Chromatography, Vol.21 No.3(2000)

Mutual Citation

Mutual Citation

Cibacron blue F3GA-attached 2µm non-porousmonodisperse silicas for affinity chromatography
Shen Li, Xiong Bohui, Cong Runzi and Wang Junde
Dalian Institute of Chemical Physics, The Chinese Academy of Science, Dalian 116012,China

Abstract
Non-porous monodisperse silica (NPS), 2µm in diameter, was modified with 3-aminopropyltriethoxysilane for immobilized of Cibacron Blue F3GA, a packing of NPS-ACB for high performance affinity chromatography was obtained. Up to 2 mg of Cibacron Blue F3GA could be attached to 1 ml of NPS
beads. There was no obvious leakage of dye from NPS-ACB. The adsorption of lysozyme varied with pH
value and ionic strengths. Furthermore, the column could be used for separation and preparation oflysozyme from hen egg white. In addition, rapid separation of α-, β-,γ-globulin is also described.
Key words : high performance affinity chromatography, non-porous monodisperse silica, Cibacron Blue
F3GA, lysozyme, α-globulin, β-globulin, γ-globulin

Introduction:
Affinity chromatography is a well known technique for theExperimental

purification of biopolymers. Conventional soft gel affinity media,Apparatus and Chemicals
Shimadzu LC-10AD liquid chromatograph with SPD-10A UV-owing to its unstable mechanical character, can not be used for rapid

VIS Detector (Japan), 7125 Injector (Rheodyne), Chromatographic

separation, which causes low mass and bioactivity recovery inwork station (Dalian Institute of Chemical Physics), 721 UV-Vis

separation process. With the development of high performance affin-Spectrophotometerity chromatography (HPAC), silica [1], metal-oxides [2] and polymers[3,4]Cibacron Blue F3GA (Sigma), 3-aminopropyltriethoxysilane

have been widely used as affinity media. Since the successful appli-(APS, Fluka), Tris (Fluka), lysozyme (Shanghai Institute ofcation of non-porous monodisperse silica in affinity chromatography

Biochemistry α, β-, γ- (-globulin (Serva).

in 1988 , the use in reversed-phase chromatography , ion-exchange
[5]

[6]

chromatography [7] and hydrophobic chromatography [8] has been
described.

Preparation of NPS-ACB
NPS was activated by acid treatment and dehydrated under aIn this study we used 2µm non-porous monodisperse silica

reduced pressure of 2mm Hg and 453K for 6h.The reaction proce-(NPS) as matrix, Cibacron Blue F3GA as ligand, to produce an affin-dure was carried out after cooling the silica to room temperature. In

ity packing. Experiments have been carried out to determine thecase of derivatization of the silica in toluene, the solvent was driedinteraction between Cibacron Blue F3GA and several proteins andwith sodium wire, and freshly distilled, and then added to the silica

enzymes.

under vacuum, in order to allow the solvent to wet completely thesurface of non-porous monodisperse particles. After that some 3-−221−

Chromatography, Vol.21 No.3(2000)

Chromatography, Vol.21 No.3(2000)

Shen Li, Xiong Bohui, Cong Runzi and Wang Jundeaminopropyltriethoxysilane was added. The reaction mixture was

The amount of APS bonded was 56.5µmol/g from elementaryaction is enhanced. Fig.4 shows that the adsorption of lysozyme

heated under reflux for 24h. The modified silica was extracted inanalysis. The amount of dye immobilized on the modified silica wasdecreases with the increased of ionic strengths. It could be said thattoluene and trichloromethane for 2 days in order to remove the2mg/ml by measuring the UV absorption at 620nm. This value wasion exchange was the dominant force between lysozyme andreaction components. The modified silica was stored underhigher than 1.2mg/ml which was obtained by Unger [5].

Cibacron Blue F3GA. So the elution could be carried out by increas-anhydrous conditions until use.

