Chapter 26

An Introduction to
Chromatographic
Separations
 What is chromatography?
• The separation of a mixture of compounds based
upon the differential partitioning of various analytes
species between a mobile phase and a stationary
phase.
What is chromatography?
 What is chromatography?

Chromatogram a plot of analyte signal as a function of elution time

 Classification of Column
Chromatographic Methods
The Effect of Migration Rates and Zone
Broadening on Resolution
 The Effect of
Migration
Rates and
Zone
Broadening
on Resolution
 Migration Rates of Solutes
Distribution Constants
• Migration rates are dependent on the magnitude of of the equilibrium
constants for the reactions by which the solutes distribute themselves
between the mobile and stationary phase.

K = cs / c m

where cs = molar concentration of the solute in the stationary phase

and cm = molar concentration of the solute in the mobile phase

• K is often referred to as the distribution coefficient, partition coefficient, or

partition ratio
 Migration Rates of Solutes
Retention Times
• The time it takes after sample injection for an analyte peak to
reach the detector is called retention time.
Where
tR = L / vavg
L = length of
column vavg = average
velocity of analyte
Dead time
 Migration Rates of Solutes
Velocity of Mobile Phase
• Velocity of mobile phase can be calculated using tM.
u = L / tM Where
L = length of
column u = average
velocity of an
Dead time unretained species
 Migration Rates of Solutes
The Relationship Between Retention Time and
Distribution Constant
• In order to relate the retention time of the solute to its
distribution constant, we express its migration as a fraction of the
velocity of the mobile phase.
Vavg = u x fraction of time solute spends in mobile phase

*
* = capacity factor or retention factor
 Migration Rates of Solutes
Relative Migration Rates: Selectivity Factor
• A fraction consisting of of the partition ratios of two retained
species on a chromatographic column; by convention the ratio of
the more strongly held species (Kb,the species that takes longer to
elute) is in the numerator. By this definition  is always greater
than 1.
 = KB/KA

 = k’B / k’A

 = [(tR)B - tM ] / [(tR)A – tM]

 Zone Broadening and Column Efficiency
The Shapes of Chromatographic Peaks
• Gaussian curves

• Random walk

• The breadth of a band increases as it moves down the column

because more time is allowed for spreading to occur.

• The zone breadth (peak width) is directly related to residence time

and inversely related to the velocity of the mobile phase
 Zone Broadening and Column Efficiency
Methods for Describing Efficiency
• The theory is based on work by Martin and Synge in which they treated a
chromatographic column as if it were a distillation column

 = L/N
theoretical plate height
N= number of theoretical plates
L= length of column

The plate theory successfully accounts for the Gaussian shape and their rate of
movement down a column, but fails to account for peak broadening in a
mechanistic way. The rate theory was developed to make up for these shortcomings.
 Zone Broadening and Column Efficiency
Methods for Describing Efficiency
• The theory is based on work by Martin and Synge in which they treated
a chromatographic column as if it were a distillation column

 = L/N

H = (LW2)/(16tR2)

N = 16 (tR/W)2

N = 5.54(tR/W1/2)2
Zone Broadening and Column Efficiency
Kinetic Variables Affecting Zone Broadening
 Zone Broadening
and Column
Efficiency
Kinetic Variables
Affecting Zone
Broadening
Mobile-Phase Flow Rate
Zone Broadening and Column Efficiency
Relationship Between Plate Height and Column
Variables - van Deemter Equation
H = A + B/u + Cu H = A + B/u +(Cs + Cm)u
 Zone Broadening and Column Efficiency
van Deemter Equation - The Multipath Term (A)
H = A + B/u + Cu
A = 2  dp

depends on particle size distribution, the

narrower the distribution the smaller the 
The smaller the particle size, the smaller the
A term
Independent of mobile phase flow rate

Also known as eddy diffusion

 Zone Broadening and Column Efficiency
van Deemter Equation - Longitudinal Diffusion (B)
Negative slope due
to this term
H = A + B/u + Cu
B/u = 2DM/u
less for LC
is related to the diffusion restriction of
packed columns, with packed columns
this value is about 0.6 and 1 for open
tubular columns
DM is the mobile phase diffusion coefficient

Inversely related to mobile

phase flow rate
Zone Broadening and Column Efficiency
van Deemter Equation - Mass Transfer (C)
H = A + B/u +(Cs + Cm)u
CS = fS(k’)df2 / DS

CM = fM(k’)dp2 / DM

f(x) is a function of x

DM is the mobile phase diffusion coefficient

DS is the stationary phase diffusion coefficient

df is film thickness

dp is particle size

Directly related to mobile phase flow rate

Zone Broadening and Column Efficiency
van Deemter Equation - Summary
H = A + B/u +(Cs + Cm)u
Zone Broadening and Column Efficiency
Effect of Particle Size on Plate Height
 Optimization of Column Performance
• A chromatographic separation is optimized varying
experimental conditions until the components of the
mixture are separated cleanly with a minimum
expenditure of time.

• Optimization experiments are aimed at

– reducing zone broadening
– altering relative migration rates of components
 Optimization of Column Performance
Column Resolution
• Resolution (RS) of a column provides a quantitative
measure of its ability to separate two analytes
Effect of Retention and Selectivity Factors
on Resolution
Column Resolution

• Resolution in terms of retention times and efficiency

• Resolution in terms of capacity factors and efficiency

Effect of Retention and Selectivity Factors
on Resolution
Column Resolution

• Resolution in terms of capacity factors, efficiency, and

selectivity factors

• Efficiency in terms of capacity factors, resolution, and

selectivity factors
Variables That Affect Column Performance
• Resolution in terms of capacity factors, efficiency, and
selectivity factor
First term related to the kinetics that lead to band broadening

Second term is a selectivity Third term depends on properties of

term that is only related to both the solute and the column
the properties of the two
solutes
Variation In Retention Factor
Variation In Retention Factor
General Elution Problem
Important Chromatographic Terms
Important Chromatographic Terms