Biochem Week 1- Separation Technqiues

Front 1

What are 5 examples of Single Stage Separation Techniques?

Back 1

1. Salting-out (eg. Ammonium Sulphate)
2. Isoelectric Precipitation
3. Organic Extraction
4. Ultrafiltration
5. Centrifugation

Front 2

What are Multi-Stage Separations?

Back 2

Simultaneous separation of many groups of molecules with high resolution. This usually requires a number of independent steps to purify the substance according to several criteria.

Front 3

What are some examples of criteria that you can purify a sample by?

Back 3

- size
- charge
- isoelectric point
- adsorption
- affinity
- electrophoresis
- chromatography
- ultra centrifugation

Front 4

What is Electrophoresis?

Back 4

Electrophoresis is the migration of ions in an electric field. It is widely used for the analytical separation of biomolecules.

Front 5

How does Electrophoresis work?

Back 5

Electrophoresis is used to separate proteins on the basis of size and/or charge.
A sample containing target proteins is loaded into a porous matrix and voltage is applied.
Proteins in the sample will migrate through the matrix at different velocities based on their varying size and charge.

Front 6

What is Centrifugation?

Back 6

Centrifugation is the process of separating substances of different densities by the use of a centrifuge.

Front 7

What can centrifugation be used for?

Back 7

- isolation of cells (and viruses)
- Separation of subcellular organelles (nucleus, ER)
- Separation of macromolecules (DNA, RNA, proteins or lipids)

Front 8

How does centrifugation work?

Back 8

- many particles/cells in a liquid suspension, given time, will settle at the bottom due to gravity (1G)
- the time required is impractical
- other particles may not separate at all unless subject to higher centrifugal force
- when a suspension is rotated at a certain Revolution Per Minute (RPM), centrifugal force causes the particles to move radially away from the axis of rotation
- the force on the particles (compared to gravity) is called Relative Centrifugal Force (RCF)
eg. RCF of 500 G means that the force applied is 500 x that of Earth's gravitational field

Front 9

What is Ultra Centrifugation?

Back 9

A high-velocity centrifuge used in the separation of colloidal or submicroscopic particles.
Ultracentrifuges are optimised for spinning a rotor at very high speeds generating centrifugal forces as high as 1000000 G.

Front 10

What types of Ultrcentrifuges are there?

Back 10

1. Preparative
2. Analytical

Front 11

What are preparative centrifuges?

Back 11

Preparative centrifuges: involve the fractionation of fine particulate samples, tissue homogenates to isolate subcellular orangelles, macromolecules, bacteria or viruses.

Front 12

What are analytical centrifuges?

Back 12

Analytical centrifuges: the physio-chemical properties of a sedimenting particle or molecular interactions of macromolecules and their possible subunits, respectively.

Front 13

What is Chromatography?

Back 13

Chromatography involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distributioin) of sample components between 2 different phases which are immiscible.
One of these phases is a 'mobile' phase and the other is a 'stationary' phase.

Front 14

What is the Distribution Coefficient (equilibrium distribution)?

Back 14

The Distribution Coefficient=[component in stationary phase]/[component in mobile phase]

The different affinity of these 2 components to stationary phase causes the separation.

Front 15

How does the distribution coefficient work in chromatography separation?

Back 15

1. Sample is placed on stationary phase
2. Mobile phase passed through the stationary phase
3. Mobile phase dissolves sample components
4. Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases.
5. Based on the distribution coefficients some components pass through the column faster than other.

Front 16

What is the Relative Fraction(RF) value?

Back 16

Due to difference in distribution coefficient of various component of mixture, there mobility in two phases differ hence, separation.
The measure of this mobility is called the Relative Fraction value.
Rf= distance traveled by substance/ distance traveled by solvent

Front 17

Define: Stationary Phase

Back 17

Stationary Phase: in some cases solid support itself from the stationary phase, while in others, it absorbs liquid phase which in turn acts as stationary phase. It is solid or liquid.

Front 18

Define: Mobile Phase

Back 18

Mobile Phase: phase which move over through stationary phase and carries sample along with it, thus resulting in the separation of its component. It is either liquid or gas.

