Capillary electrophoresis principles and applications What is

Capillary electrophoresis: principles and applications

What is electrophoresis ? ▪ Separation of solutes based on different rates of migration though an electric field through background electrolyte [running buffer]. ▪ Anions (-) move toward the anode (+) & vice versa. Charge and size influence the movement of charged particles, in opposite ways.

▪ Separation of components in a mixture in an electric field depends on velocity. ▪ v=Eq/f v V = velocity of molecule v E = electric field v Q = net charge on a molecule v F = friction coefficient

Types of electrophoresis ▪ Gel electrophoresis ▪ High resolution electrophoresis ▪ Cellulose acetate electrophoresis at alkaline p. H 8. 5 ▪ Capillary electrophoresis ▪ Citrate agar or acid agarose gel electrophoresis at PH 6. 0 ▪ Isoelectric focussing ▪ Isoelectric focusing (IEF) ▪ Immunochemical ▪ Automated High Performance Liquid Chromatography (HPLC) ▪ Pulsed field ▪ 2 -D electrophoresis ▪ Globin chain electrophoresis ▪ Capillary electrophoresis

Capillary electrophoresis: principle ▪ Capillary tube is placed between two buffer reservoir, and an electric field is applied, separation depends on electrophoretic mobility & electroosmosis. ▪ Defined volume of analysate is introduced in to the capillary by replacing one buffer reservoir with sample vial. ▪ Electrophoretic separation is measured by detector.

Capillary electrophoresis ▪ Using narrow bore tubes, CE removes the Joule heating effect, which decreases band broadening, giving faster separations than gel. ▪ CE uses tubes 20 -100 mm diameter and 20 -100 cm in length. ▪ CE is used with/without gel. Longitudinal diffusion is the main source of band-broadening. ▪ Higher electric fields result in high efficiency and narrow peaks (analyte migrates faster).

▪ All analytes travel the same distance, but the migration time (tm) for that distance is measured. ▪ Relate time to identity. ▪ Relate peak area or height to amount.

Electro-osmotic flow

Movement of Analyte Electro-osmotic flow ν=µE νEOF = [ε/4πη]ζE ν = velocity ε = dielectric Constant ζ = µ= electrophoretic mobility E = Electric field Electrophoretic mobility µ = q/[6πηr] q = charge η = solution viscosity r = radius Zeta potential Flow of migration • ν = [(μEO + μe)V]/L • V = potential L = length of capillary

Capillary electrophoresis ▪ The tube in CE is typically silica, which may be coated or uncoated. ▪ Uncoated silica lead to electro-osmosis when run at neutral or basic p. H due to de-protonation of silanol groups. ▪ In “normal polarity mode, ” a sample with many types of ions can be injected (at the + end), and they then travel in the same direction toward the negative electrode through a detector. ▪ Observed mobility will be the sum of inherent electro-osmosis plus electrophoretic mobility. ▪ These affect time, efficiency, and separation.

▪ If an analyte has a migration rate faster than electro-osmosis, it may flow in the opposite direction of the electro-osmotic flow. This is known as the “reverse polarity mode. ” ▪ Changing the degree of de-protonation (altering the p. H) of the silica will alter electro-osmotic flow. Analysis is done by injecting at the negative electrode. ▪ Using a neutral coating in the tube reduces electro-osmosis, while a positive coating will reverse direction of flow toward the positive end.

Various separation modes for CE ▪ Capillary zone electrophoresis (CZE) ▪ Non-aqueous capillary electrophoresis (NACE) ▪ Capillary gel electrophoresis (CGE) ▪ Capillary electro-kinetic chromatography (CEKC) / Capillary electrochromatography (CEC) ▪ Micellar electro-kinetic chromatography (MEKC) ▪ Micro-emulsion electro-kinetic chromatography (MEEKC)

▪ Capillary isoelectric focusing (CIEF) ▪ Capillary iso-tachophoresis (CITP) ▪ Pressurized capillary electro-chromatography (p. CEC) ▪ Affinity capillary electrophoresis (ACE) ▪ Imuno-affinity capillary electrophoresis (IACE) ▪ Nano-capillary electrophoresis (NCE) ▪ Microchip-based capillary electrophoresis (Microchip-based CE) ▪ Micro-fluidic capillary electrophoresis (MFCE)

Capillary zone electrophoresis ▪ Technique intermediary btw classical zone electrophoresis & liquid chromatography ▪ Charged molecules separated by their electrophoretic mobility in an alkaline buffer (p. H 9. 4) • High voltage protein separation in silica capillary tubes • Direct Hb detection at absorbance wavelength of 415 nm at cathodic end • Cathode to anode Hb A

