CHM510

CHAPTER 1
CHROMATOGRAPHIC SEPARATIONS

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 Course Learning Outcomes

Students should be able to:

1. Understand the general concepts and principles of
separation in chromatographic technique.
2. Understand the efficiency of separation:
a) Resolution
b) Plate theory
c) Rate theory (Van Deemter Theory)
3. Apply chromatographic equations in solving problems
4. Plot & interpret chromatogram

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 Definition of Chromatography
1. Chromatography is a separation method in which the
components in a mixture to be separated are distributed
between two phases:
◦ A stationary phase
◦ A mobile phase
2. Moves in a definite direction.
3. Separation of compounds depends on the rates at which
the components moves through the stationary phase.

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Any chromatography system is composed of three components:

1. Stationary phase - the one that stays in place inside the column
or on a solid surface (flat sheet); phase that is stationary in
chromatography

2. Mobile phase - the solvent moving through, carrying with it the

component mixture
1. GC – carrier gas (He)
2. HPLC – liquid (H2O, organic solvent)
3. SFC – supercritical fluid (CO2)

3. Mixture to be separated – targeted analyte

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The chromatographic process

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 Pack column TLC

Stationary phase (solid particles) Stationary phase fixed on a solid

in a column (HPLC) surface/plate
(thin layer chromatography, TLC)

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 Stationary phase

Cross section

Capillary column

GC oven

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 Types of chromatography

➢ Gas chromatography (GC)

➢ High performance liquid chromatography (HPLC)

➢ Thin layer chromatography (TLC)

➢ Supercritical fluid chromatography (SFC)

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 General principle of
chromatography
1. Components of a mixture are carried through the
stationary phase by the flow of a mobile phase

2. Separation occur because of differing affinities of

components with stationary phase & mobile phase.

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3. Those components that are strongly retained by the
stationary phase move slowly with the flow of mobile phase.

4. Components that are weakly held by the stationary phase

travel rapidly.

5. These differences in mobility (migration rate) will cause the

sample components separate.

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The separation process

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Migration of Solutes
sample mobile phase

A+B
B
A
packed
column B
A

B
A B
t0 t1 t2 t3 t4
detector A B
signal

time
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Chromatographic results

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 Chromatogram

➢ If a detector that responds to solute concentration is

placed at the end of the column during elution & its
signal is plotted as a function of time (or volume of
added mobile phase) a series of peak is obtained.

➢ Such plot is called chromatogram.

A record of a separation produced by a recorder or

integrator based on the signal obtained from the detector.

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➢ Peak positions (tR) used to identify components; peak
areas to determine amounts of each component/analyte.

Example of report obtained from GC:

Peak RetTime Witdth Area (pA*s) Height (pA) Area
(min) (min) (%)
B t1 0.05 327.098 754.641 6.5
A t2 0.04 218.341 167.492 4.3
C t3 0.09 4483.544 99.808 89.2

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 Common terms
❖ ELUTION: Process in which components are washed through a
stationary phase by the movement of a mobile phase

❖ ELUENT: Mobile phase

❖ ELUATE: Component leaving column

❖ SOLUTE: Solid that is dissolved in liquid

❖ ANALYTE: The chemical/substance that is determined in an

analytical procedure

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 Migration rates of solutes

The relative rates at which the 2 analytes are eluted are

determined by the magnitude of the equilibrium constant
by which the solute distribute themselves between
stationary & mobile phase.

For solute A, Amp Asp

The equilibrium constant, Kc for the distribution of species A

between mobile phase & stationary phase is called the
distribution constant/partition coefficient.

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 Principle of Chromatographic
Separation
Different distribution of analytes between the mobile and
stationary phase results in different migration rates (velocities)

[A] stationary
K=
[A] mobile

K = Partition coefficient

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 Cs
Kc =
Cm

➢ Kc is defined as the molar concentration of analyte in the

stationary phase divided by the molar concentration of the
analyte in the mobile phase.

➢ Large Kc means analyte prefer stationary phase, while small Kc

shows analyte prefer in mobile phase.

➢ The Kc for each analyte must differ to achieve separation.

➢ Kc can be manipulated by appropriate choice of mp, sp or both.

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 Retention time, tR
tR: time for the analyte to pass through the column or the
time from sample injection to detection.

Each analyte will have a different tR.

