HPLC HINTS & TIPS for
Chromatographers
Tip# 106:
Determination of HPLC Column Dead Time (T0):
Column Dead Volume or Time (AKA: Column Void/Dwell
Time) is the packed column volume divided by the flow rate and is usually expressed in
minutes. Determining T0 ("Tee zero") is necessary to find the Retention Factor
(and K1) in a separation. Ideally, it is determined by injecting a sample which is
unretained by the column and recording the retention time. It can also be calculated if
you know a few physical specifications of the column and media used. Here are some common
values based on typical HPLC column dimensions used today. At a flow rate of 1.000 ml/min
these values would also be the time in minutes.
|
COLUMN DIMENSIONS (I.D. x Length
(mm)) |
|
VOID VOLUME (ml) |
|
|
|
|
|
|
|
|
|
|
|
|
2.1 x 100 |
|
|
|
0.24 |
|
|
|
|
2.1 x 150 |
|
|
|
0.37 |
|
|
|
|
2.1 x 250 |
|
|
|
0.61 |
|
|
|
|
2.1 x 300 |
|
|
|
0.73 |
|
|
|
|
|
|
|
|
|
|
|
|
|
4.6 x 100 |
|
|
|
1.16 |
|
|
|
|
4.6 x 150 |
|
|
|
1.75 |
|
|
|
|
4.6 x 250 |
|
|
|
2.90 |
|
|
|
|
4.6 x 300 |
|
|
|
3.49 |
|
|
|
|
|
|
|
|
|
|
|
|
|
10.0
x 100 |
|
|
|
5.50 |
|
|
|
|
10.0
x 150 |
|
|
|
8.25 |
|
|
|
|
10.0
x 250 |
|
|
|
13.75 |
|
|
|
|
10.0
x 300 |
|
|
|
16.49 |
|
|
|
|
|
|
|
|
|
|
|
Note : Assumes an Average Pore
Volume of 0.70 (check with the manufacturer for actual values).
Void
Volume (ml) = (d^2 *Pi * L * Pore Volume) / 4 ; *Column Diameter & Length are in cm. |
Always measure the actual void volumn
of your specific HPLC column with a compound which is unretained by your column. For RP
applications which utilize at least 20% organic, Uracil or Thiourea are often used, but
some inorganic salts (e.g. sodium
nitrite and sodium nitrate) have also been shown to work as well.
When determining the "Void Volume", you are really measuring the void volume of
the column plus any extra-column volume from the injection volume plus all lines
connecting the injection to the column and the column to the flow cell. Note: This
is very different from the "System Dwell Volume" which also includes the volume
from the pump (or mixing valve) to the column head.
A more detailed version of this table with
other common column sizes is available at the following link: http://www.lc-ms.com/tablescharts.htm
> Bill Letter, 08/01/10.
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