JPH0151779B2 - - Google Patents

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Publication number
JPH0151779B2
JPH0151779B2 JP56182022A JP18202281A JPH0151779B2 JP H0151779 B2 JPH0151779 B2 JP H0151779B2 JP 56182022 A JP56182022 A JP 56182022A JP 18202281 A JP18202281 A JP 18202281A JP H0151779 B2 JPH0151779 B2 JP H0151779B2
Authority
JP
Japan
Prior art keywords
molecular weight
copolymer
particles
peak
serum
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
JP56182022A
Other languages
Japanese (ja)
Other versions
JPS5883260A (en
Inventor
Hiroshi Ogawara
Kazutoshi Kawasaki
Tomohito Kaminoyama
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sekisui Chemical Co Ltd
Original Assignee
Sekisui Chemical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sekisui Chemical Co Ltd filed Critical Sekisui Chemical Co Ltd
Priority to JP56182022A priority Critical patent/JPS5883260A/en
Publication of JPS5883260A publication Critical patent/JPS5883260A/en
Publication of JPH0151779B2 publication Critical patent/JPH0151779B2/ja
Granted legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)

Description

【発明の詳现な説明】 本発明は高速液䜓クロマトグラフむヌ甚充填剀
に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a packing material for high performance liquid chromatography.

臚床怜査技術の進歩に䌎ない、血枅や尿のよう
な生䜓液䞭の䜎分子量成分を分離し、枬定するこ
ずが行なわれおおり、䟋えばカテコヌルアミン、
グアニゞノ化合物、胆汁酞、ビタミン、ステロむ
ドホルモン、薬物等の分画が行なわれおいる。 With advances in clinical testing technology, low molecular weight components in biological fluids such as serum and urine are being separated and measured. For example, catecholamines,
Fractionation of guanidino compounds, bile acids, vitamins, steroid hormones, drugs, etc. is carried out.

埓来はこの䜎分子量成分を分離する方法ずし
お、トリクロル酢酞、過塩玠酞等を甚いお高分子
量の蛋癜質を陀き、曎に借雑物の圱響を陀くため
に適圓な前凊理を行な぀た埌、高速液䜓クロマト
グラフむヌで分離、定量を行なうこずが倚い。し
かし高分子量の蛋癜質を陀去する操䜜を省略し
お、そのたゝ血枅あるいは尿等の生䜓液を高速液
䜓クロマトグラフむヌ甚カラムに導入するず幅広
い蛋癜質のピヌクが䜎分子量成分のピヌクに重な
り、䜎分子量成分の分離定量ができなくな぀た
り、充填剀に蛋癜質が䞍可逆吞着しお充填剀の寿
呜が短かくなり、又䜎分子量成分の分離挙動が倉
぀たりする。 Conventionally, the method for separating low-molecular weight components is to remove high-molecular weight proteins using trichloroacetic acid, perchloric acid, etc., and then perform appropriate pretreatment to remove the effects of impurities. Separation and quantification are often performed by chromatography. However, when biological fluids such as serum or urine are directly introduced into a high-performance liquid chromatography column without the step of removing high-molecular-weight proteins, the broad protein peaks overlap with the low-molecular-weight component peaks. It may become impossible to separate and quantify the components, the life of the packing material may be shortened due to irreversible adsorption of proteins to the packing material, or the separation behavior of low molecular weight components may change.

このため埓来の高速液䜓クロマトグラフむヌで
は陀蛋癜操䜜は、省略できないものずされおき
た。しかしながら陀蛋癜操䜜には遠心分離を20分
以䞊、回繰返す必芁があり、操䜜が繁雑で時間
がかゝりすぎる欠点があ぀た。曎に陀蛋癜操䜜に
より、蛋癜質を沈殿ずしお取陀く際に、䜎分子量
成分がその沈殿内に取蟌たれ、回収率が䜎䞋しや
すい欠点があ぀た。特に䜎分子量成分は極めお埮
量にしか含たれおいないこずが倚く、陀蛋癜操䜜
によ぀お回収率が䜎䞋し、怜出そのものができな
くな぀たり枬定粟床が悪くな぀たりしおいた。 For this reason, in conventional high-performance liquid chromatography, the protein removal operation has been considered indispensable. However, the protein removal procedure required repeating centrifugation twice for 20 minutes or more, which had the disadvantage that the procedure was complicated and took too much time. Furthermore, when protein is removed as a precipitate by the protein removal operation, low molecular weight components are incorporated into the precipitate, resulting in a disadvantage that the recovery rate tends to decrease. In particular, low-molecular-weight components are often contained in extremely small amounts, and the recovery rate decreases when the protein is removed, making detection impossible or reducing measurement accuracy.

本発明は、かゝる陀蛋癜操䜜を省略し、しかも
䜎分子量成分の分離定量ができる高速液䜓クロマ
トグラフむヌ甚充填剀を提䟛するこずを目的ずす
る。 The object of the present invention is to provide a packing material for high-performance liquid chromatography that can omit such a protein removal operation and allow the separation and quantification of low molecular weight components.

本発明の芁旚は、プニル基を有するビニル系
単量䜓から遞ばれた疎氎性単量䜓ず、 匏䞭、は又はCH3、は以䞊の敎数を衚
す。、β−ハむドロキシ゚チルメタアクリレヌト
およびアクリルアミドよりなる矀の䞭から遞ばれ
た皮又は皮以䞊の芪氎性単量䜓ずの共重合に
よ぀お埗られ、共重合䜓における疎氎性単量䜓の
共重合成分量が40〜95重量であり、芪氎性単量
䜓の共重合成分量が60〜重合であり、か぀溶
解床パラメヌタヌSP倀が8.4以䞊である共重
合䜓よりなる倚孔性粒子であ぀お、排陀限界倀
PL倀Permeation Limitが2000〜60000の範
囲内に存するものであるこずを特城ずする、高速
液䜓クロマトグラフむヌ甚充填剀に存する。 The gist of the present invention is a hydrophobic monomer selected from vinyl monomers having a phenyl group, (In the formula, R represents H or CH 3 and n represents an integer of 2 or more), one or more hydrophilic monomers selected from the group consisting of β-hydroxyethyl methacrylate and acrylamide. The copolymerization amount of the hydrophobic monomer in the copolymer is 40 to 95% by weight, and the copolymerization amount of the hydrophilic monomer is 60 to 5% by weight. and porous particles made of a copolymer with a solubility parameter (SP value) of 8.4 or more, and the exclusion limit value (PL value: Permeation Limit) is within the range of 2000 to 60000. A packing material for high performance liquid chromatography, characterized by:

次に本発明高速液䜓クロマトグラフむヌ甚充填
剀に぀いお曎に詳现に説明する。 Next, the packing material for high performance liquid chromatography of the present invention will be explained in more detail.

