PHƯƠNG PHÁP điện DI ppt _ HÓA PHÂN TÍCH

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PHƯƠNG PHÁP ĐIỆN DI

(Electrophoresis)

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Lịch sử

1791 Faraday Laws of Electrolysis

1877 Helmholtz Charged Solvent Layer Closed to Surface of a Wall

1897 Nernst Properties of Small Ions

1897 Kohlrausch Kohlrausch Function describing the Order of Migration of Ions and their Concentration

1923 Kendall, Crittenden Rare Earth Metal Separation by "Ion Migraion Method"

1930 Tiselius Thesis: Moving Boundary Method for Electrophoresis of Proteins (Nobel Price 1948)

1939 Svenson Development of Zone and Displacement Electrophoresis

1950 Haglund, Tiselius Electrophoresis Tube filled with Glass Beads and Glass Powder

1955 Smithies Gel Electrophoresis

1958 Hjertén Electrophoresis in Free Solution

1967 Martin, Everaerts Displacement Electrophoresis in Glass Tube with Hydroxyethylcellulose

1967 Hjertén Elimination of Electroosmosis by Coating of Glass Tubes

1969 Giddings Non-Diffusional Model of Concentration Distribution in Free Zone Electrophoresis

1969 Virtanen Glass Capillaries 0.2 - 0.5 mm I.D

1970 Everaerts, Capillary Isotachophoresis

1970 Arlinger, Routs UV-Detection

1972 Verheggen Conductivity Detection

1979 Mikkers Use of High Voltage and TEFLON Capillaries

1981 Jorgenson Use of 75 µm I.D Open Tubular Glass Capillaries:

"High Performance Capillary Electrophoresis – HPCE"

1984 Terabe Combination of electrophoretic and chromatographic Separation:

"Micellar Electrokinetic Capillary Chromatography – MECC"

1991 Jandik, Jones Use of Surface Active Electrolyte Additives for Reversal of Electroosmotic Flow

1991 Knox "Capillary Electrochromatography – CEC"

Anion hữu cơ và vô cơ

2324

2526

27

28

29

Standard 16 phosphite 1 thiosulfate

2 bromide 17 chlorite

3 chloride 18 galacturate

4 sulfate 19 carnonate

5 nitrite 6 nitrate

20 acetate 21 ethanesulfonate

7 molybdate 22 propionate

8 azide 9 tungstate 23 propanesulfonate 24 butyrate

10.fluorophosphate 25 butyrsulfonate 11 chlorate 12 citrate

13 fluoride 14 formate 15 phospahate

26 valerate 27 benzoate 28 glutamate 29 pentanesulfonate 30 D-gluconate

Định nghĩa

 Quá trình tách các tiểu phân đã ion hóa và hòa tan hay phân tán trong dung dịch điện giải dưới tác dụng của điện trường

 Độ dịch chuyển điện di (linh độ điện di: electrophoretic mobility, μEP) phụ thuộc:

 Bản chất tiểu phân: hình dạng, kích thước, điện tích

 Dung dịch điện giải: bản chất, nồng độ, pH, độ nhớt,….

Định nghĩa

 ep(C)

µ ep (A)

A B

Moving Boundary

Electrophoresis

 μEP phụ thuộc vào E, bản chất tiểu phân, dòng bay hơi (nhiệt

Joule), dung dịch điện giải

 Tách các chất có nhỏ PLT và kích thước nhỏ, lượng mẫu ít

Điện di trên gel

Điện di mao quản (Capillary Electrophoresis)

0-±30 kV 5-150 µ A

High Voltage Power Supply

Capillary

o.d 200-400µm i.d 5-100µm (2µm)

Fused Silica, Teflon coated (RP, Ion exchange)

or filled (RP, )

Detector

UV, Fluorescence (direct, indirect);

electrochemical conductometricMS

Điện di mao quản

Liquid cooling 10m/s air

cooling w/o cooling

Dòng điện thẩm (Electroosmotic Flow)

Origin of Electroosmotic Flow:

a) Formation of negatively charged silica-surface

b) Hydrated cations at surface

c) Bulk flow of whole capillary contents towards cathode after application of electric field

A ∑i i

i

.c 1000.N e z

oriented water molecules

outer Helmholtz-Layer (diffuse) inner Helmholtz-Layer (adsorbed, rigid)

O

Si O

O O

Si O

O Si

O

O

primary H O

H H

O O O Si O

 = Potential of bulk solution [V]

µeof = Mobility of EOF [cm V sec ] 2 -1 -1

 = Dielectricity constant of electrolyte

 = Viscosity of electrolyte

Electroosmotic mobility

Linh độ điện di (Electrophoretic Mobility)

