Electrical asymmetrical flow field-flow fractionation (EAF4) is an interesting new analytical technique that separates proteins based on size or molecular weight and simultaneously determines the electrical characteristics of each population.
Journal of Chromatography A 1633 (2020) 461625 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Separation and zeta-potential determination of proteins and their oligomers using electrical asymmetrical flow field-flow fractionation (EAF4) Jaeyeong Choi a,∗, Catalina Fuentes a, Jonas Fransson b, Marie Wahlgren a, Lars Nilsson a a b Department of Food Technology, Engineering and Nutrition, Lund University, 22100 Lund, Sweden Swedish Orphan Biovitrum AB (publ.), 11276 Stockholm, Sweden a r t i c l e i n f o Article history: Received August 2020 Revised 11 October 2020 Accepted 12 October 2020 Available online 14 October 2020 Keywords: Electrical asymmetrical flow field-flow fractionation (EAF4) Electrical characteristics Zeta-potential Effective net charge Proteins Separation a b s t r a c t Electrical asymmetrical flow field-flow fractionation (EAF4) is an interesting new analytical technique that separates proteins based on size or molecular weight and simultaneously determines the electrical characteristics of each population However, until now, the research using EAF4 has not been published except for the proof-of-concept in the original publication by Johann et al in 2015 [1] Hence the methods capabilities and optimized conditions need to be further investigated, such as composition of the carrier liquid, pH stability and effect of the electric field strength The pH instability was observed in the initial method of EAF4 due to the electrolysis products when applied electric field Therefore, we have investigated and provided a modified method for rapid pH stabilization through additional focusing step with the electric field Then, the electrical properties such as the zeta-potential and effective net charge of the monomer and oligomers of three different proteins (GA-Z, BSA, and Ferritin) were determined based on their electrophoretic mobility from EAF4 The results showed that there were limitations to the applicability of separation by EAF4 to proteins Nevertheless, this study shows that EAF4 is an interesting new technique that can examine the zeta-potential of individual proteins in mixtures (or monomers and oligomers) not accessible by other techniques © 2020 The Authors Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Introduction Analytical separation for quantification and characterization of proteins is important for many applications in life science The tasks can involve separation of monomer, oligomer and aggregates of a protein as well as separation of different protein species in a mixture In addition to the amount of different protein species as well as their size and molecular weight (MW), the charge properties of proteins are important as they affect protein characteristics in relation to structure, oligomerization and aggregation The zeta-potential is one of the electrical properties which is commonly determined due to its experimental accessibility, and high importance for protein stability [2,3] The zeta-potential reflects the range over which electrostatic interaction occurs in a solution or dispersion and is related to the surface charge of a pro- ∗ Corresponding author E-mail addresses: feelcjy@gmail.com, jaeyeong.choi@food.lth.se (J Choi), catalina_csfz@hotmail.com (C Fuentes), Jonas.Fransson@sobi.com (J Fransson), marie.wahlgren@food.lth.se (M Wahlgren), lars.nilsson@food.lth.se (L Nilsson) tein and the ionic strength, among others Hence, it can be related to the inter-molecular electrostatic interaction between proteins in solution and, thus, to their physical stability The zeta-potential is commonly determined from the electrophoretic mobility [4] and the most widely utilized method of measuring zeta-potential is phase analysis light scattering (PALS) [5] As a batch-type analysis method, it provides an average value while zeta-potential values for individual components in mixtures or over broad size-range distributions cannot be obtained [1,6] A growing separation technique for proteins is asymmetrical flow field-flow fractionation (AF4) [7,8] which