Dual Electrolytic Eluent Generation for Oligosaccharides Analysis

Publication Date (Web): August 14, 2018 ... drawbacks associated with manually prepared NaOAc/NaOH eluents, and offers an easy to use, simplified oper...
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Dual Electrolytic Eluent Generation for Oligosaccharides Analysis Using High-Performance Anion-Exchange Chromatography Yongjing Chen,* Victor Barreto, Andy Woodruff, Zhongqing Lu, Yan Liu, and Christopher Pohl Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, California 94088-3603, United States

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S Supporting Information *

ABSTRACT: The research on oligosaccharides is growing and gaining in importance at a rapid pace. The efforts to understand their bioactivity and to develop new products based on oligosaccharides in biotherapeutics and food industry require effective and reliable tools for analysis of oligosaccharides. Here we present a dual electrolytic eluent generation platform for the analysis of oligosaccharides by high-performance anion-exchange liquid chromatography (HPAE) in both analytical and capillary column formats. The system consists of one eluent generator producing methanesulfonic acid (MSA) connected in series with a second eluent generator producing potassium hydroxide (KOH). Through manipulating the concentration output of both eluent generators, chromatographic performance comparable to that obtained using the conventional sodium acetate/sodium hydroxide (NaOAc/NaOH) eluents is achieved using the electrolytically generated potassium methanesulfonate/potassium hydroxide (KMSA/KOH) eluent. This platform utilizes deionized water as the only carrier stream through a single isocratic pump, overcomes the various drawbacks associated with manually prepared NaOAc/NaOH eluents, and offers an easy to use, simplified operation solution for oligosaccharides profiling with increased precision and accuracy.

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ue to their benefits and various sources of origin, oligosaccharides are important research topics. One of the categories is glycans, which are oligosaccharides covalently attached to amino acid side chains of glycoproteins.1−3 Monoclonal antibodies (mAbs) are glycoproteins produced by living cell systems. They are at the forefront of targeted therapeutics and diagnostics. Like many other proteins, mAbs are susceptible to chemical and/or enzymatic modifications that can occur during manufacture, formulation, and storage. Minor variations in glycosylation can directly affect the safety and efficacy of protein therapeutics, potentially resulting in significant differences in biological functions and clinical implications.4,5 The biopharmaceutical manufacturers use glycan distribution to indicate the stability of the process, to ensure correct and consistent structure of the glycans for achieving the desired therapeutic effect and avoiding adverse immunological reaction. Analytical techniques to characterize the glycans and monitor the batch-to-batch process consistency are therefore very important in biopharmaceutical development. Prebiotics is another category of oligosaccharides. They beneficially affect human health by selectively stimulating the growth and activity of one or a limited number of bacteria in the colon.6 Many efforts are focused on the determination of their biological activity and the understanding of the mechanisms of their metabolic effect.7,8 Therefore, the analysis of prebiotics in food has significant importance.9 © XXXX American Chemical Society

The most commonly used analytical methods for oligosaccharides analysis include hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection (FLD), after labeling the oligosaccharides with a fluorophore,10 or with mass spectrometry (MS) detection,11 capillary electrophoresis (CE) with FLD, after labeling the oligosaccharides with a fluorophore,12 or with MS detection,13 and high-performance anion-exchange chromatography of native oligosaccharides with pulsed amperometric detection (HPAEPAD).14 Among these techniques, the HPAE-PAD technique offers superior resolution of oligosaccharides15,16 and high sensitivity without the need for derivatization.17 HPAE achieves highly selective separations of oligosaccharides using a strong anion-exchange stationary phase under high-pH conditions using hydroxide-based eluents. However, hydroxide solutions are an excellent trap for atmospheric carbon dioxide (CO2), which results in the eluent being contaminated with carbonate. To minimize the carbonate contamination, the electrolytic eluent generation technology has been widely used to produces ultrapure hydroxide eluents on demand for ion chromatography (IC).18 This technology makes it possible to use only deionized water as the carrier stream, and the isocratic Received: May 31, 2018 Accepted: August 14, 2018 Published: August 14, 2018 A

