Anal. Chem. 1995,67,1047-1053
Influence of Organic Solvents in Coelectroosmotic Capillary Electrophoresis of Phenols Sonja M. Masselter and Andreas J. Zemann* Institute for Analytical Chemistry and Radiochemistry, Leopold-Franzens-UniversityInnsbruck, lnnrain 52a, A-6020 lnnsbnrck, Ausfria
The addition of organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile) as modifiers in capillary electrophoresis (CE) si@cantiy improves peak shapes and enables the separation of positional isomers of substituted phenols. A reversal of the electroosmotic flow (EOF) to reduce analysis time by migration of the anionic analytes in the same direction as the electroosmotic flow (coelectroosmotic CE) is achieved by using cetyltrimethylammoniumbromide and 1,5-dimethyl-1,5diazaundwamethylene polymethobrornide(hexadimethdne bromide, HDB, Polybrene) as EOF modifiers. The influence of various organic modjliers on EOF, electrophoretic mobilities of the analytes, and theoretical plate numbers is investigated. A high pH value of the buffer electrolytes above the pKAvalue of the phenols is chosen. The impact of various organic moditiers and the influence of the chain length of the alcohols on the separation behavior of alkyl phenols is investigated. Various applications of capillary electrophoresis (CE) for the separation of and uncharged4s5compounds have gained importance as they became suitable alternatives to wellestablished HPLC methods. The reasons for this development are multifaceted. CE requires considerably less amounts of chemicals in daily routine operation, and in most cases the separation is performed in an uncoated fused silica capillary. In traditional CE methods, anionic analytes migrates in the direction opposite to the electroosmotic flow (counterelectroosmotic methods) and are thus detected rather late. The PKA value of silanol groups at silica surfaces ( ~ 7 . 1 is ) ~low compared to the first dissociation step of the free acid H4Si04 (9.7). Although the exact ~ K A value of fused silica is difficult to determine, electroosmotic flow becomes significant above pH 4.7 By applying a high voltage through the capillary, the positively charged outer Helmholtz layer forces the contents of the capillary to move toward the cathodic side (electroosmotic flow, EOF). To reverse the direction of the electroosmotic flow, cationic buffer additives are used. This experimental setup also enables the simultaneous detection of both slow and fast anions. By dynamically coating the negatively charged inner surface of a fused silica capillary with (1) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen Th. P. E. M. J. Chromatogr. 1979, 169,11. (2) Jorgenson, J. W.; Lukacs, K D. Anal. Chem. 1981, 53, 1298. (3) Jorgenson, J. W.; Lukacs, K D. Science 1983,222,266. (4) Terabe, S.; Otsuka, K; Ichikawa, K; Tsuchiya, A; Ando, T. Anal. Chem. 1984,56, 111. (5) Otsuka, K; Terabe, S.: Ando, T. f. Chromatogr. 1987, 396, 350. (6) Hair, M. L.; Hertl, W. J. Phys. Chem. 1970, 74, 91. (7) Lambert, W. J.; Middleton, D. L. Anal. Chem. 1990, 62, 1585. 0003-2700/95/0367-1047$9.00/0 0 1995 American Chemical Society
CH3
a) CTAB
b) HDB
Figure I. Chemical structures of CTAB and HDB; n = 20-40.
