How To Freely Change the Polarity of the Stationary Phase in a Liquid

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Anal. Chem. 1997, 69, 636-642

How To Freely Change the Polarity of the Stationary Phase in a Liquid Chromatographic Column Jan T. Andersson*,† and Gu 1 nter Kaiser

Department of Analytical and Environmental Chemistry, University of Ulm, D-89069 Ulm, Germany

The concept of changing the polarity of the stationary phase in a liquid chromatographic column is developed. Three phases are presented which all contain a sulfur atom in the sulfide form. On oxidation of the sulfur atom with iodosobenzene in the packed column, a sulfoxide group is formed which is considerably more polar. This change can be performed in infinitesimally small steps and convert the phase from essentially unpolar to quite polar. The polarity change is reversible. The separation of several classes of compounds on these new phases in both the oxidized and the reduced forms is compared with that on traditional phases. Even high-performance liquid chromatography (HPLC) has a fairly limited resolving power compared to some other separation techniques like capillary gas chromatography (GC) or capillary electrophoresis. Furthermore, selective detectors are less widely available than in GC. The consequence is that coelution problems are quite common in LC separations. The traditional way to solve such a problem is to optimize the mobile phase,1 and in the last years many computer programs have appeared which facilitate the search for the optimum mobile phase. In reversed-phase LC (RP-LC) the usual binary aqueous solvent mixture can be modified by a second or even third organic component, or for certain applications, a pH buffer or additives like cyclodextrins may be employed in order to achieve the desired resolution. The temperature can also be changed, but this is a parameter that is surprisingly rarely used. It is striking that in a sense the stationary phase plays a subordinate role in the optimization strategy. In RP-LC, phases based on octadecyl-substituted silica gels dominate by far and are likely to be the first choice for most RP separations, although shorter chains are also available commercially. Phases based on other functional groups, such as phenyl or cyanopropyl, seem to be useful for special separations only. Obviously the analytical chemist is quite restricted in the choice of stationary phase, and furthermore, once that choice has been made for a certain separation, the major possibility available in the search for the necessary resolution is to vary the composition of the mobile phase. Although this situation is indeed satisfactory for many applications, it would be of great value to have a way to change the properties of the stationary phase in a way similar to the mobile phase, namely, not in discrete steps, like going from a C-8 to a †

Present address: Institute of Inorganic Chemistry, University of Mu ¨ nster, Wilhelm-Klemm-Strasse 8, D-48149 Mu ¨ nster, Germany. (1) Poole, C. F.; Poole, S. K. Chromatography Today; Elsevier: Amsterdam, 1991; pp 453.

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C-18 phase or from a C-2 to a cyanopropyl phase, but in very small steps. In effect, this would amount to the fine-tuning of the separation characteristics of the stationary phase to suit the requirements of a certain sample. In this paper, we describe a set of stationary phases that are constructed to allow such a fine-tuning of their polarity. The intention was to develop a nonpolar starting phase whose polarity can be changed at will in infinitely small steps to suit a certain application. Additionally it should be possible to perform this change in the packed column. Because each separation may require a different polarity and in many laboratories a column cannot be dedicated to only one separation, the change in polarity should ideally be reversible so that the polarity of the polar phase can be lowered to any desired value above or equal to that of the original phase. These goals were realized by synthesizing RP phases containing a sulfur atom that easily can be oxidized to a sulfoxide (or sulfone) functional group. The reduction back to the sulfide functionality is likewise easy to perform. In this paper, we present the first attempts and show that it is possible to perform such oxidations and reductions in the packed column. The phases were tested using some commonly analyzed mixtures of compounds, including polycyclic aromatic compounds, polychlorinated biphenyls, nitroaromatics, substituted phenols, and biogenic amines. EXPERIMENTAL SECTION Materials. Synthesis of the Stationary Phases. 4-Butenyl phenyl sulfide was prepared by alkylation of thiophenol with 4-bromo-1-butene: bp 110-111 °C (18 Torr); purity >99% by GC. Methyl 4-vinylphenyl sulfide and phenyl 4-vinylphenyl sulfide were prepared from methyl phenyl sulfide and diphenyl sulfide, respectively, and acetyl chloride;2 reduction of the acetophenone to the alcohol3 and thermal dehydration over alumina yielded the desired styrene:4 methyl 4-vinylphenyl sulfide bp 60-62 °C, purity >98%; phenyl 4-vinylphenyl sulfide bp 78-80 °C, purity >99%. The purity was determined through capillary gas chromatography with a flame ionization detector. The chlorodimethylalkylsilanes were synthesized through the traditional addition of chlorodimethylsilane to the unsaturated sulfide with hexachloroplatinic acid as catalyst in toluene.5 The purity was estimated through NMR spectra: 4-(phenylthio)butanodimethylchlorosilane bp 77-78 °C (0.04 Torr), purity 98%; (2) Szmant, H. H.; Palopoli, F. P. J. Am . Chem. Soc. 1950, 72, 1757-1758. (3) Szmant, H. H.; Henley, W. O., Jr. J. Org. Chem. 1954, 19, 1-3. (4) Bachmann, B. G.; Carlson, C. L. J. Am. Chem. Soc. 1951, 73, 2857-2858. (5) Capka, M.; Svoboda P.; Hetflejs, J. Collect. Czech. Chem. Commun. 1973, 38, 3830-3833. S0003-2700(96)00726-3 CCC: $14.00

