AA are ampholyte RCHCO2H NaOH NH2 HCL R R NH2-CH-COO OH RCHCO2=HH。CH-CooH N"H3 OH Anion, zwitterion,when Cation, when at at isoelectric when at high pH point (pI) low pH NOTE:peptide or protein also have both acid and base properties. They share the same property of being positively charged at low pH and negatively charged at high pH
AA are ampholyte Anion, when at high pH zwitterion, when at isoelectric point (pI) Cation, when at low pH NH2 CH COO - R H + OH - NH3 + CH COO - R H + OH - RCHCO2 H NH2 RCHCO2 - N + H3 H NaOH HCL NOTE: peptide or protein also have both acid and base properties. They share the same property of being positively charged at low pH and negatively charged at high pH
Isolectric Point (pI)of AAs anode cathode RCHCO2H + + NH2 + + RCHCO2 HO RCHCO2 H+ RCHCO2H + NH2 N"H3 N'H3 + ++ Anion in Zwitterion in pI Cation in basic sol'n. sol'n.No move acidic sol'n. to either of Electrode.And with Lowest pH溶液pl solubility pH溶液pI
Isolectric Point (pI) of AAs Anion in basic sol’n. RCHCO2 H NH2 RCHCO2 - N + H3 H + RCHCO2H N + H3 HO - RCHCO2 - NH2 + + + + + + + + + - - - - - - - - - - Zwitterion in pI sol’n. No move to either of Electrode. And with Lowest solubility Cation in acidic sol’n. pH pH溶液<pI 溶液>pI anode cathode
Note on pI The Acidity of neutral AA is stronger than its Basicity,which means the dissociation degree of reaction (i)is larger that of reaction(ii). Therefore the pH of neutral AA water solution is less than7。 H2N-CHR-CO0H+H20←→H2NCHR-CO0+H3O+() H2N-CHR-CoOH+H20←→H3N+-CHR-COOH+OH(i) therefore,[anion]>[cation],to get pI,more acid is to be added. pI of neutral AAs is around 5.6~6.3, pI of acidic AAs is around 2.8~3.2; pI of basic AAs is around 7.6~10.8
Note on pI ◼ The Acidity of neutral AA is stronger than its Basicity, which means the dissociation degree of reaction (i) is larger that of reaction(ii). Therefore the pH of neutral AA water solution is less than 7 。 H2N-CHR-COOH +H2O H2N-CHR-COO- +H3O+ (i) H2N-CHR-COOH +H2O H3N+-CHR-COOH +OH- (ii) * therefore, [anion] >[cation], to get pI, more acid is to be added. ◼ pI of neutral AAs is around 5.6~6.3， ◼ pI of acidic AAs is around 2.8~3.2； ◼ pI of basic AAs is around 7.6~10.8
Ninhydrin reaction OH RCH +RCH0+C02+3H20 OH ■Note: 1,Ninhydrin solution is made in basic solution of phosphate buffer (pH8.04). 2,The reaction products of all AAs except proline,are purpule-bule(540nm),and for proline is yellow(440nm) 3,protein and peptide also can occur this reaction due to their containing amino group. 4.This reaction can be used to quantitative and qualitative analysis,with the help of spectrophotometer,TLC
Ninhydrin Reaction RCHCOOH NH2 O O OH OH + O O N O O + RCHO + CO2 + 3H2O ◼ Note: 1, Ninhydrin solution is made in basic solution of phosphate buffer (pH8.04). 2, The reaction products of all AAs except proline, are purpule-bule(540nm), and for proline is yellow(440nm) 3, protein and peptide also can occur this reaction due to their containing amino group. 4. This reaction can be used to quantitative and qualitative analysis, with the help of spectrophotometer, TLC
Automatic AA analyzer From pH2.2 to pH 6.4 Buffer pump ☒=Sample injection lon-exchange Strip-chart recorder column Separated amino acids l几 Ninhydrin pump Photomultiplier Reaction coil Ninhvdrin Light source at 440 and 540 nm
Automatic AA analyzer From pH2.2 to pH 6.4