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hydroxide and potassium methanesulfonate eluents that are generated electrolytically. In this new system, a single isocratic pump is used to pump deionized water into an EG producing potassium hydroxide (KOH) and a second EG producing methanesulfonic acid (MSA) sequentially. By suitably manipulating the concentration output of both EGs, potassium methanesulfonate (KMSA)/potassium hydroxide (KOH) gradients of various concentrations and slopes can be generated. System performance comparable to that using manually prepared NaOAc/NaOH eluent is achieved using the electrolytically generated KMSA/KOH eluent. We demonstrate the separation of oligosaccharides in glycoprotein and food samples in both analytical and capillary separation modes.

or gradient hydroxide eluent is provided on demand by modules within the instrument. While simple monosaccharides and disaccharides can easily be separated under isocratic conditions using the potassium hydroxide eluents generated electrolytically,19 oligosaccharides generally require gradient elution in order to achieve good separations in a reasonable analysis time. Generally, a constant level of hydroxide is used to provide the necessary pH for retention while simultaneously a “competing ion” (typically sodium acetate) concentration gradient is required to facilitate elution of highly retained peaks. Three components, i.e., water, sodium hydroxide (NaOH) solution, and sodium acetate/ sodium hydroxide (NaOAc/NaOH) mixture solution, are mixed via low-pressure gradient proportioning to accomplish the aforementioned gradient conditions.20 The proper preparation and setup of the eluents to minimize the intrusion of CO2 is the key to success using this technique. First of all, the eluents must be prepared using deionized water of low resistivity (18 MΩ·cm or better), 50% (w/w) NaOH, and electrochemical-grade anhydrous NaOAc and must be filtered through a 0.2 μm nylon filter to remove any particulate contaminants. The prepared solution must be thoroughly degassed and promptly transferred to the plastic eluent bottle and immediately protected from the air using helium or nitrogen gas. The procedure requires meticulous care on multiple steps to ensure consistency in eluent composition and to minimize the carbonate intrusion throughout eluent preparation and use. The above-mentioned eluent preparation and setup only applies to a HPAE-PAD system with analytical format (i.d. >2 mm) columns. Considering that samples containing glycans are often limited, it is highly desirable to perform the separations using capillary-scale separation columns; however, it is considerably more difficult to achieve reasonably small delay volumes to enable a gradient mixing of eluents using conventional mechanical proportional pumps at the capillary flow rate, e.g., 10 μL/min. This limitation has precluded the application of HPAE-PAD to the analysis of oligosaccharides using the capillary-scale separation columns so far. Considering the above-mentioned limitations, it is preferable to operate the gradient separation of oligosaccharides using electrolytically generated eluent with lower-cost isocratic pumps. Since the introduction of the automated electrolytic eluent generation technology,18 the idea behind this innovation has been adapted to develop new electrolytic devices that incorporate ion-exchange membranes and water electrolysis to generate electrically controlled salt and pH gradients. Chen et al. demonstrated electrically controlled pH gradients by utilizing a commercial membrane-based ion-exchange suppressor used in IC21 and pH- and concentration-programmable gradients generated by a prototype electrodialytic buffer generator.22 In both cases, the devices are limited to lowpressure conditions and their performance for high-performance liquid chromatography (HPLC) applications has yet to be demonstrated. Talebi et al. described semiautomated pH gradients for profiling of monoclonal antibody charge variants, where they used base or acid generated by commercial electrolytic eluent generators (EGs) to titrate a reagent composed of low molecular weight amine to produce the buffers for separation.23 The setup requires manual preparation of the reagent, which is subject to operator error. Here we describe a viable platform to accomplish comparable separations of oligosaccharides using potassium

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PRINCIPLES The KMSA/KOH eluent is generated from the reaction of KOH generated by an electrolytic KOH EG cartridge with MSA generated by an electrolytic MSA EG cartridge. The concentrations of generated MSA and KOH are determined by the currents applied to the two EG cartridges and the flow rate through the cartridge eluent generation chambers. The two EG cartridges are connected in series, with the MSA cartridge placed on the upstream side of the KOH cartridge (Figure 1).

Figure 1. Operation principle of the dual electrolytic eluent generation (MSA−KOH configuration).

