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Band Broadening
Wherever you see this symbol, it is important to access the on-line course
as there is interactive material that cannot be fully shown in this reference
manual.
Objectives
To use the Van Deemter coefficients to illustrate how to optimise the Efficiency (N)
of chromatographic separations and to reduce Band Broadening
Content
Band Broadening
The Van Deemter Equation
Eddy Diffusion
Longitudinal Diffusion
Mass Transfer
Optimising Flow Rate
Optimising Particle Size
Minimising System Volumen
Glossary
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Band Broadening
The Van Deemter Equation
Band broadening is a phenomenon that reduces the efficiency of the separation being
carried out leading to poor resolution and chromatographic performance. This is
problematical in terms of both the quality of the separation obtained and the accuracy with
which sample components can be quantified.
The degree of band broadening (loss of efficiency) naturally increases with the age of the
chromatographic column being used, but there are measures that can be taken to slow
these processes and to optimise column and instrument conditions to ensure maximum
efficiency and minimum band broadening.
In 1956 J.J. Van Deemter derived an equation that included the main factors contributing
to column band broadening. He described the individual terms (A,B,C & D) and also
derived a composite curve which related plate height (HETP) to linear velocity of the
mobile phase flowing through the column. Whilst it is not important to necessarily know
and use the equation on a daily basis it is important to understand the terms (or factors)
that contribute to band broadening, so that we can optimise our separations. For example,
the interactive diagram opposite shows the reduction in chromatographic performance
sustained when moving from a system extra column volume of 20L to 80L.
The Van Deemter Equation and graphical representation of the contributing terms
Comparative chromatograms from HPLC systems with 20 and 80L dead volume
showing effects on efficiency and resolution.
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Eddy Diffusion
The first of the factors relating to band broadening is that of Eddy Diffusion. This is a
generic term, often used to describe variations in mobile phase flow or analyte flow path
within the chromatographic column.
Eddy diffusion itself relates to the fact that an analyte molecule, within a band of
analytes, can take one of many paths through the column. These multiple paths arise
due to inhomogeneities in column packing and small variations in the particle size of the
packing material. This multiple path effect tends to make the band of analytes broader as
it moves through the column.
Large Particles
Small Particles
Band broadening due to Eddy Diffusion (A Term) in columns with large and small particles
effects on chromatographic peak shape (Efficiency (N)).
Minimise Eddy Diffusion by:
z
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In fact, the Eddy Diffusion term in the Van Deemter equation (the A term), is often called
the packing term as it reflects the quality of column packing.
Laminar flow occurs with standard size HPLC column packing materials at flow rates up to
approximately 4mL/min. Non-laminar flow occurs when using very large particle size
packing material (>40m) and at high flow rates (>5mL/min.). This situation is also
sometimes referred to as Turbulent Flow and does not occur under normal HPLC
conditions customised HPLC equipment and columns are required for Turbulent Flow
Chromatography.
Band broadening due to Eddy Diffusion (A Term) in columns with in Laminar and Non
Laminar mobile phase flow profiles.
One other flow variation in column chromatography comes from the laminar flow profile
adopted by liquids flowing under pressure through tubes. The flow profile is said to be
laminar, where linear velocity near the inner walls of the tubing is lower than in the
centre of the column. This also tends to produce a broader band of analyte molecules.
Longitudinal Diffusion
A band of analyte molecules contained in the injection solvent will tend to disperse in
every direction due to the concentration gradient at the outer edges of the band.
This broadening factor is called Longitudinal diffusion because inside tubes, the greatest
scope for broadening is along the axis of flow. The band will broaden in all system tubing,
but the worst effects will be encountered in the column itself.
Longitudinal diffusion occurs whenever the HPLC system contains internal volumes that
are larger than necessary and some instances of this are:
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As can be seen, Longitudinal diffusion has a much larger effect at low mobile phase
velocity (flow). Therefore, using high linear velocity (high mobile phase flow with narrow
columns), will reduce the effects of this broadening factor.
Band broadening due to Longitudinal Diffusion (B Term) in columns with low and high
mobile phase linear velocity effects on chromatographic peak shape (Efficiency (N)).
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Mass Transfer
This term in the Van Deemter equation arises largely due to the fact that the stationary
phase material is porous and the mobile phase within the pores is stagnant or stationary.
The packing material is porous to allow a very large surface area for separation to occur.
