JP4388829B2 - Packing for ion exchange liquid chromatography - Google Patents
Packing for ion exchange liquid chromatography Download PDFInfo
- Publication number
- JP4388829B2 JP4388829B2 JP2004038866A JP2004038866A JP4388829B2 JP 4388829 B2 JP4388829 B2 JP 4388829B2 JP 2004038866 A JP2004038866 A JP 2004038866A JP 2004038866 A JP2004038866 A JP 2004038866A JP 4388829 B2 JP4388829 B2 JP 4388829B2
- Authority
- JP
- Japan
- Prior art keywords
- ion exchange
- filler
- liquid chromatography
- group
- phosphate buffer
- 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 - Fee Related
Links
- 238000012856 packing Methods 0.000 title claims description 29
- 238000004255 ion exchange chromatography Methods 0.000 title claims description 21
- 239000000945 filler Substances 0.000 claims description 49
- 238000005342 ion exchange Methods 0.000 claims description 25
- 239000000463 material Substances 0.000 claims description 23
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 125000000542 sulfonic acid group Chemical group 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 description 36
- 239000008363 phosphate buffer Substances 0.000 description 29
- 239000002245 particle Substances 0.000 description 25
- 239000007788 liquid Substances 0.000 description 23
- 238000011282 treatment Methods 0.000 description 21
- 238000000034 method Methods 0.000 description 20
- 239000000126 substance Substances 0.000 description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 238000005259 measurement Methods 0.000 description 12
- 239000000178 monomer Substances 0.000 description 10
- 239000003480 eluent Substances 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 229910001220 stainless steel Inorganic materials 0.000 description 8
- 239000010935 stainless steel Substances 0.000 description 8
- 239000005018 casein Substances 0.000 description 7
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 7
- 235000021240 caseins Nutrition 0.000 description 7
- 238000009210 therapy by ultrasound Methods 0.000 description 7
- 229920000642 polymer Polymers 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 238000001179 sorption measurement Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 102000001554 Hemoglobins Human genes 0.000 description 5
- 108010054147 Hemoglobins Proteins 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- -1 silica compound Chemical class 0.000 description 4
- 229920001059 synthetic polymer Polymers 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- 238000005341 cation exchange Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 238000004811 liquid chromatography Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000003505 polymerization initiator Substances 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 235000002639 sodium chloride Nutrition 0.000 description 3
- 230000008961 swelling Effects 0.000 description 3
- XFCMNSHQOZQILR-UHFFFAOYSA-N 2-[2-(2-methylprop-2-enoyloxy)ethoxy]ethyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCOCCOC(=O)C(C)=C XFCMNSHQOZQILR-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000008946 Fibrinogen Human genes 0.000 description 2
- 108010049003 Fibrinogen Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000010445 Lactoferrin Human genes 0.000 description 2
- 108010063045 Lactoferrin Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- MCMNRKCIXSYSNV-UHFFFAOYSA-N Zirconium dioxide Chemical compound O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 239000002612 dispersion medium Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229940012952 fibrinogen Drugs 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 2
- 229940078795 lactoferrin Drugs 0.000 description 2
- 235000021242 lactoferrin Nutrition 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- XQNRAQZFPXUCOT-UHFFFAOYSA-N phytene Natural products CC(C)CCCC(C)CCCC(C)CCCC(C)C=C XQNRAQZFPXUCOT-UHFFFAOYSA-N 0.000 description 2
- 230000000379 polymerizing effect Effects 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000008215 water for injection Substances 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 229920000536 2-Acrylamido-2-methylpropane sulfonic acid Polymers 0.000 description 1
- XHZPRMZZQOIPDS-UHFFFAOYSA-N 2-Methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid Chemical compound OS(=O)(=O)CC(C)(C)NC(=O)C=C XHZPRMZZQOIPDS-UHFFFAOYSA-N 0.000 description 1
- 239000004342 Benzoyl peroxide Substances 0.000 description 1
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 description 1
- 102000011632 Caseins Human genes 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 102000017011 Glycated Hemoglobin A Human genes 0.000 description 1
- 108010014663 Glycated Hemoglobin A Proteins 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000019400 benzoyl peroxide Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000003943 catecholamines Chemical class 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 239000010954 inorganic particle Substances 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000002794 monomerizing effect Effects 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000011146 organic particle Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 239000004584 polyacrylic acid Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000007613 slurry method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Description
本発明は、液体クロマトグラフィー用の充填剤に関し、特に、強度の低下を招くことなく親水性が高められたイオン交換液体クロマトグラフィー用充填剤に関する。 The present invention relates to a packing material for liquid chromatography, and more particularly to a packing material for ion exchange liquid chromatography having improved hydrophilicity without causing a decrease in strength.
イオン交換液体クロマトグラフィー法は、糖化ヘモグロビン類の分析をはじめとして、各種生体関連物質の分離分析に極めて有効な方法である。これまで、イオン交換液体クロマトグラフィー法に用いられる充填剤としては、シリカ系化合物からなる基剤にイオン交換基を導入したもの、有機合成高分子からなる架橋性粒子にイオン交換基含有化合物を反応して得られたもの(特許文献1)、あるいは架橋性単量体とイオン交換基含有化合物とを反応させて得られたもの(特許文献2、特許文献3など)などが知られている。 The ion exchange liquid chromatography method is an extremely effective method for the separation and analysis of various biological substances including the analysis of glycated hemoglobins. Up to now, the filler used in the ion exchange liquid chromatography method is the one in which an ion exchange group is introduced into a base composed of a silica compound, or the reaction of an ion exchange group-containing compound with crosslinkable particles composed of an organic synthetic polymer. And the like obtained by reacting a crosslinkable monomer with an ion exchange group-containing compound (Patent Literature 2, Patent Literature 3, etc.) are known.
上記充填剤において、イオン交換基以外の基剤部分は、膨潤や収縮を避けるために、より強く、架橋された粒子であることが望ましい。しかしながら、強度を高めようとすると、親水性が低下し、疎水性物質の非特異吸着を引き起し、測定精度が低下するという問題があった。 In the above filler, the base portion other than the ion exchange group is desirably stronger and cross-linked particles in order to avoid swelling and shrinkage. However, when the strength is increased, there is a problem that the hydrophilicity is lowered, causing nonspecific adsorption of the hydrophobic substance, and the measurement accuracy is lowered.