can be coupled online with various detectors AF4 is a sized-based separation technique which, in Brownian mode, will separate analytes according to their diffusion coefficient (i.e hydrodynamic radius) For a more detailed description of the technique, interested readers can find information elsewhere [9–11] Electrical asymmetrical flow field-flow fractionation (EAF4) is a new sub-technique of AF4 first described in 2015 [1] It is a combination of asymmetrical flow field-flow fractionation (AF4) and electrical field-flow fractionation (ElFFF) in a separation channel The combination enables separa- https://doi.org/10.1016/j.chroma.2020.461625 0021-9673/© 2020 The Authors Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) J Choi, C Fuentes, J Fransson et al Journal of Chromatography A 1633 (2020) 461625 channel spacer was a 350 μm thick Mylar spacer and a regenerated cellulose (RC) membrane (molecular weight cut-off of 10 kDa, Millipore, Bedford, USA) constituted the channel accumulation wall The actual channel thickness (w) was determined to be 309 μm from retention time of BSA in 50 mM NaNO3 using the FFFHydRad 2.2 [15] The EAF4 carrier liquid was pumped into the channel using an Agilent 1200 HPLC pump (Agilent Technologies, Waldbronn, Germany) equipped with an auto-sampler The conductivity and pH of the solvent were measured online, after passing the last detector, in flow cells which are part of the Mobility unit The carrier liquids were prepared as 50 mM NaNO3 and 50 mM phosphate buffer at pH 7.0 for BSA, and 25 mM phosphate buffer at pH 7.0 for GA-Z and ferritin experiments All experiments were performed with detector flow rate of 1.0 mL/min and constant cross-flow rate of 4.0 mL/min The channel was rinsed with the carrier liquid for 20 without cross-flow and electric field at the end of each run All EAF4 experiments were performed at room temperature The collection and processing of detector signals was performed using the ASTRA software (Wyatt Technology, Germany), and electrical data processing was performed using VISION CSH (Superon, Germany) tion based on both diffusion coefficient (based on AF4) and, to some extent, the surface charge of analytes (based on ElFFF) These two fields can be applied separately or together in EAF4 and in this study, both of them were used Ideally, EAF4 would provide charge-size dependent separation of samples with different charge or charge density, even with the same size If the charge density is different between sample components, it could potentially improve the resolution between components, in comparison to conventional AF4, due to the utilization of the electric field Another interesting aspect is that the zeta-potential could be determined for multiple components while the size distribution is simultaneously determined [1] A potential disadvantage of EAF4 is the increased number of parameters affecting the result of the separation due to the combination of the two fields applied For example, the pH in the channel can be changed by electrolysis products (i.e OH− or H+ ) from the electrodes when the electric field is applied [1,12] Obviously, such a change in pH can cause changes in the size and structure/conformation of the sample components [13,14] To date no investigation for protein characterization utilizing EAF4 has been published, except for the proof-of-concept in the original publication [1] Hence, the method’s capabilities needs to be investigated In this study, the purpose is to investigate the application of EAF4 to the separation and characterization of proteins The electric field-induced pH change in the separation channel was investigated as changes in pH may have a strong influence on protein properties and should, ideally, be minimized The second aim is to investigate whether the resolution in protein separations can be improved using the electric field The third aim is to use EAF4 for the determination of zeta-potentials of different populations (monomer and oligomers) in a protein 2.3 Theory In EAF4 without electric field (i.e AF4), the retention ratio (R) in Brownian mode is given by the general expression [7] V R= = v t0 = 6λ coth − 2λ tr 2λ (1) where V is the migration velocity of the component zone, is the average longitudinal carrier velocity, t0 