Separation of dimer acids using enhanced-fluidity liquid chromatography

Analytica Chimica Acta 449 (2001) 221–236

Separation of dimer acids using enhanced-fluidity liquid chromatography Jun Zhao, Susan V. Olesik∗ Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, USA Received 27 March 2001; received in revised form 31 July 2001; accepted 3 August 2001

Abstract The polymerization products of C18 fatty acids are complex mixtures of high dimeric, trimeric and higher molecular weight liquid acids. The current separation method for these mixtures involves a lengthy normal-phase HPLC gradient and the use of primary standards that are prepared via a semi-preparative HPLC. In this study, enhanced-fluidity liquid mobile phases, methanol/CO2 and methanol/CHF3 , were studied as a possible alternative methodology for the separation of dimer and trimer acids. This study demonstrated that enhanced-fluidity liquid chromatography provides several advantages over normal-phase HPLC with conventional solvents. These advantages included faster speed of analysis, lower consumption of organic solvents, and much improved sample throughput. The best separation of dimer acids was achieved on a silica column using a steep methanol concentration gradient. Methanol/CO2 and methanol/CHF3 mixtures showed comparable elution strength using this steep gradient on the silica stationary phase. However, these polar compounds showed a normal-phase type retention on five reversed-phase columns which indicated an unusual retention mechanism. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Dimer acid; Fluoroform; HPLC; Enhanced-fluidity liquid

1. Introduction In liquid chromatography, solvent strength and viscosity are among the most important properties of any mobile phase. An appropriate solvent strength is essential to achieve reasonable retention on a given stationary phase; low viscosity is also highly desirable, because it will allow high speed of analyses due to the fast flow rates possible. Enhanced-fluidity liquid chromatography (EFLC) addresses the above two issues in a unique way. Enhanced-fluidity liquid mixtures are defined as ∗ Corresponding author. Tel.: +1-614-292-0733; fax: +1-614-292-1685. E-mail address: [email protected] (S.V. Olesik).

common liquids, such as methanol, water, tetrahydrofuran (THF) and hexane, to which large proportions of a low viscosity, liquefied gas, such as CO2 or CHF3 , have been added. These mixtures maintain a solvent strength similar to that of the pure organic component even when as much as 40–50 mol% of a fluidity modifier is added, while the viscosity of such mixtures is substantially reduced with the addition of the liquified modifier. Improvements in chromatographic performance, such as increased efficiency, gains in speed of analysis, lower pressure drop and highly tunable solvent strength have been well documented in normal- and reversed-phases liquid chromatography [1–6], size exclusion chromatography (SEC) [7,8], chiral HPLC [9], and liquid chromatography at the critical condition for characterization of polymer

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 3 3 7 - X

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Table 1 Fundamental properties of dimer and trimer acids [3,4]

Number of carbon atoms Number of carboxyl groups Approximate MW

Monomer

Dimer

Trimer

18 1 283

36 2 565

54 3 850

functionality distribution [10–12]. In this study, we will demonstrate the utility and potential of the EFLC technique in optimizing the separation of dimer and trimer acids. Dimer and trimer acids are polymerization products of C18 fatty acids. Often derived from tall oil, oleic acid and mixed vegetable oils, these high molecular weight acids are complex mixtures of monobasic, dibasic, tribasic and also a small amount of other higher polybasic acids [13]. Table 1 lists some fundamental characteristics of the monomeric, dimeric and trimeric components of the polymerization products [14,15]. The monomer components include not only the residual C18 fatty acids after polymerization, but also any other monobasic acid which has a molecular weight close to that of the starting fatty acids [16]. The dimer components are C36 aliphatic dibasic acids. Possible structures include a long-chain dicarboxylic acid with two alkyl side chains; simple carbon–carbon bonds and cyclic rings. The composition of these structures depends on the level of unsaturation in the starting C18 fatty acids and other reaction conditions. The trimer components are C54 long-chain tricarboxylic acids. Possible structures of the trimer acid are similar to those of the dimer acid, only with more complexity due to the additional 18 carbon atoms and one carboxyl group [15]. Therefore, in any dimer or trimer acid, there are many possible isomers, such as positional and geometrical isomers of the C=C double bonds as well as other structural isomers. Although they have relatively high molecular weights, dimer and trimer acids are still liquids of a high viscosity at room temperature, which is due to the presence of many branched and cyclic isomeric structures [14]. Dimer and trimer acids, and their derivatives are often used to adjust the performance properties of industrial chemicals [15]. The application and market of these acids include, but are not limited to, paints and coatings, elastomers, lubricants, plasticizers, corrosion inhibitors, soaps and adhesives. The utility of

