Anal. Chem. 1988, 58,2233-2237
2233
Helfferich,
F. Ion Exchange; McGraw-Hill: New York, 1962; p 95.
Schik, A. A. Analytical Applications of 1,lO-Phenanthroline and Related Compounds; Pergamon Press: Oxford, 1969. Cantwell, F. F.; Puon, S. Anal. Chem. 1070, 5 1 , 623-632. Yammamoto, Y.; Tarumoto, T.; Iwamto, E. Chem. Lett. 1072, 255-258. Bidlingmeyer, 6. A.; Warren, F. V., Jr. Anal. Chem. 1982, 5 4 , 2351-2356. Sachok, E.; Deming, S. N. J . Liq. Chromafogr. 1982, 5 , 389-402. Denkert, M.; Hackzell, L.; Schili, G.; Sjogren, E. J . Chromafogr. 1981, 218, 31-43. Warren, F. V., Jr.; Bidlingmeyer, E. A. Anal. Chem. 1984, 5 6 , 47a-491. Rigas. P. G.; Pietrzyk. D. J., to be submitted for publication in Anal. Chem .
(26)
RECEIVED for review January 27,1986. Accepted May 7,1986. Part of this work was supported by Grant AM 28077 awarded by the National Institute of Arthritis, Diabetes, Digestive, and Kidney Diseases and was presented in part a t the 1984 International Chemical Congress of Pacific Basin Societies, Honolulu, HI, December, 1984, paper 0-1047 and a t the 190th American Chemical Society National Meeting, Chicago, IL, September 1985, paper 111.
Liquid Chromatography of Alkali and Alkaline-Earth Metal Ions Using Octadecylsilanized Silica Columns Modified in Situ with Lipophilic Crown Ethers Keiichi Kimura,* Hiroya Harino, Eiji Hayata, and Toshiyuki Shono* Department of Applied Chemistry, Faculty of Engineering, Osaka University, Yamada-oka, Suita, Osaka 565, Japan
Octadecyislianired silica (ODS) was coated with highly lipophilic crown ethers to obtain novel crown ether containing stationary phases for ion chromatography. The in situ modified ODs columns proved convenient and efficient for chromatographic separation of akaH and akalhreearlh metal ions. The retention times of alkali metal ions on the crown ether coated ODS are increased In the order Li' < Na' < Cs' < Rb' < K' for the 18crown-6 and 15eown-5 dwlvatives and in the order Li' < Cs' < Rb' < K' < Na' for the 12crown-4 derivatives, being reflected in the caiioncompiexing properties of the crown ether rings. Simultaneous coatlng of more than one crown ether on ODS Is possible as well, which permlts easy control of the Chromatographic behavior of the crown ether Stationary phases. The stability (durability) of the crown ether modified ODS columns is also described.
highly lipophilic crown ethers are retained strongly on octadecylsilanized silica (ODS) by powerful hydrophobic interaction. Some of them could not be eluted out from ODS columns even by using pure methanol as the mobile phase. This incident induced us to test ODS coated with highly lipophilic crown ethers for usefulness as stationary phases for liquid chromatography of ionic species. For convenience we attempted in situ coating of lipophilic crown ethers on ODS by passing coating solutions through commercially available, high-performance ODS-packed columns. We recently communicated the preliminary results concerning the use of crown ether coated ODS for chromatography (13). In this publication we wish to report in detail the liquid chromatography of alkali and alkaline-earth metal ions on ODS columns modified with lipophilic crown ethers and their analogues, 1 through 7 (Figure 1).
