Thermal Modulation for Multidimensional Liquid Chromatography

Aug 4, 2011 - gradient starting with a low elution strength mobile phase solvent for ... Compared to the nonmodulated signal, the presented thermal mo...
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Thermal Modulation for Multidimensional Liquid Chromatography Separations Using Low-Thermal-Mass Liquid Chromatography (LC) Matthias Verstraeten,† Matthias Pursch,‡ Patric Eckerle,‡ Jim Luong,§ and Gert Desmet†,* †

Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, 1050 Elsene, Belgium Dow Deutschand Anlagengesellschaft mbH, Analytical Technology Center, 77836 Rheinmunster, Germany § Dow Canada, Analytical Technology Center, Fort Saskatchewan, AB, Canada ‡

ABSTRACT: We report on a proof-of-principle experiment with a novel thermal modulation device with potential use in two-dimensional liquid chromatography (LC  LC) systems. It is based on the thermal desorption concept used in two-dimensional gas chromatography (GC  GC) systems. Preconcentration of neutral analytes eluting from the first dimension column is performed in a capillary “trap” column packed with highly retentive porous graphitic carbon particles, placed in an aluminum low-thermal-mass LC heating sleeve. Remobilization of the trapped analytes is achieved by rapidly heating the trap column, by applying temperature ramps up to +1200 °C/min. Compared to the nonmodulated signal, the presented thermal modulator yielded narrow peaks, and a concentration enhancement factor up to 18 was achieved. With a thermally modulated LC separation of an epoxy resin, it is shown that when the thermal modulation is applied periodically, the trapped and concentrated molecules can be released periodically and that the modulating interface can both serve as a preconcentration device and as an injector for the second dimension column of an LC  LC setup. Because of the thermal modulation, a high-molecular-weight epoxy resin could be adequately separated and the different fractions were identified with a GPC analysis, as well as an offline second dimension LC analysis.

A

few decades ago, multidimensional separations were introduced to the field of chromatography, which allowed a muchhigher resolving power for separating complex mixtures.1 In order to maximize the system’s peak capacity, both dimensions should preferentially have a different separation mechanism, such that the selectivities obtained on both dimensions are uncorrelated.2,3 For multidimensional gas chromatography (GC) systems, a major breakthrough was achieved in 1991, when Phillips and Liu reported on a thermal desorption modulator serving as the interface between the two dimensions.4 This first design, which is based on local resistive heating, traps the molecules eluting from the first dimension at a reduced temperature in order to concentrate the molecules locally. The molecules are then periodically remobilized by applying a high temperature, allowing them to flow into the second dimension column. The thermal modulator thus serves both as a concentration and injection device for the second dimension. Today, various thermal modulation interfaces exist for two-dimensional gas chromatography (GC  GC) systems, such as the early sweeper modulator,5 and the more-reliable cryogenic devices such as the longitudinally modulated cryogenic system (LMCS),69 the dual-stage liquid CO2 jet modulator,10 the two-stage gaseous nitrogen/heated air jet thermal modulator,11 or a similar approach with liquid nitrogen.12 In two-dimensional liquid chromatography (LC  LC) systems, the interface between the dimensions typically consists of a multiport switching valve connected to (packed) sample loops, trap columns, or alternatively a stop-flow mechanism when a longer analysis time in the second dimension is needed.1315 r 2011 American Chemical Society

The valves and loop volumes are arranged in such a way that the analytes eluting from the first column are collected and then periodically injected into the second dimension (parallel) column(s). Preconcentration only takes place when a trap column is employed in the interface, or when the concentration of the analytes is obtained at the head of the second column when, e.g., the secondary pump delivers a gradient starting with a low elution strength mobile phase solvent for normal-phase liquid chromatography (NP-LC) or reverse-phase liquid chromatography (RP-LC).14,15 Other possible preconcentration methods exist, such as solvent evaporation or reverse osmosis; however, these are not common practice.14 The objective of the present work is to construct a thermal modulator that can be used as an interface for LC  LC systems and is based on the thermal modulation principle used in GC  GC systems. In order to trap and release molecules, the retention behavior, which in LC is primarily dependent on the mobile phase, stationary phase and temperature, must be changed extremely and rapidly. In general, the retention is determined by the interactions between the stationary phase and the molecule itself. Silicabonded phases such as the C8- or C18-coated silica particles mainly interact with the hydrophobic part of the molecule. For porous graphitic carbon (PGC), a stationary phase introduced by Knox et al.,16,17 additional interactions between the delocalized π-electrons of the graphitic carbon, and the polar and aromatic Received: May 11, 2011 Accepted: August 4, 2011 Published: August 04, 2011 7053