他方、疎水性物質の非特異吸着を抑制するために、親水性の単量体を多く含有させることなどにより、親水性を高めると、上述したように膨潤や収縮が生じがちとなり、強度が低下する。従って、高流速下における分析ができなくなったり、複数の溶離液を用いた場合の平衡化が遅れたりし、測定の遅延を招くという問題があった。 On the other hand, in order to suppress non-specific adsorption of hydrophobic substances, increasing the hydrophilicity by including a large amount of hydrophilic monomers tends to cause swelling and shrinkage as described above, resulting in a decrease in strength. To do. Therefore, there is a problem that analysis at a high flow rate cannot be performed, and equilibration is delayed when a plurality of eluents are used, resulting in measurement delay.
他方、上記のような問題を解決する方法として、下記の特許文献4には、イオン交換基を有する充填剤表面に親水基を有する化合物を吸着させる方法が開示されている。 On the other hand, as a method for solving the above problems, Patent Document 4 below discloses a method of adsorbing a compound having a hydrophilic group on the surface of a filler having an ion exchange group.
しかしながら、特許文献4に記載の方法においては、吸着された化合物の充填剤表面への吸着力が弱い場合、初期状態では十分な性能を維持できたとしても、長期間使用するうちに、充填剤表面から化合物が脱離し、保持時間や測定値が変動するという問題があった。
本発明の目的は、上述した従来技術の問題点に鑑み、強度を低下させることなく、親水性を高めることができ、かつ蛋白質などが非特異的に吸着するのを抑制することができ、測定精度を高めることができ、さらに長期間にわたり性能を維持することを可能とするイオン交換液体クロマトグラフィー用充填剤を提供することにある。 In view of the above-mentioned problems of the prior art, the object of the present invention is to increase the hydrophilicity without reducing the strength, and to suppress nonspecific adsorption of proteins and the like. An object of the present invention is to provide a packing material for ion exchange liquid chromatography that can improve accuracy and maintain performance for a long period of time.
本発明は、イオン交換基を有する充填剤表面に親水基を有する複数種の化合物を吸着させたことを特徴とするイオン交換液体クロマトグラフィー用充填剤である。 The present invention is a packing material for ion-exchange liquid chromatography, wherein a plurality of types of compounds having hydrophilic groups are adsorbed on the surface of the packing material having ion-exchange groups.
本発明に係るイオン交換液体クロマトグラフィー用充填剤では、好ましくは、親水基を有する複数種の化合物の内、少なくとも1種類の化合物の等電点が4.6以下であり、か
つ少なくとも1種類の他の化合物の等電点が4.7以上である。
In the packing material for ion exchange liquid chromatography according to the present invention, preferably, the isoelectric point of at least one compound among the plurality of compounds having a hydrophilic group is 4.6 or less, and at least one compound is used. Other compounds have an isoelectric point of 4.7 or more.
本発明に係るイオン交換液体クロマトグラフィー用充填剤では、より好ましくは、等電点が4.6以下の親水基を有する少なくとも1種類の前記化合物が親水基を有する複数種の化合物の総重量中10重量%以上の割合で、かつ等電点が4.7以上の少なくとも1種類の他の化合物が親水基を有する複数種の化合物の総重量中10重量%以上の割合で用いられている。 In the packing material for ion exchange liquid chromatography according to the present invention, more preferably, at least one of the compounds having a hydrophilic group having an isoelectric point of 4.6 or less is in the total weight of a plurality of types of compounds having a hydrophilic group. At least one other compound having an isoelectric point of 4.7 or more is used in a proportion of 10% by weight or more in the total weight of the plural kinds of compounds having a hydrophilic group.
本発明に係るイオン交換液体クロマトグラフィー用充填剤では、好ましくは、上記充填剤のイオン交換基がスルホン酸基である。 In the packing material for ion exchange liquid chromatography according to the present invention, preferably, the ion exchange group of the packing material is a sulfonic acid group.
本発明に係るイオン交換液体クロマトグラフィー用充填剤では、充填剤表面に、親水基を有する2種類以上の化合物が吸着されているので、充填剤強度を低下させることなく、親水性を高めることができる。また、測定対象物、特に蛋白質または試料中の夾雑物質などの非特異吸着を抑制することができ、かつ性能を長期間にわたり維持することができる。 In the packing material for ion exchange liquid chromatography according to the present invention, two or more kinds of compounds having a hydrophilic group are adsorbed on the surface of the packing material, so that the hydrophilicity can be increased without reducing the packing material strength. it can. Further, nonspecific adsorption of a measurement object, particularly a protein or a contaminant in a sample, can be suppressed, and the performance can be maintained for a long time.
親水基を有する2種類以上の化合物の内、少なくとも1種類の化合物の等電点が4.6以下であり、かつ少なくとも1種類の他の化合物の等電点が4.7以上である場合には、性能をより長期間にわたり維持することができる。 Of the two or more compounds having a hydrophilic group, the isoelectric point of at least one compound is 4.6 or less and the isoelectric point of at least one other compound is 4.7 or more Can maintain the performance for a longer period of time.
さらに、親水基を有する複数種の化合物の総重量のうち、少なくとも1種類の等電点が4.6以下の親水基を有する化合物の使用割合が10重量%以上であり、かつ等電点が4.7以上の他の少なくとも1種類の親水基を有する化合物の使用割合が10重量%以上である場合には、性能をさらに長期間にわたり維持することができる。 Furthermore, of the total weight of the plurality of types of compounds having hydrophilic groups, at least one type of isoelectric point having a hydrophilic group having a hydrophilic group of 4.6 or less is 10% by weight or more, and the isoelectric point is When the ratio of the compound having at least one other hydrophilic group of 4.7 or more is 10% by weight or more, the performance can be maintained for a longer period.
また、充填剤のイオン交換基がスルホン酸基である場合にも、性能をより長期間にわたり維持することができる。 Further, when the ion exchange group of the filler is a sulfonic acid group, the performance can be maintained for a longer period.
本発明において、充填剤はイオン交換液体クロマトグラフィー用として用いられる充填剤であれば特に限定されず、陽イオン交換基を有するものであってもよく、陰イオン交換基を有するものであってもよい。 In the present invention, the filler is not particularly limited as long as it is a filler used for ion-exchange liquid chromatography, and may have a cation exchange group or an anion exchange group. Good.
陽イオン交換基としては、例えば、カルボキシル基、リン酸基、スルホン酸基などが挙げられ、陰イオン交換基としては、例えば、3級もしくは4級アミノ基などが挙げられる。 Examples of the cation exchange group include a carboxyl group, a phosphate group, and a sulfonate group. Examples of the anion exchange group include a tertiary or quaternary amino group.
