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) |
|
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2.1 x 100 |
|
|
|
0.24 |
|
|
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2.1 x 150 |
|
|
|
0.37 |
|
|
|
|
2.1 x 250 |
|
|
|
0.61 |
|
|
|
|
2.1 x 300 |
|
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0.73 |
|
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4.6 x 100 |
|
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1.16 |
|
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4.6 x 150 |
|
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|
1.75 |
|
|
|
|
4.6 x 250 |
|
|
|
2.90 |
|
|
|
|
4.6 x 300 |
|
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3.49 |
|
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10.0
x 100 |
|
|
|
5.50 |
|
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10.0
x 150 |
|
|
|
8.25 |
|
|
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|
10.0
x 250 |
|
|
|
13.75 |
|
|
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|
10.0
x 300 |
|
|
|
16.49 |
|
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|
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.
> Bill Letter, 08/01/10.
HINT's & TIPS
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