dimer and trimer acids is controlled by many specific physical and chemical properties, most of which are determined by the amounts of dimer and trimer components in the mixture. Therefore, the composition in terms of monomer, dimer and trimer weight percentages is critical information. A variety of chromatographic techniques has been used for the analysis of dimer and trimer acids, including SEC, thin layer chromatography (TLC), GC and HPLC [17,18]. SEC separated monomer, dimer and trimer components according to their size. Therefore no information on functionality distribution was obtained [19,20]. TLC was used to estimate the dimer acid composition as their methyl esters but the precision was poor [21]. An improved TLC–FID method was reported and recommended for process control applications [22]. Dimer methyl esters were also separated using high temperature GC on a short column [23]. However, the separation of dimer and trimer components was incomplete and sample degradation may have occurred during the analysis at high temperatures. The separation of dimer and trimer acids by the current normal-phase HPLC method [17,18] is advantageous over the previous techniques. To our knowledge, the reversed-phase separation of dimer and trimer acids has not been reported, which is probably due to their high polarity. Currently, GC and HPLC coupled with fluorescence detection are the most commonly-used methods for the analysis of free fatty acids. However, both methods require derivatization. In GC, free fatty acids are converted to their methyl esters (FAME) [24,25]; while in HPLC, they are often derivatized by some labeling agents for fluorescence detection [26,27]. Interestingly, supercritical fluid chromatography (SFC) has been used to separate fatty acids and their methyl esters on reversed-phase C18 columns previously [28,29]. Free fatty acids from C8 to C24 were separated on a C18 column using neat supercritical CO2 as the mobile phase. The reversed-phase separation was achieved using a pressure gradient. Free fatty acids from C8 to C24 were well retained on C18 columns in those studies. The best separation was achieved on a heavily end-capped ODS-silica column with wide pore (300 Å) particles [28]. Broad chromatographic bands were observed and were believed to be caused by interactions between free carboxyl groups and residual silanol groups on the surface. Separation of these

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fatty acids on other C18 columns with smaller pore structure (100 Å) and less end-capped (therefore, presumably more surface silanol groups) could not be achieved due to the much broader bands. Currently, the composition of the commercial dimer and trimer acids is analyzed by a normal-phase HPLC method, recommended by the American Oil Chemists’ Society (AOCS) [18]. The components of dimer and trimer acids are separated based on their polarity by normal-phase HPLC on a bare silica gel surface in ∼25 min, while the total run time is ∼40 min including mobile phase re-equilibration time. In addition to the gradient, primary standards are required to verify that the instrumentation is functioning properly. Primary standards are dimer acid samples whose compositions have been determined gravimetrically by a semi-preparative HPLC [18]. The semi-preparative HPLC employs a similar gradient profile as the one in the analytical method, with some adjustments to ensure successful individual fraction collections on the semi-preparative scale within 65 min. Overall the method is quite lengthy and consumes large quantities of cyclohexane and isopropyl alcohol (IPA). Therefore, there is a need for a better separation method in both analytical and preparative scales for the analysis of commercial dimer and trimer acids and the preparation of primary standards. Generally, normal-phase HPLC is not a preferred method, mainly due to some practical issues, such as long equilibration times, difficulty in applying gradients, irreproducible retention times caused by trace level of water, and consumption of large volumes of flammable organic solvents [30]. While SFC often provides a normal-phase separation, it does not share the above problems. In fact, SFC can often replace normal-phase HPLC with an improved speed of anal-

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ysis while using lower quantities of organic solvents [31]. Furthermore, EFLC, a novel separation technique between SFC and HPLC, is expected to bring advantages such as speed of analysis (from SFC) and strong solvent strength (from HPLC) together to provide an overall improved separation result. In this study, separation methods for dimer and trimer acids utilizing CO2 - and CHF3 -based high-fluidity liquid mobile phases are studied. Both reversed- and normal-phases type columns are tested, in an effort to find the most suitable stationary phase for dimer and trimer acids. Since dimer acids are mixtures of mono-, di- and tri-carboxyl acids, reversed-phase columns that are engineered to contain the least amount of surface silanol groups were tested for the separation of dimer and trimer acids. Specifically, four C18 stationary phases, including Extended C18 from Agilent Technologies (Palo Alto, CA), Luna C18 from Phenomenex (Torrance, CA), BetaBasic C18 and BioBasic C18 from Keystone Scientific (Bellefonte, PA) were used. FluoPhase WP from Keystone Scientific was also used. The detailed specifications for each column are listed in Table 2. The Extended C18 column used type B silica of ultra-high purity (≥99.995% SiO2 ) as support. The bidentate C18 bonded phase was double end-capped in order to obtain maximum deactivation of the silica support surface [32]. The combination of the bidentate C18 and double end-capping produced a highly hydrophobic surface that can greatly reduce the rate of the silica dissolution [33]. The column can be used for pH 2–11.5. It has been reported that good peak shapes for basic compounds were achieved using mobile phases containing organic buffers (such as 1-methyl-piperidine and pyrrolidine) of high pH

