Separation Techniques

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Separation Techniques
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????????????????????? A
??? A ??? B
??? C ??? D
1
2
3
1
2
3
?????????????????????
Ablk
Aunk
Astd
Reagent DW Reagent Sample Reagent Standard
??????????????????? D ???????? Ablk ???? 0
??????????? Cstd
???????? ?? Sample ????????? A ???????????????????
? B ???????????????(?????????????) ????????
???????????????
3
?????????????????????????????????????????????????
serum/plasma
Serum ???? Plasma
centrifuge
Clotted blood ???? Packed blood
Whole blood

• Proteins
• Fats
• Carbohydrates
• Minerals

??? ??? ??????? cells
??????????? ??????????????
4
???????????????????????????????
Sample
????????? ????????
??????????? ???????????????
???????????????????
?????????
????????????????????????
???????????

• ?????????????
• ??????????
• ?????????????????

?????????
5
??????????? ??????????????
?????????(separation)???????????????????
????????????????? ??????????? ?????????????

• Filtration
• Dialysis
• Gel filtration
• chromatography

• Precipitation
• Extraction

• Electrophoresis
• Ion exchange
• chromatography

• Drying (Evaporation) and
• Lyophilization

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??????????????? Separation Techniques
?????????? (Precipitation) ???????
(Extraction) ??????? (Filtration) ????????????
(Evaporation) ????????? (Dialysis) ????????????
(Chromatography) ????????????????
(Electrophoresis)
?????????? ?.?.???????? ????/????????????????????
??????????????
?????????????????? ???????????????????????? ????
211-252
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Protein precipitation (????????????????)
???????????????????
Amino acids
Peptide bond
side chain ????????????
Peptide chain (Protein)
????????????????????????? (amino acids)

• ????????????????????????? side chain
  ??????????????????????????????????????
  ????????????????????? pH ??? R
• ?????????????????? ??????????????

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??????????????????????????????? pH ??????????? R
High pH
Low pH
Zwitter ion
Isoelectric point (pI) pH ??????????????????????
??? ????? ? pH pI ???????????????????
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?????????????????????? side chain
????????????????
????????? (??? H) ????? pH ???? acid
Histidine
Arginine
Lysine
????????? (??? H) ????? pH ???? base
Aspartate
Glutamate
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Heme group
Primary structure
Secondary structure
Tertiary structure
Quaternary structure
http//www.rpi.edu/dept/chem-eng/Biotech-Environ/P
RECIP/precpintro.html
???????????????????????????????????????????
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????/?????????????????????????(???????????)
http//www.rpi.edu/dept/chem-eng/Biotech-Environ/P
RECIP/precpintro.html

• ????????????????????????????????????????????????
• ?????????????????????? (???????) ????
• ???????? (????? H-bond)

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????/?????????????????????????(???????????)

• Isoeletric Precipitation

changing the pH of the protein solution

• Use of an Organic Solvent for Precipitation

???(H2O) ????????? hydrophilic surfaces
????????? Organic solvent ????????? hydrophobic
surfaces ?????????
Organic solvent ????? dielectric constant of the
medium ????
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S is the solubility of the protein in the
organic solvent Sw is the solubility in water D
is the dielectric constant of the medium when
organic solvent is added Dw is the dielectric
constant of the water R is the gas constant T
is the absolute temperature A is a constant
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????/?????????????????????????(???????????)

• Salting out precipitation

?????????????????????????????
???????????????????????? ???? (NH4)2SO4 ??????????
???????? salting out
????????????????????????????????????????? ????????
?? ?????????????????? salting in (????????????????
???????????????????????????????? ?????)
??????????????????????????????????????????
citrate gt phosphate gt sulphate gt acetate

chloride gt nitrate gt thiocyanate
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????/?????????????????????????(???????????)

• Addition of a non-ionic polymer or metal ions

dextrans and polyethylene glycols reduce the
amount of water available to interact with the
protein
Mn2, Fe2, Co2, Ni2, Zn2, and Cd2 will bind
strongly to carboxylic acids and to nitrogenous
compounds
Ca2, Ba2, Mg2, and Pb2 will mainly bind to
carboxylic acids
Ag, Hg2, and Pb2 will bind strongly to
sulhydryl groups
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Cation precipitation
Somogyi method ??? Ba(OH)2 ZnSO4
????????????? ?????? base ?????????????????
Ba(OH)2 ?? Ba2 2 OH-
ZnSO4 ?? Zn2 SO42-
???????????????????????
Ba2 SO42- ?? Ba SO4
????????????????
Ba(OH)2 ??? ZnSO4 ?????????? mole ???????
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Anion precipitation
(Folin-Wu Method ) ??? Tungstic acid
H2SO4 Na2WO4 ?? H2WO4 Na2SO4
2H WO42- 2Na SO42-
????????????? ??????? acidic ????????????????
????????????????????????
??????? ??????????????
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??????? (extraction)
??????? like dissolve like ??????????????????????
?????????????????????? ??? ???????????????????????
???????????????????????????

