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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.

> Bill Letter, 08/01/10.


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