Table 2 Available information on particle and bonded phase characterization for five reversed-phase columnsa Zorbax Extended C18 Particle size (␮m) Pore diameter (Å) Surface area (m2 /g) Metal content (ppm) Total carbon (%) Surface coverage (␮mol/m2 )

5 80 180 Trace 12 NA

Luna C18 5.20 95 440 <55 19 3.3

BetaBasic C18

BioBasic C18

FluoPhase WP

5 150 200 Trace 13 ∼2

5 300 118 Trace 9 NA

5 300 118 Trace NA NA

a Data for Luna C18 phase was provided by Phenomenex for the specific lot; while all other data were from respective manufacturer’s catalogs as general information only.

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(pH = 11) [32]. The Luna C18 column is also stable over a wide pH range (pH = 1.5–10) [34]. In a recent study, McCalley compared the performance of conventional C18 phases with that of alternative pH resistant columns for the analysis of basic compounds [35]. Both Luna C18 and Extended C18 were among the alternative phases studied. Neither of the two phases showed superior peak shape nor efficiency for the tested basic compounds compared to conventional C18 phases or to the other phases studied. This indicated that secondary interactions may still exist to a certain extent on these two columns. Furthermore, peak shapes of basic compounds on the Extended C18 phase at pH 11 varied with buffer systems. The use of a triethylamine buffer in the mobile phase generally provided better peak shapes than using a phosphate buffer. This again suggested there may be some active sites such as free silanol groups on the surface, since triethylamine is known to behave as a silanol blocking agent to improve peak shape. Both the BetaBasic and BioBasic C18 columns are reversed-phase packing on the Betasil silica (type B silica, base deactivated) [36]. The BetaBasic C18 surface is highly deactivated and the carbon coverage is high. The BetaBasic C18 column is suitable for general reversed-phase applications, including acids, neutrals and bases. BioBasic C18 is an inert packing based on pure 300 Å pore size silica particles. It is recommended for proteins, peptides and other biological applications where moderate retention and the highest level of deactivation are needed. FluoPhase WP is a fluorinated alkyl phase that often shows increased retention and selectivity compared to C8 or C18 columns, especially for halogen-containing compounds [36]. Besides halogen-containing compounds, other compounds may also exhibit more retention and different selectivities, compared to the traditional alkyl or aromatic phases. A Keystone Betasil silica column (Bellefonte) was also used. 2. Experimental 2.1. Materials All organic solvents were used as received. Hexanes, methanol, IPA and chloroform were all HPLCgrade solvents purchased from Fisher Scientific

(Pittsburgh, PA). Glacial acetic acid (99.8%) was obtained from Mallinckrodt Chemical (Paris, KT). Distilled H2 O was deionized by a NANOpure II system (SYBRON/Barnstead, Boston, MA) with a resistivity of 17.8–18.3 M. Stearic acid was from Eastman Chemical Company (Rochester, NY). Oleic acid (>99%) was purchased from Aldrich Chemical Company (Milwaukee, WI). SFC grade CO2 (99.995%) without a helium pad was obtained from PraxAir Technologies (East Chicago, IN) and was used as received. Electronic-grade halocarbon 23 (99.95% purity fluoroform) without a helium pad was obtained from Air Products and Chemicals (Allentown, PA) and was used as received. Impurities in fluoroform were specified by manufacturer as 500 ppm of air, 0.030 ppm of acidity as HCl and 10 ppm H2 O. 2.2. Instrumentation The chromatographic system was a Gilson SF3 supercritical fluid chromatograph (Gilson Inc., Middletown, WI). The system was configured for analytical scale chromatography as described previously [37]. Either liquefied CO2 or CHF3 was pumped with pump A (model 308 with a 10 SC pump head) with a thermostated head; all organic solvents were pumped with pump B (model 306 with a 5 SC pump head). Binary mixing took place in a Gilson model 811C dynamic mixer using a 1.5 ml mixing chamber. Fixed external loop injections (20 ␮l) were accomplished using a Rheodyne model 7725i external loop injector (Rheodyne LP, Rohnert Park, CA). Columns were placed inside a Gilson model 831 temperature regulator whose temperature was maintained with a maximum deviation of less than ±0.5◦ C. An ice and water mixture was continuously circulated through the cooling coil connected to the thermostatic jacket for the pump head of pump A with a Techne Tempunit® Thermoregulator (model TU-16D) (Techne Inc., Princeton, NJ). Detection was accomplished at 210 nm using a Gilson model 151 variable wavelength UV–VIS detector. The volumetric flow rate was maintained constant at 1.0 ml/min, unless otherwise noted. Column outlet pressure, P2 , was maintained by a Gilson model 821 pressure regulator; while both column inlet pressure, P1 , and P2 were monitored and recorded by on-line electronic pressure transducers.

