Rejection of Commonly Used Electrolytes in Asymmetric Flow Field

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Rejection of Commonly Used Electrolyte in Asymmetric Flow Field Flow Fractionation: Effects of Membrane Molecular Weight Cut-off Size, Fluid Dynamics and Valence of Electrolytes Thilak K. Mudalige, Haiou Qu, and Sean W. Linder Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03749 • Publication Date (Web): 18 Jan 2017 Downloaded from http://pubs.acs.org on January 18, 2017

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Rejection of Commonly Used Electrolyte in Asymmetric Flow Field Flow Fractionation: Effects of Membrane Molecular Weight Cut-off Size, Fluid Dynamics and Valence of Electrolytes Thilak K. Mudalige*, Haiou Qu, and Sean W. Linder Office of Regulatory Affairs, Arkansas Regional Laboratory, U.S. Food and Drug Administration, 3900 NCTR Road, Jefferson, Arkansas 72079, United States

ABSTRACT: Asymmetric flow field flow fractionation (AF4) is an efficient size based separation technique for the characterization of submicron size particulates. In AF4, membranes having various molecular weight cut-off sizes are used as a barrier to retain particles while allowing carrier fluid containing electrolytes to permeate. Here, we hypothesized that electrolyte rejection by the barrier membrane lead to the accumulation of the electrolytes in the channel during operation. Electrolyte accumulation can cause various adverse effects which can lead to membrane fouling. An instrument setup containing a conductivity detector was assembled and the rejection of commonly used carrier electrolytes, such as trisodium citrate, ethylenediaminetetraacetic acid, sodium chloride and ammonium carbonate, were evaluated by varying the concentration, cross flow rate, focusing flow rate, membrane material type, and the cutoff sizes. The results showed that electrolyte rejection was increased with a decrease in electrolyte concentration and the molecular weight cut-off size (pore size) or an increase of the charge state of the anion in the 1

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carrier electrolytes. We proposed an electrostatic repulsion-based rejection mechanism and verified it with the measurement of the rejection rate while varying the electrolyte concentration in the running media.

INTRODUCTION Asymmetric flow field flow fractionation (AF4) is an efficient size-based elution and separation technique for the analysis of particulates, such as organic and inorganic nanoparticles, protein, and polymers.1-2 Large dynamic size ranges of analysis, improved recovery, and its ability to hyphenate with other instruments, such as dynamic light scattering, optical detectors, and inductively coupled plasma mass spectrometry, as well as the use of an open channel without a stationary phase, are the distinct advantages of AF4 over other size-based separation techniques.3-9 AF4 uses an open, narrow and ribbonshaped channel where one side of the channel is lined with a porous membrane.1 In the analysis process, fraction of inlet flow passes through membrane as a cross flow (also called trans membrane flow), which is applied perpendicular to the main flow (channel flow) providing a diffusion coefficient based separation mechanism.1, 10-12 In AF4, resultant size fractionation profiles is called fractogram and, a generic chromatographic term of elution is used for explanation of differential retention even though there is no stationary phase in this technique.2 The application of an ultrafiltration membrane as an accumulation wall is common practice in field flow fractionation.10, 13 A thin ultrafiltration membrane is generally laminated onto a porous fiber membrane, which acts as a physical suport.13 The accumulation wall allows dissolved electrolytes and the solvent to permeate, and it also acts as a physical barrier to retain the particulates in the channel.13 The membranes are made up of various synthetic and processed natural polymers, such as polyethelenesufone and regenerated cellulose with well controlled pore sizes. The pore size of the membrane is selected depending on the size distribution of the particulate matter in the analyte solution with the pore being smaller than the smallest analyte in order to retain particulates in the chan2