上記充填剤は、少なくとも1種類のイオン交換基を有するものであればよく、本発明においては、公知のイオン交換基を有する充填剤を用いることができる。但し、特に好ましくは、スルホン酸基を有する充填剤が用いられ、それによって性能をより長期間にわたり維持することができる。 The said filler should just have an at least 1 type of ion exchange group, and can use the filler which has a well-known ion exchange group in this invention. However, particularly preferably, fillers having sulfonic acid groups are used, so that the performance can be maintained for a longer period.
上記イオン交換基を有する充填剤の調製方法は特に限定されず、例えば、充填剤となる粒子にイオン交換基を導入する方法、あるいはイオン交換基を有する単量体を重合して充填剤となる粒子にする方法などを用いることができる。 The method for preparing the filler having an ion exchange group is not particularly limited. For example, a method for introducing an ion exchange group into particles serving as a filler, or a monomer having an ion exchange group is polymerized to obtain a filler. A method of forming particles can be used.
上記粒子としては、例えば、シリカ、ジルコニアなどの無機系粒子や高分子粒子などの有機系粒子が挙げられ、高分子粒子としては、セルロース、ポリアミノ酸、キトサンなど
の天然高分子粒子や、ポリスチレン、ポリアクリル酸エステルなどの合成高分子粒子などを挙げることができる。中でも、合成高分子粒子を用いる場合には、耐圧性及び耐膨潤性を高めるために、架橋度の高い合成高分子粒子を用いることが望ましい。
Examples of the particles include inorganic particles such as silica and zirconia, and organic particles such as polymer particles. Examples of the polymer particles include natural polymer particles such as cellulose, polyamino acid, and chitosan, polystyrene, Examples thereof include synthetic polymer particles such as polyacrylic acid esters. In particular, when synthetic polymer particles are used, it is desirable to use synthetic polymer particles having a high degree of crosslinking in order to enhance pressure resistance and swelling resistance.
粒子にイオン交換基を導入する方法としては、特に限定されず、公知の方法を用いることができる。高分子粒子の場合では、例えば、官能基を有する高分子粒子を調製した後、その後官能基にイオン交換基を有する化合物を化学反応によって導入する方法、または、イオン交換基を有する単量体と、架橋性単量体とを混合し、重合開始剤の存在下で重合し、粒子とする方法などが用いることがきる。また、特公平8−7197号公報に記載のように、架橋性重合体粒子を調製した後、イオン交換基を有する単量体を添加し、重合体粒子の表面付近にイオン交換基を有する単量体を重合させる方法を用いてもよい。また、(メタ)アクリル酸メチルや(メタ)アクリル酸エチルなどの重合性エステル化合物を架橋性単量体などと混合し、重合開始剤の存在下で重合した後、得られた粒子を加水分解処理し、エステル化合物を陽イオン交換基に変換させてもよい。 The method for introducing the ion exchange group into the particles is not particularly limited, and a known method can be used. In the case of polymer particles, for example, after preparing polymer particles having a functional group, a method of introducing a compound having an ion exchange group into the functional group by a chemical reaction, or a monomer having an ion exchange group A method of mixing particles with a crosslinkable monomer and polymerizing in the presence of a polymerization initiator to form particles can be used. In addition, as described in JP-B-8-7197, after preparing crosslinkable polymer particles, a monomer having an ion exchange group is added, and a single unit having an ion exchange group near the surface of the polymer particles is added. A method of polymerizing a monomer may be used. In addition, a polymerizable ester compound such as methyl (meth) acrylate or ethyl (meth) acrylate is mixed with a crosslinkable monomer and polymerized in the presence of a polymerization initiator, and then the resulting particles are hydrolyzed. The ester compound may be converted to a cation exchange group by treatment.
本発明で用いられる充填剤は粒子状であり、その平均粒径(直径)は、0.1〜20μmであることが好ましい。また、粒度分布は、CV値(標準偏差÷平均粒径×100)で40%以下が好ましく、15%以下がより好ましい。平均粒径が0.1μm未満では、カラム内が高圧になり過ぎ、分離不良を起こすことがあり、20μmを超えると、カラム内のデッドボリュームが大きくなり過ぎ、分離不良を起こすことがあり、また、CV値が40%を超えても、カラム内のデッドボリュームが大きくなり過ぎ、分離不良を起こすことがある。 The filler used in the present invention is in the form of particles, and the average particle diameter (diameter) is preferably 0.1 to 20 μm. Further, the particle size distribution is preferably 40% or less, more preferably 15% or less in terms of CV value (standard deviation ÷ average particle size × 100). When the average particle size is less than 0.1 μm, the inside of the column becomes too high pressure and may cause separation failure. When it exceeds 20 μm, the dead volume in the column becomes too large and may cause separation failure. Even if the CV value exceeds 40%, the dead volume in the column becomes too large, and separation failure may occur.
本発明において、「充填剤表面に親水基を有する化合物を吸着させる」とは、上記イオン交換基を有する充填剤について、下記の(1)〜(4)の方法の内、少なくとも1つの方法を施すことを意味する。すなわち、(1)〜(4)の処理は、適宜、2つ以上組み合わされてもよい。 In the present invention, “adsorbing a compound having a hydrophilic group on the surface of the filler” means at least one of the following methods (1) to (4) for the filler having an ion exchange group. It means applying. That is, two or more processes (1) to (4) may be combined as appropriate.
(1)スラリー調製時における親水化処理
イオン交換基を有する充填剤に、0.0001〜10重量%、好ましくは0.001〜5重量%の親水基を有する化合物を分散媒に溶解させたものを添加し、0.5時間以上、好ましくは1時間以上攪拌する。
(1) Hydrophilization treatment during slurry preparation A compound having 0.0001 to 10% by weight, preferably 0.001 to 5% by weight of a hydrophilic group, dissolved in a dispersion medium in a filler having an ion exchange group And stirred for 0.5 hour or longer, preferably 1 hour or longer.
(2)充填時における親水化処理
充填液中に0.0001〜10重量%、好ましくは0.001〜5重量%の親水基を有する化合物を溶解させてカラム本体に充填を行う。充填後、0.5時間以上、好ましくは1時間以上放置する。
(2) Hydrophilization treatment at the time of packing 0.001 to 10% by weight, preferably 0.001 to 5% by weight of a compound having a hydrophilic group is dissolved in the packing liquid to fill the column body. After filling, it is allowed to stand for 0.5 hour or longer, preferably 1 hour or longer.
