Introduction to Analytical Separations

1
 To Do:

1. Read Chapter 23 in Quantitative Chemical

Analysis
Chapter 23: Introduction to Analytical Separations

2. Listen to recorded lectures Lessons 1-6

3. View handwritten notes if necessary

4. Complete Canvas quizzes

2
 Topics
1. Solvent Extraction and Chromatography
a. Solvent Extraction Theory
b. Solvent Extraction pH Effects
c. Chromatography Terms and Types of
Chromatography

2. A Plumber’s View of Chromatography

3. Efficiency of Separation

4. Why Bands Spread

d.van Deemter Equation
b. Isotherms
3
 1: Solvent Extraction

• Extraction is the transfer of a solute from one phase

to another.
• The goal is to isolate or concentrate the desired
analyte, or to separate it from species that would
interfere with its analysis.
• Two liquids are immiscible if they remain in separate
phases when they are mixed in any ratio.
• Organic solvents with low polarity are immiscible
with water.
• Diethyl ether, toluene, and hexane are common
solvents that are less dense than water.
• Chloroform, dichloromethane, and carbon
tetrachloride are more dense than water.
4
 1a: Solvent Extraction: Theory

• In the figure, solute S is partitioned between two

phases: S (in phase 1) ↔ S (in phase 2)
• The partition coefficient is the equilibrium constant
for the reaction:

5
 1a: Solvent Extraction: Theory
• Suppose solute S in V1 mL of solvent 1 (water) is extracted
with V2 mL of solvent 2 (toluene). Let m be the moles of S in
the system and q be the fraction of S remaining in phase 1
at equilibrium.

• The molarity of S in phase one is: qm/V1

• The molarity of S in phase two is: (1- q)m/V2

• Substituting these into the partition coefficient expression

and rearranging for q gives:

• The fraction remaining in phase 1 after n extractions, each

with volume V2, is:

6
 1a: Solvent Extraction: Theory
EX: Solute A has a partition coefficient of 3 between
toluene and water, with three times as much in the
toluene phase.

Suppose that 100 mL of a 0.010 M aqueous solution

of A are extracted with toluene.

What fraction of A remains in the aqueous phase

(a) after one extraction with 500 mL is performed or

(b) if five extractions with 100 mL are performed?

7
EX: Solute A has a partition coefficient of 3 between toluene and water, with three
times as much in the toluene phase. Suppose that 100 mL of a 0.010 M aqueous
solution of A are extracted with toluene. What fraction of A remains in the
aqueous phase (a) after one extraction with 500 mL is performed or (b) if five
extractions with 100 mL are performed?
1b: Solvent Extraction: pH Effects
• If the solute is an acid or base, its charge changes as
the pH is changed.
• A neutral species is usually more soluble in organic
solvent than a charged species.
• Consider a basic amine whose neutral form, B, has a
partition coefficient K between aqueous phase 1 and
organic phase 2.
• Suppose the conjugate acid, BH+ (acid dissociation
constant is Ka), is only soluble in the aqueous phase 1.
The distribution coefficient, D, is:

9
1b: Solvent Extraction: pH Effects

• Substituting K = [B]2/[B]1 and Ka = [H+][B]1/[BH+]1 gives

the distribution of base between the two phases, where
a is equal to [B]aq/[B]aq + [BH+]aq:

weak base

• Distribution of acid between two phases, where aHA is

the fraction of weak acid in the form HA in the aqueous
phase:

10
1b: Solvent Extraction: pH Effects

• We use the distribution coefficient in place of

the partition coefficient in our equation for q
when dealing with a species that has more than
one chemical form.

• To extract a base into water we use a pH low

enough to convert B into BH+.

• To extract an acid into water, we use a pH high

enough to convert HA into A-.
11
1b: Solvent Extraction: pH Effects

EX: Suppose that the partition coefficient for an amine, B,

is K = 3.0 and the acid dissociation constant of BH+ is Ka =
1.0 x 10-9.

If 50 mL of 0.010 M aqueous amine are extracted with 100

mL of solvent, what will be the fraction remaining in the
aqueous phase

(a) at pH 10.00 and

(b) at pH 8.00?

