Article pubs.acs.org/ac
Increasing Flexibility in Two-Dimensional Liquid Chromatography by Pulsed Elution of the First Dimension: A Proof of Concept Simon S. Jakobsen,†,‡ Jan H. Christensen,† Sylvain Verdier,‡ Claude R. Mallet,§ and Nikoline J. Nielsen*,† †
Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark ‡ Haldor Topsoe A/S, Haldor Topsøes Allé 1, DK-2800 Kongens Lyngby, Denmark § Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, United States S Supporting Information *
ABSTRACT: This work demonstrates the development of an online two-dimensional liquid chromatography (2D-LC) method where the first dimension column is eluted by a sequence of pulses of increasing eluotropic strength generated by the LC pumps (pulsed-elution 2D-LC). Between the pulses, the first dimension is kept in a no-elution state using low eluent strength. The eluate from the first dimension is actively modulated using trap columns and subsequently analyzed in the second dimension. We demonstrate that by tuning the length and eluotropic strength of the pulses, peaks with retention factors in water, kw, above 150 can be manipulated to elute in 3−4 pulses. The no-elution state can be kept for 1−10 min with only minor changes as to which and how many pulses the peaks elute in. Pulsed-elution 2D-LC combined with active modulation tackles three of the main challenges encountered in 2D-LC and specifically online comprehensive 2D-LC: undersampling, difficulties in refocusing, and lack of flexibility in the selection of column dimensions and flow rates because the two dimensions constrain each other. The pulsed-elution 2D-LC was applied for the analysis of a basic fraction of vacuum gas oil. Peak capacity was 4018 for a 540 min analysis and 4610 for a 1040 min analysis.
N
of the separation dimensions and that they can be optimized individually, theoretically producing an infinite peak capacity at the cost of long analysis times.7 However, offline 2D-LC systems introduce difficulties in managing the large number of fractions.8 For offline 2D-LC, nc of 4000 and 7000 have been achieved in 5 and 27 h.8,9 In the online approach, each fraction from 1D is transferred to the second separation dimension (2D) and analyzed in real time. This implies that the 2 D-analysis, including reequilibration of the 2D-column, should be completed in the time it takes to collect and transfer the 1D-fraction. This requires 2D-analysis times on the subminute time scale to avoid loss of 1D-peak capacity (1nc) due to undersampling.10 The optimal number of fractions collected per 1D-peak has been found to be between 2 and 4.11−14 Another challenge is the reduction in 2D-peak capacity (2nc) caused by band broadening in 2D due to large injection volumes, high eluotropic strength of the injection plug or solvent incompatibility.15−17 To reduce the effects of injection volume, the 1D-column is normally long and narrow, operated with a low flow rate, while the 2D-column is short and wide, operated with high flow rates.17 While a large
ontarget analysis of high-complexity samples such as petroleum products and biological and environmental matrices often require analytical approaches involving chromatography to separate as many components as possible to obtain the highest level of information. The simplest way to increase peak capacity is to increase the number of theoretical plates. Therefore, great efforts have been invested into developing column stationary phase particles with diameters smaller than 2 μm, core−shell particles, stationary phases with high thermal stability, and instruments capable of withstanding backpressure of 1000 bar or more. All of these improvements translate into the use of more efficient columns which can generate a higher peak capacity. For one-dimensional liquid chromatography (1D-LC) peak capacities (nc) of 1000 to 1500 have been reached for analysis times of 600−1900 min.1,2 This appears though to be the practical limit.3,4 Another approach to achieve increased peak capacity is twodimensional liquid chromatography (2D-LC),5,6 where two (ideally) orthogonal separation mechanisms are combined. Presently, three schemes for comprehensive 2D-LC exist: offline, online (LC × LC), and stop-and-go.7 The offline scheme is conceptually simple: each fraction eluting from the first dimension (1D) is collected and stored until they are analyzed individually on the second column.7 The benefits of offline 2D-LC are the ease of combining orthogonal columns for the two dimensions, no time constraints on either © 2017 American Chemical Society
Received: March 1, 2017 Accepted: July 31, 2017 Published: July 31, 2017 8723
DOI: 10.1021/acs.analchem.7b00758 Anal. Chem. 2017, 89, 8723−8730
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Analytical Chemistry
demonstrated for the analysis of 27 nitrogen containing heterocyclic compounds as well as the basic fraction of a vacuum gas oil.