Since the presence of hydroxide is critical for both separation and detection, the concentration of the generated KOH is set to be higher than that of the generated MSA, resulting in the final product containing KMSA and KOH eluents at the desired concentrations. Each EG cartridge consists of a high-pressure eluent generation chamber and a low-pressure electrolyte reservoir. Both the chamber and the reservoir contain a platinum electrode to perform water electrolysis. As shown in Figure 1, deionized water is pumped through the MSA generation chamber with a dc current applied between the anode and cathode of the MSA EG cartridge. Under the applied electric field, water is oxidized to form H+ ions and oxygen gas at the anode in the MSA generation chamber, while water is reduced to form OH− ions and hydrogen gas at the cathode in the MSA electrolyte reservoir. The OH− ions, generated at the cathode, displace MSA− ions in the electrolyte reservoir. The MSA− ions migrate across the anion-exchange connector into the MSA electrolysis chamber, where they combine with H+ ions generated at the anode to produce MSA solution. The MSA solution along with the O2 gas generated at the anode is passed through a degas unit; then, the degassed MSA solution is directed into the KOH generation chamber. Inside the KOH B

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Analytical Chemistry EG cartridge, under the applied electric field, water is oxidized to form H+ ions and oxygen gas at the anode in the KOH electrolyte reservoir. The generated H+ ions displace the K+ ions in the electrolyte reservoir, causing the displaced K+ ions to migrate across the cation-exchange connector into the eluent generation chamber. Meanwhile, equivalent amounts of OH− ions are generated at the cathode, along with H2 gas. The generated OH− ions readily react with the H+ coming from upstream to produce water, with an equivalent amount of KMSA being produced. With excess KOH being generated, the effluent from the KOH eluent generator is a mixture of KMSA/KOH and H2 gas. The effluent then passes through another degas unit. The degassed KMSA/KOH solution is then ready to be used for separation. A different configuration based on similar operation principle, where the KOH EG cartridge is placed on the upstream side of the MSA EG cartridge, was also considered (Figure S1). The detailed description of the principle can be found in the Supporting Information. In both of the two configurations [KMSA]product = [MSA]set

(1)

[KOH]product = [KOH]set − [MSA]set

(2)

isolated from the protein, by the use of a 50 mg Hyercarb Hypersep SPE cartridge. The SPE cartridge was preconditioned with 1 mL of ACN, followed by 0.5 mL of 40% ACN/ 0.1% TFA, 0.5 mL of 5% ACN/0.1% TFA, and 0.5 mL of 0.1% TFA. After the sample was slowly loaded onto the SPE cartridge, the cartridge was washed two times with 0.5 mL of 0.1% TFA, followed by 0.5 mL of 5% ACN/0.1% TFA. Released glycans were eluted off the cartridge using 1 mL of 40% ACN/0.1% TFA and collected into a 2 mL microcentrifuge tube. The eluted glycans were dried in a SpeedVac (Savant, Thermo Scientific) and reconstituted in 100 μL of DI water before injection. HPAE-PAD Instrumentation and Conditions. The chromatographic separations were performed on a prototype Thermo Scientific Dionex ICS-6000 dual-channel HPIC hybrid (analytical−capillary) system, which consisted of a Dionex DP dual-pump (one analytical pump and one capillary pump) module, two Dionex EG eluent generator modules, a Dionex DC detector/chromatography module, and a Dionex AS-AP autosampler module. On each channel, a prototype MSA EG cartridge was connected to an EG degas cartridge, followed by a prototype KOH EG cartridge connected to a second EG degas cartridge. The gas vent ports on both of the EG degas cartridges were connected to vacuum, to remove the H2 and O2 gas, respectively, to produce gas-free eluent. On the analytical channel, the separation was achieved on a prototype guard column (1 mm × 50 mm) and a prototype separator column (1 mm × 250 mm), both of which were packed with the same resins used in Dionex CarboPac PA200 columns, at a flow rate of 0.063 mL/min at 30 °C, with an injection volume of 400 nL. On the capillary channel, the separation was achieved on a prototype guard column (0.4 mm × 50 mm) and a prototype separator column (0.4 mm × 250 mm), both of which were packed with the same resins used in Dionex CarboPac PA200 columns, at a flow rate of 0.010 mL/min at 30 °C, with an injection volume of 100 nL. The detection in both systems was identical, accomplished with an electrochemical detector equipped with a thin-layer flow cell consisted of a 1.0 mm diameter Au PTFE disposable working electrode and a Ag/AgCl reference electrode, with the titanium cell body serving as the counter electrode. Inside the flow cell, a 1 mil PTFE gasket was used to form the flow channel and a reference electrode gasket was used to reduce the dispersion and delay. The Dionex default quadruple-potential PAD waveform for carbohydrates was used for detection. A schematic diagram of the HPAE-PAD system is shown in Figure 2. The HPAE-PAD system was controlled using a prototype Thermo Scientific Dionex Chromeleon software. Although prototype EG cartridges, EG degas cartridges, and separation columns were used in this work, devices of at least

