Development of an At-Column Dilution Modulator for Flexible and

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Development of a at-column dilution modulator for flexible and precise control of dilution factors to overcome mobile phase incompatibility in comprehensive two-dimensional liquid chromatography Ying Zhuang Chen, Junjie Li, and Oliver Johannes Schmitz Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02391 • Publication Date (Web): 10 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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Analytical Chemistry

Development of a at-column dilution modulator for flexible and precise control of dilution factors to overcome mobile phase incompatibility in comprehensive two-dimensional liquid chromatography

Yingzhuang Chen1,2#, Junjie Li1,3#, Oliver J. Schmitz1,3* 1.

University of Duisburg-Essen, Applied Analytical Chemistry, Universitaetsstr. 5, 45141

Essen Germany. 2.

Key Laboratory of Phytochemical R&D of Hunan Province, Key Laboratory of Chemical

Biology & Traditional Chinese Medicine Research, Ministry of Education, Hunan Normal University, Changsha 410081, China. 3.

Teaching and Research Center for Separation (TRC), University of Duisburg-Essen,

Universitaetsstr. 5, 45141 Essen Germany.

# These authors contributed equally to this work and should be considered co-first authors Corresponding Author: Prof. Dr. Oliver J. Schmitz University of Duisburg-Essen, Applied Analytical Chemistry & Teaching and Research Center for Separation Universitaetsstr. 5, 45141 Essen, Germany Email: [email protected] Phone: +49 201 183 3950 Fax: +49 201 183 3951

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract With the combination of different mechanisms, two-dimensional liquid chromatography has brought revolutionary changes compared to the traditional one-dimensional separation, which dramatically improves the peak capacity in separation and meets the ever-increasing demand for the analysis of complex sample in different researching field, such as chemistry, medicine, etc. However, the incompatibilities between two columns due to the transport of the large sample volume and the solvent effect always limit the wide use of two-dimensional liquid chromatography. In order to resolve this problem a at-column dilution (ACD) modulator was established to overcome the solvent incompatibility in the orthogonal combination within the comprehensive two-dimensional liquid chromatography. This interface is modified from normal two-dimensional interfaces by an additional transfer pump, which realize the at-column dilution without a flow split during the transportation. Moreover, with the control of the transfer flow and the second-dimensional gradient flow, it is able to precisely regulate the at-column dilution factor and conveniently optimize the separation conditions in both dimensions. In this work, a systematic research has been done between the setups with/without the at-column interface in the combination of RPLC × HILIC and HILIC × RPLC, which proved that the at-column interface is able to resolve the solvent conflicting problem very well. Furthermore, the red 2 ACS Paragon Plus Environment

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ginseng was chosen as a real sample, to investigate the applicability of the atcolumn dilution modulator for comprehensive two-dimensional chromatography with high orthogonality. Key words: 2D-LC, at-column dilution modulator, ACD, RPLC×HILIC, LC×LC.



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1. Introduction In recent years, comprehensive two-dimensional liquid chromatography (LC×LC) has received increased attention along with the extensive study of complex systems, such as metabolomics1,2, proteomics3-6, Chinese herbal drugs7-9, industrial oils10, polymer11-13 etc. The growing interest in LC×LC is spurred by the possibility of generating much higher peak capacities in comparison with one-dimensional liquid chromatography (1D-LC)14-19. LC×LC systems are commonly consisted by two columns with a modulator responsible for collecting and transferring the fractions from the 1st dimension. In general, the difference between the separation mechanisms in two dimensions determines the utilization of two-dimensional separation space, which usually described as "Orthogonality"20. The larger the differences in separation mechanisms are, the higher the orthogonality the 2D system has, which leads to a better

separation20-22.