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A compound not-retained by the stationary phase will elute out
of the column at time tM - void time or dead time (sometimes
designated by t0)

tM:
▪ The time a non-retained compound spends in the mobile
phase
▪ The amount of time the non-retained compound spends in
the column
▪ The time taken for the mobile phase to pass through the
column

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 Adjusted retention time, tR’
t’R or tS is the time a compound spends in the stationary
phase.

t’R is the difference between tR and tM for a compound.

t’R = tR - tM

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 Average Linear Velocity (µ)/flow
rate of mobile phase

➢ Average ‘speed’ of the carrier gas in cm/sec (cm of

column traveled per second/min by a carrier gas.

➢ Influence the retention time & efficiency.

Flow rate: How much mobile phase passed / minute (mL/min).

Linear velocity: Distance passed by mobile phase per 1
min in the column (cm/min).

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Average Linear Rate

• Average Linear Velocity of analyte migration through the

column -
• Average linear rate of migration of a solute, 
L
= L = length of column (cm)
tR

• Average linear rate of movement of mobile phase molecules, u

(velocity)

L L = length of column (cm)

u=
tM tM = retention time of un-retained
peak (sec)

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 Retention/capacity factor, k’

k’ used to described the migration rate of solutes on

column.

The k’ for analyte A is defined as:

k’A = (tR)A – tM
tM

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• k’ can be derived from chromatogram.

• A large k’ (>20-30) favors good separation, but increased

elution time.

• Compound with larger Kc, will have larger kʹ & elute later.

• Good separation, 2<k’>10 (balance between elution time &

resolution).

• kʹ of a column is a direct measure of the strength of the

interaction of the solute with the stationary phase.

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Where; tM = t0

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 Selectivity factor, α

▪ α described the separation of 2 species (A & B) on the

column.

▪ α = k’B/k’A (Retention factor)

▪ k’A = [(tR)A – tM]/tM and k’B = [(tR)B – tM]/tM

▪ α = (tR)B – tM/(tR)A – tM

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 α = (tR)B – tM
B is most retained
(tR)A – tM A is least retained

▪ α always greater than 1.

▪ If α = 1, the 2 compounds cannot be separated.

▪ The higher the α, the more separation between 2

compounds or peaks.

▪ When calculating the α, species A elutes faster than B.

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 Efficiency of separation
Two factors affect how well two components are separated:

✓ Difference in retention time, tR between peaks (farther

apart, better separation)
✓ Peak widths (an efficient separation will produce narrow
peaks)

Solutes in a column spread into a Gaussian profile.

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Gaussian peak shape:
Detector response

time

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 Resolution, Rs
➢ Rs : The degree (how well) of separation of 2 peaks
➢ Although α describes the separation of peak centres, it
does not take into account peak widths (column
efficiency).
➢ Rs is preferred over α since both retention (tR) & column
efficiency (Wb) are considered in defining peak
separation.

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 Overlap of 2 peaks with different degrees of Rs

Rs=0.50 Rs=0.75

t0 2s time t0 3s time

Rs=1.00 Rs=1.50
Rs1 Complete
separation
is good

t0 4s time t0 6s time

Higher Rs, better separation

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 Rs=1.50

t0 time

Rs = 1.5 baseline separation / complete separation of two

neighboring solutes → ideal case.
Rs = 1.0 is considered adequate separation (for quantitative
analysis)
Rs < 1, compounds are overlapped or not separated (can’t
be used for quantitative analysis)

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 Column Efficiency

Column efficiency is related to :

▪ a solute’s peak width (An efficient column will produce
narrow peaks)
▪ various kinetic processes that are involved in solute
retention & transport in the column.
2 theories to describe column efficiency:
▪ Plate Theory - to measure column performance & efficiency
▪ Rate Theory - measure the contribution to band broadening

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The column efficiency is expressed as a number of:
▪ Theoretical plates, N

▪ Plate height (H)or Height Equivalent to a Plate Theory

Theoretical Plate (HETP)

H & N provide useful measures to compare the performance of

different columns for a given analyte.

Column efficiency increases with N ↑ & H ↓

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 Theoretical plates, N

▪ Theoretical plates: View column as divided into a number of

adjacent imaginary (do not really exist) segments.
▪ N is a number indicating how good a column is for a separation.
▪ N ~ few hundred to several hundred thousand.

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▪ Plate theory: Treats separation in discrete stages, more stages =
more plates.

▪ Equilibration of the solute between the stationary & mobile

phase occur in these ‘plates’.

▪ The analyte moves down the column by transfer of equilibrated

mobile phase from 1 plate to the next.

▪ Columns with high N produce narrow peak, resulting in better

separation of 2 compounds (higher column efficiency) than a
column with a lower N.

