HPLC

同

high-performance, liquid chromatography

同

高速液体クロマトグラフィー

同

high-performance, liquid chromatography

関

See High-performance liquid chromatography

Wikipedia preview

出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2013/11/06 14:52:23」(JST)

wiki ja

[Wiki ja表示]

高速液体クロマトグラフィー

高速液体クロマトグラフィー (High performance liquid chromatography) はカラムクロマトグラフィーの一種。移動相として高圧に加圧した液体を用いることが特徴。しばしばHPLCと略される。

概要

機械的に高い圧力をかけることによって移動相溶媒を高流速でカラムに通し、これにより分析物が固定相に留まる時間を短くして分離能・検出感度を高くすることを特長とする。 現在では分析物の注入から検出・定量までを一体化して自動的に行えるようにした装置を用いて、再現性の高い分析が比較的簡便に行える。分析化学や生化学で頻繁に用いられ、俗に「液クロ」（液体クロマトグラフィーの略）といえばこれを指すことが多い。

高速液体クロマトグラフィーにおいては各物質は比較的鋭いピークとして検出され、分離（他の物質のピークと明確に分けられる）および検出（鋭いピークにより高い感度が得られる）の能力が従来の液体クロマトグラフィーより良くなる。

移動相としては、カラムや装置に悪影響を与えない範囲で各種の溶媒が使用される。水や塩類の水溶液、アルコール類、アセトニトリル、ジクロロメタン、トリフルオロ酢酸などが用いられる。相溶性のある（互いに混じり合う）溶媒を混合して使用する場合が多い。

溶媒の組成に勾配を付けて（すなわち組成を連続的に変えて）溶出を行うことも多い。たとえば後述の逆相クロマトグラフィーにおいて水/メタノール勾配を使う場合、まずメタノールの少ない条件で極性の高い物質が溶出し、その後メタノールの割合を増加させてゆくに従ってより極性の低い物質が順次溶出する。これをグラジェント分析と呼ぶ。これに対し、一定組成の溶媒で分析物を溶出させる分析法をアイソクラテック分析と呼ぶ。

測定時間は測定物質および測定パラメータによって大きく変動するが、一般的には数分～数十分/回程度である。

機器構成

高速液体クロマトグラフィーの構成

• A:溶媒
• B:2溶媒切り替えバルブ
• C:ポンプ
• D:圧力ダンパー
• E:ミキサー
• F:レオダイン-マニュアルインジェクター
• G:カラム
• H:HPLCコントローラー
• I:検出器（紫外吸光度計等）
• J:検出データA/D変換装置（データ出力装置）
• K:PC（データロガー・データ解析装置として使用）
• L:プリンター

ポンプ

HPLCの心臓部とも言える機器。極めて安定した送液が出来る構造となっている。ポンプの前には、オンラインの脱気装置（デガッサー、degasser）が付いている場合が多い。また、プランジャーの前後にはチェックバルブが取り付けられており、移動相の逆流を防ぐ構造になっている。

ポンプの最大使用圧力は40 MPa程度であるが、2000年代後半には100 MPa程度での高圧送液が可能な超高速液体クロマトグラフィー(Ultra High Performance Liquid Chromatography, UHPLC)と呼ばれるシステムが登場し、シリカゲルの微粒子化と相まって、より高速・高分離能での分析が可能となった。なおUHPLCは、メーカーによって、UPLC (Waters)、UFLC（島津）などと称されている。

構造による分類

シングルプランジャーポンプ

ポンプの押し出す部分が一つのポンプ。古典的システムにおいては標準的な仕様であったが、現在は移動相脈動を軽減させるためやグラジェント分析が主流となりつつあるため、主たる移動相の送液のために用いられることは少なく、蛍光検出器のための標識試薬を送液するために用いられることが多い。但し、高い精度を要求しない分析ではこの仕様で十分事足りる、機器の価格が安い、メンテナンスが容易等の利点もあるため現在でも使用されている。

ダブルプランジャーポンプ

ポンプの押し出す部分が二つのポンプ。現在はこの方式が主流となっている。さらにダブルプランジャー方式は並流と直流の2種類が有る。

溶媒混合方式による分類

アイソクラテックポンプ

1種類の溶媒のみを送液するポンプ。安価であるが、溶媒混合比が変えられないためグラジェント分析が出来ず、分析の幅がせまい。

高圧グラジェントポンプ

2台以上のアイソクラテックポンプを繋ぎ、それぞれの流量をコントロールして溶媒混合比を変化させることによりグラジェント分析を行う。溶媒の種類ごとにポンプを用意する必要があるため、高価である。

低圧グラジェントポンプ

ポンプは1台であるが、ポンプの前に付いているバルブの切り替えによって溶媒混合比を変化させ（一般的に4種類の溶媒を混合して）グラジェント分析を行う。ポンプにより圧力がかかる前に溶媒を混合するため、こう呼ばれている。高圧グラジェントポンプに比べて安価ではあるが、溶媒の混合が甘い、気泡が発生しやすいなどの短所がある。

流量による分類

ナノ/マイクロフローポンプ

極微量の試料の分析に適した流量（nLまたはμLオーダー）での送液を行う。通常は内径1 mm以下のキャピラリカラムを接続して使用する。

分析ポンプ

通常の分析に適した流量（0.1～10 mL程度）での送液を行う。内径1～10 mm程度の分析カラムを接続して使用する。

分取ポンプ

物質の分離精製の目的に適した流量（10 mL以上）の送液を行う。大型カラムを接続して使用する。この種のポンプは分析用と比べ耐圧が低い場合が多い。

インジェクター

試料を注入する部分で、手動式(マニュアルインジェクター)と自動式(オートインジェクター)がある。

マニュアルインジェクター

現在市販されているマニュアルインジェクターはほとんどがレオダイン社の製品であり、「レオダイン」がマニュアルインジェクターの代名詞となっている。 仕組みは、2種類の流路を切り替えるという極めて単純な物である。

