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ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979
many other experiments contributed to a particularly dirty electrical environment, the improvement exhibited by the correlation mode of operation is rather remarkable.
ACKNOWLEDGMENT We are deeply indebted to Guy Preau for his technical advice and assistance.
LITERATURE CITED (1) Raymond Annino, "Signal and Resolution Enhancement Techniques in Chromatography'' in "Advances in Chromatography", Vol. 15, Marcel Dekker, New York, 1977, pp 33-63. (2) J. B. Phillips and M. F. Burke, J . Chromatogr. Sci., 14, 495 (1976).
(3) Raymond Annino and Eli Grushka, J . Chromatogr. Sci., 14, 265 (1976). (4) Raymond Annino and L. E. Bullock, Anal. Chem., 45, 1221 (1973).
(5) Gyula Gaspar, Patrick Arpino, and Georges Guiochon, J . Chromatogr.
Sci., 15, 256 (1977). (6) Gyuia Gaspar, Raymond Annino. Claire Vidal-Madjar, and Georges Guiochon, Anal. Chem.. 50, 1512 (1978) (7) 9 u l a Gaspar, Jean-Piene Ollvo, and Georges Guiochon, Chromtcgraph!a, in press. (8) Robert L. Wade and Stuart P. Cram, Anal. Chem., 44, 131 (1972). (9) Stuart P. Cram and Stephen N. Chesier, J . Chromatogr., 99, 267 (1974). (10) Raymond Annino, Joseph Franko, and Harry Keller, Anal. Chem., 43, 107 (1971).
RECEIVED for review October 25, 1978. Accepted December 1, 1978.
Combination of Size Exclusion and Normal-Phase Partition Modes in High Performance Liquid Chromatography Sadao Mori" and Akira Yamakawa Department of Industrial Chemistry, Faculty of Engineering, Mie University, Tsu, Mie 5 14, Japan
Size exclusion chromatography (SEC) and normal-phase partition chromatography (NPPC) were performed on the same column packed with polystyrene gels used for SEC of small molecules. Chloroform was used as the mobile phase in SEC in order to measure the approximate numbers of components. Then, chloroform-n-hexane mixture was used for the complete Separation. The potential utility of the SEC-NPPC technique was illustrated by application of phthalate esters, alkylbenzenes, and ketones. The combined use of SEC and NPPC on the same column may extend their advantages and compensate their disadvantages.
Recent improvements in high performance size exclusion chromatography (SEC) (conventional GPC) have advanced the separation of low molecular weight materials ( I ) . Polystyrene gels with small pore and particle sizes permitted the complete separation of n-alkanes ( I ) , phthalate esters ( Z ) , alkylbenzenes ( 3 ) ,oligostyrenes ( 4 ) , and various other materials. In the separation of these materials by "true" SEC with polystyrene gels as packing materials, it is essentially necessary to use solvents as the mobile phase which minimize the interactions between the gels and solute molecules, Le., good solvents for uncross-linked polystyrenes ( 5 ) . When solvents having solubility parameters much different from that of the polystyrene gel are used as the mobile phase, the separation mode may be regarded as liquid-solid, Le., adsorption chromatography ( 5 ) . n-Hexane has a solubility parameter less t h a n that for the polystyrene gel and the separation mode is normal-phase adsorption chromatography (henceforth referred to as NPAC) where the elution order of the homologous series is in the order of decreasing molecular weight. Chloroform and tetrahydrofuran are eluents often used for SEC with the polystyrene gel. The separation mode of the mixtures of chloroform and n-hexane is regarded as normal-phase partition chromatography (henceforth referred to as N P P C ) , the elution order being the same as SEC and NPAC. Among advantages and disadvantages in SEC, NPAC, and NPPC, those for low molecular weight materials are itemized as follows from a comparison standpoint. Advantages in SEC 0003-2700/79/0351-0382$01 0010