Cibacron Blue F3GA leakage would interfere the assay ofThe modified silica was suspended in 0.1 mol/L sodium

protein because of its UV absorption. The pH stability test showed

phosphate, at pH 8.0, containing 0.5 mol/L KCl and Cibacron Blue

that there was no significant dye leakage from pH3.0~6.0(Fig.2). In

F3GA. The reaction was carried out at 60˚C for 3h. The immobilized

spite of the higher Cibacron Blue F3GA concentration at higher ordye matrix was washed in turn with 0.1 mol/L sodium phosphate,

lower pH value, the highest dye leakage was less than 25mg/L,

methyl alcohol-water (1:1), methyl alcohol, and water until thewhich would not interfere the assay of protein.

ing the ionic strength.

washing supernatants were colorless.

The Amount of Immobilized Dye
16~60 mg NPS-ACB was solubilized in 5ml NaOH (1mol/L).
The amount of dye bonded was determined by measuring the change

Fig. 6 Chromatogram of Oval and lysozyme
Chromatographic conditions as in Fig.5
1: Oval; 2: lysozyme

in absorption at 620nm.
Fig. 4 The amount of lysozyme adsorbed at different ionic strength

pH Stability Test
NPS-ACB was suspended in 50mmol/L Tris-HCl solutions

Ovalbumin (Oval) and lysozyme from hen egg white showed

containing 0.5 mol/L KCl at different pH values and shaken fordifferent affinity to Cibacron Blue F3GA. Oval could not be retained.

24h.The solution was centrifuged and the supernatant was examinedIts retention time did not vary with injection amount. Lysozyme wasfor ligand leakage spectrophotometrically at 280nm.

Fig. 2 pH stability test

Chromatographic Procedures
NPS-ACB was placed into a stainless-steel column(4.6×50mm

Chromatographic Procedure
The interaction of protein and triazine dye is very complex. Ion

I.D.) using the downward slurry technique in water. Mobile phaseexchange and hydrophobic interaction were expected owing to thewas 50mmol/L Tris-HCl. Eluent was 50mmol/L Tris-HCl containing

presence of amino, sulphonic groups and polycylic aromatic rings in

1mol/L KCl and 20% glycol (v/v).

the dye molecule. We studied the effect of different experimental

adsorbed on the column. Its mass concentration was proportional tothe peak area(Fig.5). The mass recovery of lysozyme was more than95%.

conditions on the chromatography of lysozyme. The adsorption ofResults and Discussion

lysozyme increased with the pH value (Fig.3). Lysozyme is positive

The Character of NPS-ACB
NPS with 2µm particle size (R.S.D.<5%) was a gift of University

at pH 5.0~8.0 because of its high pI (11.0). In case of higher pH
values, dye bears higher density of negative charges and ionic inter-of Petroleum, China. The surface area was 4m /g as shown in Fig.
2

[8]

1.

Fig. 7 Chromatogram of lysozyme and hen egg white on NPS-ACB column
Chromatographic conditions as in Fig.5
a: lysozyme; b; hen egg white
Fig. 5 Calibration graph of peak area vs concentration of oval and lysozyme in 10 µl
sample.
Mobile phase: 50mmol/L Tris-HCl, pH7.8 (solution A); desorption solution:
solution A containing 1mol/L KCl and volume fraction 20% glycol; flow rate:
0.6ml/min; detection: 280nm

Oval could not bind on the column alone but could bind withdiethylamine(NPS-DEA) column. The retention time of the crude
lysozyme with and without dialysis was accordant to that oflysozyme(Fig.8).