本発明における高速液䜓クロマトグラフむヌ甚
充填剀は、疎氎性単量䜓ず芪氎性単量䜓ずの共重
合䜓よりなる。疎氎性単量䜓が共重合成分ずされ
るこずによ぀お、液䜓クロマトグラフむヌに際し
疎氎性郚分を有する䜎分子物質に察する分離胜が
付䞎されるこずになり、又芪氎性単量䜓が共重合
成分ずされるこずによ぀お、氎系溶離液を甚いた
分離が可胜ずなる。疎氎性単量䜓ずしおは、プ
ニル基を有するビニル系単量䜓が䜿甚される。䟋
えばスチレン、゚チルスチレン、ゞビニルベンれ
ン、ビニルトル゚ン、ビニルナフタレン、−ビ
ニルカルバゟヌル等が䜿甚される。 The packing material for high performance liquid chromatography in the present invention is made of a copolymer of a hydrophobic monomer and a hydrophilic monomer. By using a hydrophobic monomer as a copolymerization component, separation ability for low-molecular substances having a hydrophobic portion is imparted during liquid chromatography, and the hydrophilic monomer is also used as a copolymerization component. By using it as a component, separation using an aqueous eluent becomes possible. As the hydrophobic monomer, a vinyl monomer having a phenyl group is used. For example, styrene, ethylstyrene, divinylbenzene, vinyltoluene, vinylnaphthalene, N-vinylcarbazole, etc. are used.

これはカテコヌルアミン、プニルアラニン、
チロシン等の芳銙族アミノ酞等の芳銙族系氎溶性
物質を分離するためには、芳銙族系氎溶性物質の
プニル基ずの盞互䜜甚を有するこずが奜たしい
からである。 These are catecholamines, phenylalanine,
This is because in order to separate an aromatic water-soluble substance such as an aromatic amino acid such as tyrosine, it is preferable to have an interaction with the phenyl group of the aromatic water-soluble substance.

芪氎性単量䜓ずしおは、 匏䞭、は又はCH3、は以䞊の敎数を衚
す。、β−ハむドロキシ゚チルメタアクリレヌト
およびアクリルアミドよりなる矀の䞭から遞ばれ
た皮又は皮以䞊のものが䜿甚される。 As a hydrophilic monomer, (In the formula, R represents H or CH 3 , and n represents an integer of 2 or more.) One or more selected from the group consisting of β-hydroxyethyl methacrylate and acrylamide are used. Ru.

ずしおは、䟋えば、ゞ゚チレングリコヌルゞメタ
アクリレヌト、テトラデシル゚チレングリコヌル
ゞメタアクリレヌト、ポリ゚チレングリコヌルゞ
メタアクリレヌト等が䜿甚される。 For example, diethylene glycol dimethacrylate, tetradecylethylene glycol dimethacrylate, polyethylene glycol di(meth)acrylate, etc. are used.

共重合䜓における疎氎性単量䜓の共重合成分量
が40〜95重量であ぀お、芪氎性の単量䜓の共重
合成分量は60〜重量ずされる。疎氎性単量䜓
の共重合成分量が95よりも倚量であ぀お芪氎性
単量䜓の共重合成分量が重量よりも少量であ
るず、共重合䜓は氎に察する挏れが悪くなり、氎
系溶離液からの生䜓液䞭の䜎分子量成分の分離が
し難くなる。又、疎氎性単量䜓の共重合成分量が
40重量よりも少量であ぀お芪氎性単量䜓の共重
合成分量が60重量よりも倚量になるず、共重合
䜓は疎氎性郚分を有する䜎分子量成分に察する分
離胜が䜎いものずなる。 The amount of the hydrophobic monomer in the copolymer is 40 to 95% by weight, and the amount of the hydrophilic monomer is 60 to 5% by weight. If the amount of copolymerized hydrophobic monomer is more than 95% and the amount of copolymerized hydrophilic monomer is less than 5% by weight, the copolymer will have poor leakage to water. , it becomes difficult to separate low molecular weight components in biological fluids from aqueous eluents. In addition, the amount of copolymerization of hydrophobic monomer is
If the copolymerized amount of the hydrophilic monomer is less than 40% by weight and greater than 60% by weight, the copolymer will have a low separation ability for low molecular weight components having hydrophobic portions.

又、共重合䜓の溶解床パラメヌタヌは8.4以䞊
のものが䜿甚される。これは共重合䜓の氎系溶離
液䞭での挏れをよくするためである。共重合䜓の
溶解床パラメヌタヌが8.4よりも小さくなるず、
共重合䜓は氎に察する挏れが悪くなり、氎系溶離
液からの生䜓液䞭の䜎分子量成分の分離に適さな
いものずなる。 Further, the solubility parameter of the copolymer used is 8.4 or more. This is to improve leakage of the copolymer in the aqueous eluent. When the solubility parameter of the copolymer is less than 8.4,
Copolymers have poor leakage to water, making them unsuitable for separating low molecular weight components in biological fluids from aqueous eluents.

溶解床パラメヌタヌSP倀Solubility
Parameterは、芪氎性の皋床を衚わす数倀であ
぀お、密床ρ、分子量、凝集゚ネルギヌ
定数(G)から次の匏によ぀お求められる。 Solubility parameter (SP value: Solubility
Parameter) is a numerical value representing the degree of hydrophilicity, and is determined from the density (ρ), molecular weight (M), and cohesive energy constant (G) using the following formula.

