Supercritical-fluid chromatography (SFC) is regaining popularity in various fields of analytical chemistry owning to significant advances in instrumentation made in the past decade. However, due to the CO2 based mobile phase and the high flow rates often employed, detection of trace amounts of analytes and coupling with certain detectors or other chromatography techniques are still difficult under many circumstances.
Journal of Chromatography A 1660 (2021) 462642 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma A compound post-column re-focusing approach in supercritical fluid chromatography Mingzhe Sun a,b,∗, Peter Schoenmakers a,b a b Analytical Chemistry Group, Van’t Hoff Institute for Molecular Sciences, University of Amsterdam, 1098 XH Amsterdam, The Netherlands Centre for Analytical Sciences Amsterdam (CASA), The Netherlands a r t i c l e i n f o Article history: Received 10 September 2021 Revised 19 October 2021 Accepted 20 October 2021 Available online November 2021 Keywords: Concentration enhancement Heart-cut Re-mobilizing Signal enhancement Trapping a b s t r a c t Supercritical-fluid chromatography (SFC) is regaining popularity in various fields of analytical chemistry owning to significant advances in instrumentation made in the past decade However, due to the CO2 based mobile phase and the high flow rates often employed, detection of trace amounts of analytes and coupling with certain detectors or other chromatography techniques are still difficult under many circumstances In this study we propose a post-column re-focusing approach for SFC analysis to achieve not only signal enhancement in UV-Vis detection, but also actual concentration enhancement of the analyte By heart-cutting and transporting a selected fraction from the SFC flow into a trapping column with a flushing solvent, re-focusing of the collected analytes can be achieved by re-mobilization with another solvent once the depressurized CO2 is eliminated By carefully selecting the trapping stationary phase and the two solvents, signal-enhancement ratios between 2.2 and 6.4 were realized for four representative compounds eluting with very different percentages of SFC modifier (methanol) The actual concentration enhancement was lower (ratios between 1.7 to 2.9), because the UV response of the analytes was found to differ significantly under SFC and LC conditions © 2021 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Introduction In the past decade we have witnessed an increased proliferation of supercritical-fluid chromatography (SFC) in many application fields, thanks to significant advances in instrumentation and column technology [1] Most SFC applications utilize compressed CO2 as the major constituent of the mobile phase, with a polar organic solvent added as modifier Compared with reversed-phase liquid chromatography (RPLC) and normal-phase liquid chromatography (NPLC), which are widely used in chemical analysis at the moment, SFC type mobile phases offer a much lower viscosity, despite having a liquid-like density [2] This allows high flow rates to be used in SFC Since a low viscosity concurs with a high diffusivity, the mass transfer of analytes is also greatly enhanced and high flow rates are optimal These unique properties, combined with the possibility of using both non-polar and polar stationary phases, make SFC a viable option for the analysis of a wide range of compounds [3,4] ∗ Corresponding author E-mail addresses: m.sun@uva.nl, mingzhe.sun023@gmail.com p.j.schoenmakers@uva.nl (P Schoenmakers) (M Sun), Despite the advantages SFC offers over other chromatographic techniques, analyte band dilution arising from the high flow feature of SFC together with noise caused by fluctuations in mobile phase density can lead to poor detection limits in many applications, especially when the detector is concentration dependent or has a limited active detection volume [5–7] The high flow rate and CO2 -based SFC mobile phases also raise various technical challenges for the realization of hyphenated systems, such as the coupling of SFC with different types of mass spectrometers and on-line two-dimensional (heart-cut SFC-LC or comprehensive twodimensional SFC × LC) [8,9] To mitigate these negative effects of SFC mobile phases on detection and hyphenation, an additional analyte focusing step after the SFC separation is desirable While analyte focusing has been very rarely investigated in SFC, relevant studies in HPLC have been abundant in two main categories, viz on-column focusing and post-column re-focusing [10– 15] The generic HPLC post-column re-focusing approach involves the use of a strongly retentive trapping column installed after the analytical column to focus the analytes and of a strong solvent to re-mobilize the trapped analyte bands [12] To adopt this approach in supercritical-fluid chromatography, the trapping column must be placed after the back-pressure regulator (BPR) However, this does cause a number of complications The depressurized ef- https://doi.org/10.1016/j.chroma.2021.462642 0021-9673/© 2021 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) M Sun and P Schoenmakers Journal of Chromatography A 1660 (2021) 462642 fluent, consisting of CO2 and co-solvent flows through the trapping column at a much higher linear velocity than that in the analytical column The adiabatically expanded CO2 cools down the trapping column The combination of cold CO2 and cold co-solvent cannot be smoothly transported through the trapping column at very high velocities Besides, when the SFC mobile phase contains only a low concentration of co-solvent, analyte precipitation may take place in the BPR Finally, if a significant amount of CO2 remains in the trapping column it will have to be removed before re-mobilizing the analytes, to avoid a noisy baseline It is also highly unlikely to