Nevertheless,

increasing

the

maximum

separation

orthogonality always leads to another problem, which is the incompatibility between two dimensions23,24. This kind of incompatibility based mainly on the typical large volume re-injection and separation process in the second dimension. Normally, the injected fractions in second dimension (2nd D) has been highly diluted after the first dimension (1st D) separation, which means that the volume of 4 ACS Paragon Plus Environment

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each collected fraction must be large enough to guarantee the necessary detection limit after the 2ndD separation. In addition, a relatively long modulation time, resulting in a large collected fraction, is always necessary to realize a sufficient separation power in the 2nd D. Unfortunately, the transferred fractions often containing very high contents of strong elution solvents for the second dimension in those high orthogonality combinations. This leads to serious negative effects on the separation with a short 2nd D column, which results in peak broadening and less retention25-28. For example with a HILIC × RPLC combination, the complementary retention mechanism is useful to achieve higher peak capacity in the separation of analytes25-27. However, acetonitrile (ACN) is a strong retention solvent in HILIC, but the high content of ACN in the collected fraction from the 1st D shows a completely opposite character in RPLC of the 2nd D. The injection of a fraction with such strong elution solvent prevents focusing on the head of the 2nd D column for high or middle polar compounds and leads to a band broadening and low separation power in 2nd D. Similarly, the same situation could occur in the high orthogonality combination of RPLC × HILIC28. In order to resolve the mobile phase incompatibility, many valuable strategies have been proposed29. In these reported methods, valve based modulation method strategy is the most common, including interface with double trapping column3,30-32, vacuum assisted solvent evaporation interface33,34, loop interface with post 1st column diluting and 5 ACS Paragon Plus Environment


splitting35,36, solvent switch modulation37-39 and on-line dilution interface with bypass23,40-43, etc. Compared to the other methods, on-line dilution interface with bypass has realized the online dilution without split and a simple setup, which achieves the complete transfer of fractions. This modulator was firstly proposed by Petersson et al.23, named as Fixed Solvent Modulator (FSM), which has the similar instrumentation as the standard 2D-LC modulator system. The configuration of FSM and modulation process is shown in figure S1, and the modulator works in such a way that the 2nd D flow is split into two parts. However, due to the time difference between the mobile phases flowing through these two paths, the ratio of the different mobile phases after combining was disturbed, which leads to the baseline shift during the measurement and interferes the detection of less intensive signals. To overcome this drawback, there is an advanced modulation strategy named "ASM" raised by Stoll et al.40,42, which is very useful in 2D-LC systems. As we can see from figure S2, this modulator is designed to control the switching on/off of the bypass at each modulation period. This means that the split of 2nd D mobile phase only occurs at the stage of the fractions transfer, resulting in less negative effects on the 2nd D separation compared to the full modulation at FSM mode. Due to the inevitable limitation of the loop volume, 1st D column diameter and flow are low in LC×LC. Therefore, the dilution in the 2nd D leads to a decrease in 6 ACS Paragon Plus Environment

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sensitivity in LC×LC in comparison with one-dimensional liquid chromatography. Based on FSM modulation strategy, Chen’s group made a new improvement by replace the sample loops with short C18 trapping columns to construct a RPLC × HILIC system43,44 and the configuration is shown in figure S3. In conclusion, by ASM, FSM or FSM with trapping column mode23,40-43, the dilution of the fraction is done very conveniently. However, for a very broad range of dilution volumes, the loops or splitting column has to change/adjust manually, which is time-consuming. Therefore, it is not easy to precisely control and optimize the dilution factor during the analysis of a complex sample with analytes in wide range of polarity. In addition to modify the structure of interface, temperature dependent properties of chromatographic behavior made a promise for developing new methods to overcome the mismatch of two dimensions. Recently, a longitudinal on-column thermal modulator was developed to refocus and release analytes from the 1D column without valve switching, but a movable resistive heating sleeve on the modulation column45. The modulation process can be quickly accomplished by periodically moving the sleeve from the inlet end to the outlet end of the modulation column. In the work reported by Roy46, the 1st D separation was carried out under a temperature gradient from low to high with water as mobile phase. In this work, a new interface, called at-column dilution (ACD) modulator, based on 7 ACS Paragon Plus Environment


an application note from Waters for 1D-LC (Milford, MA, USA, 2003, 71500078010, Revision A), was developed for 2D-LC as shown in figure 1. (Figure 1) Compared to the settings of the standard two-dimensional system, a transfer pump was added for transferring the fractions. Correspondingly, the second binary gradient pump was no longer flow through the sample loop. Instead, the transfer flow and the gradient flow were combined by a T-connector and then flow through the mixer before reaching the second column. By switching the valve, the transported fraction is combined with the weak elution solvent from the second gradient pump in the mixer, which realizes the at-column dilution. Compared to the reported ASM and FSM methods, the proposed modulator enables the independently precise control of the flows in the transfer and secondary gradient pump, which means that the dilution factor of the fraction could be accurately regulated. First, to verify the proposed ACD modulator, a mixture of polar standards was used to investigate the effect of dilution factor and large volume injection on HILIC separation. Furthermore, a comprehensive RPLC × HILIC system was constructed to study the influence of the different dilution factors on the 2nd D separation. In addition, another combination, HILIC × RPLC, was also tested for a better understanding of the new ACD modulator and allows the


