Chromatography Flashcards
Who initially designed chromatography, and for what purpose?
M. Tswett (botanist); separate pigments in plants using column made of calcium carbonate
origin of the term ‘chromatography’
greek;
chroma = color graphein = to write
the 2 phases in chromatography:
mobile phase
stationary phase
What is the mobile phase? What does it do?
fluid that passes through/over the stationary phase
(liquid, gas, or supercritical fluid)
elutes out the various components in the sample (also called the ‘eluent’)
What is the stationary phase? What does it do?
column or surface - gel, solid, (or liquid distributed, bound, or immobilized on solid)
immobile (fixed) phase that interacts with compounds passing through (pushed by mobile phase)
What is the definition of chromatography?
separation based on difference in rate (speed) that components migrate through stationary phase (pushed by mobiled phase)
What is ‘elution?’
process of separating components of mixture using appropriate solvents
When a sample is passed through a chromatography column, the ____ chemicals will pass through fastest, while ____ chemicals move slowly
unretained
retained (greater affinity for column)
The results (output) diagram of chromatography is called a _____. Different compounds are shown as ______
chromatogram
different peaks
What is tR?
retention time (time between injection and max peak of analyte)
The larger the tR, the greater the ______
affinity for the column (retention)
What is u?
linear velocity of mobile phase (or non retained analyte)
The linear velocities of retained analytes ‘B’ and ‘C’ would be expressed as:
vB
vC
What does ‘L’ represent?
length of column
What is tM?
dead/void time
time needed for mobile phase to pass through
L divided by tM =
u (linear velocity of mobile phase)
What is k’B?
retention factor
time the sample component stays in stationary phase vs time it resides in mobile phase
(indicates how much longer it is held back by stationary phase vs travelling straight through with mobile phase)
How to calculate k’B: (2)
ratio of amounts in 2 phases (stationary/mobile)
(tR(B) - tM)/tM
different types of interaction between the stationary phase and compounds (4)
adsorption
partitioning (affinity based)
ion exchange (charge based)
sieving (size based)
For ____ and ____ interactions, the stationary phase is immobilized on an inert base
ion exchange
partitioning
sieving interactions will have greater retention for (larger/smaller) particles
smaller (caught up inside sorbent )
types of liquid chromatography: (5)
liquid solid (adsorption) liquid liquid (partitioning) ion exchange size exclusion (sieving) affinity (partitioning)
types of gas chromatography techniques: (2)
gas liquid (partitioning) gas solid (adsorption)
types of supercritical fluid chromatography:
SF liquid (partitioning) SF solid (adsorption)
What theory did Martin and Synge come up with?
Plate theory:
consider chromatography as a series of 2 phase extractions (without reaching actual equlibrium due to continued movement through)
What is the K distribution constant?
K = [A] in stationary phase / [A] in mobile phase
A high K distribution constant means: _____
greater distribution into the stationary phase (more affinity); longer retention
the greater the number of theoretical ‘plates’ in a column, the _____ the extraction
more efficient (better yield)
What is ‘alpha?’
selectivity factor
What is the selectivity factor?
measure of the separation of 2 components
ratio of the K distribution constants (or retention factors) for each
T/F: alpha (selectivity factor) depends on column efficiency
False; independent of column efficiency
only consider the chemistry of compounds, eluent type, adsorbent chemistry
What factors affect ‘alpha’ (selectivity factor)? (3)
chemistry of compounds
eluent type
adsorbent chemistry
‘alpha’ (selectivity factor) must be _______, other wise ______
> 1
if alpha = 1, no chance to improve column efficiency to better separate
The chromatogram peak should be a ____ shape
gaussian
The number of _______ is used to assess column performance
theoretical plates (N)
How is ‘N’ calculated?
N = 16 x (tR/W)^2
tR (retention time)
W (peak width at base)
The height of each theoretical plate is given as:
H = L/N
T/F: the theoretical plate approach does not explain peak broadening
true
What is the equation used to describe chromatographic effiency in ‘rate theory?’
Who was behind it?
H = A + B/u + C*u u (linear v of mobile phase) A: multipath coefficient B: longitudinal diffusion coefficient C: mass transfer coefficient
van Deemter
what does ‘A’ (multipath coefficient’ in rate theory describe?
describes variation in analyte flow path (molecules flow down slightly different pathways, not travelling exactly same distance)
what does ‘B’ (longitudinal coefficient’ in rate theory describe?
describes tendency of molecules to diffuse out in random directions (slightly drift in other directions rather than straight through column)
what does ‘C’ (mass transfer coefficient’ in rate theory describe?
resistance to mass transfer; analyte is trying to equilibrate between stationary and mobile phase, but not instantaneous, and mobile phase keeps moving
=> molecules leaving stationary into mobile phase will lag behind slightly
identical molecules will not elute out at the same time. This is known as ___ _____, and can be explained by ______
peak broadening
van deemter equation (A, B, C terms)
As flow velocity increases (high flow), what happens to ‘A’ factor in the van deemter equation?
remains constant
As flow velocity DECREASES, what happens to ‘C’ factor in the van deemter equation?
decreases
The C term in van deemter equation can be further divided into (2)
Cs (stationary phase)
Cm (mobile phase)
What happens to B factor in van deemter equation if flow is increased?
exponential decrease
T/F: at a high flow rate, resistance to mass transfer will have a larger impact on band broadening than the longitudinal diffusion coefficient
True
The van deemter equation for high flow?
The van deemter equation for low flow?
high flow:
H = A + B/u + C*u
low flow:
H = B/u + (Cs + Cm)*u
Describe the van deemter graph:
plot u (mobile phase velocity) vs theoretical plate height (H)
J shaped curve (optimal efficiency is at lowest point)
What is ‘resolution?’
denoted by ‘R’
characterize separation of 2 adjacent peaks
How is R calculated?
(distance from mid-peaks)/(sum of peak widths divided by 2)
or
2 x (difference in retention time)/(sum of peak widths)
A (higher/lower) resolution means better separated peaks
higher
A greater number of theoretical plates is considered to be (more/less) efficient
more
How is the plate number affected by column length?
increases with length
How does the size of the column (stationary phase and internal diameter) affect efficiency?
smaller diameter -> more efficient (more plates)
examples of non-ideal peaks (2)
tailing (stretched out slope after peak)
fronting (stretched out slope before peak)
What are some practical problems that lead to non-ideal peaks?
overload
flow rate too high
slow injection…
