2 色層分析法 Chromatography

Transcription

1 2 色層分析法 Chromatography 色層分析原理 Basic principles 極性不同的分子在兩相中有不同的分佈比例 2.2 膠体過濾法 Gel filtration 依樣本分子量之差異來做分離純化 2.3 離子交換法 Ion exchange 利用樣本分子的表面帶電性質差異來進行分離 2.4 親和層析法 Affinity chromatography 利用分子間的親和性大小不同來進行分離 2.5 HPLC 及 FPLC 改善介質的材質及吸附容量可增加速度及解析力

2 層析法演進 Historical review Martin & Synge (1952) 2 Paper partition chromatography (PPC) Round filter paper Column sample Thin layer (TLC) Development sample Long rectangle paper Sample capacity increased Larger capacity Adapted from Scope RK (1987) Protein Purification Principles and Practice p.9

3 色層分析法的基本機制 Essential mechanism 3 lp -d O +d lp 永久的偶極性 Like Dissolves Like 極性相似的兩分子間, 其親和力較強 Polar Polar Non-polar Non-polar

4 2.1 色層分析法原理 Principle of chromatography 4 A B C Nonpolar Non- Polar Two-phase Mobile phase 流動相 Polar Polar Stationary phase 固定相 樣本分子依其分子極性選擇親和兩相之一 每次分離樣本都要做一次選擇 One Plate of Separation Theoretical plate number Two-phase separation system L Sample ADSORPTION Liquid - Solid S L L Sample PARTITION Liquid - Liquid 圖 2.1

5 層析法的板數概念 One separation, one plate 5 A A B A B B B B B A B A B A A B B B A A A 100A + 100B A A A A A A A A B Polar A A B B B B B B B B B Nonpolar One plate

6 常用層析法 Common chromatographic methods 6 小分子 大分子 Partition Paper partition chromatography (PPC) Thin-layer chromatography (TLC) a) Gas chromatography (GC) b) Reverse phase Gel filtration 2.2 Reverse phase Adsorption Ion exchange TLC a) GC b) Ion exchange Affinity chromatography Hydrophobic interaction Hydroxyapatite a, b) 這兩種層析法均可進行 partition 或 adsorption, 取決於所用展開液的成分與比例 表 2.1

7 薄層層析法操作 Thin-layer chromatography 7 Thin-layer plate (Adsorption) Activated Not-activated (Partition) Sample spotting Add developer Developing

8 2.2 膠体過濾法 Gel filtration 原理概述 Basic principles 是一種 partition 層析法 膠体介質 Gel materials 是一種長鏈的大分子糖類聚合物 膠体管柱 Gel and column 管柱性質 影響因素及管柱系統 管柱操作 Column operation 裝填並操作一支膠体過濾管柱 問題及解決 Problem and solution 常見的錯誤要預先避免

9 膠体過濾法是一種 Partition 層析法 9 Stokes radius Molecular size and shape Smaller molecules are retarded Small molecules Stationary phase (liquid) Sample Mobile phase (liquid) Elute out faster Gel filtration is a partition type chromatography (liquid-liquid) Larger molecules 圖 2.2

10 各種色析膠体 Pharmacia Sephadex Sepharose Sephacryl Sephacel FPLC glucose (dextrose) agarose glucose + acrylamide cellulose Superose, Superdex Mono Q, Mono S 10 Fast Performance Liquid Chromatography Bio-Rad BioGel P BioGel A acrylamide agarose Gel material is a polymer of carbohydrate or acrylamide

11 Pharmacia (1991) Gil Filtration Principles and Methods p.35 膠体的構成 Space for filtration 11 glucose unit Sephadex Sephadex is the polymer of glucose with chemical cross-linking

12 膠体的使用範圍 Sephadex G 12 MW ranged from ten to several hundred thousands G-200 could be smashed flat easily (its backbone support is too weak) G-50 G-25 G-10 1 g 7.5 ml From powder to gel form 1 g 20 ml gel Small proteins Pharmacia (1991) Gil Filtration Principles and Methods p.37

