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
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
Web Dictionary:
Chromatography
Carrier Gas
Flow Control
Injector
Port
Column
Column Oven
Detector
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
K = Co/Cw

Co is concentration in the organic phase
(solvent)
Cw is the concentration in the aqueous
phase (water)
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
molar concentration in stationary phase
molar concentration in mobile phase

K =
PARTITION COEFFICIENT ETC.
mass in the stationary phase
mass in the mobile phase

volume of mobile phase
volume of stationary phase

concentration in stationary phase
concentration in mobile phase

K =
k =
b =
PARTITION COEFFICIENT ETC.
k = K/b
If mass = volume x concentration then:
EXAMPLE:
K = 4
b =
k =
grams in
mobile phase =
Compound A: mass = 1 mg
Vol. Mobile Phase: 1 mL
Vol. Stationary Phase: 1 mL
Compound A: mass = 1 mg
Vol. Mobile Phase: 1 mL
Vol. Stationary Phase: 2 mL
K = 4
b =
k =
grams in
mobile phase =
Mobile
Phase
Stationary
Phase
1
4

0.2
0.5
8

0.11
If the mobile phase is moving, in which
situation will compound A move faster through the
column?
PARTITIONING IN A MOBILE PHASE
1.0 mg
0.16 mg
0.83 mg 0.83 mg
0.14 mg
0.69 mg
0.69 mg
0.12 mg
0.58 mg
0.10 mg
0.08 mg 0.07 mg 0.06 mg
0.28 mg
Theoretical Plates
0.16 mg
0.03 mg
0.13 mg
0.14 mg 0.12 mg 0.08 mg
0.07 mg 0.06 mg
0.28 mg
0.13 mg 0.23 mg
0.05 mg 0.10 mg
0.23 mg
0.12 mg
0.29 mg
0.06 mg
0.29 mg
0.10 mg
0.32 mg
0.06 mg
Partitioning in a Mobile Phase
PARTITIONING IN A MOBILE PHASE
1.0 mg
0.00 mg
0.83 mg 0.83 mg
0.00 mg
0.69 mg
0.69 mg
0.00 mg
0.58 mg
0.03 mg
0.04 mg 0.07 mg 0.06 mg
0.28 mg 0.17 mg 0.05 mg 0.34 mg 0.28 mg 0.01 mg 0.00 mg 0.00 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
1.0 mg
0.00 mg
0.83 mg 0.83 mg
0.00 mg
0.69 mg
0.69 mg
0.00 mg
0.58 mg
0.03 mg
0.04 mg 0.07 mg 0.06 mg
0.28 mg 0.17 mg 0.05 mg 0.34 mg 0.28 mg 0.01 mg 0.00 mg 0.00 mg
How would you make this broad peak more narrow?
ANALYTE PEAKS IN THE MOBILE PHASE
SEPARATION OF PEAKS
RETENTION
k = (t
r
t
o
)/ t
o


Where t
r
= the retention time of the compound,
and t
o
= 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 = k
B
/k
A

the selectivity factor and is an indication of
how well the compounds will separate. Higher
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...
Units for t
r
and t
o
.?
Units for W
1/2
..?
N can be measured from the peaks on a
chromatogram..
N = 5.54
t
r

w
1/2

( )
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 ), and efficiency (how good the
column is, related to N)
RESOLUTION
R
s
= (a-1/a) (k/k+1) N

The effect on R
s
of:
increasing a?
increasing k?
increasing N?
Efficiency
Selectivity
Retention
RESOLUTION
R
s
= 2 (t
R-B
t
R-A
)/(w
b-A
+ w
b-B
)

Where: A and B are the two peaks
t
R
= retention time and
w
b
= the peak width at the base of
each peak

R
s
can also be calculated from actual
measurements of peak retention times and
measured peak widths
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 + (C
s
+ C
m
) u
u = the average linear mobile phase
velocity
A is a term expressing multipath diffusion
B/u is the term for longitudinal diffusion
C
s
is the mass transfer term in the
stationary phase
C
m
is the mass transfer term in the mobile
phase
Multipath
1
2
Flow
Direction
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.
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.
B Longitudinal Diffusion
Flow
Flow
Molecules diffuse from areas of high
concentration to areas of low concentration.
Over time.
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.
Equilibrium between the mobile and stationary
phases is never realized
Mass Transfer Terms C
s
& C
m

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 C
s
& C
m

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.
VAN DEEMTER PLOT
Linear Velocity, u
P
l
a
t
e

H
e
i
g
h
t
,

H

Multipath Term, A
Mass Transfer (both), Cu
Longitudinal diffusion, B/u
A + B/u + Cu