High or Ultra Performance LC, a matter of particle size.
Comparison of columns, and particularly the benefits of the use of smaller particle sizes is less straight forward as it looks
at a first glance. Fair comparison should take
into account other factors than the parameter plates/cm like there
time, financial and technical
constrains.
Long columns packed with 5um particles may perform equally well or better
compared to shorter column packed with (sub) 3 um particles. One of the
prominent differences is
that (sub) 3 um column can provide more separation power ( plates, resolution) per unit of time, however at the payback of an
higher backpressure. High column pressure drop require the use of a more
expensive high pressure pumps. High
pressures may also be the cause of technical difficulties like leaking or bursting
connections. The table below summarizes some LC characteristics
associated with particle size. It is obvious that longer columns require
longer runtimes. However long columns posses a higher capacity, larger
dynamic range and yield more peaks per unit of time. [c.f extremities:
100 peaks in 22 min on a 5cm 1 um column or ~5 peaks/min versus 504 peaks in
60 min on a 120cm 5 um column or ~8 peaks/min]
| particle size |
backpressure |
max. column length |
plates/cm |
max # plates |
Max peak capacity |
Typical runtime* |
| (um) |
(bar/cm column) |
at 400 bar limit |
(relative) |
at 400 bar |
at 400 bar limit |
(20 min @ 3%B/min) |
| 1 |
83 |
4.8 |
500 |
2400 |
100 |
22 |
| 1.7 |
28 |
14 |
294 |
4116 |
171 |
25 |
| 3 |
9.2 |
43 |
167 |
7181 |
299 |
35 |
| 5 |
3.3 |
121 |
100 |
12100 |
504 |
60 |
Runtime calculated for max column lengths (L
max)
at 400 bar operated at a linear velocity of 6 cm/min, an elution time window
of 20 min using a gradient of 3% B/min and a column reconditioning period
that equals the length of the column divided by the linear velocity of
the mobile phase (6 cm/min).
Small particle sizes yield lower plate heights. The relation
is linear. Also, smaller particles create an higher pressure drop across the
column. This relation is quadratic. When comparing two columns packed with
different particle sizes, d
p1
and d
p2, the plate height and pressure drop compares
according to:
HETP = dp2/dp1
ΔP = (dp1/dp2)2
|
dp |
HETP |
N |
pressure drop |
|
um |
um |
plates/cm |
bar/cm |
|
10 |
10 |
1000 |
1 |
|
5 |
5 |
2000 |
4 |
|
2 |
2 |
4000 |
25 |
However, the observed peakwidth (σpeak)
comprises three
dispersion components: σ2peak=σ2injection+
σ2column +σ2detector.
Hence, if peak broadening occurs during the injection or in the detector
(esi source) or both and are of the same order to the dispersion in the column than one would get little or no benefit
from using small particles. Rather, one would suffer from the
disadvantage, increased pressure drop. This is particularly true for
<2 um particles (6-7 times higher pressure drop compared to 5
um particles).
Comparison of the peak width obtained on two otherwise
similar columns
except particle size. Top: 3 um dp, middle and bottom, 5 um dp Biosphere
C18. The top
and bottom separations are obtained at the corresponding optimal column
head pressure (175 and 100 bar for the 3um and 5 um column, resp). The
middle bar represents the separation on a 5um column operated at a
pressure similar to the 3 um column. Peak widths are about 8 s (4σ
) or 2.5-3 s (2σ) for both 3 and 5
µm.