Front 19

What types of Chromatography exist?

Back 19

1. Liquid Column Chromatography: gel filtration, ion exchange, affinity, adsorption, reverse phase, metal binding (column)
2. Gas Liquid Chromatography (column)
3. Thin-Layer Chromatography and Paper (planar)

Front 20

How does Liquid Column Chromatography?

Back 20

A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.
With the proper solvents, packing conditions, components in the sample will travel the column more slowly than others resulting in the desired separation.

Front 21

How does Ion Exchange (Liquid Chromatography) work?

Back 21

Proteins interact with stationary phase by charge-charge interaction.
Positively charged proteins adhere to negatively charged functional groups (carboxylates, sulfates: cation exchanger)
Teriary or quaternary amines: anion exchanger
Sequential elution, change of pH
Diethyl aminoethyl (DEAE) cellulose (anion exchanger)
Carboxymethyl (CM) cellulose (cation exchanger)

Front 22

How does size exclusion/gel filtration/gel permeation(Liquid Chromatography)?

Back 22

Porous beads as stationary phase
Stroke radius; function of molecular mass and shape
Greater the stroke radius faster will be the elution
Gel is made of dextran agarose or polyacrylamide.

Front 23

How does affinity (liquid chromatography) work?

Back 23

Sensitivity of most proteins towards ligands.
Use of resins to attach ligands.
Elution by competition with soluble ligand or by disruption of interaction.

Front 24

How does Purification of Recombinant Protein Metal Binding (liquid chromatography)?

Back 24

Poly his-tag of c-terminal, binding with divalent metal ion nickel, cobalt
Eluted with imidazole
Purification of recombinant protein linked with glutathione s-transferase by glutathione matrix.

Front 25

How does Reverse Phase and Hydrophobic (Liquid Chromatography)?

Back 25

Separation of Hydrophobic Proteins
Stationary phase is non polar liquid immobilized on inert solid.
Polar mobile phase.
Polarity of mobile phase reduced to dislodge proteins.
Hydrophobic interaction chromatography phenyl sepharose and octyl sepharose is used with weak interaction to prevent denaturation.

Front 26

How does Adsorption work?

Back 26

Molecules are absorbed by stationary phase, an insoluble substance like alumina, silicon, powdered sucrose is used for elution by pure solvents(chloroform, hexane, ethyl ether)

Front 27

What is HPLC?

Back 27

High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC, is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying or purifying the individual components of the mixture.

Front 28

How does HPLC work?

Back 28

HPLC is based on other chromatographic techniques.
Employment of incompressible matrix silica or alumina micro beads.
Thin layer of stationary phase.
Mobile phase is forced through the column at a pressure of up to 5000psi.
High resolution, reduced analysis time.

Front 29

What are the advantages of HPLC?

Back 29

- high resolution
- high degree of automation
- high throughput
- sensitive detectors
- smaller sample size of quantitative anaylses
- rapid anlysis

Front 30

Preparative vs Analytical Columns?

Back 30

Preparative:
- sample purification
- removal of 'other' components
- use for analysis or for other purpose
- need to collect fractions
among most common types:
- 'desalting' columns, separating high molecular weight from very low molecular weight compounds

Analytical:
- sample
- sample preparation
- application to column
- analysis
- >result
- quantitative

Front 31

Online vs Off-Column Analysis?

Back 31

Online: The sample is injected onto column. The eluant passes through detector. Peaks detected by In-Line detector at end of column and the eluant is discarded.

Off-Column: sample injected onto column. The eluant collected in frac/ons (eg. 0.5ml, drops, or 1 min worth of eluant) then analysed.

Front 32

What 4 types of detectors exist?

Back 32

1. Electrochemical
2. Spectrophotometric
3. Fluorometric
4. Mass spectrometer

Front 33

How do you obtain a column?

Back 33

Diagnostic labs
- purchase of ready-made columns
- expensive just economical when brought in bulk

Can create/pour your own:
- Subject of next practical
- Prepare a slurry of solid phase, pour into a plastic column