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comparison with RPLC × HILIC. Finally, red ginseng, as a real sample, was chosen to demonstrate the application of RPLC × HILIC with ACD modulator.

2.

Experiments

Chemicals Vanillic acid, 2-(4-hydroxyphenylazo)benzoic acid, alpha-cyano-4-hydroxic innamic acid, l-tryptophan, nicotinic acid, urea, caffeine, bisphenol A, 4propylbenzoic acid, 4-butylbenzoic acid, raspberry ketone, 4-acetoxybenzoic acid, 3-(dimethylamino) benzoic acid, 2', 4', 6'-trihydroxyacetophenone, vanillin, nicotinamide, and indole-3-acetic acid were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). P. ginseng Red was purchased from Kronen Apotheke Wuppertal (Wuppertal, Germany). Methanol was supplied by Tedia Company Inc. (Fairfield, OH, USA) and acetonitrile (HPLC grade) was purchased from Fisher Scientific Inc. (Waltham, MA, USA). Water was purified using a Millipore system (Millipore, Milford, MA, USA). Sample preparation 17 standards (see above) were dissolved in water to form a mixed solution with concentrations of 1 mg/mL for each one.



P. ginseng Red extract solution was prepared by extracting 0.3 g dry powder in 5 mL methanol under ultra-sonication (100 W, 40 kHz, ambient temperature for 30 min). The supernatant was subsequently filtered through a 0.2 μm PTFE filter from VWR international (USA) prior to the injection into the LC×LC system. Instruments The setup involved with an Agilent two-dimensional liquid chromatography system (Agilent Technologies, Waldbronn, Germany), which consists of a 1290 Infinity binary pump (G4220A) as a 2nd D gradient pump, a 1290 Infinity High Speed pump (G7120A) as the transfer pump, a 1260 Infinity Capillary Pump (G1376A) as a 1st D gradient pump, a 1260 HIP Degasser (G4225A), a 1260 Infinity High Performance Micro Auto sampler (G1377A), a 1260 Infinity Diode Array Detector VL+ (G1315C)(1st D detector), an Agilent 1290 Infinity II Diode Array Detector (G7117B) as a 2nd D detector, two 40-μL sample loops, a Jet Weaver V35 mixer, a 1290 Infinity Valve Drive and Valve Heads (G1170A) and a 1290 Infinity Thermostatted Column Compartment (G1316C). For the at-column dilution (ACD) modulator the 1290 Infinity High Speed pump (transfer pump) was used (Figure 1). The collected fraction is transferred by the transfer pump (eg. flow 0.2 mL/min) and mix up together by a Jet Weaver V35 mixer with the high flow (eg. 2.8 mL/min) from 2nd D binary gradient pump, then 10 ACS Paragon Plus Environment

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enter the 2nd column. In general, the dilution factor is defined as the ratio of the total flow and transfer flow.

Experimental parameters In the first dimension of the RPLC × HILIC study, a C18 column (150×1.0 mm) with 2.6 μm core-shell particles (Phenomenex, CA, USA) was used. The mobile phase based on water containing 0.1% TFA (solvent A) and ACN containing 0.1% TFA (solvent B). A flow rate of 0.015 mL/min was used. In the second dimension, a Nucleoshell HILIC column (100×3.0 mm) packed with 2.7 μm particles (Macherey-Nagel, Düren, Germany) was installed. The mobile phase based on water containing 0.1% NH3 (solvent C) and ACN with 0.1% NH3 (solvent D). A total flow rate (the sum of transfer flow and 2nd D gradient elution flow) of 3.0 mL/min was used. To investigate the effect of the dilution factor for HILIC × RPLC with the standard mixture, the 2nd D gradient changes according the ratio of transfer pump flow and 2nd D gradient pump flow to keep the total gradient in 2nd D unchanged. All gradients applied in both dimensions are summarized in table S1. To study the effect of dilution factor on the 2nd D HILIC separation with the standard mixture, the 1st D column was removed to focus the research on the 2nd D separation. The mobile phase consists of 10% B and a flow rate of 0.015 mL/min 11 ACS Paragon Plus Environment