is the void time, tr is the retention time, and λ is the retention parameter At the limit λ → 0, Eq (1) can be approximated by [7,16] Materials and methods 2.1 Materials R= Sodium dihydrogen phosphate monohydrate (NaH2 PO4 H2 O), disodium phosphate dihydrate (Na2 HPO4 2H2 O), sodium nitrate (NaNO3 ), bovine serum albumin (BSA), and ferritin (equine spleen) were purchased from Sigma-Aldrich (Darmstadt, Germany) The GA-Z is a recombinant protein including GA-domain (albumin binding site) and Z-domain (target molecule binding site) consisting of 108 amino acids (MW=11.5 kDa, isoelectric point, pI=4.2) and was provided by Swedish Orphan Biovitrum AB (publ.) (Stockholm, Sweden) The carrier liquid for EAF4 and solution for sample preparation was prepared with water purified through a Milli-Q Plus purification system (Millipore Co Ltd., Billerica, USA, resistance=18.2 M /cm) t0 = 6λ tr (2) The retention parameter λ is defined by [7] λ= l D DV = = w |u0 |w Vc w2 (3) where l is the center of gravity distance from the accumulation wall of the sample zone concentration distribution, w is the channel thickness, D is the diffusion coefficient of a specific analyte, u0 is the cross-flow velocity at the accumulation wall surface, V0 is the volume of the channel (void volume), and Vc is the cross-flow rate Substituting Eq (3) into Eq (2) yields w2Vc t 6trV 2.2 Methods D= Electrical asymmetrical flow field-flow fractionation (EAF4) used in this work was an Eclipse 3+ system (Wyatt Technology, Dernbach Germany) connected with a Mobility electric field module included conductivity and pH sensor (Superon GmbH, Dernbach, Germany) The EAF4 system was coupled online with a multi-angle light scattering (MALS) detector (DAWN HELEOS II, Wyatt Technology), Agilent 1100 diode array detector (DAD, Agilent Technologies, Waldbronn, Germany) with wavelength set at 280 nm, and a differential refractive index (dRI) detector (Optilab T-rEX, Wyatt Technology) The EAF4 channel (Superon GmbH) was trapezoidal with a tipto-tip length of 26.5 cm and the inlet and outlet widths of 2.2 and 0.6 cm, respectively The two electrodes are made of platinized stainless steel and were opposed to each other in parallel in the top and bottom block respectively with a distance of 3.7 mm The which yields the relationship between tr and D, for a specific analyte Using the Stokes-Einstein equation the diffusion coefficient can be transformed into the hydrodynamic radius (Rh ) [17] Rh = (4) V kT t π ηt 0Vc r (5) w2 where k is the Boltzmann constant, T is the absolute temperature, π is the ratio of the circumference of a circle to its diameter, and η is the dynamic viscosity of the solvent The void time (t0 ) of the trapezoidal channel, can be calculated by [18] ⎛ ⎛ V0 Vc ⎝1 − t0 = ln ⎝1 + Vc Vout w b0 z − z V0 b −b L 2L −y ⎞⎞ ⎠⎠ (6) J Choi, C Fuentes, J Fransson et al Journal of Chromatography A 1633 (2020) 461625 where Vout is the detector flow rate, b0 and bL are the breadths of the trapezoid at the inlet and outlet respectively, z is the position of the focusing point, y is the area lost from the trapezoid by the tapered inlet and outlet ends, and L is the channel length When injecting a standard sample with a known diffusion coefficient (D) or hydrodynamic radius (Rh ), the actual channel thickness (w) can be determined from the retention time (tr ) of the standard sample by Eqs (4) or (5) The electrophoretic mobility (μ) is defined by [19] μ= vEP respectively The resulting zeta-potentials reported in this paper are based on Eq (13) by using the approximated Henry’s function (Eq (12)) Results and discussions 3.1 Optimization of EAF4 method for pH stability The focusing step of conventional AF4 is a sample relaxation process which is dependent on the external field (cross-flow) and the diffusion coefficient of analytes resulting in a characteristic size-dependent concentration profile in the separation channel prior to elution [23–25] EAF4 has an additional external field i.e the electric field As previously reported, the electric field of EAF4 is applied in the elution mode and is kept constant during the analysis [1] However, electrolysis by-products or components from non-Faradaic processes (e.g., ions adsorbed or accumulated on electrodes) can potentially appear