1

Broadening in chromatography (Notes adapted from Harris Quantitative Chemical Analysis)

V. L. Colvin, chemistry 330
We use the term plate height or H as a catchall term for the width of a chromatographic
band. The smaller the plate height, the narrower the band. A very small plate height is therefore
a desirable feature of a chromatographic column. It relates to the theoretical number of plates,
N, in a column through:
H
L
N =
Where L is the total length of the column. For a fixed L, if we have extremely small plate
heights then N is large which signals a great, narrow chromatography band.
Conceptually the plate height, or H, is a measure of the band spread in the column. If
you have a very thin H, then the peak is not very spread out in space. A large H, the peak is
really spreading. I like to remember what H is by imagining chopping up the column to capture
a band in space. H is proportional to the thickness of the slice that would contain most of the
analyte actually 36% to be (nearly) exact. Eventually it runs through the column it reaches
the end and travels the distance L at its elution time t
r
. The plate height here is the theoretical
plate height in this case.
Lets think about H from a practical, experimental point of view. If you had a column,
a flow rate, a target analyte and you measured the chromatogram you could measure the peak
width which you captured by measuring the standard deviation, o, of the peak - and the elution
time t
r
. You would also know the column length. You can calculate H then:
L
H
2
o
=
Plate height uses the variance, o
2
, in this equation rather than the standard deviation, o,
because variances are additive. Thus we can describe variances from multiple sources, and as
will be done next plate heights from different sources. Typically in a gas chromatography
separation H values for good columns are about 100 to 1000 microns while for liquid
chromatography systems they are only 10 microns.
We can consider plate height also theoretically. While abstract, the insights this provides
helps us design better columns and improve the selection of columns and running conditions.
The Van Deemter equation rolls many band broadening sources into one compact form:
x
x
Cu
u
B
A H + + =
Where u
x
is the linear flow rate and A, B and C are constants. This equation tells us that there
are mechanisms of band broadening that depend linearly and inversely on flow rate, and some
that are independent. In the next section I give you some standard equations for the most critical
sources of broadening without full derivations so you can examine how choices you might
make in running a column will influence your plate height. Remember you almost always
want H to be as small as possible to make for an efficient separation with highly resolved peaks.
2
1. Sources that lead to broadening independent of flow rate (A constants)
(A) You never apply your sample in a step function. There is some width in time/volume to the
initial application of sample, which is characterized by a At for the injection. This is usually
referred to as a plate height contribution from injection or sometimes application:
units length
L
H
2
inj
inj
o
=
There is a similar issue that arises from the detection process. No detector samples instantly;
samples are usually collected over some time period At for the sampling. Sometimes these
broadening issues are given to you as a volume (e.g. you injected 300 microliters onto a column).
To convert to a spread in time you need the flow rate in terms of volume per minute. Simply
multiplying the sampling or injection volume by this flow rate gives you the spread in time.
(B) Another contribution here is the possibility of multiple flow paths. Important primarily in
liquid chromatography, this leads to an intrinsic spread in the data. It becomes less severe when
bead sizes are smaller.
2. Sources that lead to broadening that depend inversely on the velocity. Diffusion is a large
factor in column broadening. Molecules randomly diffuse in space due to the concentration
gradient they move into areas of lower concentration. How quickly they do this depends on the
molecules diffusion constant in the mobile phase (hence D
m
in the equation below). You can
look up diffusion constants for molecules; the units are usually like acceleration m
2
/sec.
x
m
x
m
m
dif
diffusion
u
D
L
u
L
D
L
t D
L
H

=
|
.
|

\
|

=

= =
2
2
2
2
o

Where velocity along the direction of elution (x) is u
x
. Note: we calculated the time, t, from the
velocity and the overall column length. This equation tells us that to have small plate height we
want a velocity like a firehose, not a trickle.
3. Sources that lead to broadening that are proportional to the velocity. Another source of
broadening is equilibration. If you dont let the solute/analyte sit long enough to partition
according to the equilibrium constant (e.g. partition coefficient) between the mobile and
stationary phase then you have a distribution of partition coefficients and hence, a broad
chromatogram. This term is sometimes called the mass transfer term:

x
s
trans
transfer
u
D k
d k
L
H
+

~ =
2 '
2 '
2
) 1 (
o

In this expression we use D
s
which is the diffusion constant in the stationary phase, k the
capacity factor of the analyte, and d is the thickness of the stationary phase.
On the course website you will find and excel sheet entitled VanDeemter.xls. In this excel sheet
you can run different conditions for the Van Deemter equation and compare how hard, or easy, it
may be to minimize the plate height for a given separation.