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.
Trang 1Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/chroma
(EAF4)
Jaeyeong Choia, ∗, Catalina Fuentesa, Jonas Franssonb, Marie Wahlgrena, Lars Nilssona
a Department of Food Technology, Engineering and Nutrition, Lund University, 22100 Lund, Sweden
b Swedish Orphan Biovitrum AB (publ.), 11276 Stockholm, Sweden
a r t i c l e i n f o
Article history:
Received 1 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
Electricalasymmetricalflowfield-flowfractionation(EAF4)isaninterestingnewanalyticaltechniquethat separatesproteinsbasedonsizeormolecularweightandsimultaneouslydeterminestheelectrical char-acteristicsofeachpopulation.However,untilnow,theresearchusingEAF4hasnotbeenpublishedexcept fortheproof-of-conceptintheoriginalpublicationbyJohannet.al.in2015 [1].Hencethemethods ca-pabilitiesandoptimizedconditions needtobefurtherinvestigated,suchas compositionofthecarrier liquid,pHstabilityandeffectoftheelectricfieldstrength
The pH instabilitywas observedinthe initial methodofEAF4duetothe electrolysisproductswhen appliedelectricfield.Therefore,wehaveinvestigatedandprovidedamodifiedmethodforrapidpH sta-bilizationthroughadditionalfocusingstepwiththeelectricfield.Then,theelectricalpropertiessuchas the zeta-potential and effectivenet chargeofthe monomerand oligomers ofthree differentproteins (GA-Z,BSA,andFerritin)weredeterminedbasedontheirelectrophoreticmobilityfromEAF4.Theresults showedthattherewerelimitationstotheapplicabilityofseparationbyEAF4toproteins.Nevertheless, thisstudyshowsthatEAF4isaninterestingnewtechniquethatcanexaminethezeta-potentialof indi-vidualproteinsinmixtures(ormonomersandoligomers)notaccessiblebyothertechniques
© 2020TheAuthors.PublishedbyElsevierB.V ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/)
1 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 prop-
erties of proteins are important as they affect protein characteris-
tics in relation to structure, oligomerization and aggregation The
zeta-potential is one of the electrical properties which is com-
monly 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 solu-
tion 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 analy- sis method, it provides an average value while zeta-potential val- ues 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 on- line with various detectors AF4 is a sized-based separation tech- nique which, in Brownian mode, will separate analytes accord- ing 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/ )
Trang 2tion 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 im-
prove the resolution between components, in comparison to con-
ventional 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 simultane-
ously determined [1] A potential disadvantage of EAF4 is the in-
creased number of parameters affecting the result of the separa-
tion 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 ap-
plication 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 pro-
tein 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 Materials and methods
2.1 Materials
Sodium dihydrogen phosphate monohydrate (NaH 2PO 4H2O),
disodium phosphate dihydrate (Na 2HPO 42H2O), sodium nitrate
(NaNO 3), 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) consist-
ing of 108 amino acids (MW =11.5 kDa, isoelectric point, pI =4.2)
and was provided by Swedish Orphan Biovitrum AB (publ.) (Stock-
holm, Sweden) The carrier liquid for EAF4 and solution for sam-
ple preparation was prepared with water purified through a
Milli-Q Plus purification system (Millipore Co Ltd., Billerica, USA,
resistance = 18.2 M /cm)
2.2 Methods
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 mod-
ule included conductivity and pH sensor (Superon GmbH, Dern-
bach, 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, Ag-
ilent 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 tip-
to-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
channel spacer was a 350 μm thick Mylar spacer and a regen- erated 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 NaNO 3using the FFFHy- dRad 2.2 [15] The EAF4 carrier liquid was pumped into the chan- nel using an Agilent 1200 HPLC pump (Agilent Technologies, Wald- bronn, 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 car- rier liquids were prepared as 50 mM NaNO 3and 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 min 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 elec- trical data processing was performed using VISION CSH (Superon, Germany)
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]
R = V
v =
t0
t r = 6 λ coth 1
2 λ− 2 λ
(1) where V is the migration velocity of the component zone, < v > is the average longitudinal carrier velocity, t0 is the void time, t r is the retention time, and λis the retention parameter
At the limit λ→ 0, Eq.(1)can be approximated by [7,16]
R = t0
The retention parameter λis defined by [7]
λ= l
w= D
|u0|w= D V0
where l is the center of gravity distance from the accumulation wall of the sample zone concentration distribution, w is the chan- nel 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 V cis the cross-flow rate
Substituting Eq.(3)into Eq.(2)yields
D= w2V c t0
which yields the relationship between t r and D, for a specific an- alyte Using the Stokes-Einstein equation the diffusion coefficient can be transformed into the hydrodynamic radius ( R h) [17]
R h= V0kT
w2πηt0V c
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
ηis the dynamic viscosity of the solvent
The void time ( t0) of the trapezoidal channel, can be calculated
by [18]
t0= V0
V c
ln
⎛
⎝1 + V c
V out
⎛
⎝1 −
w
b0z−
z2b
0−b L
