1. CAPILLARY ELECTROPHORESIS:
TECHNIQUE AND APPLICATION
By Mojdeh Yousefian
Ph.D Student of Medicinal Chemistry
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2. Electrophoresis
A method used in clinical and research laboratories for
separating molecules according to their size and electrical
charge. An electric current is passed through a medium that
contains the mixture of molecules. This is due to differential
rate of movement by molecules based on their weight.
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‫مشکالت‬
1-‫در‬ ‫منفی‬ ‫و‬ ‫مثبت‬ ‫بارهای‬ ،‫انتشار‬ ‫اثر‬ ‫در‬ ‫مجددا‬ ،‫کنیم‬ ‫قطع‬ ‫را‬ ‫ولتاژ‬ ‫اگر‬
‫گردند‬ ‫می‬ ‫بر‬ ‫اول‬ ‫حالت‬ ‫به‬ ‫و‬ ‫شوند‬ ‫می‬ ‫پخش‬ ‫سیستم‬ ‫سطح‬.
2-‫می‬ ‫ایجاد‬ ‫حرارت‬ ،‫گردد‬ ‫می‬ ‫اعمال‬ ‫ولتاژ‬ ‫وقتی‬ ،‫مائی‬ ‫محلول‬ ‫یک‬ ‫در‬
‫شود‬.

5. 5
•‫بع‬ ‫سه‬ ‫مانند‬ ‫شبکه‬ ‫سیستم‬ ‫یک‬ ‫ژل‬ ‫چون‬ ‫شد‬ ‫استفاده‬ ‫مانند‬ ‫ژل‬ ‫محیط‬ ‫یک‬ ‫از‬‫است‬ ‫دی‬
‫دهد‬ ‫نمی‬ ‫فرم‬ ‫تغییر‬ ‫راحتی‬ ‫به‬ ‫و‬.‫ت‬ ‫در‬ ‫باالیی‬ ‫تماس‬ ‫سطح‬ ‫با‬ ‫محلول‬ ‫طرفی‬ ‫از‬‫ماس‬
‫شود‬ ‫می‬ ‫دفع‬ ‫بهتر‬ ‫حرارت‬ ‫و‬ ‫است‬.
‫مشکل‬ ‫رفع‬

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‫معایب‬
‫که‬ ‫است‬ ‫این‬ ‫روش‬ ‫این‬ ‫مشکل‬‫گیر‬ ‫وقت‬ ‫و‬ ‫دستی‬‫است‬.‫دلیل‬ ‫به‬:
‫آن‬ ‫کردن‬ ‫خارج‬ ‫و‬ ‫ژل‬ ‫تهیه‬
‫چاهک‬ ‫در‬ ‫نمونه‬ ‫دادن‬ ‫قرار‬ ‫و‬ ‫چاهک‬ ‫ایجاد‬
•‫خ‬ ‫از‬ ‫بعد‬ ،‫نیست‬ ‫امکانپذیر‬ ‫کار‬ ‫حین‬ ‫در‬ ‫هم‬ ‫آشکارسازی‬ ‫طرفی‬ ‫از‬‫ارج‬
‫گیرد‬ ‫می‬ ‫صورت‬ ‫ژل‬ ‫کردن‬.

7. Capilary Electrophoresis
Technique first described by Jorgensen and
Lukacs (1980’s).
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•‫ک‬ ‫را‬ ‫هایی‬ ‫جداسازی‬ ‫است‬ ‫قادر‬ ‫که‬ ‫است‬ ‫الکتروفورز‬ ‫دستگاههای‬ ‫از‬ ‫یکی‬ ‫موئینه‬ ‫الکتروفورز‬‫فقط‬ ‫ه‬
‫توسط‬HPLC‫دهد‬ ‫انجام‬ ‫باال‬ ‫تفکیک‬ ‫و‬ ‫بازدهی‬ ‫با‬ ‫و‬ ‫سرعت‬ ‫به‬ ‫را‬ ‫هستند‬ ‫انجام‬ ‫قابل‬ ‫سختی‬ ‫با‬ ‫و‬.
•‫سیستم‬ ‫از‬ ‫جداسازی‬ ‫بخش‬ ‫عنوان‬ ‫به‬ ‫تنهایی‬ ‫به‬ ‫هم‬ ‫دستگاه‬ ‫این‬CE/MASS‫کنار‬ ‫در‬ ‫هم‬ ‫و‬ ‫شود‬ ‫استفاده‬
HPLC‫کند‬ ‫عمل‬ ‫مکمل‬ ‫جداسازی‬ ‫سیستم‬ ‫یک‬ ‫عنوان‬ ‫به‬.