1. ノブをロード側にし、マイクロシリンジでサンプルループ内に試料を入れる。サンプル注入量の計量には、人の手によりシリンジで計量する方法と、サンプルループからあふれる量を注入してサンプルループにより計量する方法の2通りがある。後者の方が人による熟練度の差がないため、再現性がよい。
2. ノブをインジェクト側に切り替え、サンプルを流路に注入する。マニュアルインジェクターに電気信号を出力する機能が付いていれば、この時にインジェクション信号を検出器またはインテグレーターに送ることが出来る。

オートインジェクター

大部分のメーカーがレオダインのマニュアルインジェクターを装置に内蔵しており、サンプルをシリンジで計量し、これを切り替えて流路に注入している。メーカーによりサンプルのハンドリング方法に工夫がされており、使用する目的に応じて選択できる。大量（数十～1000以上）のサンプルの連続分析ができるように、サンプルはウェルプレートや複数本のバイアルに入れて装置内にセットするようになっている。サンプルを保冷・保温する機能がついているものもある。

カラム

混合物で構成される試料を分離する。一般にステンレス製の筒の中に、微細な真球状の多孔質シリカゲルをアルキル基等で修飾した物を充填して用いる。分取目的であれば、粉砕シリカゲルも用いられる。

シリカゲルの粒子径が小さければ小さいほどピークの分離性は良くなるが、送液に必要なポンプの圧力が高くなる。そのため、ポンプ－インジェクター間、インジェクター－カラム間の配管の耐圧を上げたり、カラム自体を比較的高温の下にさらして溶媒の粘度を下げ、抵抗を小さくする工夫をしている。

各種の高速液体クロマトグラフィーの項目にある違いは、カラムの違いである事が多いため、装置はそのままでカラムの変更で行える場合が有る。ただし、誤って不適当な溶媒を通すとカラムを破損することがあるため、切り替えを行う際には注意が必要である。

カラムオーブン

内部にカラムを収納して加熱あるいは冷却を行い、カラムの温度を制御する装置。カラムヒーターとも称する。

カラム周辺の温度の変動によって溶出時間が安定せず再現性が悪くなる場合があるため、カラム温度を一定に保つために使用する。またカラム温度を分離条件のパラメーターの一つとして積極的に利用する場合もある。

加温することが多かったため「オーブン、ヒーター」と称されるが、現在では周辺気温より低温にするための冷却機能が付いている装置も多い。また、周辺気温付近で使用する場合にも冷却機能は一定の効果がある。

ディテクター

ディテクター（検出器）としては目的とする物質の性質に応じて光学的性質（吸光度、屈折率、蛍光等）、電気化学的性質、質量分析法などを利用する装置がある。

なお、JIS K0124:2002 高速液体クロマトグラフィー通則によると、吸光光度検出器(UV/VIS検出器)、蛍光検出器(FLD)、示差屈折率検出器(RID)、電気化学検出器(ECD)、電気伝導度検出器(CD)、質量分析計(MS)、赤外分光光度計(IR)、旋光度検出器(OR)、円二色性検出器(CD)、水素炎イオン化検出器(FID)、放射線検出器(RI)、誘電率検出器、化学発光検出器(生物発光も含む)(CLD)、原子吸光分光分析装置(AA)、誘導結合プラズマ発光分光分析装置(ICP-AES)、高周波プラズマ質量分析計、熱検出器、光錯乱検出器、粘度検出器、イオン電極、超音波検出器、核磁気共鳴装置(NMR)が記載されている。

光学的性質を用いるもの

吸光度検出器 (Absorbance Detector)

物質の持つ特定波長の光を吸収する性質を利用した検出器。次のようなものが存在している。

• 紫外吸光度検出器 (Ultra-Violet Absorbance Detector)：紫外線域の光を使用する。最も一般的に使用される。
• 可視吸光度検出器 (Visible Light Absorbance Detector)：可視光を使用する。

上記2つは、一体型の物が多い。

• ダイオードアレイ検出器 (Diode Array Detector)：幅広い波長を同時に検出できる。結果は、時間、吸光度、波長の三次元で表示することも可能。

旋光度検出器(Optical Rotatory Detector)

旋光または、光学活性を持った物質の検出に使用される。

円二色性検出器 (Circular Dichroism Detector)

偏光特性を利用したもので、光学異性体の検出に使用される。

蛍光検出器 (Fluorescence Detector)

物質にエネルギーを与える（励起）ことにより発光する（蛍光）性質を利用した検出器。一般に選択性が高く高感度で、物質に特異的な検出が可能。蛍光する性質を持たない物質については、その物質を標識することにより検出が可能になる。

屈折率検出器 (Refractive Index Detector)

物質の濃度により光の通過する角度が変わることを利用した検出器。原理上グラジェント分析はできない（グラジェントによって移動相自体の屈折率が変化するため）。また、感度が低いのが欠点だが、大部分の物質に対して使用できる。

蒸発光散乱検出器 (Evaporative Light Scattering Detector)

カラム溶出液を噴霧して移動相を蒸発除去後、溶質に光を当ててその散乱光を検出する方法である、不揮発性の成分は何でも検出できる一方、揮発性の高い物質には使用出来ない。以前は感度や操作性の点で難度があり普及しなかったものの、最近では医薬分野におけるニーズ(不純物の確認)の高まりから性能も向上し、検出器として見直されてきた。グラジェント溶出法が適用できるので、糖類の検出では、示差屈折率検出器と比べ5～10倍S/N比が高くなり、特に分配法によるオリゴ糖分析に効果を発揮する。

電気化学的性質を用いるもの

主にイオン性物質の定量に威力を発揮する。

電気化学検出器 (Electro-Chemical Detector)

物質の電気化学的な性質を利用した検出器。pHの変動や酸化還元電位の変動を用いて測定を行う。

導電率検出器 (Conductivity Detector)

