General Chemistry II

Module 8
Fundamentals of Analytical
Separations

Analytical Separations
Majority of cases complex mixtures
are encountered and the analytical
chemist must:
separate
identify and
measure one or more components

Solvent Extraction

Extraction: transfer of a solute from one

phase to another
Isolate or concentrate the desired analyte
Separate it from species that would
interfere in the analysis.
Most common extraction of an aqueous
solution with organic solvent
In two phase system, one phase is usually
water and one is usually an organic solvent

Solvent Extraction

Partition coefficient (K) is equilibrium constant for

the reaction

As 2

[S]2 (1 q)m / V2
K

As1 [S]1
qm / V1
Where As1 and As2 are the activities of the solution in
phases 1 and 2, respectively and m is the total
number of moles of S, q is the fraction of S
remaining in phase 1 at equilibrium, V1 and V2 are

Fraction Not Extracted

Solvent Extraction
pH effects
Important if solute is an acid or base (e.g. B/BH+)
Usually, neutral is more soluble in organic solution
Calculate a distribution coefficient

total concentration in phase 2

D
total concentration in phase 1
K Ka
[ B ]2
D

K B

[ B ]1 [ BH ]1 K a [ H ]
Where B is the fraction of
weak base in the neutral form in phase 1
P539 for

Solvent Extraction
Extraction with a Metal Chelator
Neutral complexes are extracted into organic
phase
Charged
complexes
total concentrat
ion intypically
phase 2 stay in aqueous
D
phase
total concentration in phase 1

n
K m K an [ HL]org

K Ln [ H ]naq

Km and KL are partition coefficients

Ka is the acid dissociation constant

P541 for

Chromatography

Chromatography is a PHYSICAL
METHOD OF SEPARATION
Components to be separated
are DISTRIBUTED between TWO
PHASES
STATIONARY PHASE
MOBILE PHASE

Chromatography
Separation results from REPEATED
SORPTION/DESORPTION acts during the
movement of the sample components
along the stationary bed
A useful separation will only result when:
KINETIC FACTORS controlling zone broadening
are favorable
DISTRIBUTION CONSTANTS of the sample
components are different

Chromatography

Mobile Phase Selection

CHROMATOGRAPHY

GAS

SUPERCRITICAL
FLUID

LIQUID

Mobile Phase
Responsible for transport
May participate in the
distribution mechanism
Critical properties are DENSITY,
DIFFUSIVITY and VISCOSITY

Density
Provides an indication of the free
space between molecules and the
likelihood of intermolecular
interactions
(g / mL)
Gas 2 x 10-4 Almost no interactions
Fluid 0.1 0.8
Liquid 1
Significant interactions

Diffusivity
Provides an indication of the speed
with which particles move in a
medium
Depends on the mass of the particle,
temperature and viscosity
(cm2 / s)
Gas 0.1 1 Fast
Fluid
10-3 10-4
Liquid10-5
Slow

Viscosity
A measure of the work required to
achieve a useful mobile phase
velocity
Depends on temperature
(cP)
Gas 0.02
Low
Fluid
0.02 0.1
Liquid1
High

Stationary Phase Selection

CHROMATOGRAPHY

SOLID

ADSORPTION

POROSITY

LIQUID

COULOMBIC ABSORPTION

Types of Chromatography
Solid stationary
phase
Liquid or gaseous
mobile phase
The more strongly
a solute is
adsorbed, the
slower it travels
through the
column.

 Liquid stationary phase bonded to solid

surface (e.g. silica chromatography
column in GC)
Solute equilibrates between stationary
liquid and mobile phase (in GC this is a
flowing gas)

 Anions or cations
are attached to
solid stationary
phase, usually a
resin.
Solute ions of the
opposite charge
are attracted to
the stationary
phase by
electrostatic
force.
Mobile phase is a

Also called gel filtration

or gel permeations
chromatography
Separates molecules
by size
Larger solutes pass
through quickly
No attractive action
between solute and
stationary phase

Most selective type of chromatography

Employs specific interactions between one kind of
solute molecule and a second molecule bound to
stationary phase. Mobile phase is liquid.

http://en.wikipedia.org/wiki/Column_chromatog
raphy

Chromatogram
Contains all the useful
information from a
chromatographic
experiment
A record of the detector
response to the sample
components as a
function of the
movement of the
mobile phase

Chromatogram
Link to fundamental properties of
the sample or separation system
Peak positions provide
information about
THERMODYNAMIC PROPERTIES
Peak widths provide information
about KINETIC PROPERTIES

The chromatogram is a graph showing the

detector response as a function of elution time.