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parts of the molecules exist, resulting in an increased retention, compared to the silica-bonded phases,1618 which is needed to sufficiently trap the analytes. The influence of the mobile phase composition on the retention is very strong and is approximately given by the linear solvent strength model (LSS).18,19 However, a modification of the mobile phase in the modulation interface would require an additional mobile phase flow, which would result in dilution and band broadening. Therefore, the modulation interface is directly coupled to the first dimension column in our approach. The influence of temperature on retention is given by the Van’t Hoff equation:18,20 ln k ¼ 

ΔHR ΔSR þ þ ln ϕ RT R

Figure 1. Structure of the epoxy resin used to study the thermal modulation system.

ð1Þ

ΔHR is the molar retention enthalpy, which expresses the temperature dependence of the analytes; ΔSR is the molar retention entropy; and ϕ is the phase ratio. However, the retention enthalpy is not only a property of the molecule (larger molecules will have larger retention enthalpies, according to Martin’s rule21), but ΔHR also is dependent on the stationary phase as well as the temperature (when the heat capacity is not constant20). Zhang and McGuffin22 reported that the molar enthalpy is more negative on PGC than on the traditional silicabonded phases such as C8 or C18 (studied with a homologous series of alkylbenzenes and methylbenzenes), showing that the interactions between the molecules and the PGC stationary phase are much more sensitive to temperature. In addition, PGC is temperature stable up to 200 °C, i.e., significantly higher, compared to the traditional commercially available silica-bonded stationary phases. Because of its larger retention enthalpy, the PGC material offers the possibility to trap the molecules at room temperature and then periodically release them at a very high temperature. For this purpose, a fast heating is needed. This can be achieved using the low-thermal-mass LC device introduced by Gu et al.23 The LTM heating device consists of an aluminum heating sleeve in which a packed fused silica capillary is mounted. The resistively heated coil wrapped around the heating sleeve allows for fast heating of the system, and temperature ramps up to 1800 °C/min have been reported.23 Microbore capillary columns must be used in this system, since larger inner diameters will lead to additional band broadening, because the applied temperature ramp at the column wall induces radial column temperature gradients and radial mobile phase viscosity and velocity gradients. An estimate of the maximal inner diameter for different heating rates has been reported by Verstraeten et al.24 The present contribution investigates whether a highly retentive stationary phase (such as PGC) is suitable for the trapping of neutral molecules and whether these molecules can be adequately remobilized by applying heat pulses with the LTM heating device.

’ EXPERIMENTAL SECTION Materials, Columns, and Chromatographic Apparatus. Uracil and dimethyl phthalate were purchased from Sigma Aldrich (Munich, Germany) and dissolved in 50/50 (vol %) acetonitrile/water in a concentration of 0.2 mg/mL and 1.0 mg/ mL, respectively. An epoxy Novolac resin was dissolved in acetonitrile in a concentration of 10.0 mg/mL. The molecular structure of the epoxy resin is given in Figure 1; n is the degree of

Figure 2. Schematic representation of the thermal modulation system consisting of a three-section packed capillary column (silicaPGCsilica) and a low-thermal-mass heating sleeve connected to (a) the injector or pump and (b) the first dimension column.

