Original

Chromatography, Vol.22 No.2(2001)

Original

Isoelectric Points Estimation of Proteins by Electroosmotic Flow:
pH Relationship Using Physically Adsorbed Proteins on Silica Gel
Chaiyavat Chaiyasut and Takao Tsuda*
Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555, Japan
Received for review April 7, 2001.

Accepted May 21, 2001.

Abstract
A surface modification of the silica gel in a mini-packed polypropylene tube had been developed by physically adsorption of proteins. Severalproteins (bovine serum albumin, pepsin, globulin, β-casein) had been attempted to physically adsorb on the silica gel in a mini-packed cartridge under simple conditions. The proteins were successfully adsorbed on the silica gel, thus, minimizing the effect of the silanol groups.
The protein isoelectric point values (pI) might be determined from the pH of which the EOF is zero. The estimated pI values corresponded
very well with the literature values. The switchable EOF property of the protein adsorbed phases are fruitful for the manipulation of the magnitude and direction of EOF by selecting pH of buffer solutions and proper adsorbed protein phases. Four replicate different mini-packed cartridges were used to observe the EOF by using a simple homemade apparatus. The technique was easily to handle and did not take too muchtime consuming.
Keywords: adsorption, proteins, isoelectric points, silica gels, mini-packed cartridge

Experimental

Introduction

Instrumentation

Proteins are polyelectrolytes, and adsorption usually occurs because of the coulombic attractions between the negatively charged

A schematic diagram of an apparatus for measurements of thesurfaces and the positive charges on the protein molecules. Adsorp-EOF velocity was nearly the same as our former report [6], withtion at the surface was usually considered as a troublesome effectsome adaptation as shown in Figure 1. The apparatus consisted of ain CE, especially for the separations of proteins and peptides. The１-ml mini-packed polypropylene tube dipped in the buffer solu-

result is either tailing peaks or even complete adsorption of the pro-tion reservoir. The platinum wire electrodes were inserted to thetein to the surface. This can give rise to zone spreading, peak tail-tube and the medium reservoir. The cartridge was set vertically. Asing, and adversely affect the repeatability of the separations of pro-the level of the solution in a cartridge keep as the same as the level

teins and peptides [1-3]. However, the protein-bonded column wasof the solution in reservoir, we could avoid almost completely theused to study the selectivity in liquid chromatography [4]. At pre-effect of gravity for the estimation of EOF. The voltage power sup-sent, the adsorption effect of proteins was used as an advantage forply (DC; type R2; Toyo Roshi Ltd., Tokyo, Japan) at a constantthe separation of amino acids and was used as a chiral stationary

200 volts was applied through the approximate 1-cm long packed

phases for the enantiomers in OTCEC [5]. Proteins may strongly

bed. The total electric resistance is mostly reflected the resistanceadsorbed on the silica gel. In this work, we utilize the adsorption ofof its packed part. The approximate 200 volt/cm potential gradientprotein on silica gel in a mini-packed cartridge for the determina-

was enough to generate the EOF of the protein adsorbed phases.

tion of its pI value.

The electric current of all media used were 1-3 mA.

*Corresponding author: Fax, +81-52-735-5220;
e-mail, takao@ach.nitech.ac.jp―９１―

Chromatography, Vol.22 No.2(2001)

Procedure
The protein coated adsorbents were equilibrated with the aqueous buffer solution for about 10 min prior to measure the EOF velocity. The solvents were moved by means of a syringe in the suction mode through the luer tip side in the voltage-off mode. EOF
velocity was achieved by direct measuring the time used for theelectroosmotic volume flow movement to the 1-2 mm distance inthe upper open part of the cartridge under constant 200 volts application. All measurements were operated by using the homemade
apparatus as shown in Figure 1. EOF velocity was calculated byusing the same equation as described in our former report [6]. Each
type of protein adsorbed phases, four replicate protein adsorbed silica gel cartridges were used for the measurement of EOF. As thereproducibility of EOF measurement by this technique was alreadywell observed in our former report [6], each EOF velocity at each
pH was the average value of four EOF measurements. In this work,

