into sysmore polar tem and original mobile re-equilisolute run phases brating F 0.27 0.27 0.25 0.27 0.27 0.26 9F 1.83 1.70 1.90 29 0.86 0.67 0.82 49 2AF 5.07 3.68 5.12 5.40 4.00 5.68 2AOH a Mobile phase: hexaneimethylene chloride/methanol (4.5:1:3). Flow rate = 1.0 mL/min.
The second column shows the capacity ratios with t h e same mobile phase but after several days of use with more polar mobile phases (more MeOH). T h e capacity ratios of the G . . amine-containing solutes decreased noticeably. However, as ~ -_____ ~ T -~C 00 2C3 3C0 4 00 50C 6 30 seen in the third column of Table VIII, after passing 5.0 mL of lo-* M Cu(I1) in MeOH and equilibrating with the original JCISLUNE ,VI mobile phase, the capacity ratios, within experimental error, Figure 11. Column equilibration curves for Dithiocarbamate column are the same as initial values. Column reproducibility, then, after bonding Cu(I1). Mobile phase flow rate, 1 mL/min. Curves A, can be maintained and controlled rather easily. Tables VI1 C, E, and G are for solutes: 2-amino-9-hydroxyfluorene, 2-aminoand VI11 indicate that the amount of Cu(I1) bonded is de9-fIuorenone,4-amine9-fluorenone, and a fluorenone with mobile phase pendent upon the mobile phase used. A. Curves B, D, F, and H are for the same solutes with mobile phase A containing 5 . 5 X M CuSO,
,
~
~
~~
~
CONCLUSIONS
faster. For example, the fluorenone retention time is constant, within experimental error, almost immediately. When t h e mobile phase contained 5.5 X M CuSO,, the equilibration period is much shorter; about 200 min for the amino-9hydroxyfluorene and 2-amino-9-fluorenone. As expected, k ’ values a t equilibrium are higher when the mobile phase contains copper. Figure 11 shows a similar behavior with the dithiocarbamate-Cu(I1) column. In general, however, the equilibrium time is faster with the dithiocarbamate-Cu(I1) column. Column Reproducibility. Reproducibility studies were carried out with the diketoneCu(I1) column. In one case, after using the column with mobile phase A containing Cu(I1) (2.2 X lo-, M ) , several other mobile phases were employed over a period of several days. Upon returning to the original mobile phase i t was found that, within experimental errors, the capacity ratios were constant. Some of the results are shown in Table VII. Study of column reproducibility is shown in Table VI11 with a mobile phase made of hexane:methylene ch1oride:methanol (4.5:1:3). The first column in the table shows the original data.
Chromatography with bonded-metal ions can yield selective separations not attainable without the metals. Proper choice of the ligand to which the metal ion is bonded can affect the stability of the chromatographic column. Of course the ligand may also affect chromatographic behavior. T h e differences in the column efficiencies, t h e k’ values, and the rate of equilibration between t h e two ligands used in the present study may he due not only to t h e higher Cu(I1) content but also t o the different structure of the ligands. More exotic ligands can be used, and indeed have been used for the separation of amino acid enantiomers (18). Since the amine-Cu complex formation constants are fairly large, the efficiency of the system herein described is limited by poor mass transfer. Other metals which form weaker complexes can be used to improve efficiency. Such a n approach was studied by Cooke et al. (13). They also found that complexation with metal ions in t h e mobile phase, in conjunction with reversed phase columns, can yield efficient separations. I n our studies, the very large selectivity factors due to the presence of bonded Cu(I1) largely overcome t h e limited column efficiency in producing effective separations.
ANALYTICAL CHEMISTRY, VOL. 50,
Clearly, the potentials of using metals in chromatography show great promise, and we are now studying methods of improving column efficiency in order to apply such columns to the difficult separation of biopolymers.
(10) (11) (12) (13) (14) (15)
LITERATURE CITED
(16)
(1) S. Lam and E. Grushka, J . Chromotgr. Sci., 15, 234 (1977). (2) D. J. Weber, J . Pharm. Sci., 66, 744 (1977). (3) G. Schomburg and B. Vanach, 3rd International Symposium on Column Liquid Chromatography, Sept. 27-30, 1977, Salzburg, Austria. (4) F. G. Heifrich, Nature (London), 189. 1001 (1961). (5) H. F. Walton, Sep. Purif. Methods, 4, 189 (1975). (6) A. V. Semechkin, S. V. Rogozhin, and V. A. Davankov, J . Chromafcgr., 131, 65 (1977). (7) D. Kunzru and R. W. Frei, J . Chromatogr. Sci., 14, 381 (1974). (8) K. Yasuda, J . Chromatogr., 60, 144 (1971). (9) K. Yasuda, J , Chromatogr., 72, 413 (1972).