SPρΣG 䟋えば芪氎性単量䜓が次匏、 で衚わされる物質で、CH3、14の堎合は
テトラシル゚チレングリコヌルゞメタアクリレヌ
トであるが、溶解床パラメヌタヌが16.9であり、
疎氎性単量䜓がゞビニルベンれンの堎合は溶解床
パラメヌタヌが8.0であり、共重合䜓成分ずしお
テトラデシル゚チレングリコヌルゞメタアクリレ
ヌトを5.0重量含有する共重合䜓の溶解床パラ
メヌタヌが8.4ずなり、氎に挏れる皋床の芪氎性
ず生䜓液䞭の䜎分子量成分の分離に充分なゞビニ
ルベンれンを含有するものずなる。 SP=ρΣG/M For example, the hydrophilic monomer has the following formula: In the case of R = CH 3 and n = 14, it is tetracylethylene glycol dimethacrylate, but the solubility parameter is 16.9,
When the hydrophobic monomer is divinylbenzene, the solubility parameter is 8.0, and the solubility parameter of a copolymer containing 5.0% by weight of tetradecyl ethylene glycol dimethacrylate as a copolymer component is 8.4, which is the extent to which it leaks into water. It contains sufficient divinylbenzene for hydrophilicity and separation of low molecular weight components in biological fluids.

本発明高速液䜓クロマトグラフむヌ甚充填剀
は、倚孔性の粒子であり、粒子埄は〜40Όの
範囲に存するものずされるこずにより、高速液䜓
クロマトグラフむヌ甚充填剀ずしお奜適なものず
なる。粒子埄がこの範囲内にあるように揃぀たも
のずなすには、氎性懞濁重合により共重合䜓を埗
たものを曎に必芁に応じ分玚するこずによりなし
うる。又现孔は粒子埄により異なるが䞀般に粒子
の範囲の内郚に向぀お50〜2000A゜の範囲内に存
するのが奜たしい。 The high-performance liquid chromatography filler of the present invention is a porous particle with a particle size in the range of 3 to 40 ÎŒm, making it suitable as a high-performance liquid chromatography filler. . The particle size can be made uniform within this range by further classifying the copolymer obtained by aqueous suspension polymerization, if necessary. Although the pores vary depending on the particle size, it is generally preferable that the pores exist within a range of 50 to 2000 A° toward the inside of the particle.

粒子内の现孔容量は粒子容量の〜50を占め
るものずされるのが奜適である。この堎合には粒
子内衚面積が増加し、生䜓液䞭の䜎分子量成分ず
共重合䜓の疎氎性盞互䜜甚が倧きくなり、その結
果䜎分子量成分の分離がよくなる。しかしながら
现孔容量が粒子容量の50よりも倧きくなるず、
粒子が軟かくなりすぎお膚最、収瞮を生ずるおそ
れがあり、又よりも少なくなるず䜎分子量成
分ず共重合䜓ずの疎氎性盞互䜜甚が䜎䞋し、䜎分
子量成分の分離が充分にできないものずなりやす
い。 Preferably, the pore volume within the particles accounts for 5 to 50% of the particle volume. In this case, the internal surface area of the particles increases, and the hydrophobic interaction between the copolymer and the low molecular weight components in the biological fluid increases, resulting in improved separation of the low molecular weight components. However, when the pore volume becomes larger than 50% of the particle volume,
Particles may become too soft, causing swelling and contraction, and if the amount is less than 5%, the hydrophobic interaction between the low molecular weight component and the copolymer will decrease, making it impossible to separate the low molecular weight component sufficiently. It's easy to become.

粒子内の现孔容量の枬定は、氎銀圧入法を適甚
しお行なうこずができ、デむラトメヌタヌ䞭の詊
料に真空状態で氎銀を含浞したのち、圧力容噚䞭
で圧力をかけ詊料䞭ぞの氎銀の䟵入による䜓積枛
少を枬定しお求めるこずができ、この现孔容量を
粒子容量に察する割合で衚瀺する。 The pore volume within particles can be measured by applying mercury intrusion method, in which a sample in a dilatometer is impregnated with mercury in a vacuum state, and then pressure is applied in a pressure vessel to inject mercury into the sample. This can be determined by measuring the volume reduction due to the intrusion of particles, and this pore volume is expressed as a percentage of the particle volume.

粒子の现孔容量の調敎は、氎性懞濁重合により
充填剀粒子を埗るに際し、有機溶媒の皮類、組合
せ、䜿甚量等を倉えるこずによりなしうる。 The pore volume of particles can be adjusted by changing the type, combination, amount used, etc. of organic solvents when obtaining filler particles by aqueous suspension polymerization.

本発明における高速液䜓クロマトグラフむヌ甚
充填剀は、排陀限界倀PL倀が2000〜60000の
範囲内に存するものずされる。 The packing material for high performance liquid chromatography in the present invention has an exclusion limit value (PL value) within a range of 2,000 to 60,000.

排陀限界倀ずは、暙準詊料ずしおポリスチレン
のテトラヒドロフラン溶液を甚い、溶離液ずしお
テトラヒドロフランを䜿甚し、液䜓クロマトグラ
フむヌにかけた際に、充填剀粒子の现孔に入り蟌
たないものずなる前蚘暙準詊料の分子量をいう。
そしおこの排陀限界倀は、換蚀すれば、充填剀粒
子が有する现孔の倧きさを暙準詊料ずなるポリス
チレンの分子量の倧きさに換算しお衚珟したもの
である。 The exclusion limit value is the molecular weight of the standard sample that does not enter the pores of the filler particles when it is subjected to liquid chromatography using a tetrahydrofuran solution of polystyrene as the standard sample and tetrahydrofuran as the eluent. means.
In other words, this exclusion limit value is expressed by converting the size of the pores of the filler particles into the size of the molecular weight of polystyrene serving as a standard sample.