was used in the 1st D. Other conditions applied in the 2nd D are the same as in RPLC×HILIC described above. To study the combination HILIC × RPLC for separation of the standard mixture, a SeQuant ZIC-HILIC column (150×1.0 mm) with 3.5 μm particles (Merck, Darmstadt, Germany), and a C18 column (50×3.0 mm), filled with 2.6 μm particles (Phenomenex, CA, USA) were used. A HILIC gradient with mobile phase consists of solvents C and D was used in 1st D, and RPLC gradient with mobile phase of solvent A and B was performed in the 2nd D separation. The flows for the 1st D, the transfer and the 2nd D were 0.015, 0.2 and 1.8 mL/min, respectively, which results in a total flow in the 2nd D of 2.0 mL/min. For comparison, the traditional modulator was performed with the same column and flow in 1st D and 2nd D separation. The gradient applied in 2D-LC with ACD modulator and traditional standard modulator are show in supplement table S2. All information about the measurement of P. ginseng Red extract are listed in the supplement (Table S3 and S4). 3. Results and Discussion On-line dilution with at-column dilution (ACD) modulator The combination of RPLC and HILIC represents one of the most orthogonal approaches in two-dimensional liquid chromatography, capable to afford very 12 ACS Paragon Plus Environment

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high-resolution power due to nearly independent separation mechanisms operating on the individual stationary phases. The RPLC × HILIC coupling is a challenge and hardly straight forward to achieve, due to the incompatibility of the solvents in both dimensions. Such incompatibility mainly occurs in the early stages of the 1st D gradient, where a mobile phase with ~ 90% water was commonly used. The high water transferring fraction maybe results in non-focused peaks on the head of the 2nd D column and weaken retention of compounds. Therefore, a dilution with an organic solvent is essential before fraction transfer to achieve a peak focusing on the head of 2nd D column, resulting in good separation in the 2nd D. After precisely adjusting the settings of the transfer pump and the 2nd D binary pump, the transfer flow and the 2nd gradient pump flow can be controlled independently by using the ACD modulator (figure 1, right). Therefore, the fraction can effectively diluted by high organic solvent before transferred to the 2nd D column. To simulate the effect of the high-aqueous mobile phase at the beginning stage of the 1st D reversed phase (RP) gradient on 2nd D separation, 10% acetonitrile was used as the 1st D mobile phase. Meanwhile, the 1st D RP column was removed, allowing the 1st D sample together with the high-water mobile phase (total volume of 40 µL) to be transferred directly to 2nd D column without flow through the 1st D column. Figure 2A shows the resulting chromatograms of the mixed standards after increasing the organic content of the 10% ACN containing sample solvent by the ACD modulator. Small 13 ACS Paragon Plus Environment


dilution factor (3, 6 and 10) could significantly weaken the retention of the standards in the mixture, and serious breakthrough peaks were observed for most standards. On the other hand, high dilution factors (15 and 20) could remarkably improve the retention of these standards and the breakthrough peak was decreased obviously. A strong focus of the analytes on the head of the nucleoshell HILIC column is responsible for this effect due to the high percentage of ACN in the diluted sample solution. The chromatograms in figure 2 show that good retention of most of the compounds in the standard mixtures were obtained successfully when applied dilution factor of 15 or more. Nevertheless, a weak breakthrough peak could still be observed also with such a dilution factor. However, it must be taken into account that the ACN content in the 1st D eluent is gradually increased during analysis and the analytes in the breakthrough peak of the 2nd D HILIC separation are usually eluted from the 1st D column later in the analysis. This ensures that these analytes can also be effectively concentrated on the head of the 2nd D HILIC column after further dilution with a weak eluent for the second dimension in a real RPLC x HILIC analysis. As a summary, dilution factor no less than 15 is recommended for further optimization. (Figure 2)


