Applied Surface Science 206 (2003) 2028

Adsorption properties for urokinase on local diatomite surface

Yuxiang Yanga, Jianbo Zhangb,*, Weimin Yangc, Jieda Wua, Rongsan Chenc
a

Department of Chemistry, Tongji University, Shanghai 200092, PR China

Center for Environmental Sciences, Peking University, Beijing 100871, PR China
c
Coordination Chemistry Institute, Nanjing University, Nanjing, Jiangsu 210008, PR China
b

Received 1 October 2001; accepted 6 March 2002

Abstract
In this paper, adsorption isotherm of urokinase on two typical local diatomites were determined at 25 8C and their surface
electrical potentials (z), isoelectrical point values (IEP) were determined. The properties of diatomites, the relationship among
diatomite structure, pore-size distribution, surface z and adsorption isotherm were discussed. The adsorption equation of
urokinase was calculated from the adsorption isotherm. The adsorption mode of urokinase on diatomite surface was judged by
the conguration function a. The relationship between the amount of adsorbed urokinase and IEP value was also discussed.
# 2002 Published by Elsevier Science B.V.
Keywords: Diatomite; Urokinase; Adsorption isotherms; IEP

1. Introduction
Diatomite is a kind of non-metal that consisted of
the remains of diatoms, grew and deposited in the sea
or lake, and changed throughout with natural environmental action. Diatomite products are used in a variety
of ways, such as reinforcing, stiffening, and hardening
of organic solids, reducing adhesion between solid
surfaces, increasing adhesion, increasing viscosity,
surfactant effects, hydrophobic effects, absorbent,
catalysts, cloud seeding, and so on.
The urokinase is an important pharmaceutical material as plasminogen activators that could accelerate
thrombolysis and dissolve brin blood clots [1]. It
could be enriched from urine by using diatomite as an
absorbent. The production of urokinase by diatomite
had been found early, but the mechanism for adsorption of urokinase on diatomite has not been studied so
*

Corresponding author.

far. Study of adsorption of urokinase on the surface of

the diatomite is very important for improving urokinase production.
Based on their molecular weight, urokinase might
be classied as two species, which are high-moleculeweight urokinase (HUK) with molecular-weight of
54,000 g mol 1, and low-molecule-weight (LUK)
urokinase having two active subforms (LUK-1 and
LUK-2) with same molecular-weight of 33,000 g
mol 1. LUK-1 and LUK-2 could been separated by
QAE-Sephadex-A 25 chromatography [2].
The adsorption of urokinase on the diatomite could
be achieved by reacting of surface hydrogen bond
and surface electric charge of diatomite with OH,
NH2, C=O group or quarternary amine ion of urokinase in the solution [3,4]. The adsorption mechanism
of the urokinase on the diatomite surface has not
been studied before [5]. In this study, the biochemical method [6] was introduced to determine the
adsorption isotherm type and the adsorption mode

0169-4332/02/$ see front matter # 2002 Published by Elsevier Science B.V.