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nel. The size resolution of the method is a function of the cross flow to channel flow ratio, and the cross flow determines the liquid flow velocity through membrane pores.11 The interactions between the membrane and the analytes also effect retention time.11 The zeta potential of the membrane plays a key role on membrane particle interaction and also the retention time.14 Generally the membranes are negatively charged due to either the inherent charge or the adsorbed anions on the membrane surface.15 In most cases, the nanoparticles are also negatively charged leading to the electrostatic repulsion between the membrane and the particle, which minimizes membrane fouling. Generally, colloidal suspensions such as, nanoparticles and proteins co-exist with electrolytes as either a stabilizing agent or the byproducts of synthesis. Electrolytes are essential in the running media in AF4 to help retain particles in the channel for a long enough time to separate them based on their hydrodynamic size.9, 16 Although electrolytes are essential for the separation of particulates, the presence of excess electrolytes can cause membrane fouling as well as the aggregation of the nanoparticles during analysis.9, 14 Membrane fouling is the major limitation of AF4 in the analysis of the inorganic nanoparticles such as gold, silver and titanium dioxide.9, 14 Therefore, evaluation of the factors that affect membrane fouling are essential for the successful implementation of AF4 methodology to the analysis of nanoparticles. In this study, we systematically evaluated the electrolyte rejection of AF4 membranes as well as the impact of the electrolyte charge. In this case electrolyte rejection is defined as retention of electrolyte due to their inability of permutation trough membrane pores along with solvent molecules. As a result, the electrolytes with polyvalent anions such as ethylenediaminetetraacetic acid (EDTA), showed a higher rejection rate compared to their monovalent counterparts such as NaCl. We also found that the electrolyte rejection was a function of the cross-flow rate, membrane type, the membrane cut-off size (pore size) and electrolyte concentrations in the carrier fluids.

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EXPERIMENTAL Materials and reagents. Type I ultra-pure water (18 MΩ·cm), obtained from a Thermo Scientific Barnstead Nanopure System (Waltham, MA) was utilized for all solution preparations. Sodium Citrate dihydrate (reagent grade), ethylenediaminetetraacetic acid, 0.5M Solution/pH 8.0 (Fisher Bio Reagents) and sodium chloride (certified ACS) were purchased from Fisher Scientific (Houston, TX). Isopropanol (HPLC grade) and ammonium carbonate (ACS reagent) were purchased from Sigma-Aldrich (Saint Louis, MO). Millipore precut regenerated cellulose membrane with a molecular weight cut-off of 10 kDa (10kDaRC) and polyethylene sulfone (PES) membranes with a molecular weight cut-off of 1 kDa Pall Polyethersulfone (1KDaPES) , 5 kDa and 10 kDa Nadir PES Membrane (5kDaPES and 10kDaPES) were purchased from Wyatt Technology (Santa Barbara, CA). A surface zeta potential accessory from Malvern Instruments (Worcestershire, UK), was used for measuring the membrane surface zeta potential.9

AF4 instrumentation settings. A Wyatt Technology (Santa Barbara, CA) short channel Eclipse 4 AF4 system composed of a flow control unit and a channel compartment (short channel with 145 mm length and 350 micron spacer) was coupled to an Agilent Technologies (Santa Clara, CA) 1200 high performance liquid chromatography (HPLC) system, which contains a quaternary pump (G1311B) and an autosampler (G1329B) with a 0.1 micron hydrophilic PVDF membrane filter (EMD Millipore, Billerica, MA) after the pump. The pump was used to control the liquid flow into the channel compartment and an autosampler was used for the sample introduction. A Shodex CD-200 conductivity detector (Yokohama, Japan) was used in the downstream of AF4 system and collected the conductivity signal from the detector flow (channel flow) of the AF4. An Agilent 35900E series II analog to digital converter was used to convert the analog conductivity signal generated by the conductivity detector to the digital signal required by Agilent ChemStation (Fig4


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ure 1). Precut membranes were soaked overnight in 20% isopropanol, washed with Type I ultrapure water and equilibrated with carrier fluid inside the AF4 channel for one hour. The autosampler was used to generate the injection signal with zero volume injections.

Figure 1. Schematic illustration showing instrumental setup (CD indicates conductivity detector, A to D indicates analog to digital converter)

Conductivity measurement settings and verification. The Shodex CD-200 conductivity detector (liner range 0- 600 mS, integrator output 0-1V, cell volume 2.5 µL) was fixed to a 30°C operational temperature. The voltage output of the conductivity detector was fed in to an Agilent 35900E series II analog to digital converter. The digital signal was recorded by Agilent ChemStation software with repetition time of 0.2 second. The system was equilibrated for one hour at 30 °C with the appropriate flow rate. The manufacture claimed a linear conductivity range of 0-600 mS for the conductivity detector, and we further verified the claim by measuring the conductivity of sodium chloride solutions having known concentrations. Experiments were duplicated to verify the day-to-day variation. As a result, we confirmed the robustness of the system, and did not find any day-to-day variation (supporting information). All experimental measurements were performed within the verified linear range of conductivity and in triplicate. We calculated the relationship between the concentration of each of the electrolytes and the conductivity for all the electrolyte solutions (NaCl at pH 7.0, EDTA at pH 8.0, trisodium citrate at pH 8.1, and ammonium carbonate at pH 8.6) by measuring the conductivity of the 5