© 1997 American Chemical Society

Table 1. Physical Data for the Three Phases

phase PhBS (Kromasil) MPhS (Kromasil) DPhS (LiChrosorb) a

carbon loada (%) before after 11.5 10.8 14.3

11.9 11.1 15.0

surface coverage (µmol/m2) 3.28 3.34 2.55

Before and after end-capping.

2-[4-(methylthio)phenyl]ethanodimethylchlorosilane bp 96-97 °C (0.04 Torr), purity 98%; (4-phenylthio)phenyldimethylchlorosilane bp 152-153 °C (0.04 Torr), purity 98%. Methods. Synthesis of Bonded Phases. Kromasil (5 g) 100A 5 µm (Eka Nobel, Bohus, Sweden) or LiChrosorb Si 100, 5 µm (Merck), was dried under vacuum (0.04 Torr) for 2 h at 180 °C. It was placed in a round-bottom flask with a reflux condenser together with 25 mL of dry toluene. The chlorosilane (3.4 mmol/g of silica), dissolved in 10 mL of dry toluene, and 3 mL of pyridine were added at room temperature. The solution was next held at reflux temperature for 12 h. Thereafter, the bonded phase was filtered off, washed thoroughly with dry toluene, methanol, and acetone, and dried under vacuum at 80 °C. Elemental carbon and hydrogen analyses gave the results shown in Table 1. The spherical Kromasil gel has a surface area of 306 m2/g, and the irregular LiChrosorb 292 m2/g. For end-capping, the dry phase was suspended in dry toluene and treated under a nitrogen atmospere with hexamethyldisilazane (1 g/g of silica), refluxed for 4 h, and then treated as the phase above. Column Packing. Stainless steel columns from Latek (4 × 150 mm) or from Merck (4 × 120 mm) were equipped with zero deadvolume fittings. The silica was slurried in acetone (10 mL), sonicated for 30-60 s, and filled into the column using a Knauer pneumatic pump. The column was washed at 700 bar with filtered methanol (50 mL), aqueous ammonium nitrate (0.05 M, 100 mL) and 50 mL of deionized water. Finally, the column was conditioned with pure methanol. Oxidation Procedure. Oxidation to sulfoxide with iodosobenzene: The reagent was synthesized according to ref 6. The amount needed was calculated from the known amount of carbon (before end-capping) on the silica and the weight of stationary phase in the column. It also depends on the degree of oxidation (the percentage of sulfoxide groups) desired. The calculated amount of iodosobenzene was dissolved in 95% ethanol (45 mL) and pumped through the column at 30 °C and a flow rate of 0.2 mL/min. Finally, the phase was washed with 95% ethanol and conditioned with the desired mobile phase. Oxidation to sulfone with m-chloroperoxybenzoic acid: The calculated amount of commercially available reagent was dissolved in dichloromethane (20 mL) and pumped through the column at 25 °C and a flow rate of 0.2 mL/min. Finally, the phase was washed with dichloromethane and conditioned with the desired mobile phase. Reduction of the Sulfoxide Phase. The reduction can be effected either with commercially available Lawesson’s reagent,7 dissolved in dichloromethane or tetrahydrofuran, or with a mixture of (6) Lucas, H. J.; Kennedy, E. R.; Formo, M. W. Organic Synthesis; John Wiley: New York, 1955; Collect. Vol. III, p 483. (7) Bartsch, H.; Erker, T. Tetrahedron Lett. 1992, 33, 199-200.

