JPH08136520A - Column for liquid chromatography - Google Patents
Column for liquid chromatographyInfo
- Publication number
- JPH08136520A JPH08136520A JP6270968A JP27096894A JPH08136520A JP H08136520 A JPH08136520 A JP H08136520A JP 6270968 A JP6270968 A JP 6270968A JP 27096894 A JP27096894 A JP 27096894A JP H08136520 A JPH08136520 A JP H08136520A
- Authority
- JP
- Japan
- Prior art keywords
- column
- hydroxyapatite
- substance
- acidic
- column packing
- 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.)
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- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、医学、薬学、生化学等
の分野におけるタンパク質、核酸、糖類等の生化学物質
の分析若しくは分取等に利用可能な液体クロマトグラフ
ィー用カラムに関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liquid chromatography column which can be used for analysis or fractionation of biochemical substances such as proteins, nucleic acids and saccharides in the fields of medicine, pharmacy and biochemistry.
【０００２】[0002]
【従来の技術】ハイドロキシアパタイトをカラム充填剤
とする液体クロマトグラフィーは、１９５６年チゼリウ
スによって創案され、当初実験室的に合成されていた
が、その後大量合成した市販品が供されている。このよ
うな市販品としては、例えば湿式合成もしくは水熱合成
した板状ハイドロキシアパタイト結晶を、スプレードラ
イ法等により球状に造粒した充填剤（特開平１−２３０
４１３号公報）等が知られている。該板状ハイドロキシ
アパタイト結晶を含むカラム充填剤を用いたクロマトグ
ラフィーの吸着分離機構は、例えば酸性の生化学物質の
場合、その分子表面上の負に帯電した官能基（酸性タン
パク質のカルボキシル基、ＤＮＡのポリヌクレオチド中
のリン酸基等）により、該板状結晶のＡ面又はＢ面上に
存在するカルシウムイオンより構成される正に帯電した
吸着サイトに吸着する。一方、塩基性生化学物質の場
合、その分子表面上の正に帯電した官能基（塩基性タン
パク質のアミノ基等）により、板状結晶のＣ面上に存在
するリン酸イオンより構成される負に帯電した吸着サイ
トに吸着する。2. Description of the Related Art Liquid chromatography using hydroxyapatite as a column packing material was invented by Tizellius in 1956, and was initially synthesized in the laboratory. As such a commercially available product, for example, a filler obtained by granulating wet- or hydrothermally-synthesized plate-like hydroxyapatite crystals into spherical particles by a spray drying method or the like (Japanese Patent Laid-Open No. 1-230).
No. 413) is known. The adsorption separation mechanism of chromatography using a column packing containing the plate-like hydroxyapatite crystal is, for example, in the case of an acidic biochemical substance, a negatively charged functional group (carboxyl group of acidic protein, DNA And the like) in the polynucleotide of (1) to be adsorbed on the positively charged adsorption site composed of calcium ions existing on the A-face or B-face of the plate crystal. On the other hand, in the case of basic biochemical substances, due to the positively charged functional groups (amino groups of basic proteins, etc.) on the surface of the molecule, a negative ion composed of phosphate ions present on the C-plane of plate crystals Adsorbs on the adsorption site that is charged to the.