polymerization. Acetonitrile was purchased from VWR (Darmstadt, Germany), and HPLC-grade water was prepared in-house using a Milli-Q gradient (Millipore, Bedford, MA). The epoxy resin was separated on a Zorbax SB-C8 Microbore Rapid Resolution column (1.0  150 mm, dp = 3.5 μm) obtained from Agilent Technologies (Waldbronn, Germany). After the first dimension separation and modulation, fractions were collected and analyzed on three coupled PL Gel Mixed E columns (7.8  300 mm, dp = 3.0 μm) obtained from Polymer Laboratories (Agilent Technologies, Waldbronn, Germany). The fractions were collected three times in order to have a sufficient injection volume for GPC, and the mobile phase of the fractions was evaporated with N2 and tetrahydrofuran (THF) was added. The LC analysis of the collected fractions was carried out on a Fused Core Ascentis Express PhenylHexyl column (2.1  30 mm, dp = 2.7 μm) purchased from SigmaAldrich (Munich, Germany), which provides a different reverse-phase selectivity for polar aromatics and heterocyclic compounds, compared to alkyl stationary phases. All separation and modulation experiments were accomplished on an Agilent 1100 capillary LC system (Agilent Technologies, Waldbronn, Germany) that was equipped with a 500-nL UV flow cell detector. All measurements were done at ambient temperature. PEEKSil connection tubings were used with an internal diameter of 50 μm and a length of 550 mm (between injector and column and between column/thermal modulation device and detector). The injection volume was 150 nL. The GPC analysis of the collected fractions/epoxy resin was performed on an Agilent 7054

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Analytical Chemistry 1200 LC system (Agilent Technologies, Waldbronn, Germany) with a flow cell of 13 μL. The injection volume was 20 μL. The mobile phase was THF, with a flow rate of 0.4 mL/min. The temperature was set to 40 °C. The LC analysis of the collected fractions was carried out on an Agilent 1290 Infinity LC system (Agilent Technologies, Waldbronn, Germany) that was equipped with a 1 μL UV flow cell detector. The injection volume was 0.5 μL. The separations were performed at a flow rate of 0.5 mL/min, and a temperature of 30 °C was controlled by the oven compartment. Stainless steel tubing with an inner diameter of 120 μm was used. Both dimethyl phthalate and the epoxy resin were detected with UV radiation at a wavelength of 280 nm. Thermal Modulation Segment. The thermal modulation device is schematically represented in Figure 2. The modulation capillary was packed with three subsequent sections: the first section consisted of fully porous 10-μm silica particles (Merck LiChrosorb, purchased from Merck KGaA, Darmstadt, Germany); the second section consisted of 3040 μm PGC particles (Hypercarb, obtained from Thermo Fisher Scientific, Schwerte, Germany); and the third section consisted again of 10-μm silica particles. The Si particles were suspended in a 50/50 (vol %) chloroform/iso-propanol solution, and the PGC particles were suspended in an acetone solution. The suspensions were slurry-packed with iso-propanol in a fused silica capillary at 350 bar. All solvents used in the packing procedure were HPLCgrade, and the filling chamber and the fittings were cleaned very well after the packing of each segment to avoid the mixing of particles in the different sections. Two fused-silica capillaries (purchased from Optronis GmbH, Kehl, Germany) were used with the following dimensions: 300 μm i.d. (inner diameter), 665 μm o.d. (outer diameter), and 200 mm length (capillary 1), and 200 μm i.d., 356.3 μm o.d., and 100 mm length (capillary 2). The PGC segment had lengths of 25 and 15 mm for capillaries 1 and 2, respectively. The thermal modulation capillary (three-section packed SiO2/PGC/SiO2 capillary) was inserted into a low-thermal-mass heating sleeve (see Figure 2). The temperature ramp of the heating sleeve was controlled by the capillary column temperature control module LTM A68 (RVM Scientific, Santa Barbara, CA). More details and specifications on this module can be found in refs 2326. The aluminum heating sleeve had a length of 40 mm and an inner diameter of 790 μm, and it was insulated from the environment with thermal insulation fiber. Temperatures inside the aluminum sleeve were measured by placing a thermocouple between the heating sleeve inner wall and the fused-silica capillary. Subminiature chromel/alumel thermocouples with a diameter of 250 μm were used (Type K, Part No. B200, ThermoElectric, Waddinxveen, The Netherlands) which were connected to a 24-bit NI 9211 thermocouple input module (National Instruments, Zaventem, Belgium). The temperature signals were obtained using a NI LabVIEW SignalExpress system at a frequency of 2 Hz.

