Comprehensive on-line two-dimensional liquid chromatography × supercritical fluid chromatography with trapping column-assisted modulation for depolymerised lignin analysis

Lignin depolymerisation produces a large variety of low molecular weight phenolic compounds that can be upgraded to value-added chemicals. Detailed analysis of these complex depolymerisation mixtures is, however, hampered by the lack of resolving power oftraditional analysis techniques.

Journal of Chromatography A, 1541 (2018) 21–30 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Comprehensive on-line two-dimensional liquid chromatography × supercritical fluid chromatography with trapping column-assisted modulation for depolymerised lignin analysis Mingzhe Sun, Margareta Sandahl, Charlotta Turner ∗ Lund University, Department of Chemistry, Centre for Analysis and Synthesis, P.O Box 124, SE-22100 Lund, Sweden a r t i c l e i n f o Article history: Received 14 November 2017 Received in revised form 29 January 2018 Accepted February 2018 Available online February 2018 Keywords: Lignin Phenolic compound Supercritical fluid chromatography Trapping capacity Two-dimensional chromatography a b s t r a c t Lignin depolymerisation produces a large variety of low molecular weight phenolic compounds that can be upgraded to value-added chemicals Detailed analysis of these complex depolymerisation mixtures is, however, hampered by the lack of resolving power of traditional analysis techniques In this study, a novel online comprehensive two-dimensional reversed-phase liquid chromatography (RPLC) × supercritical fluid chromatography (SFC) method with trapping column interface was developed for the separation of phenolic compounds in depolymerised lignin samples The trapping capacities of different trapping columns were evaluated The influence of large volume water-containing injection on SFC performance was studied The relation between peak capacity and first dimension flow rate and gradient was investigated The optimized method was applied for the analysis of a depolymerised lignin sample The RPLC × SFC system exhibited high degree of orthogonality Compared with traditional loop based interface, trapping column interface can significantly shorten the analysis time and offer higher detectability, with the disadvantage of more severe undersampling in the first dimension © 2018 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction Biomass holds potential to partly replace petroleum as a raw material for production of fuels and valuable fine and bulk chemicals [1] Lignin is a naturally occurring polymer made up from phenyl-propenoid units, decorated by hydroxy- and methoxy-groups These are connected through radical polymerisation mechanisms, resulting in several types of bonds between the constituent building blocks Terrestrial biomass consists primarily of the macromolecules lignin, cellulose and hemicellulose Lignin could be an important source for the production of aromatic chemicals if well harnessed [2] One of the key steps of conversion of lignin to value-added chemicals is the depolymerisation − by selective bond cleavage – of lignin into low molecular weight phenolic compounds, which in turn can be used for chemical production Many techniques have been developed and are continuously improved for the purpose of depolymerisation [3] Due to the structural complexity of lignin and the large variance in the monomeric unit composition of different kinds of lignin, the depolymerisation products are usually very complex mixtures of aliphatic and phenolic ∗ Corresponding author E-mail address: charlotta.turner@chem.lu.se (C Turner) compounds, the compositions of which vary greatly depending on the lignin type and depolymerisation method applied [3] Gas chromatography coupled with mass spectrometry is the most widely adopted method for the analysis of lignin depolymerisation products [4] However, this technique requires a rather complicated sample preparation, usually including extraction and derivatisation, which may have preference towards certain classes of compounds if not carefully performed Also, the relatively low selectivity hinders the usage of this technique, if profiling all major components is the aim instead of analysis of only a few selected compounds High-performance liquid chromatography (HPLC) or ultrahigh performance liquid chromatography (UHPLC) have also been used in the analysis of lignin-derived compounds [5,6] However, these techniques also lack the separation power to fully resolve all major components for profiling study Multidimensional chromatography has been widely applied in various fields as it offers tremendously improved resolving power compared with their one-dimensional chromatography counterparts Lignin pyrolysis bio-oil analysis by two-dimensional gas chromatography (2DGC) has been reported in recent years [7,8] As a complementary technique to 2DGC, two-dimensional liquid chromatography (2DLC) also holds a large potential in the analysis of complex samples like lignin depolymerization mixtures It can overcome the disadvantage of 2DGC