where [MSA]set and [KOH]set are the concentrations of the MSA and KOH generated by the MSA and KOH EG cartridges, respectively. [KMSA]product and [KOH]product are concentrations of KMSA and KOH in the effluent exiting the outlet of the second cartridge. The characterization of the two configurations is discussed in the Results and Discussion section.

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EXPERIMENTAL SECTION Materials. A sialylated fetuin N-linked alditols standard was obtained from Thermo Scientific Dionex. Inulin from chicory root, IgG from human serum, and α1-acid glycoprotein (AGP) from human plasma were purchased from Sigma-Aldrich. Rapid PNGase F was purchased from New England Biolabs (NEB). Bimuno GOS powder (www.bimuno.com) was purchased from Amazon. Acetonitrile (ACN) was from Fisher Scientific, and trifluoroacetic acid (TFA) was from Pierce. Potassium hydroxide (45% w/w solution) was from J. T. Baker. Sodium acetate (anhydrous, electrochemical grade) was from Thermo Scientific. All chemicals were used as received. High-quality deionized water supplied by a Milli-Q Integral water purification system (EMD Millipore) was used to prepare all the solutions. HyperSep Hypercarb solid-phase extraction (SPE) columns (PGC, 50 mg, 1 mL) used for glycan purification were purchased from Thermo Scientific. Deglycosylation and Glycan Purification. N-Linked oligosaccharides were released from the glycoproteins as follows. The glycoprotein (0.5−1 mg) was dissolved in 145 μL of DI water in a 2 mL microcentrifuge tube. Five microliters of NEB rapid PNGase F buffer (5×) was added to the glycoprotein solution. The solution was thoroughly mixed by vortex before being placed on a heat block at 65 °C for 5 min. After that, the solution was allowed to cool to ambient temperature. Five microliters of 1:2 diluted NEB rapid PNGase F was added to the denatured glycoprotein and incubated at 50 °C for 15 min. After the reaction, the solution was cooled to ambient temperature. After the deglycosylation, the released glycans were purified from the salts and detergents used in the deglycosylation, and

Figure 2. Schematic diagram of the HPAE-PAD system with dual electrolytic eluent generation. C

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the characterization, the MSA−KOH configuration was chosen for the rest of the work. Column Format and Flow Rate Considerations. The separation of oligosaccharides typically requires NaOAc gradient in 100 mM NaOH. Depending on applications, the concentration of NaOAc can go up to as high as 500 mM. In the KMSA/KOH dual electrolytic eluent generation system, it takes an equivalent amount of KOH and MSA to generate KMSA. The total amount of KOH generated is

equivalent performance are now commercially available (the part numbers information is provided in Table S1).

