JPS58182552A - Packing agent for liquid chromatography - Google Patents

Abstract

PURPOSE:To obtain a packing agent which has excellent performances to separate a trace of containing component such as protein, etc. from a living body sample such as blood, urine etc. and to quantitatively determine it and also, is capable of fractionating at a high speed under low column pressure in case of liquid chromatography. CONSTITUTION:A packing agent for this liquid chromatography consists of a hydrophilic polymer containing a vinyl group monomer as a polymerization component. Consequently, the good separation capacity of components from an aqueous eluate is given. Further, this agent is formed by hollow and nearly globular particles. Hereby, in case of treating by the liquid chromatography after packing it in a column, a high-speed stream is obtained under low pressure and the component is separated at a high speed. Moreover, the ratio L/D of the particle diameter D to the thickness L of this agent at an optional place is set in a range of 0.06-0.45. In addition, a dent is hardly generated in the agent and a rise of pressure in the column is prevented when this agent is used in the column.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a photo-IA agent for body donation chroma dog 2 fee.

Although slow growth and melt chromatography show a remarkable progress in gh, this is largely due to the development of the filler that can withstand the linear pressure at the time of whitening.

As a conventional packing material for liquid chromatography, porous silica, which has a mechanical strength that can withstand pressure, has been introduced with an octadecyl group to give it anisotropic properties.

But ll! With the development of floor testing technology, technology to rapidly and accurately separate proteins from biological samples such as blood and urine has been explored. There was a drawback.

For this reason, there has been a search for fillers that have excellent ability to separate components such as proteins in biological samples, and synthetic molecular fillers such as methacrylic acid-divinylbenzene copolymers have been used. However, such a synthetic crow molecule full of 811K
In machining, the pressure resistance is limited and it can only withstand a pressure of about 30 m/c11, so the high speed of liquid chroma dog 2 fee cannot be achieved sufficiently, and the filler itself is easily destroyed, causing problems in durability. I had left it behind.

The present invention was made with the aim of solving the above-mentioned drawbacks of conventional liquid chromatography fillers, and its gist is to provide a hydrophilic filler containing vinyl thread monomer as a polymerization component. A hollow approximately female-shaped particle made of a polymer, wherein the particle ICDI and the flesh 71 (L) tonolt (L/D ) at any location of the hollow approximately spherical particle
t) A filler for liquid chromatography in the range of LCkO6 to α45.

Next, the packing material for liquid chromatography of the present invention will be explained in more detail.

The housing for chromatography according to the present invention is made of a hydrophilic polymer containing a w4Fi vinyl monomer as a polymerization section. The reason why polymers are made to have hydrophilic properties is to give them protection from aqueous elution waves, and for this reason, the vinyl fibers that make up the polymer are made with carboxyl groups. , acrylic acid or methacrylic acid may be used, or the following general formula % formula % (however, the formula junior 1 + R1 is the methyl method,
It is one song of n#-t2 to 18). Divinyl thread monowax is suitable for use.

xthWhen the divinyl thread monomer represented by the above general formula is used, R,, R, is a methyl group and n
When n = 14, it is tetradecyl glycol dimethacrylate, and when n = 2, it is diethylene glycol dimethacrylate, but these polymers have moderate hydrophilicity and are effective in separating proteins in blood and urine. A polymer with good properties can be obtained.

However, in the zero issue ljJ&c, the vinyl thread f of 11j
The degree of hydrophilicity can be adjusted by making it into a copolymer with a multifunctional monomer such as JLM compound, divinylbenzene, divinyltoluene, diallyl phthalate, diallyl maleate, triallyl isocyanurate, etc. .

Hydrophilic 44 is the solubility of the polymer, (lameter is &
It is preferable that the number is 4 or more.

It is a value obtained from the following formula from solubility/1 Lamécou (SP value) Fi, density (Pi, molecular weight (goods), constant cohesive energy 1k (G)).

5P=pXG/M When the solubility parameter of the polymer is smaller than &4, the polymer becomes less resistant to water and becomes unsuitable for separation from an aqueous eluent.