物質の持つ電気の流れやすさを検出する検出器。

質量分析法を用いるもの

詳しくは質量分析法の項目にゆだね、ここではLC-MSで使用される代表的なイオン化法と検出部を列挙するにとどめる。

• イオン化法
  • エレクトロスプレーイオン化法 (Electro-Spray Ionization, ESI)
  • 大気圧化学イオン化法 (Atomospheric-Pressure-Chemical Ionization, APCI)
  • 直結して使用することはできないが、マトリックス支援レーザー脱離イオン化法 (Matrix Assisted Laser Desorption Ionization, MALDI) も使用できる。
• 検出部
  • 四重極型 (Quadrupole, Q)
  • イオントラップ型 (Ion-Trap, IT)
  • 飛行時間型 (Time-of-Flight, TOF)
  • MS/MSとして上記の物の組み合わせたもの(QqQ、IT-TOF、Q-TOF等)がある。なお、ITは、シングルでもMSn分析が可能。

その他

放射線検出器

放射性同位体で標識されたサンプルを測定する。

コロナ荷電化粒子検出器 (Corona Charged Aerosol Detector, CoronaCAD)

蒸発光散乱検出器(ELSD)と同様にカラム溶出液から移動相を蒸発除去後、イオン化した窒素原子を衝突させて溶質粒子をプラスに帯電させ、コレクター（導電性の膜フィルター）で帯電粒子を検出する方式。ELSDと同じ分析条件が使用可能であり、ELSDよりも高感度であるとされている。米国ESA Biosciences社によって開発され現在はThermoFisher社が販売している。

インテグレーター

ディテクターから出力された、電気信号を記録し、そこからピークを検出、解釈を行う。結果は、感熱紙等に印字される。装置のコントロールをしないのであれば、どのメーカーの物を使用しても問題はないが、通常は、装置のコントロールも同時に行うため、同じメーカーの物を選択する。

現在では、インテグレーターとしてWindows PCを用いることが多い。このPCベースのインテグレーターの中には、メーカーが異なる装置をコントロールできる物も存在する。利点として、複数のソフトウェアの使い方を覚える必要が無いため、教育コストが下がるという点が大きい。

組み込みOSを用いて専用設計されているインテグレーター機器もある。

各種の高速液体クロマトグラフィー

順相 (normal phase) クロマトグラフィー

順相クロマトグラフィーは高速液体クロマトグラフィーにおいて最初に使われた。固定相に高極性のもの（シリカゲル）を、移動相に低極性のもの（例えばヘキサン、酢酸エチル、クロロホルムなどの有機溶媒）を用いる。分析物はより極性の高いほどより強く固定相と相互作用して溶出が遅くなる。また極性の高い物質の割合が多い移動相ほど溶出が早くなる。順相タイプは近年の逆相タイプの発展とともに使われることが少なくなったが、順相タイプは逆相タイプをはじめとする他の分離モードとは異なった特性を持つため、目的によっては非常に有効なものとなる。例えば、逆相タイプでは分離が困難なトコフェロールの異性体や保持の困難な糖類を容易に相互分析することができ、また主に水を含まない移動相を用いるので、水に難溶の脂溶性ビタミンや加水分解されやすい酸無水物などの化合物の分離に好適である。
　また近年、順相クロマトグラフィーのバリエーションとして、「親水性相互作用クロマトグラフィー(Hydrophilic interaction chromatography, HILIC)」あるいは「逆逆相クロマトグラフィー」と称する分離モードのカラムが市販されるようになった。固定相に未修飾シリカゲルあるいは表面を極性基（ジオール、アミド、スルホベタインなど）で修飾したシリカゲルを、移動相に水を含む移動相を用いることにより、逆相クロマトグラフィーでは分離が困難なアミノ酸などの極性化合物を分離することが可能である。

逆相 (reversed phase) クロマトグラフィー

前述した従来の順相タイプに対して、逆相クロマトグラフィーにおいては固定相に低極性のもの（例えばシリカゲルにアルキル基を共有結合させたもの）を、移動相に高極性のもの（例えば水や塩類の水溶液、アルコール、アセトニトリルなどの有機溶媒）を用いる。また珍しいケースではあるが、分離のための移動相pHをシリカゲルの使用範囲から外れたところに設定する必要がある場合、あるいはシリカゲル表面に残っている未反応シラノール基が分離に悪影響を及ぼし、かつそれが移動相の変更によっても解決できない場合には、固定相として樹脂を用いることがある。分析物はより極性の低いほどより強く固定相と相互作用して溶出が遅くなる。また極性の低い物質の割合が多い移動相ほど溶出が早くなる。

なお、カラムはシリカゲルに炭素鎖数18のオクタデシル基を結合させた「オクタデシル・シリカ」すなわち「ODSカラム」が最も広範に用いられる。

逆相クロマトグラフィーは、従来から低分子量物質の分析に用いられていたが、最近では核酸や蛋白質分析にも用いられている。蛋白質を分析する場合には、細孔径の大きな化学結合型シリカゲルをカラム充填剤として用い、移動相条件としては通常pH２～３あるいは中性付近で、有機溶媒量を増加させていくグラジエント溶出法が用いられる。

分子ふるい(サイズ排除)クロマトグラフィー

分子ふるいクロマトグラフィー(Size Exclusion Chromatography, SEC)はゲルろ過クロマトグラフィー(Gel Filtration Chromatography, GFC)またはゲル浸透クロマトグラフィー(Gel Permeation Chromatography, GPC)とも呼ばれ、分析物をそのサイズにより分離する。サイズの小さい分析物ほど固定相であるゲルに「引っかかり」溶出が遅くなる。分子篩を用いて行う。

イオン交換 (ion exchange) クロマトグラフィー

イオン交換クロマトグラフィーでは、無機イオンや高極性分子を電荷を利用して分離する。陽イオンタイプと陰イオンタイプの両方がある。イオン交換樹脂を利用する。

HPLCカラムメーカー

高速液体クロマトグラフィーの装置において分離を行う場であり、消耗部品である。一般的には、微細な（数μm）真球状の多孔質シリカゲルをステンレス製の管に充填したものが多い。目的、分離手法に応じて様々なタイプのHPLCカラムが存在する。下記に代表的なカラムメーカー（五十音順）とブランド名を列記する。