(3)充填後のカラムに親水基を含有する化合物の溶液を通液させることによる親水化処理
イオン交換基を有する充填剤を充填したカラムに0.0001〜10重量%、好ましくは0.001〜5重量%の親水基を有する化合物を溶解させた溶液を流速0.01〜10mL/分、好ましく0.1〜5mL/分で1分以上通液させ、通液後、0.5時間以上、好ましくは1時間以上放置する。
(3) Hydrophilization treatment by passing a solution of a compound containing a hydrophilic group through the column after packing 0.0001 to 10% by weight, preferably 0.001 in a column packed with a packing material having an ion exchange group A solution in which a compound having a hydrophilic group of ˜5% by weight is dissolved is allowed to flow for 1 minute or more at a flow rate of 0.01 to 10 mL / min, preferably 0.1 to 5 mL / min. It is preferably left for 1 hour or longer.
(4)充填後のカラムに親水基を有する化合物の溶液を注入させることによる親水化処理
イオン交換基を有する充填剤を充填したカラムに0.01〜10重量%、好ましくは0.1〜5重量%の親水基を有する化合物を溶解させた溶液を、注入量1〜1000μL、好ましくは10〜500μL、流速0.01〜10mL/分、好ましくは0.1〜5mL
/分で注入させる。注入後、0.5時間以上、好ましくは1時間以上放置する。
(4) Hydrophilization treatment by injecting a solution of a compound having a hydrophilic group into a column after packing 0.01 to 10% by weight, preferably 0.1 to 5%, in a column packed with a packing material having an ion exchange group A solution in which a compound having a weight% hydrophilic group is dissolved is injected in an amount of 1 to 1000 μL, preferably 10 to 500 μL, a flow rate of 0.01 to 10 mL / min, preferably 0.1 to 5 mL.
Infuse at / min. After the injection, it is allowed to stand for 0.5 hour or longer, preferably 1 hour or longer.
上記(1)〜(4)の処理に際しては、処理温度は15〜90℃が好ましく、より好ましくは40〜90℃で攪拌し、或いは放置することが望ましい。 In the treatments (1) to (4), the treatment temperature is preferably 15 to 90 ° C., more preferably 40 to 90 ° C., or it is desirable to leave it.
本発明により得られたイオン交換液体クロマトグラフィー用充填剤には、使用前に、測定に使用する最も溶出力の強い溶離液を、流速0.01〜10mL/分で1〜300分間通液することが望ましい。 Prior to use, the packing material for ion exchange liquid chromatography obtained by the present invention is passed through the eluent having the strongest dissolution power used for measurement at a flow rate of 0.01 to 10 mL / min for 1 to 300 minutes. It is desirable.
また、上記(1)の処理の場合には、上記(1)の処理後であって、カラムに充填する前に、測定に使用するもっとも溶出力の強い溶離液を、イオン交換液体クロマトグラフィー用充填剤重量の0.5〜100倍量添加し、1〜300分間攪拌することが望ましい。 In the case of the treatment (1) above, the eluent having the strongest elution power used for the measurement is used for ion exchange liquid chromatography after the treatment (1) and before the column is packed. It is desirable to add 0.5 to 100 times the filler weight and stir for 1 to 300 minutes.
本発明において、上記充填剤表面に吸着される親水基を有する複数種の化合物とは、アルブミン、グロブリン、カゼイン、フィチュイン、フィブリノーゲン、ヘモグロビンなどの蛋白質であり、この内2種類以上が用いられることになる。 In the present invention, the plurality of compounds having a hydrophilic group to be adsorbed on the filler surface are proteins, such as albumin, globulin, casein, Fichuin, fibrinogen, hemoglobin, two or more of this is used Will be.
また、本発明においては、上記親水基を有する複数種の化合物の内、少なくとも1種類の化合物は等電点が4.6以下のものが用いられ、かつ少なくとも他の1種類の化合物は等電点が4.7以上のものが用いられる。ここで等電点とは、蛋白質のような両性電解質において分子全体の電荷が±0になるpHである。等電点が4.6以下の親水基を有する化合物としては、カゼインやフィツインなどが挙げられる。等電点が4.7以上の親水基を有する化合物としては、アルブミン、ヘモグロビン、グロブリン、フィブリノーゲンなどが挙げられる。 In the present invention, among a plurality of types of compounds having an upper Symbol hydrophilic group, an isoelectric point of at least one compound is used those of 4.6 or less, and at least another one of the compounds equal Those having an electric point of 4.7 or more are used. Here, the isoelectric point is a pH at which the charge of the whole molecule becomes ± 0 in an amphoteric electrolyte such as a protein. Examples of the compound having a hydrophilic group with an isoelectric point of 4.6 or less include casein and phytene. The compound isoelectric point has 4.7 or more hydrophilic groups, albumin, hemoglobin, grayed b Brin, like fibrinogen and the like.
本発明においては、上記親水基を有する化合物を充填剤表面に吸着させることにより、充填剤表面が親水化される。すなわち、イオン交換基を有する充填剤表面の疎水性部分に、親水基を有する化合物が物理的あるいは化学的に吸着することにより、親水性が高められ、充填剤表面の疎水性部分と測定対象物(特に蛋白質)もしくは測定試料中の夾雑物質などとの非特異的吸着が抑制されると考えられる。 In the present invention, the surface of the filler is hydrophilized by adsorbing the compound having a hydrophilic group on the surface of the filler. That is, the hydrophilicity is enhanced by the physical or chemical adsorption of the compound having a hydrophilic group to the hydrophobic portion on the surface of the filler having ion exchange groups, and the hydrophobic portion on the surface of the filler and the object to be measured. It is considered that nonspecific adsorption with (especially protein) or contaminants in the measurement sample is suppressed.
本発明のイオン交換液体クロマトグラフィー用充填剤により測定可能な物質は、特に限定されず、従来からイオン交換液体クロマトグラフィーで分離されている物質を挙げることができる。特に、本発明により提供されるイオン交換液体クロマトグラフィー用充填剤は、カテコールアミン誘導体、ヌクレオチド、ペプチド、蛋白質などの生体関連物質の分離に好適に用いられる。 The substance that can be measured by the packing material for ion exchange liquid chromatography of the present invention is not particularly limited, and examples thereof include substances that have been conventionally separated by ion exchange liquid chromatography. In particular, the packing material for ion exchange liquid chromatography provided by the present invention is suitably used for the separation of biologically relevant substances such as catecholamine derivatives, nucleotides, peptides, and proteins.