a layer of either positively charged hemimicelles or polycations, a reversal of the EOF is achieved. With hemimicelles, two molecules of a long-chain alkyltrimethylammonium salt associate to form aggregates with the charged head groups arranged in opposite directions. Choosing a concentration below the critical micellar concentration (cmc), these hemimicelles are attached to the negatively charged silica surface and cause a change of the sign of the [-potential.* Polycations with more than one positive charge per molecule tend to stick to the wall of the capillary even at very low concentrations. These dynamic coatings are not stable as they are easily eluted at alkaline conditions. Thus, these compounds must be added to the buffer electrolyte to provide a constant supply of electroosmoticflow modifier. In this way, the negatively charged outer Helmholtz layer causes an electroosmotic flow from the cathodic to the anodic side of the capillary. Use of a power supply with switched polarity causes anionic compounds to migrate toward the detector (anodic) end of the capillary. With coelectroosmotic CE,9 anionic species are detected before the electroosmotic flow as they migrate in the same direction. This principle was applied for the fast analysis of anions.lOJ1Frequently employed electroosmoticflow modifiers used in this investigation are cetyltrimethylammonium bromide (CTAB,Figure la) and 1,s dimethyl-1,5-diazaundecamethylenepolymethobromide (hexadimethrine bromide, HDB, Polybrene; Figure lb) .12-14 Alkylammonium salts are used for the self-regulatingdynamic control of the EOF with CTAB'5 as well as in the separation of vitamins and antibiotics.I6 Initially, only pure aqueous electrolyte buffer systems were used in capillary electrophoresis. From liquid chromatography experience that organic solvents may improve separation efficien(8) Fuerstenau, D. W. f.Phys. Chem. 1956, 60, 981. (9) Jandik, P.; Bonn, G. Capillay Electrophoresis of Small Molecules and Ions; VCH Publishers, Inc.: New York, 1993. (10) Tsuda, T. J. HRC CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1987, 10,622. (11) Jandik, P.; Jones, W. R; Weston, A; Brown, P. R LC-GC 1991, 9,634. (12) Wiktorowicz, J. E. U.S. Patent 1991, 5,015,350, 1991. (13) Masselter, S. M.; Zemann, A. J.; Bobleter, 0. Electrophoresis 1993, 14, 36. (14) Terabe, S. Trends Anal. Chem. 1989, 8 (4), 133. (15) Chang, H.-T.; Yeung, E. S. Anal. Chem. 1993, 65,650. (16) Nishi, H.; Tsumagari, N.; Terabe, S. Anal. Chem. 1991, 61, 2434.
Analytical Chemistry, Vol. 67, No. 6,March 15, 1995 1047
c i e ~ , ' ~ the . ' ~ application of organic modifiers in CE became commonly accepted. This includes aliphatic alcohols and other organic solvents that are miscible with the buffer. Detailed investigations have been examined for the application of organic solvents in MECC separations of positional isomers,lg isotopically substituted compounds,z0 inorganic ions,21z2zand phthalate estersz3as well as to perform gradient elutionz4and to extend the elution range.25 Furthermore, the influence of organic modifiers on electroosmotic velocity and (-potential have been ~ t u d i e d and , ~ ~even , ~ ~pure organic systems have been introduced for CE p u r p ~ s e s However, . ~ ~ ~ ~ ~the application of solvents is not limited to counterelectroosmotic methods but can likewise be applied to coelectroosmotic application^.^^ The aim of this investigation was to develop a method for the rapid analysis of phenols with capillary electrophoresis. This class of compounds is of special interest with respect to their environmental relevance as pollutants (e.g., chlorophenols) and constituents of plant lignin (alkylphenols). Generally, phenols are weak acids and thus dissociate at high pH values, depending on their substituents. Consequently, we are dealing with anions. It is wellknown that phenolic compounds can be separated under neutral or moderately alkaline conditions with capillary zone electrop h o r e ~ i s ~and ~ , ~micellar ' electrokinetic capillary chromatogra~hy.4~5.32 It is often stated, that the only advantage of coeletroosmotic migration is to achieve rapid separations at the expense of selectivity and resolution. This is not necessarily a setback, as these parameters become significant only if the number of compounds reaches the separation capacity of the capillary. According to the Giddings- Jorgenson relation,*