in the analysis https://doi.org/10.1016/j.chroma.2018.02.008 0021-9673/© 2018 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4 0/) 22 M Sun et al / J Chromatogr A 1541 (2018) 21–30 of compounds that are comparatively non-volatile, highly polar or thermally unstable [9] One of the most important considerations in 2DLC method development is how to achieve a high degree of orthogonality in order to maximise the effective 2D peak capacity [10] This is usually done by setting the two separation mechanisms on both dimensions as orthogonal as possible In general, the most commonly used modes in LC separation include reversed-phase liquid chromatography (RPLC), normal-phase liquid chromatography (NPLC), hydrophilic interaction liquid chromatography (HILIC), size exclusion chromatography (SEC) and ion exchange chromatography (IEC) However, combinations of two reversed phase liquid chromatography (RPLC × RPLC) are still preferred, as most of the other − apparently more orthogonal combinations – such as NPLC × RPLC and HILIC × RPLC suffer from solvent incompatibility issues [11–13] In order to address the relatively low degree of orthogonality issue with RPLC × RPLC, the combination of supercritical fluid chromatography (SFC) and RPLC can be a feasible and promising option In general SFC displays a separation mechanism resembling that of NPLC [14] However, the selectivity can be tuned in a wider range as both polar and non-polar columns can be used and mobile phase temperature and pressure play important roles [15,16] The potential of SFC as the first dimension in an online SFC × RPLC system has been demonstrated by Francois and co-workers [17–19] To better harness the high efficiency and fast separation of SFC, efforts have also been made to use online multiple heart-cutting LC-SFC, in particular for pharmaceutical achiral-chiral analysis [20] Online comprehensive RPLC × SFC with traditional collection loops as the interface has also been investigated for the analysis of traditional Chinese medicine and processed biomass oil [21,22] However, compared with RPLC used as second dimension in a 2D online LC × LC system, SFC imposes stricter limitation on the amount of eluent transferred to the second dimension in a 2D RPLC × SFC design Transfer volumes within the normal range of a 2D LC × LC system can cause significant peak broadening and misshaping, as well as pressure spikes at the beginning of a second dimension run and pressure build-up with continuous 2D runs in a RPLC × SFC system [21] Consequently, a very low flow rate (0.6 This can be attributed to the fact that 4hydroxybenzoic acid lacks in alkyl moieties that can interact with C18 strongly and a significant part of the 4-hydroxybenzoic acid molecules (pKa = 4.54) were in anionic form and preferred strongly to stay in the mobile phase Compared with the other two trapping columns, the DIOL column showed apparently a lower retention of veratraldehyde Fig 3(c) showed that apparent breakthrough of veratraldehyde started to occur (0.8 min) shortly after most of it was trapped in the column (∼0.6 min) Veratraldehyde eluted at the lowest water content of the 1st dimension eluent compared with the other two compounds tested As water has good H-bonding and is regarded as a strong solvent with a DIOL column, the low retention of veratraldehyde can only be explained by the absence of hydroxyl or carboxyl groups in the veratraldehyde molecular structure, which makes it less favored to be retained by the DIOL stationary phase, compared with 4-hydroxybenzoic acid ( COOH) and acetosyringone ( OH) The phenyl-hexyl column exhibited good trapping capacity of all three compounds with veratraldehyde being slightly more retained than the other two (Fig 3(a)) This indicated that the ␲-␲ interaction of the benzene rings combined with the hydrophobic interactions with the hexyl chain can effectively retain phenolic compounds regardless of their differences in substituted functional groups The trapping capacity of the compounds largely depends on the accessibility of the benzene ring, the number of alkyl moieties and the number of double bonds on substituents 4-hydroxybenzoic acid eluted in 1st dimension C18 column at comparatively low percent of ACN owning to the two polar substituents on the benzene ring Its retention in the EC-PH trap can be attributed to the higher accessibility of the benzene ring compared with the other two compounds with more than two substitution groups For acetosyringone and veratraldehyde that eluted in 1st dimension eluent with relatively higher elution strength, their retention could be from the combinational effect of ␲-␲ interaction with the benzene rings and hydrophobic interactions with the hexyl chain It was also found out that the retention time of 4-hydroxybenzoic acid in the second dimension SFC with EC-PH trapping column is slightly lower than with the other two trapping columns This can be regarded as beneficial for performing online RPLC × SFC of lignin-derive compounds, as the length of the modulation time is largely decided by the elution of the most retained