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varying concentration of each electrolyte, and used them for the calculation of the electrolyte concentration using the conductivity detector. Conductivity measurements for operation of AF4. AF4 analysis consists of two major steps, focusing and separation. In this case, we evaluate the electrolyte accumulation during both the focusing and the separation. In the focusing step running media is injected in to the channel from both end of the channel and any particulate matter concentrated to the narrow area near the injection port, called the focusing zone. At the focusing zone, there is no net flow of liquid along the channel.9 Focusing is essential to improve resolution in the resultant fractograms in AF analysis. Here we varied the focusing parameters as described in Table 1 while maintaining a constant crossflow (2 mL/min) and channel flow (1 ml/min). In this case the AF4 flow control unit uses a fraction of fluid coming from the pump for focusing and the rest is directed to the detector. At the end of the focusing, the liquid in the channel was passed to the detector and the concentration of the accumulated electrolyte was calculated as percent increase of the original concentration with respect to channel volume (382 µL).7

Table 1. Parameters for Focusing Step

Designation

Time , Flow (mL/min)

1

Elution

2.0 min, Channel flow 1.0, Cross flow 0.0

2

Focus

10 sec to 2 min, Channel flow 1.0, Cross flow 2.0

3

Elution

8 min, Channel flow 1.0, Cross flow 0.0

During the separation step, cross flow is applied across the channel and the channel flow along the channel. Parameters listed in the Table 2 were used for measurement of the electrolyte accumulation in the separation step. Conductivity of the channel flow was measured with an online conductivity detector, 6


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and the electrolyte concentrations were calculated. Here we used the calculated relationship between the conductivity and the concentration for each electrolyte. RESULTS AND DISCUSSION In AF4 analysis, the presence of electrolytes in the carrier media is essential for the equilibration of the particulate close to the membrane which leads to longer sample retention and enhanced resolution of fractograms.9 In the absence of electrolytes, the particles will just elute after the void volume, and this phenomenon is most prominent for small charged particles.9, 16 Various asymmetric salts such as EDTA, ammonium carbonate and trisodium citrate, as well as symmetric salts such as NaCl and ammonium nitrates are used as electrolytes in AF4. Polyvalent cations are generally avoided to prevent the aggregation of negatively charged nanoparticles. The types of electrolytes and their concentrations are carefully selected to achieve the appropriate recovery and size resolution. Table 2. Parameters for separation step Method A ,Time (min), Flow (ml/min)

Method B, Time (min), Flow (ml/min)

Method C, Time (min), Flow (ml/min)

0.00 - 2.0, Channel flow 1.0, Cross flow 0.0 2.0-10.0, Channel flow 1.0, Cross flow 0.0 to 3.0



Gradient elution

10.0-18.0, Channel flow 1.0, Cross flow 3.0 to 0.0



Elution

18.0-24.0, Channel flow 1.0, Cross flow 0.0



Step

Designation

1

Elution

2

Gradient elution

3 4

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Figure 2. Focusing time dependent electrolyte accumulation in the channel as percent increase in (a) citrate, (b) EDTA and (c) NaCl concentrations for 10kDaRC membrane, and (d) citrate, (e) EDTA and (f) NaCl concentrations for 10kDaPES membrane

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Figure 3. Electrolyte concentration dependent surface zeta potential of 10kDaRC membrane and 10kDaPES membrane Evaluation of electrolyte rejection during focusing step. AF4 analysis can be divided into two major steps, which are focusing and separation.13 In the focusing step, running media is pumped into the channel from both ends and the fluid only escapes to the waste by permeating across the membrane. Sample injection is also done in the focusing step. As a general practice, the focusing step continues for a few minutes after the completion of the injection. In this step, particulate matter accumulates near the injection port as a narrow band, which leads to better resolution in the fractogram.2 During focusing, the analyte is pushed towards the membrane by the cross flow, and without any shear force along the membrane. In this step most of particles are lost due to membrane fouling.17 Additionally the electrolytes rejected from the membrane filtration will also accumulate in the channel, and may lead to further reduction of the electrostatic repulsion between the analyte and the membrane leading to an increase in interactions. To evaluate the electrolyte rejection, we quantified the accumulated electrolyte while varying the focusing time using a 2mL/min focusing flow for either 10kDaRC or 10KDaPES membrane. In this case the commonly used asymmetric electrolytes trisodium citrate and tetrasodium EDTA salt as well as the symmetric electrolyte, NaCl were examined at 0.10, 0.25, 0.50, 0.75 and 1.0 mM concentrations. The increase of electrolyte concentration in the channel vol9