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Five reversed-phase columns were used, including Extended C18 from Agilent Technologies, Luna C18 from Phenomenex, BetaBasic C18, BioBasic C18 and FluoPhase WP from Keystone Scientific. All five columns have the same dimensions, 4.6 mm×150 mm i.d., packed with 5 ␮m particles. Detailed column information is listed in Table 2. A Keystone Betasil silica column was also used, 2.0 mm × 250 mm i.d. long, packed with 5 ␮m particles. A Valco frit (0.5 ␮m bore, part No. ZUFR1F, Valco Instruments, Houston, TX) was placed in line before the analytical column to prevent particles from entering the column. All mass spectrometric experiments were performed on an API-165 (Perkin-Elmer SCIEX, Thornhill, Ont., Canada) single quadrupole mass spectrometer equipped with an ionspray source developed in-house. The in-house ionspray source was described in detail previously [38]. Briefly, a Meinhard® SB-30-A3 nebulizer was used. A length of fused silica capillary (150 ␮m o.d. × 75 ␮m i.d.) (Polymicro Technologies, Phoenix, AZ) was inserted into the nebulizer and caused a 400 ␮m protrusion past the face of the nebulizer. A model U402 union (Upchurch Scientific, Oak Harbor, MA) was used at the inlet end of the nebulizer to hold the capillary in place and to form an air-tight seal. The sample was delivered into the fused silica capillary at a flow rate of 5 ␮l/min using a model 81630, 5 ml glass syringe (Hamilton, Reno, NV) driven by a syringe pump (KD Scientific). Nitrogen gas (99.999%) was used as both nebulizer and curtain gas. The curtain gas was applied between the front and orifice plates. The ionspray high voltage was applied to the metal fitting at the outlet of the syringe. A Macintosh Power PC computer with LC Tune 2.1 and Multiview 1.2 software (Perkin-Elmer Sciex) was used for instrument control, data acquisition and data processing. 2.3. Sample preparation Three dimer and trimer acid samples, designated as samples A, B and C in Table 3, were received from Cognis Corporation (Cincinnati, OH, formerly Henkel Corporation Chemical Group). Weight compositions were also provided (Table 3) and were used for preliminary peak assignment in the separation based on the relative amounts of monomer, dimer and trimer in each sample. The monomer peak was verified by

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Table 3 Compositions of dimer and trimer acid samples provided by Cognis Corporation Sample

Monomer (wt.%)

Dimer (wt.%)

Polymer (wt.%)

A B C

5.6 3.5 1.6

79.8 93.5 23.3

14.6 3.0 75.1

individual injections of stearic acid (2 mg/ml in hexane) and oleic acid (0.5 mg/ml in hexane) standard solutions, the usual major components of C18 fatty acid monomers [39]. The retention factors of monomers were comparable to those of stearic and oleic acid. No dimer or trimer standards were available. Therefore, their peak identities were verified by off-line mass spectrometric measurements of the collected fractions following the separation, which is discussed later. Solutions of the samples (2–5 mg/ml) for chromatographic study were made by dissolving the liquid acids in hexane. These samples were further diluted to ∼0.1 mg/ml in methanol for direct mass spectrometric measurements. In fraction collection experiments, samples were dissolved in chloroform instead, in order to achieve higher concentrations (∼10 mg/ml). All samples were filtered through a fresh 0.2 ␮m syringe filter (Whatman Inc., Clifton, NJ). Solutions were sealed and stored at room temperature when not in use. Fresh sample solutions were made weekly. 2.4. Data analysis The chromatographic data were collected by UnipointTM system control software, running on a GatewayTM model E-3200 PentiumTM II based personal computer. Different sampling frequencies were used depending on the retention times of the analytes. Data were analyzed by PeakFitTM version 4.06 (PeakFit Analysis Software, Jandel Scientific, San Rafael, CA).