phenolic acids in certain modulations Thus, the EC-PH trapping column was selected for further 2D method development As the EC-PH trapping column showed only limited retention of the compounds, the presence of matrix in real samples might likely decrease the trapping capacity The impact of matrix on the trapping capacity of the chosen EC-PH column was therefore investigated For all three compounds tested, the EC-PH column exhibited similar trapping capacity for a standard mixture and a spiked depolymerised lignin sample at the collection time of 0.8 (Fig S1) However, it can be observed that the trapping capacity became relatively lower for the spiked depolymerised lignin matrix when the collection time exceeded 1.0 This trend is especially apparent for 4-hydroxybenzoic acid, which is comparatively the least retained compound on this column among the three compounds tested This can be correlated to the presence of other compounds in the matrix competing with the targeted analytes on the limited trapping sites, causing faster and more significant breakthrough at longer collection times As is shown in Fig S2, the matrix also had a slight negative impact on the trapping of compounds present at different concentrations, resulting in a relatively higher standard deviation for the 2nd dimension peak areas of the trapped compounds compared with the standard mixture When peak areas are plotted against compound concentration, the linear relationship is slightly better with the standard mixture points for all three compounds included in the study A possible way to improve the trapping capacity can be active modulation [23], which involves mixing of the first dimension eluent and a flow of weak solvent before the trapping column interface to decrease the eluent strength and increase the trapping capacity One of the major concerns when using trapping column assisted modulation is repeatability As can be seen from Table 1, the retention times of 4-hydroxybenzoic acid and acetosyringone exhibited comparable RSD values (within-day and between-day) in both dimensions The retention time RSD values of veratraldehyde in the second dimension were higher than those in the first dimension This is likely explained by the early elution of veratraldehyde in the second dimension (k < 1) However, the RSD values are still in the acceptable range The second dimension peak area RSD values of 4-hydroxybenzoic acid were higher than those of the other two compounds This can be the consequence of the limited trapping capacity of 4-hydroxybenzoic acid on the EC-PH trapping column A slight shift of the first dimension peak retention time can cause either incomplete trapping or breakthrough of the analyte, which leads to second dimension peak area fluctuation 3.2 The influence of transfer eluent volume and water content on the second dimension SFC separation It is apparent that larger-volume trapping columns can provide higher trapping capacity, which would be beneficial in a RPLC × SFC interface set-up However, a large transfer volume can have detrimental effects on the second dimension SFC separation at the same time, since a large volume of transferred organic-rich eluent can lead to distorted early eluting peaks in the SFC Furthermore, the transfer of large volumes of water-rich eluent has been proven to cause significant SFC pressure increase and accumulation at the beginning of the modulations [21] M Sun et al / J Chromatogr A 1541 (2018) 21–30 27 Fig Influence of injection volume and water content on SFC performance a) veratraldehyde in ACN:H2 O 1:9, different injection volumes; b) veratraldehyde in ACN:H2 O 1:1, different injection volumes; c) veratraldehyde in ACN:H2 O 9:1, different injection volumes; d) veratraldehyde in different ACN:H2 O mixture, ␮L injection; e) pump pressure curves of different injections See experimental section for specific chromatographic conditions In order to investigate the influence of injection volume and water content on SFC separation performance, different volumes of veratraldehyde in sample diluents of high, medium and low amount of water were injected Veratraldehyde was chosen as it elutes early in all four SFC columns screened in this study and is more prone to peak distortion caused by the sample diluent [30] As can be seen in Fig 4, injections containing relatively high amount of water lead to good peak shape up to 10 ␮L (Fig 4a) However, the peak shape started to deteriorate at 10 ␮L and ␮L respectively for injection of medium and low percentages of water (Fig 4b and c) Another general observation is that the retention time of the analyte seemed to increase with increasing injection volume between and ␮L for all three sample diluents Furthermore, for the same injection volume, increasing water content in the sample diluent seems to prolong the analytes retention Interestingly, sample diluents containing water has been proven to cause peak distortion for relatively large injection volumes in SFC, which has been attributed to demixing effect of water with mobile phase, strong solvent effect and viscous fingering [30] A