ratio between column diameters (d2/d1) alleviates the negative effect on peak capacity, it has a detrimental effect on dilution and thus the sensitivity. Strategies to overcome the challenges of increased injection band broadening cover methods using trap columns as replacements for loops,18−22 dilution prior to trapping,23,24 thermally assisted modulation,25−27 and solvent assisted modulation.28,29 Total nc for the online scheme was in the range of 500 to 1000 for analysis times less than 2 h.24,30,31 An nc of 5100 was achieved with analysis time of 200 min.32 The stop-and-go approach comprises two main techniques, stop-flow and stop-elution, for which the overall mechanism is conceptually the same: the elution from the 1D-column is stopped while a fraction is transferred and analyzed on the 2Dcolumn; subsequently, the elution from the first dimension is resumed. In the stop-flow variation, the flow is diverted to a plug and stopped. This approach reduces the time constraints on the 2D. During stop-flow, diffusive band broadening of the chromatographic peaks can be expected and with this a decrease in the efficiency of the 1D-column. However, Bedani et al.33 found no difference in additional band broadening comparing online and stop-flow size exclusion chromatography (SEC) coupled to reverse phase chromatography (RP) for stop times of 9.5 min. Fairchild et al.7 came to the same conclusion for their stop-flow strong cation exchange (SCX) × RP analysis of protein digest for stop times of 3 min. They achieved nc of 266 and 2790 for a 40 and 140 min analysis, respectively.7 The second approach is the stop-elution, most noticeably described in the multidimensional protein identification technology (MudPIT) designed for proteomics.34−38 In MudPIT, SCX and RP stationary phases are packed sequentially in a microcapillary column connected to a tandem mass spectrometer. Proteins and peptides are eluted from the SCX section by salt pulses of increasing concentrations onto the RP section. Every salt injection is followed by a RP separation. Due to the sequential nature of the column, the flow over the SCX section remains at all times but during RP-elution operation of the SCX section is in a no-elution state. The pulsed nature of MudPIT has been applied in 2D-LC setups using a SCX 1D-column and a RP 2D-column for protein identification, where 1D is eluted by an injection of a salt plug from a vial,18,22 or by generating a semi-continuous gradient with the LC-pumps.19 The aim of this work was to expand on the idea of stop-andgo 2D-LC using pulses and no-elution conditions in 1D. An online RP × RP 2D-LC method is developed in which the analytes are eluted from the first dimension by pulses of increasing eluotropic strength. Between the pulses, the 1Dcolumn is kept in a no-elution state by running a weak eluent through the column. In this state elution from 1D does not occur. The pulsed operation of the first dimension serves two purposes: first, it allows the operator to sample each peak multiple times to decrease the effect of undersampling on 1nc. Second, the time of the no-elution periods between two pulses can be extended to obtain a desired 2nc. This is done using UHPLC × UHPLC modified to include a dilution step after the 1 D to decrease the eluotropic strength of the pulses and a switching valve equipped with two trap columns. The use of active modulation prevents injection band broadening in the second dimension and allows the use of any column dimensions at any flow rate. The outcome is a flexible online 2D-LC method where the two separation dimensions can be optimized individually and the full potential of the second dimension column can be exploited. Pulsed-elution 2D-LC is
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EXPERIMENTAL SECTION Chemicals. Acetonitrile (LC-MS ultra Chromasolv, >99.9%) and water (TOC = 1 ppb, > 18.2 MΩ) purified on a Veolia Purelab Chorus ELGA system was used for mobile phases. Methanol (Chromasolv for HPLC > 99.9%) was used to dissolve the standard compounds. All solvents were from Sigma-Aldrich. Standards used were aniline (62-53-3), o-toluidine (95-53-4), 1-naphthylamine (134-32-1), N,N-dimethyl-1-naphthylamine (86-56-6), 1-aminopyrene (14062-24-9), isoquinoline (11965-3), 1-methylisoquinoline (1721-93-3), 2,6-dimethylquinoline (877-43-0), benzo[h]quinoline (230-27-3), dibenz[f,h]quinoline (217-65-2), acridine (260-94-6), benz[c]acridine (225-51-4), 7,9-dimethylbenz[c]acridine (963-89-3), 3-methylbenzonitrile (620-22-4), 1-napthonitrile (86-53-3), 9-anthracenecarbonitrile (1210-12-47), 1H-indole (120-72-9), 2,3-dimethyl-1H-indole (91-55-4), 1H-benzo[g]indole (233-34-1), 9-ethyl-9H-carbazole (86-28-2), 1,4-dimethyl-9H-carbazole (18028-55-2), and 11H-benzo[a]carbazole (239-01-0) from Sigma-Aldrich; benzonitrile (100-47-0) and 9H-carbazole from Fluka; dibenzo[a,h]acridine (226-36-8) and 7H-dibenzo[c,g]carbazole (194-59-2) from EC-JRC-IRMM; and quinoline (9122-5) from Acros Organics. Standards were of purity higher than 96% dissolved in methanol. Concentrations in the stock solutions ranged from 79.6 to 1492.0 μg/mL. Instrumentation and Chromatographic Conditions. 