.alpha.-Olefin-dialkylmaleate-based liquid separating agent

A liquid separating agent used for fractionating a liquid such as blood that contains more than one component of different specific gravities, by separating a specific component from other components by means of the specific gravity difference is characterized by consisting of as the main component an .alpha.-olefin-dialkylmaleate copolymer having a viscosity between 10,000 and 80,000 cp (at 25.degree. C.); a viscosity and specific gravity adjusting agent such as silica or clay; and wax, a surfactant and so on as required.

The present invention relates to a liquid separating agent used for 
separating components in liquids consisting of more than one component of 
different specific gravities. 
This liquid separating agent is used to fractionate a specific component 
from other components using the specific gravity difference between the 
specific component and other components of a liquid to be fractionated and 
by being interposed between these components. For this purpose, it is 
desired that the liquid separating agent have a specific gravity between 
those of said components, be flowable during centrifugal separation, and 
not flowable and stable after the operation of centrifugal separation is 
completed. 
A known conventional liquid separating agent of this kind is a gel-like 
material consisting of silicone oil, silica and a gelling agent. But a 
liquid separating agent of such a composition has problems. For example, 
because components which are mutually insoluble are mechanically mixed to 
obtain a thixotropic gel by means of a gelling agent which accelerates the 
formation of hydrogen bonds between the silica particles (specific gravity 
adjusting agent), coagulation develops due to this hydrogen bond growing 
stronger as time elapses, resulting in phase separation and poor buoyancy 
upon centrifugal separation. To prevent this, addition of a surfactant for 
avoiding phase separation has been proposed, but this presented another 
problem of hemolysis by the surfactant. 
Further, liquid separating agents of the above composition have drawbacks 
such as the tendency to change their nature by cross-linking and other 
chemical changes upon .gamma.-ray sterilization after encapsulation of a 
suitable quantity in a blood collecting tube. This degrades the liquid 
separating function, and delays blood coagulation and clot deposition by 
evaporation of low molecular components of the gel-like material rendering 
the inner surface of the tube water-repellent. Furthermore, these 
separating agents are fairly expensive due to particular raw materials 
being used. 
Another known liquid separatng agent is a gel-like material of polyester 
base. This material is not necessarily satisfactory in that it renders the 
inner surface of the tube water-repellent resulting in delay of blood 
coagulation and clot deposition. Furthermore, liquid separating agents of 
this type smell unpleasant. It is frequently observed when a 7 cc blood 
sampling tube is used that the gel material fails to be sufficiently 
fluidized due to insufficient stress during the centrifusing operation 
owing to an insufficient amount of blood being sampled. 
The present invention was made to eliminate these problems of the prior art 
and has for its object to provide a liquid separating agent that has 
excellent stability against change over long periods of time, can be 
subjected to .gamma.-ray sterilization, show virtually no 
water-repellency, is free of unpleasant odors and can be manufactured at 
comparatively low cost. 
To the above and other ends, the present invention provides a liquid 
separating agent for fractionating a liquid that contains more than one 
component of different specific gravities by separating a specific 
component by means of the specific gravity difference characterized by 
consisting of, as the main component, an .alpha.-olefin-dialkylmaleate 
copolymer having a viscosity between 10,000 and 80,000 cp, preferably 
between 40,000 and 80,000 cp (at 25.degree. C.) and viscosity and specific 
gravity adjusting agents mixed therewith.

In the present invention, the .alpha.-olefin-dialkylmaleate copolymer has 
the general formula: 
##STR1## 
where R.sub.1 stands for an alkyl group having 2 to 58 carbon atoms which 
in the copolymer molecule may either be the same kind or a combination of 
different kinds as desired; R.sub.2 and R.sub.3 are selected from the 
group consisting of methyl, ethyl, butyl, and 2-ethylhexyl groups; n is an 
integer which allows the viscosity of said copolymer to be in the range of 
40,000 to 80,000 cp (at 25.degree. C.) when using said copolymer as the 
main component. 
This .alpha.-olefin-dialkylmaleate copolymer is light yellow in color, 
transparent, odorless, inert to blood, free of blood absorption, elution 
and so on, and stable for long periods of time. It allows the inner 
surface of the blood collecting tube to remain clean since it does not 
produce any water-repellent material. It does not substantially change its 
chemical or physical nature upon sterilization with gamma rays or the 
like. 
When the liquid separation agent of the present invention is used for blood 
serum separation, the specific gravity of this 
.alpha.-olein-dialkylmaleate copolymer is selected between 1.00 and 1.038, 
preferably between 1.02 and 1.035. 
According to the present invention, aliphatic amine derivatives of smectite 
clay may be aliphatic primary amine, aliphatic secondary amine or 
aliphatic tertiary amine derivatives of smectite clay. These amine 
derivatives are already known. Among these derivatives, aliphatic tertiary 
amine derivatives of smectite clay are most desirable, examples of which 
are Bentone 34, Bentone 38, Bentone 27, and Bentone 128 (quaternary 
ammonium salts of smectite clay, products of NL Industry CO.). 