3. Results and discussion 3.1. Separation of dimer acids on reversed-phase stationary phases A minimum of 3% (v/v) of a modifier, such as methanol, was included in the sub or supercritical

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fluid, CO2 or CHF3 to ensure complete dissolution of dimer acids (especially trimer components) in the mobile phase. As a common practice, acidic modifiers, such as formic acid, acetic acid or trifluoroacetic acid (TFA) are often included in the mobile phase for the separation of acidic analytes [40]. These mobile phase additives often improve peak shape by suppressing the ionization of analytes and preventing possible interactions between analytes and surface silanol groups. In this study, 0.2 vol.% acetic acid was initially included in methanol. However, it was found later that acetic acid had minimal impact on the separation of dimer and trimer acids, even when up to 0.5 vol.% acetic acid in methanol was used. Therefore, acetic acid was not included in the mobile phases for the gradient studies. 3.1.1. Isocratic conditions An initial isocratic condition was first studied on all five reversed-phase columns. Very different results were observed for the five different columns. Table 4 shows the ranges of retention factors at the same isocratic condition for the five stationary phases. Because the peaks were broadened severely under isocratic conditions, retention ranges are reported instead of making any attempt to specify the center of mass of a given peak. The dimer acids showed minimal retention on three Keystone columns (BetaBasic C18, BioBasic C18 and FluoPhase WP), while they showed more retention on the Luna C18 column and the most retention was observed using the Extended C18 column. Since dimer and trimer acids are a group of polar compounds, low retention was expected on a typical reversed-phase stationary phase, when mobile phases of “strong” solvent strength (in reversed-phase mode) were used, such as methanol modified CO2 mixtures. An unexpected normal-phase elution order on the Extended C18 and the Luna C18 columns indicated that there might be other dominant interactions between dimer acids and the chromatographic surface in

Table 5 Comparison of retention range under different isocratic conditions on the Extended C18 column: methanol containing 0.2 vol.% acetic acid; P2 = 204 atm; T = 40◦ C; flow rate 1.0 ml/min Range of k

Monomer Dimer Trimer

Mobile phase condition: methanol vol.% (mol%) 7.0 (8.0)

10.0 (11.4)

15 (17.0)

0.53–0.61 1.68–7.31 6.38–25.8

0.45–0.63 0.88–4.03 3.56–10.0

0.41–0.57 1.03–2.03 1.50–6.65

addition to the interaction with the nonpolar bonded phases, such as interaction with residual free silanol groups. It is also possible that the separation was based on size, as monomer, dimer and trimer are in the order of increasing molecular size. The separation of sample C on the Extended C18 column was further explored by varying the concentration of the organic component. Table 5 shows the variation of retention ranges as the function of methanol modifier concentration. Typical normal-phase retention behavior was observed. In that decreasingly polar mobile phases (less methanol in mobile phase) caused more retention for dimer and trimer acids. The best separation under isocratic conditions was achieved using mobile phase composition of 85/15% (v/v), CO2 /methanol as showed in Fig. 1. Fig. 1A shows the separation of sample C, while Fig. 1B shows the separation of sample C spiked with stearic acid. Both Fig. 1A and B show very similar chromatographic bands. The much larger signal for monomer band in Fig. 1B was due to the presence of spiked stearic acid. The monomer and dimer were baseline separated but the dimer and trimer were only partially resolved. Other isocratic conditions were also tested on the three Keystone columns (BetaBasic C18, BioBasic C18 and FluoPhase WP). Since the retention of dimer acids was minimal in the isocratic condition shown in Table 4, mobile phases with “weaker” (in

Table 4 Comparison of ranges of retention factors under the isocratic condition on five different stationary phases: mobile phase composition; CO2 /methanol (0.2 vol.% acetic acid) 90/10% (v/v); P2 = 204 atm; T = 40◦ C; flow rate 1.0 ml/min Range of k

Extended C18

Luna C18

BetaBasic C18

BioBasic C18

FluoPhase WP

Monomer Dimer Trimer

0.45–0.63 0.88–4.03 3.56–10.0

0.21–0.33 0.85–2.93 2.6–6.3

0.11–1.27 0.11–1.27 0.11–1.27

0.20–1.10 0.20–1.10 0.20–1.10

0.19–1.20 0.19–1.20 0.19–1.20

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Fig. 1. Chromatograms of sample C under the isocratic condition on the Extended C18 column, mobile phase composition: CO2 /methanol (0.2 vol.% acetic acid) 85/15% (v/v); P2 = 204 atm; T = 40◦ C; flow rate 2 ml/min. (A) Sample C (3.6 mg/ml); (B) sample C (3.6 mg/ml) spiked with stearic acid (2.4 mg/ml).