’ RESULTS AND DISCUSSION Retention Modification with the Thermal Modulation Device. To study the retention behavior of the constructed

thermal modulation device, the modulation capillary was first studied without a preceding separation column and, therefore, was directly connected between the injector and the detector (see Figure 2a; capillary 1: i.d. = 300 μm, LMOD = 200 mm, LPGC = 25 mm). Figure 3a shows the elution of dimethyl

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Figure 3. Elution of the t0 marker (uracil) and dimethyl phthalate from the thermal modulation system (capillary i.d. = 300 μm, Ltot = 200 mm, LPGC = 25 mm) with a mobile phase of 30% ACN at 2 μL/min: (a) isothermal conditions: 30, 80, 120, and 160 °C (indicated on the figure) and (b) temperature-programmed conditions (4- or 6-min isothermal hold at 30 °C, inducing a trapping time of 2 and 4 min, respectively (the red and blue curves, respectively; see text); heating up to 160 at 1200 °C/min; isothermal hold of 2 min at 160 °C; and cooling to 30 °C. In overlay, the isothermal elution at 160 °C is plotted (in black). The temperature programs are represented by the dashed lines.

phthalate for different isothermal temperatures of the LTM heating sleeve around the PGC segment and for a flow rate of 2 μL/min and a mobile phase of 30 vol % ACN. It shows that the retention of dimethyl phthalate can be strongly modified by changing the temperature. A very high retention was obtained at a temperature of 30 °C (tR = 35.6 min, k0 = 7.4), while the retention decreased drastically with increasing temperature. At 160 °C, a very low retention was obtained (tR = 6.3 min, k0 = 0.5). This is consistent with the fact that the PGC stationary phase is highly retentive for the (weakly) polar and aromatic dimethyl phthalate and that the molar retention enthalpy is large on PGC (from the data of Figure 3a, a molar retention enthalpy of ΔHR = 22.7 kJ/mol can be calculated for the given conditions and assuming a linear Van’t Hoff relation, cf. eq 1). At even higher temperatures, dimethyl phthalate can be expected to almost coelute with the t0 marker. The very broad peak of dimethyl phthalate eluting at low temperatures is due to the very high residence time and dispersion in the capillary column. The high degree of peak tailing that can be noticed is most probably due to the packing quality of the capillary and the heterogeneity of the PGC surface.18 Figure 3b shows the elution of dimethyl phthalate under temperature-programmed conditions (same thermal modulation capillary and chromatographic conditions). The temperature of the LTM heating sleeve was kept at 30 °C (high retention) for 4 min (and 6 min), after which time the temperature was rapidly 7055