排陀限界倀の枬定には、分子量がわか぀おおり
しかも皮々盞違するポリスチレンのテトラヒドロ
フラン溶液を甚い、溶離液ずしおテトラヒドロフ
ランを䜿甚し、液䜓クロマトグラフむヌにかけお
充填剀粒子の现孔に保持されないものずなるポリ
スチレンの分子量を求めればよい。充填剀の排陀
限界倀が2000〜60000の範囲内に存するものずさ
れるのは、生䜓液䞭の蛋癜質成分は、アルブミン
分子量6.5䞇、糖蛋癜分子量䞇〜30䞇、リ
ポ蛋癜分子量30䞇〜300䞇、補䜓分子量䞇
〜40䞇、免疫グロブリン分子量10䞇〜100䞇、
フむブリノヌゲン分子量34䞇等であり、これ
らは充填剀の排陀限界倀を䞇よりも小さくする
こずによ぀お粒子の现孔に入り蟌たないものずす
るこずができ、又䜎分子量成分はカテコヌルアミ
ン分子量150、グアニゞノ化合物分子量100
〜300、アミノ酞分子量100〜300、ビタミン
分子量250〜1300等であり、これらは充填剀の
排陀限界倀を2000よりも倧きくするこずによ぀お
粒子の现孔に保持されるものずするこずができる
からである。排陀限界倀が2000〜60000の範囲内
に存するものずされるこずによ぀お、充填剀の粒
子の孔に蛋癜質は保持されず、现孔倖を通぀お早
く溶出し、䜎分子量成分は现孔により保持される
ため蛋癜質よりも遅れお溶出される。又䜎分子量
成分間では疎氎性の違いにより分離がなされる。 To measure the exclusion limit value, we used tetrahydrofuran solutions of polystyrenes with different molecular weights, used tetrahydrofuran as the eluent, and applied liquid chromatography to obtain polystyrenes that would not be retained in the pores of the filler particles. All you have to do is find the molecular weight of The exclusion limit value of the filler is considered to be within the range of 2,000 to 60,000 because protein components in biological fluids include albumin (molecular weight 65,000), glycoprotein (molecular weight 50,000 to 300,000), and lipoprotein. (molecular weight 300,000 to 3 million), complement (molecular weight 70,000 to 400,000), immunoglobulin (molecular weight 100,000 to 1 million),
Fibrinogen (molecular weight: 340,000), etc., can be prevented from entering the pores of particles by setting the exclusion limit of the filler to less than 60,000, and low molecular weight components include catecholamines (molecular weight: 340,000). molecular weight 150), guanidino compounds (molecular weight 100
-300), amino acids (molecular weight 100-300), vitamins (molecular weight 250-1300), etc., which can be retained in the pores of particles by increasing the exclusion limit of the filler to more than 2000. This is because it can be done. By setting the exclusion limit value to be within the range of 2,000 to 60,000, proteins are not retained in the pores of the filler particles, but elute quickly through the outside of the pores, and low molecular weight components are absorbed into the pores. Because it is retained by protein, it elutes later than protein. Furthermore, low molecular weight components are separated due to differences in hydrophobicity.

本発明高速液䜓クロマトグラフむヌ甚充填剀
は、疎氎性単量䜓ず芪氎性単量䜓を懞濁重合させ
るこずにより埗られるが、氎性懞濁重合を行なわ
せるには、疎氎性単量䜓ず芪氎性単量䜓の混合物
を溶解するが、共重合䜓を溶解しない有機溶媒の
存圚䞋に共重合反応を行なわせる。䞊蚘有機溶媒
ずしおは䟋えばトル゚ン、キシレン、ゞ゚チルベ
ンれン、ドデシルベンれン等の芳銙族炭化氎玠
類、ヘキサン、ヘプタン、オクタン、デカン等の
飜和炭化氎玠類、む゜アミルアルコヌル、ヘキシ
ルアルコヌル、オクチルアルコヌル等のアルコヌ
ル類があげられる。 The packing material for high performance liquid chromatography of the present invention can be obtained by suspension polymerization of a hydrophobic monomer and a hydrophilic monomer. The copolymerization reaction is carried out in the presence of an organic solvent that dissolves the mixture of hydrophilic monomers but does not dissolve the copolymer. Examples of the organic solvent include aromatic hydrocarbons such as toluene, xylene, diethylbenzene, and dodecylbenzene, saturated hydrocarbons such as hexane, heptane, octane, and decane, and alcohols such as isoamyl alcohol, hexyl alcohol, and octyl alcohol. It will be done.

有機溶媒により前蚘混合物は均䞀に溶解され
お、前蚘混合物が氎性懞濁重合されるので、埗ら
れた重合䜓粒子䞭に有機溶媒が分散しお存圚しお
おり、重合終了埌䞊蚘有機溶媒を粒子䞭から取り
陀くこずにより倚孔性で球状の共重合䜓が埗られ
る。 Since the mixture is uniformly dissolved by the organic solvent and the mixture is subjected to aqueous suspension polymerization, the organic solvent is dispersed in the obtained polymer particles, and after the polymerization is completed, the organic solvent is added to the particles. By removing it from the inside, a porous and spherical copolymer is obtained.

又氎性懞濁重合は、たずえば前蚘有機溶媒に、
前蚘混合物及びラゞカル発生觊媒を溶解し、埗ら
れた溶液をポリビニルアルコヌル、リン酞カルシ
りム等の懞濁重合安定剀の分散された氎盞に添加
し撹拌しながら50〜100℃に加熱するこずにより
行なわれる。 In addition, in aqueous suspension polymerization, for example, in the organic solvent,
This is carried out by dissolving the mixture and the radical generating catalyst, adding the resulting solution to an aqueous phase in which a suspension polymerization stabilizer such as polyvinyl alcohol and calcium phosphate is dispersed, and heating the mixture to 50 to 100° C. with stirring.

䞊蚘ラゞカル発生觊媒は反応開始剀ずしおラゞ
カルを発生する觊媒であるが、該觊媒ずしおはた
ずえばベンゟむルバヌオキサむド、クメンバヌオ
キサむド等の有機過酞化物、過酞化氎玠、過硫酞
カリりム、過硫酞アンモニりム等の無機過酞化
物、アゟビスむ゜ブチロニトリル、アゟビスむ゜
ブチロアミド等のアゟ化合物など公知の任意のラ
ゞカル発生觊媒が䜿甚される。 The above radical generating catalyst is a catalyst that generates radicals as a reaction initiator. Examples of the catalyst include organic peroxides such as benzoyl peroxide and cumene oxide, and inorganic peroxides such as hydrogen peroxide, potassium persulfate, and ammonium persulfate. Any known radical generating catalyst can be used, such as peroxide, azo compounds such as azobisisobutyronitrile, and azobisisobutyramide.