triphenylphosphine, iodine, and sodium iodide8 in acetonitrile. The calculated amount with a 20% excess was dissolved in the appropriate solvent (30 mL) and pumped through the column at 25 °C and a flow rate of 0.2 mL/min. After a wash with the solvent used, the column was conditioned with the desired mobile phase. After three years of extensive use of the described reagents, some attack of the iodine-containing reagent on the pump hardware was observed. The other reagents used did not seem to cause any problems. Other means of adding the reagents without pumping them through the HPLC pump are now under investigation. Chromatographic conditions: The commercial columns used for comparison were (1) octadecylsilane Hypersil ODS 5 µm, 4 × 100 mm; (2) phenylsilane Hypersil Phenyl 5 µm, 4 × 100 mm; (3) cyanopropylsilane Hypersil CPS 5 µm, 4 × 100 mm. The flow rate was 1 mL/min and the temperature 25 °C except for the phenols, which demanded 2 mL/min and 20 °C. Polycyclic aromatic hydrocarbons (PAHs) and nitroaromatics: A mobile phase of 60% methanol was used with UV detection at 254 nm. Polychlorobiphenyls (PCB): A mobile-phase gradient was used [t(0) 60, t(10) 65, and t(30) 99% methanol; for the cyanopropyl phase t(0) 30, t(10) 35, and t(30) 70% methanol]. Chloro- and nitrophenols: A mobile phase containing acetonitrile and a phosphate buffer (0.05 M KH2PO4, pH 3.25) was used with UV detection at 220 nm. The PCB sample was a mixture of equal amounts of Aroclor 1232, 1242, 1248, 1254, 1260, and 1268. Biogenic amines: A mobile phase consisting of 20% acetonitrile and 80% of a buffer (composition: 4.1 g/L sodium acetate, 4.2 g/L citric acid, 100 mg/L sodium lauryl sulfate, pH 4.3); UV detection at 280 nm. Instruments: A liquid chromatograph (Knauer Type 64) with a gradient programmer and a variable-wavelength UV/visible detector (Knauer, Type 87) were used with a Rheodyne 7125 injection valve with a 25 µL loop. The column was thermostated in a Gynkothek column oven. The data were collected on a HP 3394 A Series II Integrator and evaluated with Software Peak 96 (HP). Dead volumes were determined with thiourea and are the mean of at least three measurements. Solvents were purchased from Zinser (acetonitrile, HPLC grade) and Promochem (methanol and tetrahydrofuran, ChromAR). RESULTS AND DISCUSSION In this first report on phases with variable polarity, we will concentrate on three examples containing a sulfur atom bound to an aromatic group, namely, a phenyl butano sulfide (PhBS), a methyl phenyl sulfide (MPhS), and a diphenyl sulfide (DPhS) phase (Figure 1). (Other phases, including such with purely aliphatic sulfide chains, are at the moment also being developed.) Following their synthesis, the phases were characterized by the carbon load and surface coverage (Table 1) and by the retention characteristics on them of several classes of compounds that are commonly separated using HPLC. These compounds were chosen for their ability to interact with the stationary phase in various ways, e.g., through hydrogen-bonding and electron donor-acceptor interactions. In this way it should be possible to obtain information about the retention mechanism on the starting and the oxidized phases. (8) Olah, G. A.; Gupta, B. B. G.; Narang, S. C. Synthesis 1979, 137-138.

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Figure 1. Chemical formulas of the three ligates: (A) phenyl butano sulfide (PhBS), (B) methyl phenyl sulfide (MPhS), and (C) diphenyl sulfide (DPhS).