the resolution Rs will be best when the average mobility ji of two zones and the electroosmotic flow mobility peohave equal values but opposite migration directions. In this equation, Av/V indicates the relative velocity difference and Ap the absolute mobility difference of two different analytes, respectively, N is the separation efficiency, D is the diffusion coefficient, and Vis the applied voltage. It is assumed that only longitudinal diffusion contributes to zone spreading, whereas band-broadening effects resulting from increased temperature, wall adsorption, injection, and electrodispersion are neglected. It must be pointed out that with (17) Bourguignon, B.; Marcenac, F.; Keller, H. R.: de Aguiar, P. F.; Massart, D. L. J. Chromafogr. 1993, 628, 171. (18) Khaledi, M. G.: Strasters. J. IC;Rodgers, A. H.; Breyer, E. D. Anal. Chem. 1990, 62, 130. (19) Fujiwara, S.; Honda, S. Anal. Chem. 1987, 59, 487. (20) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1989, 61. 491. (21) Kaneta, T.; Tanaka, S.; Taga, M.; Yoshida, H. Anal. Chem. 1992, 64, 798. (22) Buchberger, W.; Haddad, P. R. J. Chromatogr. 1992, 608, 59. (23) Takeda, S.; Wakida, S.; Yamane, M.; Kawahara, A; Higashi, K. Anal. Chem. 1993, 65, 2489. (24) Balchunas, A T.; Sepaniak, M. J. Anal. Chem. 1988, 60, 617. (25) Gorse, J.; Balchunas, A. T.; Swaile, D. F., Sepaniak, M. J. HRC CC, J. High (26) (27) (28) (29) (30) (31) (32)
Resolut. Chromatogr. Chromatogr. Commun. 1988, 1 1 , 554. Schwer, C.; Kenndler, E. Anal. Chem. 1991, 63, 1801. Schutzner, W.; Kenndler, E. Anal. Chem. 1992, 64, 1991. Wahlbroehl, Y.; Jorgenson, J. W. Anal. Chem. 1986, 58, 481. Sahota, R. S.; Khaledi, M. G. Anal. Chem. 1994, 66, 1141. Gaitonde, C. D.; Pathak, P. V. J. Chromatogr. 1990, 514, 389. Smith, S. C.; Khaledi, M. G. Anal. Chem.1993, 65, 193. Khaledi. M.G.; Smith, S. C.: Strasters, J. K. Anal. Chem. 1991, 63, 1820.
1048 Analytical Chemistiy, Vol. 67,No. 6,March 15, 1995
increased analysis time t of a solute in a capillary with the length 1, the separation efficiency is reduced by the relation
N = 12/2Dt
(2)
On the one hand, both the average electrophoretic mobilities of two solutes appearing in the right denominator of eq 1 and the electroosmotic flow mobility have the same sign in coelectroosmotic techniques, which would suggest a lower resolution. On the other hand, migration times are kept considerably shorter, which compensates this disadvantage according to eq 2. A limitation of counterelectroosmotic analysis times by the mobility of the EOF has to be considered. Although run times can be shortened by optimizing buffer composition, using short capillaries, and applying high running voltages to establish a high electroosmotic flow mobility, this improvement, however, is paid for by a loss of separation efficiencythrough the formation of Joule heat. According to the literature, rapid counterelectroosmotic separations of phenolic compounds with comparable short analysis times render theoretical plate heights, which are well above the values obtained with coelectroosmotic conditions.13 At first sight, the narrow elution window of coelectroosmotic methods appear to be disadvantageous. The size of the elution window, however, is determined mainly by the number of compounds and the separation efficiency. Yet, very fast coelectroosmotic separations of phenols with an elution window below 1 min and high separation efficiencies are reported.33 A great variety of organic modfiers or mixtures thereof can be added to the buffer, and in combination with variations of pH, the selectivity becomes variable. In addition, any size of the elution window can be adjusted by a reduction of the EOF. Electroosmotic flow modifier systems should be classified not only in terms of their ability to reverse the electroosmotic flow but also with respect to distinctive features of organic solvents used to adjust the separation window. Consequently, optimized buffer electrolytes contain both electroosmotic flow reversing agents and organic solvents, depending on the specific requirements of the application and the chemical properties of the analytes. Although selected phenols can be readily separated without any organic modifier provided,4J~~~-~~ this paper demonstrates that by adding organic solvents (e.g., methanol, ethanol, 1-propanol, 2-propanol, or acetonitrile) the interactions of various phenolic compounds with the EOF moditier (e.g., CTAB or HDB) are partly suppressed. This generally causes an improvement of the selectivity and the separation efficiency by decreasing the theoretical plate heights. EXPERIMENTAL SECTION Apparatus. The analytical equipment consisted of a Quanta 4000 capillary electrophoresis system connected with a system interface module and a personal computer. Data processing was carried out with a commercial chromatography software (Maxima 820). Uncoated, narrow-bore silica capillaries (AccuSep) with an inner diameter of 50 pm, a total length of 32 cm, and an effective separation length of 24.5 cm each were used. All these devices and parts were purchased from Waters Chromatography, Division of Millipore, Milford, MA. On-column detection was performed at 254 nm. (33) Masselter, S. M.; Zemann, A J. J Chromatogr., in press.