• ??????????????????????????????????????????????????
  ?
• ????????????????
• ???????????????????????????????? ????
• ??????????????????????????????????????
• ??????????????????????????????

???????????????????????????????? ?????? ethanol,
ether, benzene, toluene, chloroform
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?????????????????????????????????
???? ???????????????????????
??????????????????????????
Separating funnel

• ????????????????????????
• ??????? micro emulsion
• ?????????????????????????????????????
  ?????????????
• ??????????????????
• ???????????????????????????????
• ??????????????????????????????????????????????????

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Soxhlet extraction
??????????????????????????????????????????????????
??
http//upload.wikimedia.org/wikipedia/commons/4/41
/Soxhlet_Extractor.jpg
http//web2.slc.qc.ca/jmc/www/Chemweb/oldchemweb/i
mages5Csoxhlet.jpg
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??????? (filtration)
??????????????????????? ??????????????????? ??????
?(????)????(filter) ???? ?????? ??????
???????????? (membrane)
???????????????????? ?????? ???????? (funnel)
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??????????????????????
http//chemsci.kku.ac.th/crystal/images/pic4.gif
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Evaporation (????????) ???????????????????????????
?????????? ????????????? ??????????????????
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Lyophilizer or freezed dryer
?????????????????????????????????????
???? ????????????(??????)?????????????????????????
?? ??????????????????????????
Freeze dryer flask
Freeze sample
Refrigerator -70C
High vacuum pump
???(????)???????????(???????)??????????
refrigerator ?????????????????????????
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Dialysis
????????????????????????????
Dialysis membrane
Buffer
Concentrated solution
At start of dialysis
At equilibrium
??????? dialysis ????????????? dialysis
????????????????
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?????????????????????????????????????
???????????????????????
?????????? Folin-Wu Method Folin-Wu Method Somogyi Method Somogyi Method ????????????? ?????????????
?????????? ???? 1 ???? 2 ???? 3,5 ???? 4,6 ???? 7 ???? 8
???????? 4.0 ml 3.5 ml 4.0 ml 3.5 ml - -
????? / ???????? - 0.5 ml - 0.5 ml - -
10 sodium tungstate 0.5 ml 0.5 ml - - - -
2/3 N H2SO4 0.5 ml 0.5 ml - - - -
0.17 M Ba(OH)2 - - 0.5 ml 0.5 ml - -
5 ZnSO4 - - 0.5 ml 0.5 ml - -
????????????????????????????????????????????????? 10 ???? ??????????????? 3,000 rpm ??? 10 ???? ?????????????????????????????????????????? ?????????????????????????????????????????????????????? Lowrys method ???????????????????? ???? Vortex mixer ??????????????????????? ??????? 5 ??????????????? 3 ?????????? 6 ??????????????? 4 ?????????? ZnSO4 ???? Ba(OH)2 ????????????????????????????????????????????????? 10 ???? ??????????????? 3,000 rpm ??? 10 ???? ?????????????????????????????????????????? ?????????????????????????????????????????????????????? Lowrys method ???????????????????? ???? Vortex mixer ??????????????????????? ??????? 5 ??????????????? 3 ?????????? 6 ??????????????? 4 ?????????? ZnSO4 ???? Ba(OH)2 ????????????????????????????????????????????????? 10 ???? ??????????????? 3,000 rpm ??? 10 ???? ?????????????????????????????????????????? ?????????????????????????????????????????????????????? Lowrys method ???????????????????? ???? Vortex mixer ??????????????????????? ??????? 5 ??????????????? 3 ?????????? 6 ??????????????? 4 ?????????? ZnSO4 ???? Ba(OH)2 ????????????????????????????????????????????????? 10 ???? ??????????????? 3,000 rpm ??? 10 ???? ?????????????????????????????????????????? ?????????????????????????????????????????????????????? Lowrys method ???????????????????? ???? Vortex mixer ??????????????????????? ??????? 5 ??????????????? 3 ?????????? 6 ??????????????? 4 ?????????? ZnSO4 ???? Ba(OH)2 ????????????????????????????????????????????????? 10 ???? ??????????????? 3,000 rpm ??? 