when an electrical current is applied, which, in turn, can cause pH-changes in the solution [26] For example, when a negative electric field is applied, the bottom and top electrodes are the anode and cathode, respectively Conversely, the opposite applies when a positive electric field is applied The electrolysis products such as OH− or H+ at the top electrode (cathode or anode) will have a larger effect on channel pH than electrolysis products from the bottom electrode as it is located under the accumulation wall membrane and its supporting frit As a result, the channel pH can be shifted which makes the use of a buffer in the carrier liquid crucial (It should be noted that using a buffer for conventional AF4-separations should, in any case, be the rule of thumb in order to have a defined pH and ionic strength) Hence, pH-changes occurring in EAF4 will also be dependent on the buffering capacity of the buffer Fig (a) and (b) show the pH vs elution time observed at various electric fields in two types of carrier liquid (50 mM NaNO3 or 50 mM phosphate buffer at pH 7.0) during an EAF4 run The pH changed rapidly during the initial after which the pH leveled off in both carrier liquids In addition, the range of changes in pH was larger in the non-buffered carrier liquid (50 mM NaNO3 ) compared to the 50 mM phosphate buffer Moreover, in Fig 1(a) and (b), the pH did not always return to the initial pH value even after 20 of channel flushing, indicating that longer channel conditioning times may be needed between runs Fig 1(c) shows the BSA fractograms and pH of repeated runs and it can be seen that the void peak of the first experiment is relatively large compared to the second experiment This is probably due to the more extensive pH-change during the first experiment using the non-buffered carrier liquid, which gave rise to changes in carrier liquid composition Very slight changes in the retention times of BSA sub-populations were observed, which could be due to the differences in pH-change Based on these results, the reproducibility in the obtained pH was investigated by changing the point where the electric field is switched on by adding an additional focusing step i.e between the initial focusing step and elution mode (Fig 2a and b) It was expected that the modified method could stabilize the pH faster compared to the initial method as the electrolysis products are already generated during the additional focusing step, rather than starting to be formed at the onset of elution This would allow for some time to equilibrate the concentration of electrolysis products and, hence, to stabilize pH The result showed that the pH stabilized faster and in more reproducible manner when using the modified method, as shown in Fig 2(c) for the NaNO3 carrier liquid and 2(d) for the phosphate buffer carrier liquid As would be expected, the effects were more pronounced for the non-buffered (NaNO3 ) carrier liquid but the re- (7) E where vEP is the drift velocity due to the electric field, and E is the electric field strength In EAF4 vEP can be calculated by [1] t ln 1+ Vf Vc vEP = v − vc = e ri out /tr − 1+ f Vc Vout Vout Ael f (8) where v is total drift velocity, vc is the drift velocity caused by the cross-flow without electric field, tri is the retention time with electric field, tr is the retention time without electric field, f is the ratio between the actual channel separation area (i.e downstream from the focusing point) and the total channel area, Vout is the detector flow rate, and Ael is the electrode area in the channel, which is identical to the total channel area The electric field strength (E) can be obtained by [1] E= I Ael kc (9) where, I is the electrical current, and kc is the specific conductivity of the carrier liquid Therefore, the electrophoretic mobility (μ) from EAF4 can be calculated through Eq (7) from at least two experiments measuring the retention times with and without the electric field The effective net charge (Z) of an analyte is defined by [20] Z= μ6 π η R h ( + κ R h ) e f ( κ Rh ) (10) where, e is the elementary charge, f(κ Rh ) is Henry’s function, and κ is the inverse of the Debye-Hückel length that is defined by 2e2 NA Ic kT κ= (11) where NA is the Avogadro number, Ic is the ionic strength, is the