Kinematic viscosity measurements were carried out with a Ubbelohde capillary viscometer Type 50110/I (Schott Gerate GmbH, Hotleim a. Ts, Germany) monitored semi-automatically (MGW Lauda Viscoboy 2, Dr. R Wobser KG, Lauda-Konigshofen, Germany), The viscosity values were Hagenbach-corrected. Densities were measured with a glass pycnometer. Reagents. All reagents were of analytical grade. Phenol standard solutions were prepared by dissolving the various phenols (Sigma Chemie Ges.m.b.H., Deisenhofen, Germany, and Aldrich-Chemie GmbH & Co. KG, Steinheim, Germany) in gradient grade methanol Wuka AG, Buchs, Switzerland). Cetyltrimethylammonium bromide (CTAB) and 1,5dimethyl-l,5diazaundecamethylene polymethobromide (hexadimethrine bromide, HDB, Polybrene) were obtained from Sigma Chemie Ges.m.b.H. The solutions were prepared from sodium tetraborate and disodium hydrogen phosphate (Merck, Darmstadt, Germany) by dissolving them in ultrapure water from a MilliQ system with a conductivity of 18 MQ (Millipore Corp., Bedford, MA). Buffers. All buffer electrolyte mixtures had a concentration of 15 mM phosphate and 1.25 mM tetraborate at pH 11. In all experiments, CTAB and HDB concentrations were 0.7 mM and 0.001% (w/v), respectively. The final pH values were adjusted with 0.5 M NaOH. In the experiments with organic solvents, the same amount of NaOH as required with the pure aqueous buffer was added. The final volume was reached through the addition of water and the respective amount of organic modifier. All buffer solutions were vacuum degassed and sonicated prior to usage. Procedure. Prior to analysis the fused silica capillary was purged for -15 min with a CTm-or HDB-free buffer electrolyte with the same composition as the running buffer (purging buffer I). After this pretreatment, the capillary was rinsed with the running buffer (purging buffer II). Between the runs, a purging sequence consisting of 1 min of buffer 1 followed by 2 min of buffer I1 was performed. The preconditioning of the capillary and the purging sequence between two runs were essential to obtain reproducible results. Sample injection was carried out hydrostatically for 5 s at 10 cm elevation. RESULTS AND DISCUSSION
With reversed electroosmotic flow conditions, run times of anions can be decreased substantially. Using high buffer pH values above the p& value of the solutes, a complete dissociation is achieved. Consequently, phenols possess a high apparent mobility when coelectroosmotic CE at alkaline conditions is performed. At the same time, the resolutions between the various zones of phenols become low, especiallywith the CTAB system (Figure 2a). This can be explained by hydrophobic interactions of the analytes with the aliphatic hexadecyl core of CTAB as well as by electrostatic effects, which cause a retention of the phenols and a broadening of peak zones. Electrostatic effects include interactions of analyte anions both with the positively charged wall and with free EOF modifier ions in solution, which have an electrophoretic mobility opposite to the phenolates. It is presumable that hydrophobic interactions are responsible for the retention in particular of higher alkylated phenols. The situation improves with HDB as EOF modifer (Figure 2b), which causes less hydrophobic interactions. This is attributed to the shorter aliphatic sections in the HDB molecule and the fact that the molar concentration of HDB is considerably less than that of "3. The likelihood of interactions of phenolates with free HDB ions is low,
8
without organic modifier
I
0
4
1
minutes
2
3
Figure 2. Coelectroosmotic capillary electrophoretic separation of a set of 12 substituted phenols without organic solvents added to the buffer electrolytes. U = 20 kV, I = 60 PA; EOF modifier, (a) 0.7 mM CTAB, (b) 0.001% (w/v) HDB. Peak identification: (1) 4-hydroxybenzoic acid, (2) 4-hydroxy-3,5-dimethoxybenzoic acid, (3) 4-hydroxy-3,5-dimethoxycinnamic acid, (4) 4-hydroxy-3,5-dimethoxybenzaldehyde, (5)3-methylpheno1, (6) 4-methylpheno1, (7) 2-methylphenol, (8) 3,4-dimethylphenol, (9) 2,3-dimethylphenol, (10) 2,6dimethylphenol, (11) 2,3,5-trimethylphenol, (12) 2,4,64rimethylphenoI, and (M) EOF marker (formamide).
