Capillary Electrophoresis and it’s practical

applications

Done by: Hussein Talal

ID No.201117011

Supervised by: prof. Ashok Kumar

Capillary Electrophoresis (CE)Definitions • Electrophoresis: is the motion of dispersed particles relative to a fluid under

the influence of a spatially uniform electric field.

• Also defined as separation technique that is base on mobility of ions in an electric field.

• Capillary electrophoresis: is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage.

Capillary Electrophoresis (CE)

Introduction

• Capillary electrophoresis is used most predominately because it gives faster results and provides high resolution separation.

• Positively charged ions migrate towards a negative electrode and negatively-charged ions migrate toward a positive electrode.

• Ions have different migration rates depending on their total charge, size, and shape, and can therefore be separated.

• The rate at which the particle moves is directly proportional to the applied electric field--the greater the field strength, the fast the mobility.

Capillary Electrophoresis (CE)

Introduction

• Neutral species are not affected, only ions move with the electric field.

• If two ions are the same size, the one with greater charge will move the fastest.

• For ions of the same charge, the smaller particle has less friction and overall faster migration rate.

• For safety reasons one electrode is usually at ground and the other is biased positively or negatively

Capillary Electrophoresis (CE)Types of electrophoresis

•Capillary electrophoresis•Native Polyacrylimide Gel Electrophoresis (PAGE)•SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)•Paper

Capillary Electrophoresis (CE)

Anode

Cathode

Capillary Electrophoresis (CE)

Basic Design of Instrumentation

Capillary Electrophoresis (CE)

Basic Design of Instrumentation• A typical capillary electrophoresis system consists of:1. a high-voltage power supply,

2. a sample introduction system,

3. a capillary tube,

4. a detector

5. an output device, and

6. a temperature control device to ensure reproducible results.

Capillary Electrophoresis (CE)

Basic Design of Instrumentation

• Each side of the high voltage power supply is connected to an electrode.

• These electrodes help to induce an electric field to initiate the migration of the sample from the anode to the cathode through the capillary tube

• The capillary is made of fused silica and is sometimes coated with polyimide

• Each side of the capillary tube is dipped in a vial containing the electrode and an electrolytic solution, or aqueous buffer.

Capillary Electrophoresis (CE)

Theory Electrophoretic Mobility

• The separation of compounds by capillary electrophoresis is dependent on the differential migration of analytes in an applied electric field.

• The electrophoretic migration velocity ( ) of an analyte toward the electrode of opposite charge is:

• Where is the electrophoretic mobility and E is the electric field strength.

The electrophoretic mobility is proportional to the ionic charge of a sample and inversely proportional to any frictional forces present in the buffer.

Capillary Electrophoresis (CE)

Theory Electrophoretic Mobility

• The frictional forces experienced by an analyte ion depend on the viscosity (η) of the medium and the size and shape of the ion. Accordingly, the electrophoretic mobility of an analyte at a given pH is given by:

• Where is the net charge of the analyte and is the Stokes radius of the analyte.

Capillary Electrophoresis (CE)

Theory Electroosmotic Flow (EOF)

• The electroosmotic flow (EOF) is caused by applying high-voltage to an electrolyte-filled capillary.

• This flow occurs when the buffer running through the silica capillary has a pH greater than 3 and the SiOH groups lose a proton to become SiO- ions.

Capillary Electrophoresis (CE)

Theory Electroosmotic Flow (EOF)

• The capillary wall then has a negative charge, which develops a double layer of cations attracted to it.

• The applied electric field causes the free cations to move toward the cathode creating a powerful bulk flow. The rate of the electroosmotic flow is governed by the following equation:

µEOF = ε/4πη *Eζ

• where ε is the dielectric constant of the solution, η is the viscosity of the solution, E is the field strength, and ζ is the zeta potential.

Capillary Electrophoresis (CE)Electroosmotic Flow (EOF)

Capillary Electrophoresis (CE)

Types of Molecules that can be Separated by CE:

• Proteins • Peptides • Amino acids • Nucleic acids (RNA and DNA)• Inorganic ions • Organic bases • Organic acids • Whole cells

Capillary Electrophoresis (CE)

The Electropherogram• Detectors are placed at the cathode since under common conditions, all

species are driven in this direction by EOF

I. Direct detection UV absorbance monitor set to a wavelength near 200nm where many

Fluorescence detector works for fluorescent analytes or fluorescent

derivatives.Electrochemical detection is sensitive to analytes that can gain or lose

electrons at an electrode Eluates can be directed into a mass spectrometer with electrospray interface to provide information on the quantity and molecular structure of analytes

Capillary Electrophoresis (CE)

The ElectropherogramII. Indirect detectors• It relies on measuring a strong signal from the background electrolytes and

a weak signal from the analytes as it passes the detector.