find one trap that accommodates the vast range of properties of compounds analysed in SFC This makes efficient and reliable analyte focusing a challenging issue in SFC The objective of the present work is to develop an SFC postcolumn re-focusing approach that is generally applicable to a wide range of analytes, with collection and subsequent trapping of an SFC peak performed in a 2D “heart-cutting” fashion We aim to evaluate the re-focusing performance of the proposed approach, the UV signal enhancement and concentration enhancement using a small number of representative compounds We also aim to eliminate all CO2 from the trapping column, using a flushing solvent, before the trapped analytes are re-mobilized by a flow of strong solvent Different combinations of trapping chemistry and solvent systems are tested as to their potential to successfully re-focus the representative compounds that vary in polarity and elute at different SFC mobile-phase compositions Fig SFC-UV system without (A) and with (B) post-column re-focusing DAD was used and spectral data from 200 to 450 nm were collected with a resolution of 1.2 nm The sampling rate was 20 Hz and the filter time was set at 0.1 s Signal data used for plotting chromatograms were collected at 280 nm, compensated by a reference signal from 400 nm to 450 nm System control and data processing were performed with Empower software (Waters) Material and methods 2.1 Chemicals, columns and equipment The four representative compounds phenanthrene, phenol, theobromine and p-coumaric acid were all purchased from SigmaAldrich (Zwijndrecht, The Netherlands) Acetonitrile (ACN) and methanol were obtained from Biosolve (Valkenswaard, The Netherlands) n-Hexane and diethyl ether were purchased from VWR (Amsterdam, The Netherlands) Ethanol was obtained from Merck (Darmstadt, Germany) All organic solvents were of HPLC grade or better Water purified using Sartorius Arium 611 UV system was used for all experiments SFC-grade carbon dioxide (4.8) was obtained from Praxair (Vlaardingen, The Netherlands) Individual standards of phenanthrene, phenol and p-coumaric acid of different concentrations were prepared in acetonitrile Standards of theobromine of different concentrations were prepared by diluting a mg/mL dimethyl sulfoxide (DMSO) solution with acetonitrile Standard mixtures of the four compounds were prepared in acetonitrile in different concentrations All standard solutions were stored at -20°C when not being used Five columns were used either as SFC separation column or trapping column in this work, viz Waters BEH (ethylene-bridged silica, 100 mm × mm i.d.; 1.7 μm particle size), Waters Torus DIOL (100 mm × mm; 1.7 μm), Waters Torus 2-PIC (2-picolylamine, 100 mm × mm; 1.7 μm), Agilent ZORBAX Eclipse Plus C18 (30 mm × 2.1 mm; 1.8 μm), and Agilent ZORBAX Eclipse Plus C18 (50 mm × mm; 1.8 μm) SFC experiments were carried out on a Waters UltraPerformance Convergence Chromatography (UPC2 ) System (Waters, Milford, MA, USA) with a binary solvent pump, an auto-sampler, a column oven, a back-pressure regulator, and a diode-array detector (DAD) A 10-μL injection loop was used for injection An additional Waters UPLC binary pump was used when a liquid flow was needed in post-column re-focusing experiments Two sampling loops of 160 μL and 230 μL were prepared to collect SFC fractions in the re-focusing experiments The SFC column oven had two channels that were employed to control the temperature of the SFC and trapping columns separately For all experiments (stand-alone SFC and SFC with post-column re-focusing), the same 2.2 SFC system design with and without post-column re-focusing Fig 1A shows the simple SFC-UV system without the postcolumn re-focusing process It is used to generate chromatograms for peak-height comparison and for acquiring UV-Vis spectra Fig 1B shows the SFC-UV system with post-column re-focusing embedded The valve is in Position to collect the peak of interest in a sample loop After the collection is completed, the valve is switched to Position Then the first step (Step 1) involves a flushing solvent to transport the collected fraction to the trapping column and to remove any remaining CO2 from the trap In the next step (Step 2), a re-mobilizing solvent flow is applied to quickly elute the trapped compounds to the UV detector After the analysis is done, the valve is switched back to Position to recondition the trapping column and prepare for the next injection 2.3 SFC separation and post-column re-focusing of four representative compounds 2.3.1 SFC separation of the four compounds The SFC separation of the four representative compounds was performed with the BEH column The gradient started with 2% methanol (with 0.1% formic acid), ramped up to 26% methanol in min, then decreased to the starting composition in after a hold at 26% The flow rate was mL/min, with a column temperature of 50°C and back pressure of 13 MPa (130 bar) To generate the SFC chromatograms of the separation, a standard mixture of the four compounds (phenanthrene, phenol and p-coumaric acid all having a concentration of 0.25 mg/mL, while theobromine was of 0.05 mg/mL) was used and the injection volume was μL All injections were performed in triplicate M Sun and P Schoenmakers Journal of Chromatography A 1660 (2021) 462642 2.3.4 Evaluation of polar trap with hexane/ethanol solvent system In order to compare the trapping performance of the three relatively polar compounds on the DIOL and 2-PIC columns, 1D-LC experiments were performed with hexane and ethanol as weak and strong solvents, respectively The mobile-phase flow rate was 0.3 mL/min, with a gradient from 100% hexane to 100% ethanol in After min, the mobile phase was held at 100% ethanol to elute strongly retained compounds Column temperature was set at 40°C The same standard mixture of the four compounds (phenanthrene, phenol, p-coumaric acid and theobromine; μL injection volume; triplicate injections) that was described in Section 2.3.1 was used in the experiments 2.3.5 Post-column re-focusing of phenol The re-focusing experiments of phenol were carried out using the 2-PIC column as the trap 30°C, 40°C and 50°C were investigated as trap temperatures The flushing solvent was hexane, delivered at 0.4 mL/min Three flushing end times were used and compared in the re-focusing experiments, i.e 2.5, 3.0, and 3.5 The re-mobilizing solvent was ethanol and three flow rates were studied, i.e 0.3, 0.4, and 0.5 mL/min The influence of solvent-switch time was also assessed, by setting it at 0.01, 0.5, and 1.0 All re-focusing experiments were done with μL injection of 0.2 mg/mL phenol solution in ACN in triplicate 2.3.6 Post-column re-focusing of p-coumaric acid The DIOL column was utilized as the trapping column for refocusing experiments of p-coumaric acid 30°C, 45°C and 55°C were used as trap temperatures Hexane at a flow rate of 0.4 mL/min was employed as the flushing solvent, while ethanol was used as the re-mobilizing solvent at three different flow rates, i.e 0.3, 0.4, and 0.5 mL/min Different end time of hexane flushing were studied and compared, i.e 4.5, 5.5, and 6.5 Also, different solvent-switch times were adopted to study its influence, i.e 0.01, 0.5 and 1.0 All re-focusing experiments were performed with μL injection of a solution of 0.05 mg/mL pcoumaric acid in ACN in triplicate Fig SFC separation of four representative compounds Refer to Section 2.3.1 for detailed SFC conditions (A) no extra connection between the column outlet and the DAD; (B) 160-μL loop placed between the column outlet and DAD; and (C) 230-μL loop placed between the column outlet and DAD Peak identity: Phenanthrene; Phenol; p-Coumaric acid; Theobromine 2.3.2 Post-column re-focusing of phenanthrene The post-column re-focusing experiments of phenanthrene were performed with the 30 mm long C18 column as the trapping column Trap temperatures of 35°C, 45°C and 55°C were tested The flushing solvent for CO2 removal was H2 O with a constant flow rate of 0.2 mL/min To study the influence of flushing time on the re-focusing performance, different end times of the flushing were employed, ranging from 2.05 to 6.05 The re-mobilizing solvent used was acetonitrile and different times were tested to switch the solvent from 100% H2 O to 100% ACN, viz 0.01 min, 0.5 and 1.0 The flow rate of the re-mobilizing solvent was also varied (from 0.12 mL/min to 0.35 mL/min) to study its effect on the re-focusing All re-focusing experiments were performed in triplicate with μL injection of 0.2 mg/mL phenanthrene solution in ACN 2.3.7 Comparison of ethanol and methanol as re-mobilizing solvents for theobromine To compare the retention of theobromine on the DIOL column with ethanol and methanol as re-mobilizing solvents, 1D-LC injections of μL of a solution of 0.1 mg/mL theobromine in acetonitrile were made The column temperature was set at 30°C The isocratic mobile phase consisted of either 100% ethanol or 100% methanol, with a flow rate of 0.4 mL/min For each experiment, triplicate injections were performed 2.3.8 Post-column re-focusing of theobromine The trapping column for post-column re-focusing experiments with theobromine as analyte was the DIOL column Only 20°C was used as the trap temperature The flushing solvent was diethyl ether, delivered at 0.5 mL/min, and three flushing end-times were investigated and compared, i.e 5.0, 6.0, and 7.0 The remobilizing flow of methanol was delivered after the flushing flow with three different solvent-switch times (0.01, 0.5, and 1.0 min) Three flow rates were studied for the re-mobilizing solvent, i.e 0.3, 0.4, and 0.5 mL/min All re-focusing experiments were performed in triplicate with 1-μL injections of a solution of 0.1 mg/mL theobromine in ACN 2.3.3 Testing the trapping system for other compounds The C18 trap and H2 O/ACN solvent system were also tested for re-focusing of the other three compounds Only the small loop (160 μL) was used for SFC peak collection The 50 mm long C18 column was used and the trap temperature was set at 55°C The flushing flow of H2 O was set at 0.2 mL/min and the end time of the flushing was 3.5 min, 5.2 and 5.6 for phenol, p-coumaric acid and theobromine, respectively The re-mobilizing flow of ACN was delivered at 0.2 mL/min with a solvent switch time of 0.01 The same standard mixture of the four compounds (phenanthrene, phenol, p-coumaric acid and theobromine; μL injection volume; triplicate injections) that was described in Section 2.3.1 was used in the experiments 2.4 Translating peak-height ratio to concentration ratio Standard mixtures of the four compounds at different concentrations were analysed (in triplicate) by SFC using the same conditions as described in section 2.3.1 Thereafter, the system was converted into a one-dimensional UPLC system with the same DAD M Sun and P Schoenmakers Journal of Chromatography A 1660 (2021) 462642 Fig (A) Re-focusing chromatogram (orange) of phenanthrene, with SFC chromatogram (black) for comparison The identities of the peaks are confirmed with UV-Vis absorption spectra (bottom) (B) Re-focusing chromatogram generated from a blank injection under the same experimental conditions as (A) (C) Overlapped re-focusing chromatograms of three repeated injections Refer to Table S-1 for specific experimental conditions used for re-focusing experiments Fig Investigation of the parameters influencing post-column re-focusing of phenanthrene: (A) sampling-loop volume; (B) H2 O flushing time; (C) trapping temperature; (D) solvent-switch time and (E) ACN flow rate Refer to Table S-2 for specific experimental settings used for re-focusing experiments in each case as used for SFC injections Phenanthrene standard solutions of