13 Pharmacia (1991) Gil Filtration Principles and Methods p.22 膠体的構成 Larger space, Stronger support for backbone 13 Acrylamide Cross-linking Sephacryl

14 洋菜膠体的成膠反應 Agar gel formation 14 Even stronger backbone Much larger space 洋菜 寒天 瓊脂 Pharmacia (1991) Gil Filtration Principles and Methods p.38, 39

15 膠体的使用範圍 Sepharose 15 MW ranged from ten thousands to several millions Sepharose is rigid Cross-linking 6% gel 2% gel Sephadex Large proteins Sephacry Pharmacia (1991) Gil Filtration Principles and Methods p.40

16 膠体過濾法的膠球 Gel filtration beads 16

17 管柱內膠体的組成區隔 Spaces in a column 17 V (total volume) t V (void volume) o V t - V o 管柱總体積 h 膠体外体積 膠体內体積 底面積 Mobile phase (L) Stationary phase (L) Adapted from Pharmacia (1991) Gil Filtration Principles and Methods p.11

18 膠体過濾的溶離圖譜 A typical chromatogram 18 A 目標酵素 (E) 最好不要落在主要蛋白質峰 (Y) 的範圍內 Blue Dextran Vo 之前不應有物質溶離出來 X E Y Enzyme activity Vit. B 12 Vt 之後不應有物質溶離出來 Z 0 Vo Ve Elution Volume (ml) Vt 圖 2.4

19 Relative amount Adapted from Pharmacia: Gil Filtration Principles and Methods 介質的粗細影響解析力 Corse Fine Superfine Poor separation Peak flattened Best separation Ve/Vt Bead size is critical to the resolution of gel chromatography

20 溶離液擴散進出膠球 Diffuse in and out bead 20 溶離液或樣本分子, 由膠体粒子外圍均勻向內或向外擴散 Sample or buffer is diffusing in and then out of the gel particle 擴散 Diffusion Dispersion 瀰散 圖 2.3A

21 溶離液對膠球的擴散或瀰散 Two types of gel 21 若膠体粒子較大, 則由外圍擴散到內部的距離長, 通過粒子所需要的時間拉長, 降低分離效果 Small particle size reduces the diffusion time and enhances its resolution Dispersion ( 瀰散 ) 方式的膠体因通透性佳, 溶離液可直接流入膠体, 不再靠擴散作用 Dispersion type gels let the sample molecules flow directly through the gel body, and have better resolution 圖 2.3B

22 粒子粗細影響溶離液流動 Bead size vs flow rate 22 膠体的顆粒越小其解析力越大但流速變慢 Smaller particles reduce the space between the beads, and prevent the turbulence as the buffer flows, but the flow rate might be retarded Adapted from Scope RK (1987) Protein Purification Principles and Practice p.192

23 Absorbance Adapted from Pharmacia: Gil Filtration Principles and Methods 不同介質的運用選擇 Sephacryl S-300 Sephacryl S-200 (10~1,500 kda) (5~250 kda) 高分子量分離較好色帶較慢溶離出來低分子量分離較好 23 0 Elution volume 色帶較快 溶離出來 Choose the gel which brings your target protein out of the column earlier

24 樣本体積的影響 Sample volume at 1% of Vt 24 Resolution 1.5 Sample volume at 1~2% of total gel volume R = a a (b+c)/2 1.0 b c 0.5 Superdex 200 Transferrin (81 kd) & IgG (160 kd) a 越大分離越開 b, c 越小越好 R = 1 表示剛好分開 Sample volume (% of Vt) Adapted from Pharmacia (1991) Gil Filtration Principles and Methods p.46

