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8/13/2019 A318Lecture12IntroChromatographicSephandout_000
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Introduction to Chromatographic
SeparationsSeparation of complex mixtures
Sample transported in a mobile phase gas, liquid, supercritical fluid
Mobile phase / sample passes over/through an immiscible stationary
phase that is fixed in place in column or on solid surface
Mobile and stationary phases chosen so that sample components will
distribute themselves between the two phases to varying degrees
Components strongly retained by the stationary phase will travel slower &
elute at longer times than those weakly retained
Each component separates into a discrete band or zone that can be
qualitatively or quantitatively analyzed
Chromatography Classifications
Introduction
26A
By means of contact:
Column Chromatography stationary phase held in narrow tube
through which mobile phase is forced under pressure (our focus)
Planar Chromatography stationary phase supported on a flat plate or
in a paper; mobile phase travels by capillary action or gravity
By mobile/stationary phase (Table 26-1):
Liquid Chromatography (LC) mobile phase is a liquid; stationary
phase can be adsorbed liquid, bound organic species, solid, ion-
exchange resin, liquid in a polymeric support
Gas Chromatography (GC) mobile phase is a gas; stationary phasecan be adsorbed liquid, bound organic species, or solid
Supercritical Fluid Chromatography (SFC) mobile phase is a
supercritical fluid; stationary phase is bound organic species
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Column Chromatography - Elution
Introduction
26A
detector
t0
t1
t2
t3
t4
packed
column
sample
A + B
mobile phase
AB
B
B
A
A
B
detector
signal
time
AB
addition of mobile
phase washes
sample through
column
B spends more
time in stationary
phase thanA
chromatogram
Chromatography Characteristics
Introduction
26A
Analyte dilution band broadening during separation leads to dilution of
analyte; requires greater detector sensitivity
concentration
A
B
t1
BA
t2
distance migrated
Chromatogram plot of detector response (concentration) vs. migration
time or mobile phase volume is used for qualitative (peak position) or
quantitative (peak area) analysis
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Migration Rates & Zone BroadeningEffect on Resolution
Introduction
26A
Improved separation changing elution time (b) or decreasing broadening
(c) can each be used
Variables that influence relative migration rates (26B) and zone broadening
(26C) can be described mathematically
detectorsignal
Time
(a) unresolved (overlapping) peaks
(b) Increase band separation
(c) decrease band width
Migration Rates of Solutes
Introduction
26B
For a single soluteA involved in transfer between mobile and stationary
phases, an equilibrium is reached whose constant K is the distribution
constant, partition ratio, or partition coefficient.
Amobile Astationary K =cScM
Linear chromatography K is constant over a wide concentration range
Retention time (tR) time for analyte to reach detector
Dead time (tM) time for unretained species to reach detector
Average linear rates for analyte (v) and mobile phase (u):
v = L/tR u = L/tM where L is length of column packing
Also,v = u X
moles of solute in mobile phase
total moles of solute
and since cV = moles
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Retention Time & Partition Coefficient
Introduction
26B
v = u Xc
M
VM
cMVM + cSVS= u X 1
1 + cSVS /cMVM
1
1 + KVS /VMv = u X
combine with definition of K to get:
which is the rate of solute migration as a
function of partition coefficient & volumes
of stationary & mobile phases
Retention factor (kA) describes migration rates of a solute on a column
kA =KAVS
VM
tR- tM
tMkA =
tR & tM obtained from expt.;
ideally 2 1
this is correct
Zone Broadening & Column Efficiency
Introduction
26C
Kinetic (rate) theory of chromatography explains peak shapes quantitatively
based on a random-walk mechanism well discuss qualitatively.
Peaks are Gaussian in shape due to thousands of transfers between mobile
and stationary phases; elution occurs only in mobile phase
- extended residence in stationary phase longer retention times
- extended residence in mobile phase shorter retention times
- longer time in column broaderpeaks; faster flow narrowerpeaks
Plate theory of chromatography provides terminology: plate height H &
number of theoretical plates N, related byN = L/H (L is column length)
plate theory based on analogy of separation to a series of distillation plates
at which solute reaches equilibrium between phases abandoned b/c it
does not account for peak broadening
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Column Efficiency / Plate Height
Introduction
26C
Plate Height is directly related to variance (2) or breadth of Gaussian peak
H =L
2
L
(L + 1)(L - 1) Analyte profile at
end of packing
Plate height contains ~34% of analyte (1 )
Based on time, variance is defined as 2, related to by = /(L/tR)
At the base, a peaks width W = 4, and thus = LW/4tR
Plate height can be defined as H =LW2
16tR2
Number of theoretical plates N = 16(tR/W)2
Using width at half height (W1/2), N = 5.54(tR/W1/2)2
H & N used by manufacturers / researchers to measure column
performance comparison can be done using same compound.
Zone Broadening / Kinetic Variables
Introduction
26C
van Deemter plot of H vs. u (linear flow rate) shows efficiency dependence
on flow rate, other variables (diffusion coefficients, particle diameter,
stationary phase thickness, retention factor, etc.) listed in Table 26-2
H,
cm
u, cm/s
AB/u
Cu
Multipath Term (A) each molecule
takes a different path through column
Longitudinal Diffusion Term (B/u)
solute molecules diffuse to regions of
lower solute concentration in front of
& behind zone; inversely proportional
to mobile phase flow rate
Mass-Transfer Terms (Cu) for Stationary Phase (CS) & Mobile Phase (CM) solute molecules require time to reach the surface of either stationary or
mobile phase, depending on thickness of each phase & diffusion
Zone Broadening reduced by using smaller particles, narrower columns,
lower temp. in GC, thinner liquid stationary phases