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CHROMATOGRAPHY LAB SESSION (different types (PRINCIPLE: equilibrium…
CHROMATOGRAPHY
LAB SESSION
2 phases
migration speed of the components: a Gaussian concentration pattern
stationary phase
liquid/solid
liquid: bound on an inert carrier material (granules or powder).
mobile phase
liquid /gas
packed in a column or spread in a tin layer over a carrier.
Ex: paper chromatography = paper: carrier
different types
Gel permeation chromatography
Ion exchange chromatography
Gas chromatography
always use a column
stationary phase = liquid/solid phase
mobile phase = gas phase
2 types of GC
gas–solid (
adsorption
) chromatography
gas–liquid (
partition
) chromatography :<3:
a sample being vaporised
injected onto the head of the chromatographic column
transported by the flow of an inert (gaseous mobile phase)
the column: contains a
not
volatile, liquid stationary phase that is adsorbed onto the surface of an inert solid.
schematic diagram of a gas chromatography
The temperature of the sample injection port and the detector
The injection volume of liquid samples: between 0.1 and 10µl
the injection volume of gaseous samples is between 1 and 10ml
inert gases: nitrogen, helium, argon, and carbon dioxide
PROCESS
Separation
= the division of the components of the sample between the gaseous and the liquid phase
Detection
1 more item...
Distribution chromatography
Adsorption chromatography
PRINCIPLE
:
equilibrium
between a stationary phase and a mobile phase
K:
distribution coefficient
Measure
concentration of eluted molecules
Plot this over time (t) = elution pattern OR CHROMATOGRAM
tm
: breakthrough time = time required for the mobile phase to traverse the column
tR,X
: retention time = total time required to elute component X
V X
: retention volume = amount of ml required to elute component X
The delay of a component with respect to the mobile phase is called the
retention
.
capacity factor k’
resolution R
: indicates whether a chromatographic system is applicable to separate certain components or not
A good quantitative and qualitative determination requires a resolution of at least 1
Column efficiency
DEFINITION: related to
band broadening
that affects the efficiency of separations
use
theoretical plate number
quantitatively describe the
efficiency of a column
Theoretical plates =
visualize
liquid-liquid counter current distribution solvent extraction system (CCD)
two phases contact each other in separate tubes and are incrementally passed along one another after equilibrium is achieved
each theoretical plate = a single equilibrium step between the mobile and the stationary phase
high efficiency
= large number of theoretical plates :red_flag:
IMPORTANT: the column height for which this number of plates is reached :red_flag:
H or HETP
L = length of the column
N = number of theoretical plates or plate number
with N can be obtained from a chromatogram from the expression
w = width of the peak on baseline
w1/2 = width of peak measured at a height of one-half of the peak height
tR = retention time
Van Deemter equation
broadening of a peak // diffusion//mass transfer
OR relation b/w HETP and elution velocity µ
Selectivity factor and capacity factor
selectivity factor α
capacity factor k'
t'R: the adjusted retention time
large capacity = good seperation but increased elution time
increased by increasing the stationary phase volume
PRACTICAL STEPS
2.2.2.1 Referential mixture
Isothermal working
alpha and R of the C14 and the C15 peak are determined. Calculate also tm and tR, k’, N and H of the C18 peak.
qualitative calibration curve
Temperature programming
:
separate as good as possible in a time as short as possible! with alpha en R should be as optimal as possible, also the retention times as low as possible
check reproducibility of the most suitable temp program
Calculate the range on the peak surface and the retention time of three injections executed with the same program
2.2.2.2 Prepare a calibration curve using an internal standard
Prepare stock solutions
thermo programming
measure standard solution and unknown sample