25 Adapted from Pharmacia (1991) Gil Filtration Principles and Methods p.59 Concentration 膠体管柱的最佳化調整 標準分離條件都可分得很好 若加快流速時間省十倍解析力下降 AB C A B C A B C 流速已加快再縮短管柱時間再縮短效果則更差但 C 沒問題 管柱長度 Reduce column height Bed height (cm) Flow rate (ml/cm 2 /h) 溶離速度 Increase flow rate Elution time (h)

26 色析管柱的形狀 10 cm 30 cm cm 3 矮胖型 Short and fat 色帶有點歪斜就會影響分離 細長型 Long and slim 色帶有點歪斜但不影響結果 10 cm cm 矮胖型管柱不能容忍分離不佳 Fat column cannot tolerate poor separation 但其流速及容量均較大 But it has better flow rate and higher capacity Adapted from Scope RK (1987) Protein Purification Principles and Practice p.195

27 液相層析管柱系統 A Buffer starts here 梯度製造器 Gradient mixer (1) 緩衝液 Buffer 轉接器 Connector reservoir 管柱 Column 膠体裝填 膠体 Gel (2) 幫浦 Pump adapter (3) (4) 監視器 Monitor (5) 記錄器 Recorder 27 分劃收集器 Fraction collector The whole family of liquid chromatography apparatus 圖 2.5

28 膠柱裝填方法 Packing column step by step 28 清洗膠体 Wash gel well 預估体積 Estimate gel volume Gel 緩衝液平衡 Equilibrated in buffer B A 靜置膠体 Gel stands o/n 溫度平衡 Temperature equilibrated 1 2 Gel Put on reservoir C 檢查管柱是否暢通 Check flow rate of empty column 暫停流洗 Stop elution X 裝填膠体 Pouring gel smoothly 繼續流洗 Keep eluting D 上清 Supernatant 沈積中 In progressing 已堆積 Sedimentation 加壓流洗 Elute under High pressure Put on adaptor E Pack gel tightly 緊密堆積 圖 2.6

29 Pharmacia (1991) Gil Filtration Principles and Methods 色析膠体的裝填 Packing column 29 一口氣倒入膠体, 勿陷入氣泡 Pour gel slurry smoothly (non-stop), avoid trapping any bubble

30 General principle for column chromatography 30 Gel selection Bead size Column size Column shape Pack tightly Flow rate Sample volume Let target protein eluted out the column earlier Finer bead has better resolution, but slower flow Use larger column size yet consider practical need Slim column for gel filtration, fat column for others Pack the gel tightly for better resolution Fast flow reduces resolution, slow brings diffusion Apply 1% of the total gel volume for sample

31 2.3 離子交換法 Ion exchange 原理概述 Basic principles 離子與固相擔体帶電基團間的爭奪戰 (Ion wars) 交換介質 Exchange materials 是帶有電荷基團的多醣長鏈聚合物 ( 膠球 ) 緩衝液與層析系統 Buffer system 緩衝液對離子交換法之影響極大 (Why?) 管柱操作方法 Column operation 如何操作一支離子交換管柱 色層焦集法 Chromatofocusing 依蛋白質等電點之差異來進行分離

32 陰離子交換法 Anion Exchange 32 Sample Solid support Stationary phase Ion exchange is an adsorption chromatography Counter ion 吸附 Cation groups Anion + Mobile phase 圖 2.7

33 質子可以吸著或脫離一基團 離子及它的產地 33 Proton: the smallest and most abundant particle in the living cell controlling the ph and the charge of a molecule Amino lone pair electrons N H H H + H + N H H Carboxylic C O O H C O O H + Ampholyte: a molecule contains both positively and negatively charged groups Proton can enter or leave a functional group relatively freely

34 選擇離子交換介質 Use buffer having only one ph unit difference to the pi of target protein Buffer ph pi Cation exchange Anion exchange (1) 環境 ph 影響帶電性質 34 (2) 蛋白質帶電性乃可變異 (3) 帶電性質決定分子行為 Net Charge of a Protein -