10 ???? ?????????????????????????????????????????? ?????????????????????????????????????????????????????? Lowrys method ???????????????????? ???? Vortex mixer ??????????????????????? ??????? 5 ??????????????? 3 ?????????? 6 ??????????????? 4 ?????????? ZnSO4 ???? Ba(OH)2 ????????????????????????????????????????????????? 10 ???? ??????????????? 3,000 rpm ??? 10 ???? ?????????????????????????????????????????? ?????????????????????????????????????????????????????? Lowrys method ???????????????????? ???? Vortex mixer ??????????????????????? ??????? 5 ??????????????? 3 ?????????? 6 ??????????????? 4 ?????????? ZnSO4 ???? Ba(OH)2 ????????????????????????????????????????????????? 10 ???? ??????????????? 3,000 rpm ??? 10 ???? ?????????????????????????????????????????? ?????????????????????????????????????????????????????? Lowrys method ???????????????????? ???? Vortex mixer ??????????????????????? ??????? 5 ??????????????? 3 ?????????? 6 ??????????????? 4 ?????????? ZnSO4 ???? Ba(OH)2
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?????????????????????????
?????????? Folin-Wu Method Folin-Wu Method Somogyi Method Somogyi Method ????????????? ?????????????
?????????? ???? 1 ???? 2 ???? 3,5 ???? 4,6 ???? 7 ???? 8
???????? - - - - 0.1 ml -
?????????????????????????? 0.1 ml 0.1 ml 0.1 ml 0.1 ml - -
???????????????? 40 mg/dl - - - - - 0.1 ml
Lowrys reagent 3.0 ml 3.0 ml 3.0 ml 3.0 ml 3.0 ml 3.0 ml
????????????????????????????????????????????????? 10 ???? ????????????????????????????????????????????????? 10 ???? ????????????????????????????????????????????????? 10 ???? ????????????????????????????????????????????????? 10 ???? ????????????????????????????????????????????????? 10 ???? ????????????????????????????????????????????????? 10 ???? ????????????????????????????????????????????????? 10 ????
1.0 M Folin-Ciocalteau Phenol reagent 0.3 ml 0.3 ml 0.3 ml 0.3 ml 0.3 ml 0.3 ml
????????????????????????????????????????????????? 30 ???? ??????????????????????????????????????? 650 ???????? ?????????????????? 0 ????????????????????????????????????????????????? 30 ???? ??????????????????????????????????????? 650 ???????? ?????????????????? 0 ????????????????????????????????????????????????? 30 ???? ??????????????????????????????????????? 650 ???????? ?????????????????? 0 ????????????????????????????????????????????????? 30 ???? ??????????????????????????????????????? 650 ???????? ?????????????????? 0 ????????????????????????????????????????????????? 30 ???? ??????????????????????????????????????? 650 ???????? ?????????????????? 0 ????????????????????????????????????????????????? 30 ???? ??????????????????????????????????????? 650 ???????? ?????????????????? 0 ????????????????????????????????????????????????? 30 ???? ??????????????????????????????????????? 650 ???????? ?????????????????? 0
???????????????????????????? 650 nm A1 A2 A3,A5 A4,A6 A7 A8
??????????????????????????????????? ???? Folin-Wu method (A2-A1) X 40 mg/dl
??????????????????????????????????? ???? Folin-Wu method (A8-A7) X 40 mg/dl
??????????????????????????????????? ???? Somogyi Method (A4-A3) X 40 mg/dl
??????????????????????????????????? ???? Somogyi Method (A8-A7) X 40 mg/dl
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???????????????? Protein ???????? Lowry Method
??????????????????????????????????????????? ?????
???????????? Cu2 ??????????????? ??? Cu-protein
complex, copper tyrosine complex ??? tryptophane
complex ??????? phospho-tungstic acid ???
phosphomolybdic acid ??? tungsten blue ???
molybdenum blue ?????????????????????????????????
????????? 650 ????????
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???????????? (chromatography)
Mobile phase
Stationary phase
????????? ?????????????????? (mobile phase)
???????? ?????????????????????????????????????????
? (stationary phase)
Planar chromatography Column chromatography
?????????????????????? mobile phase
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Mobile phase Stationary phase ???????????????? ???????? phase ??????
Liquid Solid Liquid chromatography (LSC)
Gas Liquid Gas Liquid chromatography (GLC)
Liquid Liquid Liquid liquid chromatography (LLC)