relative dielectric constant of the solvent, and is the permittivity of vacuum In EAF4, Henry’s function (f(κ Rh )) assumes a relatively simple empirical approximation described as [21] f ( κ Rh ) = 16 + 18κ Rh + 3(κ Rh )2 16 + 18κ Rh + 2(κ Rh )2 (12) The zeta-potential (ζ ) can be derived from the electrophoretic mobility (μ) by [21] ζ= 3ημ f ( κ Rh ) (13) In the limit of small analytes in relation to the DebyeHückel length, κ Rh 1, f(κ Rh ) approaches and Eq (13) reduces to the Hückel equation In the limit of large analytes in relation to the Debye-Hückel length, κ Rh 1, f(κ Rh ) approaches 1.5 and Eq (13) reduces to the Helmholtz-Smoluchowski equation Eq (13) is valid for determinations of zeta-potentials ≤|50 mV| [22] Finally, if the hydrodynamic radius (Rh ) and the electrophoretic mobility (μ) is determined from EAF4, the effective net charge (Z) and the zeta-potential (ζ ) can be calculated by Eqs (10) and (13), J Choi, C Fuentes, J Fransson et al Journal of Chromatography A 1633 (2020) 461625 Fig pH vs elution time at various electrical currents (-20 to 20 mA) in two types of carrier liquids (a) 50 mM NaNO3 , (b) 50 mM phosphate buffer at pH 7.0, and (c) BSA fractograms and pH in duplicate (in 50 mM NaNO3 ) producibility in the obtained pH was also improved for the phosphate carrier liquid when using the modified method It is clear that buffers should be employed to minimize the change in pH and subsequent effects on the sample as well as for reproducibility in pH between runs Thus, a phosphate buffer was utilized for further experiments in our study bly due to attraction between the membrane surface and analytes caused by the anode at the bottom electrode In addition, a decreased peak area was observed when the membrane was positively charged, most clearly observed at -15 mA and -20 mA electrical currents (Fig 3), which is likely to be caused by sample adsorption resulting from the opposite charge of GA-Z and the bottom electrode (anode) Accordingly, analyses should be carried out carefully to avoid sample adsorption when applying higher electrical currents in the case that the sample and the bottom electrode are oppositely charged In Brownian mode AF4 separation, the GA-Z monomer will elute before the dimer, following AF4 theory [7] In order to obtain somewhat more accurate data for the monomer and dimer, data points were taken at 10% peak height at the front as well as the tail of the peak The MW of 10% height at the front and tail were determined as 13 kDa and 25 kDa, respectively, which shows that these fractions were mainly composed of monomer (theoretical MW=11.5 kDa) and dimer (theoretical MW=23 kDa), respectively These points were, thus, used to determine the electrophoretic mobility and zeta-potential of GA-Z monomer and dimer (Table 1) The fractograms of GA-Z at -15 mA and -20 mA were excluded from the calculation of electrophoretic mobility as the retention times were the same for -10 mA to -15 mA and GA-Z was not eluted at -20 mA Most likely, strong interaction was present already at -15 mA giving rise to deviations in the relationship between drift velocity and electric field strength The results showed that the zeta-potential was -11.2 mV of the 10% height at peak front (mostly monomer) and -7.7 mV for the 10% height at peak tail (mostly dimer) of GA-Z The lower magnitude of the dimer zeta-potential could possibly be explained by that the dimer was formed through association of the Z-domains [27] The Z-domain contains a higher number of charged amino 3.2 Separation and characterization of proteins by EAF4 In a previous study it was shown that GA-Z has a fast equilibrium between monomer and dimer forms [27] Additionally, the dimer is the dominant species at pH 7.0 (approximately 82% dimer determined by small angle X-ray scattering, SAXS), and the monomer and dimer of GA-Z cannot be resolved by either AF4 or size exclusion chromatography (SEC) Therefore, it was investigated if EAF4 could increase the resolution between the monomer and dimer of GA-Z compared to conventional AF4 Fig shows the fractograms of GA-Z with different electrical currents (-15 mA to 20 mA) in 25 mM phosphate buffer at pH 7.0 The results showed that the resolution was not improved when applying the electric fields and only