35 陰離子交換膠体的 ph 變化 35 Support + OH OH - Donnan Effect + + 陰離子交換膠體附近的 ph 其實比較高 ph = 8 OH - OH - Counter ion 其實是更高 使用的緩衝液 ph 比 pi 高 1 ph ph = 7 - pi = 6 ph = 5 The ph change of the microenvironment surrounding the ion exchange gel particle + 相反用陽離子交換的情況也一樣 (ph 更低 ) 圖 2.9

36 離子取代優先順序 Displacing order of ions 36 電荷高者 (higher charge) > ++ + 離子大者 (larger ion) + > + 濃度大者 (higher concentration) change ph, NaCl gradient > + Key properties determining the order of ion replacement 圖 2.8

37 陰離子交換法分離範圍 kd pi 4 目標蛋白質 離子交換法只能分離出大約 1 ph 範圍的蛋白質 再用第二種方法縮小範圍 37 二次元電泳展開樣本大部份蛋白質之分布 The approximate range of proteins isolated by chromatography

38 Cation Exchanger Anion Exchanger 離子交換介質 Common ion exchange materials 38 陰陽強弱 Resin / Polystyrene Glycan / Cellulose (= X) Mono bead Strong Dowex-1 Dowex NR 3 TEAE-X (QAE-X) - + NR 3 Q Weak Dowex-3 IR NHR 2 DEAE-X -O(CH 2 ) 2+ NHR 2 Strong Dowex-50 -SO 3 - Phospho-X -PO 4 S Weak IRC-150 -COO - CM-X -CH 2 COO X = Sephadex, Sepharose, Sephacel or cellulose 選擇 : 陰離子或陽離子交換? 吸附力強弱? 何種材質? ( 樹脂 多糖 Monobead) 表 2.2

39 Pharmacia (1991) Ion Exchange Chromatography Principles and Methods p.24 膠体的組成與外型 Support material and bead 39 Pharmacia (1980) Separation News: 5 Cellulose Sephacel Monobead Homogeneous bead shape is critical to its resolution and flow rate

40 不同膠体的吸著容量有很大差異 40 Adapted from Pharmacia (1991) Ion Exchange Chromatography Principles and Methods p.64 Buffer: (mg / ml gel) 0.01 M Tris-HCl, ph 8.0 Lactalbumin Albumin Ferritin DEAE-Sephadex A DEAE-Sephadex A DEAE-Sepharose CL-6B DEAE-Sephacel 分子量 pi 三者吸附量之差異是可以解釋的, 如何切入思考? Each gel has different adsorption capacity toward different target proteins

41 離子交換法預備試驗 Determine the conditions 41 Enzyme + Buffer + Gel Incubation Spin Sup + Spindown ph Sup Spindown NaCl ph = M NaCl mg/ml protein Adapted from Pharmacia (1991) Ion Exchange Chromatography Principles and Methods p.70 Capacity

42 膠柱裝填方法 Packing column step by step 42 清洗膠体 Wash gel well 預估体積 Estimate gel volume Gel 緩衝液平衡 Equilibrated in buffer A B 靜置膠体 Gel stands o/n 溫度平衡 Temperature equilibrated 1 2 Gel Put on reservoir 檢查管柱是否暢通 Check flow rate of empty column 暫停流洗 Stop elution X 裝填膠体 Pouring gel smoothly 繼續流洗 Keep eluting Gel should be equilibrated completely before packing C D 上清 Supernatant 沈積中 In progressing 已堆積 Sediment 加壓流洗 Elute under High pressure Put on adaptor E 緊密堆積

43 液相層析管柱系統 A 緩衝液 Buffer 梯度製造器 Gradient mixer (1) 轉接器 Connector reservoir 管柱 Column 膠体 Gel (2) 幫浦 Pump adapter (3) (4) 監視器 Monitor (5) 記錄器 Recorder 43 分劃收集器 Fraction collector 膠体裝填 The whole family of liquid chromatography apparatus