【０００３】しかしながら、前記板状ハイドロキシアパ
タイト結晶は、形態制御されたものではなく、通常のア
スペクト比（ｃ／ａ）が３．０程度の六角柱状微結晶で
あるため、結晶のＣ面表面積は大きいが、結晶のＡ面又
はＢ面の表面積は相対的に大きくない。従って、塩基性
生化学物質を吸着するＣ面上の吸着サイトの吸着結合力
は強いが、酸性生化学物質を吸着するＡ面又はＢ面上の
吸着サイトの吸着結合力は弱くなる。このような板状結
晶をカラム充填剤に用いた場合の塩基性生化学物質と酸
性生化学物質との分離度及び各物質の吸着力を図１
（ａ）に示し、詳述する。この際分離度とは、図１にお
ける塩基性生化学物質の保持時間と、酸性生化学物質の
保持時間との差で表わすことができ、保持時間の差が大
きいほど分離度に優れていることを示す。一方吸着力
は、各々の物質の吸光度の値で表わすことができ、吸光
度が高いほどその物質に対する吸着力に優れていること
を示す。してみると、板状ハイドロキシアパタイト結晶
の場合、図１（ａ）に示されるように、酸性生化学物質
の吸着結合力は弱く（吸光度が低い）、その保持時間は
一般に短いが、塩基性生化学物質、特に等電点が７．０
を越える塩基性タンパク質等の吸着結合力は良好（吸光
度が高い）で、その保持時間は一般に長い。従って、こ
の板状結晶では、塩基性生化学物質と酸性生化学物質の
分離度は良好であるが、酸性生化学物質（酸性タンパク
質、ＤＮＡ等）の吸着結合力が弱く、故に酸性生化学物
質の吸着分離特性が劣るという欠点がある。However, since the plate-like hydroxyapatite crystal is not a morphologically controlled one and is a hexagonal columnar microcrystal having an ordinary aspect ratio (c / a) of about 3.0, the C-plane surface area of the crystal is Although large, the surface area of the A-face or B-face of the crystal is not relatively large. Therefore, the adsorption binding force of the adsorption site on the C surface for adsorbing the basic biochemical substance is strong, but the adsorption binding force of the adsorption site on the A surface or B surface for adsorbing the acidic biochemical substance becomes weak. FIG. 1 shows the degree of separation between basic biochemical substances and acidic biochemical substances and the adsorption force of each substance when such plate crystals are used as a column packing material.
It is shown in FIG. At this time, the degree of separation can be expressed by the difference between the retention time of the basic biochemical substance and the retention time of the acidic biochemical substance in FIG. 1, and the greater the difference in retention time, the better the separation degree. Indicates. On the other hand, the adsorptive power can be expressed by the value of the absorbance of each substance, and the higher the absorbance, the better the adsorptivity to the substance. As a result, in the case of plate-like hydroxyapatite crystals, as shown in Fig. 1 (a), the adsorptive binding force of the acidic biochemical substance is weak (low absorbance), and its retention time is generally short, but basic. Biochemicals, especially with an isoelectric point of 7.0
Adsorption and binding strength of basic proteins and the like exceeding 5 are good (high absorbance), and their retention time is generally long. Therefore, in this plate crystal, the degree of separation of the basic biochemical substance and the acidic biochemical substance is good, but the adsorptive binding force of the acidic biochemical substance (acidic protein, DNA, etc.) is weak, and therefore the acidic biochemical substance is However, there is a drawback that the adsorption separation property of is poor.
【０００４】そこで、このような欠点を解決するため
に、酸性生化学物質の吸着サイトを有するＡ面又はＢ面
を選択的に成長させた、即ち結晶のＡ面又はＢ面の表面
積を相対的に増大させて酸性生化学物質の吸着結合力を
強くした針状ハイドロキシアパタイト結晶を含むカラム
充填剤が提案されている。In order to solve such a drawback, therefore, the A surface or B surface having an adsorption site for an acidic biochemical substance is selectively grown, that is, the surface area of the A surface or B surface of the crystal is relatively changed. There has been proposed a column packing material containing needle-shaped hydroxyapatite crystals which has been increased to increase the adsorptive binding force of acidic biochemical substances.
【０００５】しかしながら、前記針状結晶の場合、図１
（ｂ）に示されるとおり、酸性生化学物質の吸着結合力
が強くなると共に、酸性生化学物質の保持時間が増長
し、且つ塩基性生化学物質の保持時間が減短するため、
双方の保持時間が接近してピークが重なり合う恐れが生
じ、分離度が極端に低下するという欠点がある。However, in the case of the needle-shaped crystal, as shown in FIG.
As shown in (b), since the adsorptive binding force of the acidic biochemical substance is increased, the retention time of the acidic biochemical substance is increased, and the retention time of the basic biochemical substance is shortened,
There is a drawback that the retention times of both approaches and the peaks overlap each other, resulting in extremely low resolution.
【０００６】[0006]
【発明が解決しようとする課題】従って本発明の目的
は、酸性生化学物質と塩基性生化学物質との良好な分離
度と、各物質に対する優れた吸着力とを兼備し、バラン
スの良い分離展開能を示す液体クロマトグラフィー用カ
ラムを提供することにある。SUMMARY OF THE INVENTION Therefore, an object of the present invention is to achieve a good balance of separation between an acidic biochemical substance and a basic biochemical substance and an excellent adsorption force for each substance, and to achieve a well-balanced separation. The object is to provide a column for liquid chromatography showing a developing ability.