are the following four factors: (a) As a molecule of the largest size elutes first from the SEC column in principle, the information on the size or molecular weight of the separated molecules can be obtained without difficulty. (b) As no solute elutes after the total permeation limit, it is suitable for the preliminary analysis of unknown samples. (c) Columns of 8-mm i.d. are normally used, which enables a large sample load and application of a refractive index detector. (d) Compared to the other separation modes, the separation time is short. The need for greatly increased column length in the case of insufficient separation and the high cost of columns are disadvantages in SEC. Advantages in NPAC and NPPC are (a) the capability of the increased resolution of two adjacent bands by selecting the mobile phase or the constituents and (b) less costly columns with small volume of packing materials. A lower sample load than SEC and the difficult prediction of the existence of retarded solutes in the case of unknown samples are the disadvantages in NPAC and NPPC. High performance liquid chromatography utilizes one of the four basic separation modes (adsorption, partition, ion exchange, and size exclusion) without combination of the other three modes. In SEC, for example, the information from the chromatogram is analyzed under the condition that adsorption or partition modes are not incorporated. On the other hand, the combined use of two separation modes will make it possible to extend their advantages and to compensate their disadvantages if the combination is adequate for the samples. Recently, coupled column chromatography using SEC and reversed-phase chromatography has been reported (6). In the method, SEC was used as the preliminary separation technique and some fractions which need more separation were transferred to other secondary columns(s1 for further separation. In our study, SEC and N P P C are performed on the same column packed with polystyrene gels used for SEC of low molecular weight materials. Chloroform was used in the first elution as SEC and then, the mobile phase was changed to the chloroform-n-hexane mixture as NPPC in order to obtain better separation. NPPC was performed on the SEC column (henceforth referred to as SEC-NPPC). The potential utility of the SEC-NPPC technique was illustrated by applications of three different homologous series. C 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979
383
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Chromatograms of alkylbenzenes on the same column a s in Figure 1. Flow rate: 0.48 mL/min. Attenuation: 0.64 AUFS. Sample volume: 2 pL. Mobile phase: (A) chloroform,(B) chloroform-n-hexane (6:4 v/v). Solutes: (1) n-octylbenzene, (2) ethylbenzene, (3) toluene, (4) benzene Figure 2.
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Figure 1. Size exclusion and normal-phase partition chromatograms of phthalates on the 4.5-mm i.d. column packed with Shodex A801
EXPERIMENTAL Apparatus. A Jasco (Japan Spectroscopic Co. Ltd., Hachioji, Tokyo, Japan) TRIROTAR high performance liquid chromatograph was used with a Jasco Model CVIDEC-100 variablewavelength UV detector. The detector was operated at 254 nm. Mixed solvents were prepared using a solvent programmer Model GP-ASO. Sample solutions were injected using a variable loop injector Model VL-611. Columns. Three stainless steel columns of 50-cm length and different inside diameter were used. In a 2.3-mm i.d. column, Shodex HP-255 porous polystyrene gel of 25 f 3 Irn was packed by the slurry packing procedure using methanol-water (1:l) mixed solvent under a flow rate of 0.5 mL/min first and finally 1.0 mL/min. This packing material is used for adsorption or partition chromatography. A 4.5-mm i.d. column was packed with Shodex A801 polystyrene gel of 15-20 pm. This packing material is used for SEC of small molecules. Both packing materials are composed of the same matrix, but have different physical properties ( 5 ) . The gel was suspended in a toluene-chloroform mixed solvent and packed into the column by the slurry packing procedure using the same solvent under a flow rate of 0.5 mL/min, followed by changing the solvent into chloroform and finally into chloroform-n-hexane (1:l)mixture. The final flow rate was 1.0 mL/min. A 8-mm i.d. column was a Shodex A 801 GPC column with A801 gel of about 10 pm packed by the