44 鹽梯度的兩種方式 Two ways for making gradient 44 離子鍵在鹽溶液中不易形成提高鹽濃度分別流洗蛋白質 高限溶液 低限溶液 Upper-limit Lower-limit 連續梯度 Continuous 連續梯度階段梯度 Step-wise 膠柱面 Gel surface Dead volume Eliminate dead volume 兩種方式均可, 各有特點 Both methods have their specific applications Dead volume 會破壞拉好的鹽梯度 Pharmacia 圖 2.10

45 Protein concentration Adapted from Pharmacia: Ion Exchange Chromatography Principles and Methods 連續與階段梯度比較 連續梯度 Continuous gradient 階段梯度 Step-wise gradient Continuous vs step-wise Elution volume

46 Protein concentration Adapted from Pharmacia: Ion Exchange Chromatography Principles and Methods 鹽濃度效果比較 NaCl Effect of salt concentration Elution volume

47 Relative absorption Adapted from Pharmacia: Ion Exchange Chromatography Principles and Methods 溶離体積會影響解析度 M NaCl 100 ml 0.5 M NaCl 200 ml Resolution improved but protein diluted 0.5 M NaCl 300 ml 47 Effect of elution volume Elution volume

48 Protein concentration 管柱長短的影響 5 cm column A cm column B B A 好像被拖延了 A 0.5 Effect of column length Acetate concentration (M) 0

49 Adapted from Scope RK (1987) Protein Purification Principles and Practice p.112 離子交換的梯度溶離有濃縮效果 49 Sample applied 樣本最前線會反覆吸住膠體 Salt gradient elution begins Salt elutes and brings the proteins moving forward, but proteins are adsorbed again in the front and retarded Salt gradient of ion exchange can concentrate the proteins in sample elution

50 離子交換法操作要點 Summary on operation 50 Equilibration Elution Gradient Buffer ph Non-specific Sample Used gel should be regenerated and equilibrated well Eluted with NaCl in continuous or step-wise gradient Use proper concentration and volume in elution Keep target proteins in weak charged state Wash out contaminants with low NaCl concentration Equilibrated in buffer, don t overload column capacity Dead volume Eliminate any dead volume in the column Elute through Elute target protein through column and adsorb others

51 離子交換法實例 Two-step cellulase purification FPLC (fast performance liquid chromatography) M NaCl Mono Q HR 5/5 1 ml/min 20 mm Tris, ph 7.6 Crude cellulase 2.5 mg Q M NaCl S + ++ Mono S HR 5/5 1 ml/min 20 mm acetate, ph Retention time (min) Adapted from Pharmacia (1991) Ion Exchange Chromatography Principles and Methods p

52 Protein content / Activity 離子交換法實例 DEAE Sepharose CL-6B SS SDS-PAGE 52 NaCl A typical example Elution volume

53 色層焦集法如何拉出 ph 梯度 Polybuffer 含有連續 pk a 的緩衝分子 (ampholyte) 可拉出連續 ph 梯度 Polybuffer contains ampholyte which is a mixture of chemicals having continuous pk a ? 實際結果 ph 無法拉出 ph 梯度 Volume 假如含有許 Buffering effect 多緩衝分子 9 6 If there are many buffering molecules in the buffer How chromatofocusing creates its ph gradient by Polybuffer Volume

54 2.3.5 色層焦集法 Chromatofocusing 54 其介質也是一種離子交換介質 但所使用的 Polybuffer 可拉出穩定的 ph 梯度 Shorter than Taller than Using ion exchange bead, but eluted with Polybuffer to create a ph gradient

55 Polybuffer 色析焦集法的焦集機制 陰離子交換介質 ++ 在低 ph 處帶正電被排斥 Protein at lower ph is positively charged and repelled by the beads ++ 焦集在等電點 Protein focused at its pi 超越等電點 ( 帶負電 ) 被吸附 Protein moves beyond its pi will be negatively charged and retarded Focusing mechanism for a sample protein in chromatofocusing 55