The separations on the new phases were always compared with those on traditional phases so that their usefulness could be adequately documented. In many cases, the sulfide phases themselves, without using the oxidation option, provided interesting separations different from those on commercial phases and might thus for certain separations be of great interest in their own right. Since in this first study the emphasis was on the comparison, most runs were performed under identical conditions for the various phases. In this way data could be gained on, for example, the strength of the interaction between the phases and the analytes and on the selectivities that sometimes are sensitive to the composition of the mobile phase. It must be kept in mind that this means that we did not attempt to define the conditions of optimum separation on the phases studied. It is obvious that if an effort is made to achieve an optimal separation, a better separation should easily be obtainable in many cases reported here. Such attempts are in progress and will be reported later. The inital task was to find reagents for the oxidation of the sulfide phase and reduction of the sulfoxide phase. Many reagents are known to effect these transformations, but not all of them fulfill the demands that must be met if the reactions are going to be performed within an LC column and on organic molecules bound to a silica surface. The major demands are that there must be no side reactions with the silica gel, the other organic part of the molecule, or the hardware of the system; the reactions must be reasonably fast and possible under mild conditions compatible with the chromatographic system; the reactions must be quantitative; and the oxidation should lead cleanly to the sulfoxide and not the sulfone. The requirement for a quantitative reaction follows from the demand that it should be possible to produce phases that are oxidized only to a certain desired degree and that it is easiest to determine the degree of oxidation by using an accurately measured amount of an oxidant that is known to react quantitatively with the sulfide. Several reagents were investigated with the model substances DPhS and butyl phenyl sulfide. It was difficult to find an oxidant that also in excess cleanly led to the sulfoxide and those that did required too long reaction times for complete reaction. Finally, iodosobenzene was found to be an excellent reagent9 that met all the requirements above. Not even in a large excess was a sulfone observable, and the reaction was fast (seconds to minutes) in 95% ethanol as solvent, which is ideally compatible with an RP phase. Only iodobenzene is produced as byproduct and is quickly washed out of the column. The needed amount of iodosobenzene can (9) Roh, K. R.; Kim, K. S.; Kim, Y. H. Tetrahedron Lett. 1991, 32, 793-796.

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Figure 2. Reduced plate heights for PhBS, PhBSO, PhSO2, and PhBSred (after one oxidation/reduction cycle). The test substances are PAHs chromatographed with 60% methanol at 1 mL/min.

easily be calculated from the known amount of carbon on the silica (elemental analysis after the synthesis, Table 1) and the weight of stationary phase in the column. The oxidation was carried out by simply pumping the iodosobenzene solution through the column at 0.2 mL/min at 30 °C followed by a washing step (see Experimental Section). The reduction of the sulfoxide was more difficult because most reagents are sensitive to either oxygen or moisture. Two reagents were found that proved to be equally well suited, namely, either a mixture of triphenylphosphine, iodine, and sodium iodide in acetonitrile8 or Lawesson’s reagent7 dissolved in dichloromethane or tetrahydrofuran. Again, simply pumping the solution through the column at 0.2 mL/min at 25 °C returned the sulfoxide to the original sulfide group. The sulfide functionality also offers the possibility of oxidation to the sulfone, but this reaction does not seem to be reversible. In a few cases we also investigated the chromatographic properties of a sulfone phase that was produced by pumping a solution of m-chloroperbenzoic acid in dichloromethane through the column. Although the change of functionality went very smoothly, some loss of carbon from the silica was noticeable with a concomitant change of chromatographic performance. This hydrolysis will be discussed in more detail below. Phenyl Butano Sulfide (PhBS) Phase. The general data for this phase are listed in Table 1. This phase will be discussed in most detail as the results for the other two phases were collected in the same way and can be easily related to it. The carbon load and the coverage lie in the range normally obtained for RP materials. The number of theoretical plates (as measured at k ) ∼10) was 55 500 m-1. In Figure 2, the reduced plate height is plotted as a function of the retention factor with polycyclic aromatic hydrocarbons as test substances. The usual drop in plate height with larger retention factors is observed for the starting and the sulfoxide phase. Interestingly enough, for the sulfone phase and the sulfide phase (“PhBS red.”) which resulted after an oxidation/reduction cycle (sulfide-sulfoxide-sulfide) the plate height did not depend on the retention factor. The phase in its different oxidation states was next investigated with various analytes and compared with three commercial materials, namely, an octadecyl (ODS), a phenyl, and a cyanopropyl phase.

Figure 3. Retention factor for 10 PAHs on PhBS, PhBSO, PhSO2, C-18, a phenyl, and a cyanopropyl phase. For chromatographic conditions, see Experimental Section. Compound identification: B, benzene; To, toluene; N, naphthalene; AN, acenaphthylene; MN, 2-methylnaphthalene; F, fluorene; P, phenanthrene; A, anthracene; MP, 2-methylphenanthrene; MA, 2-methylanthracene.