【０００７】[0007]
【課題を解決するための手段】本発明によれば、アスペ
クト比１０以上の針状ハイドロキシアパタイト結晶を含
むカラム充填剤層（以下カラム充填剤層Ａと称す）と、
アスペクト比２未満の板状ハイドロキシアパタイト結晶
を含むカラム充填剤層（以下カラム充填剤層Ｂと称す）
とを、直列に接続したことを特徴とする液体クロマトグ
ラフィー用カラムが提供される。According to the present invention, a column packing layer containing needle-shaped hydroxyapatite crystals having an aspect ratio of 10 or more (hereinafter referred to as column packing layer A),
Column packing layer containing plate-like hydroxyapatite crystals having an aspect ratio of less than 2 (hereinafter referred to as column packing layer B)
There is provided a column for liquid chromatography, characterized in that and are connected in series.
【０００８】以下本発明を更に詳細に説明する。本発明
の液体クロマトグラフィー用カラムは、前記カラム充填
剤層Ａと、前記カラム充填剤層Ｂとを、直列に接続した
ことを特徴とする。このように前記カラム充填剤層Ａ及
びＢを直列に接続することにより、酸性生化学物質及び
塩基性生化学物質をバランス良く分離展開させることが
できるのは、以下の吸着機構によるものと考えられる。The present invention will be described in more detail below. The liquid chromatography column of the present invention is characterized in that the column packing material layer A and the column packing material layer B are connected in series. By connecting the column packing layers A and B in series as described above, it is considered that the acidic biochemical substance and the basic biochemical substance can be separated and developed in a well-balanced manner due to the following adsorption mechanism. .
【０００９】本発明のカラムを構成する各充填剤層を、
例えば上層に前記カラム充填剤層Ｂが、下層に前記カラ
ム充填剤層Ａが配置するように直列に接続した場合、グ
ラジエント溶出によって、酸性生化学物質は、まず上層
のカラム充填剤層Ｂに吸着し、すぐに低溶媒濃度にて脱
離するが、次いで下層のカラム充填剤層Ａに再吸着し、
更に高溶媒濃度となってから下層から脱離して溶出す
る。従ってこの際、酸性生化学物質の保持時間は図１
（ｂ）及び（ｃ）に示すとおり、カラム充填剤層Ａ（針
状ハイドロキシアパタイト結晶）単独の場合と同程度の
長さとなり、しかも、吸着力も良好となる。これに対
し、塩基性生化学物質は、上層のカラム充填剤層Ｂに吸
着した後、前記酸性生化学物質が下層から脱離するより
更に高溶媒濃度になるまで保持された後に脱離し、次い
で下層のカラム充填剤層Ａに吸着することなく通過す
る。従ってこの際、塩基性生化学物質の保持時間は、図
１（ａ）及び（ｃ）に示すとおり、カラム充填剤層Ｂ
（板状ハイドロキシアパタイト結晶）単独の場合と同程
度の長さとなり、しかも吸着力も良好となる。即ち、本
発明のカラムでは、カラム充填剤層Ａとカラム充填剤層
Ｂとを直列に接続することにより、各々の充填剤層単独
使用における欠点を改善し、各物質に対する良好な分離
度と優れた吸着力とを兼備し、バランスの良い分離展開
能を示すように作用する。なお、各充填剤層を、前述の
例示とは逆に、カラム充填剤層Ａを上層に、カラム充填
剤層Ｂを下層に直列に接続することによっても同様な作
用効果が得られる。[0009] Each packing layer constituting the column of the present invention,
For example, when the column packing material layer B is connected in series so that the column packing material layer A is arranged in an upper layer and the column packing material layer A is arranged in a lower layer, the acidic biochemical substance is first adsorbed to the upper column packing material layer B by gradient elution. And immediately desorbed at a low solvent concentration, but then re-adsorbed on the lower column packing material layer A,
After a higher solvent concentration is reached, the lower layer is desorbed and eluted. Therefore, at this time, the retention time of the acidic biochemical substance is shown in FIG.
As shown in (b) and (c), the length is about the same as in the case of the column packing layer A (acicular hydroxyapatite crystals) alone, and the adsorption force is also good. On the other hand, the basic biochemical substance is adsorbed to the column packing material layer B in the upper layer, and then is retained until the acidic biochemical substance has a higher solvent concentration than that in the lower layer. It passes through without adsorbing to the lower column packing material layer A. Therefore, at this time, the retention time of the basic biochemical substance is as shown in FIGS.