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Analytical Chemistry increased to 160 °C (low retention) at a temperature ramp of 1200 °C/min. Higher temperature ramps can be obtained with the LTMLC heating module (up to 1800 °C/min23), but were not used to limit the additional band broadening due to radial temperature gradients.24 After a 2-min isothermal hold at 160 °C, the LTM heating sleeve was cooled to 30 °C via natural convection. The retention of dimethyl phthalate increased to tR = 8.1 min when delaying the heating after 4 min (red curve), a difference of ΔtR = 1.8 min, compared to the isothermal retention at 160 °C (tR = 6.3 min). The first 2 min of this delay do not influence the retention, because this is the time the analytes need to migrate through the first silica segment (packed with nonporous silica and, hence, almost nonretentive particles) preceding the PGC segment. During the last 2 min of the 4-min delay, dimethyl phthalate is then trapped on the PGC segment and only elutes off the PGC zone when the temperature is increased. The difference between the two retention times is slightly less than the trapping time of 2 min, because the analytes still migrate through the PGC section at 30 °C, although at a very low migration velocity. Similar conclusions can be drawn for the delayed heating of 6 min (blue curve). The difference in retention now is almost 4 min (ΔtR = 3.5 min), again because of the trapping time of 4 min, combined with a very slow migration through the PGC section at 30 °C. Besides these changes in retention, the peaks obtained with the delayed heating are also slightly smaller and higher than those for the undelayed isothermal elution. For the delay of 4 min, the peak width at half height is 25 s, compared to the peak width of 38 s for the isothermal elution. This observation can be explained as follows: when a peak migrates through the silica section, the front of the peak will eventually reach the PGC section and will assume a very low migration velocity there (uR,PGC), while the tail of the peak (still occupying the silica section) still migrates at a much higher velocity (uR,SiO2). This difference in migration velocities leads to the compression and concentration of the peak (because k0 PGC . 0 and k0 SiO2 ≈ 0 and, thus, uR,SiO2 ≈ u0 . uR,PGC). In case of the heating delay of 6 min, the peak width is 35 s, which is wider than the peak width for a delay of 4 min. This is in agreement with the fact that the compression is countered by the diffusion of the trapped peak on the PGC section, and, therefore, the later remobilization leads to a broader peak. In the case of the isothermal measurement at 160 °C (black curve), there is little difference in migration velocity on the PGC section, compared to the silica section (uR,PGC ≈ uR,SiO2 ≈ u0) and, therefore, no compression and concentration occur. The signal enhancement obtained by the trapping is only marginal in the example shown above. This is because the chromatographic conditions were not sufficiently well chosen to optimally benefit from the peak compression. In the employed segmented trapping capillary, again, ∼2 min were required to migrate across the last silica section, because of the relatively large capillary inner diameter (300 μm) and the low flow rate (2 μL/min). As a consequence, the gain in peak width obtained by the trapping was almost completely lost, because of the dispersion of the compressed peak in the last silica segment. Figure 3 nevertheless already indicates that a modulation capillary consisting of a three-section packed bed with PGC particles and used in combination with very rapid heating achieved by the LTMLC heating device is capable of (1) changing the peak’s retention time (peak modulation) and (2) enhance the peak’s response by concentrating the molecules at the beginning of the PGC section (investigated for neutral species). Although trapping and modulation

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Figure 4. (a) Separation of the epoxy resin on a Zorbax SB-C8 column (1.0  150 mm, dp = 3.5 μm) and corresponding pressure profile. (b) Zorbax SB-C8 column coupled with thermal modulation system (capillary i.d. = 200 μm, capillary length = 100 mm, and PGC segment length = 15 mm). The temperature inside the LTM heating sleeve and the pressure trace are shown for the following temperature program: (1) from 30 °C to 210 °C at a rate of 1200 °C/min, (2) 10 s isothermal at 210 °C, and (3) cooling with compressed air to 30 °C. This cycle is repeated every 2 min. (c) Modulated separation of the epoxy resin with the same temperature profile as that described in panel b. For all three figures, the following chromatographic conditions apply: gradient profile = 50%100% ACN in 37.5 min, and isocratic hold 100% ACN. The flow rate is 20 μL/min.

of neutral compounds is shown here with porous graphitic carbon as a stationary phase and a water/acetonitrile mobile phase, other stationary and mobile phases can be suitable for trapping, as long as the retention of the analytes (neutral, acidic, or basic) on the trapping device is significantly higher than their retention on the first dimension column. Subambient temperatures also can be used to increase the retention on the trapping device. Holm et al. demonstrated the subambient focusing of antioxidant compounds on a C18 stationary phase and neat ACN as the mobile phase.27 The trapping of acidic and basic molecules is the subject of a future study, but it can be 7056