PII: S 0 1 6 9 - 4 3 3 2 ( 0 2 ) 0 0 2 0 9 - X

Y. Yang et al. / Applied Surface Science 206 (2003) 2028

of urokinase on the surface of Zhejiang and Jilin

diatomite. The relationships among diatomite's particle size, surface structure, electrical potential (z) and
isoelectrical point (IEP) were researched, which
would provide important reference for the production
of urokinase by the diatomite adsorption method.
2. Experiment
2.1. Apparatus
DXD type micro-television electrophoresis instrument made by Jiangsu Optical Instrument Plant of
China is used to determine z and IEP of the particle
such as ore, protein, colloidal, and so on, its principal
consisted of a single tube with a television as monitor
to observe movement of the particle under various pH
condition.
2.2. Material
The silica content of Zhejiang diatomite, Jilin
diatomite, and the acid-treated Zhejiang diatomite
used in the work were 64.80, 92.75, and 86.96%,
respectively [7]. When heated, the diatomite grew
grey in color with loss of 2.91% of its weight, then
white in color with loss of 0.50% and, nally grey in
color with loss of 3.10% of its weight until 300 8C. By
BrunauerEmmettTeller (BET) multi-molecule
layer adsorption model method [8], the specic surfaces of Zhejiang and Jilin diatomite were determined
as 64.70 and 19.11 m2 g 1, respectively, the acid-treated Zhejiang diatomite's specic surface was 40.01
m2 g 1 [9]. The diatomite is principally used as an
adsorbent for its pyrogenic and large specic surface.
The diatomite ore is from Sheng county of Zhejiang
province and ChangbaiShan county of Jilin province.
The raw diatomite was roasted at 400, 800, 950 and
1150 8C and powdered with the particle diameter 160
200 mesh.
The ore was treated with 89% dilute hydrochloric
acid in the ratio of 3 g of diatomite to 50 ml of acid,
then washed with distilled water to remove the chloride ions, nally the diatomite was roasted by baking in
the oven at 110 8C. The product was ground and
sieved to a particle size of 160200 mesh before being
treated, which is called as acid-washed diatomite.

21

The acid-washed diatomite was added in 5% of

NaCl powder and 5% of Na2CO3 powder, and mixed,
then roasted at 1000 8C for 2 h, the sample called as
reux diatomite (160200 mesh) was obtained. The
reux diatomite was added to 5% of polyglycol,
octadecimal amine, cetyltrimethyl ammonium chloride, and CaO, respectively, then roasted at 50 8C in the
oven. The reux diatomite was also added to trimethylsilyl chloride, then stirred for 1 h, and ltrated
by sandy core funnel, followed by washing with
distilled water to neutralize. These samples were
called as modied diatomite.
The fresh colloidal alumina, colloidal Zr4 and
CaCl2 solutions were prepared in a ask, the reux
diatomite was immersed for 2 h, and ltrated, followed by washing with distilled water to neutralize.
Zhejiang diatomite, Jilin diatomite, urokinase sample and standard urokinase sample were rened by
Nanjing University Biochemical Plant. Three assay
reagents of urokinase (UK) including brin, plasma
clot enzyme, and brin proteolysin were made in
Beijing Bioproduct Institute.
2.3. Methods
2.3.1. Biochemical analytical method
The standard urokinase sample plasma clot enzyme
and equivalent amount of brin proteolysin mixed
solution were added into a tube that is containing
brin and barbital buffer solution, then mixed adequately. The system should be clotted within 3045 s
when the tube is kept in thermostatic bath at
37  0:5 8C [10].
The thrombus dissolution was timed at the reaction
end when little bubbles rose to half volume of the
system in the tube. The standard curve was plotted by
the thrombus dissolution time versus the logarithm of
the concentration of urokinase to determine the concentration of urokinase. The standard urokinase sample (Beijing Bioproduct Institute, China) was added
into barbital buffer solution, so standard urokinase of
concentration 60 mmol l 1 min 1 mg 1 was obtained.
2.3.2. Determination of urokinase
adsorptive properties
Zhejiang diatomite (acid-washed and raw diatomite) and Jilin diatomite (160200 mesh) were
roasted at different temperatures. Then the series of

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Y. Yang et al. / Applied Surface Science 206 (2003) 2028