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 Column with high N have;
✓higher column
efficiency
✓narrower peak at a
given tR
Than a column with a
lower N.

(Tangen method) (Peak half-height method)

▪ N is specific for each solute on a given column.

▪ Increasing tR increases N.

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▪ N depends upon the length of the column.

L
N =
HETP

How to increase N ?
1. Using longer column (L)
2. Using column with small H

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▪ N required to obtain a certain Rs :

▪ Rs can be improved by lengthening the column, thus, increasing

the N

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 Plate height (H) or Height Equivalent
to a Theoretical Plate (HETP)
▪ H or HETP is approximately the length of column in which the analyte
equilibrates between mobile & stationary phase.

▪ Compare efficiencies of columns with different lengths:

H = L/N
▪ An increase in efficiency as a decrease in the width of each sample peak,
showing that the bands of sample have not spread much as they passed
through the column.

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H is used to measure of band spreading per unit length of the column

The smaller the H, the narrower the peak width

HETP ↓, Rs ↑ (N ↑ )

❑ Greater separation occurs with:

✓Greater number of N
✓H or HETP becomes smaller

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Chromatographic Relationships

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 Why bands spread?

A band of solute spreads as it travel through a chromatographic

column

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 Zone/band broadening

➢ Band broadening reflects a loss of column efficiency.

➢ The slower the rate of mass-transfer processes (elution)
occurring while a solute migrate through a column, the
broader the band.

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❑ Mechanisms which contribute to band broadening:

✓ Multiple paths/Eddy diffusion, A

✓ Longitudinal diffusion, B
✓ Mass transfer between phases/equilibration time
between phases, C

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van Deemter equation for plate height (H):

H = A + B/v + Cv
Where,
A = Multiple paths / Eddy diffusion term
B = Longitudinal diffusion term
C = Mass transfer between phases / equilibration time
between phases term
v = flow rate or mobile phase velocity

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van Deemter equation for plate height (H) :

H = A + B/v + Cv

✓ van Deemter equation tell how the column &

flow rate affect the H

✓ The smaller the H, the narrower the bandwidth.

✓ H represents the band broadening.

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 Total Band Broadening

Each term introduces its part in the total band broadening,

therefore the sum of all of them will give the total column
plate height.

H = HA + HB + Hc

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 A term – Multiple Paths
(Eddy Diffusion)
Each molecule takes a different path through column

❑ The mobile phase moves through the column which is

packed with stationary phase.
❑ Solute molecules will take different paths of different
lengths through the stationary phase at random.
❑ This will cause broadening of the solute band.

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A term – Multiple Paths

Some paths are longer than others, molecules entering the

column at the same time (left) but eluted out at different times
(right).

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A term – Multiple Paths

A = 2λdp
❑ A is directly proportional to the packing particle diameter, dp.

❑ λ is a function of packing uniformity and column geometry

(same shape).

❑ In packed column, A ≠ 0, in open tubular column (capillary

column), A = 0.

❑ A is small when using smaller particles size & uniform size.

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❑ A is small when using smaller particles size & uniform size.
❑ Smaller dp will reduce the differences in the path lengths.

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 B term – longitudinal diffusion

Diffusion is a process in which analyte migrate from a more

concentrated to a more dilute region.

Longitudinal diffusion in column chromatography is a band

broadening process in which solute diffuse from the
concentrated center of zone to more dilute region (towards
& opposed to the direction of the flow of mobile phase).

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B term – longitudinal diffusion

High Concentration

Low Concentration

Component Molecule
Concentration

▪ Solute diffuses out from the center to the edges.

▪ Concentration of solute is lower at the edges of a band.
▪ This cause band broadening.

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B term – longitudinal diffusion

Start

Time # 1 Time # 2 Time # 3

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B term – longitudinal diffusion

B/v = 2ɣDM/v

❑ The longitudinal diffusion effect on H is inversely proportional to v

because the solute spends less time in the column at high v & less
diffusional broadening occurs.
❑ Obstructive factor,  shows that longitudinal diffusion is hindered by
the packing.
•  is lower for a packed column (ie  = 0.6) than an unpacked
(capillary) column (ie  = 1).

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B term – longitudinal diffusion

B/v = 2ɣDM/v
❑ DM = analyte diffusion coefficient in the mobile phase.

• The faster the rate of diffusion of the analyte, the greater the extent of
longitudinal diffusion → band broadening, less efficient. DM is higher in
gasses than in liquid. B term is common source of band broadening in
GC

• DM ↓ , B-term ↓, H low, peak width narrow – more efficient.

