Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,enzyme,enzyme,1,-Chymotrypsin catalyzed cleavage of the C-N,bond has a maximum rate near pH 8 that is twice,as great as the rate near pH 7 or pH 9.,最好有緩衝液存可使得反應最大,-Chymotrypsin catalyzed cleav,2,細胞培養一般步驟，資列以,Human fibroblasts,作為例子,Cultures for Human fibroblasts(HF,doubling time=24 h),A.DMEM,培養液,1.5X DMEM medium 100 ml2.100X Glutamic acid 5 ml3.100X streptomycin/penicillium 5 ml4.5.5%NaOHCO3 10-20 ml 5.Hyclone fetal calf serum(FCS,56C,30min)50 ml6.,Hepes,緩衝液,(1.5 M pH=7.4),5 ml,7.,加,ddH2O,至,500ml,細胞培養一般步驟，資列以Human fibroblast,3,The Henderson-Hasselbalch Equation,HA=H,+,+A,-,Ka=H,+,*A,-,/HA,H+=Ka*HA/A,-,-logH+=-logKa log(HA/A-),pH=pKa+log(A-/HA,Henderson-Hasselbalch Equation for an acid,可應用在處理酸鹼平衡系統並用來配製緩衝溶液。當共軛酸鹼的濃度相同時，則,pH=pKa,The Henderson-Hasselbalch Equa,4,緩衝溶液就是對於外界添加酸或鹹至溶液中，其,pH,值的變化量不大，而當溶液處於,pKa,時，則具大的緩衝能力。,下圖胺基丙酸,(alanine),水溶液在,pKa,值,2.74,及,9.8,時有最大的緩衝能力，在等電點,(PI),時緩衝能力最低，溶於水時，胺基丙酸帶有未質子化的羧基,(-COO,-,),及質子化的胺基,(-NH,3,+,),，,pH,值等於,6,，約為兩者之,pKa,中間，其滴定曲線如圖。,緩衝溶液就是對於外界添加酸或鹹至溶液中，其pH值的變化量不大,5,最好有缓冲液存可使得反应最大ppt课件,6,The Henderson-Hasselbalch Equation for a base,B+H,2,O=BH+OH,-,Kb=BH+*OH-/B,log kb=log(BH+/B)+logOH,-,log Kw log Ka=log(BH+/B)+logOH,-,),pKa-log(BH+/B)=logOH,-,-log Kw,pKa+log(B/BH,+,)=-(-logOH,-,-log Kw=-pOH+14,pH=pKa+log(B/BH,+,),The Henderson-Hasselbalch Equa,7,土壤的缓冲性能有哪些？,土壤的緩衝性質是指當外界環境產生巨大變動時，土壤仍能維持其大部分之原來性質或只是略微變動而已，而土壤具緩衝能力的多寡主要與土壤的粘粒及有機質含量有關，粘粒及有機質含量越高者，其緩衝能力越高。此緩衝性質可用來說明當添加大量石灰於土壤時，土壤的酸鹼性並不會造成巨大的改變，而施肥時也有類似的結果。,土壤的缓冲性能有哪些？,8,常用的緩衝劑叫,tris,依附錄,B BH+,的,Ka=8.075,Tris hydrochloride BH,+,Cl,-,BH,+,Cl,-,=BH,+,+Cl,-,BH,+,=B+H,+,常用的緩衝劑叫tris,9,NaOCl,溶於水中調緩衝至,pH6.2,OCl,-,/HOCl=?,HOCl=H+OCl-,pH=pKa+(OCl-/HOCl),6.2=7.53+log(OCl,-,/HOCl),OCl,-,/HOCl=10(6.2-7.53)=0.047,NaOCl溶於水中調緩衝至pH6.2,10,溶解,12.43,克的,tris(FM 121.136),及加,4.67,克的,tris chloride(FM 157.597),於,1,升水中的,pH,12.43/21.36=0.01026 M=tris=B,4.67/157.597=0.0296 M=BH+,pH=pKa+log(B/BH+),=8.075+log(0.0126/0.0296),=8.61,溶解12.43克的tris(FM 121.136)及加4.6,11,加,12mL 1.0 M HCl,入前頁的溶液中，,pH,？