different concentrations were analysed using the 30-mm C18 column with isocratic elution with 100% acetonitrile at 0.25 mL/min The column temperature was set at 55°C Phenol standard solutions of different concentrations were analysed using the 2-PIC column with isocratic elution with 100% ethanol at 0.25 mL/min The column temperature was set at 50°C Standard solutions of the other two compounds were injected into the DIOL column Isocratic elution at 0.25 mL/min was adopted for p-coumaric acid with 100% ethanol at 45°C, and for theobromine with 100% methanol at 20°C All the 1D-LC injections were performed in triplicate Results and discussion 3.1 SFC separation of the four representative compounds The four compounds selected in this study present a wide range of physio-chemical properties For example, the logarithms of their octanol-water distribution coefficients (log P) range from -0.78 to 4.46 As can be seen in Fig 2A, the four compounds elute at very different methanol percentages in the gradient SFC run Together with their varying polarities, this makes the effective trapping and re-mobilizing of all four compounds using one trapping column extremely difficult It should also be noted that severe peak broad- M Sun and P Schoenmakers Journal of Chromatography A 1660 (2021) 462642 Fig Re-focusing of the three relatively polar compounds using H2 O and ACN as flushing and re-mobilizing solvent, respectively, with the 50-mm C18 column as trap: (A) SFC chromatogram of the four compounds for comparison; (B) re-focusing of phenol; (C) re-focusing of p-coumaric acid and (D) re-focusing of theobromine Refer to Table S-3 for specific experimental settings used for re-focusing experiments in each graph (E) and (F) 1D-LC evaluation of the polar trap - hexane/ethanol system; (E) DIOL, (F) 2-PIC See Section 2.3.4 for parameter settings Peak identity: Phenanthrene; Phenol; p-Coumaric acid; Theobromine; and Background peaks ening is unavoidable once the collection loop is added between the SFC column outlet and the DAD for fraction collection Fig 2B and 2C show the broadened SFC peaks when the 160 μL and 230 μL collection loops were used, respectively The peaks are much broader than when the column outlet was directly connected to the DAD (Fig 2A) To allow for a fair comparison in this study, the re-focused peaks are always compared with the SFC peaks obtained when the column is directly connected to the DAD tent in the SFC mobile phase increases during the gradient Both the flushing and re-mobilizing solvents have to be carefully chosen to match the following criteria: (i) the flushing solvent is miscible with the re-mobilizing solvent; (ii) the flushing solvent must be a weak eluent for the analytes to ensure trapping on the stationary phase; (iii) the re-mobilizing solvent must be a strong eluent to quickly wash the analytes off the trapping column Phenanthrene eluted with approximately 3% methanol in CO2 from the SFC column C18 can be used to effectively trap the phenanthrene after transfer from the sampling loop, given the low amount of methanol present and the non-polar character of the compound Phenanthrene has a log P value of 4.46, so water is a very weak eluent and can be used as a flushing solvent to remove the remaining CO2 , without severely disturbing the band of trapped analyte Acetonitrile would be a suitable re-mobilizing solvent A typical re-focusing chromatogram is shown in Fig 3A Peak (black line) is the phenanthrene peak obtained in an SFC-UV run without the re-focusing process In the re-focusing chromatogram, the complex noisy signals from around to 3.6 originated from depressurized CO2 passing through the detector, as well as the remaining CO2 that was flushed out by water Once all the 3.2 Post-column re-focusing of phenanthrene The successful re-focusing of a compound requires that both trapping and re-mobilizing steps are efficient As the outlet of the trapping column is not pressurized, the transferred SFC fraction will undergo a phase separation when it reaches a certain point in the trapping column This leads to CO2 becoming a gas and losing its solvation power, while most of the compounds are dissolved in the precipitated (liquid) methanol The methanol phase is dispersed on the surface of the stationary phase, which must provide strong enough interactions with the analytes to retain them on the trapping column This is increasingly important for the relatively late-eluting compounds from the SFC, as the methanol con5 M Sun and P Schoenmakers Journal of Chromatography A 1660 (2021) 462642 Fig Investigation of the parameters affecting the post-column re-focusing of phenol: (A) sampling-loop volume; (B) re-focusing chromatogram of a blank injection for comparison; (C) solvent-switch time; (D) trapping temperature; (E) hexane flushing time and (F) ethanol flow rate Refer to Table S-4 for specific experimental settings used for re-focusing experiments in each graph CO2 was eliminated from the trapping column, the background returned to normal The sudden solvent switch from 100% H2 O to 100% ACN gave rise to a small peak that can be seen at around min, after which the trapped analyte was re-mobilized to the DAD by the ACN flow, generating peak The identity of the refocused peak can be verified both by matching the UV-Vis absorption spectra of peak and 2, and by comparing the re-focused chromatogram of phenanthrene with one acquired from a blank injection under the same experimental settings (Fig 3B) As can be seen from comparing Fig 3A and 3B, the CO2 noise pattern was not repeatable and varied greatly from injection to injection However, the re-focused peak presented good repeatability in terms of elution time, peak height and peak area, as shown by the overlapped re-focusing chromatograms of repeated injections of the same phenanthrene solution (Fig 3C) Some