• アジレント・テクノロジー: Zorbax
• インタクト: Unison, Cadenza, Scherzo, Presto
• Waters: XSelect, XBridge, SunFire, Atlantis
• 化学物質評価研究機構: L-column
• 関東化学: Mightysil
• クロマニックテクノロジーズ: Sunniest
• ジーエルサイエンス: InertSustain, Inertsil, MonoClad(モノリスカラム), MonoCap(モノリスキャピラリーカラム)
• シグマ アルドリッチ: Ascentis
• 資生堂: CAPCELL PAK
• 島津製作所: Shimpack
• 昭和電工: Shodex
• 信和化工: Ultron ES-OVM（逆相系キラル分離カラム）などタンパク質固定化カラム
• 住化分析センター: SUMICHIRAL（光学異性体分離用）
• ダイオネクス: Acclaim
• ダイセル: CHIRALCEL, CHIRALPAK（光学異性体分離用）
• 東京化成工業: Kaseisorb LC
• 東ソー: TSK-GEL
• ナカライテスク: COSMOSIL
• 野村化学: Develosil
• Phenomenex: Gemini, Jupiter, Luna, Onyx（モノリスカラム）
• メルク: Chromolith（モノリスカラム）, ZIC-HILIC
• ワイエムシィ: YMC-Pack
• 和光純薬工業: Wakopak

装置・システムメーカー

システムとしてポンプ、インジェクター、ディテクターまでを一貫して製造しているメーカーを挙げる。

• 島津製作所：総合分析機器メーカー。
• アジレント・テクノロジー：総合分析機器メーカー。
• Waters：総合分析機器メーカー。
• 日立ハイテクノロジーズ：総合分析機器メーカー。
• サーモフィッシャー：総合分析機器メーカー

• ダイオネクス：化学製品メーカー。イオンクロマトグラフが有名。高速液体クロマトグラフの販売、最近ではESA社を買収してCADを販売。2010年末に、サーモフィッシャーに買収された。

• ジーエルサイエンス：クロマト関連製品メーカー。逆相C18などカラムや固相ミニカラムのメーカーとして有名だが、HPLCシステムや分取システムも製造している。
• 日本分光：分光計メーカー。
• 東ソー：化学製品メーカー。各種GPCシステム、イオンクロマトグラフを製造。汎用機は2009年9月製造終了。
• 資生堂：化粧品メーカーとして有名だが、ユニークなHPLCシステムも製造している。
• エイコム：総合分析機器メーカー。hplc-電気化学検出器およびマイクロダイアリシスのメーカー。

脚注

外部リンク

• 高速液体クロマトグラフィー - 脳科学辞典

wiki en

[Wiki en表示]

High-performance liquid chromatography

A HPLC. From left to right: A pumping device generating a gradient of two different solvents, a steel enforced column and a detector for measuring the absorbance.
Acronym	HPLC
Classification	Chromatography
Analytes	organic molecules biomolecules ions polymers
Other techniques
Related	Chromatography Aqueous normal-phase chromatography Hydrophilic Interaction Chromatography Ion exchange chromatography Size exclusion chromatography Micellar liquid chromatography
Hyphenated	Liquid chromatography-mass spectrometry

A modern self-contained HPLC.

Schematic representation of an HPLC unit. (1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in "inject position", (6') Switching valve in "load position", (7) Sample injection loop, (8) Pre-column (guard column), (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.

High-performance liquid chromatography (formerly referred to as high-pressure liquid chromatography), HPLC, is a chromatographic technique used to separate the components in a mixture, to identify each component, and to quantify each component. HPLC is considered an instrumental technique of analytical chemistry (as opposed to a gravimetric technique). In general, the method involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent. Each analyte in the sample interacts slightly differently with the adsorbent material, thus retarding the flow of the analytes. If the interaction is weak, the analytes flow off the column in a short amount of time, and if the interaction is strong, then the elution time is long. HPLC has been used for medical (e.g. detecting vitamin D levels in blood serum), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and manufacturing (e.g. during the production process of pharmaceutical and biological products) purposes.

Chromatography can be described as a mass transfer process involving adsorption. HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with a sorbent, leading to the separation of the sample components. The active component of the column, the sorbent, is typically a granular material made of solid particles (e.g. silica, polymers, etc.), 2–50 micrometers in size. The components of the sample mixture are separated from each other due to their different degrees of interaction with the sorbent particles. The pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile and/or methanol) and is referred to as a "mobile phase". Its composition and temperature plays a major role in the separation process by influencing the interactions taking place between sample components and sorbent. These interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic, most often a combination thereof.

HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC typical column dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made with smaller sorbent particles (2–5 micrometer in average particle size). This gives HPLC superior resolving power when separating mixtures, which is why it is a popular chromatographic technique.

The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature the separation is performed at.

Operation

The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically microliters), into the stream of mobile phase percolating through the column. The components of the sample move through the column at different velocities, which are function of specific physical interactions with the sorbent (also called stationary phase). The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its retention time. The retention time measured under particular conditions is considered an identifying characteristic of a given analyte.

Many different types of columns are available, filled with sorbents varying in particle size, and in the nature of their surface ("surface chemistry"). The use of smaller particle size packing materials requires the use of higher operational pressure ("backpressure") and typically improves chromatographic resolution (i.e. the degree of separation between consecutive analytes emerging from the column). In terms of surface chemistry, sorbent particles may be hydrophobic or polar in nature.