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Analytical Chemistry expected that porous graphitic carbon is a suitable stationary phase. Barrett et al. reported a similar retention window for the acidic/basic compounds, compared to their un-ionized form on PGC (depending on acid/base strength and the mobile phase pH).28 Because the silica segments in the trapping capillary are used to cover the distance between the column and the PGC section and the distance between the PGC section and the detector (see Figure 2b), the PGC-trapping section can be uniformly heated and all molecules trapped on the PGC section can be thermally remobilized. This could not have been possible when the modulation capillary would be fully filled with PGC particles. Application: Modulation and Enhancement of an Epoxy Resin Separation. Figure 4a shows a typical separation of an epoxy resin (structure shown in Figure 1) on a C8 column (without modulation segment attached to it) and using a water acetonitrile gradient (from 50% to 100% ACN in 37.5 min). The four broad peaks that can be distinguished in the chromatogram correspond to the oligomer fractions with degrees of oligomerization of, respectively, n = 0 to n = 3 (see Figure 1). At t = 32 min, only a small peak elutes (n = 4), followed by a large nonseparated peak, which corresponds to the high Mw oligomer fractions (n > 4). Peak shoulders before and after the maximum of each peak can be noticed, indicating the different ortho, para, or mixed oligomer configurations. It is well-known that for a degree of polymerization of n = 0, three configurations exist (oo, op, and pp), which should normally result in three peaks. This is not the case in the obtained chromatogram, but, nevertheless, a peak shoulder can be seen before and after the peak maximum (the para configuration elutes prior to the ortho configuration). For n > 0, many more possible configurations exist, explaining the broad and continuous peak.29,30 The same epoxy separation was subsequently repeated on a system with the thermal modulation capillary connected between the C8 column and the detector (see Figure 2b). Figure 4b shows the pressure and temperature trace, and Figure 4c shows the modulated chromatogram of the epoxy resin. To improve the elution velocity after remobilization, a smaller capillary than that used in Figure 3 was used as the modulation column (capillary 2: i.d. = 200 μm, LMOD = 100 mm, LPGC = 15 mm) combined with a high flow rate of 20 μL/min (the same flow rate as that given in Figure 4a). This results in a very high linear velocity in the modulation capillary (u0 = 17.7 mm/s) and a very low dead time (t0 = 5.7 s). The temperature program applied to the LTM heating sleeve consisted of the following sequences: heating from 30 °C to 210 °C (which takes 9 s at a temperature ramp of 1200 °C/min), an isothermal hold at 210 °C for 10 s, and cooling to 30 °C by blowing compressed air over the LTM heating sleeve and the modulation capillary. The cooling is achieved within 10 s as well, and, therefore, the temperature pulse takes ∼30 s overall (see inset in Figure 4b). This temperature program is cyclically repeated every 2 min. The pressure trace exhibits behavior similar to that of the temperature. When the LTM heating sleeve heats up the modulation capillary, the viscosity of the mobile phase decreases and results in a pressure dip. The pressure drop starts 5 s later than the temperature pulse, showing that only a few seconds are needed to transfer the heat from the LTM heating sleeve to the mobile phase across the air layer and the silica capillary wall.24 When the LTM heating sleeve and capillary are cooled to 30 °C, the pressure again increases to its original value, indicating that the temperature of the mobile phase is, again, 30 °C. The total width of each pressure dip is 35 s, which is slightly larger than the

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width of a temperature pulse. Although the same temperature pulse is applied throughout the entire separation, the amplitude of the pressure dip is larger at the start of the mobile phase gradient, because the viscosity of water is more temperaturedependent than the viscosity of an organic modifier such as acetonitrile.31 The thermally modulated chromatogram of the epoxy resin separation, shown in Figure 4c, is clearly very different from the original, nonmodulated chromatogram shown in Figure 4a. For elution times larger than 28 min, the signal is perfectly modulated, with a peak arising every 2 min simultaneously with the pressure dip and, hence, also with the applied temperature pulse (neglecting the few seconds of thermal delay). Hence, the observed profile can only be explained by the fact that the analytes eluting from the C8 column are trapped and concentrated on the PGC particles in the thermal modulation capillary. Only when a temperature pulse is applied, the molecules regain a very high migration velocity and elute off the PGC segment. The fact that the peaks elute exactly with the same pattern as the heat pulses makes the thermal modulation device suitable as an injection device for the second dimension column in LC  LC applications. Figure 4c shows that this is even the case when a very strong elution solvent is pumped, since a good modulation is still obtained after t = 41 min (corresponding to the sum of the gradient time and the void time of the C8 column) when the components elute at 100% ACN. The peak width of the modulated peaks is 35 s (baseline peak width), corresponding to the length of the pressure dip and the temperature pulse. This also shows that the heating and cooling rates are the most important parameters that should be improved for further reduction of the peak width. However, already a major improvement in peak width is achieved because the signal which normally elutes continuously during the 2-min cycle time is now concentrated into a peak of 35 s. As a result of the narrowing of the peaks, an improvement in the peak response up to 18 times can be noted, similar to the signal enhancement noted in the first papers on thermal modulation in GC.4,8 The total peak area remains almost unchanged: the modulated signal has a total peak area of 35.3 AU s, comparable to the total peak area of 36.5 AU s of the nonmodulated signal (integration boundaries: t = 18 and 50 min). The small difference in total peak area can be attributed to some very-high-molecular-weight polymer fractions, which do elute off the PGC segment after t = 50 min. After a few runs, the PGC segment was regenerated by keeping the thermal modulation device at a high temperature (160210 °C) and flushing it with pure acetonitrile for 5 min, to also elute the remaining very high Mw fractions. For elution times shorter than 28 min, three main peaks can be identified (as indicated in Figure 4c). When analyzing peak (III) in more detail, a small shoulder already elutes before the major part of the peak and before the pressure dip, which indicates that the analytes present in the shoulder already started eluting off the PGC segment before the temperature pulse was applied. This can be attributed to the fact that the retention coefficient of early eluting analytes is not high, not even on PGC (in this example, the low-molecular-weight oligomer fractions). In combination with the very high linear velocity in the modulation capillary (u0 = 17.7 mm/s), it hence does not take a long time before the analytes elute off the relatively short PGC segment (LPGC = 15 mm). This is even more pronounced for the peaks indicated as (I) and (II), because they have an even lower retention on the PGC. This shows that the length of the PGC segment should be 7057