Figure 1. Schematic diagram of an apparatus for measurements ofthe EOF velocity of the protein adsorbed silica gel in amini-packed polypropylene cartridge.

the reproducibility of the adsorption of proteins on silica gel in amini-packed cartridge was then observed as the %RSD of the EOF
velocity of four different cartridges.
Results and Discussion
EOF as a function of pH

Cartridge packing, mobile phases and reagents
The packing supports were silica gel (LC-Sorb, 45 µm diame-

The plots of EOF as a function of pH for the protein adsorbed

ter, Lot no 401025, Chemco. Ltd., Osaka, Japan). The packing pro-silica gel cartridge were shown in Figure 2 and 3. The effect ofcedure was the same as in the former report [6]. Mobile phases

having both positive and negative charges on the protein adsorbed

used were the mixtures of the citric acid and disodium hydrogensurface can be illustrated by the measurement of the EOF in the ex-

phosphate of constant ionic strength (0.01) containing 1 mg/ml

periment. From Figure 2 and 3, it is apparent that both magnitudeβ-casein). All aqueousand direction of EOF change with the pH of the contacted medium.

buffer solutions were degassed by ultrasonic bath prior to use. TheAt the zero EOF, the phases exhibited no EOF, indicating that theproteins used were bovine serum albumin (BSA), pepsin, globulin

surface of the phases had a net zero charges: this pH is referred toand β-casein. All reagents were purchased from Wako Pure Chemi-

as the isoelectric point (pI) of the surface of the protein adsorbed

cal Industries, Osaka, Japan.

phases. EOF direction is from the cathode to the anode if the ad-protein solution (0.4 mg/ml in case ofsorbed surface becomes positive charges as observed in the region
below the surface isoelectric point. In the region of cathodic EOF

Modification of the packed silica gel with proteins
Silica gel packed polypropylene tubes were activated by rins-

above the surface pI, the proteins bear negative charges and theing with 0.5 N sodium hydroxide and deionized water for 30 min

EOF direction is reversed. The magnitude of EOF increases withand 15 min, respectively. The protein adsorption phase was gener-decreasing pH in the anodic EOF region and incresing pH in theated by filling the mini-packed cartridge with the 10 mg/ml protein

cathodic EOF region, according to the amount of charged func-solution (4 mg/ml β-casein solution owing to low solubility) andtional groups of coated protein stationary phase.

keeping for 20 min. The solution was then flushed away by suction
Estimation of isoelectric points of proteins

with syringe via luer tip end for 5 min. Cartridge was kept drying
in an oven at constant 37 degree for 20 min. Then filling the mini-Isoelectric point is the pH at which the protein has zero net

packed cartridge again with the protein solution, keeping for 20charge. In protein adsorbed silica gel, EOF is suppressed at about amin, flushing solution away for 5 min and drying cartridge at 37

surface isoelectric point. The zero EOF was estimated by curve-degree for 20 min. The physically adsorbed proteins on silica gel

fitting the EOF velocity at different pH values. According to ourpacked cartridge was then used to observe the EOF at various pH

former research [6], EOF velocity (vosm) is related to the total sur-buffer medium containing protein solutions.

face charge density (σt ), regardless of its source, as expressed as―９２―

Chromatography, Vol.22 No.2(2001)

Chaiyavat Chaiyasut and Takao Tsuda

mC sinhvosm ＝ σt ＝ σn ＋Σσfunctional group, ii＝1m＝σn ＋Σeγfunctional group, iKa,functional group, i/ (Ka,functional group, i＋aH)
i＝1C equals ∆E (ε/η) (exp(-κx)) (2kT/e) (500 π/εRTc)−1/2，where ∆E,ε, η, κ, x, k, T, e, R and c are the applied voltage at unit length,permittivity, viscosity, inverse Debye length, radius of the counterion, Boltzmann constant, temperature, elementary charge, gas constant and concentration of ions, respectively. C is an approximate
constant value under a given set of conditions if the applied voltage
is constant. σn, σfunctional group, i, e, γfunctional group, i, Ka,functional group, i, aH and mare naturally induced surface charge density, functional group surface charge density, the elementary charge, the number of surfacefunctional groups per unit area, acid dissociation constant of thefunctional groups, activity of hydrogen ion and number of functional groups of proteins, respectively.
In case of protein adsorbed silica gel, the phases contained