Common mobile phases used include any miscible combination of water with various organic solvents (the most common are acetonitrile and methanol). Some HPLC techniques use water-free mobile phases (see Normal-phase chromatography below). The aqueous component of the mobile phase may contain acids (such as formic, phosphoric or trifluoroacetic acid) or salts to assist in the separation of the sample components. The composition of the mobile phase may be kept constant ("isocratic elution mode") or varied ("gradient elution mode") during the chromatographic analysis. Isocratic elution is typically effective in the separation of sample components that are not very dissimilar in their affinity for the stationary phase. In gradient elution the composition of the mobile phase is varied typically from low to high eluting strength. The eluting strength of the mobile phase is reflected by analyte retention times with high eluting strength producing fast elution (=short retention times). A typical gradient profile in reversed phase chromatography might start at 5% acetonitrile (in water or aqueous buffer) and progress linearly to 95% acetonitrile over 5–25 minutes. Periods of constant mobile phase composition may be part of any gradient profile. For example, the mobile phase composition may be kept constant at 5% acetonitrile for 1–3 min, followed by a linear change up to 95% acetonitrile.

The chosen composition of the mobile phase (also called eluent) depends on the intensity of interactions between various sample components ("analytes") and stationary phase (e.g. hydrophobic interactions in reversed-phase HPLC). Depending on their affinity for the stationary and mobile phases analytes partition between the two during the separation process taking place in the column. This partitioning process is similar to that which occurs during a liquid–liquid extraction but is continuous, not step-wise. In this example, using a water/acetonitrile gradient, more hydrophobic components will elute (come off the column) late, once the mobile phase gets more concentrated in acetonitrile (i.e. in a mobile phase of higher eluting strength).

The choice of mobile phase components, additives (such as salts or acids) and gradient conditions depends on the nature of the column and sample components. Often a series of trial runs is performed with the sample in order to find the HPLC method which gives adequate separation.

Types

Partition chromatography

HILIC Partition Technique Useful Range

Partition chromatography was the first kind of chromatography that chemists developed. The partition coefficient principle has been applied in paper chromatography, thin layer chromatography, gas phase and liquid-liquid applications. The 1952 Nobel Prize in chemistry was earned by Archer John Porter Martin and Richard Laurence Millington Synge for their development of the technique, which was used for their separation of amino acids. Partition chromatography uses a retained solvent, on the surface or within the grains or fibers of an "inert" solid supporting matrix as with paper chromatography; or takes advantage of some coulombic and/or hydrogen donor interaction with the stationary phase. Analyte molecules partition between a liquid stationary phase and the eluent. Just as in Hydrophilic Interaction Chromatography (HILIC; a sub-technique within HPLC), this method separates analytes based on differences in their polarity. HILIC most often uses a bonded polar stationary phase and a mobile phase made primarily of acetonitrile with water as the strong component. Partition HPLC has been used historically on unbonded silica or alumina supports. Each works effectively for separating analytes by relative polar differences. HILIC bonded phases have the advantage of separating acidic, basic and neutral solutes in a single chromatographic run.

The polar analytes diffuse into a stationary water layer associated with the polar stationary phase and are thus retained. The stronger the interactions between the polar analyte and the polar stationary phase (relative to the mobile phase) the longer the elution time. The interaction strength depends on the functional groups part of the analyte molecular structure, with more polarized groups (e.g. hydroxyl-) and groups capable of hydrogen bonding inducing more retention. Coulombic (electrostatic) interactions can also increase retention. Use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times.

Normal–phase chromatography

Normal–phase chromatography was one of the first kinds of HPLC that chemists developed. Also known as normal-phase HPLC (NP-HPLC) this method separates analytes based on their affinity for a polar stationary surface such as silica, hence it is based on analyte ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole type of interactions) with the sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase (e.g. Chloroform), and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity. The interaction strength depends not only on the functional groups present in the structure of the analyte molecule, but also on steric factors. The effect of steric hindrance on interaction strength allows this method to resolve (separate) structural isomers.

The use of more polar solvents in the mobile phase will decrease the retention time of analytes, whereas more hydrophobic solvents tend to induce slower elution (increased retention times). Very polar solvents such as traces of water in the mobile phase tend to adsorb to the solid surface of the stationary phase forming a stationary bound (water) layer which is considered to play an active role in retention. This behavior is somewhat peculiar to normal phase chromatograhy because it is governed almost exclusively by an adsorptive mechanism (i.e. analytes interact with a solid surface rather than with the solvated layer of a ligand attached to the sorbent surface; see also reversed-phase HPLC below). Adsorption chromatography is still widely used for structural isomer separations in both column and thin-layer chromatography formats on activated (dried) silica or alumina supports.

Partition- and NP-HPLC fell out of favor in the 1970s with the development of reversed-phase HPLC because of poor reproducibility of retention times due to the presence of a water or protic organic solvent layer on the surface of the silica or alumina chromatographic media. This layer changes with any changes in the composition of the mobile phase (e.g. moisture level) causing drifting retention times.

Recently, partition chromatography has become popular again with the development of HILIC bonded phases which demonstrate improved reproducibility, and due to a better understanding of the range of usefulness of the technique.

Displacement chromatography

The basic principle of displacement chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities. There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentration .

Reversed-phase chromatography (RPC)

A chromatogram of complex mixture (perfume water) obtained by reversed phase HPLC

For more details on this topic, see Reversed-phase chromatography.

Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been surface-modified with RMe₂SiCl, where R is a straight chain alkyl group such as C₁₈H₃₇ or C₈H₁₇. With such stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily (early in the analysis). An investigator can increase retention times by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more organic solvent to the eluent. RP-HPLC is so commonly used that it is often incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RP-HPLC to qualify drugs before their release.

RP-HPLC operates on the principle of hydrophobic interactions, which originates from the high symmetry in the dipolar water structure and plays the most important role in all processes in life science. RP-HPLC allows the measurement of these interactive forces. The binding of the analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand on the stationary phase. This solvophobic effect is dominated by the force of water for "cavity-reduction" around the analyte and the C₁₈-chain versus the complex of both. The energy released in this process is proportional to the surface tension of the eluent (water: 7.3×10⁻⁶ J/cm², methanol: 2.2×10⁻⁶ J/cm²) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding a less polar solvent (methanol, acetonitrile) into the mobile phase to reduce the surface tension of water. Gradient elution uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis.