electric field-dependent shifts in the retention time of GA-Z was observed In the positive electric field, the retention time of GA-Z decreased from approximately 5.8 to 5.5 which could be due to an increased electrostatic repulsion between the membrane surface and the analytes (GAZ pI=4.2), caused by the increased negative surface charge of the membrane with increasing positive electrical current Moreover, no significant difference in the retention time of the peak maxima between 10 mA and 15 mA were observed For this reason, positive electrical current higher than 15 mA was not used Contrariwise, with the negative electrical current, the retention time of GA-Z increased from 5.8 to 6.2 min, which is proba4 J Choi, C Fuentes, J Fransson et al Journal of Chromatography A 1633 (2020) 461625 Fig Separation methods for EAF4 (a) initial method, (b) modified method pH vs elution time with electrical current at -10 mA for two types of carrier liquids (c) in 50 mM NaNO3 , and (d) in 50 mM phosphate buffer at pH 7.0, performing two runs using both the initial and modified method respectively Table Electrical properties of GA-Z, BSA, and Ferritin from EAF4 From EAF4 Electrophoretic mobility1 μ (μmcm/(Vs)) Proteins GA-Z2 BSA3 Ferritin3 10% height at front Peak maxima 10% height at tail Monomer Dimer Trimer Monomer Dimer -0.614 -0.577 -0.420 -0.164 -0.159 -0.229 -0.383 -0.280 ± ± ± ± ± ± ± ± 0.120 0.120 0.075 0.012 0.002 0.044 0.044 0.009 Theoretical Zeta-potential (mV) Effective net charge -11.2 ± 2.1 -10.5 ± 2.2 -7.7 ± 1.4 -3.2 ± 0.2 -3.1 ± 0.1 -4.4 ± 0.9 -6.9 ± 0.8 -5.0 ± 0.2 -1.62 -1.70 -1.76 -0.77 -1.08 -1.88 -3.01 -3.50 ± ± ± ± ± ± ± ± 0.31 0.35 0.31 0.01 0.02 0.36 0.35 0.11 Net charge at pH 7.0 -10 -16 ~ 18 [29,30] - The values include the standard error of the mean (±) based on two replicates The theoretical net charge of GA-Z at pH 7.0 was calculated based on the sequence [31] The values of electrical properties from EAF4 were calculated at peak maxima of monomer and oligomers acids compared to the GA-domain At pH 7.0, the Z-domain and the GA-domain have 14 and negatively charged amino acids, respectively Thus, it is possible that the lower magnitude of the zeta-potential for the dimer is due to that the negatively charged amino acids of the Z-domain were shielded to a larger extent The zeta-potential of the peak maximum (i.e a mixture of dimer and monomer) was determined as -10.5 mV Fig shows the fractograms of BSA and Ferritin with different electrical currents The retention times of BSA and Ferritin were decreased with positive electric field, and increased with negative electric field No change in resolution between monomers and oligomers was clearly observed when the electric field was applied Similarly, as for GA-Z mentioned, repulsion or attraction between the analytes and the accumulation wall membrane is likely the cause for the change in retention time as the pH of carrier liquid was above pI of both proteins (Ferritin pI=4.1-5.1 [31] and BSA pI=4.5–5.5 [32]) The MW of monomer and dimer of BSA without electric field (0 mA) were determined as 66.2 kDa and 130.8 kDa, respectively However, the MW determined for the monomer and dimer of BSA were different depending on the electric field applied For example, the MW decreased when negative field was applied, and increased when the positive field was applied (Table 2) Probably, the differences observed in the determination of MW was due to the elec5 J Choi, C Fuentes, J Fransson et al Journal of Chromatography A 1633 (2020) 461625 tric field-dependent change in pH which would slightly affect the dn/dc value of BSA as the solvent composition was changed Hence, the utilization of the electric field introduces an uncertainty for the determination of MW The zeta-potentials and electrophoretic mobility of monomers and oligomers of BSA and Ferritin were determined from peak maxima for the respective species and are shown in Table The zeta-potentials and electrophoretic mobility of BSA were determined as -3.2 mV and -0.164 μmcm/V−1 s−1 for monomer, -3.1 mV and -0.159 μmcm/V−1 s−1 for dimer, and -4.4 mV and -0.229 μmcm/V−1 s−1 for trimer, respectively The determined zeta-potentials of the monomer and dimer of BSA were similar, while that of the trimer was slightly higher The