diatomite were added into the urokinase solution

(pH 6:6) with original concentration (C0) in asks
and stirred for 1 h. After reaching the equilibrium and
ltrated the mixing solution by sandy core funnel, the
equilibrium concentration (C) of urokinase was determined by biochemical method mentioned above. The
amount of adsorbed urokinase can be calculated according to the equation G C0 CV=m, in which V (ml)
is the volume of urokinase solution, m (mg) the weight
of diatomite, C0 (mol l 1 min 1) original concentration
of urokinase, C (mol l 1 min 1) equilibrium concentration, and G (mmol l 1 min 1 mg 1) adsorptive amount
of urokinase. In this paper, the specic activity was used
to represent the concentration of urokinase solution (the
unit was mmol l 1 min 1). The adsorption isotherms
were plotted by G versus C (the specic activity). The z
of the diatomite was determined by DXD type microtelevision electrophoresis instrument, then IEP value
was acquired from the curve of z versus pH. To determine the adsorption mode of urokinase on diatomite
surface, the saturated adsorption amounts to urokinase
of HUK and urokinase of LUK on upper diatomite were
determined by biochemical method as mentioned in
Section 2.3.1.
2.3.3. Adsorption isotherm equation of urokinase on
diatomite surface
The adsorptive amount (G) of urokinase corresponding to different equilibrium concentration (C)
in adsorption isotherm of urokinase (in Figs. 26) was
found out, respectively, saturation adsorption amount
(G1) of Zhejiang and Jilin at temperature intervals
was determined also [11]. Dening surface coverage
degree y G=G1 the curve of log[y/(1 y)] versus
log C was plotted. The adsorption isotherm equation
of urokinase on Zhejiang diatomite, acid-washed Zhejiang and Jilin diatomite surface was acquired [12,13]:

  
y
1
log

log KC;
1 y
b
y
KC1=b
i:e:
1 y
where K is adsorption equilibrium constant, and b
adsorption function chain number in urokinase.
When Jilin diatomite was roasted at 1150 8C, the
adsorption isotherm turned to type I, then C/y versus C
plot was made, and the equation C=y kC b was
gained, in which k and b were all constants.

3. Results and discussion

3.1. The relationship between diatomite structure
and adsorption isotherm of urokinase
According to the previous work [14], pH 5:5 is
suitable for adsorption of urokinase on diatomite. At
this pH, the diatomite adsorbed maximum amount of
urokinase. When considering urokinase had the
greater activity at neutrality [1], pH 6:6 was chosen
by using distilled water as solvent for this work.
Diatomite is a kind of pyrogenic silica powder. The
HUK molecule of 54,000 molecule-weight with double link of about 2 nm diameter, and 29.7 nm length
[15], were easy to penetrate into the pores and be
adsorbed on the surface of inner pores or outside
surface of diatomite. The adsorption was classed as
multi-molecular adsorption [3].
The LUK-1 and LUK-2 could been identied [2] by
Edman degradation, the rst seven cycles of Edman
degradation. Steps of the unreduced LUK-1 gave a
double sequence with practical equivalent amounts of
the phenylthiohydantoin-amino acids. However, the
seven cycles of Edman degradation of LUK-2 showed
a single sequence homologous with one of the two
sequences identied in LUK-1. Thus, LUK-1 consists
of two polypeptide chains, and LUK-2 consist of only
a single chain. According to Li [15], LUK-1 is of about
2 nm diameter and 18.2 nm length, and LUK-2 is of
about 1 nm diameter and 29.7 nm length.
At ambient temperature, the adsorption isotherm of
urokinase on Zhejiang and Jilin diatomite surface were
classed as type V and II, respectively (Figs. 1 and 2).
The reason is that the structure of Zhejiang diatomite
presented single cylindrical or double joint cylindricals with capillaries of different sizes arranged on the
cylinder wall and the structure of Jilin diatomite
presented disc shape with capillaries of different sizes
arranged inside the disc. The pore structure of these
diatomites (Zhejiang and Jilin) was determined by
using mercury porosimetry. The micropores of Jilin
diatomite centered round 500700, 520 nm, and
were smaller than that of Zhejiang diatomite apparently [10]. The amount of urokinase adsorbed on Jilin
diatomite was less than that on Zhejiang diatomite,
because HUK, LUK-1 and LUK-2 are easy to adsorb
on micropore's inner surface of the cylinder. As for
disc shaped diatomic structure, only LUK-2 with

Y. Yang et al. / Applied Surface Science 206 (2003) 2028

23

Fig. 1. Adsorption isotherm of urokinase on diatomite surface.

single link could be adsorb on micropore inner surface, so HUK and LUK-1 with double link was
adsorbed on the disc surface. The specic surface
area of Jilin diatomite (19.11 m2 g 1) was smaller
than that of Zhejiang diatomite (64.70 m2 g 1). When
equilibrium concentration reached more than 80
mmol l 1 min 1, urokinase condensed on disc surface
resulted in the increase of adsorption amount (as
shown in Figs. 1 and 2).