Polycyclic Aromatic Hydrocarbons. These compounds are very frequently analyzed with liquid chromatographic techniques. They are nonpolar and give good peak shapes on well-packed columns. In this study, a set of compounds with one, two, and three rings was used together with some methyl derivatives. The retention factors in Figure 3 compare very well with those on an ODS phase so that the same retention mechanism appears reasonable. There is no major change in elution order on the polar columns but all compounds elute faster. The one elution reversal of a pair of compounds can be observed for acenaphthylene and 2-methylnaphthalene since on the sulfone phase they elute in the reverse order with baseline separation compared to the sulfide phase; on the sulfoxide phase they coelute. Another pair for which a peak reversal is observable after the oxidation is naphthalene and cumene; on the sulfoxide phase they elute in the opposite order.10 From these two examples it seems that methylated derivatives respond more sensitively to the degree of oxidation of the stationary phase than nonalkylated aromatics do in that their retention factors decrease more strongly. Nitroaromatics. Nitrosubstituted aromatics are electrondeficient and can take part in electron donor-acceptor interactions with electron-rich groups. Nine compounds, from toluene to 2,4,6trinitrotoluene (TNT), were tested on PhBS, ODS, a phenyl, and a cyanopropyl phase, and the resulting retention factors with methanol as the organic modifier are tabulated in Table 2. On the sulfide phase, the compounds elute in order of the number of nitro groups, with toluene first and TNT last. The only coelution occurs between 2,6- and 3,4-dinitrotoluene. The same nine compounds are separated in a totally different order on the ODS phase, with the most polar compound, TNT, eluting first, and the most hydrophobic compound, toluene, last. The elution orders on the two phases point to different retention mechanisms: charge-transfer processes dominate on the electron-rich PhBS and partition on the ODS phase. This elution order is known from other phases that also can act as electron donors with nitroaromatics.11 The cyanopropyl phase shows a more complicated behavior; here TNT elutes in the middle of the field. Probably (10) Michel, F. Diploma Thesis, University of Mu ¨ nster, Mu ¨ nster, Germany, 1995; p 53. (11) Welsch, T.; Dornberger, U.; Lerche, D. J. High Resolut. Chromatogr. 1993, 16, 18-26.

the permanent dipole of the cyano group leads to dipole-dipole interactions with the analytes. The phenyl phase shows very poor selectivity. The interactions on the PhBS phase are obviously stronger than on the other phases since a higher concentration of methanol was needed in the mobile phase for elution within a reasonable time. The donor-acceptor interactions on PhBS are strongly influenced by the choice of organic modifier of the eluent. Aqueous mixtures of modifiers other than methanol were also investigated and were chosen to give similar elution power according to Snyder.12 Acetonitrile led to a broad unresolved peak of eight of the compounds, and only TNT was well separated. Tetrahydrofuran produced several sharp peaks in a narrow elution region with 2,3-dinitrotoluene as the first-eluting, but two pairs of compounds showed coelution. TNT was much more strongly retained than the rest of the analytes. Oxidation of the PhBS to 50% led to the phase designated PhBSO0.5 in Table 2. The other two separations listed are on 100% sulfoxide and on 100% sulfone phases. Since the chromatograms were recorded with the same mobile phase composition on the four sulfur phases, the shorter retention times clearly reflect weaker interactions as the stationary phase becomes more polar. Although the phenyl group in the stationary phase is now electron deficient and thus should not be in a position to donate π-electrons to electron-deficient eluates, on the whole, the same retention order prevails (albeit at shorter retention times). This effect is now being investigated in more detail. However, a different selectivity is noticeable for some of the compounds: 2,3- and 3,4dinitrotoluene shift toward the end of the elution range when the stationary phase becomes more polar. Polychlorobiphenyls. PCBs are common environmental pollutants and occur as mixtures too complex to be resolved into individual compounds by HPLC. Figure 4 shows that the sulfurcontaining phase, and in particular the oxidized phase, leads to a better group separation than either C-18 or a cyanopropyl phase. A closer study of the individual peaks was performed with reference mixtures. When collected fractions were analyzed using GC/MS, it could be shown that the same elution order of PCBs is found on all columns, namely, according to the number of chlorine atoms. The baseline separation on the sulfur phases greatly facilitated the cutting of fractions. Chlorophenols. Since the sulfoxide group is capable of forming strong hydrogen bonds, we expected different selectivites on PhBS and PhBSO for a set of phenols. However, no additional selectivity could be registered for either of those phases compared to C-18. It is plausible that hydrogen bonds do contribute to the retention of phenols, since most of them exhibited slightly longer retention times on PhBSO than on PhBS under identical conditions; this is in contrast to all cases discussed so far, for which the retention times are shorter on the polar phase. Nitrophenols. A set of eight di- and trinitrophenols and dinitromethylphenols was used. Contrary to the chlorophenols, the degree of oxidation of the stationary phase influenced the selectivities considerably, as depicted in Figure 5, possibly because of the additional dipole-dipole interactions which are possible with the nitrogroups. The compounds reacted individually on the change of polarity of the stationary phase with either an increase or a decrease in retention times. A test mixture made up of 10 (12) Snyder, L. R. J. Chromatogr. Sci. 1978, 16, 223-234.