本発明に係るイオン交換液体クロマトグラフィー用充填剤は、ステンレスなどからなるカラム本体に充填され、液体クロマトグラフィー用カラムを構成する。充填方法は特に限定されず、公知の方法を用いることができる。例えば、湿式法(スラリー法)を用いる場合、イオン交換液体クロマトグラフィー用充填剤を溶離液に用いる溶媒などの分散媒に分散させ、カラム本体にパッカーなどを経由して圧入することにより充填することができる。 The packing material for ion exchange liquid chromatography according to the present invention is packed in a column main body made of stainless steel or the like to constitute a liquid chromatography column. The filling method is not particularly limited, and a known method can be used. For example, when a wet method (slurry method) is used, packing is performed by dispersing a packing material for ion exchange liquid chromatography in a dispersion medium such as a solvent used as an eluent and press-fitting the column body through a packer or the like. Can do.
本発明のイオン交換液体クロマトグラフィー用充填剤を用いた液体クロマトグラフィーによる分析において、使用する溶離液は特に限定されず、公知の溶離液を用いることができる。例えば、リン酸、硝酸、塩酸、過塩素酸などの無機酸及びその塩、酢酸、リンゴ酸、酒石酸、コハク酸、クエン酸などの有機酸及びその塩等を含む各種緩衝液を用いること
ができる。また、溶離液には、水酸化ナトリウム、水酸化カリウムなどの塩、アセトン、アセトニトリル、ジオキサン、メタノールもしくはエタノールなどの有機溶媒を添加してもよい。
In the analysis by liquid chromatography using the packing material for ion exchange liquid chromatography of the present invention, the eluent to be used is not particularly limited, and a known eluent can be used. For example, various buffer solutions containing inorganic acids such as phosphoric acid, nitric acid, hydrochloric acid, perchloric acid and salts thereof, organic acids such as acetic acid, malic acid, tartaric acid, succinic acid, citric acid and salts thereof, and the like can be used. . In addition, a salt such as sodium hydroxide or potassium hydroxide, an organic solvent such as acetone, acetonitrile, dioxane, methanol or ethanol may be added to the eluent.
次に、具体的な実施例及び比較例を挙げることにより本発明をより具体的に説明する。なお、本発明は以下の実施例に限定されるものではない。 Next, the present invention will be described more specifically by giving specific examples and comparative examples. In addition, this invention is not limited to a following example.
〔実施例1〕
(充填剤の調製)
2−アクリルアミド−2−メチルプロパンスルホン酸(単量体A:東京化成工業社製)200g、ジエチレングリコールジメタクリレート(架橋性単量体B:新中村化学社製)400g及びベンゾイルパーオキサイド(重合開始剤:和光純薬社製)1.5gを混合し、2.5Lの4重量%ポリビニルアルコール(日本合成化学社製)水溶液に分散させた。これを窒素雰囲気下で攪拌しながら昇温し、80℃で8時間重合した。重合後、洗浄・分級して平均粒径8μmの充填剤を得た。
[Example 1]
(Preparation of filler)
200 g of 2-acrylamido-2-methylpropanesulfonic acid (monomer A: manufactured by Tokyo Chemical Industry Co., Ltd.), 400 g of diethylene glycol dimethacrylate (crosslinkable monomer B: manufactured by Shin-Nakamura Chemical Co., Ltd.) and benzoyl peroxide (polymerization initiator) : Wako Pure Chemical Industries, Ltd.) 1.5 g was mixed and dispersed in 2.5 L of 4 wt% polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd.) aqueous solution. This was heated with stirring in a nitrogen atmosphere and polymerized at 80 ° C. for 8 hours. After polymerization, washing and classification were performed to obtain a filler having an average particle size of 8 μm.
(親水化処理)
上記充填剤0.7gを170m mol/Lリン酸緩衝液(pH5.7)10mLに分散させ、5分間超音波処理した後、よく攪拌した。
(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 10 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.
次に、0.1重量%BSA(和光純薬社製、等電点4.9)と0.1重量%カゼイン(和光純薬社製、等電点4.6)を含む170m mol/Lリン酸緩衝液(pH5.7)を20mL添加し、80℃の恒温水槽中で24時間ゆるやかに攪拌した後、恒温水槽から取出し、室温になるまで放置した。その後、3000rpm×10分の遠心操作を行った後、上清を除去し、次に300m mol/Lリン酸緩衝液(pH8.5)を5mL添加した。再度3000rpm×10分の遠心を行った後上清を除去した。次に、170m mol/Lリン酸緩衝液(pH5.7)を30mL添加し、ゆるやかに攪拌し、親水化処理された充填剤分散液を得た。この全量をステンレス製空カラム(直径4.6mm×長さ35mm)を接続してなるパッカー(梅谷精機社製)に注入した。パッカーに送液ポンプ(サヌキ工業社製)を接続し、圧力200kg/cm2で充填剤を定圧充填した。 Next, 170 mmol / L containing 0.1 wt% BSA (manufactured by Wako Pure Chemical Industries, Ltd., isoelectric point 4.9) and 0.1 wt% casein (manufactured by Wako Pure Chemical Industries, Ltd., isoelectric points 4.6). After adding 20 mL of a phosphate buffer (pH 5.7) and gently stirring in a constant temperature water bath at 80 ° C. for 24 hours, the sample was taken out from the constant temperature water bath and left to reach room temperature. Then, after centrifuging at 3000 rpm × 10 minutes, the supernatant was removed, and then 5 mL of 300 mmol / L phosphate buffer (pH 8.5) was added. After centrifugation again at 3000 rpm × 10 minutes, the supernatant was removed. Next, 30 mL of 170 mmol / L phosphate buffer (pH 5.7) was added and gently stirred to obtain a hydrophilic dispersion-treated filler dispersion. The whole amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected to a stainless steel empty column (diameter: 4.6 mm × length: 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer, and the filler was filled at a constant pressure with a pressure of 200 kg / cm 2 .
〔実施例2〕
(充填剤の調製)
実施例1と同じ充填剤について以下の親水化処理を施した。
[Example 2]
(Preparation of filler)
The following hydrophilization treatment was performed on the same filler as in Example 1.
(親水化処理)
上記充填剤0.7gを170m mol/Lリン酸緩衝液(pH5.7)10mLに分散させ、5分間超音波処理した後、よく攪拌した。
(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 10 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.
