CA1145737A - Filling composition for use in liquid chromatography - Google Patents

Filling composition for use in liquid chromatography

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Publication number
CA1145737A
CA1145737A CA000345165A CA345165A CA1145737A CA 1145737 A CA1145737 A CA 1145737A CA 000345165 A CA000345165 A CA 000345165A CA 345165 A CA345165 A CA 345165A CA 1145737 A CA1145737 A CA 1145737A
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Prior art keywords
liquid chromatography
serum protein
copolymer
bead
hydroxymethylated
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CA000345165A
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French (fr)
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Masaaki Nakao
Fumio Kodama
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Kureha Corp
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Kureha Corp
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26Synthetic macromolecular compounds
    • B01J20/261Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26Synthetic macromolecular compounds
    • B01J20/264Synthetic macromolecular compounds derived from different types of monomers, e.g. linear or branched copolymers, block copolymers, graft copolymers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26Synthetic macromolecular compounds
    • B01J20/265Synthetic macromolecular compounds modified or post-treated polymers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282Porous sorbents
    • B01J20/285Porous sorbents based on polymers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/3092Packing of a container, e.g. packing a cartridge or column
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206Organic carriers, supports or substrates
    • B01J20/3208Polymeric carriers, supports or substrates
    • B01J20/321Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268Macromolecular compounds
    • B01J20/3272Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274Proteins, nucleic acids, polysaccharides, antibodies or antigens

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)

Abstract

FILLING COMPOSITION FOR USE IN LIQUID CHROMATOGRAPHY
ABSTRACT OF THE DISCLOSURE:
Disclosed herein is a bead-like substance comprising a hydroxymethylated copolymer of styrene and divinylbenzene having a serum protein adsorbed or adhering thereonto, suit-able for filling the column used in liquid chromatography in the field of analysis, particularly in clinical examinations.

Description

BACKGROUND OF THE INVENTION:
The present invention relates to a novel filling compo-sition for use in the column of a liquid chromatograph, and to the liquid chromatography particularly suitable for clinical examinations, using the filling composition.
It is extremely important for the diagnosis of diseases and the establishment of the guiding principle of treatments of the disease to obtain information on the morbid state by analysing the properties and the components of the patient's physiological specimens such as plasma, serum, cerebro-:-~ spinal fluid, urine, etc.
Hitherto, such information has been obtained by . various chemical and biochemical analytic means, howeve~r ln the cases of elucidation of the morbid state of multifarious cases, a:method of examination for obtaining exact information of the morbid state is required.
~ . Particularly in cases of the diseases of the kidney :~ and of the liverj according to the complicatedness of the morbid statæ, ther.e:has been a keen request for the development of new -~ 20 indications~ That is, as the indications conventionally utilized : of the morbid state, various determined values are mentioned, for : . ~ example in the renal disease, the values representing the renal functions such as creatinine,uric acid and electrolytes in blood, protein and sugar in urine and pH of urine, and in the hepatic : disease, the values representing the hepatic functions such as ~hA . eiviti of bioenz~mes, ~ Instance, ~OT, GPT, LDP anG L~P, . ,. :
. ~
~ .
: ~
;

11~5737 and`the components of bloods, for instance, protein and bilirubin.
These data of various determined values are playing important roles and are actually utilized for establishing the dia-gnosis and the guiding principles for treatment in their own ways.
However, recently it has been begun to point out that there is an intimate relationship between the uremic toxins and the morbid state of renal diseases, and the above-mentioned values determined by the conventional methods are not able to confirm the presence of the above-mentioned toxins, and moreover, no simple methods for detecting and confirming the toxins have yet been developed. Also in the hepatic diseases, in spite of the suggestion of the presence of various substances by abnormal metabolism and of the etiopathogenic substances, there are no suitable methods for detecting and confirming such substances at present.
In short, the present analytical methods of chemistry and biochemistry are not sufficient, although they are useful.
And accordingly, the development of a suitable method for detecting and confirming the above-mentioned substance for supplying newer indications of the morbid state is keenly expected.
In consideration of the above-mentioned status quo, the utilization of the liquid chromatography which is one of analyti-cal means based on the different principles from those of chemical and biochemical analytical methods has recently attracted the specialists' attention. Even thermally and chemically unstable ~substances sr possibly determined by the liquid chromatography " ll'~i 73~