slightly higher zeta-potential for the trimer is likely to be related to the trimer structural properties but it is difficult to draw any conclusion regarding this observation The electrophoretic mobility for monomer and dimer of BSA was previously reported as -2.66 μmcm/V−1 s−1 and -3.77 μmcm/V−1 s−1 by EAF4 [1], which is approximately 15 times higher than the values determined in this study The 50 mM phosphate buffer at pH 7.0 for carrier liquid used in our study was closer to the pI of BSA than the carrier liquid used in a previous study (10 mM phosphate buffer at pH 8.0) [1], which could result in lower electrophoretic mobility Another reason for the lower electrophoretic mobility in our study is that the high conductivity (ionic strength) of the carrier liquid decreased the electric field strength at a given electrical current (Eq (9)) In our results, the retention time shift between runs in absence or presence of the electric field was lower than the previously reported results, (i.e lower vEP , Eq (8)) Therefore, it is presumed that we obtained lower electrophoretic mobility (Eq (7)) The zeta-potentials of Ferritin were determined to be -6.9 mV for the monomer and -5.0 mV for the dimer, respectively Similar to GA-Z, it is reasonable to suspect that the lower zeta-potential of the dimer is related to shielding of charges when in the dimeric form The theoretical net charge of GA-Z and BSA at pH 7.0 are 10 and -16 ~ -18, respectively [28,29] However, the effective net charge determined from EAF4 was much lower than the theoretical values (see Table 1) The deviation probably, in part, arose from the low electrophoretic mobility resulting in a low effective net charge (Eq (10)) Nevertheless, the approach can be advantageous when used for relative comparison between analytes rather than as absolute value It is important to emphasize that zeta-potential and surface charge reflects considerably different properties The zeta-potential is dependent on the charge at the surface but is influenced by several other parameters This means that the zeta-potential will be strongly dependent on the ionic strength of the surrounding solution as it will influence the Debye-Hückel length (Eq (11)) as well as, for instance, pH Thus, the zeta-potential will decrease strongly with increasing ionic strength and experimental determination of the zeta-potential becomes sensitive to already small differences in the ionic strength It should be noted that results with similar trend were observed for the analyzed samples (GA-Z, BSA, and Ferritin) i.e when positive current was applied, the retention time of the samples decreased This is expected because the samples (pI range 4.1 to 5.5) were negatively charged under these running conditions (i.e close to physiological pH) The negative charge of the cathode at the bottom electrode gave rise to repulsion between the sample and the surface of the membrane resulting in shorter retention time Conversely, it is expected that the retention time and separation behavior increase when negative electric filed was applied It could be thought that increment of the electrical current (higher electric field) would allow for higher resolution Nevertheless, this did not Fig Fractograms of GA-Z with different electrical currents in 25 mM phosphate buffer at pH 7.0 Fig EAF4 fractograms with different electrical currents (a) BSA in 50 mM phosphate buffer at pH 7.0, and (b) Ferritin in 25 mM phosphate buffer at pH 7.0 Table The molecular weight (MW) of the monomer and dimer of BSA determined from EAF4-MALS (using dn/dc=0.185 mL/g) Electrical current (mA) Molecular weight of BSA at peak maxima Monomer (kDa) Dimer (kDa) -20 -10 10 20 63.3 64.2 66.2 66.3 67.8 121.7 125.3 130.8 131.8 141.5 J Choi, C Fuentes, J Fransson et al Journal of Chromatography A 1633 (2020) 461625 occur during the analysis of the samples The elution behavior did not show significant improvements when a negative current is applied (see Figs and 4) and at excessive electrical current, adsorption or immobilization to the accumulation wall membrane could instead be observed An underlying limitation for the applicability of EAF4 to proteins seems to be the relatively narrow window for EAF4 method parameters This applies both for higher ionic strengths (approx.