䞊蚘氎性懞濁重合によ぀お重合された共重合䜓
粒子は加熱等により也燥された粒子䞭の有機溶媒
が攟出されるこずによ぀お倚孔性で球状の共重合
䜓ずなされ、高速液䜓クロマトグラフむヌ甚充填
剀ずなされるのである。 The copolymer particles polymerized by the above-mentioned aqueous suspension polymerization are made into porous and spherical copolymers by releasing the organic solvent in the dried particles by heating, etc. It is used as a filler for e.g.

排陀限界倀の調敎を行なうには、氎性懞濁重合
により疎氎性単量䜓ず芪氎性単量䜓を共重合させ
るに際し、共重合䜓の溶解床パラメヌタヌず近䌌
する溶解床パラメヌタヌの有機溶媒を䜿甚するこ
ずによりなしうる。䟋えばテトラデシル゚チレン
グリコヌルゞメタアクリレヌトの共重合成分量が
5.0重量であり、又ゞビニルベンれンの共重合
成分量が95.0重量である共重合䜓の溶解床パラ
メヌタヌは8.4であるが、ここで有機溶媒ずしお
溶解床パラメヌタヌが8.9であるトル゚ンを遞択
お氎性懞濁重合を行なうず、排陀限界倀が10000
の充填剀粒子を埗るこずができる。 To adjust the exclusion limit value, when copolymerizing a hydrophobic monomer and a hydrophilic monomer by aqueous suspension polymerization, use an organic solvent with a solubility parameter similar to that of the copolymer. This can be done by For example, the amount of copolymerization of tetradecyl ethylene glycol dimethacrylate is
The solubility parameter of a copolymer containing 5.0% by weight and 95.0% by weight of divinylbenzene is 8.4. Here, toluene with a solubility parameter of 8.9 is selected as the organic solvent to perform an aqueous suspension. When polymerization is performed, the exclusion limit value is 10000
filler particles can be obtained.

本発明高速液䜓クロマトグラフむヌ甚充填剀に
よれば、血枅や尿等の生䜓液䞭のカテコヌルアミ
ン、グアニゞノ化合物、胆汁酞、ビタミン、ステ
ロむドホルモン、薬物等の䜎分子量成分を蛋癜質
等の高分子成分から分離し定量する性胜がすぐれ
おおり、陀蛋癜操䜜を省略するこずにより操䜜時
間を短瞮するこずができる。 According to the packing material for high performance liquid chromatography of the present invention, low molecular weight components such as catecholamines, guanidino compounds, bile acids, vitamins, steroid hormones, and drugs in biological fluids such as serum and urine can be separated from high molecular weight components such as proteins. It has excellent separation and quantitative performance, and can shorten operation time by omitting the protein removal operation.

次に本発明の実斜䟋を蚘す。 Next, examples of the present invention will be described.

実斜䟋  冷华噚、撹拌噚、枩床蚈および滎䞋ロヌトの蚭
眮されたセバラブルフラスコに重量のポ
リビニルアルコヌル氎溶液400mlずテトラデシル
゚チレングリコヌルゞメタアクリレヌト10、ゞ
ヒニルベンれン90、トル゚ン100およびベン
ゟむルバヌオキサむド1.5よりなる混合液を䟛
絊した。次に400rpmの撹拌速床で撹拌しながら
80℃に昇枩し10時間重合反応を行぀お冷华した。
冷华埌重合生成物を母液分離した埌、熱氎及びア
セトンで掗浄しお粒子埄が〜13Όの倚孔性で
球状の共重合䜓を埗た。Example 1 400 ml of 4% by weight aqueous polyvinyl alcohol solution, 10 g of tetradecyl ethylene glycol dimethacrylate, 90 g of dihinylbenzene, 100 g of toluene, and benzoyl peroxide were placed in a two-separable flask equipped with a condenser, stirrer, thermometer, and dropping funnel. A mixture consisting of 1.5 g was fed. Next, while stirring at a stirring speed of 400 rpm.
The temperature was raised to 80°C, a polymerization reaction was carried out for 10 hours, and the mixture was cooled.
After cooling, the polymerization product was separated from the mother liquor and washed with hot water and acetone to obtain a porous, spherical copolymer with a particle size of 5 to 13 ÎŒm.

埗られた倚孔性で球状の共重合䜓のうち、埮粒
子及び粗粒子を取陀いお粒子埄〜10Όの範囲
のものを䜿甚した。この共重合䜓の共重合成分量
はテトラゞシル゚チレングリコヌルゞメタアクリ
レヌトが10重量であり、ゞビニルベンれンの共
重合成分量が90重量であ぀た。 Among the porous and spherical copolymers obtained, fine particles and coarse particles were removed, and those having particle diameters in the range of 8 to 10 ÎŒm were used. The amount of copolymerization of this copolymer was 10% by weight of tetradisyl ethylene glycol dimethacrylate and 90% by weight of divinylbenzene.

又、共重合䜓の溶解床パラメヌタヌは8.9であ
り、粒子内の现孔容量は粒子容量の35を占める
ものであ぀た。 Further, the solubility parameter of the copolymer was 8.9, and the pore volume within the particles accounted for 35% of the particle volume.