without being denaturation, and multiple components can be determined with a relatively small amount of the specimen, and accordingly, in principle, the applicability of the liquid chromatography in the fields of medicine and clinic is large enough. However, in order to put the liquid chromatography into practical use in the above-mentioned fields, there have been problems to be solved concerning the selection of the filling material, the establishment of conditions for separation in the chromatography and the pre-treatments adopted in accordance with the necessity.
For instance, Chang et al.l) tried to detect the above-mentioned toxins appearing in the blood of the patient suffering from the renal disease by using the liquid chromatography with columns filled with porous beads of cross-linked dextran.
As a result, Chang et al. found that in the-serum of the patient suffering from the-renal disease different from the normal serum there aré some substances which give specific peaks in the chromatogram obtained by their liquid chromatography, and suggested that the peaks include some harmful substances.
However, there are defects in the method of Chang et al., that is, the specific peaks obtained by the method are extremely broad and the method necessitates a relatively large amount of the serum specimen of 2 to 3 ml and moreover, it takes 4 to 7 hours in treating one specimen.

)T.M.S. Chang et al. "Trans. Amer. Artif. Int. Organs", Vol. XX, page 364 (1974).

Accordingly, the method is not satisfactory as the clinical method of examination.
On the other hand, Furst 2)proposes a method of analysis of serum specimens by the high-speed liquid chromatography, how-ever, although Furst's method was successful in shortening the time period for analysis, there is a defect of appearing many peaks over-lapping each other in the obtained chart of the chromatography to make the separation and identification of each component difficult. In addition, either of these methods has not been tried with an intention of applying to clinical examina-tion. That is, although the analysis of bio-specimens by liquid chromatography has been tried, even if it is possible to detect some specific peaks in the chromatogram of a specimen from the patient suffering from a disease of the liver or the kidney, the result does not provide information on the relationship between the progress of the morbid state and the appearance of the peaks in the chromatogram. Accordingly, both methods of Chang and Furst have not been put into practical use.
We, in consideration of the above-mentioned status quo, have studied the methods for effectively examining clincially the physiological specimens from patients of, particularly, renal diseases, etc., and as a result, we have found that on the appli-cation of the liquid chromatography using a specified filling )P. Furst, "Clinical Nephrology", Vol 5 (4), page 198 (1976) ~:14573;'' composition in its columns to such physiological specimen, it is possible to separate and detect the peaks in the chromatogram, which have relationship to the progress of the morbid state of the above-mentioned diseases within a short time period with only a small amount of the specimen.
Accordingly, the object of the present invention is to offer a novel filling composition for use in the column used for the liquld chromatography, particularly suitable for clinical examinations.
;10 Another object of the present invention is to offer the chromatographical technique using the column utilizing the above-mentioned filling composition, particularly the technique of high-speed li~uid chromatography. The other objects of the present invention will be made clear from the following description.
¦ Thus, the present invention provides a filler composition for use in a column for liquid chromatography used for clinical examination, comprising a bead-like hydroxymethylated copolymer of styrene and divinylbenzene having a serum protein absorbed or adhered thereonto in an amount of 0.1 to 1% by weight of the bead-like hydroxymethylated copolymer on the dry basis.
In another aspect, the invention includes a method of ~liquid ohromatography comprising using a column filled up with a substance formed by adsorping or adhering of a serum protein on-"''~! ~ to a bead-like hydroxymethylated copolymer of styrene and di-~J vinylbenzene.
BRIEF EXPLANATION OF DRAWINGS:
l .
~ Figure 1 of the drawings shows chromatograms of serum".~
specimens obtained by the high-speed liquid chromatography in ~ Example l, and (a) is the chromatogram of normal serum, and -~30~ ~ tb)~is the chromatogram of the serum of a patient suffering from ~ it~ ~
;f ~ renal failure.

~ ~ - 5 -Figure 2 of the drawings also shows chromatograms of serum specimens obtained by the high-s?eed liquid chromatography in Example 3, and (a) is the chromatogram of normal serum, and (b) is the chromatogram of the serum of a patient suffering from liver disorder, and (c) is the chromatogram of the same patient ', ~

-- 5a -;~ , ~ 1145~73~

; after recovering from the liver disorder.

