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Instrumental Analysis II

Band broadening and column efficiency

Compiled by: S. Louw and K.M. Kalili


List of content: Band broadening and column
efficiency

– Tailing and fronting


– Quantitative description of column efficiency
– Kinetic variables affecting column efficiency
• The Van Deemter equation
Band broadening and column efficiency
• All solutes experience band broadening in function of time!

• If band broadening is larger than band separation  peak


overlap (no separation)!
• Ideally, band separation should be greater than band
broadening
• The efficiency of a chromatographic column is affected by the
amount of band broadening that occurs as a compound
passes through the column
Chromatographic peaks
• Chromatographic peaks can be:
– Symmetrical (Gaussian distribution)  Ideal
– Asymmetrical (either fronting or tailing) can be due to
instrument dead-volume effects, stationary phase
adsorptive effects or quality of the column packing
Gaussian

Tailing Fronting

a b

 When smaller quantities are moving faster than the larger quantities
 Fronting peak
 When smaller quantities of the solute are retained strongly than the
larger quantities  Tailing peak
• Please note that the theory and definitions are based on
the fact that we assume all chromatographic peaks to
have a Gaussian distribution, i.e. “tailing” and “fronting”
are assumed to be minimal
Quantitative description of column efficiency
• Quantitative measures of column efficiency:
– Plate height (H) or height equivalent to a theoretical
plate (HETP)
– Plate count or number of theoretical plates, N
• The two are related by the equation:
𝐿
𝑁=
𝐻
where L is the length of the column

 N is directly proportional to L
• Longer column provides higher N (efficiencies)
 N is inversely proportional to H
• Smaller H provides higher N
Theoretical plates:
• Martin and Synge defined these terms,
by looking at the processes in a
chromatographic column as if it were
similar to a distillation column which
consists of numerous discrete, but
adjacent narrow layers called
theoretical plates.
• At each plate, equilibration of the
solute between mobile and stationary
phase take place
• Movement of the solute was then
treated as a stepwise transfer of
equilibrated mobile phase from one
plate to the next
N and H
• The plate count (N) and the plate height (H) are
widely used in literature and by experts in the field
of chromatography
• They are very useful quantities for comparing
separation power of different separations and
efficiencies among columns
• The plate theory successfully accounts for the
Gaussian shape of chromatographic peaks and
their rate of movement down a column
Definition of plate height, H
• If we assume a Gaussian peak,
then the extent of band
broadening is given by its
standard deviation, s, or its
variance, s2
• The efficiency of a column can
be defined as the variance per
unit length of column:
𝜎2
𝐻=
𝐿
• The plate height can be
thought of as the length of
𝐿
column that contains a fraction 𝐼𝑓 𝑤𝑒 𝑠𝑢𝑏𝑠𝑡𝑖𝑡𝑢𝑡𝑒 𝐻 𝑖𝑛𝑡𝑜 𝑡ℎ𝑒 𝑁 =
𝐻
of analyte that lies between L 𝐿 2
and L - s 𝑡ℎ𝑒𝑛 𝑁 = 2
𝜎
See Skoog et al, Appendix 1 for more information on standard deviation etc.
Calculating the number of theoretical plates

• From the formula of


the Gaussian curve,
it can be shown that
w1/2 = 2.35s and w =
4s

𝑡𝑅2 16𝑡𝑅2 5.55𝑡𝑅2


𝑁= 2 = 2
=
𝜎 𝑤 2
𝑤1/2
• Because the experimental determination of H and N are
based on Gaussian chromatographic peaks, the
calculations are considered estimates
Kinetic variables of column efficiency
• Band broadening reflects the loss of column efficiency
• The slower the rate of mass-transfer processes occurring
while a solute migrates through a column, the broader the
band at the column exit
– Mass-transfer: movement of solute between the two
phases
• The effect of mobile-phase flow rate
– The extent of band broadening depends on the length
of time the mobile phase is in contact with the
stationary phase, which in turn depends on the flow
rate of the mobile phase
LC vs GC
• Plate heights for LC much
smaller than those in GC
• However, LC columns longer
than 25 cm are impractical
(pressure becomes too high)
• GC columns can be up to 60 m
and longer
 much greater number of
theoretical plates than LC
and hence superior column
efficiency
 GC capable of faster and
higher efficiency separations,
but not necessarily at the
same time
Components of a chromatographic system
Injector