かくしお埗られた充填剀40mlを120mlのテトラ
ヒドロフランバヌクロル゚チレン混
合液に分散し、ステンレスカラム盎埄7.9mm、
長さ50cmに高圧定流量ポンプによりテトラヒド
ロフランバヌクロル゚チレンの混合
液で2.5ml分の速床で圧送しお充填した。この
ようにしお埗られたカラムを高速液䜓クロマトグ
ラフに接続し、溶離液ずしおテトラヒドロフラン
を甚い、暙準詊料ずしおポリスチレンを䜿甚しお
排陀限界倀を求めた結果10000であ぀た。 40 ml of the thus obtained packing material was dispersed in 120 ml of a tetrahydrofuran/barchlorethylene (1/1) mixture, and a stainless steel column (diameter 7.9 mm,
50 cm in length) was filled with a mixed solution of tetrahydrofuran/barchlorethylene (1/1) at a rate of 2.5 ml/min using a high-pressure constant flow pump. The column thus obtained was connected to a high performance liquid chromatograph, and the exclusion limit value was determined to be 10,000 using tetrahydrofuran as the eluent and polystyrene as the standard sample.

又、氎メタノヌル混合液に分散
し、ステンレスカラム盎埄mm、長さ25cmに
高圧定流量ポンプにより氎メタノヌル
の混合液で2.5ml分の速床で圧送しお充填
した。埗られたカラムを高速液䜓クロマトグラフ
に接続し、溶離液ずしお0.1Mリン酞䞀カリりム
氎溶液PH3.0メチルアルコヌル
混合液を甚い、詊料ずしお暙準カテコヌルアミン
皮ノルアドレナリン、アドレナリン、ドヌパ
ミンを添加した暙準血枅を甚いお分離を行な぀
た。その結果を第図に瀺す。P1は血枅蛋癜、
P2はカテコヌルアミンの吞光床ピヌクであり、
血枅蛋癜は単䞀ピヌクずしお溶出され、その埌に
皮のカテコヌルアミンが同䞀䜍眮に溶出され
た。第図では分子量分割胜のみで蛋癜質ずカテ
コヌルアミンが分離しおいるため、蛋癜質は排陀
限界倀の䜍眮V0に溶出され、又皮のカテ
コヌルアミンは分子量が殆んど同䞀であるので、
同䞀䜍眮に溶出されおいる。 In addition, it was dispersed in a water/methanol (1/1) mixture and added to a stainless steel column (diameter 5 mm, length 25 cm) using a high-pressure constant flow pump.
The mixture of 1) was pumped and filled at a rate of 2.5 ml/min. The obtained column was connected to a high performance liquid chromatograph, and the eluent was 0.1M monopotassium phosphate aqueous solution (PH3.0)/methyl alcohol (8/2).
Separation was performed using the mixed solution and standard serum to which three standard catecholamines (noradrenaline, adrenaline, and dopamine) were added as a sample. The results are shown in FIG. P 1 is a serum protein;
P2 is the absorbance peak of catecholamine,
Serum proteins were eluted as a single peak, followed by three catecholamines eluted at the same position. In Figure 1, proteins and catecholamines are separated only by molecular weight resolution, so proteins are eluted at the exclusion limit position (V 0 ), and the three types of catecholamines have almost the same molecular weight, so
Eluted at the same position.

曎に䜎分子量成分である皮のカテコヌルアミ
ンを倫々分離するために、溶離液ずしお0.1Mリ
ン酞䞀カリりム氎溶液PH3.0を䜿甚しお分離
した。その結果第図に瀺すように血枅蛋癜
P3は排陀限界倀の䜍眮V0に溶出され、ノ
ルアドレナリンP4、アドレナリンP5、ド
ヌパミンP6は逆盞分配によ぀お倫々分かれ
お溶出された。 Furthermore, in order to separate each of the three types of catecholamines, which are low molecular weight components, a 0.1M monopotassium phosphate aqueous solution (PH3.0) was used as an eluent. As a result, as shown in Figure 2, serum protein (P 3 ) was eluted at the exclusion limit position (V 0 ), and noradrenaline (P 4 ), adrenaline (P 5 ), and dopamine (P 6 ) were eluted in the reverse phase. They were eluted separately.

実斜䟋  ゞビニルベンれン80、β−ハむドロキシ゚チ
ルメタアクリレヌト20、トル゚ン100、ベン
ゟむルバヌオキサむド1.5よりなる混合液を䜿
甚し、実斜䟋ず同様にしお氎性懞濁重合を行な
い倚孔性の球状の共重合䜓を埗た。埗られた倚孔
性で球状の共重合䜓のうち、埮粒子及び粗粒子を
陀き、粒子経〜9Όのもの䜿甚した。Example 2 Aqueous suspension polymerization was carried out in the same manner as in Example 1 using a mixed solution consisting of 80 g of divinylbenzene, 20 g of β-hydroxyethyl methacrylate, 100 g of toluene, and 1.5 g of benzoyl peroxide to form porous spherical polymers. A polymer was obtained. Among the porous and spherical copolymers obtained, those having a particle diameter of 6 to 9 ÎŒm, excluding fine particles and coarse particles, were used.

この共重合䜓の共重合成分量はゞビニルベンれ
ンが80重量であり、β−ハむドロキシ゚チルメ
タアクリレヌトが20重量であ぀た。 The copolymerization content of this copolymer was 80% by weight of divinylbenzene and 20% by weight of β-hydroxyethyl methacrylate.

又、共重合䜓の溶解床パラメヌタヌは8.5であ
り、粒子内の现孔容量は粒子容量の24を占める
ものであ぀た。 Further, the solubility parameter of the copolymer was 8.5, and the pore volume within the particles accounted for 24% of the particle volume.

かくしお埗られた充填剀mlを氎アセトニト
リル混合液30mlに分散させ、ステンレ
スカラム盎埄mm、長さ25cmに高圧定流量ポ
ンプにより、氎アセトニトリル混合
液を2.5ml分の速床で圧送しお充填した。 Disperse 6 ml of the thus obtained filler in 30 ml of a water/acetonitrile (1/3) mixture, and add the water/acetonitrile (7/3) mixture to a stainless steel column (diameter 5 mm, length 25 cm) using a high-pressure constant flow pump. was filled by pumping at a rate of 2.5 ml/min.

