Transcript Introduction to Chromatography

INTRODUCTION TO
CHROMATOGRAPY
HISTORY

The Russian botanist Mikhail
Tswett coined the term
chromatography in 1906 to
describe his experiments in
separating different colored
constituents of leaves by passing
an extract of the leaves through a
column
Chromatography
Web Dictionary:
Analytic technique to discover chemical components:
a method of finding out which components a gaseous
or liquid mixture contains that involves passing it
through or over something that absorbs the different
components at different rates
Chromatography
Injector
Port
Flow Control
Detector
Column
Column Oven
Carrier Gas
Recorder
CHROMATOGRAPHY COLUMNS
Packed Column:
Typical HPLC columns but some gas
chromatography columns also (especially older columns). The
columns are packed with tiny particles.
Capillary Column:
Typical gas chromatography column
which consists of a small diameter tube coated on the inside with
stationary phase.
CHROMATOGRAPHY THEORY
PARTITION COEFFICIENT
Remember from the solvent lecture……….
K = Co/Cw
Co is concentration in the organic phase
(solvent)
Cw is the concentration in the aqueous
phase (water)
PARTITION COEFFICIENT
K = Co/Cw
Co is concentration in the organic phase (solvent)
Cw is the concentration in the aqueous phase (water)
K=
molar concentration in stationary phase
molar concentration in mobile phase
PARTITION COEFFICIENT
K=
k =
b=
ETC.
concentration in stationary phase
concentration in mobile phase
mass in the stationary phase
mass in the mobile phase
volume of mobile phase
volume of stationary phase
PARTITION COEFFICIENT
ETC.
If mass = volume x concentration then:
k = K/b
EXAMPLE:
Compound A: mass = 1 mg
Vol. Mobile Phase:
1 mL
Vol. Stationary Phase: 1 mL
Mobile
Phase
Stationary
Phase
K=4
b= 1
k= 4
grams in
mobile phase = 0.2
Compound A: mass = 1 mg
Vol. Mobile Phase:
1 mL
Vol. Stationary Phase: 2 mL
K=4
b = 0.5
k =8
grams in
mobile phase = 0.11
If the mobile phase is moving, in which
situation will compound A move faster through the
column?
PARTITIONING
IN A
MOBILE PHASE
Theoretical Plates
0.83 mg
0.69
0.83 mg
0.69 mg
0.58
0.16 mg
0.14 mg
0.12 mg
1.0 mg
0.28 mg
0.10 mg
0.08 mg
0.07 mg
0.06 mg
Partitioning in a Mobile Phase
0.13 mg
0.13 mg
0.23
0.23 mg
0.29
0.29 mg
0.32
0.16
0.03 mg
0.14 mg
0.05
0.12 mg
0.06
0.10 mg
0.06
0.28 mg
0.08 mg
0.07 mg
0.06 mg
PARTITIONING
IN A
MOBILE PHASE
0.83
0.00 mg
mg
0.69
0.83
0.00 mg
mg
0.58
0.69
0.01 mg
mg
0.05 mg
0.17 mg
0.28 mg
0.34
0.28 mg
0.00 mg
0.00 mg
0.00 mg
0.03 mg
0.04 mg
0.07 mg
0.06 mg
1.0 mg
Note: These equilibrium steps to do not actually take
place in the column, it is a continuous process.
ANALYTE PEAKS
IN THE
MOBILE PHASE
0.83
mg
0.00 mg
0.69
0.83
0.00 mg
mg
0.58
0.69
mg
0.01 mg
0.05 mg
0.17 mg
0.28 mg
0.34
0.28 mg
0.00 mg
0.00 mg
0.00 mg
0.03 mg
0.04 mg
0.07 mg
0.06 mg
1.0 mg
How would you make this broad peak more narrow?
ANALYTE PEAKS
IN THE
MOBILE PHASE
SEPARATION
OF
PEAKS
RETENTION
k = (tr – to)/ to
Where tr = the retention time of the compound,
and to = the dead time
Higher values of k mean the analyte will stay
in the column longer. The longer it stays, the
more time there is for the peak will widen.