3.2. The effect of acid-treatment, roasting

temperature, and particle size on the adsorption
isotherm type of urokinase
The adsorption isotherm changed from type V to III
for acid-washed Zhejiang diatomite (as shown in
Fig. 1). This was because the acid-washed macropores
were centered round 2.817.50 mm, whereas, the raw
ones were around 1.342.81 mm [10]. It was, thus,

Fig. 2. Adsorption isotherm of urokinase on Jilin diatomite surface.

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Y. Yang et al. / Applied Surface Science 206 (2003) 2028

Fig. 3. Adsorption isotherm of urokinase on Zhejiang diatomite surface.

evident that pore diameter increased after washed with

acid, which is favorable for adsorption. When comparatively high equilibrium concentration was attained,
urokinase was adsorbed on inner pore surface in monolayer, and then was condensed inside the inner pore.
The average of pore diameter of acid-washed Jilin
diatomite after washing increased from 120 to 140 nm
[16], it also lead to the adsorption isotherm turning
into type III isotherm. The adsorption isotherm type
unchanged and moved towards lower equilibrium

concentration obviously, and also the total adsorption

amount increased, when Zhejiang diatomite was
roasted at 400 and 800 8C, respectively (Fig. 3). This
was because average pore diameter and surface z of
the diatomite gradually increased after roasted. When
Zhejiang diatomite was roasted at over 950 8C,
adsorption isotherm turned from type V to III (as
shown in Fig. 4), and the adsorption amount decreased
abruptly as the specic surface decreased. When
equilibrium concentration was comparatively high,

Fig. 4. Adsorption isotherm of urokinase on surface of Zhejiang diatomite roasted at 950 8C.

Y. Yang et al. / Applied Surface Science 206 (2003) 2028

25

Fig. 5. Adsorption isotherm of urokinase on surface of Jilin diatomite roasted at 950 8C.

urokinase condensed in the inner pore surface owing

to the smaller micropore diameter (6.007.67 nm)
inside Zhejiang diatomite [9].
When Jilin diatomite was roasted at 400 and
800 8C, adsorption isotherm type unchanged, and
yet classed as type II (as shown in Fig. 2). Those
three curves reached mono-layer adsorption at urokinase equilibrium concentration with 80, 45 and 37
mmol l 1 min 1, respectively, which was concerned
with surface electrical charge with z of the diatomites.
Surface z of Jilin diatomite gradually increased [6]
with the increase in roasting temperature, which
affected on the adsorption of urokinase. As a consequent, the equilibrium concentration for reaching
saturation mono-molecular layer decreased. When
Jilin diatomite was roasted at 950 8C, the adsorption
isotherm turned from type II to III (as shown in Fig. 5),
and the adsorptive amount of urokinase decreased
strikingly with the decrease of specic surface of
the diatomite. When roasted at 1150 8C, the adsorption isotherm type of Jilin diatomite varied evidently
(as shown in Fig. 6), and was obviously different from
that of Zhejiang diatomite. Because the micropore on
disc surface of roasted Jilin diatomite melted and
collapsed, so the adsorption of urokinase took place
only on the outside surface of the diatomite.
The adsorption isotherm type of Zhejiang diatomite
unchanged as particle size of the diatomite varied,
classed still as type V (as shown in Fig. 7), and moved

towards higher equilibrium concentration with

increasing particle size. In this paper, urokinase IEP
value was 9.09.5, so the amino acid in protein chain
all took the form of acid ionization, and became