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RESULTS AND DISCUSSION Configuration and Characterization of Dual Electrolytic Eluent Generation. There are two basic ways to mix the electrolytically generated MSA and KOH to produce KMSA and KOH: (1) connecting the two EGs in parallel and mixing the two flows passing through the EGs with a tee to generate the product eluent; (2) connecting the two EGs in series so that the reaction takes place in the second EG. In arrangement (1), if one pump is employed, the flow needs to be equally split into two flows of equal flow rate with one tee, and then the two flows need to be merged with another tee to ensure that the reaction between MSA and KOH is precisely controlled in a quantitative manner. The challenge lies in the difficulty to maintain the equal resistance on the downstream side and the design of the tee to precisely split the flow. The alternative is to employ two pumps to carry the two flows at the equal flow rate through the two cartridges followed by a tee to mix the two flows. The drawback is the added cost and complexity due to the second pump. In arrangement (2), it requires only one pump, therefore simplifying the system setup. In arrangement (2), in order to decide whether to place the MSA EG cartridge on the upstream side of the KOH EG cartridge or vice versa, the KMSA/KOH concentration gradients generated under both configurations were examined. The test was conducted using both analytical and capillary IC systems, where an MSA EG cartridge and a KOH EG cartridge were connected in series either in the MSA−KOH configuration (the MSA EG placed upstream of the KOH EG) or KOH−MSA configuration (the KOH EG placed upstream of the MEA EG). In those experiments, only one EG degas cartridge placed after the second EG cartridge was used. At the outlet of the second EG degas cartridge, a piece of 0.001 in. i.d. PEEK tubing was used to create the backpressure of above 2000 psi. The degassed effluent was directed into a conductivity detector, which monitored the conductivity of the eluent and therefore indicated the concentration change in the KMSA/KOH gradient. During the tests, the difference between the concentrations of the two cartridges ([KOH] − [MSA]) was kept constant at 20 mM throughout the entire gradient program. The test result shows that the delay of concentration change is consistently longer with the KOH−MSA than with the MSA−KOH configuration. The delay time of the concentration change is about 3 min for the MSA−KOH configuration and about 4 min for the KOH−MSA configuration, during the ascending (20 mM change) and descending (40 mM change) step changes. The response time of the increasing slope gradient of 30 mM change over 7 min is 3.5 and 4.4 min for the MSA− KOH configuration and the KOH−MSA configuration, respectively. The descending concentration step change of 30 mM is shown to be 3 min for the MSA−KOH configuration and 4 min for the KOH−MSA configuration, respectively. Similar results were obtained using the Dionex capillary EG cartridges (data not shown). More detailed interpretation of the characterization (including Figure S2) can be found in the Supporting Information. The overlays of conductivity traces from seven consecutive runs show excellent reproducibility of eluent generation (Figure S3). On the basis of the results from

[KOH]total_generated = [KOH]set = [KMSA]product + [KOH]product (3) −

Considering that methanesulfonate (MSA ) is a stronger competing ion than acetate ion (OAc−) (as demonstrated in Figure S4), we designed prototype KOH and MSA EG cartridges capable of generating up to 200 mM KOH and 200 mM MSA, respectively, at a flow rate of 0.063 mL/min for separations which are performed using 1 mm i.d. anionexchange columns. For the capillary-scale separation at 10 μL/min, the existing commercially available capillary KOH and MSA EG cartridges have the capacity to generate KOH and MSA at concentrations up to 200 mM. They were therefore used to generate the eluents for separations using 0.4 mm i.d. capillary columns without modification. Comparison of Performance between Manually Prepared NaOAc/NaOH Eluent and Electrolytically Generated KMSA/KOH Eluent. Two representative separations were chosen to demonstrate the comparable performance generated using the manually prepared NaOAc/NaOH eluent and the electrolytically generated KMSA/KOH eluent. Sialylated fetuin N-linked alditols are the alditol form of sialylated complex type N-linked oligosaccharides from a mammalian plasma glycoprotein. In Figure 3A, the eluent is manually prepared 20−150 mM NaOAc gradient in 100 mM NaOH over 60 min, followed by 5 min of column wash to remove potential sample matrix contamination. In Figure 3B,

Figure 3. Gradient separation of 50 μM sialylated fetuin N-linked alditols. (A) Manually prepared NaOAc/NaOH eluent. Gradient: 20 mM NaOAc/100 mM NaOH to 150 mM NaOAc/100 mM NaOH during 0−60 min, 500 mM NaOAc/100 mM NaOH during 60−65 min, re-equilibration during 65−80 min. Flow rate: 0.5 mL/min. Injection volume: 5 μL. (B) Electrolytically generated KMSA/KOH eluent. Overlay of four consecutive injections. Gradient: 15 mM KMSA/136 mM KOH to 64 mM KMSA/136 mM KOH during 0− 50 min, 80 mM KMSA/90 mM KOH during 50−60 min, 100 mM KMSA/100 mM KOH during 60−65 min, re-equilibration during 65−80 min. Flow rate: 0.063 mL/min. Injection volume: 0.4 μL. D