The inorganic fine powder used as the viscosity and specific gravity 
adjusting agent in the present invention may be suitably selected from 
calcined silica, precipitated silica and so on. 
The structure-forming agent used in the present invention is added for 
maintaining the gel state of the liquid separation agent, and can be 
selected as desired according to the liquid to be fractionated. In the 
separation of blood serum, for example, 
dimethylpolysiloxane-polyoxyalkylene copolymer (e.g., trade names SH-3771, 
SH-190, and SH-192 of Toray Silicone Co., Ltd.) or Carbitol (e.g., 
ethyldiglycol) and the like may be used. 
In the present invention, in addition to the .alpha.-olefin-dialkylmaleate 
copolymer, the viscosity and specific gravity adjusting agent and the 
structure-forming agent, a surfactant (e.g., polyoxyethylene-hydrogenated 
caster oil monolaurate, polyoxy-ethylene-hydrogenated caster oil 
tri-isostearate or the like) may be added as required. 
Compositions of the liquid separating agent of the present invention for 
serum separation are shown in the following tables 1 and 2. In these 
tables, the .alpha.-olefin-dialkylmaleate copolymer (A) is an 
n-.alpha.-olefin-dimethylmaleate copolymer average molecular weight 
3,000-4,000; specific gravity 1.027-1.038 (at 25.degree. C.); viscosity 
40,000-70,000 cp (25.degree. C.) of a combination of .alpha.-olefin 
components with carbon atom numbers 12 and 14, respectively; the copolymer 
(B) is an n-.alpha.-olefin-dimethylmaleate copolymer average molecular 
weight 2,000-3,000; specific gravity 1.005 (28.degree. C.); viscosity 
10,000-15,000 cp (28.degree. C.) of a combination of .alpha.-olefin 
components with carbon atom numbers 6 and 8, respectively; and the 
copolymer (C) is an n-.alpha.-olefin-dimethylmaleate copolymer of 
.alpha.-olefin components with an average carbon atom number between 30 
and 60. 
TABLE 1 
______________________________________ 
(examples of serum separation agent compositions) 
Unit: parts by weight 
Composition number 
1 2 3 4 5 6 
______________________________________ 
.alpha.-olefin-dialkylmaleate 
copolymer 
(A) 100 100 100 100 50 -- 
(B) -- -- -- -- 50 100 
(C) -- -- 1.0 3.0 -- -- 
Aerosil R-972* -- 2.0 2.0 1.0 
Aerosil 200** 1.0 1.0 -- -- -- -- 
Benton 38*** 1.0 1.0 1.0 3.0 
Benton 34*** 1.0 
Benton 27*** 3.0 
Benton 128*** 2.0 
______________________________________ 
*hydrophobic silica fine powder of average particle size 16 m.mu., 
apparent specific gravity about 60 g/l, (product of NIPPON AEROSIL CO., 
LTD.) 
**hydrophilic silica fine powder of average particle size 12 m.mu., 
apparent specific gravity about 60 g/l, (product of NIPPON AEROSIL CO., 
LTD.) 
***quaternary ammonium salts of smectite clay, (products of NL Industry 
CO., U.S.A.) 
TABLE 2 
______________________________________ 
(examples of serum separation agent compositions) 
Unite: parts by weight 
Composition number 
7 8 9 10 11 12 
______________________________________ 
.alpha.-olefin-dialkylmaleate 
copolymer 
(A) 100 100 100 100 50 -- 
(B) -- -- -- -- 50 100 
(C) -- -- 5.0 5.0 -- -- 
Aerosil R-972* 6 -- 7.0 5.0 7.0 7 
Aerosil 200** -- 6 -- -- -- -- 
Surfactant*** 0.5 1.0 1.0 -- 1.0 3.0 
Structure-forming 
agent**** 0.18 0.04 -- 0.2 0.5 0.6 
______________________________________ 
*see Table 1 
**see Table 1 
***Polyoxyethylenehydrogenated caster oil monolaurate, (product of NIHON 
EMULSION CO., LTD.) 
****SH3771, (product of Toray Silicone Co., Ltd.), 
dimethylpolysiloxanepolyoxyalkylene copolymer with specific gravity 
1.060-1.080 (20.degree. C.) and viscosity 260-280 cp. 
Next, the preparation method of the liquid separating agent of the present 
invention will be described. 
First, the manufacturing method of the .alpha.-olefin-dialkylmaleate 
copolymer is low polymerization of ethylene to obtain an n-.alpha.-olefin. 