DETAILED DESCRIPTION OF THE INVENTION:
The filling composition for use in the column of the liquid chromatography according to the present invention comprises the bead-like substance consisting of a hydroxymethylated copolymer of styrene and divinylbenzene, to which serum protein has been adsorbed or is adhering.
The filling composition of tke present invention is prepared as follows:
(a) The preparation of the bead-like substance comprising a hydroxymethylated copolymer of styrene and divinylbenzene:
For instance, a monomeric mix~ure of styrene and divinylbenzene is polymerized in suspension in a non-solvent, for instance, in water in the presence of a polymerization initiator to obtain a bead-like copolymer of styrene and divinylbenzene.
By bringing the bead-like copolymer into reaction with formal-dehyde, a hydroxymethylated copolymer of styrene and divinyl-benzene is obtained. The commercialized bead-like copolymer of styrene and divinylbenzene hitherto available for use in the column of the high-speed chromatography as the filler may be applied as well as in the following step.
The particle size of the bead-like copolymer and its degree of hydroxymethylation are possibly selected respectively in accordance with the purpose of the liquid chromatography, however, the former i acually S ~o 50 mioron in diameter and the latter ~ ~ : : , ~ ~ - 6 -,.'~ ,, .
.

is usually 0.05 to O.S.
A (b) Adsorption ~ adherence of serum protein onto the bead-like oxymethylated copolymer of styrene and divinylbenzene:
~7J~bG/~f~
A serum protein, for instance, albumin and- ~ lin, obtained from a broad range of mammals such as mankind, cattle, horse, dog or sheep is dissolved in a solvent mentioned as follows, and the solution is brought into contact with the above-mentioned bead-like hydroxymethylated copolymer under agitation and then the system is separated into the liquid and the solid to obtain a hydroxymethylated copolymer of styrene and benzene having a serum protein adsorbed or adhering thereonto. The above-mentioned contact process is èxecuted by immersing the above-mentioned hydr-oxyme~hylated bead-like copolymer into the solution of serum protein or by passing the solution of serum protein through a column filled with the bead-like hydroxymethylated copolymer. In addition, adsorption or adhesion is automatically completed only by bringing the above-mentioned two substances into contact with each other because of the adsorbing facility of the hydroxy-methylated copolymer for the serum protein.

The above-mentioned solution of serum protein used for adsorption or a &esion is obtained by dissolving the protein into water, a buffer solution or a buffer solution containing a salt such as sodium perchlorate or an organic solvent such as methanol, ethanol, propanol and dioxan. In cases where the concentration of the serum protein in the above~mentioned solution is high, the adsorption or adhesion of the serum protein to the 1:~573~7 hydroxymethylated copolymer is completed within a short time period, however, the state of the adsorption or adhesion tendsto be non-uniform, while in cases where the above-mentioned concentra-tion is low, it takes a long period of time for the completion of adsorption or adhesion. In such circumstances, the concentration of the serum protein in the above-~entioned solution is preferably 0.2 to 5% by weight.
As is mentioned above, after having the serum protein adsorbed or adhering onto the bead-like hydroxymethylated copolymer of styrene and divinylbenzene, the filllng composition of the present invention is obtained by washing well the bead-like copolymer thus treated. In order to apply the filling composition into the liquid chromatography, the filling composition is filled into the column for use in the chromatographical analysis.
However, since the interparticle coagulative tendency has been raised by the adsorption of the serum protein, the uniform fill-ing of the filling composition into the column has come to be difficult. Care should be paid on this fact. In order to avoid the troubles on the filling of the column with the filling composition of the present invention, it is recommended that the hydroxymethylated copolymer not yet having adsorbed the serum pro tein is in advance filled into the column and after equipping the filled column onto the conventional appz~atus of the liquid chromatography, the solution of the serum protein is passed through the column. According to the above-mentioned technique, the time required for the serum protein to be adsorbed or to 114573~ 1 adhere onto the bead-like hydroxymethylated copolymer becomes longer, while on the other hand there is a merit of simplification of the preparatory operation for the analysis.
On the treatment for adsorption or adhesion of the serum protein onto the bead-like hydroxymethylated copolymer, the tempera-ture is kept at a degree at which denaturation of the serum protein does not occur, that is, 5 to 70C, preferably at 20 to 40C.
The pH of the solution of the serum protein may be in the range in which the denaturation ana coagulation of the serum protein do not occur, and is usually selected suitably from the range of 5 to 9, however, it is natural to avoid the isoelectric point of the serum protein. In addition, on the preparation of the solution of the serum protein, the use of a buffer solution having the same pH value as the pH value of the moving phase used for the liquid chromatography carried out by using the column filled with the filling composition of the present invention facilitates the maintenance of the steady state of the solution of the serum protein during the operation of the examination and is suitable for the purpose.
Accordingly, the adsorption or adhering of the serum protein onto the bead-like hydroxymethyl2ted copolymeris preferably carried out under the same conditions as far as possible to those at the time of examination, in considering the stability of the filling composition of the present invention.
The amount of the serum protein adsorbed or adhering onto the bead-like hydroxymethylated copolymer of thepresent inven-tion varies slightly corresponding to the kinds of the bead-like hydroxymethylated copolymer and those of the serum protein for use in the liquid chromatography, however, usually in the range of 0.1 to 1% by weight of dry matter, preferably 0.2 to 0.5~ by weight of dry matter.
The column filled with the filling composition of the present invention is possibly used for examination of various physiological specimens after installing the column onto any commercial apparatus for liquid chroma_ography or onta any other apparatus having the same function as ~ove, and the thus instal-led apparatus is able to separate and detect the components of the specimen as the peaks which have relationships to the progress of the morbid state. In addition, the physiological specimens mentioned in the present invention include the-blood compone~ts A - Iyp~Ph~ c~5e~fes~
such as serum and plasma, cérebrospinal fluid, lymph~; asai~
urine, etc.
In the next place, the method of examination utilizing the filling composition of the present invention, particularly the method of examination of physiological specimens by the high-speed liquid chromatography can be executed under the following conditions, however, the followings only illustrate the mode of its execution referring to examples no, being limited in them-selves.
As the moving phase for use in the column filled with the filling composition of the present invention, water, a buffer - 1~573~