次に、0.1重量%ラクトフェリン(和光純薬社製、等電点7.2)と0.1重量%カゼイン(和光純薬社製、等電点4.6)とを含む170m mol/Lリン酸緩衝液(pH5.7)20mL添加し、80℃の恒温水槽中で24時間ゆるやかに攪拌した後、恒温水槽から取出し、室温になるまで放置した。その後、3000rpm×10分の遠心を行った後上清を除去した。次に、そこに300m mol/Lリン酸緩衝液(pH8.5)を5mL添加し、再度3000rpm×10分の遠心を行った後、上清を除去した。次に、170m mol/Lリン酸緩衝液(pH5.7)を30mL添加し、ゆるやかに攪拌し、親水化された充填剤分散液を得た。この全量をステンレス製空カラム(直径4.6mm×長さ35mm)を接続してなるパッカー(梅谷精機社製)に注入した。パッカーに送液ポンプ(サヌキ工業社製)を接続し、圧力200kg/cm2で充填剤を定圧充填した
。
Next, 170 mmol / l containing 0.1 wt% lactoferrin (Wako Pure Chemical Industries, Ltd., isoelectric point 7.2) and 0.1 wt% casein (Wako Pure Chemical Industries, Ltd., isoelectric point 4.6). After adding 20 mL of L phosphate buffer (pH 5.7) and gently stirring in a constant temperature water bath at 80 ° C. for 24 hours, it was taken out from the constant temperature water bath and left to reach room temperature. Then, after centrifuging at 3000 rpm × 10 minutes, the supernatant was removed. Next, 5 mL of 300 mmol / L phosphate buffer (pH 8.5) was added thereto, and after centrifugation again at 3000 rpm × 10 minutes, the supernatant was removed. Next, 30 mL of 170 mmol / L phosphate buffer (pH 5.7) was added and gently stirred to obtain a hydrophilic filler dispersion. The whole amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected to a stainless steel empty column (diameter 4.6 mm × length 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer, and the filler was filled at a constant pressure with a pressure of 200 kg / cm 2 .
〔実施例3〕
(充填剤の調製)
実施例1と同じ充填剤について以下の親水化処理を施した。
Example 3
(Preparation of filler)
The following hydrophilization treatment was performed on the same filler as in Example 1.
(親水化処理)
上記充填剤0.7gを170m mol/Lリン酸緩衝液(pH5.7)30mLに分散させ、5分間超音波処理した後、よく攪拌した。
(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 30 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.
次に、その全量をステンレス製空カラム(直径4.6mm×長さ35mm)を接続したパッカー(梅谷精機社製)に注入した。パッカーに送液ポンプ(サヌキ工業社製)を接続し、圧力200kg/cm2で定圧充填した。次に、上記カラム内に0.05重量%BS
A(和光純薬社製、等電点4.9)と0.05重量%カゼイン(和光純薬社製、等電点4.6)を含む170m mol/Lリン酸緩衝液(pH5.7)を、室温において送液ポンプ(LC−9A、島津製作所社製)により流速2mL/分で30分間通液した。
Next, the entire amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected with a stainless steel empty column (diameter: 4.6 mm × length: 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 200 kg / cm 2 . Next, 0.05 wt% BS in the column.
170 mmol / L phosphate buffer (pH 5.7) containing A (Wako Pure Chemical Industries, Ltd., isoelectric point 4.9) and 0.05 wt% casein (Wako Pure Chemical Industries, Ltd., isoelectric point 4.6). ) At room temperature by a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation) at a flow rate of 2 mL / min for 30 minutes.
その後、60℃の恒温水槽中で168時間放置し、恒温水槽から取出し、室温になるまで放置した。その後、上記カラム内に300m mol/Lリン酸緩衝液(pH8.5)を室温において送液ポンプ(LC−9A、島津製作所社製)により流速2mL/分で30分間通液した。引き続き、上記カラム内に170m mol/Lリン酸緩衝液(pH5.7)を室温において送液ポンプ(LC−9A、島津製作所社製)により流速2mL/分で30分間通液した。 Then, it was left to stand in a constant temperature water bath at 60 ° C. for 168 hours, taken out from the constant temperature water bath and left to reach room temperature. Thereafter, 300 mmol / L phosphate buffer (pH 8.5) was passed through the column at a flow rate of 2 mL / min for 30 minutes at room temperature using a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation). Subsequently, 170 mmol / L phosphate buffer (pH 5.7) was passed through the column at room temperature for 30 minutes at a flow rate of 2 mL / min using a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation).
〔実施例4〕
(充填剤の調製)
実施例1と同じ充填剤について以下の親水化処理を施した。
Example 4
(Preparation of filler)
The following hydrophilization treatment was performed on the same filler as in Example 1.
(親水化処理)
上記充填剤0.7gを170m mol/Lリン酸緩衝液(pH5.7)30mLに分散させ、5分間超音波処理した後、よく攪拌した。
(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 30 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.
次に、その全量をステンレス製空カラム(直径4.6mm×長さ35mm)を接続してなるパッカー(梅谷精機社製)に注入した。パッカーに送液ポンプ(サヌキ工業社製)を接続し、圧力200kg/cm2で定圧充填した。次に、上記カラム内に0.05重量%
ラクトフェリン(和光純薬社製、等電点7.2)と0.05重量%カゼイン(和光純薬社製、等電点4.6)を含む170m mol/Lリン酸緩衝液(pH5.7)を、室温において送液ポンプ(LC−9A、島津製作所社製)により流速2mL/分で30分間通液した。
Next, the entire amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected to an empty stainless steel column (diameter 4.6 mm × length 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 200 kg / cm 2 . Next, 0.05 wt% in the column
170 mMole / L phosphate buffer (pH 5.7) containing lactoferrin (Wako Pure Chemical Industries, Ltd., isoelectric point 7.2) and 0.05 wt% casein (Wako Pure Chemical Industries, Ltd., isoelectric point 4.6). ) At room temperature by a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation) at a flow rate of 2 mL / min for 30 minutes.
その後、60℃の恒温水槽中で168時間放置し、恒温水槽から取出し、室温になるまで放置した。その後、上記カラム内に300m mol/Lリン酸緩衝液(pH8.5)を室温において送液ポンプ(LC−9A、島津製作所社製)により流速2mL/分で30分間通液した。引き続き、上記カラム内に170m mol/Lリン酸緩衝液(pH5.7)を室温において送液ポンプ(LC−9A、島津製作所社製)により流速2mL/分で30分間通液した。 Then, it was left to stand in a constant temperature water bath at 60 ° C. for 168 hours, taken out from the constant temperature water bath and left to reach room temperature. Thereafter, 300 mmol / L phosphate buffer (pH 8.5) was passed through the column at a flow rate of 2 mL / min for 30 minutes at room temperature using a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation). Subsequently, 170 mmol / L phosphate buffer (pH 5.7) was passed through the column at room temperature for 30 minutes at a flow rate of 2 mL / min using a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation).