(Plate shaped hydroxyapatite crystal) The length is about the same as the case of using it alone, and the adsorption force is also good. That is, in the column of the present invention, by connecting the column packing material layer A and the column packing material layer B in series, the drawbacks of using each packing material layer alone are improved, and good separation and excellent separation for each substance are achieved. It also functions as an adsorption force and exhibits a well-balanced separation and development ability. It is to be noted that, similar to the above example, the same effects can be obtained by connecting the column packing layer A to the upper layer and the column packing layer B to the lower layer in series, which is the reverse of the above example.
【００１０】前記カラム充填剤層Ａは、アスペクト比が
１０以上、好ましくは１５以上の針状ハイドロキシアパ
タイト結晶を含む。この際アスペクト比とは、ハイドロ
キシアパタイト１次結晶のｃ軸方向の結晶子径を長軸と
し、ａ軸又はｂ軸方向の結晶子径を短軸とした場合の相
対比（ｃ／ａ）のことであり、このようなｃ軸方向の結
晶子径は、５０〜２０００ｎｍ、ａ軸方向の結晶子径は
１０〜１００ｎｍに調整するのが好ましい。The column packing material layer A contains needle-like hydroxyapatite crystals having an aspect ratio of 10 or more, preferably 15 or more. At this time, the aspect ratio is a relative ratio (c / a) when the crystallite diameter in the c-axis direction of the hydroxyapatite primary crystal is the major axis and the crystallite diameter in the a-axis or the b-axis direction is the minor axis. It is preferable to adjust the crystallite diameter in the c-axis direction to 50 to 2000 nm and the crystallite diameter in the a-axis direction to 10 to 100 nm.
【００１１】前記針状ハイドロキシアパタイト結晶を調
製するには、例えば湿式合成法により得られるハイドロ
キシアパタイトスラリーに、ＥＤＴＡ等を添加した後、
水熱処理する方法、湿式合成法により得られるハイドロ
キシアパタイトスラリーに塩酸を添加溶解し、ｐＨ＝４
〜６に調整した後、水熱処理する方法等の公知の方法に
より得ることができる。To prepare the acicular hydroxyapatite crystals, for example, EDTA or the like is added to a hydroxyapatite slurry obtained by a wet synthesis method,
Hydrochloric acid is added to and dissolved in a hydroxyapatite slurry obtained by a hydrothermal treatment method or a wet synthesis method to obtain pH = 4.
After adjusting to ~ 6, it can be obtained by a known method such as hydrothermal treatment.
【００１２】前記カラム充填剤層Ａを調製するには、前
記針状ハイドロキシアパタイトを、好ましくは粒径１〜
１００μｍの球状凝集体に造粒し、カラムに充填する方
法等により得ることができる。該造粒された球状凝集体
の比表面積は、１０〜１００ｍ2／ｇであるのが好まし
い。In order to prepare the column packing material layer A, the acicular hydroxyapatite, preferably having a particle size of 1 to
It can be obtained by, for example, a method of granulating into a spherical aggregate of 100 μm and packing in a column. The specific surface area of the granulated spherical aggregates is preferably 10 to 100 m 2 / g.
【００１３】また前記カラム充填剤層Ｂは、アスペクト
比が２未満、好ましくは 未満の板状ハイドロキシア
パタイト結晶を含む。この際板状ハイドロキシアパタイ
ト結晶のｃ軸方向の結晶子径は、１〜２００μｍ、ａ軸
方向の結晶子径は１０〜１００μｍに調整するのが好ま
しい。The column packing layer B also contains plate-like hydroxyapatite crystals having an aspect ratio of less than 2, preferably less than 2. At this time, it is preferable to adjust the crystallite diameter in the c-axis direction of the plate-like hydroxyapatite crystal to 1 to 200 μm and the crystallite diameter in the a-axis direction to 10 to 100 μm.
【００１４】前記板状ハイドロキシアパタイト結晶を調
製するには、例えば湿式合成法により得られるハイドロ
キシアパタイトスラリーに、塩化ナトリウム等を添加し
た後、水熱処理する方法、湿式合成法により得られるハ
イドロキシアパタイトスラリーに水酸化ナトリウムを添
加溶解し、ｐＨ＝１０〜１４に調整した後、水熱処理す
る方法等の公知の方法により得ることができる。To prepare the plate-like hydroxyapatite crystals, for example, a method of hydrothermal treatment after adding sodium chloride or the like to a hydroxyapatite slurry obtained by a wet synthesis method or a hydroxyapatite slurry obtained by a wet synthesis method is added. It can be obtained by a known method such as a method of adding and dissolving sodium hydroxide to adjust the pH to 10 to 14, and then performing hydrothermal treatment.