A-: Anions

Syringaldehyde2-(p-Hydroxyphenyl)acetic acid Benzoic acidVanillic acid

4-Hydroxybenzoic acid

Overlay of Migration of Charged Ions and Molecules with EOF

a) Cations to Cathode (Detection before EOF).

b) Neutral Moleculese (Detection together EOF).

c) Anions to Anode (Detection for |µAnion| < |µeof| after EOF; no Detection for |µAnion| > |µeof |)

C+: Cations

Linh độ điện di (Electrophoretic Mobility)

Dissociation of Weak Electrolytes

Nguyên tắc của điện di mao quản vùng

(Capillary Zone Electrophoresis, CZE)

µ obs(A ) = µ EOF + µ EP(A )

µ obs(C ) = µ EOF - µ EP(C )

electroosmotic flow

Separation principle of MEKC

-Thông số thực nghiệm trong CE

Analyte

geometry molecular weight, structure

pKA

ionic strength

effective charge

1 mannuronic acid (n.a.) 2 glucuronic acid (3.20)

3 galacturonic acid (3.48)4 gluconic acid (3.76)5 N-acetylneuraminic acid (2.60)6 fructose (12.03)

7 rhamnose (n.a.)8 glucose (12.35)9 galactose (12.35)10 2-deoxy-D-ribose (12.65)11 sucrose (12.51)

A Zemann, D.T Nguyen,

G Bonn

Electrophoresis 18 (1997) 1142

12

4

EOF

6 75

89

3

1011

Conditions:

Capillary: uncoated fused silica, i.d 50µM; L=32cm, l=24.5cm; Electrolyte: 6mM sorbate, 0.001% HDB, pH12.1; Injection: 3sec hydrostatic (10cm); Detection: indirect UV @ 254nm; Instrumentation: WATERS Quanta 4000; U=-10kV, I=29,2µA, T=amb

A Zemann, D.T Nguyen,

G Bonn

Electrophoresis 18 (1997) 1142

Conditions:

Capillary: 20 µm I.D.; 375 µm O.D.; l=60 cm; U=30 kV;

I=1.3 µA

counter-electroosmotic CE

3 4

5

6

7 8

1 formaldehyde2 acetaldehyde3 benzaldehyde4 crotonaldehyde

5 m-tolualdehyde

6 acetaldehyde7 propioaldehyde8 butyraldehyde9 valeraldehyde10 hexaldehyde11 acetone12 butanone

Anion hữu cơ và vô cơ

2324

2526

27

28

29

Standard 16 phosphite 1 thiosulfate

2 bromide 17 chlorite

3 chloride 18 galacturate

4 sulfate 19 carnonate

5 nitrite 6 nitrate

20 acetate 21 ethanesulfonate

7 molybdate 22 propionate

8 azide 9 tungstate 23 propanesulfonate 24 butyrate

10.fluorophosphate 25 butyrsulfonate 11 chlorate 12 citrate

13 fluoride 14 formate 15 phospahate

26 valerate 27 benzoate 28 glutamate 29 pentanesulfonate 30 D-gluconate

Detection, UV @ 195 ± 5 nm (bubble cell 200 µm).

AMP amprenavir; RTV ritonavir; SQV saquinavir; NFV nelfinavir; IDV indinavir

3TC DDC

IDVEOF

protease and reverse transcriptase inhibitors

Group 1 : Chemical structure of CDs

Lisinopril (LI)

NCOOH

H N

O

HOOC

N H

S NH

O Cl

Group 1 : ACE inhibitors and diuretics

• Optimized electrophoretic conditions

Electrophoretic conditions: 60 mM orate buffer at pH 8.6; fused-silica capillary (57 cm x 50 µ m i.d., 48.5 cm); injection: 5s at 50 mbar; 18 kV; 25 o C; detection wavelength: 214 nm

Separation principle of MEKC

e t +

Hệ đệm: dung dịch dinatri tetraborat 25 mM pH 9,3; 50 mM SDC Cột mao quản: silica nung chảy 72/80,5 cm x 50 µm

Nhiệt độ cột: 25 oC, điện thế: 30 kV

Lượng mẫu tiêm: 50 mbar x 3 s; Bước sóng phát hiện: 210 nm

Application – Multi-components

Conditions: Background electrolyte 10 mM borate, 10 mM phosphate, pH 9.2, 5% ACN, 50 mM SDS Capillary 39.5/48 cm,

50 µm I.D Temperature 25oC Detection 210 nm Applied voltage 20 kV Injection 50 mbar x 20 sec