Structural properties of the analyte molecule play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic bonds, such as S-S and others) is retained longer because it is non-interacting with the water structure. On the other hand, analytes with higher polar surface area (conferred by the presence of polar groups, such as -OH, -NH₂, COO^– or -NH₃⁺ in their structure) are less retained as they are better integrated into water. Such interactions are subject to steric effects in that very large molecules may have only restricted access to the pores of the stationary phase, where the interactions with surface ligands (alkyl chains) take place. Such surface hindrance typically results in less retention.

Retention time increases with hydrophobic (non-polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single C-C-bonds elute later than those with a C=C or C-C-triple bond, as the double or triple bond is shorter than a single C-C-bond.

Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca. 1.5×10⁻⁷ J/cm² per Mol for NaCl, 2.5×10⁻⁷ J/cm² per Mol for (NH₄)₂SO₄), and because the entropy of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. This technique is used for mild separation and recovery of proteins and protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC).

Another important factor is the mobile phase pH since it can change the hydrophobic character of the analyte. For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH. Buffers serve multiple purposes: control of pH, neutralize the charge on the silica surface of the stationary phase and act as ion pairing agents to neutralize analyte charge. Ammonium formate is commonly added in mass spectrometry to improve detection of certain analytes by the formation of analyte-ammonium adducts. A volatile organic acid such as acetic acid, or most commonly formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column effluent. Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is a fairly strong organic acid. The effects of acids and buffers vary by application but generally improve chromatographic resolution.

Reversed phase columns are quite difficult to damage compared with normal silica columns; however, many reversed phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases as these will destroy the underlying silica particle. They can be used with aqueous acid, but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment. RP-HPLC columns should be flushed with clean solvent after use to remove residual acids or buffers, and stored in an appropriate composition of solvent. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good test for the metal content of a column is to inject a sample which is a mixture of 2,2'- and 4,4'- bipyridine. Because the 2,2'-bipy can chelate the metal, the shape of the peak for the 2,2'-bipy will be distorted (tailed) when metal ions are present on the surface of the silica.^{[citation needed]}..

Size-exclusion chromatography

For more details on this topic, see size-exclusion chromatography.

Size-exclusion chromatography (SEC), also known as gel permeation chromatography or gel filtration chromatography, separates particles on the basis of molecular size (actually by a particle's Stokes radius). It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of the purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.

This technique is widely used for the molecular weight determination of polysaccharides. SEC is the official technique (suggested by European pharmacopeia) for the molecular weight comparison of different commercially available low-molecular weight heparins.

Ion-exchange chromatography

For more details on this topic, see Ion-exchange chromatography.

In ion-exchange chromatography (IC), retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Solute ions of the same charge as the charged sites on the column are excluded from binding, while solute ions of the opposite charge of the charged sites of the column are retained on the column. Solute ions that are retained on the column can be eluted from the column by changing the solvent conditions (e.g. increasing the ion effect of the solvent system by increasing the salt concentration of the solution, increasing the column temperature, changing the pH of the solvent, etc...).

Types of ion exchangers include:

• Polystyrene resins – These allow cross linkage which increases the stability of the chain. Higher cross linkage reduces swerving, which increases the equilibration time and ultimately improves selectivity.
• Cellulose and dextran ion exchangers (gels) – These possess larger pore sizes and low charge densities making them suitable for protein separation.
• Controlled-pore glass or porous silica

In general, ion exchangers favor the binding of ions of higher charge and smaller radius.

An increase in counter ion (with respect to the functional groups in resins) concentration reduces the retention time. A decrease in pH reduces the retention time in cation exchange while an increase in pH reduces the retention time in anion exchange. By lowering the pH of the solvent in a cation exchange column, for instance, more hydrogen ions are available to compete for positions on the anionic stationary phase, thereby eluting weakly bound cations.

This form of chromatography is widely used in the following applications: water purification, preconcentration of trace components, ligand-exchange chromatography, ion-exchange chromatography of proteins, high-pH anion-exchange chromatography of carbohydrates and oligosaccharides, and others.

Bioaffinity chromatography

For more details on this topic, see Affinity chromatography.

This chromatographic process relies on the property of biologically active substances to form stable, specific, and reversible complexes. The formation of these complexes involves the participation of common molecular forces such as the Van der Waals interaction, electrostatic interaction, dipole-dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, biospecific bond is formed by a simultaneous and concerted action of several of these forces in the complementary binding sites.

Aqueous normal-phase chromatography

Aqueous normal-phase chromatography (ANP) is a chromatographic technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution using reversed-phase solvents.^{[citation needed]}

Isocratic and gradient elution

A separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic (meaning constant composition). The word was coined by Csaba Horvath who was one of the pioneers of HPLC.^{[citation needed]},

The mobile phase composition does not have to remain constant. A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution. One example is a gradient starting at 10% methanol and ending at 90% methanol after 20 minutes. The two components of the mobile phase are typically termed "A" and "B"; A is the "weak" solvent which allows the solute to elute only slowly, while B is the "strong" solvent which rapidly elutes the solutes from the column. In reversed-phase chromatography, solvent A is often water or an aqueous buffer, while B is an organic solvent miscible with water, such as acetonitrile, methanol, THF, or isopropanol.

In isocratic elution, peak width increases with retention time linearly according to the equation for N, the number of theoretical plates. This leads to the disadvantage that late-eluting peaks get very flat and broad. Their shape and width may keep them from being recognized as peaks.

Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower (and taller) peaks for most components. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks "sharper"), which is important in trace analysis. The gradient program may include sudden "step" increases in the percentage of the organic component, or different slopes at different times – all according to the desire for optimum separation in minimum time.

In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change – that is, the peaks elute in the same order. In gradient elution, the elution order may change as the dimensions or flow rate change.^{[citation needed]}

The driving force in reversed phase chromatography originates in the high order of the water structure. The role of the organic component of the mobile phase is to reduce this high order and thus reduce the retarding strength of the aqueous component.