1D Instrumentation. A Waters (Milford, United States) Acquity UPLC I-class system composed of a binary solvent manager, a flow-through-needle sample manager, a column manager, and a PDA detector was used for the 1D experiments. We used a Cortecs UPLC C18 column (30 × 2.1 mm; 1.6 μm, 90 Å, Waters). The column temperature was maintained at 25.0 °C. The mobile phases were A: water and B: acetonitrile. A flow rate of 0.2 mL/min was used. The settings for the PDA detector were a sampling frequency of 20 Hz with a filter constant of 0.05 s and a resolution of 1.2 nm over the wavelength range 190−400 nm. Generation of Pulses Using the UHPLC Pump. To investigate if the pump could deliver precise pulses the column was replaced with a zero dead volume union. The initial and final mobile phase composition was 95:5 water: acetonitrile. The pulse length and amplitude was from 0.09 to 0.44 min and from 10% acetonitrile to 100% acetonitrile. Manipulation of Gradient Peak Pattern. To evaluate if the peak pattern for an analyte could be manipulated to elute in 3− 4 pulses, a test set consisting of aniline (1), isoquinoline (2), 3methylbenzonitrile (3), 1H-benzo[g]indole (4), 1,4-dimethyl9H-carbazole (5), benz[c]acridine (6), and 7,9-dimethylbenz[c]acridine (7) was analyzed under gradient pulse conditions. The pulse gradient had an amplitude increase of 1% B/pulse. The initial mobile phase and the mobile phase in the 1 min noelution time between pulses was 99:1 A:B. The pulse length was varied from 0.3 to 0.5 min to find a pulse length that would result in elution in 3−4 pulses. With the optimal pulse length setting and the same pulse gradient as above, the time of the no-elution period was changed from 1 to 10 min to evaluate if the 1D could be kept in a no-elution state for extended periods of time without changing the peak pattern or the pulse positions in which the peaks elute. 8724
DOI: 10.1021/acs.analchem.7b00758 Anal. Chem. 2017, 89, 8723−8730
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For the setup with 2D cycle time of 10.4 min, the dilution pump was set to deliver 100% water at 1.8 mL/min from 0 to 1.5 min again to dilute the high eluotropic strength pulse. From 1.5 to 2.0 min the flow rate was lowered to 0.1 mL/min and kept at 0.1 mL/min until 9.4 min, after which it was increased again to 1.8 min over 0.5 min (until 9.9 min) and held at 1.8 mL/min until 10.4 min. The system was controlled by Waters Masslynx 4.1. The data was exported as CDF files and processed using an in-house Matlab-R2015b (The MathWorks, Natick, MA, 2015) script. Gas Oil Samples. For this study, one hydrocracking feedstock was analyzed. It was a blend of a straight-run vacuum gas oil (VGO) and a coker gas oil (CGO). The blending ratio was 90/10 with a basic N content of 432 ppm and a total N content of 1357 ppm. Sample Preparation. The neutral and basic nitrogen compounds was separated and preconcentrated by solid phase extraction (SPE) using a silica SPE (Chromabond SiOH, 2g, Machery-Nagel, Düren, Germany) prior to analysis. The SPE cartridge was preloaded with 5 mL heptane. The sample (0.3 g) was diluted with 2 mL heptane and loaded to the top of the SPE cartridge. The nonretained hydrocarbon matrix was flushed out of the SPE cartridge using 10 mL of heptane. The compounds containing neutral nitrogen were eluted using 10 mL of a 80:20 heptane:dichloromethane solution, and the compounds containing basic nitrogen were last eluted using 5 mL of acetone. Each fraction was evaporated to dryness under a stream of inert nitrogen at 60 °C and reconstituted in 0.5 mL of acetone. MS Detection. The basic fraction of vacuum gas oil was detected using a Waters Synapt G2-S quadrupole-time-of-flight detector equipped with a z-spray ESI source. The source was operated in positive ion mode and parametrized as follows: capillary voltage 0.5 kV, sampling cone 40 V, source temperature 120 °C, desolvation gas temperature 500 °C, cone gas flow 100 L/h, desolvation gas flow 1000 L/h. The m/ z-axis was calibrated using sodium formate clusters and a fifth order polynomial fit; the instrument operated in resolution mode (resolving power 20.000) with a leucine enkephaline lock spray pulsed every 30 s. The scan time was 0.25 s; the interscan delay was 0.014 s, and data retrieved in centroid mode between m/z 50 and 1000. Calculation of Peak Capacity. The peak capacity in the second dimension (2nc) was determined from the average width at half height (w1/2h) according to eq 1:
2D instrumentation. The 2D-LC setup used the same hardware as described above and, in addition, two binary solvent managers. One was used to add a dilution flow into the effluent from the first column via a 50 μL mixer (Waters) and the second was used to elute the 2D-column. An electrically actuated Vici 10-port 2-postion high-pressure switching valve was used as the modulation interface between the two dimensions. The switching valve was configured in backflush mode. Two Xbridge BEH C18 direct connect HP columns (30 mm × 2.1 mm, 10 μm, 130 Å, Waters) were installed into the switching valve and used as traps. A schematic presentation of the system is shown in Figure 1.