plausible explanation for the good peak shape with large volume of high water composition sample injections in this study could be that for this short column with relatively high mobile phase flow rate, the water containing injection solvent plug travels through the column in a very short time, so that the extent of demixing of water in the mobile phase and viscous fingering is minimal Another very important factor that has to be considered for 2D RPLC × SFC application is the pressure increase caused by large volume injection of a water containing solution Fig 4e shows that immediately after the injection, a pressure increase occurs before the normal pressure ramp caused by the gradient (red circle) And the more water the injection solvent contains, the higher the pressure increase is Moreover, Fig 4d shows that with the same injection volume, retention is decreased with less water injected Our hypothesis on these findings is that a temporary water layer is formed on the diol stationary phase at the front end of the column after the sample plug reaches the inlet of the column The thickness of the water layer is determined by the amount of water injected This suggested water layer could perti- nently increase the retention power of the stationary phase which might be one of the reasons why retention increases with increasing injection volume for the same sample diluent and with increasing water content in sample diluent of the same injection volume Also, the mobile phase channels in the column are narrowed by the water layer, which causes a pressure spike right after injection The system pressure difference among different injections might be another cause of different retention times observed, considering the compressibility of supercritical CO2 Although it is not the focus of this study, the effect of water as sample diluent on SFC certainly deserves a thorough and systematic investigation in the future Based on these results, trapping columns with larger volume than 10 ␮L are not favored in the 2D RPLC × SFC system, even though it may offer higher trapping capacities Also, the usage of the selected trapping column in this study requires that the first dimension LC separation should be controlled in a way that most of the analytes are eluted before the gradient reaches very high organic solvent percentage Furthermore, the gradient in the first dimension should be set as gradual as possible to avoid mismatch of retention times of different fractions of the same peak 3.3 First dimension RPLC peak capacity study The influences of flow rate and gradient time/void time (tg /t0 ) on the first dimension RPLC peak capacity were investigated As can be seen in Fig 5(a), the first dimension peak capacity shows a seemingly positive logarithmic relationship with the ratio between gradient time and void time, when the flow rate was kept unchanged at a relatively low value This indicated that when the first dimension is only running at a relatively low flow rate, the increase of gradient time is unnecessary for elevating the peak capacity after a certain point as only limited increase of peak capacity can be achieved with much lengthened analysis time Although the first dimension LC still has to be run at low flow rates considering the limited trapping capacity of the trapping column, the importance of increasing the flow rate as much as possible is illus- 28 M Sun et al / J Chromatogr A 1541 (2018) 21–30 Fig Influence of: (a) the gradient time/void time ratio (tg /t0 ) and (b) flow rate on the first dimension LC peak capacity of lignin phenolic compound standard mixture Conditions: mobile phase A: H2 O, B: ACN (a) Linear gradient from 10 to 70% B at different speed, flow rate was kept at 0.05 mL/min (b) Linear gradient from 10 to 70% B with constant tg /t0 of 12, with varied flow rates trated in Fig 5(b) When the ratio between gradient time and void time was kept constant, peak capacity increases nearly proportionally with the flow rate in the tested range (Fig 5(b)) Although higher flow rates can lead to higher first dimension peak capacity, this impact can be gradually evened out by the increasing undersampling effect This is because the first dimension peaks will become narrower with higher flow rates, but the modulation time has to be long enough to make sure all the compounds transferred in one modulation are eluted before the next modulation Taking this and the trapping column retention capacity into consideration, 0.05 mL/min was finally selected as the first dimension flow rate and the first dimension LC method was then improved accordingly 3.4 Second dimension column screening and 2D method development Preliminary experiments (data not shown) revealed that phenolic compounds in depolymerised lignin samples showed a wide variety of retention behavior in SFC analysis due to their structural complexity In order to achieve complete elution of analytes in every modulation and taking into consideration the results of the trapping column test, 0.8 was chosen as the modulation time It has been reported that a large volume (>5 ␮L) water-containing