>50 mM) which will quench the electric field and limits the investigation of therapeutic proteins in formulation or proteins under physiological conditions as well as for higher electric fields as outlined above Additionally, as the electrolyte concentration increases at higher ionic strengths, the value of zeta-potential falls due to the shielding effect of the increased concentration of counter ions which causes a strong decay in the electric potential arising from analyte charges editing, Project administration, Supervision Lars Nilsson: Conceptualization, Writing - review & editing, Supervision, Project administration Acknowledgments The research in this study was performed with financial support from Vinnova-Swedish Governmental Agency for Innovation Systems and the Swedish Research Council within the NextBioForm Competence Centre (grant number 2018-04730) SOLVE Research & Consultancy AB, Lund, Sweden is gratefully acknowledged for providing access to the EAF4 instrumnet Swedish Orphan Biovitrum AB, Stockholm, Sweden are acknowledged for providing the GA-Z protein and information regarding the protein 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proteins such as ionic strength and buffer composition Another challenge is of course that many proteins are relatively small and have relatively low number of charges which limits the effect of the electric field It is likely that the method would be more suitable for application to larger or highly charged analytes such as large proteins, polyelectrolytes and charged nanoparticles Nevertheless, the results show that EAF4 made it possible to determine differences in zeta-potential between monomers and oligomers This shows that EAF4 is an interesting technique for probing zeta-potentials in protein mixtures (or oligomer mixtures) yielding valuable information which is otherwise not accessible by other techniques It could also be a possibility to apply EAF4 to research questions where protein charge properties are changed as a result of binding or interacting with other molecules Contributor roles taxonomy (credit) The contribution of each author who have participated in “Separation and zeta-potential determination of proteins and their oligomers using electrical asymmetrical flow field-flow fractionation (EAF4)” based on CRediT is shown in the table below 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 Jaeyeong Choi: Investigation, Validation, Writing - original draft, Writing - review & editing, Visualization Catalina Fuentes: Validation, Writing - review & editing Jonas Fransson: Resources, Writing - review & editing Marie Wahlgren: Writing - review & J Choi, C Fuentes, J Fransson et al Journal of Chromatography A 1633 (2020) 461625 ´ ˙ M Trojanowicz, A Wilk, P Garstecki, R Hołyst, Net [20] J Szymanski, E Pobozy, charge and electrophoretic mobility of lysozyme charge ladders in solutions of nonionic surfactant, J Phys Chem B 111 (19) (2007) 5503–5510 https://doi 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Eisenberg (Eds.), Advances in Protein Chemistry, 45, Academic Press, 1994, pp 153–203 https://doi.org/10.1016/S0065-3233(08) 60640-3 [30] L.P Kozlowski, IPC – isoelectric point calculator, Biol Direct 11 (1) (2016) 55 https://doi.org/10.1186/s13062- 016- 0159- [31] P Arosio, T.G Adelman, J.W Drysdale, On ferritin heterogeneity Further evidence for heteropolymers, J Biol Chem 253 (12) (1978) 4451–4458 PMID 659425 [32] R Kun, M Szekeres, I Dékány, Isothermal titration calorimetric studies of the pH induced conformational changes of bovine serum albumin, J Therm Anal Calorim 96 (3) (2009) 1009–1017 https://doi.org/10.1007/s10973- 009- 0040- ... mobility of monomers and oligomers of BSA and Ferritin were determined from peak maxima for the respective species and are shown in Table The zeta-potentials and electrophoretic mobility of BSA... determination of proteins and their oligomers using electrical asymmetrical flow field-flow fractionation (EAF4)? ?? based on CRediT is shown in the table below Declaration of Competing Interest The... result of binding or interacting with other molecules Contributor roles taxonomy (credit) The contribution of each author who have participated in ? ?Separation and zeta-potential determination of proteins