FSCI 5355 Flashcards
Capillary Electrophoresis and Chromatography (CE) is one of the most powerful liquid phase separation techniques because of:
High speed
High efficiency
Different selectivity
Ability to sample small volumes
What is CE?
Movement of ionic species in conductive media under applied electric field.
Basic premise of electrophoretic separation:
Different charged species move at different velocity and can be separated.
Complex interplay between analyte, ions, capillary surfaces and additives in electrolyte.
Electroomosis and electromootic flow:
Observed when electric field is applied to conductive solution in capillary with fixed charges on its interior wall.
Potential difference close to wall called:
Zeta potential
Innermost layer close to capillary surface is static and called:
Helmholtz (stern) Layer
Second layer more diffuse layer and migrates in direction of cathode called:
Outer Helmholtz Plane (OHP)
During CE migration, associated solvent is drags molecules, which creates:
Electromootic flow
During CE migration, force propelling liquid originates at charged surface, causes flow to move in ____like fashion as opposed to when pressure pumps liquid.
This difference in flow profile main reason for CE efficiency.
Plug
Modes of separation (ZE)
Zone electrophoresis is most widely used mode and simplest form of CE:
- Capillary filled w/buffer
- Separation occurs because solutes migrate to discrete zones at different velocities
- Neutral compounds cannot be separated
Modes of separation
Electrophoretic Chromatography (EC):
Allows separation of neutral compounds w/addition of additive.
Mode of Separation
Sieving Electrophoresis (SE)
- Separation based on differences in size and shape of charged analytes
- Basis for DNA Generation Sequencing
Modes of separation
Electrochromatography:
- Combination of CE and HPLC
- Heterogeneous phase inside capillary
Modes of separation
Isoelectric Focusing:
- High resolution technique
- Used to separate proteins
- Acids and based
Modes of separation
Isotachophoresis is when injection of the sample is between leading electrolyte and terminating electrolyte:
When voltage is applied, formation of zones is proportional to concentration of analyte.
Injection/ Main advantage of CE:
- injection of extremely small volumes
- electric voltage applied to end of capillary, while immersed in DNA sample
- Negative charge draws sample into capillary
Interpretations of electropherograms
Two thresholds:
Analytical - average fluorescent signal converted to peak height
Stochastic - signal levels that cannot be used
Chromatography:
Powerful and versatile technique that is routinely used for single chemical species.
Classification of Chromatographic Techniques
Physical set/up of stationary phase:
Planar (thin layer)
Column based
Chromatographic technique mobile phase:
Gas, liquid, supercritical fluid
Chromatography further subclassified by separation mechanism:
Adsorption Partition Ion exchange Size exclusion Affinity
Liquid chromatography based on:
Adsorption
Gas chromatography based on:
Partition mechanism
Ion exchange chromatography separation based on:
Charge
Ion exchange chromatography:
Cation retains cations with
Negative charge
Anion exchange:
Retains anions with positive charge
Column types:
Packed column
- Fill with small particles
Open Tubular Capillary Column:
- Wall coated
- Support Coates
- Adsorption (solid particles attach to wall)
Chromatography Distribution Equilibria is:
Chromatography separation depend on distribution of sample components between stationary phase and mobile phase.
Retention time is:
Time taken for each analyte to move from injection into column until detection.
Un-retained compounds elute first:
Void time
Tr= tr - tm
Subtract void time from retention time.
Spectroscopy is:
Study of interaction between matter and electromagnetic radiation.
Mass Spectroscopic Principles:
Ionizer, Mass analyzer and detector
What does a mass spectrum do?
Give info on chemical structures.
General schematic of mass spectrum:
- Creation of ions
- Separation of ions
- Detection of ions