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In RPLC × HILIC with ACD modulator, the dilution factor can be easily adjusted over a wide range by the flow of the transfer and 2nd gradient pumps. However, a high dilution factor of the transferring fraction might lead to an increase of the transfer time in 2nd D. Table S1 shows the change of transfer time by adjusting the dilution factor from 3 to 20, when a sample loop of 40 µL was used. Obviously, the higher the dilution factor, the longer the transfer time. In LC×LC, commonly a modulation time of 2 min or less is used, thereby, the separation time has to be reduced by the transfer time and the equilibrium time. In figure 2, it could be easily observed that the breakthrough peak at dead time move to higher retention time with increasing dilution factor, resulting in a shorter 2nd D separation window. Therefore, it is essential to determine the minimum dilution factor under the premise that the sample was well separated on the 2nd D column. The chromatograms in figure 2 show that a dilution factor no less than 15 is essential for a good separation of most of the compounds in the standard mixtures. In addition, table S5 shows that a transfer time of 12 s is required for a dilution factor of 15. Figure 2a-e demonstrates that even smaller breakthrough peaks could be observed with a dilution factor of 20. However, the dilution factor of 20 means a longer transfer time of 16 s, which accounts in 13.3% of the total analysis time (120 s), leading to a serious decrease of the separation window. Therefore, an optimized dilution factor of 15 (10% of the total analysis time) was used for good 15 ACS Paragon Plus Environment


separation of the given standard mixture, which is corresponding to 0.2 mL/min of transfer flow and 2.8 mL/min of 2nd D gradient pump flow. Optimization of RPLC × HILIC system with the ACD modulator for standard mixture analysis To verify the results obtained in above section, the effect of dilution factor on RPLC × HILIC separation of the mixed standards was further investigated in detail and shown in Figure 4S. An optimized RPLC gradient with 15 µL/min flow in the 1st D, and several dilution factors from 3 to 20 were applied. The details about the gradients are summarized in table S1. As shown in figure S4d and e, with dilution factors of 15 and 20, respectively, nearly all standards were well separated with good peak shapes, which is consistent with the results shown in figure 2. Serious peak tailing were observed for individuals, and might be attributed to a non-special interaction with the stationary phase. Similar to the results with the dilution factor of 15 and 20, no obvious breakthrough peak was observed and most analytes were well separated by a dilution factor of 10 (figure S4c). However, the results in figure 2c demonstrate that the standards could not be well retained and separated on the 2nd D Nucleoshell HILIC column with the dilution factor of 10. Nevertheless, in the first dimension of a RPLC separation, the polar analytes are eluted with high water mobile phase at the beginning of the gradient, and later, the 16 ACS Paragon Plus Environment

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less polar analytes eluted with a higher organic mobile phase of the gradient. In other words, the later the fraction was collected, the higher the organic solvent content have, therefore, both, polar and less polar, analytes could be well diluted according to the difference of their polarity under given dilution factor of 10, resulting in well focused analytes on the head of the 2nd HILIC column. By using the dilution factors of 3 and 6, the peaks of some analytes split significantly during the 2nd D HILIC separation, dividing into a non-retained and retained part. Such peak-splitting phenomenon was also observed in the reported HILIC × RPLC system equipped with a trapping column modulator47. Peak splitting could be explained by mismatch of two dimension. Because the high content of strong solvent (for 2nd D) in transfer fraction, which is a weak solvent in 1st D, prevents the focusing effect of all analytes on the head of the 2nd D column, which causes some analytes eluted rapidly with the strong solvent of the fraction and form a breakthrough peak. In figure S4, by comparing the 2D contour plot with different dilution factors, it could be clearly observed that the spot intensity of the retained part for each analyte is increasing with the increase of the dilution factor (up to dilution factor of 10) and the non-retained part would almost disappear. This result also indicates that the dilution is closely related to the detection sensitivity and a high dilution factor is beneficial to achieve a high sensitive detection. In conclusion, the results also demonstrate that the dilution 17 ACS Paragon Plus Environment