parameters that may potentially influence the final refocusing results have been investigated in this study Two sampling loops (160 μL and 230 μL) were compared to investigate whether the volume of the collected SFC fraction affected the trapping As can be seen in Fig 4A, the size of the sampling loop hardly affected the height of the re-focused peak There was a shift in elution time, because of the different dwell volumes of the loops The invariable peak height in Fig 4A indicated that the trapping process was successful Both peaks were equally high and equally broad, despite the broader starting profile (before trapping) that resulted from using a larger loop (see Fig 2) The 160-μL loop was then picked for the other experiments Compared with samplingloop size, an increase in H2 O flushing time brought a clear increase in peak height (Fig 4B) However, the improvement is not dramatic when considering the longer time needed Possibly, the more-efficient removal of the SFC co-solvent leads to a sharper H2 O/ACN front at the elution stage The trap temperature certainly plays an important role, not only during the trapping process, but also during re-mobilization As shown in Fig 4C, increasing the trapping temperature led to higher re-focused peaks, especially when the temperature was changed from 45°C to 55°C An increase in the solvent-switching time resulted in only a slight decrease in the height of the re-focused peaks (Fig 4D) The peak-compression effect normally encountered in gradient-elution HPLC was not observed here, but somewhat sharper peaks were obtained with a faster transition from the flushing solvent to the re-mobilizing solvent A change in the flow rate of the re-mobilizing solvent flow rate greatly influenced the area of the re-focused peak, as expected, but led to very small changes in peak height (Fig 4E) This means that the concentration of phenanthrene at the top of the re-focused peak was almost unchanged, regardless of the varying ACN flow rate A high re-mobilizing flow is preferred to shorten the analysis time, as long as column pressure is not a concern M Sun and P Schoenmakers Journal of Chromatography A 1660 (2021) 462642 Fig Investigation of the parameters influencing the post-column re-focusing of p-coumaric acid: (A) sampling loop volume; (B) re-focusing chromatogram of a blank injection for comparison; (C) trap temperature; (D) hexane flushing time; (E) ethanol flow rate and (F) solvent-switch time Refer to Table S-5 for specific experimental settings used for re-focusing experiments in each graph nol, p-coumaric acid and theobromine To reduce the probability of losing analytes during the flushing step, n-hexane was selected as the flushing solvent Acetonitrile was replaced by ethanol for solvent-miscibility reasons Before using these NPLC systems in refocusing experiments, 1D-LC experiments with hexane and ethanol as weak and strong solvents were performed on the DIOL and 2PIC columns to compare their trapping performance for the three polar compounds (Fig 5E and 5F) When subjected to the same solvent gradient, phenol and theobromine presented very similar retentions on the two columns p-Coumaric acid was much-more retained on the 2-PIC column than on the DIOL column, while displaying much more severe peak broadening For the sake of peak width and analysis time, the 2-PIC column was selected as the trapping column for re-focusing of phenol and the DIOL column was used for re-focusing of p-coumaric acid and theobromine 3.3 Re-focusing of phenol, p-coumaric acid and theobromine with the C18 trap The implementation of the C18 trapping with H2 O and ACN as flushing and re-mobilizing solvent, respectively, did not provide good performance for the three more polar compounds, despite the use of a longer C18 column for trapping As shown in Fig (BD), only very small re-focused peaks could be observed for phenol and p-coumaric acid, while theobromine was not trapped at all This was to be expected, as these compounds were transferred to the trapping column with higher amounts of methanol (phenol, p-coumaric acid and theobromine eluted with approximately 8%, 18% and 21% methanol in CO2 , respectively) In these cases, the methanol may lead to partial or total breakthrough of the compounds from the trapping column Furthermore, water is no longer an appropriate flushing solvent as the compound polarity increases, especially for theobromine, which is more soluble in H2 O than in most organic solvents While the H2 O flow removed the remaining CO2 from the trap, the analytes could also be flushed away 3.5 Post-column re-focusing of phenol The system consisting of the 2-PIC trapping column and nhexane and ethanol as flushing and re-mobilizing solvents, respectively, was evaluated for the re-focusing of phenol As displayed in Fig 6A, two peaks close to each other appeared after the background returned to normal from the CO2 noise A blank injection revealed that the second peak was the re-focused phenol peak, while the first one was most likely caused by the fast solvent 3.4 Evaluation of polar trap with hexane/ethanol solvent system Polar stationary phases (DIOL and 2-picolylamine, 2-PIC) were then considered in order to achieve more effective trapping of phe7 M Sun and P Schoenmakers Journal of Chromatography A 1660 (2021) 462642 Fig (A) 1D-LC comparison of methanol and ethanol as re-mobilizing solvents for theobromine (B) Re-focusing chromatogram of a blank injection Investigation of the parameters influencing the post-column re-focusing of theobromine: (C) sampling-loop volume; (D) diethyl-ether flushing time; (E) MeOH flow rate and (F) solvent-switch time Refer to Table S-6 for specific experimental settings used for re-focusing experiments in each graph Table Overall best re-focusing conditions and enhancement ratios for the four representative