NH3 R0 or NCH3 3 R00 positive polar group

[3]. The diatomite, with IEP value about 2, ionized in
the form of alkali, so its surface had large negative
electrical charge. The surface z of Zhejiang diatomite
decreased with increasing particle size [6], so the
amount of adsorptive urokinase reduced accordingly.
3.3. Adsorption mode of urokinase on diatomite
surface
The molecular weights of HUK and LUK in urokinase were M1 54,000 and M2 33,000 g mol 1,
respectively. The saturation adsorption amounts
for HUK and LUK on diatomite surface were As1
and As2, respectively, in which As1 K 1 M1a ; As2
K 1 M2a [12,13]. K1 is a constant, and a represents
conguration function of urokinase on the interface
of solventadsorbent. According to the equation,
the conformational modes of the urokinase in the
adsorbed mono-layer could be proved.
The two equations were divided and the logarithm
was made as follows:
a

log As1 =As2

log M1 =M2

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Y. Yang et al. / Applied Surface Science 206 (2003) 2028

Fig. 6. Adsorption isotherm of urokinase on surface of Jilin diatomite roasted at 1150 8C.

when (1) a 0, urokinase molecule lain at on diatomite surface; (2) a 1, urokinase chain attached
only at mono- or multi-point; (3) 0 < a < 0:1, urokinase chain attached as spheroids with radius equaling
rotation radius; (4) a 0:5, a random coil with segments attached to the surface of diatomite; (5)
0:5 < a < 1, multi-layer adsorption, urokinase chain
attached on diatomite surface tightly, then adsorbed
ulteriorly on the urokinase layer by forming a chain
loop [17].

According to this equation, the molecular conformational functions of urokinase which adsorbed on the
surface of Zhejiang, Jilin and acid-washed Zhejiang
diatomite were acquired. So, it was obtained that
conformational mode of urokinase adsorbed in Zhejiang diatomite probably belonged to multi-layer
adsorption, owing to a 0:7; urokinase adsorbed
on Zhejiang diatomite (roasted at 400 and 800 8C)
as spheroids with radius equaling rotation radius,
owing to a 0:066, and urokinase on the surface

Fig. 7. The effect of particle size on the adsorption isotherm of urokinase on Zhejiang diatomite.

Y. Yang et al. / Applied Surface Science 206 (2003) 2028

of Zhejiang diatomite (roasted at 9501150 8C) as a

random coil owing to a 0:5. But the conformational
mode of urokinase that adsorbed in Jilin diatomite was
spheroids owing to a 0:065. Adsorbed in Jilin diatomite (roasted at 1150 8C), the urokinase might be
attached either by mono- or multiple-points, owing to
a  1. Conformational mode of urokinase that
adsorbed in acid-washed Zhejiang diatomite was analogous to that in Zhejiang diatomite.
3.4. The effect of isoelectric point of diatomite on
adsorption of urokinase
In the solution that has the pH value of 6.6, diatomite adsorbed urokinase by forming ionic binding of
surface BSiO with NH3 R0 or NCH3 3 R00
positive polar groups. The amount of adsorptive urokinase was concerned with IEP value, pore structure,
specic surface, adsorption point number and conguration of diatomite.
IEP value of Zhejiang diatomite (both acidwashed and raw) which was roasted at 400 8C
attained minimum value (as shown in Table 1), so
did the highest acid strength and maximum amount
of adsorptive urokinase. When the roasting temperature rose to >400 8C, the acid strength and the
amount of adsorptive urokinase decreased as IEP
value increased.
For Jilin diatomite, IEP value gradually increased
with the rising of roasting temperature, which resulted
in the strength of acid site and the amount of adsorptive urokinase decreased. The amount of adsorptive
urokinase attained maximum value when the diatomite was roasted at 400 8C, then gradually decreased
at the roasting temperature over 400 8C, because the