Figure 1. Schematic presentation of the pulsed-elution 2D-LC setup. A dilution flow is introduced into the effluent from the first dimension to decrease the eluotropic strength and allow refocusing of the analytes on the trap column prior to injection into 2D.
In 2D we used an Acquity HSS PFP column (100 × 2.1 mm; 1.8 μm, 100 Å, Waters). The standard F-value for the 1D and 2 D columns is 60.7 according to the hydrophobic subtraction model.39 Separations were performed using mobile phase A:water and mobile phase B: acetonitrile. The flow rate for 1D was 0.2 mL/min, and the temperature was 25.0 °C. The pulse gradient was 1% B/pulse starting at 1% B and finishing at 100% B; the number of pulses was 100 and the pulse length was 0.4 min. The initial mobile phase and the mobile phase in the noelution time was 99:1 A:B. The no-elution time was 5 or 10 min, resulting in 2D cycle times of 5.4 or 10.4 min (pulse length + no-elution time). Total analysis time was 540 and 1040 min, respectively. In 2D, the flow rate was 0.4 mL/min at 40.0 °C. In the case with 5 min no-elution time, the 2D-gradient went from 5 to 95% B in 4 min. From 4.0 to 4.1 min, the mobile phase was changed back to 5% B, and the re-equilibration time was from 4.1 to 5.4 min at 5% B. In the experiment with 10 min noelution time, the 2D-gradient was from 5 to 95% B in 0−9 min, back to 5% B from 9.0 to 9.1 min, and re-equilibration of the column from 9.1 to 10.4 min with 5% B. This was repeated for each 2D cycle. For the setup with 5.4 min 2D cycle time, the pump to facilitate active modulation was set to deliver 100% water at 1.8 mL/min from 0 to 1.5 min to dilute the high eluotropic strength pulse. From 1.5 to 2.0 min, the flow rate was lowered to 0.1 mL/min and kept at 0.1 mL/min until 4.4 min, followed by an increase to 1.8 min over 0.5 min (until 4.9 min) and held at 1.8 mL/min until 5.4 min.
2
nc = 1 +
tG 1.7w̅ 12 h
(1)
An important setting for pulsed-elution 2D-LC is how many fractions, f, to collect from the first dimension as well as the number of fractions in which the analyte should elute. This determines the peak capacity in the first dimension. For a given 1 nc, this can be expressed as8,40 1
nc =
f N
(2)
where N is the average number of pulses in which one analyte elutes. The predicted peak capacity was calculated according to eq 3: 2D
8725
nc,pred ≈ 1nc 2nc
(3) DOI: 10.1021/acs.analchem.7b00758 Anal. Chem. 2017, 89, 8723−8730
Article
Analytical Chemistry Eq 3 does not take undersampling of the first dimension into account, and the undersampling factor from Davis, Stoll, and Carr14 was used to correct the first dimension peak capacity, assuming a 4σ peak width: 1 1
n′c =
1
nc = β
nc 3.424 N2
1+
(4)
Thus, by combining eqs 2 and 4, the corrected peak capacity of the first dimension can be written as: f
1
n′c =
2
N + 3.424
(5)
As the number of pulses in which each analyte elutes changes across the chromatogram, eqs 2 and 5 were corrected for this nonuniform distribution. This was done by calculating 1nc and 1 n′c for individual parts of the chromatogram, where N was calculated as the average for the analytes eluting in the given section and summed as demonstrated in eqs 6 and 7: 1
nc,weighted =
f1 N1̅
+
N2̅
+ ···
f1
1
n′c,weighted =
f2
Figure 2. Experimental pulse profiles. The programmed pulse lengths were A: 0.09 min, B: 0.18 min, C: 0.26 min, D: 0.35 min, and E: 0.44 min. For each pulse length, the initial mobile phase was 95:5 water: acetonitrile. Amplitudes were programmed to be 100% (black solid line), 90% (red round dotted line), 80% (orange square dotted line), 70% (green dashed line), 60% (blue dash-dotted line), 50% (purple long dashed line), 40% (pink long dash-dotted line), 30% (brown long dash-double-dotted line), 20% (indigo, bold solid line), and 10% acetonitrile (blue, bold round dotted line). The intensity was recorded at 195 nm, and the % acetonitrile was calculated using a calibration curve of increasing acetonitrile steps (see Supporting Information, S1).
