Serparation techniques and Principles of Chromatography

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Jeffrey Vongmany 11067648

Components of a sample mixture partition between 2 phases. What are these phases called?

Mobile phase: moves the simple mixture over the...

Stationary phase* during chromatography.

components of mixture pass through systems are different rates and become separated in time... why ?

because they are separated due to differences in the way they interact with the two phases.

Separation is based on 5 distinct separating mechanisms. List the 5 mechanisms:

1. partitioning
2. adsorption
3. Size exclusion
4. Ion-exchange
5. Affinity.

why not chromatography?

some instruments are expensive and not easily portable.
often preliminary bench 'work up' is required to avoid contamination.

ALL chromatographic systems contain: (three things)

-stationary phase
-mobile phase
- sample molecules

Movement of molecules is determined by the balance between two forces. What are the two forces?

impelling force of the mobile phase. (carries with it molecules that it is attracted to)

retarding force of the stationary phase:
holds back molecules with which it interacts.

what is partition chromatography?

type of liquid-liquid or liquid-gas chromatography utilising a liquid stationary phase and liquid (or gas) mobile phase.

are solute molecules decomposed by chromatographic separation ?

nope, they are not.

1. what is the stationary phase of partition chromatography
and
2. what is the mobile phase

3. how do they interact

1. sorbed solvent held on the surface or within the grains or fibres of an inert solid supporting matrix.

2. sample molecules carried in liquid in liquid chromatography and gas in in gas-liquid.

3. they equilibrate (partition) between stationary and mobile.

what is retiontion and how can it be describedf quantitatively ?

retention depends on a sample molecule's escaping tendency into the mobile phase versus its solubility in the stationary phase

described by the partition coefficient. (ratio of solubilities in two phases)

solute in mobile phase / solute in stationary

the components of partition chromatography mobile phase is always a solvent mixture whereby:

one component is polar: taken up by the polar matrix to form stationary phase. the component of the polar solvent is the retarder.

non polar travels around stationary phase. sample molecules in this solvent dont spend time in the stationary phase - this component of the mixture is the mover.

Adsorption chromatography is...

the solute in the liquid or gas phase interacts with adsroption sites on a solid surface.

what kind of chemical interaction do the polar groups on the solid form wit hthe sample dissolved in organic solvent?

hydrogen dipolar interactions.

what is gradient elution?

using solutions of increasing polarity. competing bond with adsroption sites.

How does separation of two solutes occur in adsorption chromatography.

solute that is more polar with be attracted to stationary phase. will move slower along the column compared to the solute less attracted to stationary phase.

Ion-exchange chromatography is

where retention occurs by the attraction between groups on stationary phase with opposide charge to sample molecules.

describe the component and it's purpose for ion exchange chromatography:

insoluble, but solvent permeable polymer matrix (e.g. cellulose) chemically modified to introduce ionisable groups (e.g. -COOH).

what can be used to enhane seperation?

elution is affected by pH or salt gradients that can enhance separation.

an increase in salts in eluant buffer, displaces solute by competing ions.

ion exchange media are classified according to..

whether the attached ionisable group is strongly or weakly acidic or basic.

DEAE cellulose and CM-cellulose are popular for

chromatographic separation of proteins (it is a mild and non-denaturing procedure).

describe the ion exchange seperation principle...

the surface group will attract the opposite charged solute and will retain that solute. the similarly charge solute will pass through the column.

Gel permeation chromatography/ size exclusion chromatography is good for ?

use of mild, non-denaturing conditions. suitable for separating proteins of different molecular masses.

how is retention brought about in affinity chromatography?

biospecific interaction using a ligand molecule chemically coupled to a dextran or cellulose matrix. perfect for complex biological mixtures!!!

how can you elute a affinity chromatography mixture?

displacement with lagand molecules in free solution. however, the analyte then eluted as complex with ligand. better to elute by change of pH to weaken binding.

what are important parameters to consider in chromatographic seperation?

retention time and peak width. which are measured in the same units.

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