Extensive studies about use of crown ethers in liquid chromatography of cationic species, especially, alkali and alkaline-earth metal ions, have been made so far. In most cases polymeric crown ether resins (1-6) and silica gels on which crown ether moieties are immohilized through covalent bonding (7-10) or which are coated with polymeric crown ether (11, 12) have been applied to the stationary phases for the ion chromatography. The crown ether stationary phases are quite different in chromatographic behavior from conventional "ionic" cation exchangers. For instance, the elution order of alkali metal ions in the "ionic" cation exchangers follows the ionic size (Li+ < Na+ < K+< Rb+ < Cs+ in the retention time), not being varied from one cation exchanger to another. In contrast, the retention behavior of the crown ether stationary phases depends on the type of the immobilized crown ether moiety. This variable elution order on the crown ether stationary phases is attractive for chromatographic analyses of the cationic species. Hence, various cation-specific stationary phases may be designed for liquid chromatography of ionic species by selecting the immobilized crown ether moiety. The crown ether immobilized stationary phases, however, might not be easily available for most analytical chemists due to some difficulty with the syntheses. During synthesis and purification of crown ether derivatives incorporating long aliphatic chain($, we have found that the
Materials. Dodecyl-18-crown-6,-15-crown-5,and -12-crown-4,
EXPERIMENTAL SECTION 1 through 3, were synthesized by modification of the procedure in the literature (14). Dodecanoyloxymethyl crown ethers 4 and 5 were prepared by the reaction of dodecyl chloride with the corresponding hydroxymethyl crown ethers (15) in refluxing chloroform in the presence of triethylamine for 20 h. A similar reaction of chlorocarbonylbenzo-18-crown-6(16) and dioctylamine afforded N,N'-dioctylcarbamoylbenzo-l8-crown-6,6.All of the lipophilic crown ether derivatives were isolated as oil or wax after purification by preparative reversed-phase liquid chromatography (ODS, CHC13/MeOH). They were identified by 'H NMR and mass spectroscopy and elemental analysis. The data for the elemental analysis are as follows: Calcd for 1, CUH,Os: C, 66.63; H, 11.18. Found C, 66.70; H, 11.32. Calcd for 2, CzzH440$ C, 68.00; H, 11.41. Found: C, 67.69; H, 11.66. Calcd for 3, C&IN04: C, 69.72; H, 11.70. Found: C, 70.05; H, 11.98. Calcd for 4, C2.Ha08: C, 63.00; H, 10.15. Found: C, 62.73; H, 10.27. Calcd for 5, C23H4407:C, 63.86; H, 10.25. Found: C, 64.56; H, 10.53. C, 68.36; H, 9.91; N, 2.42. Found: C, Calcd for 6, C33HS7N07: 68.01; H, 10.34; N, 2.51. Oligo(oxyethy1ene)mono-p-nonylphenyl ether 7 (average polymerization degree of 18, Tokyo Kasei) is commerically available and was employed as received. The metal salts are of analytical grade. Water was distilled and deionized. Methanol was purified by double distillation. Chromatography. The high-performance liquid chromatograph system employed here consists of a pumping system (Milton Roy 0369-57 or Waters Associates 6000A), a sample injector (Kyowa Seimitsu KHP-UI-130A or Waters Associates U6K), and
0003-2700/86/0358-2233$01.50/00 1986 American Chemical Society
2234
ANALYTICAL CHEMISTRY, VOL. 58, NO. 11, SEPTEMBER 1986 05 04
03 1
R =C12H25 , n: 3
2
R'Cq2H25
3
R=C,2H25 , n = 1
4
R=C11H23C~CH2 , n = 3
5
R ' C I ~ H ~ ~ C ~ ,CnH= ~2
I
6
LLI
3
2
>
n= 2
C9Hl9 @ t t O t H 7 (n=l8)
02 01
oc A 30 80 60 40
Flgure 1. Lipophilic crown ethers employed for this study
30
80
60