manufacturer (Showa Denko Co. Ltd., Minato-ku, Tokyo, Japan). These packing materials and the column were provided by courtesy of Hikari Kogyo Co. Ltd., Chuo-ku, Tokyo, Japan. Samples a n d Procedure. All compounds used as solutes (samples) were guaranteed reagents purchased from several chemical supply houses and wed as received. They were phthalate esters, alkylbenzenes, and ketones as follows: dimethyl- (DMP), diethyl- (DEP), dibutyl- (DBP), and di-n-nonyl- (DNP) phthalates, benzene, toluene, ethyl- and n-octyl benzenes, acetone, methyl ethyl- (MEK), methyl isobutyl- (MIBK),and diisobutyl(DIBK) ketones. A sample concentration was selected between 0.1 and 0.5% (w/v) to obtain suitable responses. Sample injection volumes, flow rates, and pump pressure are indicated in the figure captions. The mobile phase was chloroform, n-hexane, and mixtures of the two. All measurements were performed at room temperature. RESULTS AND DISCUSSION Figure 1 shows the chromatograms of phthalates achieved on SEC and SEC-NPPC using the 4.5-mm i.d. column. Figure 1A is the example of SEC and the phthalates were sufficiently resolved to make a tentative identification based on molecular size. However, more complete separation was accomplished by adding n-hexane to the mobile phase. Figures 1B-1E are t h e examples of SEC-NPPC and the increasing n-hexane content in t h e mobile phase resulted in increasing t h e res-
I
I l
polystyrene gel. Flow rate: 0.48 mL/min. Attenuation: 0.64 A U F S . Sample volume: 10 pL. Mobile phase: (A) chloroform (pump pressure, 18 atm), (B) chloroform-n-hexane (9.1 v/v), (C) (8:2),(D) (7:3),(E) ( 5 5 ) (pump pressure, 10 atm). Solutes: (1) DNP, (2) DBP, (3)DEP, (4) DMP
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Figure 3. Chromatograms of ketones on the same column as in Figure 1 Flow rate: 1.0 mL/min. Attenuation: 0.08 AUFS. Sample volume:
10 pL. Mobile phase: (A) chloroform, (B) chloroform-n-hexane (9.1 v/v), (C)(8:2),(D) (7:3),(E) (6:4). Solutes: (1) DIBK, (2) MIBK, (3)MEK + acetone
2 j L + 5 $ 7 5 ? * 1 # : 3 1 E.J-.~Y
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Normal-phase adsorption chromatograms on the 2.3-mm i.d. column packed with Shodex HP-255. Flow rate: 0.48 mL/min. Attenuation: 0.16 AUFS. Sample volume: 5 pL. Mobile phase: n-hexane (pump pressure 18 atm). Solutes: (A) phthalate esters, (€3) alkylbenzenes ( 1 . n-octylbenzene, 2. benzene, toluene, and ethylFigure 4.
benzene), (C) ketones olution of t h e adjacent peaks. With t h e mixture of chloroform-n-hexane of the same volume content, all the phthalates were separated almost completely (Figure 1E). Figures 2 and 3 are t h e chromatograms of alkylbenzenes and ketones on the 4.5-mm i.d. column, respectively. Figures 2A and 3A are t h e examples of SEC and Figures 2B and 3B t o 3E are those of SEC-NPPC. T h e increased resolution of alkylbenzenes by SEC-NPPC is predominant. The insufficient resolution of ketones by SEC (Figure 3A) was somewhat improved by adding n-hexane t o t h e mobile phase, though peaks of acetone and M E K were still unresolved. Phthalate esters were completely separated on t h e NPAC system employing the 2.3-mm i.d. column using n-hexane as a n eluent. The result is shown in Figure 4 together with those of alkylbenzenes and ketones. Similar separation on SEC as in Figure 4 could be done using two Shodex A801 S E C col-
384
ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979
Table I. Peak Half-Width and the Resolution of Adjacent Peaks of Phthalate Esters on the SEC-NPPC System mobile phase DNP DBP DEP DMP chloroform half-width, 0.18 0.17 0.18 0.17 mL resolution' 1.04 0.61 0.64 chloroform-n-hexane half-width, 0.18 0.18 0.13 0.17 9:l mL resolution 1.18 0.59 0.94 chloroform-n-hexane half-width, 0.19 0.19 0.19 0.20 8:2 mL resolution 1.24 0.77 0.94 chloroform-n-hexane half-width, 0.21 0.20 0.21 0.21 7 :3 mL resolution 1.26 0.90 0.90 chloroform-n-hexane half-width, 0.24 0.23 0.21 0.23 5:5 mL resolution 1 . 3 3 1.12 0.93 a Resolution = 1.177 ( V 2 - V l ) / ( W ( o , j l i , W(o.5)2). elution volume ( m L ) ; Wti(o.5) = peak half-width.