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Table 1. GPC Molecular Weight Data (Mp) and Degree of Polymerization (n) of the Modulated Peak Fractions fraction

Mp (g/mol)

n

F1

634

2

F2 F3

808 966

3 4

F4

1123

5

F5

1286

6

F6

1480

7

F7

1621

79

F8

1712

79

F9

1820

79

Figure 5. Gel permeation chromatography (GPC) of (a) the collected fractions F1F9 (the corresponding fractions are indicated in the inset) and (b) the entire epoxy resin (the corresponding degree of polymerization (n) is given in the inset) on three coupled PL gel mixed E columns (7.8 mm  300 mm, dp = 3 μm), using tetrahydrofuran (THF) as the mobile phase at 0.4 mL/min and 40 °C.

Figure 6. Offline analysis of the collected fractions F1F9 on a Fused Core Ascentis Express PhenylHexyl column (2.1 mm  30 mm, dp = 2.7 μm). Gradient program: 60% to 100% ACN in 1.0 min, isocratic hold for 0.7, and 0.85 min column re-equilibration. The flow rate is 0.5 mL/min.

adjusted to trap the early eluting components as well, but the high linear velocity should be maintained in order to elute the analytes quickly after the thermal remobilization (see the discussion in the section entitled “Retention Modification with the Thermal Modulation Device”). The inset in Figure 4c shows that, when a longer PGC segment is used (45 mm), the low MW epoxy oligomers are trapped. Also, subambient temperatures or a trapping support with a higher surface area/packing density can be employed to trap low-molecular-weight molecules. Some very minor fluctuations in the modulated signal can also be seen every 2 min when the temperature pulse is applied (even during the void time of the C8 column). This is a complex effect of the mobile phase temperature, pressure, and flow rate changes on the UV detector intensity, which however does not influence the chromatographic performance. Also, a note should be made on the possible deactivation of the PGC particles resulting in a reduced retention of solutes. The PGC particles can be reactivated by a pure dioxane wash, whereupon the original retention strength is restored.16 To further analyze the chromatographic performance of the system, gel permeation chromatography (GPC) analysis were carried out to characterize the different modulated peaks, which were collected during the 35 s of elution (the fractions F1F9 are indicated by arrows in Figure 4c). Figure 5a shows an overlay of the GPC analysis of the nine fractions, and Table 1 gives the corresponding molecular weight Mp of the peak maxima and the degree of oligomerization of the fractions. The molecular weight Mp (obtained with a linear universal calibration) of each peak of

