Phương pháp điện di

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 descri

Phương Pháp Điện Di (Electrophoresis)

PGS.TS Nguyễn Đức Tuấn Bộ môn Phân Tích – Kiểm Nghiệm Khoa Dược – Đại học Y Dược TPHCM

Phương Pháp Điện Di

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

5 nitrite 20 acetate 6 nitrate 21 ethanesulfonate 7 molybdate 22 propionate

8 azide 23 propanesulfonate 9 tungstate 24 butyrate

10.fluorophosphate 25 butyrsulfonate 11 chlorate 26 valerate

12 citrate 27 benzoate 13 fluoride 28 glutamate 14 formate 29 pentanesulfonate 15 phospahate 30 D-gluconate

W.R Jones, P Jandik

J Chromatogr 546 (1991), 445

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,  EP ) phụ thuộc:

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

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

Phân loại

 Điện di dung dịch tự do (Moving

Boundary Electrophoresis)

  EP phụ thuộc vào điện trường E,

bản chất tiểu phân

Phân loại

 Điện di vệt (điện di vùng:

Zone Electrophoresis)

giấy, cellulose acetat, gel

agar, gel polyacrylamid

  EP phụ thuộc vào E, bản

chất tiểu phân, dòng bay

hơi (nhiệt Joule), dung

dịch điện giải

nhỏ và kích thước nhỏ,

lượng mẫu ít

Điện di trên gel

Điện di mao quản (Capillary Electrophoresis)

High Voltage Power Supply

0-±30 kV 5-150  A

(0-100%)

Pt Electrodes

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 conductometric

MS

Điện di mao quản

50µm 12µm

363µm

Fused Silica Capillary

Hydrodynamic flow profile and chromatographic peak form a) pressure driven

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





i

i i

N

T k

.

1000

.

2 2

Y2= Potential of bulk solution [V]

µeof = Mobility of EOF [cm2V-1sec-1]

 = Dielectricity constant of electrolyte

 = Viscosity of electrolyte

Electroosmotic mobility

Linh độ điện di (Electrophoretic Mobility)

) (

) (

F

E ep

F Force frictional

F Force electric

4-Aminopyridine

N: Neutral Molecules

Benzylalcohol Phenol

A - : Anions

Syringaldehyde 2-(p-Hydroxyphenyl)acetic acid Benzoic acid

Vanillic 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 |)

Linh độ điện di (Electrophoretic Mobility)

Dissociation of Weak Electrolytes

Nguyên tắc của điện di mao quản vùng

(Capillary Zone Electrophoresis, CZE)

electroosmotic flow

Separation principle of MEKC

Thông số thực nghiệm trong CE

molecular weight, structure

pKA

ionic strength

effective charge

capillary wall

capillary length high voltage V

field strength E=V/L

solvation

Electrolyte

Instrument

Carbohydrat

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)

hydrostatic (10cm); Detection: indirect UV @ 254nm; Instrumentation: WATERS Quanta 4000; U=-10kV, I=29,2A, T=amb

analysis of soft drinks

1 fructose 2

hydrostatic (10cm); Detection: indirect UV@254nm; Instrumentation: WATERS Quanta 4000; U=-22kV, I=34,4A, T=amb

A Zemann, D.T Nguyen,

G Bonn

Electrophoresis 18 (1997) 1142

counter-electroosmotic CE

Hợp chất carbonyl

1 formaldehyde 2 acetaldehyde

3 benzaldehyde 4 crotonaldehyde

5 m-tolualdehyde

6 acetaldehyde 7 propioaldehyde 8 butyraldehyde 9 valeraldehyde 10 hexaldehyde 11 acetone 12 butanone

Anion hữu cơ và vô cơ

5 nitrite 20 acetate 6 nitrate 21 ethanesulfonate 7 molybdate 22 propionate

8 azide 23 propanesulfonate 9 tungstate 24 butyrate

10.fluorophosphate 25 butyrsulfonate 11 chlorate 26 valerate

12 citrate 27 benzoate 13 fluoride 28 glutamate 14 formate 29 pentanesulfonate 15 phospahate 30 D-gluconate

W.R Jones, P Jandik

J Chromatogr 546 (1991), 445

Các thuốc kháng HIV

Capillary, L=48.5 cm, leff=40 cm, 50 µm; electrolyte, 16 mM phosphate, 0.001% HDB, pH 2.5