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 Effect of mobile phase flow rate on H for GC and LC

B term is common source of band broadening in GC,

DM higher
H for HPLC smaller

Velocity, VGC is larger

Velocity, VGC is larger than VLC H for HPLC smaller than H for GC
➢ GC faster analysis time than HPLC ➢ HPLC more efficient (B term)

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 C term – mass transfer between
phases
The term Cv comes from the finite (or a certain amount) time
required for solute to equilibrate between mobile &
stationary phase.

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C term – mass transfer between phases

❑ The equilibrium between the mobile & stationary phase is established

so slowly that a chromatographic column always operates under non-
equilibrium conditions.

❑ So, analyte molecules at the front of a band are swept ahead without
equilibrating with the stationary phase & those at the trailing edge are left
behind for a longer time in the stationary phase

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C term – mass transfer between phases

❑ If the velocity of the mobile phase is high & the analyte

has a strong affinity for the stationary phase, then the
analyte in the mobile phase will move ahead of the
analyte in the stationary phase.

❑ The band of analyte is broadened.

❑ The higher the velocity of mobile phase, the worse the

broadening becomes.

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 Mass transfer coefficient
(CS & CM)

Hmass transfer = Cv = (Cs + CM)v

▪ CMv = the mobile phase mass transfer term.
▪ Csv = the stationary phase mass transfer term.

❑ The mass-transfer effect on H is directly proportional to v because the solute

residence time is longer at low v, the deviation from equilibrium is less &
band broadening or H is smaller.

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 Mass transfer in
stationary phase (Csv)

Csv = (fS(k’)df2/DS)v

▪ df = stationary phase film thickness (most important factor)

▪ Ds = diffusion coefficient of the solute in the film
▪ A complex function of fs(k’) of the retention factor k’.

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❑ Decreasing df, reduces H & increase efficiency because solute can diffuse
faster from the farthest depth of the stationary phase into the mobile
phase.

❑ Eg.
▪ With thick films, molecules must on the average travel farther to reach the
surface; with smaller Ds, they travel more slowly.
▪ The consequence of both factors lower rate of mass transfer & an increase
in H.

Csv is less if the df is smaller, or the solute DS is larger

= H↓, efficiency ↑

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 Mass transfer in
mobile phase (CMv)

CMv = (fM(k’)dp2/DM)v
▪ dp = particle diameter of packing
▪ DM = mobile phase diffusion coefficient

▪ CMv is less if dp is smaller (hence greater surface area) or DM is larger

▪ Small particles (dp) reduce the distance solute must diffuse in the mobile
phase.

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 Note:

Both longitudinal & mass transfer broadening depend upon

the rate of diffusion of analyte molecules but the direction
of diffusion in the 2 cases is different.

❑ Longitudinal broadening arises from the tendency of

molecules to move in directions that tend to parallel the
flow.

❑ Mass transfer broadening occurs from diffusion that

tends to be at right angles to the flow.

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 The extend of longitudinal
broadening is inversely
related to flow rate.

For mass transfer, the faster the mobile phase moves, the less
time there is for equilibrium to be approached.

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 Van deemter plot
❑ Determine the optimum mobile phase flow rate to obtain the minimum H
(maximum efficiency).

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❑ The minimum in the curve indicates the flow velocity which will give the
maximum efficiency in that particular column.

❑ Using a carrier gas flow which is higher than the optimum will cause some
loss of efficiency.

❑ However, this may be a satisfactory compromise, since it will also reduce

the tR, & so may shorten the time required for an analysis.

❑ Using a flow slower than the optimum is, however, never a good idea,
since it lengthens the analysis time & also causes a loss in efficiency.

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Why optimization of flow rate of the mobile phase is one of
the important steps in chromatographic analysis?

➢ For B/v, high flow rate will decrease the term & thus reduces band
broadening.

➢ But for Cv, low flow rate is required to give the molecules enough
time to reach equilibrium.

➢ Thus, an optimum flow rate is high enough to reduce diffusion &

low enough to give enough time for achieving equilibrium.

Ooooo…

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Decrease in flow rate will
increase diffusion, H will
increase

Increase in flow rate – not

enough time for equilibrium –
inefficient mass transfer, H
increases

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THANK YOU FOR YOUR ATTENTION
THANK YOU
Q & A SESSION
CHM 510
ANALYTICAL SEPARATION METHODS

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