2L − y
V0
⎞
⎠
⎞
Trang 3where V out is the detector flow rate, b0 and b L are the breadths of
the trapezoid at the inlet and outlet respectively, 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 co-
efficient ( D) or hydrodynamic radius ( R h), the actual channel thick-
ness ( w) can be determined from the retention time ( t r) of the
standard sample by Eqs.(4)or (5)
The electrophoretic mobility ( μ) is defined by [19]
μ= vEP
where v EPis the drift velocity due to the electric field, and E is the
electric field strength
In EAF4 v EPcan be calculated by [1]
vEP = v−vc = e t riln
1 +f V c
V out
/ t r
− 1 + f V c
V out
V out
A el f (8)
where v is total drift velocity, v cis the drift velocity caused by the
cross-flow without electric field, t riis the retention time with elec-
tric field, t ris 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, V out is the detec-
tor flow rate, and A elis 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
A el k c
(9) where, I is the electrical current, and k cis 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 measur-
ing the retention times with and without the electric field
The effective net charge ( Z) of an analyte is defined by [20]
Z = μ6 πηR h
e
(1 + κR h)
where, e is the elementary charge, f( κR h) is Henry’s function, and
κ is the inverse of the Debye-Hückel length that is defined by
κ =
2 e2N A I c
where N A is the Avogadro number, I cis the ionic strength, is the
relative dielectric constant of the solvent, and 0is the permittivity
of vacuum
In EAF4, Henry’s function ( f( κR h)) assumes a relatively simple
empirical approximation described as [21]
f( κR h)= 16 + 18 κR h+ 3 ( κR h)2
The zeta-potential ( ζ) can be derived from the electrophoretic
mobility ( μ) by [21]
ζ= 3 ημ
In the limit of small analytes in relation to the Debye-
Hückel length, κR h1, f( κR h) approaches 1 and Eq (13) reduces
to the Hückel equation In the limit of large analytes in rela-
tion to the Debye-Hückel length, κR h1, f( κR h) 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 ( R h) 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),
respectively The resulting zeta-potentials reported in this paper are based on Eq.(13)by using the approximated Henry’s function ( Eq.(12))
3 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 characteris- tic size-dependent concentration profile in the separation chan- nel 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 support- ing 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 depen- dent on the buffering capacity of the buffer
Fig.1(a) and (b) show the pH vs elution time observed at var- ious electric fields in two types of carrier liquid (50 mM NaNO 3
or 50 mM phosphate buffer at pH 7.0) during an EAF4 run The pH changed rapidly during the initial 5 min 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 NaNO 3) com- pared 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 min of channel flushing, indicating that longer channel condi- tioning 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 rel- atively 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 al- ready 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 re- producible manner when using the modified method, as shown in Fig.2(c) for the NaNO 3 carrier liquid and 2(d) for the phosphate buffer carrier liquid As would be expected, the effects were more pronounced for the non-buffered (NaNO 3) carrier liquid but the re-
Trang 4Fig 1 pH vs elution time at various electrical currents (-20 to 20 mA) in two types of carrier liquids (a) 50 mM NaNO 3 , (b) 50 mM phosphate buffer at pH 7.0, and (c) BSA fractograms and pH in duplicate (in 50 mM NaNO 3 )
producibility in the obtained pH was also improved for the phos-
phate 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
3.2 Separation and characterization of proteins by EAF4
In a previous study it was shown that GA-Z has a fast equi-
librium 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 3 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 ap-
plying 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
min to 5.5 min which could be due to an increased electrostatic
repulsion between the membrane surface and the analytes (GA-
Z 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 be-
tween 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 min to 6.2 min, which is proba-
bly due to attraction between the membrane surface and analytes caused by the anode at the bottom electrode In addition, a de- creased peak area was observed when the membrane was posi- tively charged, most clearly observed at -15 mA and -20 mA elec- trical currents ( Fig.3), which is likely to be caused by sample ad- sorption resulting from the opposite charge of GA-Z and the bot- tom electrode (anode) Accordingly, analyses should be carried out carefully to avoid sample adsorption when applying higher electri- cal 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 mo- bility and zeta-potential of GA-Z monomer and dimer ( Table1) 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 al- ready at -15 mA giving rise to deviations in the relationship be- tween 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 mag- nitude 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
Trang 5Fig 2 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 NaNO 3 , and (d) in 50 mM phosphate buffer at pH 7.0, performing two runs using both the initial and modified method respectively
Table 1
Electrical properties of GA-Z, BSA, and Ferritin from EAF4
Proteins
Electrophoretic mobility 1μ
( μm cm/(V s)) Zeta-potential 1 (mV) Effective net charge 1 Net charge at pH 7.0