the fraction corresponds to a degree of polymerization between n = 2 and n = 9, although the last fractions have a very broad molar mass distribution and, therefore, are difficult to characterize. However, when carefully analyzing the chromatogram of fraction F3, a shoulder can be noticed with the same elution time as the peak maximum of fraction F2. This indicates that fraction F3 not only contains a resin with a degree of oligomerization of n = 4, as well as n = 3. This is most probably the orthoconfiguration of n = 3, which elutes after the mixed and paraconfigurations and is trapped together with the resin n = 4 and therefore coelutes in fraction F3. The same can be seen for fraction F4, which shows a smaller shoulder with the same elution time as the peak maximum of fraction F3. Because the number of possible configurations increases with increasing degree of oligomerization, the distributions become broader and the very high Mw fractions (F6F9) are not easy to distinguish. The GPC chromatogram of the entire sample is shown in Figure 5b. When comparing this to the GPC data of the modulated peaks (Figure 5a), it can be noted that, without modulation, only a degree of polymerization up to n = 5 can be distinguished, whereas the modulation allows for a characterization up to n = 7, because of the signal enhancement of the highmolecular-weight polymer fractions. Figure 6 shows the offline LC analysis of the collected fractions on a PhenylHexyl column. Although the high-molecularweight polymer resin was not separated on the nonmodulated system, separated fractions corresponding to the different modulated peaks could be obtained, because of the signal enhancement 7058

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Analytical Chemistry of the thermal modulation device. Triplets can be seen for fraction F1, corresponding to the para, mixed, and ortho configurations. Fraction F3 shows a quartet for which the first peak coelutes with the last peak from fraction F2, indicating the ortho configuration of the resin with a degree of polymerization of n = 3.

’ CONCLUSIONS Trapping and releasing of neutral analytes was achieved using the low-thermal-mass liquid chromatography (LC) setup for ultrafast heating, combined with the highly retentive porous graphitic carbon (PGC) as the stationary phase. When the analytes elute from the first dimension column, they are trapped on the PGC and locally concentrated due to the difference in migration velocity on the different stationary phases. When, subsequently, a very fast temperature pulse is applied, the molecules are remobilized and by repeating this cyclically, the thermal modulation interface can serve as a concentration and an injection device for a second dimension separation column. This was demonstrated with the separation of an epoxy resin. An improvement in signal enhancement up to 18 times was achieved for the modulated high-molecular-weight polymer resins, allowing the second dimension analysis by gel permeation chromatography (GPC) or LC. This was demonstrated with an off-line analysis of the high-molecular-weight epoxy oligomers. With the GPC analysis, fractions up to a degree of oligomerization of n = 7 could be identified, compared to n = 5 for the nonmodulated signal. With a subsequent off-line LC analysis, indications of the different para, mixed, and ortho configurations were now observed, whereas this was totally impossible without the modulation interface. ’ AUTHOR INFORMATION Corresponding Author

*Tel.: (+32) (0)2 629 32 51. Fax: (+32) (0)2 629 32 48. E-mail: [email protected].

’ ACKNOWLEDGMENT M.V. gratefully acknowledges a research grant from the Research FoundationFlanders (FWO Vlaanderen). Mark Schure is thanked for the stimulating and helpful discussions on this work. Gisela Jeschek and Romina Hammes are thanked for the technical help. Agilent Technologies (Hans-Georg Weissgerber, Tom Vandegoor, Gerard Rozing, Monika Dittmann and Helmut Schulenberg-Schell) is kindly thanked for the experimental support (providing some columns and the capillary LC system), as well as the stimulating discussions on this project. Thermo Fisher Scientific (Markus Foerster) is acknowledged for the donation of Hypercarb material. ’ SYMBOL LIST dp = particle diameter [μm] F = flow rate [μL/min] ΔHR = retention enthalpy [J/mol] i.d. = inner diameter [m] k = retention coefficient kSiO2 = retention coefficient in the SiO2 segment kPGC = retention coefficient in the PGC segment LMOD = length of the total modulation capillary [m] LPGC = length of the PGC segment of the modulation capillary [m]

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Mp = molecular weight of the GPC peak maximum [g/mol] Mw = molecular weight n = degree of polymerization o.d. = outer diameter [m] R = universal gas constant tR = retention time [s] T = temperature [K] u0 = linear velocity [m/s] uR = migration velocity of the analyte [m/s] uR,SiO2 = migration velocity of the analyte in the SiO2 segment [m/s] uR,PGC = migration velocity of the analyte in the PGC segment [m/s]

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