. So the column could be used to remove most of theIn addition, α-globulin could not be retained by the packing,oval in the when preparation of lysozyme from hen egg white. Fig.7

while β- and γ-globulin could be adsorbed on the column. γ-Globulin

shows that most component of hen egg white does not bind to this

could be eluted by 1 mol/L KCl and 20% glycol (v/v), while β-packing and eluted in the breakthough peak. Lysozyme was boundglobulin was not able to be eluted by the same eluent(Fig.9). Theand eluted by 1 mol/L KCl and 20% glycol (v/v). The purity of crudedifference in affinity interaction could be used to separate the three

Zn or Mg
2+Fig. 3 The amount of lysozyme adsorbed at different pH valuelysozyme was monitored using the non-porous silica-2+[10]Fig. 1 Scanning electron micrograph of 2 µm non-porous silica beads−222−−223−

Chromatography, Vol.21 No.3(2000)

Chromatography, Vol.21 No.3(2000)

Shen Li, Xiong Bohui, Cong Runzi and Wang Jundeaminopropyltriethoxysilane was added. The reaction mixture was

The amount of APS bonded was 56.5µmol/g from elementaryaction is enhanced. Fig.4 shows that the adsorption of lysozyme

heated under reflux for 24h. The modified silica was extracted inanalysis. The amount of dye immobilized on the modified silica wasdecreases with the increased of ionic strengths. It could be said that

toluene and trichloromethane for 2 days in order to remove the2mg/ml by measuring the UV absorption at 620nm. This value wasion exchange was the dominant force between lysozyme andreaction components. The modified silica was stored underhigher than 1.2mg/ml which was obtained by Unger [5].

Cibacron Blue F3GA. So the elution could be carried out by increas-anhydrous conditions until use.

Cibacron Blue F3GA leakage would interfere the assay ofThe modified silica was suspended in 0.1 mol/L sodium

protein because of its UV absorption. The pH stability test showed

phosphate, at pH 8.0, containing 0.5 mol/L KCl and Cibacron Blue

that there was no significant dye leakage from pH3.0~6.0(Fig.2). InF3GA. The reaction was carried out at 60˚C for 3h. The immobilized

spite of the higher Cibacron Blue F3GA concentration at higher ordye matrix was washed in turn with 0.1 mol/L sodium phosphate,

lower pH value, the highest dye leakage was less than 25mg/L,

methyl alcohol-water (1:1), methyl alcohol, and water until thewhich would not interfere the assay of protein.

ing the ionic strength.

washing supernatants were colorless.

The Amount of Immobilized Dye
16~60 mg NPS-ACB was solubilized in 5ml NaOH (1mol/L).
The amount of dye bonded was determined by measuring the change

Fig. 6 Chromatogram of Oval and lysozyme
Chromatographic conditions as in Fig.5
1: Oval; 2: lysozyme

in absorption at 620nm.
Fig. 4 The amount of lysozyme adsorbed at different ionic strength

pH Stability Test
NPS-ACB was suspended in 50mmol/L Tris-HCl solutions

Ovalbumin (Oval) and lysozyme from hen egg white showed

containing 0.5 mol/L KCl at different pH values and shaken fordifferent affinity to Cibacron Blue F3GA. Oval could not be retained.

24h.The solution was centrifuged and the supernatant was examinedIts retention time did not vary with injection amount. Lysozyme wasfor ligand leakage spectrophotometrically at 280nm.

Fig. 2 pH stability test

Chromatographic Procedures
NPS-ACB was placed into a stainless-steel column(4.6×50mm

Chromatographic Procedure
The interaction of protein and triazine dye is very complex. IonI.D.) using the downward slurry technique in water. Mobile phaseexchange and hydrophobic interaction were expected owing to thewas 50mmol/L Tris-HCl. Eluent was 50mmol/L Tris-HCl containing

presence of amino, sulphonic groups and polycylic aromatic rings in

1mol/L KCl and 20% glycol (v/v).

the dye molecule. We studied the effect of different experimental

adsorbed on the column. Its mass concentration was proportional tothe peak area(Fig.5). The mass recovery of lysozyme was more than95%.

conditions on the chromatography of lysozyme. The adsorption ofResults and Discussion

lysozyme increased with the pH value (Fig.3). Lysozyme is positive

The Character of NPS-ACB
NPS with 2µm particle size (R.S.D.<5%) was a gift of University

at pH 5.0~8.0 because of its high pI (11.0). In case of higher pH
values, dye bears higher density of negative charges and ionic inter-of Petroleum, China. The surface area was 4m /g as shown in Fig.
2[8]

1.