(17) (18)
NO. 9, AUGUST 1978
1353
L. A. Sternson and W. J. DeWitte, J , Chromatogr., 137, 305 (1977). L. A. Sternson and W. J. DeWitte, 138, 229 (1977). F. K. Chow and E. Grushka, Anal. Chem., 49, 1756 (1977). N. H. C. Cooke, R. L. Viavattene, R. Eksteen, W. S. Wong, G. Davies, and B. L. Karger, Submitted to J . Chromatogr. D. E. Leyden and G. W. Luttrel, Anal. Chem., 47, 1612 (1975). F. A. Cotton and G. Wilkinson, "Advanced Inorganic Chemistry", Interscience, New York, N.Y.. 1962, p 229. R. Belcher and A. J. Ntitten, "Laboratory Manual of Quantitative Inorganic Analysis", Butterworth, London, 1955, p 244. W. K. AI-Thamir, R. J. Laub, and J. H. Purnell. J . Chromatogr., 142, 3 (1977). V. A. Davankov and A. V. Semechktn, J . Chromatogr., 141, 313 (1977).
RECEIVED for review February 13, 1978. Accepted May 12, 1978. We thank NIH for supporting the present work under grant GM-20846.
High Performance Liquid Chromatographic Study of the Retention and Separation of Short Chain Peptide Diastereomers on a C, Bonded Phase Eugene P. Kroeff' and Donald J. Pietrzyk" Chemistry Department, The University of Iowa, Iowa City, Iowa 52242
The effects of peptide structure and stereochemistry and eluent pH and composition on the retention of short chain peptides and peptide diastereomers on a C8 bonded phase are evaluated by HPLC. The peptides are highly retained in acidic and basic solution and are at a minimum retention at the isoelectric pH. Peptide retention is influenced by the hydrophobicities of the amlno acld subunits and by the position of the hydrophobic amino acid subunits in relation to the charged sites. I t is suggested that the different conformations for peptide diastereomers have slight differences in hydrophobicity, and this influences their retention order. Several examples which illustrate these parameters and the scope of separating peptide diastereomer mixtures by HPLC are described.
T h e separation of short chain peptides has traditionally been accomplished by ion-exchange or thin-layer chromatography (1). During the past year, high performance liquid chromatography (HPLC) employing nonpolar (reverse) stationary phases has been shown to be very effective in bringing about these separations (2-6). Reverse phase HPLC is particularly attractive since it is fast, highly reproducible, and requires a minimum of sample preparation. Easily prepared eluting solvents such as buffered aqueous or aqueous-alcohol or -acetonitrile mixtures are used. Furthermore by using a variable wavelength detector (200 to 220 nm) detection of any amino acid or peptide is possible without resorting to derivitization. Several nonpolar stationary phases have been used to effect peptide separations. Short chain peptides containing as many as six nonpolar amino acid (AA) subunits, as well as larger hormone peptides were separated on a 5-pm octyl-silica bonded phase type stationary phase (2). Retention studies 'Present address, Eli Lilly and Company, Indianapolis, Ind. 46206. 0003-2700/78/0350-1353$01.00/0
of pharmacologically important nonapeptides were performed on microparticulate octyl- and octadecyl-silica columns (3). An HPLC procedure for their determination in dosage forms on these columns has also been reported ( 4 ) . Peptide retention and separation studies have been carried out on columns containing organic copolymers as the stationary phase. In this laboratory, Amberlite XAD-2 and 4 (polystyrene-divinylbenzene copolymers) have been used to study the retention and separation of' AA, peptides, and AA derivatives (5,6). Retention of several dipeptides on XAD-2 was also reported in gravity flow column chromatography ( 7 ) . Other copolymer stationary phases used in AA and peptide retention studies were Poropak Q ( 8 ) and Poragel P N and P S (9). Procedures have been reported for the separation of certain diastereomeric peptides and their derivatives by paper (IO), thin-layer (11, 12), and ion-exchange chromatography (13). A tripeptide chemically bonded to silica was used as a stationary phase in HPLC for the separation of peptides (14,15) and diastereomeric peptides (16). Recently, ion-exchange columns were used to separate diastereomeric tetrapeptides (17)and D- and L-amino acids as the 1,-D and L-L dipeptides after reaction with t e r t - but y 1oxy car bony 1- L-leuc ine - N hydroxysuccinimide ester (18). XAD-2 and XAD-4 columns have also been used to separate diastereomeric peptides (6, 7). Although the XAD copolymers showed promise as stationary phases for the separation of diastereomeric peptides and are stable over the entire pH range unlike the bonded stationary phases, they do not generally provide efficiencies that are comparable to those readily achieved with the bonded stationary phases ( 5 , 6). This is particularly true when compared to the microparticulate bonded phases. Therefore, the purpose of this paper is to describe results of our experiments which were designed to illustrate those factors which influence the retention of diastereomeric di- and tripeptides on a bonded reverse stationary phase, of the CB type. With C 1978 American Chemical Society