This is then separated into fractions of carbon atom numbers, for example, 
of 4, of 6, of 8 and 10, of 12 and 14, of 16 and 18, and of 30 to 60 by 
fractionating distillation. In accordance with the specific gravity of the 
liquid to be fractionated, these fractions may be used either singly or in 
combination, and a fraction with carbon atom numbers of 12 and 14, or of 6 
and 8 is preferable for use in serum separation from the standpoints of 
viscosity and specific gravity. The selected fraction is subjected to 
copolymerization with a maleic diester in the conventional manner to 
obtain the desired product. 
Using this .alpha.-olefin-dialkylmaleate copolymer with a viscosity between 
10,000 and 80,000 cp, preferably between 40,000 and 80,000 cp (25.degree. 
C.) as the base, an aliphatic amine derivative of smectite clay, a 
viscosity and specific gravity adjusting agent such as fine silica powder, 
a structure-forming agent and a wax consisting of an 
.alpha.-olefin-maleic-diester copolymer are aded as required. The mixture 
is kneaded using either a roll mill, a grinding mill, a planetary mixer or 
the like. 
The liquid separating agent thus prepared should preferably has a 
viscosity, for use in serum separation for example, between 250,000 and 
800,000 (25.degree. C.) and a specific gravity between 1.035 and 1.055. 
All of the components in the previous tables are thixotropic, showing 
flowability upon application of a centrifugal force or the like and 
staying in the normal condition as a stable uniform gel otherwise. 
Furthermore, because this liquid separating agent does not contain a 
gelling agent such as those which are used conventionally, it is not apt 
to coagulate in time as stated previously, nor does it foster phase 
separation or deterioration of buoyancy. Its specific gravity is 
comparatively high being close to that of blood serum, so that even if 
phase separation does take place the base layer does not float suspended 
in the serum. 
EXAMPLE 
As shown in FIG. 1, about 1.7 ml of each liquid separating agent 
composition 1 above (numbers 1 to 12) was put it the bottom of a 10 cc 
blood collecting tube 2. A polyester unwoven cloth 3 coated with 1-5 mg of 
diatomaceous earth (e.g., Caper Flattery Sand, trade name WG-200, for 
Kyoritsu Ceramic Materials Co., Ltd.) or micro-glass powder, was then 
placed at a slant in each blood collecting tube. Each tube was then 
stopped with a butyl rubber plug 4, and the tubes were placed under 
reduced pressure. Then a blood sample was placed in each blood collecting 
tube and allowed to stand for 7 to 8 minutes. As a result, the 
diatomaceous earth dispersed in the blood upon the introduction of the 
blood, and it accelerated blood coagulation together with the nonwoven 
cloth 3. Adequate coagulation was thus attained within this short time. 
Each blood collecting tube was placed in a centrifuge for 10 minutes at 
700-1,000 G, and the liquid separating agent compositions were stably 
distributed between the serum and the clot layers. This state is shown in 
FIG. 2. Because the liquid separating agent is thixotropic and has a 
specific gravity between that of the blood serum 5 and that of the blood 
clots 6, it stays between the blood serum 5 and blood clot 6, forming a 
gel that separates these two layer. Since the diatomaceous earth and the 
unwoven cloth 3 have higher specific gravities, they were not included in 
the layer of blood serum 5. Thus, blood serum 5 obtained was of high 
purity with no entrainment of fibrin. This blood serum 5 was readily 
collected from the blood collecting tube by decantation or by suction. 
Because the smectite clay particles are not light-transmitting, their state 
of dispersion can easily be checked by means of a microscope etc., making 
quality control easier. Because the aliphatic amine derivatives of 
smectite clay increase viscosity, the structure-forming agent which was 
conventionally essential can be eliminated. The use of wax in the above 
example is especially effective for prevention of phase separation of the 
liquid separating agent. Because the .alpha.-olefin-dialkylmaleate 
copolymer is used as the main component in the present invention, the 
manufacturing cost is very low, being about one third of that of liquid 
silicone. 
When being used as a serum separator, the liquid separating agent does not 
form a water-repellent film in the blood collecting tube by releasing 
water-repellent substances and consequently does not cause delay of blood 
coagulation. Because the blood collecting tube is made of glass, it 
accelerates coagulation when contacting blood at the surface, so that it 
is necessary to keep the inner surface clean. Accordingly, compared with 
conventional liquid separating agents which have the drawback of forming a 
water-repellent film, the time needed for collecting blood serum is 
shortened. This is even more effective in combination with the use of the 
diatomaceous earth and unwoven cloth. The time saving can amount to as 
much as 30 minutes. 
When encapsulation in a blood collecting tube is performed, sterilization 
is desirable. In clinical tests, no chemical or physical changes that 
cause adverse effects were found after applying gamma-ray sterilization. 
In the above examples, use of the liquid separating agent of the present 
invention for serum separation was described, but it is to be understood 
that similar applications in other liquid separations are possible.