solution or the buffer solution containing some salt component such as sodium perchlorate or an organic solvent such as methanol, ethanol, isopropyl alcohol, dioxan, etc. is preferable. Parti-cularly preferable material as the moving phase is a phosphate buffer, and by using the phosphate buffer as the moving phase, extremely finely separated peaks are available in the chromato-gram.
In addition, since the filling composition of the pre-sent invention contains the protein and accordingly there is a probability of suffering from undesira~ie transformations such as the degradation by microorganisms d17~ing the long time period of using the column filled with the filling composition of the present invention, it is preferable in those cases to add a minute amount of an anti-microbial agent such as sodium azide, etc. to the moving phase.
The temperature at which the examination is carried out by using the liquid chromatography utilizing the filling composi-tion of the present invention is 20 to 40C. The amount of the specimen required for carrying out the analysis by the above-mentioned apparatus may be 1 to 15 microliter.
The liquid chromatography according to the present invention is provided with a detector comprising an ultra-violet spectrophotometer and an ordinary spect-ophotometer, however, other than t'nose mentioned, a differential refractometer, a fluorescence spectrophotometer, an infrared spectrophotometer, a radiation detector, a polarograph or a conductometer may be ~ 1i~3~

optionally utilized after careful selection.
In addition, in order to quantify the obtained data, the peak area on the chromatogram is possibly quantified by connecting a data-treating machine to the above-mentioned detector.
The liquid chromatography utilizing the filling composi-tion of the present invention is not only applicable to the clinic examination but also, for instance, to separately collect-ing fractions after filling into a larger column.
The present invention is explained as follows by refer-ring to the Examples, however, the sco?e of the present invention is not to be limited within Examples.
Example 1:
- After filling a hydroxymethylated bead-like (10 to 15 microns in diameter) copolymer of styrene and divinylbenzene prepared by a well known process into a stainless-steel column of 4 mm in-diameter and 50 cm in length by an ordinary technique, the column was installed onto a high-speed liquid chromatograph provided with an ultraviolet detector.
Into the thus installed column, a phosphate buffer of pH of 7.4 was introduced at a rate of 1.2 ml/min and after confirming that the indications of the recorder and of the integrating planimeter became stabilize~, an aqueous 10% solution of human serum albumin was introduced l-=o the column from the specimen-inlet. After repeating the introduction of the solution of albumin to obtain the fixed height of the indication on the recorder or the fixed value of the indication of area on the 11~S737 integrating planimeter, the treatment of the bead-like filler with the above-mentioned solution of albumin was completed.
Then, using the above-mentioned high-speed liquid chromatograph provided with the thus prepared column in which the bead-like filling had ad'sorbed or a &ere~'to the human serum albumin completely, the examination of the serum of a normal person and the serum of a patient of chronic renal failure under dialytic treatment was carried out.
As the results of the examina~ion which took 30 minutes, lo the charts shown in Fig. 1 illustratins the peaks a, (b), (c), d, e, (f) and (g) were obtained, the paren~hesized peaks appearing only on the chart of the serum of the pztient of chronic renal failure.
The mutualseparation of the peaks appearing only on the chart taken on the serum of t~e patient of chronic renal failure ''and their ~uantificàtion suggest the application of the quantified values of these peaks as the other values of clinical examination than the conventional values of conventional clinical examination.
For comparison, the same procedure of the high-speed liquid chromatography was carried out except that the filling of the column was carried out using the same bead-like hydroxymethy-lated copolymer of styrPne and divinylbenzene, however, without having adsorbed or adhering the serum zl_umin, on the serum of the patient of chronic renal failure under dialytic treatment.
The result showed that (1) the peak a was lower than that o~taincd by using the filling composition of the present '' "

, :114573'7 invention and (2) the separation of the peaks b, _, d, etc. was insufficient.