• ??????????????????
• Adsorption chromatography
• Partition chromatography
• Ion exchange chromatography
• Gel filtration chromatography
• Affinity chromatography

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Adsorption chromatography
Solute1
Solute2
?????????????????????? mobile phase
Mobile phase
Stationary phase ?????????????
stationary phase ????????? solute1 ??? solute2
??????????
Stationary phase Use for separate
Alumina Small organic compounds and lipids
Silica gel Amino acid, lipids, carbohydrate
Fluorisil (magnesium silica) Medium size Lipids
Calcium phosphate (hydroxyapatite) Protein, polynucleotides
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Thin layer chromatography
a
c
b
Start point
Retardation factor (RF)
???????????????????? RF ???????
RF
RF
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Partition chromatography
???? paper chromatography
34
??????????
sample
35
Gel filtration chromatography or
Molecular Exclusion Chromatography
36
http//www.hmds.org.uk/figures/fitc_sep.jpg
http//genchem.chem.wisc.edu/demonstrations/Images
/23biochem/sizeexclusion.jpg
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???????????????????????? Column
chromatography ????????????????????????? ????????
?? (????????????) ????????????????
????????? ????????????????
Fraction collector
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????????????????????????????????????? column
????????????????
Blue dextran ?????? 2 ??????????
Dinitrophenyl lysine (DNP lysine) ?????? 30 ??????
Blue dextran ?????????????? ?????????? blue
dextran ????? ????? void volume (Vo)
DNP lysine ??????????????????? ?????????? DNP
lysine ????? ????? total volume (Vt)
?????????? ?????????????????? ????? elution
volume (Ve)
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Relative migration (K)
Log Mr
Relative migration
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Ion exchange chromatography
Matrix
Ion exchanger

• Anion-exchanger
• ?????????
• Cation-exchanger
• ??????????

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Affinity Chromatography
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Gas liquid chromatography
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Chromatogram
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??????????????????????
Electrophoresis

-
?????????????
???????? buffer
? ???????????????????????? Q ???????????????? K
???????? R ?????????????????? ? ??????????????
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?????????????????? electrophoresis

• Power supply ???????????????????????????????
• ??????????? ??????????????????
• Electrophoresis chamber ?????? buffer ???
• support media
• Support media ???? ??????, cellulose acetate
• starch gel, agarose gel, polyacylamide gel
• Buffer ????????? pH ??????????????? ??????????
• ??????????????????
• Staining dye ??????????????????????????????????
• ?????????? ???? Coomassie brilliant blue R-250
• ????????????

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Type of electrophoresis
???????????????????????????
Paper electrophoresis Cellulose
electrophoresis Starch gel electrophoresis Agarose
gel electrophoresis Polyacrylamide gel
electrophoresis
Polyacylamide gel ?????????? cross-link ???
acrylamide ??? methylene-bis acrylamide ?????
ammonium persulfate ???? ??????? ??? TEMED
?????????????????????????????????????????????????
cross-link
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http//www.funsci.com/fun3_en/exper1/exper1_21.gif
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(No Transcript)
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http//porpax.bio.miami.edu/cmallery/150/protein/
electrophoresis.jpg
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??? Desitometer scan ?????????????????????????????
??? protein
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Agarose gel electrophoresis
http//a32.lehman.cuny.edu/molbio_course/gelel.GIF
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Polyacrylamide gel electrophoresis
(Discontinuous electrophoresis)
http//web.siumed.edu/bbartholomew/images/chapter
6/F06-21.jpg
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http//classes.ansci.uiuc.edu/ansc438/Milkcompsynt
h/proteingelx.jpg
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Polyacrylamide gel electrophoresis Non-denaturin
g polyacrylamide gel electrophoresis ????????????
???????????????????????????????????? ????????????
? ????? ?????????????? Denaturing
polyacrylamide gel electrophoresis ??????????????
???????????????????????????????? ????????????????
??????????????????? ??? SDS (sodium dodecsyl
sulfate) ???????? SDS polyacrylamide gel
electrophoresis ????????????????????????
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Two dimensional electrophoresis
??????????????????????? 1
???????? support media ?? 90 ????
??? ??????????????????????? 2
??????????????????????????????????????????????