Capillary electrophoresis Flashcards
What is electrophoresis?
The movement of charged particles in a suspending fluid induced by an electrical field.
How was convection overcome in electrophoresis?
Anti-convective media such as agarose/polyacrylamide gels was added, and also narrow capillary tubes which have less conductance (generate less heat; convection) was added.
What does stacking gels in SDS-PAGE accomplish?
It concentrates samples which will increase the resolution of separation
What forces are involved in electrophoresis?
ElectroSTATIC force, friction, and Electrophoretic retardation force (from counterions in solution).
What are the 4 methods of sample loading in electrophoresis?
Hydrodynamic with pressure, electrokinetic with an electric power, vacuum or siphoning to suck it in.
How does separation occur in electrophoresis?
Separation results from electroPHORETIC & electroOSMOTIC mobility.
Electrophoretic flow occurs from the movement of charged molecules in the electric field and depends on their charge and size whereas NEUTRAL molecules are unaffected and don’t move.
Electroosmotic flow (EOF) occurs from the flow of the solvent (water) towards the cathode. Cations will “drag” solvent along as they move towards the cathode and the bulk solvent flow created carries the anions and neutral compounds towards the cathode.
The ELUTION ORDER is cations, neutrals, and then anions.
How is electroosmotic flow achieved?
The capillary walls are with silanoyl groups which are readily ionised at >pH 3 and the extent of ionisation increases with pH (where the peak of ionisation is at pH 4). The water molecules will bind to Si and form SiOH, and when ionised it becomes an anionic SiO- compound which is attracted to the cations in the system. This creates the double electrical layers of cations (fixed and diffused). When a high-voltage electrical current is applied, the diffuse layer of cations becomes attracted to the cathode through electrostatic attraction. Since the cations are hydrated, the water molecules will follow along as the cations migrate towards the cathode. The dragging of water towards the cathode creates the bulk solvent flow in that direction which carries the analytes (cations, anions, neutral molecules) to the cathode and passes the detector. Hence, the electroosmotic flow is achieved. There is also a potential difference between the fixed and diffuse layers called zeta, and zeta is proportional to the electroosmotic flow where it will increase with pH and decrease with increased ionic strength. Therefore, more electroosmotic flow will increase the number of cations mobilised for detection.
How are anions separated in electrophoresis?
Anions are separated based on their relative migration variance, which is a balance of the anodal electrostatic attraction and the cathodal EOF.
What is Capillary electrochromatography (CEC)?
Capillary electrochromatography is similar to a High-Performance Liquid Chromatography (HPLC) system where both use SMALL BEADs but instead of pressure, but it uses ‘electroosmosis-driven’ high-performance separations which MOBILIZES the analytes with EOF and separates them based on their ELECTROPHORETIC MOBILITY and ability to INTERACT with the stationary & mobile phase.
When the analytes pass the spectrophotometric detector their ABSORBANCE and TIMINGs are noted.
CEC allows the use of longer columns with smaller beads which results in less peak broadening with better resolution.
When is CEC best applied to achieve 3-10x separation efficiency?
When PHASE PARTITIONING is the key separation factor (hydrophilic to hydrophobic), or when using NEUTRAL molecules, WEAK acids/bases as highly-charged species will be accelerated/decelerated in the mobile phase and produce inaccurate results.
How does Capillary Isoelectric Focussing (CIEF) work?
Separates proteins based on their ISOELECTRIC POINT.
Relies on CARRIER AMPHOLYTES with ZWITTERIONIC molecules (-ve & +ve) that when placed in a fluid with an electrical field generate a pH GRADIENT. Proteins will then MIGRATE towards their pI (isoelectric point) and stop moving.
Analytes must be allowed to FOCUS by suppressing the EOF with wall coatings to cover the SiOH layer.
The mobile phase maintains the electric field and PUMPs OUT the stationary analytes past the detector which DETECTS using a laser/camera system that allow the ANALYSIS of analytes in relation to their biological & pharmaceutical information.
CIEF is particularly used for the separation of protein isoforms which have a similar net charge or mass-to-charge ratio, but different pI.
How does Capillary Gel Electrophoresis (CGE) work?
CGE separates macromolecules that cannot be separated based on mass: charge ratios such as SDS-saturated proteins, nucleic acids, and compounds with uniform mass: charge ratio.
The analytes are mobilised in a fluid by an electrical field, and as they migrate in the opposite charge direction they are filtered by a POLYMERIC GEL based on SIZE as they pass through their pores. They are then detected by a laser/camera system and analysed for their biological information.
What is multiple myeloma?
Malignancy of plasma cells in the marrow.
What is the order of proteins detected in CE for protein serum?
Gamma-globulin > beta2 > beta1> alpha2 > alpha1 > albumin
Gamma(γ)-globulins: Immunoglobulins (Ig) & C-reactive protein (CRP)
Beta(β)2-globulins: C3 complement.
Beta(β)1-globulins: Transferrin & some LDL (Hemopexin)
Alpha(α)2 -globulins: α2-macroglobulin, haptoglobin, β-lipoprotein.
Alpha(α)1-globulins: α1-antitrypsin &
α1-antichymotrypsin (acid glycoprotein)
Albumin: A single protein.
What do biclonal gammopathies indicate?
Symptomatic disease.