【００１５】前記カラム充填剤層Ｂを調製するには、前
記板状ハイドロキシアパタイト結晶を好ましくは粒径１
〜１００μｍの球状凝集体に造粒し、カラムに充填する
方法等により得ることができる。該造粒された球状凝集
体の比表面積は、１０〜１００ｍ2／ｇであるのが好ま
しい。To prepare the column packing layer B, the plate-like hydroxyapatite crystals preferably have a particle size of 1
It can be obtained by, for example, a method of granulating into spherical aggregates of ˜100 μm and packing in a column. The specific surface area of the granulated spherical aggregates is preferably 10 to 100 m 2 / g.
【００１６】本発明の液体クロマトグラフィー用カラム
において、前記カラム充填剤層Ａ及びＢの配合割合は、
Ａ：Ｂ＝１：１０〜１０：１が好ましく、特に１：２〜
２：１が望ましい。In the liquid chromatography column of the present invention, the mixing ratio of the column packing layers A and B is
A: B = 1: 10 to 10: 1 is preferable, and especially 1 to 2
2: 1 is preferable.
【００１７】本発明の液体クロマトグラフィー用カラム
は、前記カラム充填剤層Ａと前記カラム充填剤層Ｂとを
直列に接続したものであって、精製すべき生化学物質と
しては、トリプトファン、アルブミン、リゾチーム、チ
トクロムＣ等のアミノ酸やタンパク質;一本鎖ＤＮＡ、
二本鎖ＤＮＡ等の核酸等を好ましく挙げることができ
る。The liquid chromatography column of the present invention comprises the column packing material layer A and the column packing material layer B connected in series, and the biochemical substances to be purified are tryptophan, albumin, Amino acids and proteins such as lysozyme and cytochrome C; single-stranded DNA,
Nucleic acids such as double-stranded DNA can be preferably mentioned.
【００１８】[0018]
【発明の効果】本発明の液体クロマトグラフィー用カラ
ムは、針状ハイドロキシアパタイト結晶を含むカラム充
填剤層と、板状ハイドロキシアパタイト結晶を含むカラ
ム充填剤層とを直列に接続しているので、酸性生化学物
質及び塩基性生化学物質の分離度を良好なものとすると
共に、各物質に対して優れた吸着特性を示す。従って種
々の生化学分析等に利用できる。INDUSTRIAL APPLICABILITY The liquid chromatography column of the present invention has a column packing layer containing needle-shaped hydroxyapatite crystals and a column packing layer containing plate-shaped hydroxyapatite crystals connected in series. It provides good separation of biochemical substances and basic biochemical substances, and exhibits excellent adsorption characteristics for each substance. Therefore, it can be used for various biochemical analysis and the like.