2
N1̅ + 3.424
(6)
+
f2 2
+ ···
N2̅ + 3.424
the one at 100% B. The dispersion of the pulse can be mitigated by use of smaller volume-mixers or by adding another pump. Although the observed amplitude does not correspond to the programmed, there is an observable difference in the amplitude between two pulses with increasing % B for all five pulse lengths. Furthermore, there is a difference in observed amplitude for different pulse lengths at fixed amplitude. The use of smaller volume-mixers or an additional pump was not pursued here. Manipulation of Gradient Peak Pattern. The majority of both 1D and 2D-LC methods use gradient elution to yield sufficient separation of both weakly and strongly retained compounds in a reasonable time and to augment the peak capacity due to narrower peaks in gradient elution compared to those in isocratic elution.43 In pulsed-elution 2D-LC the application of gradient elution in the first dimension has another important feature; it can be used to tune the number of pulses in which each compound elutes, and we can thereby address the issue of undersampling of 1D.11−14 In Figure 3, a pulsed gradient of 1% B/pulse was applied with a flow rate of 0.2 mL/min with a no-elution time between the pulses of 1 min where the mobile phase composition is 99:1 A:B with different pulse lengths. Figure 3A shows the results with a pulse length of 0.3 min. For aniline (1), isoquinoline (2), and 3-methylbenzonitrile (3), a 0.3 min pulse length is too wide to elute the compounds in 3 pulses. Furthermore, the compounds elute between pulses (in the no-elution time) and thus may be difficult to modulate properly. This can be seen as the zero baseline level between the slices is not reached. Verstraeten et al.26 used thermal modulation and experienced elution in the no-elution time before the temperature pulse was applied for compounds with a low retention factor, k′. They solved the problem by choosing a column length that for their collection time and flow rate did not experience breakthrough. For 1Hbenzo[g]indole (4) and 1,4-dimethyl-9H-carbazole (5), the modulation is near optimal according to undersampling theory, i.e. 3−4 samplings per peak. But for benz[c]acridine (6) and
(7)
where f1 and f 2 are the number of pulses in each of the sections of the chromatogram and N1 and N2 are the average number of pulses in which each peak elutes in the sections of the chromatogram. The total peak capacity of the 2D-system that can be predicted and practically obtained then becomes:
■
2D
nc,pred ≈ 1nc,weighted 2nc
(8)
2D
nc,prac ≈ 1n′c,weighted 2nc
(9)
RESULTS AND DISCUSSION 1D Analysis. Generation of Pulses Using the UHPLC Pump. To develop the pulsed-elution 2D-LC method, an evaluation of the UHPLC pump module’s ability to deliver precise pulses was performed. Figure 2 shows the pulse profiles generated using pulse amplitudes of 10−100% B in increments of 10% for each of the five tested pulse lengths. The pulses were programmed in the gradient table to be rectangles, but as Figure 2 shows, the experimental profiles deviate from this shape for all pulse lengths. For the pulse profiles with 0.09, 0.18, and 0.26 min lengths, the experimentally observed amplitudes never reached the programmed amplitude, which can be observed on the secondary vertical axis. This deviation from the rectangular shape and the lower-than-expected amplitude can be explained by gradient dispersion in the high-pressure mixing chamber.41,42 The volumes of the pulses with lengths 0.09, 0.18, and 0.26 min correspond to 18, 36, and 52 μL, respectively, which is smaller than or equal to the volume of the mixer (50 μL) in the UHPLC system. The volume of the mixer will at no time be completely full with the mobile phase of the pulse, and no plateau can be observed due to the mentioned dispersion. For the pulses with length 0.35 and 0.44 min, the corresponding volumes are 70 and 88 μL. A plateau at the programmed value can be observed for all amplitudes except 8726
DOI: 10.1021/acs.analchem.7b00758 Anal. Chem. 2017, 89, 8723−8730
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Analytical Chemistry
Figure 3. Effect on peak pattern by changing the pulse length in gradient elution. The pulse gradient was from 1 to 100% B with an increase of 1% B/pulse. The pulse length was in A: 0.3 min, B: 0.4 min, and C: 0.5 min. The no-elution time between each pulse was 1 min at 99:1 A:B. The chromatogram is the sum of the extracted wavelengths 230, 250, and 280 nm. Numbering of the compounds can be found in Table 1. Artifacts related to software limitations were removed. The procedure is described, and raw data are given in the Supporting Information, Section S3.