Table 1: Effluent Time and Relative Area of Peaks of Normal Serum and Serum of Renal Failure in Liquid Chromatography Relative Area of Peaks of Specimen ~ I l a b c d e f g .
Normal Serum 9895 _ _ 323 52 _ Serum of 8046 1301293 ~S-3 1001505 38 Effluent time 4.7 6.9 7.8 8.9 10.6 11.8 25.4 of each peak to tmin~ _ 16.5 Example 2:
Using the same high-speed liquid chromatograph equipped with the column filled with the filling composition obtained by the procedures shown in Example 1, the transition of the morbid state of a patient with the patient's history of from slight renal failure through hospitalization to the treatment with dialysis was : followed by the chromatographic examination of the patient's serum as well as by the determination of BUN --d creatinine. The results are shown Ln Table 2.
As is seen in Table 2, corresponding to the aggravation ~ 114~737 of the values of conventional clinical examination such as BUN
and creatinine in the serum, the number of the abnormal peaks appearing in the chromatogram increased indicating the usefulness of the method according to the present invention.

Table 2: Progress of Morbid State of a Patient of Renal Failure and Results of Chromatography of the Patient's Serum Relative Area of Each Peak Values of Days of clin cal examn. Clinical .~ observation b c d e f g ~ (mg/dl) findings 0 72 _ a3a 47l _ _10l ~ ~

7 57 _ 736 446 _ _101 8.3 48 78 116 434 514 21 116 105 9.2 aggravation 56 95 214 534 618 70 191 104 9.9 hospitalization 63 104 234 770 626 170 168 102 10.8 preparation for dialysis 144 243 730 61Q 191 388 102 10.8 77 151 328 588 589 222 545 103 10.5 _ " ____ : Example 3:
A female JCL-SD rat was made ,~ be a model of the liver disorder by administratlon of 1,000 mg/kg of D-galactosamine and : its serum was sampled before the administration tnormal stage), during the morbid state and during its recovering period, and the ,~ ~ .
~ , ' ~ - 15 --:
" ' ' ~
- .
. . . . .

1~73~

specimens of the serum were examined by the high-speed liquid chromatograph shown in Example 1 provided with the column filled with the filling composition of the present invention. As the results, a chart shown in Fig. 2 and the relative values of area of the peaks on the chromatogram shown in Table 3 were obtained.
Separately, biochemical analyses were carried out on GOT, GPT and bilirubin, the results being shown also in Table 3.
As is shown in Fig. 2 and Table 3, in the serum of the rat administered with D-galactosamine to be a model of the liver disorder, the values of GOT, GPT and b:l;rubin were clearly abnormal after administration as compared to the values before the administration and after the recovery has begun. Just corre-sponding to the transition of the above-mentioned values, each of the peaks of more than 5 minutes of the effluent time showed a conspicuous change.
" ..
Table 3: Result of Chromatography of Rat in Model Liver Disorder with Results of Clinical Examination SpecimenRelative Area of each Peak of Nos. Data of Clinical .
of serum Examirlation (m~/dl) .
1 2 3 4 5 6 7 8 9GOT GPT Bilirubin .

state34156 :37.37 132211 7~3 - 246128100 70 less than .
At liver 35292 41155 30 202 38 1075 512 795 4560 2260 3.9 disorder . .
At reco- l vering379113i 122 651808 78 _ 56 113 540 520 0.5 .
.
Effluent : time of 4.56.3 6.87.4 8.8 10.6 13.8 15.4 19.1 - ~14573~

As is possibly understood from the results of the above-mentioned examination, by the application of the high-speed liquid chromatography utilizing the filler composition of the present invention, a chromatographic pattern corresponding to the transi-tion of the morbid state is available also in the case of the liver disorder. On the other hand, the examination of the above-mentioned specimens of the serum by the same chromatography, however, with the column filled with the bead-like oxymethylated copolymer of styrene and divinylbenzene without adsorbed or adher-ing the human serum albumin gave a char_ in which, as is shown in the later part of Example 1, the peak 1 was lower in height and the separation of the peaks 2, 3, 4.,7 a-nd 8 was insufficient.
,L

Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A filler composition for use in a column for liquid chromatography used for clinical examination, comprising a bead-like hydroxymethylated copolymer of styrene and divinylbenzene having a serum protein absorbed or adhered thereonto in an amount of 0.1 to 1 % by weight of the bead-like hydroxymethylated copolymer on the dry basis.
2. The filler composition according to claim 1, wherein the amount of said serum protein absorbed or adhered onto said bead-like hydroxymethylated copolymer is in the range of 0.2 to 0.5 % by weight of said bead-like hydroxymethylated copolymer on the dry basis.
3. The filler composition according to claim 1, wherein said serum protein is selected from the group consisting of albumin, globulin and a mixture thereof.
4. The filler composition according to claim 1, wherein the adsorption or the adhesion of said serum protein is carried out by passing a solution of said serum protein through a column for chromatography, in which said bead-like hydroxymethylated copolymer of styrene and divinylbenzene has been filled up.
5. The filler composition according to claim 4, wherein said solution of said serum protein is a solution comprising dissolving said serum protein into a solvent selected from the group consisting of water, a buffer solution, a buffer solution having a salt added thereinto, or a buffer solution having an organic solvent added thereinto.
6. The filler composition according to claim 4, wherein the concentration of said serum protein in said solution of said serum protein is in the range of 0.2 to 5 by weight.
7. A method of liquid chromatography comprising using a column filled up with a substance formed by adsorping or adhering of a serum protein onto a bead-like hydroxymethylated copolymer of styrene and divinylbenzene.
8. The method of liquid chromatography according to claim 7, wherein said liquid chromatography is a high-speed liquid chromatography.
9. The method of liquid chromatography according to claim 7, wherein said liquid chromatography is used for examination of a physiological specimen.
10. The method of liquid chromatography according to claim 9, wherein said physiological specimen is the blood, the cerebrospinal fluid, the ascites or the urine.
CA000345165A 1979-02-07 1980-02-06 Filling composition for use in liquid chromatography Expired CA1145737A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP13161/79 1979-02-07
JP1316179A JPS55106357A (en) 1979-02-07 1979-02-07 Filler for liquid chromatography

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CA1145737A true CA1145737A (en) 1983-05-03

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CA000345165A Expired CA1145737A (en) 1979-02-07 1980-02-06 Filling composition for use in liquid chromatography

Country Status (6)

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JP (1) JPS55106357A (en)
CA (1) CA1145737A (en)
DE (1) DE3004356C2 (en)
FR (1) FR2448718A1 (en)
GB (1) GB2042557B (en)
IT (1) IT1150076B (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE457797B (en) * 1982-01-13 1989-01-30 Kureha Chemical Ind Co Ltd PROCEDURES FOR PREPARING POROEST SUBSTRATE INCLUDING PEARLS THAT HAVE METHYLOL GROUPS OF CO-POLYMS BASED BY THE CIRCUIT BOARD AND APPLICATION OF THE SUBSTRATE BY SCIENCE CHROMATOGRAPHY
JPS58147647A (en) * 1982-02-26 1983-09-02 Kureha Chem Ind Co Ltd Carrier for analizing and manufacture thereof
JPS58120607A (en) * 1982-01-13 1983-07-18 Kureha Chem Ind Co Ltd Carrier having protein adsorbing function and its preparation
DE19605003A1 (en) * 1996-01-30 1997-08-07 Abion Ohg Sorption material has pores down to submicron size

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3442819A (en) * 1965-03-26 1969-05-06 Mount Sinai Hospital Research Molecular sieve coated particulate adsorbent and processes using same
FR2388584A1 (en) * 1977-04-26 1978-11-24 Mo Khim T Prepn. of sorbent for extracting toxins from blood - by modifying surface of anion exchanger, active carbon or di:vinylbenzene-styrene! copolymer with blood serum albumin

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DE3004356C2 (en) 1982-04-01
IT8019759A0 (en) 1980-02-07
GB2042557B (en) 1983-01-26
JPS55106357A (en) 1980-08-15
DE3004356A1 (en) 1980-08-14
FR2448718B1 (en) 1985-03-01
FR2448718A1 (en) 1980-09-05
GB2042557A (en) 1980-09-24
JPS6331737B2 (en) 1988-06-27
IT1150076B (en) 1986-12-10

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