【００１９】[0019]
【実施例１】水酸化カルシウム７．３７ｇと８５重量％
リン酸６．８９ｇとを湿式合成した２重量％ハイドロキ
シアパタイトスラリー５００ｍｌに、塩化ナトリウム５
０ｇを添加し、２００℃、２ＭＰａ、５時間の条件で水
熱処理して板状のハイドロキシアパタイト１次結晶を得
た。この結晶のアスペクト比をＸ線回折法により測定し
たところ１．７であった。また、Ｃ軸方向の結晶子径は
１００ｎｍであった。次に、水酸化カルシウム７．３７
ｇと８５重量％リン酸６．８９ｇとを湿式合成した２重
量％ハイドロキシアパタイトスラリー５００ｍｌにＥＤ
ＴＡ２５ｇを添加し、２００℃、２ＭＰａ、５時間の条
件で水熱処理して針状のハイドロキシアパタイト１次結
晶を得た。この結晶のアスペクト比をＸ線回折法により
測定したところ１２．９であった。また、Ｃ軸方向の結
晶子径は３００ｎｍであった。得られた板状及び針状双
方のハイドロキシアパタイト１次結晶を、スプレードラ
イ法により造粒した後、９００℃、１時間焼成処理し、
それぞれ平均粒径６μｍの球状凝集体を得た。４φ×１
５０Ｌｍｍのステンレス製カラム２本に、これら２種類
の結晶から構成されるカラム充填剤を各々別々に充填し
た。次にこの２本のカラムをジョイントで直列に接続
し、液体クロマトグラフ装置に取り付けた。次いでトリ
プトファン、アルブミン、リゾチーム及びチトクロムＣ
の４種の生化学物質混合水溶液をカラム内に注入し、吸
着させた後、１０〜３５０ｍＭＫＨ2ＰＯ4水溶液のリニ
アグラジエント（３０分）で展開溶出試験（流速：０．
２ｍｌ／分、検出：ＵＶ（２８０ｎｍ））を行なった。
その結果を図１（ｃ）に示す。また最も保持時間が接近
する酸性生化学物質のアルブミンと塩基性生化学物質リ
ゾチームとの分離度を算出し、比較した。その結果を表
１に示す。これらの結果より、本発明のカラムは、酸性
化学物質及び塩基性化学物質の両方が良好にバランス良
く吸着分離されていることが判った。Example 1 7.37 g of calcium hydroxide and 85% by weight
500 ml of a 2 wt% hydroxyapatite slurry obtained by wet synthesis with 6.89 g of phosphoric acid was mixed with 5 ml of sodium chloride.
0 g was added, and hydrothermal treatment was performed under the conditions of 200 ° C., 2 MPa, and 5 hours to obtain plate-shaped hydroxyapatite primary crystals. The aspect ratio of this crystal was measured by an X-ray diffraction method and found to be 1.7. The crystallite diameter in the C-axis direction was 100 nm. Next, calcium hydroxide 7.37
ED in 500 ml of a 2 wt% hydroxyapatite slurry obtained by wet-synthesizing g and 85 wt% phosphoric acid 6.89 g.
25 g of TA was added, and hydrothermal treatment was performed under the conditions of 200 ° C., 2 MPa, and 5 hours to obtain acicular primary hydroxyapatite crystals. When the aspect ratio of this crystal was measured by the X-ray diffraction method, it was 12.9. The crystallite diameter in the C-axis direction was 300 nm. The obtained plate-shaped and needle-shaped hydroxyapatite primary crystals were granulated by a spray drying method, and then calcined at 900 ° C. for 1 hour,
Spherical aggregates each having an average particle size of 6 μm were obtained. 4φ x 1
Two 50 Lmm stainless steel columns were separately filled with column packings composed of these two types of crystals. Next, the two columns were connected in series with a joint and attached to a liquid chromatograph. Then tryptophan, albumin, lysozyme and cytochrome C
After injecting four kinds of biochemical substance mixed aqueous solution of No. 3 into the column and adsorbing the mixture, a development elution test (flow rate: 0. 0%) was performed with a linear gradient (30 minutes) of 10 to 350 mM KH 2 PO 4 aqueous solution.
2 ml / min, detection: UV (280 nm)).
The result is shown in FIG. The degree of separation between albumin, which is an acidic biochemical substance having the closest retention time, and lysozyme, a basic biochemical substance, was calculated and compared. Table 1 shows the results. From these results, it was found that both the acidic chemical substance and the basic chemical substance were adsorbed and separated in a good balance in the column of the present invention.
【００２０】[0020]
【比較例１】実施例１で得られた板状ハイドロキシアパ
タイトの平均粒径６μｍの球状凝集体を、４φ×３００
Ｌｍｍのステンレス製カラムに充填し、実施例１と同様
な方法で展開溶出試験を行なった。その結果を図１
（ａ）及び表１に示す。これらの結果より、酸性生化学
物質のアルブミンと塩基性生化学物質のリゾチームとの
分離度は良好であるが、酸性生化学物質の吸着結合力が
悪いことが判った。[Comparative Example 1] The spherical agglomerates of the plate-like hydroxyapatite having an average particle size of 6 µm obtained in Example 1 were 4φ x 300.
It was packed in an Lmm stainless steel column, and a development dissolution test was conducted in the same manner as in Example 1. The result is shown in Figure 1.
It shows in (a) and Table 1. From these results, it was found that although the degree of separation between albumin which is an acidic biochemical substance and lysozyme which is a basic biochemical substance is good, the adsorptive binding force of an acidic biochemical substance is poor.
