Analytical methods Flashcards
What does qualitative analysis study?
It defines the identity of the compound. We don’t want to know specific amounts, but the nature of the sample.
What does quantitative analysis study?
Defines the quantity of the compound
Explain solvent extraction
Separation funnels separate two layers of immiscible (liquids unable to mix) liquids.
This involves an aqueous phase (= hydrophilic or water-based) and organic phase (= hydrophobic, e.g. dichloromethane or ethyl acetate). When the two liquids are mixed, they form two distinct layers, which can be drawn off from the funnel by using the tap at the bottom.
Partitioning:
If a third component is introduced into the funnel and the components are shaken, the chemical compound will distribute itself between the two liquids. Different types of compounds show affinity for either hydrophobic or hydrophilic solvent.
How do we determine affinity with the distribution ratio
K=concentration in aqueous phase over concentration in organic phase.
if K > 1, the compound will preferentially partition into the aqueous phase
if K < 1, the compound will preferentially partition into the organic phase
How does Chromatography work?
Chromatography utilises the differential partitioning of chemical species between two immiscible phases (solid, liquid or gas). In general, chromatography uses a stationary phase and a mobile phase to achieve separation
What is molecular exclusion chromatography used for?
It is used to fractionate protein mixtures.
What is size exclusion chromatography?
small molecules which will fit into the holes will remain on the column for longer and have larger retention times.
larger analytes which do not fit into the holes will pass through the column faster and thus have shorter retention times
Separation based on charge
To separate out compounds on the basis of electrical charge we would use a stationary phase that attracts species of a particular charge, allowing both neutral species and species carrying an opposite charge to pass through the column more quickly.
Separation based on affinity
We can use selective binding properties of antibodies to separate molecule from the mobile phase. A particular antibody can be immobilized on the stationary phase of a column. The antibody then binds only to the particle to which it has a complementary shape and retains that particle on the column.
Gas chromatography
liquid and gas chromatography are powerful tools for separating out complicated mixtures of compounds, including those extracted from biological systems.
the mobile phase is an inert carrier gas, while the stationary phase most often is a liquid (GLC) or rarer, a solid (GSC).
High performance liquid chromatography (HPLC)
reversed-phase HPLC is a powerful analytical technique and is able to separate analytes with subtle differences in their physical properties, including structural isomers and diastereoisomers.
Electrophoresis
Gel electrophoresis separates charged macromolecules through a combination of charge, size and shape. Proteins are separated on a gel made of polyacrylamide upon addition of the anionic surfactant sodium dodecylsulfate (SDS-PAGE). The nucleic acid electroporesis is conducted on an agarose gel.
Principle of isoelectric focussing (IEF)
Another method for separating polypeptides and proteins couples to a gel based method that separates molecules on the basis of charge. Isoelectric focussing (IEF) separates proteins using a gel containing a pH gradient
each protein will move until its isoelectric point is reached.
2D gel electrophoresis
In two-dimensional gel electrophoresis, proteins are first separated by isoelectric focussing (IEF) according to their pI. The gel is then rotated by 90°, and then subjected to SDS-PAGE. Thus, complex protein mixtures can be separated into individual proteins.