compounds achieved in this study Phenanthrene Phenol Interface Flushing & re-mobilizing conditions Loop size: 160 μL Valve pos to 2: 0.75 Trapping column: 30 mm C18, 55°C Flushing solvent: H2 O, - 3.05 min, 0.2 mL/min Re-mobilizing solvent: ACN, 3.06 - 5.06 min, 0.2 mL/min Trapping column: 2-PIC 50°C Flushing solvent: Hexane, - min, 0.4 mL/min Re-mobilizing solvent: Ethanol, 3.5 - 5.5 min, 0.5 mL/min Trapping column: DIOL 45°C Flushing solvent: Hexane, - 5.5 min, 0.4 mL/min Re-mobilizing solvent: Ethanol, 5.51 - 7.5 min, 0.3 mL/min Trapping column: DIOL 20°C Flushing solvent: Diethyl ether, - min, 0.5 mL/min Re-mobilizing solvent: Methanol, 6.01 - min, 0.3 mL/min size: 230 μL Valve pos 1.69 size: 160 μL Valve pos 3.36 p-Coumaric acid Loop to 2: Loop to 2: Theobromine Loop size: 160 μL Valve pos to 2: 3.67 switch from hexane to ethanol The bigger loop gave rise to a higher re-focused peak height, as evidenced by Fig 6A An increase in the solvent-switch time brought a very slight increase in the height of the re-focused peak (Fig 6C) Similar to the trend observed with re-focusing of phenanthrene, an increase in the trap temperature led to an increase in the height of the re-focused phenol peak, although the effect is much weaker than in the case of phenanthrene (Fig 6D) Practically no difference was observed when the hexane flushing time was varied (Fig 6E), which could be attributed to the poor solvation power of hexane for phenol By UV peak height enhancement Concentration enhancement 6.4 times 2.6 times 6.4 times 2.2 times 3.2 times 2.9 times 2.2 times 1.7 times employing a higher re-mobilizing ethanol flow, a slight increase in the height of the re-focusing peak was achieved in a shorter analysis time (Fig 6F) 3.6 Post-column re-focusing of p-coumaric acid Although the 2-PIC column provided the strongest retention of p-coumaric acid, it was not deemed a good option for re-focusing, because re-mobilizing the trapped compound with ethanol in a sharp band would be extremely difficult Therefore, the DIOL col8 M Sun and P Schoenmakers Journal of Chromatography A 1660 (2021) 462642 umn was chosen, together with hexane and ethanol as the flushing and re-mobilizing solvents The re-mobilizing p-coumaric acid peak was observed after the background peak that resulted from the solvent switch (Fig 7A and 7B) The use of sampling loops of two different volumes did not yield any apparent differences in the height of the re-focused peak The 160-μL loop was used for the rest of the tests, as it required a shorter total analysis time Surprisingly, changes in the trap temperature, hexane flushing time and ethanol flow rate did not induce any clear differences in the re-focused peak (Fig 7C-E) A shorter solvent-switching time (immediate switch or 0.5-min gradient) yielded slightly better refocusing performance than the longer gradient (1.0 min; Fig 7F) pounds, which might lead to a significantly broadened trappedcomponent band Also, ethanol could possibly be ineffective in remobilizing the trapped compound, as the solubility of theobromine in ethanol is limited Methanol displays much higher solubility of theobromine than ethanol [16] Therefore, methanol was investigated as the re-mobilizing solvent for theobromine To avoid solvent immiscibility issues, it was combined with diethyl ether as the flushing solvent 1D-LC experiments were performed to compare the elution of theobromine using methanol and ethanol from the DIOL column As can be seen in Fig 8A, methanol eluted the compound faster and in a narrower band One benefit of using diethyl ether as the flushing solvent could be that it can potentially remove precipitated methanol from the transferred SFC fraction However, its high volatility is a concern for pumping, as can be observed in Fig (B-F) A comparison of Fig 8B and 8C revealed that the re-focused peak eluted right after the noisy baseline returned to normal Fig 8D shows that this was the case, regardless of the diethyl-ether flushing time Thus, it was evident that the fluctuations in the baseline originated from the pumping of diethyl ether They disappeared right after the solvent switch to methanol and before the elution of the re-focused peak 3.7 Post-column re-focusing of theobromine The DIOL trapping - hexane/ethanol flushing/re-mobilizing system was also evaluated in the re-focusing of theobromine However, the results from the preliminary runs were not satisfactory The re-focused peaks were too broad This was not totally unexpected, as theobromine eluted with the largest concentration of methanol from the SFC column among the four test com- Fig (A) SFC/LC UV response ratio (PHE - Phenanthrene; PH - Phenol; COU - p-Coumaric acid; THEO - Theobromine) (B) Compound UV spectra in SFC-type and LC-type solvents The SFC UV signal and spectra for each compound were measured under their elution conditions in SFC The LC UV signal and spectra for each compound were measured under the optimized re-mobilizing conditions described in Table 1, except that 0.25 mL/min was used for all analytes M Sun and P Schoenmakers Journal of Chromatography A 1660 (2021) 462642 To minimize the problems related to solvent volatility, only 20°C was later used as trap temperature to study the factors influencing the re-focusing performance The volume of the sampling loop did not lead to any significant variations in the re-focusing performance (Fig 8C), with the smaller loop yielding slightly higher re-focused peaks The diethylether flushing time had almost no effect on the re-focusing of theobromine (Fig 8D), likely due to the limited elution strength of diethyl ether, which caused the theobromine band in the trapping column to remain unaltered Similar to what was observed with the other compounds, re-mobilizing flow rate and solventswitch time hardly had any impact on the re-focused peak height (Figs 