40
100
80
60
41
MeOH CONTENT ( '/a1
a conductivity detector (LDC Conductomonitor). Chromatograms were processed with a computing integrator (Shimadzu Chromatopac-ElA). Chromatography was carried out at a flow rate of 1 mL min-' unless otherwise specified. The sample size was generally 50 pL and the concentrations were 0.04 mol L-' for each alkali metal salt and 0.02 mol L-l for each alkaline-earth metal salt. Thin-layer chromatography was run with reversed-phase TLC plates (Merck HPTLC RP-18, 10 X 10 cm) and methanol/water developing solvent. Coating of Crown Ethers on ODs. The coating was performed by passing an appropriate coating solution of crown ether through a ODs-packed column at a flow rate of 1mL min-' (17). Commercially available packed columns (6 mm id. X 10 cm column length, 5 pm spherical ODS, YMC-Pack A-311) were employed throughout this study. Appropriate amounts of crown ethers were dissolved in methanol/vater mixtures (about 200 mL), the compositions of which were 60/40(v/v) for 3,4,5, and 7,50/50 (v/v) for 1,2, and 6, and 1/3 mixture. The coating solutions were filtered with membrane filters (Teflon,0.5 pm pore size) and then degassed by sonication. After the coating of ODS the solvent of the effluent was evaporated and the residual crown ether was weighed. The coating amount of crown ether on ODS was calculated by subtracting the residual crown ether weight from the initial one. RESULTS AND DISCUSSION Coating of Lipophilic 18-Crown-6 Derivatives. High lipophilicities of crown ethers are required for stable coating on ODS through hydrophobic interaction. Highly lipophilic 18-crown-6 derivatives employed here are the crown ethers carrying one or two long aliphatic chains, 1,4, and 6. As the solvent of the coating solution, methanol/water mixtures in which the crown ethers are barely soluble were chosen for effective coating. Under these coating conditions almost all of the crown ether molecules in the coating solution were retained on ODS. Higher methanol content of the coating solvent was also attempted, which was expected to allow the more homogeneous coating of crown ether on ODs. Moreover, circulation of the coating solution through the packed column was carried out for the coating. Nevertheless, these attempts did not improve the efficiency of the resulted crown ether modified ODS columns. Liquid chromatography of alkali and alkaline-earth metal ions was performed using the ODS column modified in situ with dodecyl-18-crown-6 1 and methanol/water mixtures as the mobile phase. Selection of the mobile phase for the liquid chromatography is very important since high methanol content in the mobile phase may cause severe stripping of the crown ether from ODs. An examination of the mobile phase by reversed-phase thin-layer chromatography indicated that the use of higher than 60% methanol content in the mobile phase leads to easy removal of the crown ether from ODS (Figure 2). Less than 60% methanol content, therefore, was adopted as the mobile phase for the ion chromatography. The results for the chromatography of alkali metal halides and thiocyanates on a ODS column loaded with 0.12 mmol of l are given as retention times in Table I. Use of pure water as the mobile phase allowed only slight separation of the alkali metal ions. Increasing methanol content in the mobile phase gen-
Flgure 2. Correlation between R, value and methanoVwater composition in developing solvent for reversed-phase thin-layer chromatography of lipophilic crown ethers 1 (a), 2 (b), and 3 (c). For details see Experimental Section.