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Analytical Chemistry the eluent is electrolytically generated 15−64 mM KMSA gradient in 136 mM KOH over 50 min, followed by 10 min of isocratic elution of 64 mM KMSA/90 mM KOH, and 5 min of column wash. During the optimization of the separation condition, it was found that higher concentration of KOH with KMSA/KOH eluent was required to obtain comparable resolution obtained with NaOAc/NaOH eluent. As the eluting strength of MSA− ion is stronger than that of OAc− ion, an increased pH as a result of the increased OH− concentration favors the ionization and the retention of the glycan anions. The combination of the stronger eluent strength and the increasing retention leads to the resolution comparable to that using the NaOAc/NaOH eluent. During 50−60 min, the [KOH]product is decreased while the [KMSA]product is increased to facilitate the elution of strongly retained glycans. Despite a slight difference in retention times, the chromatograms in Figure 3, parts A and B, show excellent resolution of the major sialylated glycans, and also similar resolution of the minor peaks between the trisialyated and the tetrasialyated glycans. Figure 3B shows overlays of four consecutive injections. The relative standard deviations (RSDs) of the retention time and peak area for peak 1 are 0.09% and 0.82%, respectively, for peak 2, 0.05% and 0.61%, respectively, and for peak 3, 0.04% and 0.72%, respectively. Inulin is a prebiotic dietary fiber. Figure S5 shows chromatograms of inulin (overlays of four consecutive injections) using the manually prepared NaOAc/NaOH eluent and the electrolytically generated KMSA/KOH eluent. The RSDs (n = 4) of peak retention time at around 10, 20, and 30 min are within 0.39% and 0.16% for NaOAc/NaOH eluent and KMSA/KOH eluent, respectively. Despite a slight different selectivity of the minor peaks, the entire profiles of inulin obtained using the two methods are very similar, both showing 119 major peaks. The retention time and peak area data are summarized in Table S2. Other Applications and Reproducibility. Galactooligosaccharides (GOS) are another important prebiotic. According to previous work on characterization of GOS,24 GOS samples typically contain high amounts of glucose, galactose, and lactose, and complex structures of oligosaccharides with increasing degrees of polymerization (DP). In this work, the separation of the entire GOS profile was achieved using a linear gradient of 20−70 mM KMSA in constant 70 mM KOH over 45 min. The profile shows that the distribution of peak response is skewed toward the smaller carbohydrates, DP1−DP3, which are a mixture of glucose, galactose, lactose, and maltotriose. To visualize the peaks of increasing DPs up to DP13, the enlarged section of the Bimuno GOS chromatograms is shown in Figure 4A. After the separation, a 10 min wash of 100 mM KMSA/100 mM KOH was applied to remove the contaminants which were present in the samples. The retention time RSDs of DP4, DP6, DP8, DP10, and DP12 from six consecutive injections are within 0.10% (Table S3). Figure S6 shows an equivalent performance using the manually prepared NaOAc/NaOH eluent and the electrolytically generated KMSA/KOH eluent. AGP is an important example of a naturally occurring Nlinked plasma glycoprotein. Using 5−42 mM KMSA gradient in a constant 135 mM KOH eluent, the human AGP glycans were eluted based on their increasing negative charge, in the order of increasing retention time. Within each charge group, structures are further separated on the basis of size and sequence of monosaccharides which form the chain and the

Figure 4. Overlaid HPAE-PAD chromatograms obtained using electrolyically generated KMSA/KOH eluent in analytical mode: (A) oligosaccharides in 25 mg/L Bimuno GOS (n = 6); (B) glycans released from human AGP (n = 3); (C) glycans released from human IgG (n = 3). The peaks denoted with asterisks are artifact peaks. A water blank chromatogram in Figure S7 shows the artifact peaks associated with the gradient.