V=
umns of 8 mm i.d. X 50 cm length (2). However, total separation time by SEC was about 26 min under a flow rate of 1 mL/min. T h e cost of the SEC columns is about ten times that of the other two columns. Sample load on a 4.5-mm i.d. column increases 3.8 times that on a 2.3-mm i.d. column, but an eluent consumed by adsorption chromatography employing a 4.5-mm i.d. column would be about 5 times that by SECN P P C with the same column dimensions. This value was calculated by considering column size and the retardation of sample elution due t o adsorption (compare the elution time of D M P in Figure 1E and Figure 4A). Hence, combination of SEC and N P P C would have proved advantageous over a single usage of SEC, NPAC, or NPPC. Benzene, toluene, and ethylbenzene were not separated on the NPAC system employing a 2.3-mm i.d. column (Figure 4B). However, their elution volumes determined individually were not identical ( 5 ) and they could be separated by SEC-NPPC. A longer column is required for further separation. T h e case of acetone and MEK was the opposite. Though acetone and MEK could be separated on the NPAC system (Figure 4C), they were hardly separated on the SEC system (Figure 3A) and SEC-NPPC system (Figure 3B-3E). Partial separation of adjacent peaks on SEC and NPAC systems are required for the success of the SEC-NPPC technique. Separation on the SEC system is the first requisite. Table I gives peak half-width and the resolution of adjacent peaks for phthalate esters on the SEC-NPPC system. T h e resolution increases with increasing n-hexane content, followed by the increase of peak half-width. T h e space between adjacent peaks must increase enough t o compensate for the decrease of the resolution accompanied by the increase of peak width. Figure 5 illustrates the results of SEC and NPPC of phthalate esters on the Shodex A801 GPC column of 8-mm i.d. Separation of the esters by SEC on one column was insufficient, but separation by SEC-NPPC was complete. However, the number of theoretical plates of the column decreased rapidly after this experiment, because the column was packed under the swollen state and the addition of nonsolvents such as n-hexane resulted in shrinkage of the gel in the column. In the SEC-NPPC step, the gel must be packed into the column with the solvent including n-hexane. In our experiment, the gel was packed with a mixture of chloroform and n-hexane (55). Table I1 indicates the reproducibility of the SEC-NPPC technique by successive change of the mobile phase in the order of chloroform, chloroform-n-hexane (5:5), chloroform, and finally chloroform-n-hexane ( 5 : 5 ) . Consistent
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Figure 5. Size exclusion and normal-phase partition chromatograms of phthalate esters on the Shodex A801 GPC column of 8-mm i.d. Flow rate: 1.0 mL/min. Attenuation: 0.16 AUFS. Sample volume: 100 FL. Mobile phase: (A) chloroform (pump pressure 30 atm), (B) chloroform-n-hexane (7:3 v/v) (pump pressure 21 atm). Solutes: (1) DNP, (2) DBP, (3) DEP, (4) DMP
Table 11. Reproducibility in SEC-NPPC Technique elution mL run no. mobilephase DNP DBP DEP DMP N b 1 chlornform 3.57 3.88 4.06 4.25 7700 2 chloroform-n- 4.05 4.58 5.00 5.34 6300 hexane (5: 5 ) 3 chloroform 3.56 3.89 4.11 4.27 7600 4 chloroform-n- 4.02 4.52 4.97 5.29 6700 hexane ( 5 : s ) a Average in triplicate measurements. Relative average deviation is within 0.8%. Number of theoretical plate measured b y benzene.
elution volumes and numbers of theoretical plates prove that this technique is the reproducible and powerful method in high performance liquid chromatography. The technique of SEC-NPPC will be proved to be effective when unknown samples are separated. T h e SEC technique provides information about the number of components in the unknown sample. As the elution order of solutes in SEC and NPPC will not reverse each other, the number of components in the unknown sample can be estimated precisely by SEC-NPPC and the solute eluted behind will not be overlooked. T h e preparation of more rigid polystyrene gels for small molecules will increase the effectiveness of the SECNPPC technique, because the n-hexane content can be increased more than 50%. The SEC-NPPC technique will be applied to the separation of mixtures of several compound types besides homologues.
LITERATURE CITED A . Krishen and R . G. Tucker, Anal. Chem., 49, 898 (1977). S. Mori, J . Chromatogr., 129, 53 (1976). M. Popl, J. Coupek. and S. Pokorny. J . Chromatogr., 104, 135 (1975). Y. Kato, S. Kido, H. Watanabe. M. Yarnamoto, and T. Hashimoto, J . App/. Polym. Sci., 19. 629 (1975). (5) S. Mori, Anal. Chem., 5 0 , 745 (1978). (6) E. L. Johnson, R. Glmr, and R. E. Majors, J . Chromatogr.. 149, 571 (1978).
(1) (2) (3) (4)
RECEI\FDfor review September 11,1978. Accepted November 14, 1978.