Parameters

Theoretical

HPLC separations have theoretical parameters and equations to describe the separation of components into signal peaks when detected by instrumentation such as by a UV detector or mass spectrometer. The parameters are largely derived from two sets of chromatagraphic theory: plate theory (as part of Partition chromatography), and the rate theory of chromatography / Van Deemter equation. Of course, they can be put in practice through analysis of HPLC chromatograms, although rate theory is considered the more accurate theory.

They are analogous to the calculation of retention factor for a paper chromatography separation, but describes how well HPLC separates a mixture into two or more components that are detected as peaks (bands) on a chromatogram. The HPLC parameters are the: efficiency factor(N), the retention factor (kappa prime), and the separation factor (alpha). Together the factors are variables in a resolution equation, which describes how well two components' peaks separated or overlapped each other. These parameters are mostly only used for describing HPLC reversed phase and HPLC normal phase separations, since those separations tend to be more subtle than other HPLC modes (e.g. ion exchange and size exclusion).

• Void volume is the amount of space in a column that is occupied by solvent. It is the space within the column that is outside of the column's internal packing material. Void volume is measured on a chromatogram as the first component peak detected, which is usually the solvent that was present in the sample mixture; ideally the sample solvent flows through the column without interacting with the column, but is still detectable as distinct from the HPLC solvent. The void volume is used as a correction factor.
• Efficiency factor (N) practically measures how sharp component peaks on the chromatogram are, as ratio of the component peak's area ("retention time") relative to the width of the peaks at their widest point (at the baseline). Peaks that are tall, sharp, and relatively narrow indicate that separation method efficiently removed a component from a mixture; high efficiency. Efficiency is very dependent upon the HPLC column and the HPLC method used. Efficiency factor is synonymous with plate number, and the 'number of theoretical plates'.
• Retention factor (kappa prime) measures how long a component of the mixture stuck to the column, measured by the area under the curve of its peak in a chromatogram (since HPLC chromatograms are a function of time). Each chromatogram peak will have its own retention factor (e.g. kappa1 for the retention factor of the first peak). This factor may be corrected for by the void volume of the column.
• Separation factor (alpha) is a relative comparison how well two neighboring components of the mixture were separated (i.e. two neighboring bands on a chromatogram). This factor is defined in terms of a ratio of the retention factors of a pair of neighboring chromatogram peaks, and may also be corrected for by the void volume of the column. The greater the separation factor value is over 1.0, the better the separation, until about 2.0 beyond which an HPLC method is probably not needed for separation.

Resolution equations relate the three factors such that high efficiency and separation factors improve the resolution of component peaks in a HPLC separation.

Internal diameter

The internal diameter (ID) of an HPLC column is an important parameter that influences the detection sensitivity and separation selectivity in gradient elution. It also determines the quantity of analyte that can be loaded onto the column. Larger columns are usually seen in industrial applications, such as the purification of a drug product for later use. Low-ID columns have improved sensitivity and lower solvent consumption at the expense of loading capacity.

• Larger ID columns (over 10 mm) are used to purify usable amounts of material because of their large loading capacity.
• Analytical scale columns (4.6 mm) have been the most common type of columns, though smaller columns are rapidly gaining in popularity. They are used in traditional quantitative analysis of samples and often use a UV-Vis absorbance detector.
• Narrow-bore columns (1–2 mm) are used for applications when more sensitivity is desired either with special UV-vis detectors, fluorescence detection or with other detection methods like liquid chromatography-mass spectrometry
• Capillary columns (under 0.3 mm) are used almost exclusively with alternative detection means such as mass spectrometry. They are usually made from fused silica capillaries, rather than the stainless steel tubing that larger columns employ.

Particle size

Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles (very small beads). These particles come in a variety of sizes with 5 µm beads being the most common. Smaller particles generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared.

This means that changing to particles that are half as big, keeping the size of the column the same, will double the performance, but increase the required pressure by a factor of four. Larger particles are used in preparative HPLC (column diameters 5 cm up to >30 cm) and for non-HPLC applications such as solid-phase extraction.

Pore size

Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics, especially for larger analytes. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside.

Pump pressure

Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flow rate. Pressure may reach as high as 40 MPa (6000 lbf/in²), or about 400 atmospheres. Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use much smaller particle sizes in the columns (<2 μm). These "Ultra High Performance Liquid Chromatography" systems or RSLC/UHPLCs can work at up to 100 MPa (15,000 lbf/in²), or about 1000 atmospheres. The term "UPLC" is a trademark of the Waters Corporation, but is sometimes used to refer to the more general technique.

See also

• History of chromatography
• Csaba Horváth
• Ion chromatography
• Column chromatography
• Capillary electrochromatography
• Micellar liquid chromatography

References

Further reading

• L. R. Snyder, J.J. Kirkland, and J. W. Dolan, Introduction to Modern Liquid Chromatography, John Wiley & Sons, New York, 2009.
• M.W. Dong, Modern HPLC for practicing scientists. Wiley, 2006.
• L. R. Snyder, J.J. Kirkland, and J. L. Glajch, Practical HPLC Method Development, John Wiley & Sons, New York, 1997.
• S. Ahuja and H. T. Rasmussen (ed), HPLC Method Development for Pharmaceuticals, Academic Press, 2007.
• S. Ahuja and M.W. Dong (ed), Handbook of Pharmaceutical Analysis by HPLC, Elsevier/Academic Press, 2005.
• Y. V. Kazakevich and R. LoBrutto (ed.), HPLC for Pharmaceutical Scientists, Wiley, 2007.
• U. D. Neue, HPLC Columns: Theory, Technology, and Practice, Wiley-VCH, New York, 1997.
• M. C. McMaster, HPLC, a practical user's guide, Wiley, 2007.

External links

• Liquid Chromatography at the Open Directory Project

UpToDate Contents

全文を閲覧するには購読必要です。 To read the full text you will need to subscribe.