56 環境影響分子的帶電性質 Isoelectric point, pi + + Buffer ph Net Charge of a Protein - 56 圖 7.1

57 抹香鯨肌紅蛋白 (pi = 8.2) 追過馬肌紅蛋白 (pi = 7.4) 57 The early protein might be caught up by a later protein in chromatofocusing Pharmacia

58 2.4 親和層析法 Affinity chromatography 原理概述 Basic principles 要有一對具有高親和性的分子 親和吸著劑 Affinity adsorbent 有很多親和性吸著劑可利用 金屬螯合層析法 Metal chelating 利用分子與金屬的吸引力 疏水性層析法 Hydrophobic interaction 利用分子間的疏水性引力 液相分配 Liquid partition 分子在兩液相間的分配比例不同

59 親和層析法的四項要素 Four essential factors 59 配体 Ligand (A) 耦合反應 (3) Coupling Reaction 雜質 contaminants B A (2) Specific Binding Substance (B) (1) Solid Matrix B B Solid matrix = Sepharose B B 溶離 Elution (4) (1) 固相擔體 (2) 專一配對 (3) 耦合反應 (4) 溶離方法 B 圖 2.12

60 親和層析法的作用機理 How it works 60 A X (1) (2) (3) Sample B A Washing A Elution B 固相擔体 Solid support 親和基團配体 ligand X B 圖 2.11

61 典型的親和層析操作 Typical elution pattern 61 Protein Activity ph 2.05 * * 超出管柱容量 Elution volume

62 專一性結合力量的構成因素 Specific interactions 62 I. Conformational Match: Van der waals interaction 兩分子間因構形互補所造成的吸引力是由凡得瓦爾力所貢獻 II. Interaction Forces: (1) Hydrogen bond (2) Hydrophobic interaction (3) Electrostatic interaction (4) Van der Waals interaction + Kd =O H-N- + - Two types of forces contribute to the specific affinity between proteins

63 構形互補所造成的吸引力 Van der Waal interactions A vdw vdw B 凡得瓦爾鍵數目夠多即足以造就親和力 非極性電子分布均勻 有時電子分布偏向一邊 -d +d 造成短暫耦極 -d +d -d +d vdw 產生短暫凡得瓦爾力 +d -d +d -d 也有可能轉另一方向 63 引發鄰近原子耦極 between non-polar surfaces 可能回到原來的狀態

64 生化分子的反應基團 Some functional groups 64 氰基 -C N 醛基 -HC=O 酸基 -COOH 胺基 -NH 2 醇基 -OH 硫醇基 -SH 醚 -O- 烷基 -CH 3 Inert The functional groups in a living cell are mild in reactivity

65 可與各種配体基團反應的介質 65 Pharmacia 配体基團親和性介質反應基團反應方式 CNBr-activated Sepharose 4B -C N 直接反應 -NH 2 CH-Sepharose 4B 或其活化型 -COOH N-OH-succinimide 加 EDC* 直接反應 Epoxy-activated Sepharose 6B oxirane 直接反應 -COOH AH-Sepharose 4B -NH 2 加 EDC* -OH Epoxy-activated Sepharose 6B oxirane 直接反應 Epoxy-activated Sepharose 6B oxirane 直接反應 -SH Thiopropyl-Sepharose 6B -S-S-R DTT 活化 Activated Thio-Sepharose 4B -G-S-S-R 直接反應 * EDC = N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide HCl Affinity materials are designed to react with functional groups on ligands 表 2.3