27

maximum specic surface of Jilin diatomite reached at

400 8C.
As for modied diatomite, IEP value varied with
modier, accompanying with the amount of adsorptive
urokinase variation. When the surface of diatomite
was modied, respectively, by Ca2, Zr4, CaO, and
colloidal alumina as well as organic compound, the
capacity of adsorptive urokinase on the surface of
diatomite would change accordingly with IEP value
varying as shown in Table 2. When the surface of the
ux calcinated Jilin diatomite was modied by Ca2,
IEP value was kept constant at 2.41. The increase of
trace Ca2 on the surface (undetected by XPS) took
``binding point'' effect on urokinase and, so the
adsorption capacity for urokinase increased from
29.5 to 31.2 mmol l 1 min 1 mg 1.
When the surface of the ux calcinated diatomite was
modied, respectively, by Zr4, and colloidal alumina,
though IEP value increased owing to the adsorption of
Zr4, and Al13O4(OH)24(H2O)127 on diatomite surface [10], metal ion adsorbed on diatomite surface
took ``binding point'' effect on urokinase, so that the
capacity of adsorbing urokinase increased from 29.5 to
37.1 mmol l 1 min 1 mg 1, and 29.5 to 35.9 mmol l 1
min 1 mg 1, respectively. After the ux calcinated
diatomite was calcinated by adding CaO, IEP value
decreased from 2.41 to 1.92 mmol l 1 min 1 mg 1, and
the capacity of adsorbing urokinase also increased from
29.5 to 30.4 mmol l 1 min 1 mg 1.
When the surface of the ux calcinated diatomite
was modied, respectively, by polyglycol and octadecimal amine, OH or NH2 groups on the surface of
diatomite could be increased, which promoted
binding combination with OH, NH2 or CO groups
in urokinase, so the capacity of adsorbing urokinase

Table 1
Relationship between the amount of adsorptive urokinase and IEP of diatomite
Roast
temperature (8C)

Zhejiang diatomite
IEP

G (mmol l

Unroasted
400
800
950
1150

1.93
1.85
1.97
1.96
2.52

760
2430
2370
500
51.6

Acid-washed Zhejiang diatomite

1

min

G of equilibrium concentration at 65 mmol l

mg )

min

IEP

G (mmol l

2.40
1.92
1.95

2.19

620
2600
1380

44.9

mg 1.

min

Jilin diatomite
1

mg )

IEP

Ga (mmol l

1.84
1.94
2.01
2.08
2.10

407
800
400
180
37.0

min

mg 1)

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Y. Yang et al. / Applied Surface Science 206 (2003) 2028

Table 2
Relationship between saturation amount of adsorptive urokinase and IEP value of Japanese diatomite, the ux calcinated Jilin diatomite as
well as it's modied diatomite
Diatomite

IEP

G1 (mmol l

Japanese diatomite
Flux calcinated Jilin diatomite
Ca2 modified
CaO modified
Zr4 modified
Colloidal alumina modified
Polyglycol modified
Octadecimal amine modified
Cetyltrimethyl ammonium modified
Trimethylsilyl chloride modified

2.0
2.41
2.41
1.92
7.41
7.50
2.30
9.46
Positive electrical charged
Electrical neutral

26.0
29.5
31.2
30.4
37.1
35.9
37.0
33.8
25.5
36.2

increased (as shown in Table 2). When the surface

of the ux calcinted diatomite was modied by
cetyltrimethyl ammonium chloride, it had repulsive
interaction with positive groups in urokinase for
the positive electrical charge on particle surface,
so the capacity of adsorbing urokinase decreased
from 29.5 to 25.5 mmol l 1 min 1 mg 1. When the
surface of the ux calcinated diatomite was modied by trimethylsilyl chloride, although particle surface appeared electrically neutral, adsorption of
urokinase on the surface of diatomite particle
depended on its protein chain conguration, so the
capacity of adsorbing urokinase increased (as shown
in Table 2).
4. Conclusion
At ordinary temperature, adsorption isotherms of
urokinase on Sheng county, Zhejiang province diatomite and ChangbaiShan county, Jilin province diatomite surface belonged to type V and II, respectively.
When the diatomite was roasted or acid-washed, the
adsorption isotherm type all changed into type III.
Adsorption isotherm type was concerned with the
surface structure, pore structure, and surface z of
diatomite. When diatomite was roasted at 400 8C,
the IEP decreased to minimum and adsorption amount
of urokinase reached maximum value. After diatomite
was modied, adsorption amount also changed. The
adsorption mode of urokinase on diatomite surface
was also determined, which was consistent with
y=1 y KC1=b equation.

min

mg 1)

Acknowledgements
The project is supported by National Nature
Science Foundation of China (29201004).
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