Chromatography Flashcards
chromatography
chromatography refers to the separation
of a mixture of compounds which comes into contact with two phases: the stationary phase and
the mobile phase, and how these two phases
move in relation to each other.
the stationary phase and the mobile phase
the stationary phase
the material that interacts with the compound dependent on its physical and chemical properties
the mobile phase
the phase that the compounds will be dissolved in
The components of the sample become distributed between either of
the two phases, depending on their physical or chemical properties. The interaction between the two
phases is differential (i.e. is not the same for each component), so the compounds move at different
speeds and so are separated.
Analytical chromatography
Chromatographic separation can be used both quantitatively, to determine how much of a substance
there is, and qualitatively, to separate the individual components for identification. Both techniques
are called analytical chromatography.
preparative chromatography
It can also be used to separate and purify a complex mixture to
give a pure (or purer) compound without (or with fewer) contaminants.
it is used widely in the industrial preparation of pure proteins,
All chromatographic methods have the following common elements:
a stationary phase
a mobile phase
a mechanism to make the sample components move relative to the stationary phase
a way of adding a sample to be separated
a method for detecting the sample.
paper chromatography technique
used to separate the different pigments of the in the marker
1. draw a line with the marker on the chromatography paper
2. place the chromatography paper into a beaker with a small volume greater
water will start to rise up the paper due to capillary action
the ink will start to separate into different pigments this is because they have different properties :
some are heavier
some are light
some are more soluble in water
ALL THE DIFFERENT PIGMENTS TRAVEL AT DIFFERENT SPEEDS
paper chromatography technique
analysis of results
measure the maximum distanced travelled by the water and at each different pigment starting at the the line drawn on the paper
calculate an Rf Value for each colour of pigment by using the formula : Rf = DP/DW
CHROMATOGRAPHIC MECHANISMS
The components of a sample can be separated in two ways:
• through their chemical properties, such as how hydrophilic (water loving), hydrophobic (water
hating), chemical functionality (do they have a specific chemical signature), or polar (with acharge distribution or separation within the compound) they are
• through their physical properties, for example their size.
adsorption
ion exchange,
size exclusion,
affinity
adsorption,
where components selectively sorb onto the surface of a material with
compatible chemistry
ion exchange
where components are separated by charge
size exclusion
components are separated by size;
affinity
components are separated according to their affinity for the stationary phase (e.g. antibodies) that specifically target certain compounds in the
mixture (e.g. antigens), retaining them and allowing everything else through.
ADSORPTION
the solutes in the mixture adsorb to a greater or lesser extent onto sites on the surface of the stationary phase
the mobile phase is either gas (gas chromatography GC) or a liquid (in liquid chromatography LC)
the solutes will adsorb onto the surface of the stationary phase after first displacing a solvent molecules
depending on the strength of the interaction (non-covalent , ionic) they will later desorb in an equilibrium governed process
PARTITION CHROMATOGRAPHY
stationary phase is a liquid supported on an inert solid (often silica)
BONDED- PHASE CHROMATOGRAPHY
If the liquids are chemically bonded to the solid support it is bonded phase chromatography
NORMAL PHASE CHROMATOGRAPHY
Stationary phase is polar and the mobile phase is non polar solvent
non polar compounds will tend to remain in the mobile phase and come off in the column (eluted) eariler than the polar compounds which are attracted to the more polar stationary phase
REVERSE PHASE CHROMATOGRAPHY
polar mobile phase
non polar stationary phase
most commonly used chromatographic separation method
the non polar stationary phase can be obtained by attaching non polar chains of hydrocarbons
Solid-phase Extraction
solid-phase extraction
(SPE), a separation process by which compounds are dissolved or suspended in a liquid mixture, and
then separated from other compounds in the mixture according to their physical and chemical
properties.
ion-exchange or bonded-phase the most common
The stationary phase (a powdered solid such as silica with chemically tailored surface properties) is contained in a cylindrical glass or plastic column between two frits (porous but inert pieces of glass or
plastic that hold the stationary phase in place).
he sample solution is loaded onto the cartridge where the compound (or compounds) of interest
bind (adsorb) to the stationary phase. The loading is generally performed under a slight vacuum (using
a vacuum pump to pull the sample through) to speed up the process.