The filler for liquid chroma dog 2 feed at 1'1K is made of hollow, approximately spherical particles. Since the filler is made of hollow, approximately spherical particles, when the column Kff, a is subjected to liquid droplet chromatography, a high Ui and a high concentration can be obtained at a low pressure, and high-speed separation is possible.

Even if the packing material is approximately spherical and soft particles, if it is not hollow, the column pressure will increase significantly if you try to obtain the same flow rate when performing chromatography using a packed column. If it is held to the same extent as when it is hollow, the separation performance is likely to be impaired.

Furthermore, the packing material for liquid chromatography in the present invention has a particle size (■) at an arbitrary location and a wall thickness (ratio (L) to Ll).
/D) is in the range of α06 to α45. Here, the term "arbitrary location" refers to any location arbitrarily selected, and for approximately whale-shaped particles, (L/D
) means that there are no locations where the value is smaller than α06 or larger than α45.

When the ratio (L/D') of particle size p) to wall thickness becomes smaller than (106), packing 11J tends to be unstable, and when the liquid is subjected to chromatography after filling the column, only 1. Due to the shadow, the self-peel becomes wider. Also, if the ratio (L/D) of grain It (to) and wall thickness W) becomes larger than a45, when liquid is applied to the column after filling the column. If the same flow rate is to be obtained, the column pressure will increase significantly, and if the column pressure is lowered, the separation performance will be impaired.

The filler for liquid chromatographies of the present invention can be obtained by aqueous suspension polymerization of the above-mentioned monomers. The polymerization reaction is carried out in the presence of a diluent with poor compatibility with.

If methacrylic acid or diethylene glycol dimethacrylate is used as the monomer, a mixture of toluene/n-octatool can be used as the Fi diluent.

Aqueous suspension polymerization is described in Example LF! A radical generating catalyst is dissolved in a mixture of monomer m and a diluent, and polyvinyl alcohol,
Stable Sm polymerization of calcic phosphoric acid 5 etc. 1iIIO is separated into 9, added to the aqueous phase, stirred and then heated to SO ~ 10. Catalysts that generate radicals include organic hydroxides such as benzoyl peroxide and cumene oxide, inorganic peroxides such as aqueous peroxide and ammonium persulfate, azobisisobutyronitrile, and azobisisobutyroamide. and other azo compounds are used.

The polymer is washed with water, dried, and then classified to make the particle size uniform.

The optical loading for liquid chromatography of the present invention has excellent performance in separating and quantifying a single amount of protein, etc., from biological samples such as blood and urine. Then ^0 minutes in a row can be done.

Contains 11 to 21 volumes of a 400% polyvinyl alcohol aqueous solution and 60v1 of diethylene glycol dimethacrylate to a 21-volume cellulose membrane.) Luene/n-octano! Mixed liquid (mixing ratio 2/1) 40
and benzoyl/(-okinide L5f) and stirred at a stirring speed of 400-17 minutes.
The mixture was heated to 0°C KiI# and a mold synthesis reaction was carried out for 10 hours. After cooling,
The polymerization product is separated from the mother liquor, washed with hot water and acetone, and the particle size is approximately spherical with a diameter of 7-15. j#lL'-
A diethylene glycol dimethacrylate copolymer was obtained.

This 0@m-shaped copolymer particle O scanning electron micrograph is 41
It is shown in Fig. and II Snow Map. The particle 11 (2) that can be applied to any part of the approximately spherical copolymer particle, the wall thickness (transformation), and the O ratio (
L/D) Fi is about Q14.

The substantially spherical zero copolymer particles obtained in this way were classified and the particle sizes were made almost the same as OJ! 1!04g of isf.

Water was added in fractions into a stainless steel column (diameter 7.11 length z s am ) using a high-pressure constant flow pump.
It was filled by pumping at a speed of 1/min.

The column obtained in this way was connected to a high-performance liquid chromatography device, and an ultraviolet variable fractionation photometer (I!) was used as a detector.
! 411 mmg), 50 ws M above the eluent
Phosphate buffer pH 6.5 and 200 nM phosphate buffer
As a result of separation analysis using H6,3 and normal human lysed blood as a sample, a case chromatogram with a no-hike turn as shown in the 8th wAK was obtained.