12
EX: Suppose that the partition coefficient for an amine, B, is K = 3.0 and the acid
dissociation constant of BH+ is Ka = 1.0 x 10-9. If 50 mL of 0.010 M aqueous amine
are extracted with 100 mL of solvent, what will be the fraction remaining in the
aqueous phase (a) at pH 10.00 and (b) at pH 8.00?
We use the distribution
coefficient in place of the
partition coefficient in our
equation for q when
dealing with a species
with more that one
chemical form.
EX: Suppose that the partition coefficient for an amine, B, is K = 3.0 and the acid
dissociation constant of BH+ is Ka = 1.0 x 10-9. If 50 mL of 0.010 M aqueous amine
are extracted with 100 mL of solvent, what will be the fraction remaining in the
aqueous phase (a) at pH 10.00 and (b) at pH 8.00?
EX: Suppose that the partition coefficient for an amine, B, is K = 3.0 and the acid
dissociation constant of BH+ is Ka = 1.0 x 10-9. If 50 mL of 0.010 M aqueous amine
are extracted with 100 mL of solvent, what will be the fraction remaining in the
aqueous phase (a) at pH 10.00 and (b) at pH 8.00?
 1c: Chromatography Terms

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. 16
 1c: Types of Chromatography
Adsorption – solid sp and a liquid
or gas mp. Solute adsorbed onto
surface of solid sp. More strongly
adsorbed = longer on column.

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. 17
 1c: Types of Chromatography
Molecular exclusion (size
exclusion) – no attraction
between analyte and sp. Liquid or
gaseous mp moves analyte
through column. Large molecules
move quickly because excluded
from pores. Small 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. 18
 2: A Plumber’s View of Chromatography

Consider a column with an inner diameter of 0.60 cm (r = 0.30

cm) and the mobile phase occupies 20% of the column volume.

• Each cm of the column length has a volume of πr2 x length =

π(0.30cm)2(1 cm) = 0.283 mL, of which 20% or 0.0565 mL is
the mobile phase.

• The volume flow rate, 0.30 mL/min for example, tells us how
many milliliters of solvent per minute travel through the column.

• The linear flow rate tells us how many centimeters are traveled
in 1 min by the solvent. The linear flow rate corresponding to
0.30 mL/min is 5.3 cm/min.
19
 2: A Plumber’s View of Chromatography

• A chromatogram is a graph that shows the detector

response as a function of elution time.

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

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

• Retention volume, Vr, is 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, tm20.
 2: A Plumber’s View of Chromatography

• The adjusted retention time, t’r, for a retained solute

is the additional time required to travel the length of
the column, beyond that required by the solvent.
'
t t r  t m
r

21
 2: A Plumber’s View of Chromatography

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

components is the ratio of their adjusted retention
times:
'
t
α r2
'
t r1

• The greater the relative retention, the greater the

separation between the two components.

• Always expressed as greater than one.

22
 2: 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:
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 time and
retention factor for each solute, the relative retention, and the unadjusted relative 23
retention.
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
time and the retention factor for each solute, the relative retention, and the
unadjusted relative retention.
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
time and retention factor for each solute, the relative retention, and the
unadjusted relative retention.
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
time and retention factor for each solute, the relative retention, and the
unadjusted relative retention.
 2: A Plumber’s View of Chromatography

Relation Between Retention Time and the Partition

Coefficient:

• The retention factor can 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.
27
 2: A Plumber’s View of Chromatography

• Relation of retention time to partition coefficient:

• Because tr' is proportional to k and proportional to K, the

relative retention can also be expressed as:

• The greater the ratio of the partition coefficients, the

greater the separation between the two components in
the mixture – the physical basis of chromatography.
28
 3: 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.

Resolution
• Solute moving through a chromatography column tends to
spread into a Gaussian shape with standard deviation σ.
“Random Walk Model”
• A symmetric spread of velocities around the mean value results
with a standard deviation of σ. 29
 3: Efficiency of Separation
• The longer the solute spends passing through the
column, the broader the band becomes.
• Common measures of the breadth are:
1. Width w1/2 measured at a height equal to one half
of the peak height.
2. Width at the baseline between tangents drawn at
the steepest parts of the peak.

30
 3: Efficiency of Separation
• Column 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.
• In chromatography, the resolution of two peaks from
each other is defined as:

31
 3: Efficiency of Separation
Diffusion
• One main cause of band spreading is diffusion – the
net transport of solute from a region of high
concentration to a region of low concentration caused
by the random movement of molecules.