66 親和性介質的反應基團 Functional groups 66 CNBr - OH - O - C N NH 2 - Protein NH - O - C N - O - C - NH - Protein CNBr activated Sepharose - NH -C-C-C-C-C-C- NH 2 - NH -C-C-C-C-C-C- COOH Amino Carboxylic O - NH -C-C-C-C-C-C- C - O-N - O-C-C-C-O-C-C-C-C-O-C-C - C OH spacer arm effect O O O N-Succinimide Epoxy

67 各種親和性介質及其專一性基團 67 Pharmacia 配体 (A) 親和性分子 (B) 說明 抗体 對應之抗原 免疫吸著劑, 大多自行合成 基質或抑制劑 對應之酵素 酵素的專一性結合 Protein A 部分 IgG 單株抗体純化 Con A 醣蛋白 對 α-d- 葡萄糖 甘露糖基有專一性 Heparin 凝血蛋白等 Heparin Sepharose CL-6B Chelator (NTA)-Ni 2+ His-tag 表現蛋白質 (His) 6 與兩價陽離子間的親和力 Oligo (dt) mrna Oligo (dt)-cellulose Cibacron-Blue NAD(P) + 結合酵素 Blue Sepharose CL-6B AMP 或 ADP 等 同上 5'AMP-, 2', 5'ADP-Sepharose 4B 單糖及其衍生物 Lectin 用來純化 lectin Affinity adsorbents with special affinity ligands 表 2.4

68 金屬螯合層析法 Using metal-chelating affinity 68 Transition metals 過渡元素 Tag His 6 Expressed protein imidazole Solid support C-COOH -O-C-C-C-O-C-C-C-N C-COOH OH OH Ni d orbiter elution His His His Protein Metal Chelate Affinity Chromatography 圖 2.13

69 親和層析法實例 Materials used in an example 69 Stryer L (1995) Biochemistry 4/e CH 2 OH OH O O CH 2 OH OH O Chitin (solid support) O Trypsin (target) NH NH 2 O=CH 3 glutaraldehyde CHOM (ligand, bait) 雞蛋粘多糖蛋白 Chicken ovamucoid Trypsin inhibitor

70 以親和層析法純化 Trypsin purification 70 CHOM Disc-PAGE SDS-PAGE Chitin Trypsin A B C&D 以膠体電泳檢定親和層析操作過程各步驟的樣本 Check by PAGE A B C = D 相同的樣本卻有不同的色帶?

71 Hydroxyapatite 吸著劑 氫氧基磷灰石人骨的主要成分 71 (Ca 5 (PO 4 ) 3 OH) 2 磷酸鈣陶土 可選擇 NaCl 或磷酸等不同溶離條件 Elution by NaCl or phosphate gradient leads to different results 具有非常多樣的吸附特性有負電荷磷酸也有鈣離子 University of Liverpool, ChemTube3D (left) Bio-Rad CHT (right)

72 蛋白質表面的極性或非極性分布 72 Hydrophobic area Activie site 蛋白質表面的非極性區域多寡不同可利用來作為分離純化的手段 (HIC) Superxoide dismutase (SOD) Stryer L (1995) Biochemistry 4/e Fig

73 疏水性及反相層析法 Hydrophobic interaction 73 C 18 = -C 18 H 37 溶離液由極性轉成非極性 ( 或相反 ) HIC (liquid-solid) Reversed phase chromatography (liquid-liquid) Polar solvent Polar solvent Non-polar solvent Polar area Non-polar area Hydrophobic group (solid) Non-polar area (liquid) Polar phase Using non-polar groups as a stationary phase Using ion-exchanger C6 = -C 6 H 13 Phenyl = -C 6 H 6 有各種不同應用方式最常被使用在 HPLC 圖 2.14

74 Protein concentration Ethylene glycol (%) Hydrophobic interaction chromatography(hic) 74 BA = beta amylase Ammonium sulfate (% sat.) BA 蛋白質表面非極性區域很小 由極性到非極性 Elution volume (ml) 的梯度 Adapted from Pharmacia - Hydrophobic Interaction Chromatography 非極性區較大