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Table 2. Retention Factors for Nitrotoluenes on Seven Stationary Phasesa stationary phaseb

T

2NT

3NT

4NT

2,3DNT

2,4DNT

2,6DNT

3,4DNT

TNT

PhBS (60) PhBSO0.5 (60) PhBSO (60) PhBSO2 (60) C-18 (50) Phenyl (40) CN (30)

3.08 2.29 1.72 1.14 7.22 3.05 1.57

3.57 2.78 2.14 1.58 3.79 3.41 2.23

4.22 3.26 2.48 1.72 4.51 3.61 2.33

3.97 3.07 2.34 1.69 4.13 3.53 2.25

4.87 4.37 3.71 2.87 2.60 3.94 5.49

7.11 5.42 4.09 2.87 2.99 3.15 2.85

5.41 4.14 3.13 2.29 2.86 3.42 2.78

5.34 4.70 3.91 3.14 2.31 3.83 4.82

11.94 8.19 5.73 4.27 2.36 3.01 2.79

a

Key: T, toluene; NT, nitrotoluene; DNT, dinitrotoluene; TNT, 2,4,6-trinitrotoluene. b Values in parentheses are percent methanol.

Table 3. Retention Factors for Eight Biogenic Amines on Three Stationary Phasesa stationary phase

NE

PhBS PhBSO C-18

5.26 8.29 4.28 5.74 4.29 5.75

E

DNBA 10.08 6.79 10.32

NM

DA

MET EPIN

12.76 16.12 19.73 8.48 10.31 11.40 10.49 13.64 16.51

22.31 12.91 18.39

3MT 45.57 23.26 46.93

a Key: NE, norepinephrine; E, epinephrine; DHBA, 3,4-dihydroxybenzylamine; NM, normetanephrine; DA, dopamine; MET, methanephrine; EPIN, epinine; 3-MT, 3-methoxytyramine. Mobile phase: see Experimental Section.

Figure 4. Chromatogram of an Aroclor mixture on three commercial phases and on variously oxidized PhBS phases. For chromatographic conditions, see Experimental Section.

Figure 5. Retention times for eight nitrophenols on variously oxidized PhBS phases.

out of the 11 EPA phenols showed that they were all separated with only one semiresolved pair (2-chloro- and 2-nitrophenol). The pair 4-nitro- and 2,4-dinitropenol can be a problem but they were separated by ∼4 min on the PhBS phase. Again, a complete optimization was not carried out, but the many combinations possible by changes in the composition of the stationary as well 640 Analytical Chemistry, Vol. 69, No. 4, February 15, 1997

as the mobile phase and pH effects for the acidic phenols could well mean that an even better separation may be achieved. Biogenic Amines. Amines derived from phenylalanine and tryptophan are important analytes in clinical studies. Here we used a synthetic mixture of eight such amines (Table 3). On C-18, a less than satisfactory separation was obtained which easily was improved by using the PhBS phase. Without seriously compromising the resolution, the oxidized phase gave even better chromatographic results, resulting in a reduction of analysis time by nearly half. Again, the mobile phase was not optimized; e.g., a gradient elution could easily be used to speed up the elution of 3-methoxytyramine. Methyl Phenyl Sulfide (MPhS) Phase. The structure of this ligate is shown in Figure 1, and the data for the column used for the studies are listed in Table 1. Structurally it is quite similar to PhBS but the sulfur atom is situated in a more exposed position, and it was therefore of interest to see whether this easier access to the functional group for the eluates leads to modified properties of the phase. The chromatographic properties of this phase were investigated with the same test substances as above. The separations were generally very similar to those described for PhBS. On MPhS, there is a stronger contribution to the retention by each nitro group in the nitrotoluenes than on PhBS, which we interpret as evidence for a somewhat stronger charge-transfer interaction with MPhS. That the retention times are comparable could to some degree be a result of the slightly lower carbon content of MPhS. In general it seems that the position of the sulfur (sulfoxide) group in the ligate is not critical. Diphenyl Sulfide (DPhS) Phase. This phase contains more carbon than the previous phases but shows a slightly lower ligate density (Table 1). The phase in its reduced and oxidized forms showed the same selectivites for PAHs as the previous phases. The nitrotoluenes eluted in the same order as on PhBS. On DPhSO, the retention order agreed with that on the other sulfoxide phases but the contribution of a nitro group to the