factor of 10 was not enough for the standards on the proposed RPLC × HILIC system due to the slight presence of breakthrough peak. In this case, a dilution factor of 15 is essential to achieve good separation and the highest sensitive detection. The application of at-column dilution (ACD) modulator in HILIC × RPLC 2D-LC system for standard mixtures analysis In principle, at-column dilution (ACD) modulation could also be used in 2D-LC system coupling with two separation mode in the order of HILIC-RPLC, which usually used in many reported works24,26,40,47. Therefore, the HILIC × RPLC system equipped with at-column dilution (ACD) modulator was also examined by using the same standard mixtures as test sample. Figure 3 shows the comparison of the contour plots achieved with traditional standard modulation and ACD modulation for the analysis of the mixed standards. Strong breakthrough can be found at the dead time in TS modulation. In addition, by using the traditional standard modulation, most of the analytes are separated with a serious peak fronting. This could be attributed to an insufficient focusing of analytes on the head of 2nd RPLC column. (Figure 3)


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Once ACD modulation was applied, the separation was significantly improved without any breakthrough and no peak fronting due to sufficient dilution of the transferred fractions with high aqueous mobile phase at the beginning of each 2nd D gradient cycle. Compared to the results as shown in figure S4d and e, a better separation was achieved by using HILIC × RPLC system. This might be attributed to the relative low polarity of standards analytes, which has stronger retention and better separation performance on RPLC column than on HILIC column. This result demonstrated that a HILIC × RPLC coupling was more suitable for the analysis of less polar compounds. RPLC × HILIC versus HILIC × RPLC for the analysis of polar analytes After the optimization of the separation condition, a slightly worse separation can be obtained by RPLC × HILIC (Figure S5) than HILIC × RPLC (Figure S6) for the standard mixtures analysis. However, the order RPLC × HILIC coupling offers more advantages compared to HILIC × RPLC. First of all, quick separation could be realized due to low back operating pressure of HILIC. In figure 3 and figure S5 operating pressure on the 2nd D Nucleoshell HILIC column (2.7 μm, 100×3.0 mm) was lower than 500 bar with a total flow of 3 mL/min during the entire analysis. However, more than 600 bar was observed on RPLC column (2.6 μm, 50×3.0 mm) when total flow of 2 mL/min was applied. Therefore, a higher flow could be used 19 ACS Paragon Plus Environment


to speed up the separation with HILIC as the 2nd D separation mode. In addition, a relatively high flow allows to shorten the fraction transfer time if the dilution factor kept constant. Secondly, better sensitivity could be achieved by coupling with ESIMS due to a high organic content in the mobile phase, which could enhance the ionization efficiency of ESI-MS. Furthermore, a longer 2nd column could be applied to yield better separation. Finally yet importantly, polar compounds can be dissolved easily in water, which is more suitable for sample preparation and 1st D RPLC separation in RPLC × HILIC. Nevertheless, the potential risk of so-called longer equilibrium of HILIC may have a negative effort on the 2nd D separation, however, the high 2nd D flow rate and the additional short equilibrium time at the end of each 2nd D gradient cycle would accelerate the balance of 2nd D HILIC column before the start of the next 2nd D injection. Take a step back, complete equilibrium of the 2nd D column is hard to achieve in virtually any twodimensional system due to the short modulation time. However, this concern is superfluous if the separation result is acceptable, which has been clearly proved in the previous section. RPLC × HILIC with ACD modulation for the analysis of red ginseng extract To investigate the application of proposed ACD modulation with a real sample, red ginseng, a typical traditional Chinese medicine made of white ginseng by 20 ACS Paragon Plus Environment

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steaming at a high temperature, was chose to further evaluated the proposed system. Up to date, many methods has been developed to separate the chemical constituents in ginseng, which mainly performed on single RPLC or HILC48-51. However, due to the existence of many structurally similar compounds, the limited separation capability of a single separation mode seriously affected the separation of those compounds, resulting in the difficulty of constituents identification. Therefore, it is meaningful to develop more sufficient separation method for such complex sample. Red ginseng mainly consists of saponin compounds with a hydrophobic gonane steroid nucleus and several polar glycosyl units. It could be separated by both, RPLC and HILIC mode, due to its coexistence of hydrophilicity and hydrophobicity. Therefore, the 2D-LC system RPLC × HILIC could be a good choice to separate those compounds. For 2D-LC analysis, the optimization mainly focused on the effect of the dilution factor on the separation performance. As shown in figure S7, separation with dilution factors of 0 (no dilution), 3 and 13 were applied. Under optimized dilution condition, the 2D-LC system was connected with a TOFMS to identify the chemical constituents in red ginseng by comparison with literature. As a further improvement, the analysis time was prolonged to 100 min to get better separation, and the used gradient for both dimensions are shown in Table S4. In figure 4 the comparison of RPLC × HILIC with traditional standard (TS) 21 ACS Paragon Plus Environment