Table I. Retention Times of Alkali Metal Salts in Liquid Chromatography on ODS Column Modified with Dodecyl-18-crown-6 1" retention time, min, for following MeOH/H20 cation
Li+
counteranion CI-
BrISCNNa+
Cl-
BrISCN-
K+
Cl-
BrISCN-
Rb+
Cl-
BrIcs+
Cl-
BrI-
20/80
30/70
40/60
50/50
1.8 1.8 1.9 2.0
1.8 1.8 1.9 2.0
1.8 1.8 2.0 2.0
1.8 1.9 2.0 2.0
1.9 1.9 2.0 2.2
1.9 1.9 2.0 2.2
2.0 2.0 2.2 2.4
2.1 2.2 2.3 2.5
2.2 2.4 2.8 3.7
2.4 2.6 3.2 4.1
3.0 3.2 4.3 5.2
4.5 4.8 5.7 6.7
2.0 2.1 2.4
2.0 2.2 2.6
2.4 2.5 3.1
3.1 3.1 3.7
2.0 2.0 2.3
2.0 2.0 2.3
2.1 2.2 2.7
2.5 2.5 2.8
"Coating amount of crown ether: 0.12 mmol. For other details see Experimental Section. erally enhances the retention of the metal ions on the stationary phase and improves the chromatographic separation. The 1-coated ODS possesses high affinity for K+,the retention time decreasing in the order K+ > Rb+ > Cs+ > Na+ > Li+. This retention behavior is definitely reflected in the cationcomplexing property of the 18-crown-6 ring (18). The chromatographic retention of the metal ions is also affected by the kind of the counteranion. High polarizability of the counteranion generally augments the cation complexation of the crown ether, in turn increasing the retention times as is the case for ion chromatography using the previous covalently bonded crown ether stationary phases. Alkaline-earth metal ions were also chromatographed on the crown ether coated ODS and the retention time was increased in the order Mg2+ 5 Ca2+< Sr2+< Ba2+. Typical chromatograms for the ion chromatography using the 1-modified column are given in Figure 3, which shows excellent separation of five alkali metal ions. Also, Mg2+/Ca2+group, Sr2+,and Ba2+can be separated on this column, although Mg2+and Ca2+are hard to separate under these chromatographic conditions. The lower coating amounts of 1 (less than 0.1 mmol) suppressed the chromatographic retention on the resulting stationary phases. The much higher coating amount did not afford remarkable im-
ANALYTICAL CHEMISTRY, VOL. 58, NO. 11, SEPTEMBER 1986
2235
Table 11. Retention Times of Alkali Metal Salts in Liquid Chromatography on ODS Columns Modified with Dodecyl-15-crown-5and -12-crown-4" retention time, min
countercrownether 2
anion
Li+
Na+
K+
Rb+
Cs+
c1-
2.0 2.0 2.0 2.1
2.3 2.4 2.5
4.8 5.2
4.2 4.5
5.7
4.9
2.3 2.4 2.5
2.9
8.2
2.4 2.5
2.0 2.0 2.3 2.5
2.0 2.0 2.2
2.0 2.1
BrI-
SCN3 0 1 2 3 . 4 5 6
0 1 2 3 4 5
RETENTION TIME(min)
c1BrISCN-
1.9 2.0 2.0
2.0
3.0 3.5
1.9
"Coating amount of crown ether: 0.18 mmol for 2,0.43 mmol for
Figure 3. Liquid chromatography of alkali and alkaline-earth metal halides on ODS coated with dodecyl-18-crown-6, 1: coating amount of crown ether, 0.12 mmol; mobile phase, MeOH/H,O (50150); counteranbn, bdide for alkali metal ions and bromide for alkaline-earth metal
3; mobile phase, MeOH/H20 (50/50).
ions.
-2 > I-
IV
3
P
0 1 2 3 4 5 6 7
z
0
0 1 2 3 4 5
RETENTION TIME (min)
V
0
1
2
3
0
1
2
RETENTION TIME (min) Figwe 4. Effect of flow rate on liquid chromatography of alkali metal
Iodides using 1-coated ODS. provement on the chromatography. Distinct peak broadening was even observed in the chromatograms on the heavily coated ODS columns, thus resulting in diminution of the column efficiency. The same retention order of the metal ions was found for liquid chromatography using ODS columns modified with the other 18-crown-6 derivatives, 4 and 6. The crown ether, 6, should be more favorable than 1 for the coating with regard to column stability due to the higher lipophilicity. The use of 4 and 6, however, did not give any better result than 1 in the liquid chromatography of the metal ions on the modified ODS column. For example, serious peak broadening was observed on the stationary phases with the 18-crown-6 derivatives except l. The reason for the difference in the column efficiency between 1 and the other 18-crown-6 derivatives is not yet understood. Rapid, but still excellent chromatographic separation of alkali metal ions on the 1-coated ODS was realized with higher flow rates than 1mL min-' as shown in Figure 4. Even with a flow rate of 3 mL min-', five alkali metal ions can be separated almost completely within 2 min. Coating of Lipophilic 15-Crown-5 a n d 12-Crown-4 Derivatives. ODS columns were similarly modified with lipophilic 15-crown-5and 12-crown-4 derivatives, 2,3, and 5. The results for the liquid chromatography of alkali metal ions on the 2- or 3-coated ODS using methanol/water (50/50) as the mobile phase are presented in Table I1 and Figure 5. The retention times are in the order K+ > Rb+ > Cs+ 1 Na+ > Li+ for the 15-crown-5 system and Na+ > K+ > Rb+ 1 Cs+ 2 Li+ for the 12-crown-4 system. The elution orders generally
Figure 5. Uquld chromatographyof alkali metal iodides on ODs coated with dodecyl-15-crown-5, 2 (a), or dodecyl-12-crown-4, 3 (b). The chromatographic conditions are as given in Table 11.