【００２１】[0021]
【比較例２】実施例１で得られた針状ハイドロキシアパ
タイトの平均粒径６μｍの球状凝集体を、４φ×３００
Ｌｍｍのステンレス製カラムに充填し、実施例１と同様
な方法で展開溶出試験を行なった。その結果を図１
（ｂ）及び表１に示す。これらの結果より酸性生化学物
質及び塩基性生化学物質の吸着結合力は良好であるが、
分離度が悪いことが判った。Comparative Example 2 The spherical agglomerates of the acicular hydroxyapatite having an average particle size of 6 μm obtained in Example 1 were 4φ × 300.
It was packed in an Lmm stainless steel column, and a development dissolution test was conducted in the same manner as in Example 1. The result is shown in Figure 1.
It shows in (b) and Table 1. From these results, the adsorptive bond strength of acidic biochemical substances and basic biochemical substances is good,
It was found that the degree of separation was poor.
【００２２】[0022]
【表１】 [Table 1]
【図１】図１（ａ）は、板状ハイドロキシアパタイト結
晶を含むカラム及び実施例１の展開溶出試験の結果を示
すグラフである。図１（ｂ）は、針状ハイドロキシアパ
タイト含むカラム及び比較例１の展開溶出試験の結果を
示すグラフである。図１（ｃ）は、板状ハイドロキシア
パタイト結晶と針状ハイドロキシアパタイト結晶とを含
むカラム及び比較例２の展開溶出試験の結果を示すグラ
フである。FIG. 1 (a) is a graph showing the results of a development dissolution test of a column containing plate-like hydroxyapatite crystals and Example 1. FIG. 1 (b) is a graph showing the results of the deployment elution test of the column containing acicular hydroxyapatite and Comparative Example 1. FIG. 1 (c) is a graph showing the results of the deployment dissolution test of a column containing plate-like hydroxyapatite crystals and needle-like hydroxyapatite crystals and Comparative Example 2.
Claims (1)
シアパタイト結晶を含むカラム充填剤層と、アスペクト
比２未満の板状ハイドロキシアパタイト結晶を含むカラ
ム充填剤層とを、直列に接続したことを特徴とする液体
クロマトグラフィー用カラム。1. A column packing layer containing acicular hydroxyapatite crystals having an aspect ratio of 10 or more and a column packing layer containing plate-shaped hydroxyapatite crystals having an aspect ratio of less than 2 are connected in series. A column for liquid chromatography.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6270968A JPH08136520A (en) | 1994-11-04 | 1994-11-04 | Column for liquid chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6270968A JPH08136520A (en) | 1994-11-04 | 1994-11-04 | Column for liquid chromatography |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH08136520A true JPH08136520A (en) | 1996-05-31 |
Family
ID=17493543
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6270968A Withdrawn JPH08136520A (en) | 1994-11-04 | 1994-11-04 | Column for liquid chromatography |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH08136520A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999015251A1 (en) * | 1997-09-22 | 1999-04-01 | Organo Corporation | Method and equipment for chromatographic separation |
| JP2001287903A (en) * | 2000-04-05 | 2001-10-16 | Asahi Kasei Corp | Method for producing needle-like apatite particle |
| JP2008069041A (en) * | 2006-09-14 | 2008-03-27 | Okayama Univ | Apatite composite body and method of producing the same |
| JP2011079697A (en) * | 2009-10-06 | 2011-04-21 | Hyogo Prefecture | Spherical hydroxyapatite and method for producing the same |
-
1994
- 1994-11-04 JP JP6270968A patent/JPH08136520A/en not_active Withdrawn
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999015251A1 (en) * | 1997-09-22 | 1999-04-01 | Organo Corporation | Method and equipment for chromatographic separation |
| US6331250B1 (en) | 1997-09-22 | 2001-12-18 | Organo Corporation | Method and equipment for chromatographic separation |
| JP2001287903A (en) * | 2000-04-05 | 2001-10-16 | Asahi Kasei Corp | Method for producing needle-like apatite particle |
| JP2008069041A (en) * | 2006-09-14 | 2008-03-27 | Okayama Univ | Apatite composite body and method of producing the same |
| JP2011079697A (en) * | 2009-10-06 | 2011-04-21 | Hyogo Prefecture | Spherical hydroxyapatite and method for producing the same |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A300 | Withdrawal of application because of no request for examination |
Free format text: JAPANESE INTERMEDIATE CODE: A300 Effective date: 20020115 |