modulator (without dilution) and with ACD modulator (dilution factor of 13) were demonstrated. (Figure 4) The contour plot of the extracted ions with m/z 799.4849 and 945.5428 clearly showed that the dilution factor of 13 leads to sharper peaks, better separation and increasing sensitivity in 2nd D. More than fifty analytes were determined in a single analysis. With the comparison to the MS/MS fragments of those peaks with the reported data48,50,52-54, about 20 saponin compounds were identified successfully. The results were listed in figure S8a-m. 4. Conclusion Within the study, a new ACD modulator has been developed, which successfully realized the at-column dilution without splitting 1st D effluent. In addition, the dilution factor can be conveniently controlled and optimized so that the separation in the second dimension is also sufficient. The results showed that the incompatibility problem in comprehensive two-dimensional chromatography with high orthogonality, such as the combination of RPLC × HILIC and HILIC × RPLC, could be improved. Especially with RPLC × HILIC, the gradient of the second dimension needs a high content of organic phase (eg. between 97 and 50 %) and the ACD modulator allows the dilution factor (injection time of the second 22 ACS Paragon Plus Environment

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dimension) to be adjusted in a wide range and hardly influences the gradient of the second dimension. Thus, the dilution factor can be well adapted to the variation of the modulation time. For example, a small dilution factor (less injection time) can be used for short modulation times (e.g. less than 1 min). Alternatively, when switching to a larger modulation time such as 4 min, commonly used in LC + LC36, assures that the transported fractions are sufficiently diluted for a better focusing effect on the head of the 2nd D column. In future, to reduce the loss of sensitivity because of the µLCxLC approach (combination of a 1 mm column in the 1st and a 3 mm column in the 2nd dimension with a relatively high flow rate (> 2.5 mL/min)), we will use a LCxLC system with 2.1 mm columns in the 1st and 3 mm columns in the 2nd dimension (with the same 2nd D flow rate) to increase the sensitivity of the analysis. In conclusion, this new interface might be able to promote the widespread use of the chromatographic platforms with high orthogonality for the analysis of complex samples. Acknowledgements This work was financially supported by the China Scholarship council (CSC).



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Figure 1. Scheme of the LC×LC system with traditional standard modulator (left) and at-column dilution (ACD) modulator (right).


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figu

Figure 2. Effect of the dilution factor on 2nd D HILIC separation for mix standards with increasing dilution factor from 3 (a) to 20 (e); 1st D mobile phase: 10%ACN(A).



Figure 3. HILIC× RPLC contour plot of the mixed standards with traditional standard (TS) modulator and at-column dilution (ACD) modulator. Peak identification of mixture standards shown in figure S5.


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Figure 4. 2D contour plot of RPLC × HILIC with traditional standard modulator (TS) and ACD modulator for MS analysis of red ginseng. a, f show the TICs; b, g the EIC at m/z 799.4849; c, h the peaks at m/z 799.4849; d, i the EIC at m/z 945.5428 and e, j the peaks at m/z 945.5428 of the RPLC × HILIC analysis with the traditional standard modulator (TS) and the ACD modulator, respectively.



TOC


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HILIC× RPLC contour plot of the mixed standards with traditional standard (TS) modulator and at-column dilution (ACD) modulator. Peak identification of mixture standards shown in figure S5. 179x142mm (150 x 150 DPI)

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2D contour plot of RPLC × HILIC with traditional standard modulator (TS) and ACD modulator for MS analysis of red ginseng. a, f show the TICs; b, g the EIC at m/z 799.4849; c, h the peaks at m/z 799.4849; d, i the EIC at m/z 945.5428 and e, j the peaks at m/z 945.5428 of the RPLC × HILIC analysis with the traditional standard modulator (TS) and the ACD modulator, respectively. 358x180mm (150 x 150 DPI)

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Development of an At-Column Dilution Modulator for Flexible and

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