seem to be governed by the cation-complexing properties of the respective crown ether rings (18). On the 3-coated ODs, K+ is eluted faster than Na+, although the former ion is equivalent to or slightly greater than the latter one in the stability constant for the 12-crown-4 complexes. The Na+ selectivity over K+ of the 3-coated ODS might be explained by formation of 2:1 (crown ether/cation) complexes as expected in the ion chromatography on the poly(l2-crown-4)modified silica (9). On the 2- coated ODS, Na+ and Cs+ were hard to separate from each other under our chromatographic conditions. This is also the case for the separation of Li+, Rb+, and Cs+ on the 3-coated ODs. On the liquid chromatography of alkaline-earth metal ions on either of the 2- or 3-coated ODS,the retention times followed the order of the ionic size as observed in the 18-crown-6chromatographic system. Since 12-crown-4 derivatives generally possess weak cation-complexing abilities as compared to 15-crown-5 and 18-crown-6 derivatives, a high coating amount of crown ether (0.43 mmol) was required in the 3-coated ODS to obtain such chromatographic separation as shown in Figure 5. The higher coating amount did not improve the chromatographic separation of the metal ions any more. The 3-coated ODS, however, is specific for Na+ on chromatography. In this respect this crown ether stationary phase is distinct from the 18-crown-6 and 15-crown-5systems. An ODS column modified with the other 15-crown-5 derivative, 5, was quite similar to that with 2 in chromatographic behavior. Coating of Lipophilic Oligo(oxyethy1ene). Lipophilic oligo(oxyethy1ene) derivatives such as 7, which are inexpensive nonionic surfactants, are considered as noncyclic crown ether derivatives. Cross-linked polystyrene resins on which oligo(oxyethy1ene)s are immobilized through covalent bonding have been applied to stationary phases for liquid chromatography of alkali metal ions (19). Mixtures of thiocyanates of Li+, Na+,
2236
ANALYTICAL CHEMISTRY, VOL. 58, NO. 11, SEPTEMBER 1986
0 1 2 3 4
0 1 2 3 4
(mid Flgure 6. Liquid chromatography of alkali metal thiocyanates using lipophilic oligo(oxyethylene), 7: (a) ODs column modified with 7 (0.12 mmd), mob& phase MeOH/H20 (40160);(b) unmodified ODS column, mobile phase MeOH/H,O (40/60) containing 7 of 5.2 mmol L-'.