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ume (382 µL) as percentage of running media electrolyte concentration was graphed against the focusing time (Figure 2).7 Figures 2a, b, and c represent electrolyte accumulation of the 10kDaRC membrane as function of focusing time and concentration of citrate, EDTA and NaCl in the running media respectively. Figures 2d, e, and f represent electrolyte accumulation of the 10kDaPES membrane as function of focusing time and concentration of citrate, EDTA, and NaCl in the running media respectively. As indicated in the graphs, at a crossflow of 2 ml/ml, it takes up to five channel volumes (382 µL) to reach saturation of the membrane and equilibrate the electrolytes in the channel.7 The channel saturation time is also a function of the original electrolyte concentration in the running media with a higher electrolyte concentrate reaching equilibrium faster. Further, accumulation of electrolyte is concentration dependent with lower concentrations having higher rejection percentages.

Proposed mechanism of electrolytes rejection. The mechanism of electrolyte rejection by a charged membrane is a very complicated process, but recent literature has simplified it into two components, which are electrostatic rejection and steric rejection.18-19 In this case, the smallest cut-off size of the membrane used in this study is 1000 Daltons, which is much larger than the size of a hydrated electrolyte, therefore steric rejection of hydrated salt ions can be ignored.20-21 Even for nanofiltration membranes, electrostatic rejection plays a major role in the retention of the charged species.22

To evaluate the electrostatic repulsion of the anions by the charged mem-

brane and pores, membrane zeta potential was measured while varying the electrolyte concentration (Figure 3). All membranes investigated in this study have a negative zeta potential, and the zeta potential values decreased with the increase of electrolyte concentration as expected. The negative zeta potential is due to a negatively charged membrane. The membranes charge can be generated by the deprotonation of functional groups or the adoption of anions on the surface, the latter case is more prominent in synthetic membranes such as PES.15, 23 As indicted in Figure 4B, electrostatic repulsion resists electrolytes passing 10


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through the membrane while cross flows pushes particles through the membrane.24 The partial rejection of electrolytes is due to electrostatic repulsion. Mukherjee and coworker studied the effect of zeta potential on the rejection of electrolytes, and note that an increased zeta potential due to fluoride ion implantation resulted in increased rejection.25 The negatively charged membrane rejects anions and cations will be retained to maintain charge balance of the total electrolytes. As seen in Figure 2, the percentage of rejection decreases with an increase in salt concentration, because increased electrolyte concentration shields electrostatic repulsion, thus allowing more ions to pass through the charged pore. In the nanofiltration studies, the electrostatic rejection mechanism is demonstrated by varying electrolyte concentration and measuring the rejection rate.19 In the presence of electrostatic rejection, the percent rejection decreases with increasing electrolyte concentration as seen in this study. We can speculate that the rejection of electrolytes causes an increase in electrolyte concentration near membrane, the accumulated electrolytes then diffuse away from the vicinity of the membrane towards the fresh solution, and eventually, the rate of electrolytes getting into the channel equals to the rate of electrolytes passing through the membrane. The equilibrium condition will depend on the cross flow rate, and in our studied condition, it takes about 1 minute or about five equivalents of channel volumes . Further, NaCl solution having a monovalent anion of Cl- is shown to have lower rejection compared to multivalent citrate and EDTA. This can be explained with the charge-dependent electrostatic repulsion by the charged membrane.25

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Figure 4. (a) Schematic illustration of channel fluid flow profile during the focusing, (b) Schematic illustration of anion rejection by negatively charged pore and membrane.

Figure 5. Cross flow dependent increase in electrolyte concentration in the separation as percent increase in (a) citrate, (b) EDTA and (c) NaCl concentrations for 10KDaRC membrane and (d) citrate, (e) EDTA and (f) NaCl concentrations for 10KDaPES membrane. Lines 1 and 3 represent 0.5 mM and 1.0 mM concentration of electrolyte respectively with a cross flow gradient up to 3 mL/min, and lines 2 and 12


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4 represent 0.5 mM and 1.0 mM concentration of electrolyte respectively with a cross flow gradient up to 4 mL/min gradient.