due to the small concentration of HDB and its strong a€6nity to the capillary wall, as described by Wiktorowicz.12 In addition, the structure of the dynamic coating may influence the type of interaction,as CTAB operates on hemimicelles, whereas the HDB type coating is rather coil-shaped. However, spread zone widths in the separation with HDB as EOF modifier suggest electrostatic interactions of the phenolates with the capillary wall. The negative effect of analyte-EOF modifer interactions can be overcome by adding organic solvents to the buffer electrolyte. This results in peak zones with higher theoretical plate numbers and improved selectivities in the electropherogramsof substituted phenols.'3 Furthermore, the flow velocity of the EOF decreases with an increasing amount of organic solvent. These effects are of multifarious origin. The addition of organic solvents results in major changes of physicochemical,electrochemical and chemical properties of the running buffer, as well as of the analyte molecules and the EOF modifier. Likewise, the alteration of the pH value in the presence of organic solvents and its influence on the analytes has to be considered. The investigatedphenols are likely dissociated in the solvent systems as long as the pH of the buffer exceeds the pK,. Note that throughout the pH range of 10-12 in systems with organic modifiers, separation efficiencies are higher than in pure aqueous systems.33 Influence of Organic Modifiers on I, c, and 5. The electroosmotic mobility pea, which describes the magnitude and the direction of the bulk flow in the capillary is defined by (3) Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
1049
2.5 +methanol ethanol - c .1-propanol - - 2-propanol -0acetonitrile
- -
2.0
1.5
1 .o
0.5
I
-/ 0
10
20
30
40
50
-Z-propanol/HDB -acetonitrile/HDB
0
25
20
in which E is the applied electric field, 17 and E are the absolute viscosity and the relative permittivity of the buffer solution, respectively, and 5 represents the electrokinetic potential between the bulk solution and the electric double layer on the capillary wall. A determination of the absolute viscosities of electrolyte buffers with various contents of organic modifer was performed. The viscosities of the acetonitrile buffers remain more or less constant over the investigated concentration range. In contrast, the systems with the aliphatic alcohols show significant dependencies of the viscosities on the modifier concentration (Figure 3). This effect is well-known from liquid chromatography experience.%The comparison of absolute buffer viscosities shows a more s i m c a n t increase in the systems with protic solvents (methanol, ethanol, propanol). This is consistent with the binary water-alcohol systems. The deviations between the CTAB and HDB systems were negligible. A direct measurement of the (-potential of the inner surface of the silica capillary was not performed in this investigation. However, the alteration of the relative permittivities can be discussed in relation to the ratios of organic modifer in the aqueous buffers. It is known that in binary systems consisting solely of water and organic solvents the relative permittivities decrease compared to pure water. The formation of ion pairs consisting of analyte compounds and EOF modifier is not likely as the decrease of the relative permittivity is within limits unless a significant concentration of organic modifier in the buffer system is reached.35With organic modifier concentrationsabove 50% (v/ v), peak broadening is observed that is likely due to the formation of ion pairs. Several explanationsof the impact of organic modifiers on the 5-potential are given by Reijenga et al.,36and similar effects are presumably responsible for the alteration of the electroosmotic flow in coelectroosmotic CE applying organic modifiers (Figure 4). EOF mobilities are not solely dependent on changes of the viscosities and relative permittivities of the buffers. Moreover, the 5-potential is influenced by the addition of organic modifier. Above 30% (v/v) methanol a marked reduction of the EOF in the CTAB system is observed which can be attributed both to a (34) Handbuch der HPLC; Unger, IC K, Ed.; GITVerlag: Darmstadt, Germany, 1989. (35) Longhi, P.; Mussini, T.; Rondinini, S. Anal. Chem. 1986,58,2290. (36) Reijenga, J. C.; Aben, G. V. A; Verheggen, Th. P. E. M.; Everaerts, F. M.]. Chromatogr. 1983,260, 241.
1050 Analytical Chemistiy, Vol. 67, No. 6, March 15, 7995
30
35
-me!hanol/HDB 0 methanol/CTAE
- -
40
1 45
%organic modifier (vlv)
% organic modifier (vlvl
Figure 3. Absolute viscosities of buffer systems with various organic solvents at 25 "C. For details, refer to Experimental Section.