retention was lower than on the other two phases. We ascribe this to weaker charge-transfer and stronger hydrophobic interactions between the eluates and the stationary phase. This could also be observed for alkylated PAHs, which on this phase showed a stronger relative retention with respect to their parent compounds than on the previous phases. Desired Degree of Oxidation. It is desirable to be able to select the degree of oxidation of the phase since any value between 0 and 100% can be experimentally useful. Such a mixed phase will obviously have properties intermediate between those of the two pure phases. In order to demonstrate that it is possible to obtain a predetermined intermediate value simply by varying the amount of oxidant used, we first used 0.5 equiv of oxidant in order to obtain 50% oxidation. In practically all cases, the retention factor for the analytes changed to the mean value of the retention factors on the two pure phases. In a second experiment, from a plot of the retention factor versus the percentage of sulfoxide groups in the PhBS phase, a calculation showed that 2,3- and 2,6-dinitrotoluene would have precisely the same retention times for an oxidation degree of 35.6%. This amount of oxidant was weighed in and the phase oxidized. The experimentally determined retention data showed that the two compounds coeluted, precisely as predicted. We have not looked into the longitudinal distribution of the sulfoxide groups within the column for cases with less than 100% oxidized groups. In the early part of the column we expect a complete oxidation due to the fast kinetics of the reactions, and at the end of the column, the stationary phase will most probably be in the unchanged, reduced form. Thus, there is a gradual change along the column from oxidized to reduced phase. The question arises whether this will give rise to other selectivities than if, for instance, two columns, each packed with a different form of the stationary phase, were coupled in series. We have not experimentally investigated this for two reasons. First, we find it more convenient to use one column in which any desired ratio of oxidized to reduced phase can be generated at will; this is hardly possible when two columns are coupled. Second, we would not expect a significant difference in separation in the two situations. In liquid chromatography it seems to be an uninvestigated question, but in gas chromatography it is known that if two columns containing different stationary phases are coupled together, the chromatograms will not be significantly different from the case when the phases are mixed in one column.13 The differences that occasionally can be observed in GC follow from the changing carrier gas velocity along the coupled columns, which has an influence on the plate height of the columns. Due to the much lower compressibility of liquids than of gases, this difference in mobile-phase velocity should be negligible in HPLC. The experiment described above, in which the retention of 2,3and 2,6-dinitrotoluene was determined on a 100% oxidized and a 100% reduced phase, and the retention on a 35.6% oxidized phase was calculated through a linear combination and then experimentally verified, also indicates that the longitudinally varying distribution of oxidized and reduced sulfur functionalities does not pose any problems. Hydrolysis. An advantage of the phases described here is that the oxidation to the sulfoxide is reversible so that a polar (13) Guiochon, G.; Guillemin, C. L. Quantitative gas chromatography for laboratory analyses and on-line process control; Elsevier: Amsterdam, 1988; p 177.

phase can be returned to a more nonpolar phase. Thus, a column once prepared in a more polar state is not dedicated to a certain separation but can be used for other cases that require a less polar stationary phase. We investigated the three phases with this aspect in mind to see if the oxidation/reduction cycle changes the properties of the phases. The ligates are bonded to the silica gel through siloxane bonds that are known not to be completely resistant to hydrolysis and possibly also to other reactions which might take place concurrently with the oxidation and reduction. The loss of ligates should be detectable both through a lower carbon content of the oxidized/reduced phase and chromatographically through shorter retention times under identical conditions. Since loss of ligates means an increase in the number of free silanol groups, interactions of basic compounds with the acidic silanols should be more pronounced if ligates are lost. The end-capped PhBS phases were most thoroughly investigated. One phase showed a decrease of carbon from 11.9 to 10.8% after an oxidation/reduction cycle with extensive retention measurements both before and after each chemical reaction. Another phase was prepared in the same way (with end-capping) and contained 11.6 and 11.3% C after the oxidation/reduction cycle with only a few retention measurements. Separate experiments showed that the trimethylsilyl groups from the end-capping are more sensitive to hydrolysis than the ligates.10. The oxidation procedure contributes about as much to the loss as the reduction with triphenylphosphine/iodine/iodide does. Initial experiments with isopropyl instead of methyl substituents on the silicon atoms to give sterically hindered ligates14 show much enhanced stability toward such losses, and those phases will be investigated in more detail in the future. The other phases were more prone to ligate loss after a complete cycle. For MPhS, the carbon content decreased from 11.1 to 9.1% and for DPhS from 15.0 to 12.9%. For the aromatic compounds (benzene to 2-methylanthracene), the retention factors were between 10 and 24% lower after the cycle than before, which we ascribe to loss of carbon. Since the retention factors are lower on the reduced sulfoxide phases than on the original sulfide phase, it must be shown that the lower k values are not due to an incomplete reduction. Since the selectivities after the reduction were the same as before the oxidation (particularly noticeable for the pair acenaphthylene/2methylanthracene, which shows coelution on the sulfoxide phase), we conclude that the reduction was complete, as verified in the initial model experiments with BPhS. Basic compounds such as the toluidines showed a more pronounced tailing on the phases after oxidation/reduction, and the retention times (compared with toluene as a neutral compound) were longer. An unexplained observation was that the loss of carbon seems to occur only on the first oxidation/reduction cycle. This is illustrated in Figure 6, which shows the retention factors for a series of nitrotoluenes on a PhBS phase as a function of the treatment of the phase. The k values are lowered as a result of the oxidation and then again grow when the stationary phase is reduced back to the sulfide state but not quite to the original value. If this phase is repeatedly treated with reducing agent, no further change is noticed. Oxidation of the phase returns the retention factors to the previous values found on the column in the correspondingly oxidized state. A final reduction back to the (14) Kirkland, J. J.; Henderson, J. W. J. Chromatogr. Sci. 1994, 32, 473-480.

