Introduction to Analytical Separations

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 Chromatography
Chromatography operates on the same principle as extraction, but one
phase is held in place while the other moves past it.

• The mobile phase (MP) (solvent flowing through the column) is

either a liquid or a gas.

• The stationary phase (SP) (the one that stays in place inside the
column) can be:

1. A viscous liquid chemically bonded to the inside of a silica

capillary column.
2. A viscous liquid coated onto the surface of solid particles that
are made of silica and packed in the column.
3. The solid silica particles themselves.
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What is Chromatography?

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 What is Chromatography?

• The partitioning of
solutes between the
mobile and stationary
phases gives rise to
separation.

• Columns are either

packed – filled with
stationary phase
particles, or open
tubular (capillary) –
hollow capillary with
stationary phase coated
on the inside walls.
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 Types of Chromatography

Adsorption – Solute adsorbed

onto surface of solid SP.

Partition – Liquid SP is bonded to

a solid surface (silica SiO2).
Solute equilibrates between the
SP and MP.

Ion-exchange – anions (-SO3-) or

cations (-N(CH3 are covalently
attached to the solid SP (resin).
Solute ions of opposite charge are
attracted to the SP by electrostatic
attraction. The MP is a liquid.

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 Types of Chromatography

Molecular exclusion (size

exclusion) – Liquid or gaseous
MP moves analyte through
column. Large molecules move
quickly, small ones are retained in
the pores

Affinity – most selective.

Specific interactions between
analyte and a second molecule
covalently attached to the SP.
Example: antibody on SP that
reacts with only one protein in the
sample.

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 A Plumber’s View of Chromatography

• Chromatogram - graph that shows the detector

response as a function of elution time

• Retention time, tr, for each component - the time

between injection of the mixture onto the column and
when that component reaches the detector

• Retention volume, Vr, - the volume of mobile phase

required to elute a particular solute from the column

• The time required for an unretained solute (methane)

to travel through the column is called the dead time, tm 7
 Retention Parameters

• The adjusted retention time, t’r, - the additional time

required to travel the length of the column, beyond
that required by the solvent

t  tr  tm
'
r

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 A Plumber’s View of Chromatography

• The relative retention (separation factor), a, for two

components is the ratio of their adjusted retention
times: '
tr 2
α '
t r1
Always expressed as greater than one

• The greater the relative retention, the greater the

separation between the two components

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 A Plumber’s View of Chromatography

• For each peak in the chromatogram, the retention factor, k, is

the time required to elute that peak minus the time tm required
for mobile phase to pass through the column, expressed in
multiples of tm:
tr  tm
k
tm

• The longer a component is retained by the column, the greater

the retention factor.

• EX: A mixture of benzene, toluene, and methane was injected into a gas
chromatograph. Methane gave a sharp spike in 42 s, whereas benzene
required 251 s and toluene was eluted in 333 s. Find the adjusted retention
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time and retention factor for each solute, and the relative retention.
 Retention Time & Partition Coefficient

• The retention factor can also be expressed as:

csVs
k
cmVm
• The ratio cs/cm is the ratio of the concentrations of
the solute in the two phases, if the column is run
slowly enough to maintain equilibrium, this ratio is
the partition coefficient, K.

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 Efficiency of Separation
Two factors contribute to how well compounds are
separated by chromatography:
1. The difference in elution time between the peaks –
the farther apart, the better their separation.
2. How broad the peaks are – the wider the peaks, the
poorer their separation.

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 Resolution
• Resolution tells us how far apart two bands are
relative to their widths. It is a measure of the ability
of the column to separate two solutes.

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Example

1.17

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 Plate Height (H)
Plate Height: A Measure of Column Efficiency
• If solute has traveled a distance x at the linear flow
rate ux (m/s), then the time it has been on the column
is t = x/ux therefore:

• Plate height, H, is the proportionality constant

between the variance, σ2 of the band, and the
distance it has traveled, x.

• The smaller H is, the better the separation 15

 Number of Theoretical Plate (N)
H = σ2/x
σ = standard deviation of the Gaussian band
x = distance traveled

For a solute emerging from a column of length L,

the number of plates, N, in the entire column is the
length L divided by the plate height.

• Also recall that x = L and σ = w/4 (because w =

4σ).
L Lx L2 16 L2
N  2  2  2
H   w 16
 Number of Theoretical Plate (N)
• The number of plates on a column:

• If we use the width at half-height:

• For two closely spaced, symmetric peaks, resolution is

governed by the Purnell equation:
N   1  k 2 
Resolution   
4   1  k2 

EX: A solute with a retention time of 407 s has a width at the

base of 13.0 s on a column 12.2 m long. Find the number of
plates and plate height.
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Useful Equations

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 Why Bands Spread
• A band of solute spreads as it travels through the
column and emerges at the detector with standard
deviation a.
• The variance is additive.

• Broadening outside the column:

Variation due to injection or detection

Variance due to connecting tubing

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 van Deemter Equation
• Plate height, H, is proportional to the variance of a
chromatographic band
• The van Deemter equation tells us how the column
and flow rate affect plate height

Contributions to plate height are:

Packed columns: A, B, and C terms.

Open tubular columns: B and C
Capillary electrophoresis: B
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Why Bands Spread

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 Multiple Flow Paths (A)
• The A term in the van Deemter equation
• Some flow paths are longer than others
• Molecules entering simultaneously exit at different
times

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 Longitudinal Diffusion (B)
• Longitudinal diffusion is greater for gases than
liquids, therefore the optimum flow rate for GC is
much higher than in LC.
2 Dm L
• The variance from diffusion is:   2 Dm t 
2

ux

• Plate height due to longitudinal diffusion:

2 2 Dm B
H longitudinal diffusion   
L ux ux

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 Equilibration Time (C)
Finite Equilibration Time Between Phases
• Solute must diffuse from the mobile phase to the
surface of the stationary phase for equilibration to
occur.

• The time required depends on the distance the solute

must travel to reach the stationary phase, and
inversely on how fast it diffuses. 24
 Equilibration Time (C)

• Plate height due to finite equilibration time:

H mass transfer  Cu x  (Cm  Cs )u x

• For gas chromatography in an open tubular column:

Mass transfer 1  6k  11k 2 r 2

in mobile Cm 
phase: 24(k  1) Dm
2

2
Mass transfer 2k d
in stationary Cs 
phase: 3(k  1) Ds
2

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 Asymmetric Peaks
• A Gaussian band shape results when the partition
coefficient K = cs/cm is independent of the
concentration of the solute on the column.
• A plot of cs versus cm is called an isotherm.

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 Overload in Chromatography

• Peak fronting: occurs when solute becomes more soluble in

the stationary phase due to overload, and the stationary phase
begins to resemble the solute.

• Peak tailing: occurs when small quantities of solute are

retained more strongly than large quantities due to high
concentration of solute.

• Salinization reduces tailing by blocking the hydroxyl

groups with nonpolar trimethylsilyl groups.

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