9. • CE is electrophoresis performed in a capillary tube.
• It is the most efficient separation technique available for the
analysis of both large and small molecules.
• These separations are facilitated by the use of high voltages, which
may generate electroosmotic and electrophoretic flow of buffer
solutions and ionic species, respectively, within the capillary.

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•‫جنس‬‫لوله‬‫از‬‫سیلیکا‬‫است‬.‫سیلیکا‬‫اسیدی‬‫است‬.‫در‬PH‫اسیدی‬‫پروتونه‬‫است‬‫ولی‬‫در‬PH
،‫قلیایی‬‫دپروتونه‬‫شده‬‫و‬‫دارای‬‫بار‬‫منفی‬‫خواهد‬‫شد‬.
•‫دیواره‬‫بار‬‫منفی‬‫دارد‬‫چون‬‫داخل‬‫آن‬‫گروههای‬‫سیالنول‬‫هستند‬.
•،‫سیلیکا‬‫هدایت‬‫حرارتی‬‫باال‬‫دارد‬.
•‫برای‬،‫کارخانه‬‫تولید‬‫لوله‬‫با‬‫قطر‬‫میکرومتر‬‫توسط‬‫سیلیکا‬‫راحتتر‬‫است‬.
•‫سیالنول‬‫هایی‬‫که‬‫به‬‫دیواره‬‫درونی‬‫لوله‬‫متصل‬‫شده‬‫اند‬‫باعث‬‫کاهش‬EOF‫می‬‫شوند‬‫و‬‫از‬
‫جذب‬‫آنالیت‬‫جلوگیری‬‫می‬‫کنند‬‫و‬‫همچنین‬‫باعث‬‫ثابت‬‫ماندن‬PH‫می‬‫شود‬.

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12. ‫اگر‬‫در‬‫نمونه‬‫بار‬‫منفی‬‫داشته‬،‫باشیم‬EOF‫را‬‫می‬‫توان‬‫برعکس‬‫کرد‬.‫با‬‫پوشش‬‫دادن‬‫سیلیکا‬
‫توسط‬‫کاتیونیک‬‫دترجنت‬‫ها‬‫مثل‬‫تترادسیل‬‫تری‬‫متیل‬‫آمونیوم‬،‫یک‬‫سطح‬‫باردار‬‫مث‬‫بت‬‫خوب‬
‫ایجاد‬‫می‬‫شود‬‫چون‬‫تشکیل‬‫دو‬‫الیه‬‫می‬‫دهد‬‫که‬‫الیه‬‫رویی‬‫حاوی‬‫بار‬‫مثبت‬‫و‬‫الیه‬‫زیری‬‫حاو‬‫ی‬
‫بار‬‫منفی‬‫است‬.‫بعد‬‫جای‬‫الکتروها‬‫را‬‫می‬‫توان‬‫عوض‬‫کرد‬‫و‬‫دتکتور‬‫را‬‫در‬‫سمت‬‫مخالف‬‫قر‬‫ار‬
‫داد‬.

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Capillary Zone Electrophoresis (CZE)
• CZE also known as free solution CE (FSCE).
• It is the simplest form of CE (what we’ve been talking about).
• The seperation mechanism is based on differences in the charge and
ionic radius of the analytes.
• Capillary is empty from gel.
• Important parameters are regulation of PH, types of buffer and ionic
strength.

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• Electrochromatography is a hybrid of HPLC & CE.
• It is applicable to the seperation of neutral species or charged species.
• A polar solvent is usually driven by electroosmotic flow through a capillar
packed with a reversed phase HPLC packing.
• Seperation depend on distribution of the analyte species between the mobile
phase and the stationary phase held on the packing.
• It is expensive.
• Higher selectivity due to combination of chromatography and electrophoresis.
• It is more efficient than MEKC.
Capillary Electrochromatography (CEC)

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Capillary Gel Electrophoresis (CGE)
• CGE uses seperation based on the difference in solute size as a particle
migrate through the gel.
• Gels prevent the capillary walls from absorbing then solute.

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Capillary Isoelectric Focusing (CIEF)
• Electrophoresis run in a solution buffered at constant PH.
• CIEF is a technique for separating different molecules by differences in their
isoelectric point (pI).
• Protein in isoelectric poin has no net charge.

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Capillary Isotachophoresis (CITP)
• CITP is a focusing technique based on the migration of the sample components
between leading and terminating electrolytes.
• Solutes having mobilities intermediate to those of the leading and terminating
electrolytes stack into sharp, focused zones.

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• Lower instrument costs compared to HPLC
• Small sample size (nL) compared to (µl) in HPLC
• Significantly greater speed than HPLC (1-15min)
• High resolution due to electroosmotic flow
• Automaticity
• Coupled with mass

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Disadvantage
• Can not do preparative scale seperation
• Reproducibility problems

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References:
H Whatley - Clinical and Forensic Applications of Capillary …, 2001 –
Springer