Separation Methods

An Introduction to
Chromatographic
Separations

Chromatography
Chronology
column chromatography
paper chromatography
gas-liquid chromatography
thin layer chromatography (TLC)
high-pressure liquid chromatography
(HPLC - high-performance liquid
chromatography)

Parts of Column

column
support
stationary phase
mobile phase

Parts of Column
Column
copper tubing
stainless steel tubing
glass tubing

Parts of Column
Support
finely divided solids
ground firebrick
alumina, specially treated

walls of column for capillary columns

Parts of Column
Stationary Phase
stationary phase evenly dispersed on
surface of support
column chromatography
non-volatile, viscous liquids dispersed evenly on
surface of support

Parts of Column
Stationary Phase
stationary phase evenly dispersed on
surface of support
planar chromatography
porous paper in paper chromatography
finely ground solid spread evenly on glass or plastic
plate for tlc (thin-layer chromatography

Parts of Column
Mobile Phase
sample mixture carried through
stationary phase by mobile phase
non-reactive gas in glc (gas-liquid
chromatography, gc)
non-reactive liquid in llc (liquid-liquid
chromatography, lc)

Linear Chromatography
Cs
K = ----Cm
K is T dependent
Cs => conc. in stationary phase
Cm => conc. in mobile phase

Linear Elution Chromatography

Theories of
Elution Chromatography
some zone broadening
zone separation

Theories of
Elution Chromatography
N = L/H
where n => number of plates
L => length of column
H =>height equivalent to a
theoretical plate
(HETP)

Rate Theory of
Chromatography
zone shapes (Gaussian curve)
Why?

Rate Theory of Chromatography

Rate Theory of Chromatography

Rate Theory of Chromatography

Rate Theory of
Chromatography
W=4
where

W =>width of peak at baseline

=>standard deviation
expressed as time

Rate Theory of
Chromatography
H = 2/L
where

=>standard deviation
expressed as length

Rate Theory of
Chromatography
thus
L
L
L2
N = ---- = ------- = ------2
H
2/L

Rate Theory of
Chromatography
and

= -----L/tR
where

tR =>retention time
L/tR => rate of travel of band

Rate Theory of
Chromatography
therefore,
LW
= --------4tR

Rate Theory of
Chromatography
and
LW2
H = ---------16tR2
N = 16 (tR/W)2

Sources of Zone Broadening

classical van Deemter Equation
H = A + B/u + Cu
where A => eddy diffusion
B => longitudinal diffusion
u => flow rate
C => mass transfer

Eddy Diffusion
A = 2dR
where =>packing factor
dR =>
average diameter of
particle
caused by many pathways
minimized by careful packing

Longitudinal Diffusion
B = 2DM
where =>
DM =>

obstruction factor
diffusion coefficient of
solute in the
mobile phase

minimized by lowering temperature of

column oven and decreasing flow rate

Mass Transfer
qR (1 - R)df2
dR2
C = -------------------- + ----------DS
DM
where

df => film thickness of

stationary phase (most
important factor)
dR =>diameter of support
particle

Mass Transfer
qR (1 - R)df2
dR2
C = -------------------- + ----------DS
DM
where

R =>retention ratio: tM/tR

DS =>diffusion coefficient for
solutes in stationary phase

Mass Transfer
qR (1 - R)df2
dR2
C = -------------------- + ----------DS
DM
where

DM =>diffusion coefficient for

solutes in mobile phase
q & =>constants for
column

Sources of Zone Broadening

modernized version
H = B/u + CSu + CMu
where CS =>
coefficient of mass
transfer in the
stationary phase
coefficient of mass
CM =>
transfer in the
mobile phase

Sources of Zone Broadening

Separations on Columns
Z
Rs = ------W
where Rs =>
W =>
Z =>

column resolution
av. width of peaks
time separation,
difference of
retention time

Separations on Columns
Z
2((tR)y - (tR)x)
Rs = ------- = ------------------W
Wx + Wy
where

(tR)x =>
(tR)y =>

retention time,
component x
retention time,
component y

Separations on Columns
(tR)y - (tR)x
(N)1/2
Rs = ------------------ * --------4
(tR)y

Separations on Columns

Separations on Columns

Separations on Columns

EXAMPLE:
Substances A and B were found to have
retention times of 6.4 and 14.4 min,
respectively, on a 22.6 cm column. An
unretained sample of air passed through the
column in 1.30 min. The widths of the peak
bases were 0.45 and 1.07 min. Calculate the:
(a.) column resolution

EXAMPLE:
Substances A and B were found to have
retention times of 6.4 and 14.4 min, respectively,
on a 22.6 cm column. An unretained sample of
air passed through the column in 1.30 min. The
widths of the peak bases were 0.45 and 1.07 min.
Calculate the: (a.) column resolution
2((tR)y - (tR)x) 2(14.4 - 6.4)
Rs = ----------------- = ---------------- = 10.5
(0.45 + 1.07)
Wx + Wy