linkages between them. The structures with the same monosaccharides units but differing in one linkage are separated with good resolution. Figure 4B shows the overlay of three chromatograms. The RSDs of the retention time of the five labeled peaks are within 0.09% (Table S4). The reader is referred to a comparable separation of a similar sample using manually prepared eluent.25 Recombinant mAbs represent the largest group of therapeutic proteins as a major new class of drugs. So far all approved therapeutic mAbs are immunoglobulins G (IgGs).26 Using the dual electrolytic eluent generation platform, the uncharged glycans are separated using a shallow KMSA gradient from 0.3 to 1 mM in a constant 65 mM KOH. From 24 to 43 min, a simultaneous linear KMSA gradient from 1 to 28 mM and linear KOH gradient from 65 to 90 mM is introduced to separate the charged glycans. The pH change caused by the KOH gradient results in a very slight increase of baseline. The observed rise of the baseline during 35−55 min was found to be associated with the sample matrix rather than eluent generation (an overlay of the sample and water blank is shown in Figure S7). The monosialylated glycans are eluted in the window of 40−45 min. With the KOH concentration held at 90 mM, the KMSA concentration is raised to 42 mM to elute the disialylated glycans in the 50−55 min window. During 55−60 min, 100 mM KMSA/100 mM KOH is used to wash the column before the eluent concentration is set to initial condition for 15 min prior to next injection. The glycan profile (three overlaid chromatograms) shown in Figure 4C matches the separation in previously published work.16 The E

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CONCLUSION In summary, the dual electrolytic eluent generation platform provides a unique and convenient way to generate high-purity KMSA/KOH eluent for the separation of oligosaccharides. The performance is demonstrated to be comparable to that using the conventional manually prepared NaOAc/NaOH eluent. It provides ease of use and simplified operation while providing consistent performance that potentially enhances lab-to-lab reproducibility. Where the eluent concentration and gradient conditions need to be optimized frequently for each different type of sample, this automated eluent generation significantly reduces the time required for method development. Since the platform requires only one isocratic pump exposed only to deionized water, it increases the lifetime of pump seals and piston, and it also enables the capillary separation of oligosaccharides.

retention time RSDs of the neutral and sialyated glycans are within 0.16% (Table S5). The dual electrolytic eluent generation platform enables the separation of oligosaccharides to be performed using HPAEPAD in the capillary mode. Parts A−C of Figure 5 show the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b02436. Description of operational principle of the KOH−MSA configuration, part numbers of the commercially available devices which provide equivalent performance as the prototypes used in this work, interpretation of characterization, demonstration of eluting strength of competing ions, additional example of performance comparison between the two eluent systems, retention time and peak area data, overlay of blank and human IgG chromatograms, and long-term retention time stability data (PDF)

Figure 5. Overlaid HPAE-PAD chromatograms obtained using electrolyically generated KMSA/KOH eluent in capillary mode: (A) 5 mg/mL inulin (n = 7); (B) glycans released from human AGP (n = 3); (C) glycans released from human IgG (n = 5). Peaks denoted with asterisks are artifact peaks associated with the gradient.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 408-481-4173. E-mail: yongjing.chen@thermofisher. com.

separation of inulin, glycans from human AGP, and glycans from human IgG, respectively. The chromatograms show profiles comparable to that on the analytical mode with excellent reproducibility. Note that, for the same sample, the concentration of the gradients used for the separation on the capillary column is slightly different from that on the analytical column. This is likely due to the slight variation in system delay volume (the delay between the two cartridges in particular) and column capacity between the analytical and capillary systems. Long-Term Stability. The stability of the system performance was evaluated through monitoring the separation of GOS over a month. After running the GOS sample on the first day, the system was dedicated to other applications and tests until day 20, when the test of GOS separation was resumed. The separation of GOS was run about every other day to monitor the retention stability of DPs. The loss of retention on day 42 over day 1 was found to be 5.1%/41 days (3.6%/month) for DP5, 4.5%/41 days (3.2%/month) for DP6, and 3.6%/41 days (2.6%/month) for DP7, respectively. A plot showing the trend of retention times is shown in Figure S8. The result indicated reasonable long-term stability of column performance with the use of dual electrolytic eluent generation.

ORCID

Yongjing Chen: 0000-0002-5297-7572 Christopher Pohl: 0000-0003-2529-3201 Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.analchem.8b02436 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.8b02436 Anal. Chem. XXXX, XXX, XXX−XXX