1
RETENTION TIME
and K+ seem separable on the oligo(oxyethy1ene)-immobilized resins. This opens up a possibility of use of the lipophilic oligo(oxyethy1ene)sas the coating reagent on ODS for liquid chromatography of metal ions. However, since the noncyclic crown ethers have low cation-complexingabilities as compared to the "cyclic" ones, the ODS with oligo(oxyethy1ene) 7 afforded only poor separation of even alkali metal thiocyanates on the chromatography (Figure 6). The higher coating amount hardly promotes the retention of the metal ions on the resulting stationary phases. Another problem in the oligo(oxyethy1ene)system is rather high water solubility of the coating reagent. The lipophilic oligo(oxyethylene), 7, is easily removed from ODS during chromatography. We also tried liquid chromatography of the alkali metal thiocyanates on unmodified ODS using methanol/water solutions of 7 as the mobile phase. As a result some improvement on the chromatography separation of the metal ions was found with the oligo(oxyethy1ene)-containing mobile phase, as also illustrated in Figure 6. Better separation between Li+ and Na+ could not be attained by changing the composition of methanol/water or the oligo(oxyethy1ene) concentration in the mobile phase. ODS columns with higher theoretical plates or greater column length might bring about improvement of the chromatographic separation by the oligo(oxyethylene)-0DS system. Simultaneous Coating of Different Crown Ethers. One of the advantages for this in situ coating of ODS is ease of coating and removal of the crown ethers. The crown ethers on ODS can be flushed out readily with methanol or methanol/chloroform mixtures from the ODS column, which is usually reused without any other special treatment. Another is simultaneous coating of more than one crown ether derivative, which thereby enables us to regulate the separation on the liquid chromatography of ionic species. We attempted simultaneous coating of dodecyl-l&crown-6 and -12-crown-4, 1 and 3, which are quite different in the cation-complexing property as already mentioned. Let us think about chromatographic separation of Li+, Na+, and K+, which are likely to coexist in environmental and biological systems, on a crown ether modified ODS column. On the chromatography of samples containing the same concentrations of Li+, Na+, and K+ on the 1-coated ODS, the peaks for Li+ and Na+ are too close to each other and K+ is eluted too slowly (Figure 7). If a larger quantity of Li+ than Na+ is contained in the samples or vice verse, the two ions are hard to separate on this modified column. Additional coating of the 12-crown-4 derivative, which possesses high affinity for Na+, are expected to improve the separation among the three alkali metal ions. Actually, a more favorable chromatographic separation was realized by the simultaneous coating of 1 (0.12 mmol) and 3 (0.43 mmol) on ODS (Figure 7). In comparison with the chromatogram on ODS with only 1, Li+ and Na+ are
2
3
4
5
6
I
,
,
,
,
,
0
1
2
3
4
5
RETENTION TIME (min)
Flgure 7. Comparison of liquid chromatographic separations of alkali metal iodides between single and binary coating of lipophilic crown ethers on ODs: (a) W S column with 1 (0.12 mmol); (b) ODS column with 1 (0.12 mmol) and 3 (0.43 mmoi); mobile phase, MeOH/H20 (50/50). 1.5
I
V c
d4 0.5
I
0.0 8-
RFCI
1 0.5
0
U
0.1
A
O
0 4
0
4
5
IO
"
I
0
-
A
A
15
NdCl
KCI Nal L K I
20
VOLUME OF ELUTION(U
Flgure 8. Stability of ODS column modified with dodecyl-18-crown-6 on liquid chromatography of Na+ and K+: coating amount, 0.12 mmol of 1; mobile phase, MeOH/H,O (40160).