8E-F) fine if only a single fraction is selected for further analysis, as in commonly applied heart-cut 2D approaches However, it is a drawback for multiple-heart-cut approaches This drawback may feasibly be overcome by using a multiple-loop collector to store fractions [17] However, this will unavoidably prolong the total analysis time The drawback is even greater for comprehensive 2D chromatography, where many fractions must be collected and modulation times must be kept short Besides the use of columns packed with sub-2-μm particles to achieve highly efficient and fast separations, another current trend in SFC is the use of mobile phase containing high concentrations (more than 50%) of modifier [18,19] This type of mobile phase that is intermediate between supercritical and liquid solvents extend the range of compounds that can be analysed by SFC systems The use of the post-column re-focusing approach proposed in this work under such conditions will be extremely challenging, as a very high trapping capacity would be needed to successfully retain the compounds In such a situation, the design may have to be modified to include an extra solvent-dilution step before trapping, similar to the concept of active solvent modulation in 2D-LC [20] The main purpose of this work was to propose a strategy for achieving SFC post-column re-focusing, with emphasis on the possibility of nuclear-magnetic-resonance (NMR) spectroscopy as a quantitative structure-elucidative detector for SFC NMR can be a powerful chemical analysis tool when coupled with LC separations [21] However, one of the biggest challenges of this hyphenation is the sample dilution in the LC mobile phase, which makes the detection of low-abundant analytes very difficult [22] This issue is even more challenging in the on-line coupling of SFC with NMR, as much higher flow rates are often employed than in (U)HPLC [6] A controlled-expansion type SFC-NMR interface has been proposed in recent years for analyte re-focusing after SFC separation and before NMR detection [6,23,24] The basic idea was to retain the compounds in small amount of precipitated SFC modifier, while letting the CO2 expand in a controlled fashion In comparison, the postcolumn re-focusing approach presented minimizes the effects of the SFC modifier on the NMR measurements and eliminates the need to use deuterated modifiers in SFC The influencing parameters assessed were not exhaustive Other parameters may potentially improve the re-focusing performance, such as the length of the trapping column and direction of the remobilizing flow (forward flush or backflush) Such factors may be reconsidered if the proposed post-column re-focusing approach is adopted in real applications in future work 3.8 Peak height enhancement and concentration enhancement of all compounds Table summarizes the parameters employed to obtain the best overall re-focusing performances for the four compounds achieved in this study, together with the UV peak height and concentration enhancement ratio As a compound’s UV-detector response at a specific concentration may vary greatly in different solvents, a straightforward translation of peak-height enhancement to concentration enhancement may be erroneous To accurately calculate the concentration enhancement brought by the post-column re-focusing approach, calibration lines (peak area vs concentration) were obtained for the four compounds under SFC and LC elution conditions, respectively (Figures S1 and S2) After correction with the different flow rates employed in LC and SFC, the UV response differences of the compounds in SFC and LC type mobile phase were revealed For example, a phenanthrene fraction of the same concentration showed an approximately twice as high UV response in 100% acetonitrile than in supercritical CO2 with a small amount of methanol (Fig 9A) The response difference can be partially attributed to a shift in compound UV absorption spectra (Fig 9B) For phenanthrene, an obvious red shift can be observed when the solvent changed from SFC-type to LCtype, which enhances the absorption at 280 nm It should be noted that the signal-enhancement ratios can change drastically if signals are determined at different wavelengths that lead to maximum absorbance under SFC and LC elution conditions, respectively Conclusions, limitations and perspectives A trapping approach has been developed to achieve SFC postcolumn re-focusing of compounds of a wide polarity range Adequate re-focusing can be achieved for relatively non-polar compounds by using a C18 trapping column, combined with water as flushing solvent and acetonitrile as re-mobilizing solvent For effective trapping and focusing of more polar compounds, polar stationary phases were proven successful, in combination with n-hexane or diethyl ether as flushing solvent and ethanol or methanol as remobilizing solvent The effects of flushing time, sampling-loop size, trap temperature, re-mobilizing flow rate and solvent switch time were studied In most cases the effects of these parameters were found to be negligible or small UV peak height enhancement ratios of 6.4, 6.4, 3.2, 2.2 were achieved for phenanthrene, phenol, p-coumaric acid and theobromine, respectively Concentration enhancement ratios of 2.6, 2.2, 2.9, 1.7, respectively, were obtained for the same four analytes, taking into consideration differences in UV absorption between SFC and LC conditions Since most SFC separations are completed within 5-15 mins with contemporary instruments and columns, one obvious limitation of the current post-column re-focusing design is its low speed Practically, only one or two peaks can be re-focused if one sampling loop is used for collection of the SFC eluents This is perfectly Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper CRediT authorship contribution statement Mingzhe