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Figure 6. Retention factors for nitrotoluenes on PhBS in dependence on the treatment of the stationary phase. “red” is treatment of the phase with triphenylphosphine, iodine, and sodium iodide. The relative retention was measured against toluene.

sulfide state restores the retention factors exactly as they were after the first oxidation/reduction. The loss of carbon that is noticeable on the first oxidation/reduction is consequently limited to the first treatment of the phase with reagents. It therefore seems that there might be two kinds of ligates, one that is fairly easily lost on treatment with both the oxidation and the reducing agent and one kind that is stable to those agents and that makes up the largest part of the ligates. This phenomenon is currently being studied. We have demonstrated above that it is possible to vary the polarity of the stationary phase within wide boundaries without ever saying what the “polarity” of a stationary phase is. It seems to be a somewhat undefined concept with little more meaning than that there is a polar functional group in the ligate bonded to the silica gel. This functional group can be anyone of several of very different chemical character and still be labeled “polar”. The sulfone group has a dipole moment of ∼3.9 D, as high as that of the cyano group, but that this dipole moment alone is not sufficient to explain the separations achieved on the sulfone phases is obvious when the separations on the sulfone and the cyano phases are compared. Polar groups can be involved in interactions other than those arising from dipole-dipole or dipole-induced dipole interactions, e.g., hydrogen bonds, and these can be of different strength for different dipoles. Added to that, in the present case the cyano dipole is bound to a short hydrocarbon chain (propyl) while the sulfone group is attached via a longer chain, leading to a situation where interactions of the hydrophobic kind may play a role. It is altogether not surprising that different polar phases, however that polarity is defined, can show different separation characteristics.

liquid chromatography. They fulfill the following important requirements: (1) A controlled change of polarity is possible in infinitely small steps. (2) The polarity change is reversible. (3) The change of polarity can be carried out in the packed column. The separations used as an illustration hint at mixed separation mechanisms for analytes with functional groups, and this can be used to advantage to optimize a separation. It should be repeated that the separations in this work were not optimized for each analyte mixture and phase but rather kept constant for an easy comparison with traditional phases. The phases should have their greatest potential in their ability to give a fine-tuned change in polarity. The substance classes used here as examples were selected to contain different functional groups so that a first impression of the properties of the phases could be gained. Although the separations presented did not reveal dramatic changes in selectivities, several improvements were found. We are convinced that as these phases are explored in more detail, the true potential of being able to change the polarity of the stationary phase to nearly any desired degree in infinitely small steps will be revealed and that the principle of tuning the stationary phase to the requirement of the sample will find useful applications.

CONCLUSIONS The phases presented here show properties that should make them of interest as a complement to traditional RP phases for

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ACKNOWLEDGMENT Financial support from the Deutsche Forschungsgemeinschaft is gratefully acknowledged as is the partial support from the Fonds der chemischen Industrie. This paper is dedicated to Prof. Dr. Wolfgang Jeitschko on the occasion of his 60th birthday. Received for review July 23, 1996. Accepted December 4, 1996.X

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Abstract published in Advance ACS Abstracts, January 15, 1997.