normal-phase mode) solvent strength, including the CO2 /methanol (0.2 vol.% acetic acid) composition of 93/7, 95/5, 97/3% (v/v) were employed in order to increase analyte retention and potentially achieve resolution. However, the separation of dimer acids was still not achieved due to the minimal increase in retention for the BetaBasic C18 and BioBasic C18 columns. For the FluoPhase WP column, partial resolution for dimer acids was achieved. The calculated retention ranges using 97/3% (v/v) CO2 /methanol (0.2 vol.% acetic acid) are: 0.12–0.22 for monomer, 0.22–0.48 for dimer and 0.45–0.90 for trimer. A similar trend was also observed on the Luna C18 column: the more polar the mobile phase, the less retention for these dimer acids. However, separation was not achieved. Overall, the unexpected normal-phase behavior for polar dimer and trimer acids on these reversed-phase columns may be associated with polar functionalities in the reversed-phase packing material (such as residual silanol groups). 3.1.2. Gradient conditions Since isocratic conditions did not provide a satisfactory separation, pressure (or density) gradient and

composition gradients were studied. Several pressure gradients were applied to all five columns, with the mobile phase composition of CO2 /methanol held at 97/3% (v/v). However, separation was not achieved, except that resolution between monomer and dimer was obtained on the FluoPhase WP column. The highest resolution chromatogram of samples A and C using pressure gradient conditions are shown in Fig. 2. Composition gradients (such as methanol from 3 to 30% in 10 min) were also used. The separation on the FluoPhase WP column showed the best results, although the dimer and trimer peaks were only partially resolved as showed in Fig. 3. Specifically, Fig. 3A–C show the separation of samples A–C under the above composition gradient. This is expected, since under the isocratic conditions, the retention of the dimer and trimer acids showed the most variation with the mobile phase composition on the FluoPhase WP column. However, baseline separation of the dimer and trimer components was not achieved, due to the very broad chromatographic bands and close retention factors of dimer and trimer components.

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Fig. 2. Chromatograms of separation of dimer acid samples under the pressure gradient condition on the FluoPhase WP column, mobile phase composition: CO2 /methanol 97/3% (v/v) P2 from 102 to 204 atm in 5 min; T = 40◦ C; flow rate 1 ml/min. (A) Sample C (3.6 mg/ml); (B) sample A (2.8 mg/ml).

Fig. 3. Chromatograms of separation of dimer acid samples under the composition gradient condition on the FluoPhase WP column, mobile phase composition: CO2 /methanol gradient, methanol from 3 to 30% in 10 min; P2 = 204 atm; T = 40◦ C; flow rate 1 ml/min. (A) Sample A (3.6 mg/ml); (B) sample B (2.9 mg/ml); (C) sample C (2.8 mg/ml).

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Table 6 Comparison of retention range under the isocratic condition on the Betasil silica column, IPA containing 0.2 vol.% acetic acid; P2 = 204 atm; T = 50◦ C; flow rate 0.50 ml/min Range of k

Monomer Dimer + trimer

Mobile phase condition: IPA vol.% (mol%) IPA containing 0.2 vol.% acetic acid 5.0 (3.1)

10.0 (6.4)

20.0 (13.3)

0.57–1.4 2.4–5.3

0.25–0.87 1.2–4.0

0.32–0.57 0.57–1.19

3.2. Separation of dimer acids on the silica column Since complete separation of dimer acids was not achieved on the reversed-phase columns, efforts were turned back to normal-phase studies using a silica column.

3.2.1. Isocratic conditions Isocratic conditions were tested initially. The modifier was IPA containing 0.2 vol.% acetic acid, instead of methanol, simply because is was also used in the AOCS method on a silica column. The flow rate was 0.50 ml/min. Table 6 shows the measured retention

Fig. 4. Chromatograms of separation of dimer acid samples under the isocratic condition on the Betasil silica column; mobile phase condition: CO2 /IPA (0.2 vol.% acetic acid) 90/10% (v/v); P2 = 204 atm; T = 50◦ C; flow rate 0.50 ml/min. (A) Sample A (3.6 mg/ml); (B) sample B (2.9 mg/ml); (C) sample C (2.8 mg/ml).

