Introduction to Chromatography

Theory

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 The Theory of Chromatography
• Plate theory - older; developed by Martin &
Synge

• Rate theory - currently in use today

 Plate Theory - Martin & Synge
1954 Nobel Laureates
• View column as divided into a number (N)
of adjacent imaginary segments called
theoretical plates

• within each theoretical plate complete

equilibration of analytes between stationary
and mobile phase occurs
 Plate Theory - Martin & Synge
1954 Nobel Laureates
• Significance?
Greater separation occurs with:
– greater number of theoretical plates (N)
– as plate height (H or HETP) becomes smaller

• L = N H or H = L / N
where L is length of column, N is number
of plates, and H is height of plates
 N can be Estimated
Experimentally from a
Chromatogram
• N = 5.55 tr2 / w1/22 = 16 tr2 / w2
where:
tr is retention time;
w1/2 is full width at maximum
w is width measured at baseline
 Choice of Column Dimensions
• Nmax = 0.4 * L/dp

where:
N - maximum column efficiency
L - column length
dp - particle size

• So, the smaller the particle size the higher the

efficiency!
 Efficiency Relative to Analysis
Time
today
90 mm L
3 um
today
150 mm L
N 5 um
1970’s
300 mm L
10 um

Analysis Time, min

10 100
First Important Prediction of
Plate Theory

Bandspreading - the width of bands

increases as their retention time
(volume) increases
 Problem:
• A band exhibiting a width of 4 mL and a
retention volume of 49 mL is eluted from a
column. What width is expected for a band
with a retention volume of 127 mL eluting
from the same analyte mixture on the same
column?

• ANS: 10.4 mL
Second significant prediction of
plate theory

The smaller HETP, the narrower the

eluted peak
 Plate Theory - Practical
Considerations
• Not unusual for a chromatography column
to have millions of theoretical plates

• Columns often behave as if they have

different numbers of plates for different
solutes present in same mixture
 Rate Theory
• Based on a random walk mechanism for the
migration of molecules through a column

• takes into account:

– band broadening
– effect of rate of elution on band shape
– availability of different paths for different
solute molecules to follow
– diffusion of solute along length
 Van Deemter Equation
• H=Aν 1/3
+ B/ν + C ν

where:

H is HETP (remember want a minimum!)

ν is mobile phase velocity
A, B, and C are constants
 Van Deemter Equation
• H=Aν 1/3
+ B/ν + C ν
– first term - rate of mobile phase movement
through column (often just a constant)

– second term - longitudinal solute diffusion;

solute concentration always lower at edges of
column so solute diffuses longitudinally

– third term - equilibration is not instantaneous

 Resolution
• Ideal chromatogram exhibits a distinct
separate peak for each solute

• reality: chromatographic peaks often

overlap

• we call the degree of separation of two

peaks:
• resolution = peak separation
average peak width
 Resolution
• Resolution = ∆ tr / wavg

• let’s take a closer look at the significance of

the problem:
 Resolution
• So, separation of mixtures depends on:

– width of solute peaks (want narrow)

efficiency

– spacing between peaks (want large spacing)

selectivity
 Example
• What is the resolution of two Gaussian
peaks of identical width (3.27 s) and height
eluting at 67.3 s and 74.9 s, respectively?

• ANS: Resolution = 2.32