well-separated and K+ is eluted a little faster in the binary coating system. This example indicates that the simultaneous coating of lipophilic crown ethers is a convenient, promising way to improve separation in the ion chromatography on crown ether stationary phases. Ternary systems of lipophilic crown ethers for the ODS coating would be also possible. Column Stability. A most important factor in ion chromatography with crown ether-ODS columns is stability or durability of the columns, since the lipophilic crown ethers are adsorbed mainly by hydrophobic interaction. Definitely, the stability of the modified ODS columns depends on lipophilicities of the crown ethers and the methanol content in the mobile phase. We followed changes in the capacity factor and the height of theoretical plate (HETP) with volume of eluent, in the chromatography of Na+ and K+ salts using the 1 (0.12 mmo1)-coated ODS column and methanol/water (40/60)as the mobile phase. Figure 8 shows that except in the early stages there is no significant change in the factors even after elution with 20 L of the eluent, which takes more than 330 h in a flow rate of 1mL min-'. The initial changes, especially decrease in HETP, might be due to rearrangement of the crown ethers adsorbed on ODS. The phenomenon, however, does not seem to be a serious problem on the chromatographic analysis of the metal ions. The crown ether coated ODS is, therefore, considered rather stable under this chromatographic condition. We tried to draw calibration curves (correlation between the peak area and ion concentration) for Na+ and K+ determination by chromatography
Anal. Chem. lQSS, 58, 2237-2241
under identical condition as a stability check. The calibration curves exhibited good linearity in a wide concentration range from about 0.5 to lo00 Fg mL-' at both of the ions. Thus, the coating of lipophilic crown ethers on ODS is quite promising for high-performance liquid chromatography of ionic species. Registry No. 1, 83255-15-6; 2, 74649-89-1; 3, 102725-12-2; 4, 102725-13-3; 5, 102725-14-4; 6, 102725-15-5; 7, 26027-38-3; Li, 7439-93-2; Na, 7440-23-5; Cs, 7440-46-2; Rb, 7440-17-7; K, 7440Ba, 7440-39-3; Mg, 7439-95-4; Sr, 7440-24-6; Ca, 7440-70-2.
LITERATURE CITED Blasius, E.; Janzen, K.-P.; Adrian, W.; Klautke, 0.; Lorschelder, R.; Maurer, P A . ; Nguyen, V. B.; Nguyen-Tlen, T.; Schoiten, G.; Stockemer, J. 2.Anal. Chem. 1077. 284, 337-360. Blasius. E.; Janzen, K.-P.; Klein, W.; Klotz, H.; Nguyen, V. B.; NguyenTien, T.; Pfeiffer, R.; Scholten, G.; Simon, H.; Stockemer, H.; Toussaint, A. J . Chromatogr. 1080, 201, 147-166. Blasius. E.; Janzen, K.-P.; Keller, M.; Lander, H.; Nguyen-Tien, T.; Schoken, G. TaLnfa 1080, 27, 107-126. Kutchukov, P.; Ricard, A,; Quivoron, C. Eur. folym. J . 1080, 78, 753-758. Yagi, K.; Sanchez, M. C. Makromol. Chem., RapM Commun. 1081, 2 , 311-315.
2237
(6) Frere, Y.; Gramain, P. Makroml. Chem. 1082, 183, 2163-2172. (7) Nakajlma, M.; Kimura, K.; Shono, T. Anal. Chem. 1083, 5 5 , 463-467. (8) Nakajima, M.; Kimura, K.; Shono, T. Bull. Chem. SOC.Jpn. 1083, 56, 3052-3056. (9) NakaJima. M.; Kimura, K.; Hayata, E.; Shono, T. J . Liq. Chromatogr. 1084, 7, 2115-2125. (10) Blasius, E.; Janzen, K.-P.; Simon, H.; Zender, J. Fresenlus' 2.Anal. Chem. 1085, 320, 435-438. (11) Igawa, M.; Saito, K.; Tsukamoto, J.; Tanaka, M. Anal. Chem. 1081, 5 3 , 1942-1944. (12) Igawa, M.; Salto, K.; Tanaka, M.; Yamabe, T. BunsekiKagaku 1083, 3 2 , E137-E141. (13) Kimura, K.; Hayata, E.; Shono, T. J . Chem. SOC.,Chem. Commun. I084, 271-272. (14) Bowsher, 8. R.; Rest, A. J.; Main, €3. G.J . Chem. SOC.,Dalton Trans. 1084, 1421-1425. (15) Czech, B. Tetrahedron Lett. 1080, 21, 4197-4198. (16) Bourgoin, M.; Wong, K. H.; Hui, J. Y.; Smid, J J . Am. Chem. SOC. 1075, 9 7 , 3462-3467. (17) Cassidy, R. M.; Elchuk, S. Anal. Chem. 1082, 5 4 , 1558-1563. (18) Izatt, R. M.; Bradshaw, J. S.; Nlelsen, S. A.; Lamb, J. D.; Christensen, J. J.; Sen, D. Chem. Rev. 1085, 8 5 , 271-339. (19) Fujita, H.; Yanagida, S.; Okahara, M. Anal. Chem. 1080, 5 2 , 869-875.