Group 2 : Chemical structure of CDs

Metoprolol (METO)

Propranolol (PRO)

HH3C N CH3

O

CH3

OH3C

Cl

Amlodipine (AM)

Nifedipine (NI)

Group 2 : β -blockers and Ca channel antagonists

• Optimized electrophoretic conditions

Electrophoretic conditions: 10% methanol in 100 mM tris buffer at pH 12.0 containing 100 mM SDC; fused-silica capillary (57 cm x 50 µ m i.d.,

48.5 cm); injection: 5s at 50 mbar; 25 kV; 25oC; detection wavelength: 225 nm

nm

240 260 280 300 320 340

mAU

0 2.5 5 7.5 10 12.5

15 17.5

20

(2)

ATE METO

NI PRO AM

Group 3 : Chemical structure of CDs

Atorvastatin (ATOR) O

O

OHH

CH3

H C3CH3

H3C

CH3

Group 3 : Statin derivatives

• Optimized electrophoretic conditions

Electrophoretic conditions: 15% methanol in 15 mM borate buffer at pH 8.0 containing 50 mM SDC; fused-silica capillary (57 cm x 50 µ m i.d.,

48.5 cm); injection: 5s at 50 mbar; 30 kV; 30oC; detection wavelength:

SIM

Application – Natural products

Flavonoid (rutin, isoquercitrin, quercitrin,

kaempferol, quercetin, etc)

50 mM SDS P.G Pietta, et al; J Chromatogr (549)

Application – Optical purity testing of drugs

• Use area percentage method for purity testing

of drugs as in HPLC

• Normalize peak areas with migration times

• Identify impurities above apparent levels of 0.1%

Application – Dexchlorpheniramine maleate

Optical purity testing of dexchlorpheniramine maleate by CE with β -CD

Conditions: Capillary: 76.5 cm (68 cm effective length) x 50 µ m I.D.; Background electrolyte: 0.05 M Tris buffer pH 3.5 + 5 mM β -CD; Detection: 214 nm; Applied voltage: 20 kV; Injection: 50 mbar x 10 sec.; Temperature: 25oC.

1 Pseudoephedrine HCl (IS)

2 Levochlorpheniramine maleate

3 Dexchlorpheniramine maleate

Chemical structure of drug substances

* 2

N CH3

O 1O

N1

O

NH2

COOCH 2CH3 Cl

CH3

CH3OOC 4

3

4 5 6

N

1 O N N

OH F

O O

2

* 3

4 5

6 7 8

10 9

Promethazine

S N

N 2

* CH3 CH3

CH3

1

10

Effect of the CD types and their concentrations on Rs

Electrophoretic conditions: 50 – 100 mM tris-phosphate buffer pH 2.5

– 3.0, 20% methanol (for propranolol) or 25% acetonitrile (for nefopam); capillary (63.5 cm x 50 µ m i.d., 54 cm); λ = λ max of each

Rs

compound; 25 o C; 20 kV; injection: 5s at 50 mbar.

Electropherograms for the chiral separation of enantiomers

0123456

Electropherograms for the chiral separation of enantiomers

Electropherograms for the chiral separation of enantiomers

Nefopam

30 mM

β -CD

30 mM HP- β CD

-20 mM HB- β -CD

Optimized electrophoretic 2.5, 25% MeCN;

capillary 25 o C; 20 kV; injection: 5s

conditions: 50 mM tris-phosphate buffer pH (63.5 cm x 50 µ m i.d., 54 cm);

λ = 275 nm,

at 50 mbar.

Electropherograms for the chiral separation of enantiomers

Optimized elect rophoretic

2468

Ofloxacin

Optimized electrophoretic conditions:

50 mM tris-phosphate buffer pH

5 10 15 20 25

min.

02468

10 mAU 30

mM HP- β - CD

-02468

Electropherograms for the chiral separation of enantiomers

Optimized ele ctrophoretic

Tris-phosphate; 63.5 cm x 50 µ m i.d., 54 cm; 25 o C; 20 kV; injection:

mAU

20 mM HB- β -CD

8

610

min.

02468

mAU

30 mM HB- β -CD

5s at 50 mbar KET : 50 mM BGE pH

S-amlodipine drug substance

CZE and MEKC can be used for drug analysis as a complementary or alternative method to HPLC

Advantage

relatively short time

• MEKC is especially powerful for the separation of complex mixtures because of its high resolution

• Direct enantiomer separation also can be successful

using chiral selectors

Disadvantage

• For much wider use it is still desirable for the precision in quantitative analysis

to be improved to be comparable to those in HPLC