Figure 4. Effect of changing the no-elution time between pulses on peak pattern and peak position. 1: aniline, 2: isoquinoline, 3: mtolunitrile, 4: 1H-benzo[g]indole, 5: 1,4-dimethylcarbazole, 6: benz[c]acridine, and 7: 7,9-dimethylbenz[c]acridine. Chromatographic conditions: pulse gradient of 1% acetonitrile/pulse with pulse length 0.4 min. The composition in the no-elution time between pulses is 99:1 A:B at a flow rate of 0.2 mL/min. The chromatogram is summed over 230, 250, and 280 nm. The lower chromatogram in each panel is the obtained gradient pulse profile recorded at 195 nm. Artifacts related to software limitations are removed. The procedure is described and raw data are given in the Supporting Information, Section S3.
7,9-dimethylbenz[c]acridine (7), the pulses are too narrow and the signal from the analytes is distributed in >4 pulses. In Figures 3B and 4C, the pulse length is increased to 0.4 and 0.5 min, respectively. This increase in pulse length decreases the number of pulses in which the analytes elute. The optimal pulse length was 0.4 min. Here, aniline elutes as a single peak; isoquinoline and 3-methylbenzonitrile elute in two pulses and in between the pulses, and 1H-benzo[g]indole (4), 1,4dimethyl-9H-carbazole (5), and benz[c]acridine (6) elute in three pulses. Lastly, 7,9-dimethylbenz[c]acridine (7) elutes in four pulses. If the pulse length is increased to 0.5 min, all
analytes but benz[c]acridine (6) and 7,9-dimethylbenz[c]acridine (7) elute in too few pulses. If a method with a uniform peak pattern along the entire chromatogram is desired, the pulse lengths can be modified from being constant throughout the entire chromatogram to instead increase in pulse length. However, to demonstrate the feasibility of the pulsed-elution 2D-LC concept, a simple pulse gradient that 8727
DOI: 10.1021/acs.analchem.7b00758 Anal. Chem. 2017, 89, 8723−8730
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Analytical Chemistry Table 1. Overview of the Pulse Position in Which the Analytes Elute no-elution time between pulses kw 1 2 3 4 5 6 7
aniline isoquinoline 3-methylbenzonitrile 1H-benzo[g]indole 1,4-dimethyl-9H-carbazole benz[c]acridine 7,9-dimethylbenz[c]acridine
S
6 32 51 154 351 455 975
2.121 3.454 2.709 3.270 3.261 2.934 3.015
1 min a
1−2 16−17b 25−26b 44−46 56−57 63−65 72−75
5 min
10 min
c
1c 5−6b 8−10d 40−42 56−58 63−66 72−75
1 8−10d 14−16d 43−44 56−57 63−66 72−75
Elutes between pulse 1 and 2. bElutes in the two pulses and between them. cElutes before pulse 1. dDoes not elute in the first pulse, but in the noelution time. Experimental determinations of kw and S are described in the Supporting Information, Section S5.