• A fluorescence ion with the same sign of charge as the analytes is added to background electrolyte to provide a steady background signal.

Capillary Electrophoresis (CE)

The Electropherogram

• The general layout of an electropherogram:

Capillary Electrophoresis (CE)

The Electropherogram vs. Chromatogram

Capillary Electrophoresis (CE)

Capillary Electroseparation Methods

• There are six types of capillary electroseparation methods available:

1. capillary zone electrophoresis (CZE),2. capillary gel electrophoresis (CGE),3. micellar electrokinetic capillary chromatography (MEKC), 4. capillary electrochromatography (CEC),5. capillary isoelectric focusing (CIEF), and 6. capillary isotachophoresis (CITP).

Capillary Electrophoresis (CE)

Capillary Electroseparation Methods• They can be classified into continuous and discontinuous systems as shown

in the Figure

• A continuous system has a background electrolyte acting throughout the capillary as a buffer.

• A discontinuous system keeps the sample in distinct zones separated by two different electrolytes.

Capillary Electrophoresis (CE)

Capillary Zone Electrophoresis (CZE)

• Capillary Zone Electrophoresis (CZE), also known as free solution capillary electrophoresis, it is the most commonly used technique of the six methods.

• A mixture in a solution can be separated into its individual components

quickly and easily.

• The separation is based on the differences in electrophoretic mobility, which is directed proportional to the charge on the molecule, and inversely proportional to the viscosity of the solvent and radius of the atom.

• The velocity at which the ion moves is directly proportional to the electrophoretic mobility and the magnitude of the electric field

Capillary Electrophoresis (CE)

Capillary Gel Electrophoresis (CGE)

• Capillary Gel Electrophoresis (CGE) is the adaptation of traditional gel electrophoresis into the capillary using polymers in solution to create a molecular sieve also known as replaceable physical gel.

• This allows analytes having similar charge-to-mass ratios to also be resolved by size.

• This technique is commonly employed in SDS-Gel molecular weight analysis of proteins and in applications of DNA sequencing and genotyping.

Capillary Electrophoresis (CE)

Micellar Electrokinetic Capillary Chromatography (MEKC)• Electrokinetic Chromatography (EKC): a family of electrophoresis techniques

named after electrokinetic phenomena, which include electroosmosis, electrophoresis and chromatography.

• Micellar Electrokinetic Capillary Chromatography (MECC OR MEKC) is a mode of electrokinetic chromatography in which surfactants are added to the buffer solution at concentrations that form micelles.

• The separation principle of MEKC is based on a differential partition between the micelle and the solvent (a pseudo-stationary phase). This principle can be employed with charged or neutral solutes and may involve stationary or mobile micelles.

• MEKC has become valuable in the separation of very hydrophobic pharmaceuticals from their very polar metabolites.

Capillary Electrophoresis (CE)

Micellar Electrokinetic Capillary Chromatography (MEKC)

Capillary Electrophoresis (CE)Micellar Electrokinetic Capillary Chromatography (MEKC)

Capillary Electrophoresis (CE)

Capillary Electrochromatography (CEC)• Capillary Electrochromatography (CEC) is a hybrid separation method

• CEC couples the high separation efficiency of CZE with the selectivity of HPLC

• Its most useful application appears to be in the form of on-line analyte concentration that can be used to concentrate a given sample prior to separation by CZE

Capillary Electrophoresis (CE)Capillary Electrophoresis vs. High Performance Liquid Chromatography (HPLC) 1. CE has a flat flow, compared to the pumped parabolic flow of the HPLC. The flat flow results in narrower peaks and better resolution. (See Fig. ) 2. CE has a greater peak capacity when compared to HPLC

3. HPLC is more thoroughly developed and has many mobile and stationary phases that can be implemented. 4. HPLC has more complex instrumentation, while CE is simpler for the operator. 5. HPLC has such a wide variety of column lengths and packing, whereas CE is limited to thin capillaries. 6. Both techniques use similar modes of detection.

7. Can be used complementary to one another.

Capillary Electrophoresis (CE)

Capillary Electrophoresis versus High Performance Liquid Chromatography (HPLC)

Capillary Electrophoresis (CE)

Capillary Electrochromatography (CEC)

Capillary Electrophoresis (CE)

Capillary Isoelectric Focusing (CIEF)

• Allows amphoteric molecules, such as proteins, to be separated by electrophoresis in a pH gradient generated between the cathode and anode.

• This technique is commonly employed in protein characterization as a mechanism to determine a protein's isoelectric point.

• Isoelectric Point: is the pH at which a particular molecule (protein) or surface carries no net electrical charge.