EXAMPLE:
Substances A and B were found to have
retention times of 6.4 and 14.4 min, respectively,
on a 22.6 cm column. An unretained sample of
air passed through the column in 1.30 min. The
widths of the peak bases were 0.45 and 1.07 min.
Calculate the: (b.) the av. no. of plates in the
column
N = 16 * (tR/W)2
for component A
NA = 16 * (6.4/0.45)2 = 3.2 x 103 plates

EXAMPLE:
Substances A and B were found to have
retention times of 6.4 and 14.4 min, respectively,
on a 22.6 cm column. An unretained sample of
air passed through the column in 1.30 min. The
widths of the peak bases were 0.45 and 1.07 min.
Calculate the: (b.) the av. no. of plates in the
column
for component B
NB = 16 * (14.4/1.07)2 = 2.9 x 103 plates

EXAMPLE:
Substances A and B were found to have
retention times of 6.4 and 14.4 min,
respectively, on a 22.6 cm column. An
unretained sample of air passed through the
column in 1.30 min. The widths of the peak
bases were 0.45 and 1.07 min. Calculate the:
(c.) the plate height
H = L/N
for component B
H = L/NB = (22.6 cm)/(2.9 x 103 plates)
= 7.8 x 10-3cm/plate

EXAMPLE: Substances A and B were found to

have retention times of 6.4 and 14.4 min,
respectively, on a 22.6 cm column. An
unretained sample of air passed through the
column in 1.30 min. The widths of the peak
bases were 0.45 and 1.07 min. Calculate the:
(d.) the length of column required to achieve a
resolution of 1.5
N1 (Rs)12
--- = -------- = ((Rs)1/(Rs)2)2
N2 (Rs)22

EXAMPLE: Substances A and B were found to have

retention times of 6.4 and 14.4 min, respectively, on a
22.6 cm column. An unretained sample of air passed
through the column in 1.30 min. The widths of the
peak bases were 0.45 and 1.07 min. Calculate the:
(d.) the length of column required to achieve a
resolution of 1.5
N1
(Rs)12
--- = -------- = ((Rs)1/(Rs)2)2
N2 (Rs)22
where
N1 = (NA + NB)/2 = (3.2 x 103 + 2.9 x 103)/2
= 3.1 x 103 plates
R1 = 10.5

EXAMPLE: Substances A and B were found to have

retention times of 6.4 and 14.4 min, respectively, on a
22.6 cm column. An unretained sample of air passed
through the column in 1.30 min. The widths of the
peak bases were 0.45 and 1.07 min. Calculate the:
(d.) the length of column required to achieve a
resolution of 1.5

N2 = ((Rs)2/(Rs)1)2 * N1 = (1.5/10.5)2 *
3.1 x 103 plates
N2 = 63 plates

EXAMPLE: Substances A and B were found to have

retention times of 6.4 and 14.4 min, respectively, on a
22.6 cm column. An unretained sample of air passed
through the column in 1.30 min. The widths of the
peak bases were 0.45 and 1.07 min. Calculate the:
(d.) the length of column required to achieve a
resolution of 1.5

Hav = (HA + HB)/2 = ((7.8 + 7.0)x 10-3)/2

= 7.4 x 10-3 cm/plate
L = Hav * N2 = (7.4 x 10-3cm/plate)(63 plates)
= 0.46 cm

EXAMPLE: Substances A and B were found to have

retention times of 6.4 and 14.4 min, respectively, on a
22.6 cm column. An unretained sample of air passed
through the column in 1.30 min. The widths of the
peak bases were 0.45 and 1.07 min. Calculate the:
(d.) the length of column required to achieve a
resolution of 1.5

(e)
(tR)1
(Rs)12
-------- = ---------- = ((Rs)1/(Rs)2)2
(tR)2
(Rs)22
(tR)2

= ((Rs)1/(Rs)2)2 * (tR)1

EXAMPLE: Substances A and B were found to have

retention times of 6.4 and 14.4 min, respectively, on a
22.6 cm column. An unretained sample of air passed
through the column in 1.30 min. The widths of the
peak bases were 0.45 and 1.07 min. Calculate the:
(d.) the length of column required to achieve a
resolution of 1.5

(e)
(tR)2 = ((Rs)1/(Rs)2)2 * (tR)1
(tR)2 = (1.5/10.5)2 * (10.4 min)
= 0.13 min = 7.8 sec

Applications of Chromatography
Qualitative Analysis
Quantitative Analysis

Analyses Based on Peak Height

Analyses Based on Peak Areas
Calibration and Standards
The Internal Standard Method
The Area Normalization Method