Sun: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Project administration Peter Schoenmakers: Resources, Writing – original draft, Writing – review & editing, Supervision, Funding acquisition Acknowledgement The research is part of the SFC-NMR project that is funded by the Dutch Research Council (NWO) in the framework of Technology Area COAST (project 053.21.115), and the MANIAC project that is funded by NWO in the framework of the Programmatic Technology Area PTA-COAST3 of the Fund New Chemical Innovations 10 M Sun and P Schoenmakers Journal of Chromatography A 1660 (2021) 462642 (project 053.21.113) The authors thank Prof Arno Kentgens and Dr Fleur van Zelst for inspiring scientific discussions and suggestions on the SFC-NMR coupling [11] A.F.G Gargano, M Duffin, P Navarro, P.J Schoenmakers, Reducing dilution and analysis time in online comprehensive two-dimensional liquid chromatography by active modulation, Anal Chem 88 (2016) 1785–1793 [12] J De Vos, G Desmet, S Eeltink, A generic approach to post-column refocusing in liquid chromatography, J Chromatogr A 1360 (2014) 164–171 [13] J Slobodnik, H Lingeman, U.A.T Brinkman, Large-volume liquid chromatographic trace-enrichment system for environmental analysis, Chromatographia 50 (1999) 141–149 [14] L Griffiths, R Horton, Optimization of LC-NMR III - Increased signal-to-noise ratio through column trapping, Magn Reson Chem 36 (1998) 104–109 [15] M.J Mills, J Maltas, W.J Lough, Assessment of injection volume limits when using on-column focusing with microbore liquid chromatography, J Chromatogr A 759 (1997) 1–11 [16] J.L Zhong, N Tang, B Asadzadeh, W.D Yan, Measurement and correlation of solubility of theobromine, theophylline, and caffeine in water and organic solvents at various temperatures, J Chem Eng Data 62 (2017) 2570–2577 [17] M Pursch, S Buckenmaier, Loop-based multiple heart-cutting two-dimensional liquid chromatography for target analysis in complex matrices, Anal Chem 87 (2015) 5310–5317 [18] M.R Silva, F.N Andrade, B.H Fumes, F.M Lancas, Unified chromatography: Fundamentals, instrumentation and applications, J Sep Sci 38 (2015) 3071–3083 [19] S.V Olesik, Enhanced-fluidity liquid chromatography: connecting the dots between supercritical fluid chromatography, conventional subcritical fluid chromatography, and HPLC, Lc Gc N Am (2015) 39–44 [20] D.R Stoll, K Shoykhet, P Petersson, S Buckenmaier, Active solvent modulation: A valve-based approach to improve separation compatibility in two-dimensional liquid chromatography, Anal Chem 89 (2017) 9260–9267 [21] G.S Walker, T.N O’Connell, Comparison of LC-NMR and conventional NMR for structure elucidation in drug metabolism studies, Expert Opin Drug Met (2008) 1295–1305 [22] M.V.S Elipe, Advantages and disadvantages of nuclear magnetic resonance spectroscopy as a hyphenated technique, Anal Chim Acta 497 (2003) 1–25 [23] S van Meerten, F van Zelst, K Tijssen, A Kentgens, An optimized NMR stripline for sensitive supercritical fluid chromatography-nuclear magnetic resonance of microliter sample volumes, Anal Chem 92 (2020) 13010–13016 [24] F.H.M van Zelst, S.G.J van Meerten, A.P.M Kentgens, Characterising polar compounds using supercritical fluid chromatography-nuclear magnetic resonance spectroscopy (SFC-NMR), Faraday Discuss 218 (2019) 219–232 Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2021.462642 References [1] C West, Current trends in supercritical fluid chromatography, Anal Bioanal Chem 410 (2018) 6441–6457 [2] L Novakova, A.G.G Perrenoud, I Francois, C West, E Lesellier, D Guillarme, Modern analytical supercritical fluid chromatography using columns packed with sub-2 mu m particles: A tutorial, Anal Chim Acta 824 (2014) 18–35 [3] H.K Vlckova, V Pilarova, P Svobodova, J Plisek, F Svec, L Novakova, Current state of bioanalytical chromatography in clinical analysis, Analyst 143 (2018) 1305–1325 [4] C West, E Lemasson, S Bertin, P Hennig, E Lesellier, An improved classification of stationary phases for ultra-high performance supercritical fluid chromatography, J Chromatogr A 1440 (2016) 212–228 [5] L Akbal, G Hopfgartner, Hyphenation of packed column supercritical fluid chromatography with mass spectrometry: where are we and what are the remaining challenges? Anal Bioanal Chem 412 (2020) 6667–6677 [6] F.H.M van Zelst, S.G.J van Meerten, P.J.M van Bentum, A.P.M Kentgens, Hyphenation of supercritical fluid chromatography and NMR with in-Line sample concentration, Anal Chem 90 (2018) 10457–10464 [7] T.A Berger, B.K Berger, Minimizing UV noise in supercritical fluid chromatography I Improving back pressure regulator pressure noise, J Chromatogr A 1218 (2011) 2320–2326 [8] D Guillarme, V Desfontaine, S Heinisch, J.L Veuthey, What are the current solutions for interfacing supercritical fluid chromatography and mass spectrometry? J Chromatogr B 1083 (2018) 160–170 [9] I Francois, A.D Pereira, F Lynen, P Sandra, Construction of a new interface for comprehensive supercritical fluid chromatography x reversed phase liquid chromatography (SFC x RPLC), J Sep Sci 31 (2008) 3473–3478 [10] V Pepermans, J De Vos, S Eeltink, G Desmet, Peak sharpening limits of solvent-assisted post-column refocusing to enhance detection limits in liquid chromatography, J Chromatogr A 1586 (2019) 52–61 11 ... mobile phase contains only a low concentration of co-solvent, analyte precipitation may take place in the BPR Finally, if a significant amount of CO2 remains in the trapping column it will have to... Fig Investigation of the parameters in? ??uencing the post-column re-focusing of p-coumaric acid: (A) sampling loop volume; (B) re-focusing chromatogram of a blank injection for comparison; (C) trap... if only a single fraction is selected for further analysis, as in commonly applied heart-cut 2D approaches However, it is a drawback for multiple-heart-cut approaches This drawback may feasibly