32
 3: Efficiency of Separation
• If solute starts in an infinitely sharp layer with m
moles per unit cross-sectional area of the column
and spreads by diffusion as it travels, then the
Gaussian profile of the band is described by:

Broadening of chromatography
band by diffusion:

• Comparing this equation to the formula for a

Gaussian peak, the standard deviation of the band is:

33
 3: Efficiency of Separation
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.
34
 3: Efficiency of Separation
• Plate height is approximately the length of
column required for one equilibration of the solute
between the mobile and stationary phases.
• It is different for different analytes on the same
column due to differences in diffusion
coefficients.
• The smaller the plate height, the narrower the
band and the better the separation.
• Plate heights are ~0.1-1 mm for gas
chromatography, 10 mm for high-performance
liquid chromatography, and <1 mm for capillary
electrophoresis. 35
 3: Efficiency of Separation
• Since plate height, H = σ2/x where σ is the
standard deviation of the Gaussian band and x is
the 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).
2 2
L Lx L 16 L
N  2  2  2
H   w
36
 3: Efficiency of Separation
• The number of plates on a column:

• If we use the width at half-height:

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.
37
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.
 3: Efficiency of Separation
• For two closely spaced, symmetric peaks, resolution is
governed by the Purnell equation: N   1 k 2 
Resolution   
4   1  k2 
• Resolution is proportional to the square root of N, so doubling
the column length increases the resolution by 2 1/2.

EX: Two solutes have a relative retention of a= 1.08 and

retention factors k1 = 5.0 and k2 = 5.4. The number of
theoretical plates is nearly the same for both compounds. How
many plates are required to give a resolution of 1.5? Of 3.0? If
the plate height is H = 0.5 mm in gas chromatography, how long
must the column be for a resolution of 1.5?
39
EX: Two solutes have a relative retention of a= 1.08 and retention
factors k1 = 5.0 and k2 = 5.4. The number of theoretical plates is nearly
the same for both compounds. How many plates are required to give a
resolution of 1.5? Of 3.0?
EX: If the plate height is H = 0.5 mm in gas chromatography, how long
must the column be for a resolution of 1.5?
3: Efficiency of Separation

42
 4a: Why Bands Spread: 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
43
 4a: Why Bands Spread: van Deemter Equation

Longitudinal Diffusion
• Solute molecules
diffuse away from the
concentrated center
of the band in both
directions due to the
concentration
gradient.
• This type of diffusion
is inversely
proportional to the
mobile phase flow
rate.
44
 4a: Why Bands Spread: van Deemter Equation

• 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

45
 4a: Why Bands Spread: van Deemter Equation

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.
46
 4a: Why Bands Spread: van Deemter Equation

• Plate height due too 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  2
phase: 24(k  1) Dm

2
Mass transfer 2k d
in stationary Cs  2
phase: 3(k  1) Ds
47
 4a: Why Bands Spread: van Deemter Equation

Multiple Flow Paths

• The A term in the van Deemter equation.

• Some flow paths are longer than others.

48
 4a: Why Bands Spread: van Deemter Equation

Advantages of Open Tubular Columns

• Higher resolution
• Shorter analysis time
• Increased sensitivity
The radius of the column must be small and the stationary
phase must be as thin as possible.
• Disadvantage of Open Tubular Columns
• Lower sample capacity

49
 4b: Why Bands Spread: Isotherms
• A Gaussian bandshape 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.

50
 4b: Why Bands Spread: Isotherms
• When too much solute has been applied to the
column, the solute becomes more and more
soluble in the stationary phase, so much so that the
stationary phase begins to resemble the solute.

• The front of an overloaded peak has gradually

increasing concentration.

• Overloading produces a gradual rise and an abrupt

fall of the chromatographic peak.

• The peak is said to “front”/”fronting”.

51
 4b: Why Bands Spread: Isotherms
• When small quantities of solute are retained more
strongly than large quantities, it leads to a long “tail” of
gradually decreasing concentration after the peak.
• The peak is said to “tail”/”tailing”.
• Sites that bind solute strongly lead to tailing.
• Silica surfaces of columns and stationary phase
particles have hydroxyl groups that form hydrogen
bonds with polar solutes.
• Silanization reduces tailing by blocking the hydroxyl
groups with nonpolar trimethylsilyl groups.

52