The Theory of HPLC

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
i manual.
Aims and Objectives

Aims and Objectives

Aims

To illustrate and explain the principle of Band Broadening in HPLC

To define the Van Deemter equation and explain the terms of the equation
To demonstrate the effects of Eddy Diffusion, Longitudinal Diffusion and Mass
Transfer on the Efficiency of Chromatographic Peaks

Objectives

At the end of this Section you should be able to:

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 3
The Van Deemter Equation 3
Eddy Diffusion 4
Longitudinal Diffusion 5
Mass Transfer 7
Optimising Flow Rate 9
Optimising Particle Size 10
Minimising System Volumen 11
Glossary 12

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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 Selecting well packed columns

z Using smaller stationary phase particles
z Using particles with a narrow size distribution

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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:

z Tubing length too long

z Tubing that is too wide (internal diameter)
z Tubing joined by unions
z Incorrectly connected Zero Dead Volume fittings
z Using the wrong column nuts and ferrules
z Using a detector flow cell that has a large internal volume

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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)).

Minimise Longitudinal Diffusion by:

z Using higher mobile phase flow rates

z Keep system tubing short and as a narrow as possible
(careful with back-pressure) (<0.12mm i.d. is ideal)
z Use correct nuts, ferrules and fittings wherever possible

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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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Large Stationary Phase Particles

Greater possible pore distance for analyte diffusion

Diffusion time increased
Differences in diffusion times out of the pore are amplified
Peak efficiency decreases (peak broadens)

Small Stationary Phase Particles

Reduced possible pore distance for analyte diffusion

Diffusion time decreased
Differences in diffusion times out of the pore are reduced
Peak efficiency increases (peak becomes narrower)

Minimise Mass Transfer effects by:

z Using smaller (diameter) stationary phase particles

z Using lower mobile phase flow rates
z Heating the column (at higher temperatures the diffusion
processes are speeded up and the differences in elution time
from the particle pore are reduced)

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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 Optimising Flow Rate

Lets look at the composite Van Deemter curve and equation once again this time using
real world examples - to investigate options for minimising band broadening and
optimising peak efficiency.

During the previous pages you have seen that the mobile phase velocity and the particle
size of packing material used, have a fundamental effect on efficiency (and therefore band
broadening).

Although the change in

i particle size does not appear to
Van Deemter relationship between mobile phase
flow rate and peak efficiency in HPLC
The On-line course contains an interactive
experiment in which the eluent flow rate can be
changed to alter the HETP parameter
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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 Optimising Particle Size

Another important factor to consider regarding band broadening is the particle size of the
stationary phase used. We have seen several instances where the use of smaller
diameter particles is advantageous to both efficiency and resolution.

i 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
Van Deemter relationship between stationary phase
be achieved.
particle size and peak efficiency in HPLC
When using smaller
diameter column packing
The On-line course contains an interactive experiment
materials, system
in which the effects of changing particle diameter (dp)
backpressure should be
on efficiency (band broadening) and resolution are
presented carefully observed as
increased backpressure
results from the use of small
silica particles

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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 Minimising System Volumen

As previously discussed, systems containing large extra-column volume (tubing, unions,

flow cell etc.), show low efficiency and hence broadened chromatographic peaks and loss
of resolution.

You can use the slider bar to investigate the effects of extra column volume. You may
want to consider how you could reduce these volumes on your system. Typical ways
might include: reduce tubing length and internal diameter / reduce the number of unions
between tubing / fit column end fittings appropriate to the column type being used / reduce
the injection loop volume / reduce the detector flow cell volume.

i The system extra column

volume can have a critical effect
on efficiency and therefore
resolution of a separation
You need to also consider the
column dead volume, and if in
any doubt about the quality of
the column packing, use a new
column. Column voids from
within columns over time adding
very large dead volumes to the
system
Extra column dead volumes
below 20L should be easily
achievable on modern HPLC
systems
You should consider the
volume of the detector flow cell
as being a major contributor to
extra column volume, along with
any high pressure mixing
chambers on the HPLC pump
System dead volume
Van Deemter relationship between extra column becomes more critical as the
dead volume and peak efficiency in HPLC internal diameter, (and to a
lesser extent particle size), of
the column used are reduced

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 cross-
section of the chromatographic bed or column. It can be calculated from the column flow-
rate 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.

Incorrect ferrule and tubing fitting

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