Chromatography Flashcards
What is Chromatography?
Chromatography is the process of separation of components from a mixture of compounds
the partition (division/separation) of the compounds in the mixture between a stationary phase and a mobile phase
How can chromatography be used?
qualitatively to assess purity - all move to the same distance = cannot tell how many analytes there are
preparatively to purify compounds - separates analytes
it can be used quantitatively with GC or HPLC with an internal standard - find out how many analytes there are
What are the stationary and mobile phase?
the stationary phase is fixed in place - solid packing material
the mobile phase carries the compounds (analytes) along - liquid or gas
Why are analytes (compounds) separated in chromatography?
analytes are separated due to their different distributions between stationary and mobile phases
this distribution is an equilibrium
What is the partition coefficient?
the distribution of analytes through the 2 phases is an equilibrium
it is the equilibrium constant
it gives a numerical measure of the distribution of analytes in the mixture
A[stationary]/ A[mobile]
What is TLC?
Thin Layer Chromatography
normal phase chromatography or reverse phase chromatography
How does TLC work?
stationary phase = uses a solid backing material - silica gel or alumina (polar)
mobile phase = uses a mixture of organic solvents (non-polar)
unknown mixture is spotted onto the plate near the bottom (at least 1 cm away)
tank prepared with mobile phase
plate is placed in the tank
solvent moves up the plate due to capillary action
once the solvent level reaches the top, the analytes can be seen
How can analytes be seen on the plate after movement?
it can be seen under UV light or chemical staining
Fluorescent light
What is normal phase chromatography?
stationary phase is polar due to OH groups = silica gel or alumina
mobile phase is non-polar = mixture of organic solvents
used for non-polar separations
strong interactions between non-polar mobile phase and mixture
polar analytes will interact strongly with the stationary phase and be found near the bottom of the plate whereas non-polar analytes will move up faster
What is reverse phase chromatography?
stationary phase is non-polar = C-18 modified silica gel
mobile phase is polar = water can be used, polar organic solvents
used for polar separations
strong interactions between polar mobile phase and mixture
non-polar analytes will interact strongly with the stationary phase and be found near the bottom of the plate whereas polar analytes will move up faster
What is the Rf value?
distance moved by analytes/ distance moved by solvent
affected by polarity of the solvent
not affected by the size of the plate
What is column chromatography?
can be normal phase or reverse phase
glass columns are be packed with different stationary phases (silica gel, alumina, sephadex, reverse-phase C-18 silica) - one at a time
add sample to the top
sample is absorbed onto the stationary phase
solvent is added and it washes down the column
separates the analytes into components
What is column chromatography separation based on?
polarity - using normal or reverse phase chromatography
size - uses sephadex as a stationary phase
How can column chromatography be analysed?
components collected (fractions/eluates) can be analysed via TLC to find out their contents and purity
TLC is usually used to determine whether separation has been successful
analysis with NMR, MS, UV and/or IR
What is flash chromatography?
can be normal phase or reverse phase
Glass columns can be pressurised with air to speed up the movement through the column
Compounds can be detected by UV or ELSD (Evaporative light scattering detector)
What are the advantages and disadvantages of flash chromatography?
advantage - speed of separation = faster
disadvantage - loss of resolution = poorer separation of components
What is HPLC and UHPLC?
high performance liquid chromatography
ultra high performance liquid chromatography
can be normal phase or reverse phase
can use small columns - analytical
can use large columns - preparative or semi-preparative
What is the difference between HPLC and UHPLC?
UHPLC can be run as HPLC but HPLC cannot be run as UHPLC
UHPLC can use smaller particles
-better resolution
better separation
What is gas chromatography?
mobile phase must be an inert, dry and oxygen free gas - compounds could react with oxygen or water
stationary phase is
-packed column = packed with finely ground diatomaceous earth (porous rock) coated with a high boiling liquid (usually waxy polymer)
-capillary column - stationary phase lines the wall of the thin glass tube which supports the liquid phase
How is gas chromatography used?
used to separate the components
must be passed through mass spectrometer, flame ionisation or thermal conductivity to identify components or give molecular ions/formula
How does gas chromatography work?
gas is released and flow controller controls how quickly gas flows through
gas enter the column over with the sample that is injected in via in the injector port
column oven is heated
- lower bp compounds vaporise first and move alone mobile phase
- higher bp compounds remain in liquid phase longer = longer retention time (stationary phase is liquid)
some compounds interact differently with the stationary phase
- some are more soluble than others
- non-volatile compounds cannot be used = won’t vaporise
- large compounds cannot be used = takes too long ti vaporise/ needs higher temperature
What is retention time in gas chromatography?
time from injecting the sample to detect of the analytes
How can analytes be detected in gas chromatography?
thermal conductivity
thermal conductivity
- electrically heated source is maintained at constant power
- temperature of the source depends on the TC of surrounding gases
- the source is usually a thin wire made of platinum or gold.
- the resistance within the wire depends upon temperature, which is dependent upon the thermal conductivity of the gas
- measures change in carrier gas TC caused by presence of sample
How can analytes be detected in gas chromatography?
flame ionisation
flame ionisation
-organic compounds burn and produce ions
- ions can be attracted to either the anode (negative) or cathode (positive) of the detector, producing a small current that can be picked up
-the more the ions, the larger the current and the larger the peak.
mass spectrometry
What are the properties of each type of chromatography? preparative quantitative qualitative normal or reverse
all can be normal phase or reverse phase
qualitative - pure or not (all move the same distance)
quantitive - how many different analytes there are/concentration
preparative - isolate and purify
analytical - separates
TLC - qualitative only
Column/Flash - qualitative or quantitative, preparative
HPLC - qualitative or quantitative, preparative with preparative column
GC - qualitative or quantitative