Evaluation of electrolyte rejection during separation step. To evaluate the effect of the cross flow rate on the electrolyte rejection, the increase in the channel electrolyte concentration was measured while varying the cross flow rate.

For 10kDaRC and

10kDaPES membranes, cross flow program A and B (Table 2) were used. The cross flow was linearly increased from 0 to 3 or 4 mL/min and then linearly decreased to 0 mL/min while maintaining channel flow at 1mL/min. The channel flow was directed to the conductivity detector and the variation of the electrolyte concentration was measured. In this case, 0.5 mM and 1.0 mM concentrations of sodium citrate, EDTA and NaCl were used as electrolytes in the running media. A gradual increase of channel electrolyte concentration was observed with the increase of cross flow up to a maximum with further increase in cross flow resulting in a decreasing rejection rate (Figure 5). When the cross flow is increased, the mass flow rate across the membrane (across the pores), the total amount of electrolytes, and the total volume of carrier fluid in the system are also increased, which forces a higher percentage of electrolytes to pass through the membrane by overcoming the electrostatic repulsion. The higher mass flow works against the diffusion of rejected electrolyte from membrane to center of the channel. A recent molecular dynamics simulation has shown an increase in electrolyte permeation of nanoporus membranes with an increase of cross flow rate.26 When we decreased the cross flow rate, the same phenomenon was observed. A maximum was observed at the same cross flow rate for each electrolyte regardless of the cross flow gradient (0 to 3 or 0 to 4). As observed in the focusing step, in the separation step, NaCl showed the lowest rejection rate compared to citrate and EDTA.

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Figure 6. Cross flow dependent increase in electrolyte concentration in the separation as percent increase in (a) citrate, (b) EDTA and (c) NaCl concentrations for 1kDaPES membrane and (d) citrate, (e) EDTA and (f) NaCl concentrations for 5kDaPES membrane. For 1kDaPES membrane lines 1 and 2 represent 0.5 mM and 1.0 mM electrolyte concentration with a cross flow gradient up to 2 mL/min. For 5kDaPES membranes lines 1 and 3 represent 0.5 mM and 1.0 mM electrolyte concentration respectively with a cross flow gradient up to 3 mL/min, and lines 2 and 4 represent 0.5 mM and 1.0 mM electrolyte concentration respectively with a cross flow gradient up to 4 mL/min.

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Effect of molecular weight cut off size on electrolytes rejection. The effect of the pore size was evaluated with the application of the cross flow gradient method for 5kDaPES membrane and 1kDaPES membranes. Due to the increased back pressure and limited system capabilities, we applied cross flow up to 2 mL/min for 1kDaPES membrane using program C in Table 2. The same trend discussed for 10kDaPES and 10kDaRC was observed for both the 1kDaPES and the 5kDaPES membranes. In the case of 1kDaPES membrane, we applied the maximum cross flow rate of 2mL/min but the system did not reach a maximum point of electrolytes concentration. This study clearly indicates that for any given cross flow, the rejection rate was the highest for the smallest pored membrane of PES (Figure 5 and 6). This trend can be explained by the electrostatic rejection of anions, where in the smallest pored membrane, the electrolytes has to overcome a higher energy barrier because negatively charged charge ions must pass very close to negatively charged pore wall thereby facing a greater electrostatic repulsion compared to a larger pore size.24

Effect of anion charge on electrolytes rejection. To evaluate the effect of the charge state of the anionic electrolyte, the rejection rates were compared by varying the anionic charge state while maintaining the positive counter ion at +1 charge state. In this study, citrate ions in trisodium citrate remain in -3 charge state at measured pH of 8.1, EDTA remain the same charge state of -3 at pH of 8.0 and it is well known that the chloride ion can have only state of -1 charge state at neutral pH of 7.0. We also analyzed ammonium carbonate, another commonly used electrolyte in AF4 analysis.

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Figure7. Crossflow rate dependent percent increase in ammonium carbonate in the channel for (a) 10kDaRC, (b) 10kDaPES (c) 1kDaPES and (d) 5kDaPES membranes. . For the 1kDaPES membrane, line 1 represents 0.5 mM (NH4)2CO3 while line 2 represent 1.0 mM both with a cross flow gradient up to 2 mL/min. For 10kDaRC, 10kDaPES and 5kDaPES membranes lines 1 and 3 graphs represent 0.5 mM and1.0 mM of (NH4)2CO3 respectively with a cross flow gradient up to 3 mL/min while lines 2 and 4 represent 0.5 mM and1.0 mM of (NH4)2CO3 respectively with a cross flow gradient up to 4 mL/min gradient.