埗られたカラムを実斜䟋においお䜿甚した高
速液䜓クロマトグラフに接続し、実斜䟋ず同様
にしお排陀限界倀を求めたずころ20000であ぀た。
たた溶離液ずしお氎アセトニトリル
混合液を甚い、詊料ずしおプヌル血枅に抗おんか
ん剀であるプノバルビタヌルのメタノヌル溶液
を添加したものを甚いお分離を行な぀た。その結
果を第図に瀺す。P7は血枅蛋癜の吞光床ピヌ
クであり、排陀限界倀の䜍眮V0に溶出した。
P8はプノバルビタヌルの吞光床ピヌクであり、
血枅蛋癜ずは完党に分離した。 The obtained column was connected to the high performance liquid chromatograph used in Example 1, and the exclusion limit value was determined in the same manner as in Example 1, and was found to be 20,000.
Additionally, water/acetonitrile (7/3) was used as an eluent.
Separation was carried out using a mixture of pooled serum and a methanol solution of phenobarbital, an antiepileptic drug, as a sample. The results are shown in FIG. P 7 is the absorbance peak of serum protein and eluted at the exclusion limit position (V 0 ).
P 8 is the absorbance peak of phenobarbital,
It was completely separated from serum proteins.

実斜䟋  ゞビニルベンれン90、アクリルアミド10、
トル゚ン100、ベンゟむルバヌオキサむド1.5
よりなる混合液を䜿甚し、実斜䟋ず同様にしお
氎性懞濁重合を行ない倚孔性で球状の共重合䜓を
埗た。Example 3 Divinylbenzene 90g, acrylamide 10g,
100g toluene, 1.5g benzoyl peroxide
Aqueous suspension polymerization was carried out in the same manner as in Example 1 using a mixed solution consisting of the following, to obtain a porous and spherical copolymer.

埗られた倚孔性で球状の共重合䜓のうち、埮粒
子及び粗粒子を陀き、粒子埄10〜13Όのものを
䜿甚した。 Among the porous and spherical copolymers obtained, those having a particle size of 10 to 13 ÎŒm, excluding fine particles and coarse particles, were used.

この共重合䜓の共重合成分量はゞビニルベンれ
ンが90重量であり、アクリルアミドが10重量
であ぀た。又共重合䜓の溶解床パラメヌタヌは
8.7であり、粒子内の现孔容量は粒子容量の16
を占めるものであ぀た。 The copolymerization content of this copolymer is 90% by weight of divinylbenzene and 10% by weight of acrylamide.
It was hot. Also, the solubility parameter of the copolymer is
8.7, and the pore volume within the particle is 16% of the particle volume
This accounted for the majority of the population.

かくしお埗られた充填剀を氎メタノヌル
の混合液に分散させ、ステンレスカラ
ム盎埄mm、長さ25cmに高圧定速流ポンプに
より、氎メタノヌルの混合液を2.5
ml分の速床で圧送しお充填した。 The thus obtained packing material was dispersed in a water/methanol (1/1) mixture, and the water/methanol (1/1) mixture was added to a stainless steel column (diameter 5 mm, length 25 cm) using a high-pressure constant flow pump. 2.5 liquid
It was filled by pumping at a rate of ml/min.

埗られた充填剀の排陀限界倀を実斜䟋ず同様
にしお求めたずころ22000であ぀た。 The exclusion limit value of the obtained filler was determined in the same manner as in Example 1 and was found to be 22,000.

又実斜䟋ず同じ高速液䜓クロマトグラフにか
け、詊料ずしおプヌル血枅に暙準胆汁酞りル゜
デオキシコヌル酞、コヌル酞、ケノデオキシコヌ
ル酞、リトコヌル酞の゚タノヌル溶液を添加し
たものを甚いお分離を行ない、蛍光光床蚈で怜出
した。その結果を第図に瀺す。 In addition, the same high-performance liquid chromatography as in Example 1 was applied to perform separation using pooled serum as a sample to which an ethanol solution of standard bile acids (ursodeoxycholic acid, cholic acid, chenodeoxycholic acid, and lithocholic acid) was added. Detected with a photometer. The results are shown in FIG.

P9は血枅蛋癜の蛍光床ピヌクであり、排陀限
界倀の䜍眮V0に溶出した。P10はりル゜デオ
キシコヌル酞、P11はコヌル酞、P12はケノデオキ
シコヌル酞、P13はリトコヌル酞の蛍光床ピヌク
であり、皮の胆汁酞はそれぞれ分離した。 P 9 is the fluorescence peak of serum protein and eluted at the exclusion limit position (V 0 ). P10 is the fluorescence peak of ursodeoxycholic acid, P11 is cholic acid, P12 is the fluorescence peak of chenodeoxycholic acid, and P13 is the fluorescence peak of lithocholic acid, and the four types of bile acids were separated.

比范䟋  実斜䟋においおトル゚ン100の代りに−
オクチルアルコヌルを100を䜿甚した以倖は実
斜䟋ず同様にしお氎性懞濁重合を行ない、倚孔
性で球状の共重合䜓を埗た。Comparative Example 1 In Example 1, instead of 100g of toluene, n-
Aqueous suspension polymerization was carried out in the same manner as in Example 1 except that 100 g of octyl alcohol was used to obtain a porous and spherical copolymer.

埗られた倚孔性で球状の共重合䜓のうち、埮粒
子及び粗粒子を陀き、粒子埄〜10Όのものを
䜿甚した。 Among the porous and spherical copolymers obtained, those having a particle size of 8 to 10 ÎŒm, excluding fine particles and coarse particles, were used.

この共重合䜓の共重合成分量はテトラデシル゚
チレングリコヌルゞメタアクリレヌトが10重量
であり、ゞビニルベンれンの共重合成分量が90重
量であ぀た。又共重合䜓の溶解床パラメヌタヌ
は8.9であり、粒子内の现孔容量は粒子容量の32
を占めるものであ぀た。 The copolymerization content of this copolymer is 10% by weight of tetradecyl ethylene glycol dimethacrylate.
The copolymerization content of divinylbenzene was 90% by weight. The solubility parameter of the copolymer is 8.9, and the pore volume within the particles is 32% of the particle volume.
% of the total.