Capillary Gel Electrophoresis ▪ Used for size & shape separation. ▪ Separation based on differences in solute size. ▪ Detection is by UV absorbance of chromohore. ▪ DNA sequencing ▪ Protein analysis

Capillary Iso-electric Focusing ▪ Depends on PH buffer gradient ▪ The capillary is coated inside with an ampholyte , when the field is applied, will create a p. H gradient. ▪ Molecules migrate under influence of electric feild ▪ Uses: – Separation of proteins – Peptides – Amino acids – Drugs ▪ Not useful for chiral compounds

Affinity capillary electrophoresis (ACE) ▪ uses a biologically active compound in the running buffer. ▪ Adv : Measure specific interaction of anylate with ligand (receptor, antibodies , etc) ▪ ACE can separate chiral analytes.

Immunoaffinity capillary electrophoresis (IACE) ▪ Combine immunoassay and CE ▪ Three step procedure: – Bio-selective absorption – Subsequent recovery of compounds from immobilised affinity ligand – Separation of enriched compounds ▪ Rapidly emerging : analysis of low- abundance biomarkers ▪ Uses : – DNA analysis – Pharmacological – Forensic

Advantages of Capillary electrophoresis ▪ Simple ▪ Automated ▪ High efficiency of separation ▪ Short analysis time ▪ Low sample volume ▪ Ease of operation ▪ Ability to separate both charged and non-charged molecules ▪ Different mechanisms for selectivity ▪ Low cost ▪ Use aqueous rather organic solvents hence environment friendly

Disadvantages of CE ▪ Aged , improperly stored blood samples – degradation products ▪ Abnormal Hb – use other means of identification ▪ Migration of Hb variant close to Hb. A – underestimation of Hb A & variant + overestimation of Hb. A 2 ▪ Sensitivity & resolution limits

Capillary Electrophoresis V/S High Performance Liquid Chromatography (HPLC) ▪ Advantages: – Automated, utilise less staff time and permit processing of large batches. – Very small sample sufficient for analysis : 5μl. – Quantification of normal and variant Hb available in every sample. ▪ Disadvantages – Hb A is separated in to its component fractions of A 0 and A 1 ( subdivides in to several peaks) – Various abnormal and normal Hb can have same retention time – Hb. E and Hb Lepore co-elute with A 2. – Retention time of glycosylated and other derivatives of Hbs can be same as Hb. A 0 and A 2.

Separates Hb. S, A, A 2, F, S, C, D and G

A) Iso-electric focusing, B) Cation exchange HPLC, C) Capillary Electrophoresis

Capillary electrophoresis V/S cellulose acetate electrophoresis ▪ Labor-intensive. ▪ Inaccurate in quantification of low-concentration variants (Hb. A 2) and in detection of fast variants (Hb. H, Hb Barts). ▪ The precision and accuracy for Hb A 2 using scanning of electrophoretic gels is poor (in comparison to HPLC).

Applications ▪ Hemoglobin electrophoresis : abnormal Hb detection and characterization ▪ Immuno-typing : monoclonality ▪ Protein electrophoresis [capillary protein (E) 6] ▪ High resolution (HR) : multifraction human serum proteins ▪ Carbohydrate deficient transferrin : chronic alcohol abuse ▪ Molecular diagnosis : – DNA sequencing : ▪ Analysis of DNA fragment length / restriction patterns/ microsatellites ▪ Analysis of single strand polymorphism

– Diagnosis of neoplastic disorders ▪ ▪ ▪ Loss of heterozygosity Microsatellite instability Monoclonality assay Analysis of tumor related mutations Single nucleotide polymorphisms – Diagnosis of hereditary disease and prenatal testing – Diagnosis of infectious disease ▪ Pharmaceutical and biopharmaceutical applications ▪ Forensic applications

Abnormal hemoglobin detection by using CZE Hb move from Cathode to anode

Capillary serum protein electrophoresis : high resolution (HR) ▪ Designed for multifraction human serum proteins : – Albumin – α 1 acid glycoprotein (oromucoid) – α 1 antitrypsin – Haptoglobulin – Transferrin – C 3 complement – CRP – Gamma globulins Can be quantified

α 1 Antitrypsin deficiency

Uses ▪ Intravascular hemolysis ▪ Nephrotic syndrome (↓albumin, α 1 acid glycoprotein, transferrin and haptoglobin) ▪ Nutritional problems: dec in albumin levels

Capillary immunotyping ▪ Serum sample is mixed with individual specific antisera Ag-Ab complex is rapidly formed in liquid medium ▪ Treated samples are electrophoresed interpretation is accomplished by comparing reference pattern ▪ Used for immunoglobulin quantification and detect monoclonality ▪ Adv : – No sample incubation is required – Alternative to immunofixation – Allows easy identification of monoclonal peaks