Capillary Electrophoresis (CE)

Biomedical applications of capillary electrophoresis

1. Analysis of complex sample matrices. Simple anions and cations*

2. Optimization for separation conditions of endogenous compounds

3. Enzyme activity assay by CE

4. Protein-drug binding assay

5. Diagnosis of metabolic disorders by CE*

6. Monitoring of drugs in body fluids

Capillary Electrophoresis (CE)

Biomedical applications of capillary electrophoresis

1-Analysis of complex sample matrices. Simple anions and cations

• Complex mix. of anions, cations or mixtures of anions with other analytes present e.g. in tissue culture media may serve as typical examples

• Separation is performed using capillary ionic electrophoresis (CIE)

• Separations are carried out in fused silica columns

• Separation times are usually short (2-10) mins.

Capillary Electrophoresis (CE)

Anions and cations that have been characterized by CIE and UV Detection• Inorganic anions: Thiocyanate, Sulphide, Iodide, Borate, Hypochlorite• Organic anions: Acetate, Formate, Glutarate, Glucoronate, Benzoate• Metals: Lithium, Sodium, Calcium, Iron, Zinc, Lead

Capillary Electrophoresis (CE)

Biomedical applications of capillary electrophoresis

2-Diagnosis of metabolic disorders by CE• Application of CE to the determination of several metabolites related to

Diabetes Mellitus has been focus recently.

• Diabetes Mellitus is considered a class of diseases that exhibits intolerance to glucose.

• Typically, high levels of glucose are found in blood, which promote the glycosylation of hemoglobin, giving the so called glycated hemoglobin (HbA1c).

• The folowing Figure illustrates the separation of HbA1c among other hemoglobins and variants using capillary isoelectric focusing (cIEF) with chemical mobilization.

Capillary Electrophoresis (CE)

Diagnosis of metabolic disorders by CE

Capillary Electrophoresis (CE)

Nutritional applications of CE

• Fructose, glucose, maltose and sucrose are present in a variety of foods and their quantitative determination allows the evaluation of sample authenticity, quality control of food products and possible microbiological alterations during product storage.

• By raising the electrolyte pH, carbohydrates can acquire a negative charge and be easily characterized by CE under indirect detection.

Capillary Electrophoresis (CE)

Pharmaceutical applications of CE: Detecting bacterial/microbial contamination quickly using CE • Current methods for determination of microbial contamination require

several days.

• Direct innoculation (USP) requires a sample to be placed in a bacterial growth medium for several days, during which it is checked under a microscope for growth or by turbidity measurements.

• False positives are common (simply by exposure to air)

• Techniques like ELISA, PCR, hybridization are specific to certain microorganisms

Capillary Electrophoresis (CE)Detection of Bacterial Contamination with CE Method• A dilute cationic surfactant buffer is used to sweep microorganisms out of the

sample zone and a small plug of “blocking agent” negates the cells’ mobility and induces aggregation

• Method detects whole bacterial cellls

Capillary Electrophoresis (CE)

Detection of Bacterial Contamination with CE

• Single-cell detection of a variety of bacteria

• Why is CE a good analytical approach to this problem?

1. Fast analysis times (<10 min)2. Readily miniaturized

Capillary Electrophoresis (CE)

Advantages and Disadvantages of CEAdvantages

• Offers new selectivity, an alternative to HPLC • High separation efficiency (105 to 106 theoretical plates) • Small sample sizes (1-10 ul) • Fast separations (1 to 45 min)

Disadvantages• Cannot do preparative scale separations• Low concentrations and large volumes difficult

“Sticky” compounds• Species that are difficult to dissolve• Reproducibility problems

Capillary Electrophoresis (CE)

Summary• CE is based on the principles of electrophoresis

• The speed of movement or migration of solutes in CE is determined by their charge and size. Small highly charged solutes will migrate more quickly then large less charged solutes.

• Capillary electrophoresis is a modern separation technique, with vast acceptance in the academic and industrial communities.

• The technique is yet to reach its full potential, despite its excellent analytical performance, diversity of applications and relatively simple instrumentation.

Thank you

References:1-http://www.scielo.br/scielo.php?pid=S0103-50532003000200016&script=sci_arttext2-http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Capillary_Electrophoresis

3-http://www.sciencedirect.com/science/article/pii/S0003269705005956

4-Journal of Chromatography B, 656 (1994) 3-27Biomedical applications of capillary electrophoresisZ. Deyl* @, F. Tagliarob, I. MikSik”“Institute of Physiology, Academy of Sciences of the Czech Republic, Videiiskri 1083, 14220 Prague 4, Czech RepublicInstitute of Forensic Medicine, University of Verona, Policlinico Borgo Roma, 37134 Verona, Italy

http://www.analyt.natureblink.com/publikace/biomed_rev.pdf