-
1-propanol/HDE 4ethanollHDB
Figure 4. Dependence of the electroosmotic flow mobilities of various solvent systems on the organic solvent concentration of the buffer electrolyte. U = -20 kV; EOF mobilities without organic modifier, 2.58 (0.001% (w/v) HDB) and 5.60 (0.7 mM CTAB).
dynamic equilibrium in which organic modifier and CTAB ions are adsorbed at the silica and to structural changes of the hemimicelle itseK38 With constant running voltage, a decrease in produced current is observed when organic modifiers are added to the buffer. This is due to a variation of the conductance of the electrolyte, as the ionic strength is reduced with increasing contents of organic modifer, which directly influences the EOF. At this point, the type of EOF modifier has to be taken into account, whether we are dealing with hemimicelles or polycations. Organic Modifiers and CTAB. Alkyltrimethylammonium bromide based EOF modifiers are easily affected by buffer additives as the reversal of the EOF is dependent on the formation of hemimicelles. This occurs if the surfactant concentration exceeds the critical hemimicelle concentration. It is known from the literature that short-chainaliphatic alcohols at low concentrations usually have little influence on the critical micelle concentration or the micellar dissociation degree.39 However, at higher concentrations, methanol affects both the critical hemimicelle concentration and the ionization degree of CTAB hemimicelles as well as ion pair formation of CTAB hemimicelles with corresponding buffer anions.38 The electropherogramsin Figure 5 are a continuation of Figure 2a, which shows the situation in the pure aqueous system. It is obvious that the elution window is increased, the selectivity is markedly improved, and eventually a baseline separation is achieved in the CTAB system upon addition of methanol. A plot of the corresponding electrophoretic mobilities of the phenols vs the percentage of methanol demonstrates that with increasing concentrations of methanol the electroosmotic flow mobility is reduced and some phenols increase their electrophoretic mobilities (Figure 6). The latter effect is more obvious with methylated phenols, which have almost no effective electrophoretic mobility in the pure aqueous system, although the pH of the buffer exceeds the pK,. At 40% (v/v) methanol, the methylated phenols are separated and partly manifold their effective mobilities. Since phenolic compounds, especially alkylated species, show high solubilization rates in CTAB micelles,4Oit is likely that interactions (37) Benz, N. J.; Fritz, J. S. J. Chromatogr. 1994,671, 437. (38) Zana, R; yiv, S.; Strazielle, C.: Lianos, P.J. Colloidhtelface Sci. 1981,80, 208.
(39)Treiner, C. J. Colloid Inte$uce Sci. 1983,93, 33.
40% vlv organic modifier
methanol (CTAB)
J
It
0
2
-___________
water
lo4 I
I
..
2 ir----
(-_--
_ ----*-- _ _ _ ______*--------------*-- - _ _ _ _ _ _
1-
0
10
20
ethanol
1-propal DI
minutes 6
4
Figure 5. Coelectroosmotic capillary electrophoretic separation of phenols with 0.7 mM CTAB as EOF modifier and methanol as organic modifier. U = -25 kV; (a) 20% methanol, l = 57 PA; (b) 30% methanol, I = 48pA; (c) 40% methanol, I = 40pA. Peak identification as in Figure 2. [cm2V'S''] x
methanol
Figure 7. Dependence of the electroosmotic flow mobility and the effective electrophoretic mobilities of phenols on the chain length of homologous aliphatic alcohols at a concentration of 40% (v/v). Identification as in Figure 2.
30
40
% methanol (vh)
Figure 6. Dependence of the electroosmotic flow mobility and effective electrophoretic mobilities of phenols on the methanol concentration of the buffer with CTAB as EOF modifier. Identification as in Figure 2.
of phenolates with CTAB ions and hemimicelles are of both electrostatic and hydrophobic natures. This elucidates the low electrophoretic mobilities of the alkylated phenols in alcohol-free buffers, although the concentration of CTAB is kept below the cmc, which is 1 mM in pure aqueous systems.41 With other aliphatic alcohols (ethanol, 1-propanol,2-propanol) or acetonitrile added to the buffer, no significant improvements (40) Menger, F. M.; Portnoy, C. E. 1.Am. Chem. SOC.1988,122,154. (41) Mukerjee, P.; Mysels, K. J. Critical Micelle Concentrations of Aqueous Surfactant Systems; National Standard Reference Data System, National Bureau of Standards, 1971; Vol. 36, p 57.