Fig. 7 Chromatogram of lysozyme and hen egg white on NPS-ACB column
Chromatographic conditions as in Fig.5
a: lysozyme; b; hen egg white
Fig. 5 Calibration graph of peak area vs concentration of oval and lysozyme in 10 µl
sample.
Mobile phase: 50mmol/L Tris-HCl, pH7.8 (solution A); desorption solution:
solution A containing 1mol/L KCl and volume fraction 20% glycol; flow rate:
0.6ml/min; detection: 280nm

Oval could not bind on the column alone but could bind withdiethylamine(NPS-DEA) column. The retention time of the crude
lysozyme with and without dialysis was accordant to that oflysozyme(Fig.8).

. So the column could be used to remove most of theIn addition, α-globulin could not be retained by the packing,oval in the when preparation of lysozyme from hen egg white. Fig.7

while β- and γ-globulin could be adsorbed on the column. γ-Globulin

shows that most component of hen egg white does not bind to thiscould be eluted by 1 mol/L KCl and 20% glycol (v/v), while β-packing and eluted in the breakthough peak. Lysozyme was boundglobulin was not able to be eluted by the same eluent(Fig.9). Theand eluted by 1 mol/L KCl and 20% glycol (v/v). The purity of crudedifference in affinity interaction could be used to separate the threeZn or Mg
2+Fig. 3 The amount of lysozyme adsorbed at different pH valuelysozyme was monitored using the non-porous silica-2+[10]Fig. 1 Scanning electron micrograph of 2 µm non-porous silica beads−222−−223−

Chromatography, Vol.21 No.3(2000)

Fig. 8 Chromatogram of crude lysozyme on NPS-DEA column
Mobile phase: 0.5mol/L NaCl in 50mmol/L Tris-HCl (pH8.0, adjusted withHCl); flow rate: 0.7ml/min; detection: 280nm
a: lysozyme; b: crude lysozyme, without dialysis; c: crude lysozyme, withdialysis

Fig. 9 Chromatogram of α, β, γ-globulin
Chromatographic conditions as in Fig.5
a: α-globulin; b: β-globulin; c: γ-globulin3) Tuncel, CDenizli A, Purvis D, et al. J Chromatogr., 1993, 634:globulins rapidly.

161-1684) Dominic C. Nash, Graham E. McCreath, Howard A. Chase.
J.Chromatogh., 1997, 758: 53-64

Conclusion
In this paper, non-porous silica was used as matrix, Cibacron5) Anspach B, Unger K K, et al. J Chromatogr., 1988, 457: 195-204

Blue F3GA as ligand to attain an affinity packing NPS-ACB. The

6) Wang Junde, Lei Xiaoling. Chemical Reagents, 1998, 20 (5) :amount of ligand attached reached 2mg/ml. The stability test of pH

277-281indicated that this packing is stable. It could be used to preparation of7) Unger K K, Jilge G, et al. Chromatographia, 1986, 22: 379-384

lysozyme from hen egg white in a small scale and rapid separation of8) Dong Peng, Zou Ying. J University of Petroleum China (Natrualα-, β-, γ-globulin.

Science Edition), 1992, 2: 2-39) Wang Junde, Shang Zhenhua, Yu Yunlu. High Performance

References:
1) Liu Guoquan, Down-Stream Technology-Cell Cultrue, Separa-Liquid Chromatography, Beijing, China Petrochem Press, 1992:

tion and Purification, Analysis and Detection, Beijing, Chemical

10) Feng Wenke. Biophysics and Biochemistry, 1993, 14 (2): 186-107-108

Industry Press, 1993: 232-235

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2) Wirth H J, Eriksson K O, Holt P., et al. J Chromatogr, 1993, 646:

[Originally published in Sepu, 17, 427-430 (1999).]

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