a
increases with 1% B/pulse and with a fixed pulse length of 0.4 min was chosen for further experiments. Changing the No-Elution Time. In Figure 4 and Table 1, the effect of increasing the no-elution time between each pulse from A: 1 min to B: 5 min and C: 10 min is presented. In the upper plots of Figures 4A−C, the sums of the extracted wavelengths 230, 250, and 280 nm are shown. The lower chromatograms in Figures 4A−C are the extracted wavelength chromatograms (EWC) at 195 nm, which show the pulse gradient. Figure 4C shows that the baseline drifts over time and then abruptly falls to zero again. The drops are due to software limitations. It was only possible to program 20 pulses in the software, and thus, spanning the full gradient requires five runs. For poorly retained compounds such as aniline (1), isoquinoline (2), and 3-methylbenzonitrile (3), both the pulse position and the peak pattern changes when the no-elution time is increased. This is due to movement in the no-elution time. For intermediately retained compounds such as 1H-benzo[g]indole (4) with kw ≈ 150 (data for linear solvent strength modeling is in the Supporting Information, Section S5), the position changes slightly from elution in pulse 44 to elution in pulse 40 when changing the no-elution time from 1 to 10 min, but the distribution of the signal between three pulses is maintained. Thus, the mitigation of 1D undersampling is not compromised. It can be observed for 1,4-dimethyl-9H-carbazole (5) (kw = 351), benz[c]acridine (6) (kw = 455), and 7,9-dimethylbenz[c]acridine (7) (kw = 975) that the pulse in which the analyte starts to elute does not change, even though the time between each pulse is increased. For example, 7,9-dimethylbenz[c]acridine starts to elute in pulse 72 when the no-elution time between each pulse is 1, 5, and 10 min. However, it can be observed that the relative peak heights for an analyte changes. If the starting time of the sampling is shifted slightly due to the extended no-elution time, this can cause a shift in the relative heights for the analyte. The modulation of the eluate from the 1 D is normally done in the valve interface. This study shows that it is possible to modulate the eluate directly on the 1Dcolumn, and by keeping the 1D in a no-elution state, it is possible to increase the 2D-analysis time. However, as the volume of the pulse is large (80 μL) and as the gradient progresses, the eluotropic strength of the pulses increases, which potentially can cause additional band broadening in the 2 D due to poor refocusing.15−17 To overcome this limitation, active modulation23,24 was applied, as illustrated in Figure 1. Active Modulation. Comprehensive 2D-LC normally uses loops to collect the eluate from the 1D, and the maximal 2Danalysis time is then determined by the time it takes to fill one loop. Here, active modulation where the two loops are replaced by two C18 trap columns and a dilution flow of 1.8 mL/min is
used. In the Supporting Information, Table S2, the peak widths of the neutral analytes are presented. No increase in peak width is observed for the analytes eluting late in 1D, even though the eluting pulse from 1D is 80 μL, and the eluotropic strength of the mobile phase in the pulse is high. Thus, an effective refocusing on the trap occurs due to the dilution because the retention factor of the analyte under the new conditions is increased. For the analytes with kw < 150 that elute between pulses, active modulation prevents losses as the mobile phase in the no-elution time is led to the trap where the analyte is retained. Pulsed-Elution 2D-LC. Pulsed-elution 2D-LC with 5 min no-elution time was tested on a fraction from a vacuum gas oil containing the basic nitrogen-containing compounds. In this case, positive ion mode electrospray time-of-flight mass spectrometry was used for detection. Figure 5 shows the extracted ion chromatograms of C7-C9 alkylated quinolines. Each line in Figure 5 represents one cycle: one pulse in 1D and a linear gradient in 2D. For this proof of concept, RP was used in both dimensions, and orthogonality of the two dimensions was not further optimized. However, in spite of the correlations between the retention mechanisms in the two dimensions, it can be observed that isomeric pairs of C7quinolines as well as C8-quinolines that coelute in 1D are resolved in 2D. Peak Capacity. A pulsed-elution 2D separation of a 27component mixture was used to estimate the practical peak capacity (Figures S7 and S8). Due to the fixed pulse length, the number of pulses in which each peak elutes changes. The chromatogram in Figure S7 was therefore divided into two sections to calculate the practical peak capacity. For the chromatogram with a no-elution time of 10 min, the first section was pulse 1 to 20, where analytes elute in 1.6 pulses on average. The second section was from pulse 21 to 100, where analytes elute in 3.4 pulses on average. Using eqs 6 and 7, this results in a predicted 1D peak capacity 1nc,weighted of 36.0 (first section 12.5, second section 23.5) and a corrected 1D peak capacity 1n′c,weighted of 28.7 (first section: 8.2 s section: 20.5). In Figure S7, it can be observed that some of the peaks are wide in 2 D, e.g. analyte 9 (acridine). The mobile phase of the second dimension was not pH-controlled, which results in excessive band broadening for some of the basic analytes. Therefore, only peaks arising from neutral analytes were used for the calculations of 2nc. The second dimension peak capacity was calculated according to eq 1 and was found to be 160, resulting in 2Dnc,pred of 5765. 2Dnc,prac was 4610 with peak production rate of 4.5 peaks per minute for the 1040 min (17.33 h) analysis. The loss of 1nc in the first section due to undersampling is 35%, and in the second section, the loss is 12%. However, the loss is 8728