injection into SFC in an online comprehensive RPLC × SFC system can result in significant accumulative pressure increase at the beginning of every modulation depending on the water content, the injection volume and the SFC stationary phase [21] This was claimed to be caused by water accumulation before the narrow column inlet Based on these findings and the internal volume of the trapping column in this study (∼10 ␮L), different SFC columns with relatively polar stationary phases and slightly wider internal diameter (3.0 mm i.d.) were screened as compared with the ones used in the previous study (2.1 mm i.d.) [21] Fig shows the screening results of the four columns The performances of all columns were quite similar, with analyte peaks distributed over large portions of the separation space The BEH column exhibited a slightly lower coverage factor of 0.64, compared with the other three columns (f = 0.70 for DIOL, 0.72 for 2-PIC and 0.68 for 1-AA) This indicates that for the separation of ligninderived phenolic compounds in a 2DLC system with reversed-phase LC separation in the first dimension, ␲-␲ and H-bonding interaction in the second dimension SFC can both provide high degrees of orthogonality The comparatively lower coverage factor of the BEH column could be related to the ethylene bridged hybrid particle which decreases the H-bonding capability of the silica stationary phase However, one advantage of the BEH column is that it had the lowest pressure spike at the beginning of each modulation (20 bar compared with 30 to 40 bar for the other three columns) This observation could also be related to the hypothesis of water layer formation on the stationary phase As ethylene-bridged silica is far less acidic than normal silica, the weakened hydrogen bonds between the stationary phase and H2 O can reduce the thickness of the water layer The highly orthogonal separation demonstrated by the combination of RPLC and SFC indicated that hydrophobic interaction which determines the elution of the first dimension RPLC plays a weak role in deciding the compound elution of the second dimension SFC The DIOL column was picked for further experiments as it provided relatively better separation of the early eluting peaks from the first dimension, medium pressure spike and Fig RPLC × SFC second dimension column screening Sample: 40 lignin phenolic compound standard mixture Chromatographic conditions can be found in the Experimental section See Table S1 for peak identities M Sun et al / J Chromatogr A 1541 (2018) 21–30 29 Fig 2D separation of a lignin depolymerised sample with the final RPLC × SFC methods with interface using A: two trapping columns; B: two collection loops Chromatographic conditions can be found in the Experimental section See Table S1 for peak identities Due to the software limitation of the home-built instrument, only limited number of modulations could be programmed and performed for one analysis Consequently, 2D analysis was only applied with the maximum number of modulation allowed by the software during the periods of time when most compounds eluted in both A and B, including the same fractions of peaks eluting from the 1st dimension column comparatively better second dimension peak shape and less noisy background For the improvement of the 2D separation, different co-solvents, column temperatures, back pressures and gradients were tested for the second dimension SFC Additionally, the second dimension gradient was tuned so that it started already in the previous modulation, in order to compensate for the comparatively large gradient delay volume of the system The final RPLC × SFC method was then applied for the separation of a lignin depolymerised sample (Fig 7A) 3.5 Comparison of trapping columns and loops in the RPLC × SFC system For the comparison of using trapping columns and collection loops as interface in the RPLC × SFC system, the developed method was modified by replacing trapping columns with loops of similar volume (∼10 ␮L) Even though the modulation time was kept the same, the first dimension LC flow rate had to be significantly decreased (4 times) considering the amount of eluent transferred into the second dimension The comparison of 2D chromatograms generated from using collection loops and trapping columns can be visualized in Fig In general, the combination of RPLC and SFC shows high degree of orthogonality The coverage factor was calculated to be 0.79 and 0.77 for trapping column interface and loop interface respectively The usage of trapping column in the interface reduced the total analysis time by times, as compared to interface with loops Four compounds could be detected and tentatively identified both with the trapping column interface and collection loop interface based on the separation of the mixture of the 40 standards However, vanillic acid (peak 11) can only be detected with the usage of trapping columns in the interface This demonstrated another advantage of using trapping columns: some analytes of relatively low concentration can be detected as they are concentrated in the trapping