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ranges at different mobile phase conditions. Fig. 4 shows the resultant separations of samples A–C under the isocratic condition. Resolution between monomer and dimer was obtained. However, the trimer was never observed as a peak on the chromatograms under these conditions. It is possible that the trimer components may have eluted as a very broad band that was embedded under the tailing dimer peak. In order to elute trimer with a good peak shape, solvent strength must be increased accordingly. 3.2.2. Gradient conditions In the isocratic separation on the silica column, IPA containing 0.2 vol.% acetic acid was used as the organic component in the mobile phase. However, isocratic separation was not satisfactory due to the close elution of dimer and trimer. Both methanol and IPA are commonly-used organic modifiers in SFC. Their eluotropic strength parameters, ε 0 on silica surface are similar (methanol: 0.70 and IPA: 0.60). However, IPA has a viscosity of 2.4 cP, while methanol’s viscosity is only 0.55 cP at 25◦ C [41]. The much greater viscosity of IPA is not desirable if a high proportion of organic component is needed in the mobile phase, such as in this gradient study. The column pressure drop is directly proportional to the

mobile phase viscosity [42] therefore the maximum flow rate for a column under a particular mobile phase composition is controlled by the viscosity of the mobile phase. Methanol was used in the gradient study, in an effort to achieve higher flow rates. Various pressure gradients were attempted but the trimer peak was still absent. In order to increase the solvent strength of the mobile phase before trimer acid elutes as a broad band, a very steep composition gradient with a linear rate of 24 vol.%/min was used. The final composition of 65% (v/v) methanol was strong enough to elute the most polar components in a relatively short time. Fig. 5 shows the separation of sample A under this optimized gradient profile which is listed in Table 7. The monomer and dimer eluted with good resolution in the first 5 min and the steep gradient prevented the trimer from eluting as a broad band. The trimer peak eluted with good peak shape within 2 min when 65 vol.% methanol was present in the mobile phase. The mobile phase was quickly ramped back to the initial condition in 2 min. The last 6 min of the gradient were adequate to allow the mobile phase to reach equilibrium and be ready for the next injection. The overall analysis time is ∼18 min. Due to the high mass transport rate and the low viscosity of enhanced-fluidity liquids, higher optimum

Fig. 5. Chromatogram of sample A under the gradient condition on the Betasil silica column; P2 = 170 atm; T = 50◦ C; flow rate 1.0 ml/min. The gradient condition is described in Table 7.

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Table 7 Optimized gradient mobile phase condition for the separation of dimer acids using the Betasil silica column Time (min)

Flow (ml/min)

CO2 (vol.%)

Methanol (vol.%)

P2 (atm)

Profile

0 5.0 7.5 10.0 12.0 18.0

1.0 1.0 1.0 1.0 1.0 1.0

95 35 35 35 95 95

5 65 65 65 5 5

170 170 170 170 170 170

Step Linear Step Linear Step Step

linear velocity is observed in EFLC than in HPLC [43]. Therefore, higher flow rates can be used without losing much efficiency and resolution. In this study, the maximum pressure drop during the gradient was measured to be ∼204 atm (3000 psi), when the mobile phase reached a CO2 /methanol composition of 35/65% (v/v).

Therefore, a higher flow rate than 1.0 ml/min may be applied on the same column to further reduce the analysis time, corresponding to a pressure drop of 306 atm (4500 psi), provided the resolution is still maintained. Fluoroform-based mobile phases were also tried. The exact same gradient condition as used with

Fig. 6. Negative ionspray mass spectra of samples A and C.

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CO2 -based mobile phases was shown in Table 7 for a direct comparison. Very similar results were obtained, whether CO2 or CHF3 was used. The retention time for the dimer and trimer peaks increased by ∼1 min when using fluoroform. Since such a steep composition gradient was used, the solvent strength of the mobile phase during the run should be controlled mainly by the percentage of organic component methanol [44]. Pressure, temperature and even the nature of supercritical fluid contributed only secondary effects.

3.3. Fraction collection and ionspray mass spectrometry of dimer acids Fig. 6 shows the mass spectra of samples A and C with a concentration of ∼0.1 mg/ml in methanol using negative ionspray ionization, with the m/z range scanned from 200 to 1000. Negative ionspray mode was chosen due to the acidity of these samples. The mass spectra illustrates the complexity of the samples. There are at least three regions for both samples,

Fig. 7. Ionspray mass spectra for fraction collections of sample A.

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corresponding to the monomer (∼283), dimer (∼565) and trimer (∼850) as expected. In addition, there are some relatively strong signals in the m/z region around 200–350, which may be due to impurity or degradation products, etc. The trimer signal is very weak for sample A as expected, since its trimer concentration was low (∼15%). Therefore, it is expected that the verification of fraction collection for trimer in sample A will be difficult. Sample C showed a stronger trimer signal due to its much higher trimer concentration (∼75%). Sample B

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was not studied due to its extremely low monomer and trimer concentrations. 3.3.1. Fraction collection To confirm the peak identity for dimer and trimer peaks, off-line mass spectrometry was carried out. Fraction collection of chromatographic peaks was performed first. The SFC system was modified in house to allow fraction collection. An ISCO 260D syringe pump (ISCO, Lincoln, NE) was used to provide a makeup

Fig. 8. Ionspray mass spectra for fraction collection of sample C.