Column Chromatogram

Mobile phase Detector


delivery unit
Origin of band broadening
Pump A
Mixer Injector Column Detector
Pump B

Chromatographic efficiency is affected by: Computer


 Band broadening originating from the injector
 Band broadening originating from the column
 Band broadening originating from the detector
The observed peak variance (σ2obs) is the sum of variances from all
contributing mechanisms. Variance is additive:
s2(total) = s2(injector) + s2(column) + s2(detector) + s2(extra-column volume)
(minimal) (minimal)
Major contributor Requires optimisation
Theory of band broadening
• The efficiency of chromatographic columns can be approximated
by the Van Deemter equation:
𝐵
𝐻 = 𝐴 + + 𝐶𝑢
𝑢

Directly proportional to the flow rate


Inversely proportional to the flow rate
Independent of flow rate
• Processes that contribute to band broadening are:
– Multiple flow paths (𝐴)
– Longitudinal diffusion (𝐵 𝑢)
– Mass-transfer (𝐶𝑢)

(cm/s)
Multiple flow paths or eddy dispersion term (A)
A-term
𝐻=𝐴 + 𝐵𝑢 + 𝐶𝑢

Plate height
 Broadening due to multiple flow paths
 Independent of the flow rate
 Theoretically: A = 2dp ;  = packing Mobile phase velocity

efficiency, dp = particle diameter


 For packed columns: A is constant for a specific particle size
 For capillary columns: A = 0
Longitudinal diffusion term (B/u)
𝐻 = 𝐴 + 𝐵𝑢 + 𝐶𝑢 B-term

Plate height
 Broadening due to diffusion of analyte
in the mobile phase
 Diffusion: movement of molecules
Mobile phase velocity
from the region of high concentration
to a region of low concentration
 Theoretically: B = 2DM ; DM = diffusion
coefficient of analyte in the mobile
phase
 Inversely proportional to the flow rate
 Faster mobile phase velocity (u) leads
to smaller B/u less time available for
diffusion to take place
 Smaller DM leads to smaller B
Resistance to mass transfer term (Cu)
𝐵
𝐻 =𝐴+ 𝑢
+ 𝐶𝑢

 Band broadening due to finite equilibration of molecules


between the mobile phase and the stationary phase
 Directly proportional to the flow rate

Slow
Stat. phase equilibration Stat. phase
Mobile phase Mobile phase
Resistance to mass-transfer term (Cu)
• The mass transfer term, Cu, is
actually the sum of two terms: Cu = CSu + Cmu
– the stationary phase mass-
transfer term, CSu, which is
linear, is
𝐶𝑆 𝑢 ∝ 𝑑𝑓2 𝐷𝑆
– and the mobile phase mass-
transfer term, Cmu, which bears
a complex dependency on
mobile phase velocity, is
– For packed columns:
𝐶𝑀 𝑢 ∝ 𝑑𝑝2 𝐷𝑀
– For open tubular columns:
𝐶𝑀 𝑢 ∝ 𝑑𝑐2 𝐷𝑀
where dc is the column diameter
Interpretation of a van Deemter curve
uopt : optimal mobile phase
velocity

Hmin: minimum plate height

Hmin 𝐿
Recall: 𝑁 =
𝐻

(cm/s)

uopt
 Ideally, one should work at Uopt i.e. the velocity which
delivers the minimum plate height (Hmin)  the
contribution of each term is minimal, i.e. higher N
(efficient separation)
Example: the effect of particle size dp on H
References
• D. A. Skoog, F. J. Holler and S. R. Crouch, Principles of
Instrumental Analysis, Chapter 26
• Analytical Chemistry 2.0 by David Harvey (electronic revision
of Modern Analytical Chemistry by David Harvey)

Further reading:
• D. C. Harris, Quantitative Chemical Analysis
• H. H. Willard, L. L. Merritt, Jr., J. A. Dean, F. A. Settle, Jr.,
Instrumental Methods of Analysis