of separation efficiencies can be achieved with phenolic compounds as analytes. Moreover, EOF mobilities decrease even with small amounts of added solvent. Organic Modifiers and HDB. Generally, HDB allows a greater variety of organic modifiers to be used. The reduction of the EOF with increasing amount of alcohol is due both to hydrophobic interactions of the alkyl chain of the alcohol with the methylene groups of the HDB and to increasing viscosities of the buffer systems. 1-Propanol has a more significant influence on the EOF than 2-propanol, although viscosities of corresponding 2-propanol systems are higher. Particularly with 1-and 2-propanolthe differences in the relative permittivities are assumed to be rather small, as described with binary alcohol-water 1-Propanol shows its EOF reducing effect expressed by eq 1rather by altering the (-potential through interactions with HDB than by a viscosity increase or altered permittivities. The influence of the first three homologous alcohols at concentrations of 40% (v/v) compared to an aqueous electrolyte on the electroosmotic flow and on the effective electrophoretic mobilities of the phenols is shown in Figure 7. The shift of water to methanol is more significant than that between the alcohols. Ethanol and methanol show similar effects, although hydrophobic interactions with shorter alkyl chain lengths and hydrogen bonds with water become a considerable mechanism directly influencing the Helmholtz double layer. In contrast to the EOF, the effective electrophoretic mobilities of the investigated phenols show only little dependence on the chain length of the alcohols (Figure 7). The phenolic acids and the aldehyde slightly reduce their mobility, whereas alkylated phenols remain almost constant. 2-Propanol serves more like methanol rather than like 1-propanol. In the case of acetonitrile, the aprotic character of the solvent has less influence on the adsorption of water on the surface of the modified capillary. Relative permittivities of binary acetonitrile-water mixtures decrease less than the corresponding alcohol-water systems.43Acetonitrile as a polar molecule has a (42) Longhi, P.; Mussini, T.; Rondinini, S. Anal. Chem. 1986,58, 2290. (43) Rondinini, S.; Longhi, P.; Mussini, P. R.; Pozzi, M.; Tiella, G. Anal. Chim. Acta 1988,207, 211.
Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
1051
EOF 11 12
I
n
12
8
a) without organic modifier 4 2
, ...-ihanol 4 J U Ut$l.. Y Un U U U
I’; I1
4I
40% vlv organic modifier 1
h
.. 0
2
4
I
minutes 6
Figure 8. Influence of various organic solvents at 40% (v/v) concentration on the coelectrophoretic separation of phenols with 0.001% (w/v) HDB. U = -30 kV. Peak identification as in Figure 2.
low affinity for the EOF modifier and thus less iduence on the modified surface. This corresponds with the observation that below 30% (v/v) no reduction of the EOF occurs. Thus a selective adjustment of the elution window is possible by reducing the EOF through the addition of an appropriate organic solvent. In Figure 8a-e, the effects of various organic modifiers on the separation of selected phenols is demonstrated. The organic solvents influence both the EOF and the electrcphoretic mobilities. The order of migration remains the same, independent of the type of organic modifier. Without an organic modifier, 2- and Cmethylphenol coincide. With acetonitrile, two substances are distinguishable, and with alcoholic modifiers, a separation of all the phenols is achieved. To demonstrate the direct influence of the organic modifer on the effective electrophoretic mobilities, an overlay of two electropherograms is shown in Figure 9. In this particular case, the running voltage was adjusted to obtain identical elution windows. Using an aqueous buffer (Figure 9a), the peak zones are closer together, apparentlywith lower resolutions than reached in buffer systems with organic solvents. With 40% (v/v) 2-propanol as organic modifier, the running voltage has to be chosen considerably higher to obtain equal currents and an adequate EOF mobility. At the same time the resolution of the peak zones improve, with all methylphenol isomers being separated (Figure 9b). It is shown that improved resolutions, selectivities, and separation efficiencies are due to the presence of the organic modifer rather than to the reduced EOF and the increased separation window. Figure 10 depicts the influence of Zpropanol on the EOF and the effective electrophoretic mobilities of the 1052 Analytical Chemistry, Vol. 67, No. 6,March 15, 1995
2
3
4
5
minutes 6
Figure 9. Coelectroosmotic capillary electrophoretic separation of phenols with 0.001% (w/v) HDB as EOF modifier and (a) without organic modifier and (b) with 40% (v/v) 2-propanol. (a) U = -10 kV, I = 21 PA; (b) U = -30 kV, I = 29 PA. Peak Identification as in Figure 2. 5-
,~cltp [cm’V’S”]
0
x
10
io4
20
30
40
50
% 2-propanol (vlv)
Figure 10. Dependence of electroosmotic flow and the effective electrophoretic mobilities of phenols on the concentration of P-propanol in the buffer with 0.001% (w/v) HDB as EOF modifier. U = -20 kV. Identification as in Figure 2.