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Fig. 9. Ionspray mass spectrum for mobile phase collected as blank.

flow of pure methanol at a flow rate of 0.20–0.40 ml/min via a Valco Tee (0.25 mm bore, part No. ZT1C, Valco Instruments, Houston, TX). This additional modifier flow met the effluent downstream the UV–VIS detector and allowed the solutes to remain in the liquid phase, even after CO2 expansion due to the depressurization of the mobile phase. The addition of this makeup flow was critical. The makeup flow improved the recovery yields, as well as eliminated the risk of plugging associated with high sample loading. The chromatographic bands for monomer, dimer and trimer were collected using the following steps. Prior to fraction collection, each collection vial was filled with 2 ml methanol. After injection of samples onto the column, UV signal at 210 nm was observed closely. The switch valve was manually controlled to allow fraction collection based on the change in the intensity of the UV signal. 3.3.2. Ionspray mass spectrometry of dimer and trimer acids Fig. 7 shows the mass spectra of fraction collections of monomer, dimer and trimer peaks for sample A (concentration of ∼20 mg/ml). Fig. 8 shows the mass spectra for fraction collections of monomer, dimer and trimer peaks for sample C (concentration of ∼9 mg/ml). For sample A, the dimer peak was verified by the unique presence of m/z region at ∼560;

fraction collection of trimer peak showed a very weak signal at the m/z region at ∼850 due to its low concentration of trimer. For sample C, both dimer and trimer peak collections were verified by the unique presence of their characteristic m/z region of ∼560 and ∼850, respectively. However, unexpectedly intense signals in all of the fractions for sample C were observed in the 200–350 m/z region. A blank run was perform using the same gradient and only mobile phase was collected as blank solution. The mass spectrum for the blank solution is shown in Fig. 9. The same instance signals over the 200–350 m/z range were observed for the blank which suggested that these signal were probably due to impurities from the SFC system. 4. Conclusions Dimer and trimer acids were well separated on a silica column using a very steep concentration gradient using enhanced-fluidity liquid mobile phases. This steep gradient was necessary to elute trimers with good peak shape. Furthermore, enhanced-fluidity liquids, methanol/CO2 and methanol/CHF3 mixtures, showed comparable elution strength in this steep gradient for the separation of dimer acids on the silica stationary phase. However, these polar compounds showed a normal-phase type retention on all five

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reversed-phase columns under study, indicating an unusual retention mechanism. UV–VIS detection was used mainly due to its simplicity and availability. However, it will not be suitable for quantitation for dimer acid samples due to the lack of primary standards and also lack of strong absorption bands of some components, especially monomers. The previously reported ELSD method was based on the assumption that the response factors of the monomer, dimer and trimer components are same. SFC and EFLC has been proven to work well with evaporative laser light scattering detection [11,12]. Off-line mass spectrometry was used to verify the peak identity. It is expected that SFC or EFLC coupled with mass spectrometry should be a powerful technique for the direct analysis of dimer acids. EFLC provided several advantages over the commonly-used conditions for HPLC, such as a faster speed of analysis due to the higher flow rates enabled by the low viscosity of these fluids; consumption of much less organic solvents; and finally much improved sample throughput. The scale-up of the SFC/EFLC separation should be a viable method to replace the semi-preparative HPLC method, in order to obtain dimer and trimer primary standards. The available phase diagram and solvent strength information was useful for the method development undertaken in this study [45,46]. Some of the studied mobile phases were in the supercritical fluid region, such as the initial mobile phase condition in the composition gradient: methanol/CO2 (5 vol.% or 3.1 mol% of methanol) and methanol/CHF3 (5 vol.% or 8.2 mol% of methanol) at P > 170 atm and T = 50◦ C; while others were under sub-critical conditions. In a single chromatographic run, the mobile phase started from the supercritical fluid region, such as supercritical CO2 and CHF3 with only 5 vol.% of modifier addition; then it immediately traveled to the enhanced-fluidity liquid region, where large proportions of organic solvent methanol and a liquefied gas CO2 or CHF3 were combined to achieve liquid-like solvent strength.

Acknowledgements The authors thank Gilson Inc. for the loan of Gilson SF3 supercritical chromatograph for this study. We

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