column before transferred into the second dimension This is not possible with traditional loop interfaces However, the total conditional peak capacity obtained with trapping column is 277, which is lower than that with loops (340) This is caused by more severe undersampling (ˇ = 0.26 for trapping column; ˇ = 0.57 for loops) when trapping column is used as interface What is also worthy to be pointed out is that even though no wrap-around peaks were observed during the 2nd dimension optimization with the standard mixture, as the variety of compounds in the real depolymerised lignin sample is very wide, a few peaks eluted at the end of or after the end of one modulation If these wrap-arounds are to be eliminated, the gradient has to end with a high percent of co-solvent or an extra hold-up time at a relatively high percent of co-solvent has to be added Either way, the 2nd dimension separation or the trapping of the compounds would have to be significantly compromised Therefore, in this study the 2nd dimension gradient was set in a way that the number of wrap-around peaks were reduced to minimum and the remaining wrap-around peaks all eluted very early in the next modulation before the solvent peak without overlapping with the peaks in the following modulation period In our previous research we have developed an ultra-high performance supercritical fluid chromatography method (UHPSFC) for the analysis of lignin-derived phenolic compounds [31] The same depolymerised lignin sample in this study was also analysed using the UHPSFC method (see chromatogram in Fig S1) In comparison, the RPLC × SFC method developed had a more than three times higher peak capacity than the UHPSFC method (277 for RPLC × SFC and 81 for UHPSFC) The disadvantage of the RPLC × SFC method is the longer analysis time, which was times longer than that of the UHPSFC method In order to shorten the 2D analysis time without a sacrifice of the peak capacity, future work should focus on developing more effective interface sample focusing techniques to allow higher flow rate to be used in the first dimension and achieve faster SFC separation to remedy the undersampling issue Conclusion A novel comprehensive online two-dimensional RPLC × SFC method was developed for the analysis of depolymerised lignin samples with trapping column assisted modulation A Phenyl-hexyl trapping column was picked based on a trapping capacity evaluation of different trapping columns Although the RSD values were within acceptable ranges, the repeatability of the trapping column assisted RPLC × SFC system was shown to be undermined by the limited trapping capacity Active modulation or a more thorough search for columns specifically designed for the retention of small phenolic compounds could potentially improve the system No demixing effects were observed when injecting large volumes of water containing samples in SFC when using a high flow rate and a short column, possibly due to fast travel of intact sample diluent plug through the column Water as sample diluent turned out to enable good peak shapes, even at as large injection volumes as 10 ␮L When the first dimension LC flow is kept relatively low (e.g ≤0.05 mL/min in this study), an increase in the first dimension flow rate can raise the first dimension peak capacity more 30 M Sun et al / J Chromatogr A 1541 (2018) 21–30 than an increase in gradient time RPLC × SFC showed a high degree of orthogonality for all four SFC columns screened, with coverage factors ranging from 0.64 to 0.72 for the standard mixture of 40 compounds and 0.79 for a real depolymerised lignin sample with the diol SFC column A comparison between trapping column assisted modulation and traditional loop based modulation revealed that the former has the advantage of shorter analysis time and better detectability due to the analyte concentrating effect However, the disadvantage lies in the fact that higher first dimension flow rates led to more severe undersampling Acknowledgement The authors would like to thank the Swedish Research Council Formas (2016-00604) and the Swedish Foundation for Strategic Research (SSF, RBP 14-0052) for financial support of this work We thank Professor Gunnar Lidén for the kind help in revising the manuscript We are grateful to Joseph Samec and Maxim Gulkin for providing the depolymerised lignin samples Appendix A Supplementary data Supplementary data associated with this article can be found, in the online 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A novel comprehensive online two-dimensional RPLC × SFC method was developed for the analysis of depolymerised lignin samples with trapping column assisted modulation A Phenyl-hexyl trapping. .. two-dimensional liquid chromatography has to our knowledge never been applied for the analysis of depolymerised lignin samples In the present study, a comprehensive 2D RPLC × SFC method was developed for. .. supercritical fluid chromatography, J Chromatogr A 1191 (2008) 21–39 [17] I Francois, P Sandra, Comprehensive supercritical fluid chromatography x reversed phase liquid chromatography for the analysis