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constitutes 20% of the chromatogram. For the chromatogram where the 2D cycle time was 5.4 min (Figure S8), the first section was from pulse 1 to 24. Here, analytes eluted in 1.9 pulses, and the second section was from pulse 25 to pulse 100, where analytes eluted in 2.8 pulses on average, giving a corrected 1D peak capacity of 31.9. The 2D peak capacity was 125.9. This gave 2Dnc,prac of 4018 and a peak production rate of 7.4 peaks/min. The 2D-gradient time for the 540 min analysis is 4 min, and the 2D-gradient time in the 1040 min analysis is 9 min, a 2.25 fold increase in available gradient time, but the increase in 2nc is only a factor 1.28. Gilar et. al4 derived a relationship between nc and gradient time based on the linear solvent strength model, which could be simplified to8
nc = 1 +
atG b + tG
(10)
This can be fitted to experimentally obtained peak capacities from gradient analysis, and it can be observed that the achievable peak capacity increases rather steeply and then goes toward a limit. When optimizing the peak capacity in 2D-LC, the goal is either to obtain a target peak capacity in the shortest possible time or to reach the highest peak capacity in a given time.8 This was investigated for offline 2D-LC by Horváth, Fairchild, and Guiochon.8 They found that for a given target peak capacity there is an optimum sampling rate which can be lower than the recommended for comprehensive online 2DLC. This optimum sampling rate depends on the characteristics of both columns and the target peak capacity. For the current pulsed-elution 2D-LC setup, a sampling rate of 1.6 across the entire chromatogram would result in a 1n′c of 40 compared to 1 n′c 27 if the sampling rate had been 3.4 for the entire chromatogram with corresponding 2Dnc,prac of 6423 and 4335, respectively. For a given target 2Dnc,prac, the same number of fractions could be collected for the two 1n′c; a decreased 2Dgradient time could be used for the smaller sampling rate, or fewer fractions could be collected with the same 2D-gradient time to save time and still obtain the target 2Dnc,prac. Optimization, primarily balancing the peak capacity obtained in each of the two dimensions, of the pulsed-elution 2D-LC was not the scope for this study but might be beneficial in future work. Also, a head-to-head comparison of pulsed-elution 2DLC with comprehensive online 2D-LC for different types of samples and scopes is recommended.
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CONCLUSIONS Until now, the use of pulses in LC has primarily been applied for separation of samples into a few fractions. The present study is a proof of concept where the possibility to use a large number of pulses in online comprehensive 2D-LC to elute the 1 D is demonstrated. In combination with active modulation, the pulsed-elution of the 1D increases the flexibility of 2D-LC separation, as the two separation dimensions are independent of each other. Key features of the pulsed-elution 2D-LC method are the following: The pulse gradient in 1D can be programmed such that analytes with kw >150 can be manipulated to elute in 2−4 pulses, thereby minimizing the effect of undersampling on 1nc. Less retained analytes can still be manipulated to elute in more than one pulse, but some elution between pulses is inevitable. Between each pulse in 1D, there is a no-elution time. This time can be increased from 1 to at least 10 min with no changes in
Figure 5. Extracted ion chromatograms of alkylated quinolines from the basic fraction of a vacuum gas oil analyzed by pulsed 2D-LC with positive ion mode electrospray-time-of-flight detection. Top is the sum of the [M + H]+ ions of C7−C9-quinolines: m/z 228.17, m/z 241.18, m/z 256.20 ± 0.05. Second: C7-quinolines. Third: C8-quinolines. Fourth: C9-quinolines. Z-axis display intensity in arbitrary units.
counteracted by the narrower 1D peaks in the first section compared to those in the second section of the chromatogram (1.6 pulses/peak vs 3.4 pulses/peak), making space for almost twice as many peaks. The first section contributes with 28% of the total first dimension peak capacity even though it only 8729
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peak pattern for analytes with kw > 150, only minor changes in the pulse position for analytes with kw = 150, and no changes in peak pattern or pulse position for analytes with kw > 350. This allows the analyst to choose freely 2D-analysis time to obtain a desired 2nc. Each pulsed eluting from 1D is actively modulated by dilution and trapping. This reduces the additional band broadening in 2D from poor refocusing and removes the need for high flow rates in 2D such that any column dimensions and flow rates can be chosen for 2D without sensitivity loss due to dilution. In summary, the pulsed-elution 2D-LC approach combined with active modulation tackles three of the main challenges in 2D-LC, namely undersampling, difficult refocusing, and time- and column dimensionality constraints.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b00758. Supporting figures and additional experimental and analytical details (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Simon S. Jakobsen: 0000-0003-4526-0618 Author Contributions
N.J.N. and J.H.C. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by Haldor Topsoe A/S. The authors of this work would like to thank Giorgio Tomasi for his help with creating the Matlab scripts to process and visualize the data and Asger B. Hansen from Haldor Topsoe A/ S for fruitful discussion.
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