As a result of collecting and identifying each peak portion of the detection pattern, P is hemoglobin A1 a + b % p.

are hemoglobins Alc, P, a, and other hemoglobins, and the flow rate LeWIl/min and the column pressure were 30 l/min. The above separation and analysis was repeated 500 times, but no phenomena such as pressure increase occurred.

Example 2 Tetradecyl z f to Glucco with 2I capacity
V fuglycol dimethacrylate 100v and toluene/n-octatool mixture (mixing ratio 1/1) mixture 4
0f was added, and the mixture was heated to 80° C. with moderate stirring at a rate of 4001 ml/min to carry out a 10 hour type reaction. After cooling, the polymerization product was separated from the mother liquor and washed with hot water and acetone to obtain approximately spherical electropolymer particles. Next, classification was performed.

A scanning electron micrograph of this approximately spherical polymer particle is shown in FIG. In addition, the ratio (L/D) between the particle size p) and the wall thickness (t) at any location of the OW&spherical polymer was approximately α08.

The approximately spherical polymer particles were packed into a stainless steel column (inner diameter 7.9, length 5Ocs+) in the same manner as in Example 1, and gel permeation chromatography was performed using dextran as a sample. As a result of separation analysis using a refractometer, dextran of each molecular weight was eluted in descending order of molecular weight, and the exclusion limit was 200,000, which was 69 times.

Comparative Example 1 In Example I, toluene/n-octatool bulk metal 1
(Mixing ratio 2/1) #'i except that the amount used was 6or
A copolymer was obtained by sequentially performing polymerization, washing, and classification in the same manner as in Example 1, but as shown in Figures 4 and 5.*
A part of the wAO was deformed in a concave manner, and as shown in No. 68, the fracture surface was also unstable in terms of wall thickness.

The thus obtained copolymer particles were packed into a stainless steel column in the same manner as in Example 1, and tested using the same measuring device and under the same separation conditions as in Example 11. As a result, a liquid with a pattern as shown in Figure 8 was obtained. A chromatogram was obtained. When this copolymer particle was used, the separation performance was poorer than that in JiH1111, and the column pressure at a flow rate of LOrnl/min was 28 F/min.

Comparative Example 2 In JHRJ I, polymerization, washing, and classification were performed sequentially in the same manner as in Fi Example 1 except that the lid used for toluene/n-octanol mixture (mixing ratio 2/1) was changed to 207 to produce a copolymer. Obtained. Scanning electron micrographs of this copolymer are shown in Figures 6 and 7.
Fracture 11 in the figure [As shown in the figure, it was not a hollow particle.

In addition, a stainless steel column was filled in the same manner as in Example 1, but the pressure increased significantly, causing deformation and breakage of the packing material.

[Brief explanation of drawings]

FIG. 1 is a scanning electron micrograph of a hollow approximately spherical particle in Example 1, FIG. 2 is a scanning electron micrograph showing an enlarged fracture surface of the same, and FIG. It is a scanning electron micrograph showing an enlarged view of a fractured surface of a substantially spherical particle. FIG. 4 is a scanning electron-machine gun photograph of particles in Comparative Example 1, FIG. 5 is a scanning electron micrograph showing an enlarged view of the same as the above, and FIG. It is a scanning electron micrograph showing an enlarged view of the fracture surface on Figure tj1. Figure 8 shows the liquid chromatogram in Example 1, and Figure 9 shows the liquid chromatogram in Comparative Example IK. Leu breasts

Claims (1)

[Scope of Claims] Hollow O11 spherical particles made of a hydrophilic polymer having a vinyl monomer as a polymerization component, wherein the hollow substantially spherical particles have a particle 11G) at any location of the hollow substantially spherical particles. Thickness (
The ratio (L/D) to aOSh (L4S01!I
IK Existing Filling Agent for Case A Matog 9 Fee 1 Patent Requested Category 1m1131, characterized in that the vinyl monomer has a carboxyl group.
Filler 1 for Kneaded Chromatoguff 4 The vinyl monomer has the following formula (iiL, where R1 and R□ are methyl groups, n
Fig~Spring at 180 alignment) Patent I characterized by being strained by K
Filling agent for liquid chroma dog feed according to item 1 of the scope of IIIt