,當強酸加入弱鹼時，幾乎完全反應至,BH,+,B +H+=BH,+,tris From HCl,moles,始,0.1026 0.012(0.012L*1M),末,0.1026 0 0.0296,-00.012 +0.012,0.0906 0.0416,pH=pKa+log(0.0906/0.0416)=8.41,若加入沒有緩衝能力的液體則,pH=1.93,加12mL 1.0 M HCl入前頁的溶液中，pH？,12,最後體積為,250,毫升，,pH 7.6,，內含,10,克的,tris hydrchloride(BH,+,，,FW=157.597,BH+,的,pKa=8.075),，須添加多少毫升的,0.5M NaOH,。,10/157.597=0.0635 mol,BH,+,+OH,-,=B,始,0.0635 x 0,末,0.0635-x 0 x,pH=pKa+log(mol B/mol BH+),7.6=8.075+log(x/(0.0635-x),10-0.475=x/(0.0635-x)=0.335 x=0.0159 mol,0.0159/y=0.5 y=0.0318 L=31.8 mL,最後體積為250毫升，pH 7.6，內含10克的tris h,13,A buffer resists changes in pH because the added,acid or base is consumed by the buffer.As the buffer,is used up,it becomes less resistant to changes in pH.,A buffer resists changes in pH,14,最好有缓冲液存可使得反应最大ppt课件,15,pH=pKa maxmum buffer capacity,The useful pH range of a buffer,is usually considered to be pKa1,pH value,pH=pKa maxmum buffer capacityT,16,練習,HA=H+A-,Ka=10-5 HA+A-=1,0.01 H+OH-,加入 新的,pH,形成。,pH=pH,的改變,例如：混合,0.0383mol,的,A-,及,0.9617 mol,的,HA,起始,的,pH3.6,加人,0.01 mol,的,OH-,則,pH,的改變,pH=3.705,3.705-3.6=,0.105,練習 HA=H+,17,最好有缓冲液存可使得反应最大ppt课件,18,細胞培養一般步驟，資列以,Human fibroblasts,作為例子,Cultures for Human fibroblasts(HF,doubling time=24 h),A.DMEM,培養液,1.5X DMEM medium 100 ml2.100X Glutamic acid 5 ml3.100X streptomycin/penicillium 5 ml4.5.5%NaOHCO3 10-20 ml 5.Hyclone fetal calf serum(FCS,56C,30min)50 ml6.,Hepes,緩衝液,(1.5 M pH=7.4),5 ml,7.,加,ddH2O,至,500ml,細胞培養一般步驟，資列以Human fibroblast,19,溴甲酚綠 在,pH,小於,4.7,為黃色，大於,4.7,為藍色,acid-base indicator,pH=pKa maxmum buffer capacity,溴甲酚綠 在 pH小於4.7為黃色，大於,20,瑞香草酚藍，,pH,小於,1.7,為紅色，,1.7-8.9,為黃色，,大於,8.9,為藍色,瑞香草酚藍，pH小於1.7為紅色，1.7-8.9為黃色，,21,以,Thymol blue,為例,Transition range Acid Base,color,Thymol blue 1.2-2.8 Red Yellow,8.0-9.6 Yellow Blue,R=Y-+H+pH=pK1+log(Y-/R),當,pH=1.7(=pk1)1:1,比例,green,R/Y-,大於等於,10/1(pH=0.7)red,Y-/R,大於等於,10/1(pH=2.7 yellow,pH8-9.6,為綠色 小於,8,為黃色 大於,9.6,為藍色,以Thymol blue 為例,22,