Injection, 20 sec @ 10 mbar; standard

concentration, 5 ppm;

Separation, -30 kV Detection, UV @ 195 ± 5 nm (bubble

IDV EOF

mAU (195nm)

Nguyen D.T., A Zemann

J Chromatogr A, 922 (2001) 313 – 320

protease inhibitors

Các thuốc kháng HIV

DLV ABC NVP

3TC DDC

IDV EOF

12

16

20

Capillary, L=42.5 cm, leff=34 cm, i.d.=50 µm

Electrolyte, 16 mM H3PO4, 0.001% HDB, pH 2.2

Injection, 20 sec @ 10 mbar;

standard concentration, 5 ppm;

Separation, U=-30 kV Detection, UV (bubble cell 200

µm)

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

NVP nevirapine; DLV delavirdine; ABC abacavir; 3TC lamivudine; DDC zalcitabine

D.T Nguyen, A Zemann Journal of Chromatography A,

982 (2002) 153 – 161

protease and reverse transcriptase inhibitors

Group 1 : Chemical structure of CDs

• ACE inhibitors

Captopril (CAP)

Enalapril (ENA)

Lisinopril (LI)

• Diuretics

Hydrochlorothiazide

(HCT)

Furosemide (FURO)

N HS

COOH

CH3

O

H N

N

CH3O

O

H3C

H N

N COOH

H2N

O HOOC

N H

S NH

Group 1 : ACE inhibitors and diuretics

• Optimized electrophoretic conditions

Electrophoretic conditions: 60 mM orate buffer at pH 8.6;

nm

240 260 280 300 320 340

mAU

0 2 4 6 8 10 12

HCT

LI ENA FURO CAP

Separation principle of MEKC

MECC – Các NSAID

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

Group 2 : Chemical structure of CDs

Metoprolol (METO)

Propranolol (PRO)

• Calcium channel antagonists

H

O CH3O

H3C

O

H3C

Cl O

Amlodipine (AM)

Nifedipine (NI)

Group 2 :  -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  m i.d., 48.5 cm); injection: 5s at 50 mbar; 25 kV; 25oC; detection wavelength:

15 17.5

20

(2)

ATE METO

NI PRO

AM

Group 3 : Chemical structure of CDs

• Statin derivatives

Lovastatin

(LOV)

Simvastatin (SIM)

Atorvastatin (ATOR)

OOHOH

2+ , 3H2O

2

NH

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  m i.d., 48.5 cm); injection: 5s at 50 mbar; 30 kV; 30oC; detection wavelength:

10 ATOR

LOV SIM

Application – Natural products

mM Tween 20 I Bjornsdottir, et al; J Pharm Biomed Anal

(13) 687 Amphetamines and related

substances 25 mM CTAB + 11% DMSO + 1% ethanol V.C Trenerry, et al; J Chromatogr A (708)

169 Cocaine and related

substances 50 mM CTAB + 7.5% ACN V.C Trenerry, et al; Electrophoresis (15)

103

Chromatogr (645) 366

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

Background electrolyte: 0.05 M Tris buffer pH 3.5 + 5 mM  -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

* OH

Propranolol

*

Brompheniramine Ketoconazole

N N

Cl Cl

* *

1 2 3 4 5

4'

N

O NH2COOCH2CH3Cl

CH3

CH3OOC

1

3 4 5 6

2

3 4

N O

N N

CH3 CH3

OH F

O O

1 2

*

3

4 5

6 7 8

10 9

Promethazine

S N

Effect of the CD types and their concentrations on R s

Electrophoretic conditions: 50 – 100 mM tris-phosphate buffer pH 2.5 – 3.0, 20% methanol (for propranolol) or 25% acetonitrile (for

R s

Electropherograms for the chiral separation of enantiomers

Optimized electrophoretic conditions: 50 mM tris-phosphate buffer pH

Electropherograms for the chiral separation of enantiomers

Optimized electrophoretic conditions: 50 mM tris-phosphate buffer pH

Electropherograms for the chiral separation of enantiomers

Optimized electrophoretic conditions: 50 mM tris-phosphate buffer pH

Electropherograms for the chiral separation of enantiomers

Electropherograms for the chiral separation of enantiomers

S-amlodipine tablet

Conclusion

CZE and MEKC can be used for

drug analysis as a complementary

or alternative method to HPLC

Conclusion

Advantage

• One-run separation of every kind of drug, including cationic, neutral and anionic is possible within a 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