〔比較例1〕
(充填剤の調製)
実施例1と同じ充填剤を用い、以下のようにして充填を行ったが、親水化処理は行わなかった。
[Comparative Example 1]
(Preparation of filler)
Filling was performed as follows using the same filler as in Example 1, but no hydrophilization treatment was performed.
上記充填剤0.7gを170m mol/Lリン酸緩衝液(pH5.7)30mLに分散させ、5分間超音波処理した後、よく攪拌した。 0.7 g of the above filler was dispersed in 30 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.
次に、その全量をステンレス製空カラム(直径4.6mm×長さ35mm)を接続してなるパッカー(梅谷精機社製)に注入した。パッカーに送液ポンプ(サヌキ工業社製)を接続し、圧力200kg/cm2で定圧充填した。 Next, the entire amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected to an empty stainless steel column (diameter 4.6 mm × length 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 200 kg / cm 2 .
〔比較例2〕
(充填剤の調製)
実施例1と同じ充填剤について以下の親水化処理を施した。
[Comparative Example 2]
(Preparation of filler)
The following hydrophilization treatment was performed on the same filler as in Example 1.
(親水化処理)
上記充填剤0.7gを170m mol/Lリン酸緩衝液(pH5.7)10mLに分散させ、5分間超音波処理した後、よく攪拌した。
(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 10 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.
次に、0.2重量%カゼイン(和光純薬社製、等電点4.6)を含む170m mol/Lリン酸緩衝液(pH5.7)を20mL添加し、80℃の恒温水槽中で24時間ゆるやかに攪拌した後、恒温水槽から取出し、室温になるまで放置した。その後、3000rpm×10分の遠心により上清を除去し、そこに300m mol/Lリン酸緩衝液(pH8.5)を5mL添加し、再度3000rpm×10分の遠心により上清を除去した。そこへ170m mol/Lリン酸緩衝液(pH5.7)を30mL添加し、ゆるやかに攪拌した後、全量をステンレス製空カラム(直径4.6mm×長さ35mm)を接続してなるパッカー(梅谷精機社製)に注入した。パッカーに送液ポンプ(サヌキ工業社製)を接続し、圧力200kg/cm2で定圧充填した。 Next, 20 mL of 170 mmol / L phosphate buffer (pH 5.7) containing 0.2% by weight casein (manufactured by Wako Pure Chemical Industries, Ltd., isoelectric point 4.6) is added, and in a constant temperature water bath at 80 ° C. After gently stirring for 24 hours, it was taken out from the constant temperature water bath and left to reach room temperature. Thereafter, the supernatant was removed by centrifugation at 3000 rpm × 10 minutes, 5 mL of 300 mmol / L phosphate buffer (pH 8.5) was added thereto, and the supernatant was removed again by centrifugation at 3000 rpm × 10 minutes. After adding 30 mL of 170 mmol / L phosphate buffer (pH 5.7) and stirring gently, a packer (Umeya) connected to an empty stainless steel column (diameter: 4.6 mm × length: 35 mm). Into Seiki Co.). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 200 kg / cm 2 .
〔比較例3〕
(充填剤の調製)
実施例1と同じ充填剤について以下の親水化処理を施した。
[Comparative Example 3]
(Preparation of filler)
The following hydrophilization treatment was performed on the same filler as in Example 1.
(親水化処理)
上記充填剤0.7gを170m mol/Lリン酸緩衝液(pH5.7)30mLに分散させ、5分間超音波処理した後、よく攪拌した。
(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 30 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.
次に、その全量をステンレス製空カラム(直径4.6mm×長さ35mm)を接続したパッカー(梅谷精機社製)に注入した。パッカーに送液ポンプ(サヌキ工業社製)を接続し、圧力200kg/cm2で定圧充填した。次に、上記カラム内に0.1重量%フィツ
イン(和光純薬社製、等電点3.5)を含む170m mol/Lリン酸緩衝液(pH5.7)を、室温において送液ポンプ(LC−9A、島津製作所社製)により流速2mL/分で30分間通液した。
Next, the entire amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected with a stainless steel empty column (diameter: 4.6 mm × length: 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 200 kg / cm 2 . Next, a 170 mmol / L phosphate buffer solution (pH 5.7) containing 0.1 wt% phytene (manufactured by Wako Pure Chemical Industries, Ltd., isoelectric point 3.5) in the above column was fed at room temperature to a liquid feed pump ( LC-9A (manufactured by Shimadzu Corporation) was passed for 30 minutes at a flow rate of 2 mL / min.
その後、60℃の恒温水槽中で168時間放置し、恒温水槽から取出し、室温になるまで放置した。その後、上記カラム内に300m mol/Lリン酸緩衝液(pH8.5)を室温において送液ポンプ(LC−9A、島津製作所社製)により流速2mL/分で30分間通液した。引き続き、上記カラム内に170m mol/Lリン酸緩衝液(pH5.7)を室温において送液ポンプ(LC−9A、島津製作所社製)により流速2mL/分で30分間通液した。 Then, it was left to stand in a constant temperature water bath at 60 ° C. for 168 hours, taken out from the constant temperature water bath and left to reach room temperature. Thereafter, 300 mmol / L phosphate buffer (pH 8.5) was passed through the column at a flow rate of 2 mL / min for 30 minutes at room temperature using a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation). Subsequently, 170 mmol / L phosphate buffer (pH 5.7) was passed through the column at room temperature for 30 minutes at a flow rate of 2 mL / min using a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation).
(使用開始直後の同時再現性の評価)
グリコHb(ヘモグロビン)コントロールレベル2(国際試薬社製)を200μLの注
射用水で溶解した後、希釈液(0.1重量%トリトンX−100)を含有するリン酸緩衝液(pH7.0))で100倍に希釈したものを測定試料として用い、以下の条件でヘモグロビンA1cの測定を10回連続行い、同時再現性の比較を行った。再現性の評価は、A1c値とA1c成分の保持時間を測定することにより行った。結果を下記の表1及び表2に示す。
(Evaluation of simultaneous reproducibility immediately after use)
Glyco-Hb (hemoglobin) control level 2 (made by Kokusai Reagent Co., Ltd.) was dissolved in 200 μL of water for injection, and then diluted with phosphate buffer (pH 7.0) containing 0.1 wt% Triton X-100) Using the sample diluted 100 times as the measurement sample, hemoglobin A1c was continuously measured 10 times under the following conditions, and the simultaneous reproducibility was compared. The reproducibility was evaluated by measuring the A1c value and the retention time of the A1c component. The results are shown in Tables 1 and 2 below.
