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