investigated phenolic species. Generally, the values decrease with increasing amount of propanol, in particular with concentrations up to 20% and above 35-40% (v/v). The presence of organic modifiers iduences the effective electrophoretice mobilities by altering the mass to charge ratio, which increases both selectivity and efficiency. This effect is more marked with 1- and 2-propanol. Theoretical plate numbers increase with increasing amount of organic mod6er. By o p h b ing the percentage of organic modifier and the running voltage, up to 73 500 theoretical plates can easily be achieved, which
Table 1. Separation Efficiency Expressed by Theoretical Plate Numbers per Effective Separation Length and Heights Equivalent to a Theoretical Plate of Selected Phenols Performed with Various Amounts of Organic Modifiers'
I OCA)
theor plate numbers 4-hydroxy-3,5 4-hydroxy-3,5 dimethoxybenzoic dimethoxybenz3-methylphenol 2,3-dimethylphenol 2,4,6trimethylphenol acid aldehyde
HDB no organic modifier 40%methanol 40%ethanol 40% l-propanol 40%2-propanol 40%acetonitrile
10 25 30 30 30 30
39 37 33 31 30 85
26 200 (9.35) 29 400 (8.33) 31 600 (7.75) 28 900 (8.48) 38 700 (6.33) 17 200 (14.2)
29 200 (8.39) 36 500 (6.71) 38 200 (6.41) 35 000 (7.00) 41 900 (5.85) 12 000 (20.4)
47 300 (5.18) 47 800 (5.13) 61 500 (3.98) 57 300 (4.28) 65 700 (3.73) 16 900 (14.5)
32 100 (7.63) 45 100 (5.43) 67 600 (3.62) 57 100 (4.29) 74 500 (3.29) 17 200 (14.3)
29 200 (8.39) 35 000 (7.00) 60 800 (4.03) 57 300 (4.28) 74 500 (3.29) 31 400 (7.80)
20 25
28 39
75 200 (3.26) 41 700 (5.88)
47 500 (5.16) 39 200 (6.25)
56 100 (4.37) 38 200 (6.41)
39 200 (6.25) 28 400 (8.63)
22 500 (10.9) 16 700 (14.7)
CTm 40%methanol 40%methanol a
Values in parentheses are for HETP bm).
represents heights equivalent to a theoretical plate (HETP) of less than 3.5 pm (Table 1). A direct comparison of CTAB- and HDBbased EOF moditiers demonstrates lower theoretical plate numbers in particular of higher methylated phenols in the CTAB system, which is due to analyte-EOF moditier interactions. The lower number of theoretical plates in the case of acetonitrile and methanol is partly due to the formation of Joule heat. In particular with acetonitrile the produced electrical power is substantially higher than in alcoholic or pure aqueous buffer system. Summing up, coelectroosmotic capillary electrophoresis can be considered a powerful tool in the fast separation of phenolic compounds. Moreover, by adding organic modifiers, a great
variety of running conditions can be established to optimize separations. This enables the separation of positional isomers of phenols with both short times and high separation efficiencies, which otherwise is not easily achieved with comparable counterelectroosmotic methods and commercial standard equipment. Received for review August 30, 1994. Accepted December 19, 1994.@ AC940873+ @
Abstract published in Advance ACS Abstracts, February 1, 1995.
Analytical Chemistv, Vol. 67, No. 6, March 15, 1995
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