Retention
Retention = Column + Adjusted
Time
Hold-up
Retention
Time
Time

tR

= tM

+ tR

tM = time sample spends in the mobile phase

(same for all compounds)
tR = time sample spends in the stationary
phase
(different for all separated compounds)

Column Hold-up Time

Time required by the mobile
phase entering the column to
reach the detector
It is equivalent to the volume of
streaming mobile phase contained
in the column
It should be corrected for
extracolumn volumes

Retention Factor (Capacity

Factor)
More fundamentally important than the
absolute retention time
Represents the ratio of the time spent by
the solute in the stationary phase to
the time it spends in the mobile phase

k = tR / tM = (tR tM) / tM

Separation (selectivity)
factor
Relative retention of two adjacent
peaks in a chromatogram

= tR2 / tR1 = k2 / k1
Provides a measure of the selectivity of
the chromatographic system
Constrained to have values >1

Distribution constant
Distribution = Retention X
Phase
Constant
Factor
Ratio

K = k
= volume of mobile phase /
volume (or surface area) of
stationary phase

General Elution Problem

Sample components have a narrow range
of distribution constants
Preferred separation approach
Isothermal (constant temperature) in GC
Isocratic (constant mobile phase
composition) in LC
Isopycnic (constant density) in SFC

General Elution Problem

Sample components have a wide range

of distribution constants
Fixed separation conditions
Long separation times
Poor separation of early eluting peaks
Poor detectability of late eluting peaks
due to band broadening

General Elution Problem

Sample components have a wide range of
distribution constants

Preferred separation modes

GC

Temperature
Flow

LC

Mobile phase composition

Flow
Temperature

SFC

Density
Composition
Temperature

Band Broadening
For Gaussian peaks
the PLATE
NUMBER is given
by
N = (t

t)

t is the band variance

Band Broadening
Peak width measurements are
normally used for convenience
N = a (tR / w)
a=4
a = 5.54
a = 16

w = w w = inflection point
i i
w=w
w=w

w = half height
h

w = at base

Band Broadening
The PLATE HEIGHT (H) is related to the
plate number by

H=L/ N
where L is the column length

Rate Theories
Factors Contributing to band
broadening:
Flow Anisotropy (Eddy Diffusion)

Longitudinal Diffusion
Resistance to Mass Transfer
Extracolumn Sources

Flow Anisotropy
Results from flow
inequalities in a packed
bed
Local velocities vary
considerably within
interparticle channels in
pressure driven systems
due to parabolic flow
Because of radial diffusion
molecules experience
many streamlines during
their passage down the
column acting to reduce
flow anisotropy

CHAPTER 22: Figure 22.20

Flow Anisotropy
Uncompensated flow inequalities result in a
contribution to band broadening that would
not exist for an ideal column
HE = 2dp
Use particles of the smallest practical size
Use particles with a narrow size distribution
Pack column homogenously
Does not contribute to band broadening in opentubular columns

 Flux , the number of moles of solute

crossing each square meter per minute
is proportional to the concentration
gradient.
dc
J D
dx
Diffusion coefficient (D)
measures the rate at
which a substance
moves randomly from a
region of high
concentration to one of
lower concentration.

Longitudinal Diffusion
Results from the tendency of a solute band
to diffuse away from regions of high
concentration to regions of lower
concentration

HL= 2DM / u
Always important for Gases
May be negligible for liquids
High values of u minimize HLbut adversely
affect resistance to mass transfer

Resistance to Mass
Transfer
Results from the
limitations of diffusion
in the mobile and
stationary phases as a
transport mechanism
to move analyte
molecules to the
boundary region
between phases

Resistance to Mass
Transfer
CHAPTER 22: Figure 22.18

Resistance to Mass Transfer

Important experimental variables
Retention factor
Stationary phase film thickness (GC)
Diffusion coefficients in the mobile and
stationary phases
Mobile phase velocity
Column internal diameter (GC)
Particle size (LC)

Mobile Phase Velocity

The mobile phase linear velocity is
more important than flow rate for
fundamental studies
For compressible mobile phases the
local velocity at any position in the
column depends on:
Flow resistance of the column
Ratio of the column inlet to outlet pressure

Van Deemter Equation

H= A + B/u + Cu
A = flow anisotropy
B = longitudinal
diffusion
C = resistance to mass
transfer
Minimum value for the
plate height exists at
an optimum velocity

Resolution

Resolution
RS = 1 corresponds to 94% peak
separation
RS = 1.5 corresponds to baseline
separation

Resolution
RS = 1 corresponds to 94% peak
separation
RS = 1.5 corresponds to baseline
separation

Resolution And Column

Properties

Resolution is proportional to L
Separation time is proportional to L
N increases with decreasing column
diameter for GC
N increases with decreasing particle
size for LC

Resolution And Column

Properties

Resolution And Column

Properties

Resolution And Column

Properties

Resolution And Column

Properties