測定条件
システム : 送液ポンプ:LC−9A(島津製作所社製)
オートサンプラー:ASU−420(積水化学工業社製)
検出器:SPD−6AV(島津製作所社製)
溶離液 : 第1液:170m mol/Lリン酸緩衝液(pH5.7)
第2液:300m mol/Lリン酸緩衝液(pH8.5)
溶出法 : 0〜3分は第1液を、3〜3.2分は第2液を、3.2〜4分は第1
液を流した。
流速 : 1.0mL/分
検出波長 : 415nm
試料注入量: 10μL
Measurement conditions System: Liquid feed pump: LC-9A (manufactured by Shimadzu Corporation)
Autosampler: ASU-420 (manufactured by Sekisui Chemical Co., Ltd.)
Detector: SPD-6AV (manufactured by Shimadzu Corporation)
Eluent: First liquid: 170 mmol / L phosphate buffer (pH 5.7)
Second solution: 300 mmol / L phosphate buffer (pH 8.5)
Elution method: 1st liquid for 0 to 3 minutes, 2nd liquid for 3 to 3.2 minutes, 1st liquid for 3.2 to 4 minutes
The liquid was poured.
Flow rate: 1.0 mL / min Detection wavelength: 415 nm
Sample injection volume: 10 μL
(実検体の負荷におけるカラム劣化による測定値の変動の評価(耐久性試験))
健常人血をNaF採血し、溶血希釈液(0.1%トリトンX−100を含有するリン酸緩衝液(pH7.0))で溶血し、150倍に希釈したものを負荷検体とし、1日に300検体を測定した。測定の前後でグリコHbコントロールレベル2(国際試薬社製)を200μLの注射用水で溶解した後、希釈液(0.1%トリトンX−100を含有するリン酸緩衝液(pH7.0))で100倍に希釈したものを測定試料として、上述した測定条件に従ってヘモグロビンA1cの測定を10回連続で行い、その平均値を測定値とした。本試験の評価は、A1c値(%)と、A1c成分の保持時間(秒)により行った。結果を下記の表3及び表4に示す。
(Evaluation of changes in measured values due to column deterioration under actual sample load (durability test))
Healthy human blood was collected from NaF, hemolyzed with a hemolysis dilution (phosphate buffer (pH 7.0) containing 0.1% Triton X-100), and diluted 150 times as a load sample. 300 samples were measured. Before and after the measurement, Glyco Hb Control Level 2 (made by Kokusai Reagent Co., Ltd.) was dissolved in 200 μL of water for injection, and then diluted with a diluent (phosphate buffer (pH 7.0) containing 0.1% Triton X-100). Using a sample diluted 100 times as a measurement sample, hemoglobin A1c was measured 10 times continuously according to the measurement conditions described above, and the average value was taken as the measurement value. This test was evaluated based on the A1c value (%) and the retention time (seconds) of the A1c component. The results are shown in Tables 3 and 4 below.
Claims (2)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2004038866A JP4388829B2 (en) | 2004-02-16 | 2004-02-16 | Packing for ion exchange liquid chromatography |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2004038866A JP4388829B2 (en) | 2004-02-16 | 2004-02-16 | Packing for ion exchange liquid chromatography |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2005227237A JP2005227237A (en) | 2005-08-25 |
JP4388829B2 true JP4388829B2 (en) | 2009-12-24 |
Family
ID=35002051
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2004038866A Expired - Fee Related JP4388829B2 (en) | 2004-02-16 | 2004-02-16 | Packing for ion exchange liquid chromatography |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP4388829B2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4511847B2 (en) * | 2004-02-16 | 2010-07-28 | 積水化学工業株式会社 | Method for measuring hemoglobin A1c |
-
2004
- 2004-02-16 JP JP2004038866A patent/JP4388829B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
JP2005227237A (en) | 2005-08-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP4740036B2 (en) | Method for measuring hemoglobins | |
JP2010164579A (en) | Method of measuring hemoglobin | |
JP3927322B2 (en) | Method for producing packing material for liquid chromatography | |
JP3352645B2 (en) | Filler for liquid chromatography and measurement method using the same | |
JP4231165B2 (en) | Packing for liquid chromatography | |
JP5041733B2 (en) | Method for measuring hemoglobins | |
JP4388829B2 (en) | Packing for ion exchange liquid chromatography | |
JP4511847B2 (en) | Method for measuring hemoglobin A1c | |
JP4860133B2 (en) | Method for analyzing glycated hemoglobin | |
JP2001228133A (en) | Measuring method of hemoglobins | |
JPH11295286A (en) | Hemoglobins measuring method | |
JP4094776B2 (en) | Hemolysis reagent and method for measuring hemoglobin using the same | |
JP4268730B2 (en) | Method for producing packing material for liquid chromatography | |
JP3429679B2 (en) | Method for measuring stable hemoglobin A1c | |
JP2012073184A (en) | Method for measuring hemoglobins | |
JP2001183356A (en) | Measuring method for hemoglobins | |
JP2001221788A (en) | Measuring method for hemoglobin and the like | |
JP2005233905A (en) | Packing for ion exchange liquid chromatography | |
JPH087198B2 (en) | Quantitative method for glycated hemoglobin | |
JP5378623B2 (en) | Packing material for ion exchange liquid chromatography and analysis method of glycated hemoglobin | |
JP5522772B2 (en) | Column filler and method for producing column filler | |
JP4074393B2 (en) | Packing for liquid chromatography | |
JP4109395B2 (en) | Method for measuring hemoglobins | |
JP3990713B2 (en) | Packing material for ion exchange liquid chromatography and analysis method of glycated hemoglobin | |
JP3975017B2 (en) | Method for measuring hemoglobin by liquid chromatography |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20061205 |
|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20090423 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20090610 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20090807 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20090909 |
|
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20091005 |
|
R151 | Written notification of patent or utility model registration |
Ref document number: 4388829 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R151 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121009 Year of fee payment: 3 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121009 Year of fee payment: 3 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131009 Year of fee payment: 4 |
|
LAPS | Cancellation because of no payment of annual fees |