Figure 2. Plots of EOF velocity and pH for pepsin adsorbed silica
gel phases (curve A) and for BSA adsorbed silica gel
phases (curve B) at constant 200 applied voltage. The
electrolyte solutions were citric acid-disodium hydrogenphosphate (ionic strength 0.01) containing 1 mg/ml protein solutions. The error bars showed the deviation ofEOF velocity using four replicate cartridges.

many different charged groups of protein. Therefore, the relationship between the EOF velocity and activity of hydrogen ion was in
the rational form with multiparameters. The zero EOF could be estimated from the fitting procedure between the EOF velocity andactivity of hydrogen ion. The reliability of the fitting procedure
was indicated by the confidence coefficients [6], which were 99.5
% (p<0.005) in this paper. It confirmed very well with our former
research [6].
From Figure 2 and 3, the titration curve shape of the plot ofEOF and pH was achieved. The shape of the curves between theEOF velocity and pH resembles the titration curves with different
slope and asymptote. Each curve in Figure 2 and 3 approached theasymptote at different pH. This might be the result of the different
charged groups, compositions, and distribution of charged side
chains of proteins. The pI values of the protein adsorbed on the silica gel might be estimated from the pH at zero EOF. In this report,
the estimated pI values of the pepsin, BSA, β-casein and globulin
were 3.2, 4.6, 4.5 and 5.0, respectively. These estimated pI values

Figure 3. Plots of EOF velocity and pH for β-casein adsorbed silica gel phases (curve A) and for globulin adsorbed silica
gel phases (curve B). The electrolyte solutions were thesame as in Figure 2 except containing 0.4 mg/ml βcasein solutions. Other explanations were the same as inFigure 2.

Table 1. Comparison of pI values of proteins from experiment andliterature (from refs.7-9).

―９３―

Chromatography, Vol.22 No.2(2001)

corresponded very well with the literature pI values [7-9] as com-means of benefaction for preparation of the physically adsorbed

pared in Table 1. From the experiment result, the outer part of theproteins on silica gel in a mini-packed cartridge. The data pre-coated proteins on silica gel and the proteins in the solution give

sented in this report indicated the successful adsorption of proteins

the same contribution to the pI estimation.

on silica gel. Effective protein stationary phases are obtained without interference from the existence of silanol groups. The adsorption protocols are promising and will become more useful on theAdsorption of proteins on silica gel
Macromolecular substances often show greater tendencies toseparation of compounds; i.e., proteins, amino acids and chiraladsorb to any surface than do low-molecular weight compounds.

compounds. We believe this method minimizing the effect of theThe reason is that macromolecules especially proteins have manysilanol groups. Therefore, the pI values could be obtained from themore binding sites and accordingly can be adsorbed by multipoint

relationship between EOF and pH of the medium. This technique, aattachment [10, 11]. Furthermore, the binding sites can be stereose-surface modification has been developed, which yields adsorbed

lective in nature, making them potentially useful in the separation

protein phases with switchable EOF (anodal/cathodal). Because ofof chiral molecules [12]. Therefore, we have been attempted tothe adsorption of proteins on the phases, the net charges of thephysically adsorb protein to silica gel in a mini-packed cartridge.

phases can be varied from positive to negative by changing the pH

The successful adsorption of proteins on silica gel could be illus-of the contacted buffer, enabling the manipulation of EOF. This

trated by analyzing the relationship between EOF and pH of solu-

was the same as the description of the chemical modification of thetions. As in our former report [6], the in-situ pKa of silanol groups

inner surface of the fused silica capillary by adsorption within a mini-packed cartridge were between 4.0 and 4.3. The effect ofsurface-active reagents (cetyltrimethylammonium bromide) [13,the existence of silanol groups could be seen from the inflection