As the analyte molecules move through the stagnant mobile phase to reach the surface of
the packing material, they do so by diffusion only. Analyte molecules entering the pore,
those that dont enter the pore and those that penetrate more deeply into the pore, will all
be held up at that point to different extents causing a broadening of the band. This is the
C term.
Mass Transfer (C Term) band broadening processes in the pore structure of stationary
phase particles
Further, the analyte residence time in (or on) the stationary phase is also variable again
causing a variation in elution time and band broadening. These effects may be minimised
by reducing the size (diameter) of the packing material particle size to make the pores as
shallow as possible. The effects of mass transfer are also lower at lower linear velocity of
the mobile phase.
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Effect of mobile phase linear velocity (top) and stationary phase particle size (bottom) on
Mass Transfer effects and peak shape (Efficiency (N))
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Although
the change in
particle size does not appear to
have a marked effect on the
efficiency and resolution of this
separation, you should study the
results carefully
Reducing particle size (even
when using traditional column
internal diameters), has a
marked effect on plate height,
which can be usefully used to
improved the resolution of
separations, especially where
selectivity options have failed to
produce baseline separated
peaks
The plate heights obtained
with very small particles are
extremely low. By combining
short columns with traditional
internal diameter (0.46cm), and
small diameter silica packing
material (1.8m) very short
analysis times may be achieved.
When using smaller diameter
column
packing
materials,
system backpressure should be
carefully observed as increased
backpressure results from the
use of small silica particles
Starting with an investigation of mobile phase flow (linear velocity) you should see that
the mobile phase flow rate can have an effect on band broadening, plate height and
hence resolution. Its important to note that there is an optimum flow rate for each
separation (the minimum point on the curve corresponding to the lowest plate height).
Whilst this effect is noticeable, it is minor in comparison with other variables affecting
resolution and therefore flow rate would be used to fine tune a separation only.
Of course, if you have enough efficiency (and resolution) to run at higher flow rates then
this will make the analysis time shorter however, care should be taken not to exceed the
pressure limit of your HPLC pump!
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It should be noted that whilst smaller diameter particles are favourable there is a trade
with system backpressure. Small particles may be used in conjunction with higher mobile
phase flow rates to produce very fast analyses, but system backpressure needs to be
carefully monitored.
The use of smaller diameter silica particles (<2m), with traditional column internal
diameters (i.e. 4.6mm), means extra-column dead volume is less important than when
using narrow bore columns. This can be an advantage when fast HPLC analysis is
required, without having to change the analytical system hardware to very low dead
volume, or very high pressure versions.
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Important:
It is straightforward to measure the extra column volume of a system:
Remove the column and join the tubing with a Zero Dead Volume union
Inject 10L of 100% strong solvent (acetonitrile works with UV at 200nm) or a
solution of 1% acetone (monitor at 265nm)
The apex retention time (tR) of the baseline perturbation due to the passage of the
solvent gives the extra column hold up time of the system (expressed in minutes)
Multiply this time by the flow rate (in millilitres per minute) to obtain the extra
column volume (in millilitres)
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Apex solvent retention time used to determine extra column volume of an HPLC system.
Glossary
Linear Velocity The linear velocity of the mobile phase across the average crosssection of the chromatographic bed or column. It can be calculated from the column flowrate at column temperature (Fc ), the cross-sectional area of the column (Ac ) and the
interparticle porosity ( ):
= Fc /(Ac)
In practice the mobile phase velocity is usually calculated by dividing the column length
(L) by the retention time of an unretained compound (tM)
= L/tM
System Extra Column Volume the volume of the HPLC system from the point of
injection to the point at which the sample passes out of the detector, MINUS the column
dead volume. This typically includes the injection loop, any tubing and unions as well as
the detector flow cell where appropriate.
Tubing Unions Fittings which allow the coupling of two pieces of tubing in HPLC. May
be made from Stainless Steel or and inert polymeric material such as PEEK (polyether
ether ketone).
Zero Dead Volume refers to a fitting in which the nut, ferrule and tubing exactly match
the internal volume of the fitting, leaving no dead space. This dead volume tends to
cause band broadening in HPLC by artificially holding up analyte molecules in the
stagnant (not moving) phase that resides in the dead volume within the fitting.
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Dead Volume
Wrong column nuts and ferrules - each column manufacturer uses particular end fitting
types. Specially design stainless nuts and ferrules must be used in order to obtain a
satisfactory zero dead volume union with the column. Some workers use PEEK fittings
that deform to fit the internal geometry columns from any manufacturer. However, these
fittings do loose their plasticity and should be regularly replaced to avoid band
broadening.
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