chromatography Flashcards
chromatography
what are three types
analytical technique used to separate and identify compounds/components in a mixture based on the Rf values (TLC) or retention times (GC/CC)
thin layer chromatography
gas chromatography
column chromatography
Rf value meaning and eq
Rf is the retention factor. it is the ratio of the distance travelled by spot : distance travelled by solvent front on the gas or plastic plate in TLC
therefore eq is Rf = distance travelled by spot / distance travelled by solvent front
greatest it can be is 1
it identifies compounds because each one has a unique one
the Rf value changes based on temperature, what solvent is used, different plate types in TLC, different type of chromatography. so any comparison must be made under same conditions
retention time
time it takes for a compound to travel through a chromatography column and be removed at detector by being washed with a solvent (eluted)
elution
verb - elute
it is the process of extracting one material from another (separating a component from a mixture) by washing with a solvent. its the whole process of the chromatography, the compounds moving through the solvent and hence being separated by their differential solubility in the mobile phase and retention in the stationary phase
the fluid used to elute a substance
what is chromatography based on
aka, what about chromatography separates the components of a mixture?
separation depends on the balance between solubility in the moving phase and retention by the stationary phase
so all chromatography includes an s phase and m phase so can eparate compounds based on their solubility… and retention… .
what does our understanding of what chromatography is based on mean for evaluating retention times and Rfs
substances are separated according to relative affinity to stationary and mobile phase. if they are more attracted to, soluble in, stationary phase, their retention time will be longer/ their Rf value will be smaller
if they are more attracted to, soluble in, mobile phase, their retention time will be shorter/ their Rf value will be larger
what is the mobile phase
solvent
molecules can move through it
if you have a greater affinity for, solubility in, solvent you will move further/faster
what is the stationary phase
solid (CC) or liquid on solid support (GC)
molecules can’t move through. they adsorb?
if there is higher retention by stationary phase, then compounds will travel less distance
stationary phases of TLC, GC AND CC
TLC
plate coated with solid of SiO2 or Al2O3
COLUMN C
solid powder of SiO2 or Al2O3.
GAS C
high bp liquid on solid. on the inside of the column, the liquid is immobilised inside the column.
what is thin layer chromatography, describe the set up
glass or plastic plate coated with solid SiO2 or Al2O3 powder.
s phase = solid
m phase = solvent the sample is dissolved in
what happens if samples are colourless [3]
- revealed by UV light. shine it on it. (because component of s phase is normally fluorescent) the sample spots will be unfluroesced therefore visible
- amino acid chromatagrams shown by ninhydrin. gives them colour
- iodine vapour. shows up as browny spots
if the solvent is non polar then…
compounds that have greater affinity, higher solubility in it, so large Rf value, are (likely to be) non polar
and vv; if a solvent is polar and theres high Rf value, that means there was high attraction and compound is polar
and vvvv; applies to s phase i think
column chromatography set up
glass column/tube packed with stationary phase of solid SiO2 or Al2O3 powder.
non polar organic solvent as m phase,, is added to cover powder
theres a plug at the bottom to prevent stuff leaking out
sample, dissolved in minimal solvent, is poured into top. and the retentin time is recorded, time taken to reach bottom. not analysing how far but how long it takes to reach same point.
CC is more __. why?
preparative
it separates compounds out so can get pure sample
gas liquid chromatography set up
column packed with a solid
OR
packed with a solid coated by a liquid that is immobilised
^^the s phase options
the m phase is an inert gas that is passed through the column. under pressure at high temps.
the samples analyses partition between the gaseous mobile and the liquid stationary phase.
a detector transmits signals to a display processor, where peaks represent a compound in the mixture passing through a detector. area under peaks shows relative quantities of compounds present
how are samples prepped in each type
TLC
dissolved in solvent
GAS C
dissolved in solvent and vaporised
COLUMN C
dissolved in minimal solvent
how do they account for the solvents in each type
TLC
solvent front is used to calculate Rf, so its movement is required
GAS C
solvent would make a reading on display and in MS afterwards, but because solvent is carefully chosen, they know what it is and can ignore it. it is chosen to be volatile so you can ignore its peak in the display of the GC, and acts almost like a solvent front
COLUMN C
?
what is mass spectrometry
- analytical technique used to identify and quantify chemical substances by measuring the mass-to-charge ratio of ions.
- involves sorting gaseous ions in electric and magnetic fields, so can determine a sample’s chemical composition.
- results are typically presented as a mass spectrum, which plots current intensity against mass-to-charge ratio
steps of mass spectrometry
- vaporisation
- ionisation. this is so can be accelerated by magnetic field and be discharged at detector plate
a. it is done by either electron impact:
b. or electrospray ionisation - accelerated using magnetic field towards detector plate.
- drift/deflection - they are accelerated so all have same KE, but bc different atomic masses (bc isotopes/different elements), have different velocities. they are deflected more by magnetic field if lighter. so lighter the quicker go to detector plate, unless mag field too strong then discharged in walls and led out. so need to have less strong mag field for atomically light samples, and vv
- detection - so they reach detector plate at different times
- data - relative abundance on y and m/z on x
electrospray ionisation
dissolved in polar solvent
led though capillary needle
needle is surrounded by an electrode so a high voltage is applied to a sample in a polar solvent
sample molecule gains proton. M + H+ –> MH+
why is GLC often put together with MS?
it is a powerful analytical technique to identify compounds but the sample must be pure, you can’t just put the mixture in it
so by associating it with GC, compounds are separated using the chromatography and then analysed using mass spec
the SiO2/Al2O3 powder is
polar
PRAC 12