1 The values include the standard error of the mean ( ±) based on two replicates
2 The theoretical net charge of GA-Z at pH 7.0 was calculated based on the sequence [31]
3 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 9 negatively charged amino acids, re-
spectively 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.4shows 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 nega-
tive 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 liq- uid 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 ( Table2) Probably, the differ- ences observed in the determination of MW was due to the elec-
Trang 6Fig 3 Fractograms of GA-Z with different electrical currents in 25 mM phosphate
buffer at pH 7.0
Fig 4 EAF4 fractograms with different electrical currents (a) BSA in 50 mM phos-
phate buffer at pH 7.0, and (b) Ferritin in 25 mM phosphate buffer at pH 7.0
Table 2
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)
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 1 The zeta-potentials and electrophoretic mobility of BSA were deter- mined as -3.2 mV and -0.164 μm cm/V−1s−1 for monomer, -3.1
mV and -0.159 μm cm/V−1s−1 for dimer, and -4.4 mV and -0.229
μm cm/V −1s −1for 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 μm cm/V −1s −1 and -3.77
μm cm/V−1s−1 by EAF4 [1], which is approximately 15 times
higher than the values determined in this study The 50 mM phos- phate 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 re- sult in lower electrophoretic mobility Another reason for the lower electrophoretic mobility in our study is that the high conductiv- ity (ionic strength) of the carrier liquid decreased the electric field strength at a given electrical current ( Eq.(9)) In our results, the re- tention time shift between runs in absence or presence of the elec- tric field was lower than the previously reported results, (i.e lower
v EP, Eq.(8)) Therefore, it is presumed that we obtained lower elec- trophoretic 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 Table1) 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 ab- solute 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 sev- eral other parameters This means that the zeta-potential will be strongly dependent on the ionic strength of the surrounding solu- tion 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 pos- itive current was applied, the retention time of the samples de- creased 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 bot- tom electrode gave rise to repulsion between the sample and the surface of the membrane resulting in shorter retention time Con- versely, it is expected that the retention time and separation be- havior 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
Trang 7occur during the analysis of the samples The elution behavior did
not show significant improvements when a negative current is ap-
plied (see Figs.3and 4) and at excessive electrical current, adsorp-
tion 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 lim-
its the investigation of therapeutic proteins in formulation or pro-
teins under physiological conditions as well as for higher electric
fields as outlined above Additionally, as the electrolyte concentra-
tion 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
4 Conclusion
In this study, we investigated methods for rapid stabilization of
pH in EAF4 to maintain high reproducibility of results It was con-
firmed that using an additional focusing step including the electric
field gives a more rapid stabilization of pH Furthermore, the re-
sults show that it is crucial to use appropriate buffers as carrier
liquids to avoid large pH-changes during analyses
In addition, no significant increase in resolution between
monomers and oligomers was observed for the investigated pro-
teins EAF4 was also used to determine the electrical properties
based on electrophoretic mobility, such as zeta-potential and effec-
tive net charge, over the size distribution of the investigated pro-
teins There are limitations for the applicability of EAF4 to proteins
such as ionic strength and buffer composition Another challenge
is of course that many proteins are relatively small and have rela-
tively low number of charges which limits the effect of the electric
field It is likely that the method would be more suitable for appli-
cation to larger or highly charged analytes such as large proteins,
polyelectrolytes and charged nanoparticles Nevertheless, the re-
sults 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 pro-
tein 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 “Sep-
aration and zeta-potential determination of proteins and their
oligomers using electrical asymmetrical flow field-flow fractiona-
tion (EAF4)” based on CRediT is shown in the table below
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial 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 &
editing, Project administration, Supervision Lars Nilsson: Concep- tualization, Writing review & editing, Supervision, Project admin- istration
Acknowledgments
The research in this study was performed with financial sup- port 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 pro- viding 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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... in “Sep-aration and zeta-potential determination of proteins and their
oligomers using electrical asymmetrical flow field-flow fractiona-
tion (EAF4)? ?? based on CRediT is shown... 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... retention time as the pH of carrier liq- uid 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