• 1. 異常ヘモグロビン症の検査室診断 laboratory diagnosis of the hemoglobinopathies
• 2. 小児および思春期におけるビタミンD不足および欠乏 vitamin d insufficiency and deficiency in children and adolescents
• 3. 骨代謝の生理学および生化学的マーカー bone physiology and biochemical markers of bone turnover
• 4. 先天性代謝異常：個々の障害の発見 inborn errors of metabolism identifying the specific disorder
• 5. 小児における非結核性抗酸菌の皮膚および軟部組織感染症の概要 overview of nontuberculous mycobacterial skin and soft tissue infections in children

English Journal

• Structural elucidation of olive pomace fed sea bass (Dicentrarchus labrax) polar lipids with cardioprotective activities.

• Nasopoulou C, Smith T, Detopoulou M, Tsikrika C, Papaharisis L, Barkas D, Zabetakis I.SourceLaboratory of Food Chemistry, Faculty of Chemistry, National and Kapodistrian University of Athens, Athens 15771, Greece.
• Food chemistry.Food Chem.2014 Feb 15;145C:1097-1105. doi: 10.1016/j.foodchem.2013.08.091. Epub 2013 Aug 31.
• The purpose of this study was to structurally characterise the polar lipids of sea bass (Dicentrarchus labrax), fed with an experimental diet containing olive pomace (OP), that exhibit cardioprotective activities. OP has been added to conventional fish oil (FO) feed at 4% and this was the OP diet, h
• PMID 24128590

• Comparison of cytotoxic extracts from fruiting bodies, infected insects and cultured mycelia of Cordyceps formosana.

• Lu RL, Bao GH, Hu FL, Huang B, Li CR, Li ZZ.SourceAnhui Agricultural University, Hefei, 230036 Anhui, China.
• Food chemistry.Food Chem.2014 Feb 15;145C:1066-1071. doi: 10.1016/j.foodchem.2013.09.001. Epub 2013 Sep 13.
• A resazurin method was employed to test and compare cytotoxicity of extracts from fruiting bodies, insects and cultured mycelia of Cordyceps formosana against Chinese hamster ovary (CHO) cells. Results showed that the cultured mycelia had much stronger cytotoxicity than that of the fruiting bodies a
• PMID 24128585

• Combined determination and confirmation of ethylenethiourea and propylenethiourea residues in fruits at low levels of detection.

• López-Fernández O, Rial-Otero R, Cid A, Simal-Gándara J.SourceNutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Sciences, University of Vigo, Ourense Campus, E-32004 Ourense, Spain.
• Food chemistry.Food Chem.2014 Feb 15;145C:1002-1010. doi: 10.1016/j.foodchem.2013.09.023. Epub 2013 Sep 12.
• In this work, a new method for the determination of ethylenethiourea (ETU) and propylenethiourea (PTU) in fruits and vegetables is presented. Different extraction and purification techniques, including matrix solid phase dispersion (MSPD) and solid-liquid extraction (SLE), followed by a clean-up ste
• PMID 24128576

Japanese Journal

• Determination of the ratio between mercaptalbumin and nonmercaptalbumin by HPLC with fluorescence probe specifically binding to albumin

• Ohyama Kaname,Kishikawa Naoya,Matsuo Aya,Imazato Takahiro,Ueki Yukitaka,Wada Mitsuhiro,Nakashima Kenichiro,Kuroda Naotaka
• Journal of Pharmaceutical and Biomedical Analysis 88, 170-173, 2014-01-25
• … A simple and selective HPLC-fluorescence (FL) method with FL probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1. …
• NAID 120005343838

• Characterization of phase-II conjugation reaction of polycyclic aromatic hydrocarbons in fish species : Unique pyrene metabolism and species specificity observed in fish species

• Ikenaka Yoshinori,Oguri Mami,Saengtienchai Aksorn,Nakayama Shouta M. M.,Ijiri Shigeho,Ishizuka Mayumi
• Environmental Toxicology and Pharmacology 36(2), 567-578, 2013-09-00
• … Metabolic activity, particularly conjugation, was examined in fish by analyzing pyrene (a four-ring, polycyclic aromatic hydrocarbon) metabolites using high-performance liquid chromatography (HPLC) with fluorescence detector (FD), a mass spectrometry (MS) system, and kinetic analysis of conjugation enzymes. … Identification of pyrene metabolites by HPLC/FD and ion-trap MS indicated that the major metabolites were pyrene glucuronide and pyrene sulfate in all 14 species. …
• NAID 120005341794

• Scale up of Cerrena unicolor Laccase Production

• Rola Beata,Karaśkiewicz,Majdecka Dominika,Mazur Iwona,Bilewicz Renata,Rogalski Jerzy,Ohga Shoji,大賀 祥治
• Journal of the Faculty of Agriculture, Kyushu University 58(2), 231-238, 2013-09-00
• … The obtained preparations compared by ion–exchange HPLC, electrophorese and cyclic voltammetry gave no significant differences in their properties. …
• NAID 120005326647

Related Links

• 高速液体クロマトグラフィー - Wikipedia

HPLCの心臓部とも言える機器。極めて安定した送液が出来る構造となっている。ポンプ の前には、オンラインの脱気装置（デガッサー、degasser）が付いている場合が多い。 また、プランジャーの前後にはチェックバルブが取り付けられており、移動相の逆流を ...

Related Pictures

---

★リンクテーブル★

リンク元	「high performance liquid chromatography」「高速液体クロマトグラフィー」「high-pressure liquid chromatography」
関連記事	「HP」

「high performance liquid chromatography」

　　[★]

• 高速液体クロマトグラフィー、高速液クロ

関

high-pressure liquid chromatography、HPLC

「高速液体クロマトグラフィー」

　　[★]

英

[[]]

同

HPLC

関

[[]]

-HPLC

関

See High-performance liquid chromatography

同

HPLC

「high-pressure liquid chromatography」

　　[★]

• 高圧液体クロマトグラフィー

関

high performance liquid chromatography、HPLC

「HP」

　　[★]

• Helicobacter pylori
• ハプトグロビン haptoglobin
• 遺伝性膵炎 hereditary pancreatitis
• 過敏性肺臓炎 過敏性肺炎 hypersensitivity pneumonitis