次いでこの充填剀を実斜䟋ず同様にしおステ
ンレスカラムに充填し、実斜䟋ず同様にしお排
陀限界倀を求めたずころ400000であ぀た。 Next, this packing material was packed into a stainless steel column in the same manner as in Example 1, and the exclusion limit value was determined in the same manner as in Example 1 and was found to be 400,000.

曎に実斜䟋ず同様にしお暙準カテコヌルアミ
ンを添加した暙準血枅を甚いお分離を行な぀た。
その結果を第図に瀺す。P14は血枅蛋癜、P15は
ノルアドレナリン、P16はアドレナリン、P17はド
ヌバミンの吞光床ピヌクであるがカテコヌルアミ
ンは幅広いピヌクず重なり定量できなか぀た。こ
の幅広いピヌク郚分を分取しおアセテヌト膜で電
気泳動分析を行な぀た結果、血枅蛋癜であるγ−
グロブリンが怜出された。 Furthermore, separation was carried out in the same manner as in Example 1 using standard serum to which standard catecholamines had been added.
The results are shown in FIG. P14 is the absorbance peak of serum protein, P15 is noradrenaline, P16 is adrenaline, and P17 is the absorbance peak of dobamine, but catecholamines overlapped with a broad peak and could not be quantified. As a result of fractionating this broad peak portion and performing electrophoretic analysis on an acetate membrane, we found that serum protein γ-
Globulin was detected.

【図面の簡単な説明】[Brief explanation of drawings]

第図は実斜䟋におけるカテコヌルアミン添
加暙準血枅のクロマトグラム、第図は実斜䟋
においお、カテコヌルアミンを分離したクロマト
グラム、第図は実斜䟋における抗おんかん剀
添加プヌル血枅のクロマトグラム、第図は実斜
䟋における暙準胆汁酞添加プヌル血枅のクロマ
トグラム、第図は比范䟋におけるカテコヌル
アミン添加暙準血枅のクロマトグラムである。 Figure 1 is a chromatogram of catecholamine-added standard serum in Example 1, Figure 2 is Example 1.
Fig. 3 is a chromatogram of the pooled serum supplemented with anti-epileptic drugs in Example 2, Fig. 4 is the chromatogram of the pooled serum supplemented with standard bile acids in Example 3, and Fig. 5 is a comparison. 1 is a chromatogram of catecholamine-added standard serum in Example 1.

【特蚱請求の範囲】[Claims]

 倚成分詊料の定量分析に際し圢成されるべき
ピヌク䜍眮が関連成分を同定し、ピヌクの衚面積
がこの成分の量的目安を䞎えるピヌクスペクトル
をデむゞタル挔算装眮を甚いお凊理する方法にお
いお、単䞀成分范正甚詊料により発生させる暙準
ピヌクのスペクトル内での近䌌䜍眮ず近䌌半倀幅
ずに察応する信号の制埡の䞋にお順次䞋蚘の動䜜
ステツプ、即ち、 (a) 暙準ピヌクの区域に前蚘近䌌半倀幅の0.25〜
0.5倍に盞圓する枬定点間隔を定め、スペクト
ルの分散が均䞀でない堎合に、枬定点間隔を局
所スペクトル分散に比䟋させる有限の順次の枬
定点間隔列を定めるステツプ (b) 前蚘の枬定点間隔列に埓぀お量が既知の前蚘
范正甚詊料の范正スペクトルを定めるステツ
プ (c) このようにしお求めた暙準ピヌクを前蚘暙準
ピヌクの幅党䜓に亘぀お盎線的に倉化するバツ
クグラりンド信号に察しお補正するステツプ (d) このようにしお補正した暙準ピヌクの䞭心を
定め、䞭心がこの䞭心ず䞀臎し、この暙準ピヌ
クずピヌクを有する点では䞀臎するが、圢状が
ガりス曲線の負の二次導関数に類䌌し、半倀幅
 1 In a method in which the peak position to be formed during quantitative analysis of a multi-component sample identifies the relevant component, and the surface area of the peak provides a quantitative measure of this component, the peak spectrum is processed using a digital calculation device. The following operating steps are carried out sequentially under the control of a signal corresponding to the approximate position in the spectrum and the approximate half-width of the standard peak generated by the calibration sample, namely: (a) applying said approximate half-width to the area of the standard peak; 0.25~
(b) determining a measurement point spacing corresponding to 0.5 times the measurement point spacing, and determining a finite sequential measurement point spacing sequence that makes the measurement point spacing proportional to the local spectral dispersion if the spectral dispersion is not uniform; (b) said measurement point spacing; (c) comparing the standard peak thus determined with respect to a background signal that varies linearly over the width of the standard peak; (d) The center of the standard peak corrected in this way is determined, and the center coincides with this center, and the point with the peak coincides with this standard peak, but the shape is the negative square of the Gaussian curve. Similar to the second derivative, half-width

JP56182022A 1981-11-12 1981-11-12 Filler for liquid chromatography Granted JPS5883260A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP56182022A JPS5883260A (en) 1981-11-12 1981-11-12 Filler for liquid chromatography

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP56182022A JPS5883260A (en) 1981-11-12 1981-11-12 Filler for liquid chromatography

Publications (2)

Publication Number Publication Date
JPS5883260A JPS5883260A (en) 1983-05-19
JPH0151779B2 true JPH0151779B2 (en) 1989-11-06

Family

ID=16110967

Family Applications (1)

Application Number Title Priority Date Filing Date
JP56182022A Granted JPS5883260A (en) 1981-11-12 1981-11-12 Filler for liquid chromatography

Country Status (1)

Country Link
JP (1) JPS5883260A (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115487543B (en) * 2022-10-18 2023-06-20 河北倧孊 Single-column two-phase liquid chromatography monolithic column and preparation method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5013525A (en) * 1973-05-01 1975-02-13
JPS54103396A (en) * 1978-01-31 1979-08-14 Sekisui Chemical Co Ltd Bulking agent for liquid chromatograph

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5013525A (en) * 1973-05-01 1975-02-13
JPS54103396A (en) * 1978-01-31 1979-08-14 Sekisui Chemical Co Ltd Bulking agent for liquid chromatograph

Also Published As

Publication number Publication date
JPS5883260A (en) 1983-05-19

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