SELECTIVITY
a = kB/kA
the selectivity factor α and is an indication of
how well the compounds will separate. Higher α
means larger difference in retention time and
more separation
EFFICIENCY
Efficiency is a factor that is typically used to
describe peak width.
High Efficiency - narrow peaks
EFFICIENCY
The term that is generally used to describe
column efficiency is “number of theoretical
plates” or N
N = L/H
Where: L =column length
H = plate height (both in the same units)
N IN PRACTICAL TERMS...
N can be measured from the peaks on a
chromatogram..
N = 5.54
( )
2
tr
w1/2
Units for tr and to….?
Units for W1/2 …..?
RESOLUTION
The purpose of chromatography is to separate or
resolve compounds. The separation or distance
between two peaks is known as their resolution
and is a function of the 3 factors discussed
previously: retention (the time it takes for the
analytes to elute, related to k), selectivity (how
different the analytes are from each other and
related to α), and efficiency (how good the
column is, related to N)
RESOLUTION
Efficiency
Rs = ¼ (a-1/a) (k/k+1) N½
Selectivity
Retention
The effect on Rs of:
increasing a…?
increasing k…?
increasing N…?
RESOLUTION
Rs can also be calculated from actual
measurements of peak retention times and
measured peak widths
Rs = 2 (tR-B – tR-A)/(wb-A + wb-B)
Where:
each
A and B are the two peaks
tR = retention time and
wb = the peak width at the base of
peak
RESOLUTION
With a resolution value of 1.0, two peaks that
overlap by about 4%. Values less than 1.0
indicate peaks that overlap, while at a
resolution of 1.5, the peaks are considered fully
separated.
GOING
BACK TO
N….
N = L/H
The value of N is greatly dependent on the value
of H.
The value of H depends primarily on four factors:
1) the velocity of the mobile phase,
2) eddy diffusion or multipath diffusion,
3) the diffusion of the compound in the mobile phase
4) the transfer of the compound between the stationary phase
and the mobile phase.
H - Theoretical Plate Height
H = A + B/u + (Cs + Cm) u
u = the average linear mobile phase
velocity
A is a term expressing multipath diffusion
B/u is the term for longitudinal diffusion
Cs is the mass transfer term in the
stationary phase
Cm is the mass transfer term in the mobile
phase
A
Multipath
Flow
Direction
1
2
The amount of spreading is affected by
the nature of the column material and
how well the column is packed. This
factor is generally proportional to the
particle size of the packing material. This
factor must be taken into account for
packed columns, but for capillary
columns, this term is not needed since
there are no particles.
Pathways of two molecules during
elution. Distance traveled by molecule 1
is longer than that traveled by molecule
2, thus molecule 1 will take longer to
elute.
B
Longitudinal Diffusion
At low velocities longitudinal diffusion has a negative effect on
resolution, but this effect is negligible at higher velocities. This
term is very important in gas chromatography as diffusion
coefficients in gasses are orders of magnitude higher than in
liquids. In liquid chromatography, this term is typically close to
zero relative to the other terms.
Molecules diffuse from areas of high
concentration to areas of low concentration.
Flow
Over time….
Flow
Mass Transfer Terms Cs & Cm
Equilibrium between the mobile and stationary
phases is never realized
It takes time for analytes to move from the mobile phase
into the stationary phase. Because no equilibrium is
reached, some of the analytes are swept ahead of the of
the main band.
It also takes time for molecules to move back out of the
stationary phase, and some of the analyte molecules will
be left behind by the rapidly moving mobile phase.
Mass Transfer Terms Cs & Cm
The faster the mobile phase moves, the less time
there is for equilibrium between the phases and
the mass transfer effect on peak broadening is
directly related to mobile phase velocity.
Plate Height, H
VAN
DEEMTER PLOT
A + B/u + Cu
Mass Transfer (both), Cu
Multipath Term, A
Longitudinal diffusion, B/u
Linear Velocity, u