RECEIVED for review February 18,1986. Accepted April 17, 1986.
Mechanism of the Reversed-Phase High-Performance Liquid Chromatographic Separation of D- and L-Valine Using Copper( I I) and L-Aspartyl-L-phenylalanine Methyl Ester J. M. Broge' and D. L. Leussing* Department of Chemistry, The Ohio State University, Columbus, Ohio 43210
Moblle and statlonary phase Interactions exlstlng In the reverse-phase separatlon of D and L-vallne wHh an eluant contalnlng Cu( I I ) and asparlame have been studled In depth. Dlastereomerlc mbted ligand complexes formed In the solutlon phase have a negligible Influence on the separatlon. Enantiomerlc dkrhnlnatlon occurs solely on the statlonary phase.
TheMmberofmolesofCu(1I)andasparlameadsorbedonto the Cl0 packing are described by Langnuk tsothenns In wtdch HA, Cu(A)+, and Cu(A), compete for a given number of shes (A- = aspartame-). Observed retentlon tlmes are well-reproduced by a model assuming the reactlons HVal, Cu(A), + Cu(A)(Val), H+, HVal,, Cu(A),, Cu(A),(Val), -t H', and Cu(Val),, Cu(A),, 4 Cu(A),Cu(Val),.
+
+
+
+
The use of complexes formed between chiral ligands and metal ions to perform chromatographic separations and analyses of amino acid enantiomers has attracted considerable attention in recent years (I). One approach has been to covalently bond a chiral ligand to the solid support of the chromatographic column (2-5). Another has been to add a chiral ligand and metal ion to the eluant phase (6-18). This latter method is particularly attractive because the eluant is easily prepared by mixing solutions containing the metal ion and ligand and adjusting the pH to an appropriate value. A conventional column, usually reverse phase, may be used. Two limiting mechanisms have been invoked to account for the separations obtained by using chiral eluant phases. At 'Present address: General Mills, Inc., James Ford Bell Technical Center, 9OOO Plymouth Ave. N, Golden Valley, MN 55427.
one limit the chiral complex is considered to be adsorbed onto the solid support with separation occurring entirely on the stationary phase via a ligand exchange mechanism (1,6,11, 19). Along this line, Karger (6,10,13) and Davankov (11)and their co-workers have designed ligands with long alkyl side chains that have been successfully used to effect separations of amino acid enantiomers and their derivatives. At the other limit, separation is thought to occur mainly in the eluant phase through the formation of diastereomeric mixed ligand complexes which have different stabilities (20). Mixed ligand complex formation perturbs partition between moving and stationary phases resulting in different rates of movement of the enantiomers through the column. Separations effected by using small chiral ligands that contain relatively low proportions of hydrocarbon, such as proline and histidine, are generally considered to operate through this second limiting mechanism. Recently Zare has employed solution phases containing Cu(I1) and L-histidine to separate amino acid enantiomers using zone electrophoresis (21). An intriguing reagent, which may operate through both mechanisms, is the Cu(I1) complex of L-aspartyl-L-phenylalanine methyl ester (aspartame) (7). Originally this reagent was thought to operate via mobile phase interactions (7), but later experiments using analogous L-aspartyl-c-hexylamine derivatives led to the conclusion that stationary phase reactions were important (15). We report here the results of an in-depth study of the separation of D- and L-valine using mobile phase solutions of Cu(1I) and aspartame. Stability constants of solution species have been determined as well as the amounts of Cu(I1) and aspartame adsorbed on to an octadecylsilane stationary phase as a function of solution composition. A relationship between solution and stationary
0003-270018610358-2237$01.50/0 0 1986 American Chemical Society