14]. An additional aspect of such behavior is that, in principle, atpoint of the plot between EOF and pHs at which the inflection

any pH the magnitude and direction of EOF can be adjusted by se-

point is nearly the same as the pKa value of the silanol groups. In

lecting the proper adsorbed protein phases. Other physically ad-this work, there were no inflection points at the pH around the pKa

sorbed macromolecules and quaternary compounds on other phases

values of the silanol groups as shown in Figure 2-3 for all protein

are now under investigated.

adsorbed silica gel phases. For different kinds of the proteins used
Acknowledgments

for the physically adsorption on silica gel, the inflection points
were around the surface pI values. These inflection points repre-C. C. gratefully acknowledges the Ministry of Education, Sci-

sented only for the pI values of the proteins. No other inflection

ence, Sports and Culture, Japan for the grant-in-aid (No. B(2)points caused by the existence of silanol groups observed, thus, it12440209) supported as a part of the research in Japan.

implied the successful of physically coated proteins on silica gel ina mini-packed cartridge in this work and the amount of the ad-References

sorbed proteins is enough for the pI estimation.

[1]

Hjerten, S. J. Chromatogr. 1985, 347, 191-198.

[2]

Green, J. S.; Jorgenson, J. W. J. Chromatogr. 1989, 478, 63-[3]Landers, J. P. Handbook of Capillary Electrophoresis; CRC:

[4]

Haginaka, J.; Murashima, T.; Seyama, C. J. Chromatogr.

According to our data concerning the adsorption effect of cetyl

70.

trimethy ammonium bromide (CTAB) on silica gel, there is an evidence of the incomplete adsorption effect during the running ex-

Boca Raton, Florida, 1993.

periment if we use the running buffer containing no CTAB. Therefore, for more reliability and complete adsorption of proteins of all

1994, 666, 203-210.

the running experiments, it is better to add the protein in the run[5]ning electrolytes. The concentration we used in this experiment isLiu, Z.; Zou, H.; Ni, J. Y.; Zhang, Y. Anal Chim Acta. 1999,378, 73-76.

the same as used in the former references experiments [10, 11].
[6]

The reproducibility of the EOF velocity of four different repli-Chaiyasut, C.; Takatsu, Y.; Kitagawa, S.; Tsuda, T. Electrophoresis 2001, 22, 1267-1272.

cate physically protein adsorbed silica gel in a mini-packed car[7]tridge was found to be within 1.6% RSD. This technique was suc-Neurath, H.; Bailey, K. The Proteins; Academic Press: NewYork, 1954; Vol 2, Part A.

cessful diminish the existence effect of the free silanol groups of[8]the silica gel.

Anfinsen, C. B.; Edsall, J. T.; Richards, F. M. Advances inProtein Chemistry; Academic Press: New York, 1985; Vol37.

Conclusion[9]The adsorption effect of the protein on surface, usually consid-Malamud, D.; Drysdale, J. W. Anal. Biochem. 1978, 86,620-647.

ered to be a troublesome effect, has been successfully utilized as a―９４―

Chromatography, Vol.22 No.2(2001)

Chaiyavat Chaiyasut and Takao Tsuda[10] Yang, J.; Hage, D. S. Anal. Chem. 1994, 66, 2719-2725.

[13] Tsuda, T. J. Liq. Chromatogr. 1989, 12, 2501-2514.

[11] Barker, G. E.; Russo, P.; Hartwick, R. A. Anal. Chem. 1992,[14] Tsuda, T. in Handbook of Capillary Electrophoresis;
Lander, J.P., Ed.: CRC: Boca Raton, Florida, 1993, Chapter

64, 3024-3028.

22.

[12] Camilleri, P. Capillary Electrophoresis; CRC: Boca Raton,Florida, 1998.

―９５―