The carbonate ions mostly stay in the form of a singly charged HCO3- state at a pH of 8.6. Ammonium carbonate also shows the same general trend as the other electrolytes with the rejection rate comparable to that of NaCl (Figure 7). These findings indicate that the anionic charge state strongly effect the rejection rate while also supporting the electrostatic rejection mechanism.24

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Possible effect of electrolyte rejection on membrane fouling. In the focusing step, carrier fluids enter the channel from both ends, permeate through the membrane, and pass into the waste line. During this process, the entire concentrated electrolyte moves along the fluid towards the focusing zone while losing a fraction of the fluid to the waste. With the consideration of liquid flow profiles during focusing, we can expect the highest electrolyte concentration in the focusing zone. The diffusion rate of the electrolyte away from the membrane, mass liquid flow velocity, and the membrane rejection rate will determine the actual concentration distribution of the electrolytes during focusing. In the separation step, electrolytes rejected by membrane may stay close to the membrane due to the application of the cross flow, however diffusion will try to equilibrate the electrolyte concentration by moving it away from the membrane. The channel flow will also carry the concentrated electrolyte solution away from the channel. The liquid mass flow, membrane rejection rate, and the diffusion rate will determine the electrolyte distribution in channel during separation. In AF4, the analyte spends most of the time in the vicinity of the membrane. During the focusinjection and the focusing steps, the analytes concentrate in the focusing zone close to the membrane and reach an equilibrium distance from the membrane based on the membrane particle interactions, particle size, and cross flow rate.11 The electrostatic and the Van der Waal interactions are prominent membrane-particle interactions.14 All these interactions are distance dependent and the repulsive forces drastically increase with the reduction of the membrane-particle distance.27-28 The electrostatic repulsion is a function of the particles size, particle zeta potential and membrane zeta potential, as well as the concentration and valance of the surrounding electrolytes.29-31 An increase in the surrounding electrolyte concentration reduces the electrostatic repulsion ultimately leading to membrane fouling by the analytes. Higher cross flow also forces the particles towards the membrane reducing the membrane-particle distance, and may cause membrane fouling. In our previous study, we found that the membrane fouling was worse for a 5kDaRC membrane compared to the 10 or 30 kDaRC membrane with the 30 kDa 17 ACS Paragon Plus Environment

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membrane having the least amount of fouling.9 But other factors in addition to the electrolyte rejection explored here may also have an effect on the membrane fouling.

CONCLUSION Here, we proposed and evaluated the electrolyte rejection by the membrane in AF4. An instrument setup with a conductivity detector was assembled and the functionality was validated. As a result of the experimentation, electrolyte rejection was observed to be a function of the membrane cut-off size, electrolyte concentration, electrolyte charge state, and cross flow rate. A possible electrostatic rejection mechanism was proposed that supported a previously discussed concentration dependent rejection mechanism. The effect of electrolyte rejection on the membrane fouling correlated with the previous findings. We are currently evaluating methodology to visualize electrolyte rejection through the application of fluorescent or colored electrolytes, as well as the effects of electrolyte rejection on membrane fouling of various metallic nanoparticles.

ASSOCIATED CONTENT Supporting Information Available: linearity verification of conductivity detector and membrane zeta potential measurement procedure. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Telephone: 1- 870 543 4665 Notes The authors declare no competing financial interest.

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ACKNOWLEDGEMENT These studies were conducted using the Nanotechnology Core Facility (NanoCore) located on the U.S. Food and Drug Administration’s Jefferson Laboratories campus (Jefferson, AR), which houses the FDA National Center for Toxicological Research and the FDA Office of Regulatory Affairs Arkansas Regional Laboratory. We thank Dr. Marilyn Khanna, Crystal Ford, Dr. Desiree Van Haute, Dr. Andrew Fong and Dr. Siyam M. Ansar for their support. The views expressed in this document are those of the researchers and should not be interpreted as the official opinion or policy of the U.S. Food and Drug Administration, Department of Health and Human Services, or any other agency or component of the U.S. government. The mention of trade names, commercial products, or organizations is for clarification of the methods used and should not be interpreted as an endorsement of a product or manufacturer.

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Rejection of Commonly Used Electrolytes in Asymmetric Flow Field

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