Translator for HPLC HINTS and TIPS for Chromatographers

Sunday, May 1, 2011

Determination of HPLC Column Dead Volume / Dead Time (T zero):

Column Dead Volume, Column Dead Time or Column Void Volume are all the same terms we apply to find the packed column internal volume (divided by the flow rate and usually expressed in minutes for the Void Time). You must know what this value is BEFORE starting to run an HPLC method or perform liquid chromatography. The value for volume changes for different column dimensions.

Are you peaks or samples eluting at or near the column void volume? If so, no chromatography has taken place and no HPLC method has been developed. People with no chromatography training or experience often make this mistake and create methods with little or no peak retention. Make sure you have retention for a long enough time on the column. How do you know how long is long enough? Calculate the Column Void Volume first, and then, the K prime value for your sample. The K prime should be at least 1.5 (>2.0 is the accepted standard) for the method to be useful.

Determining  the column void time or T0 "Tee Zero", as we call it,  is necessary to find the Resolution, Separation Factor and Capacity Factor (K1) in a chromatography separation. Ideally, it is determined by injecting a sample which is unretained (passes right through) by the column & mobile phase. It can also be calculated for spherical, bare or coated silica supports if you know a few physical specifications of the column and media used. You should first calculate it, then measure it (the values should be close).

Here is short list of typical HPLC column dimensions and their associated void volumes for fully porous silica supports. At a flow rate of 1.000 ml/min these values would also be the same as the void 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

                       Void Volume (ul) = (d^2 *Pi * L * Pore Volume) / 4 ;
                                  *Column Diameter & Length are in mm.

[Note : Assumes an Average Pore Volume of 0.70 (70%) on a non-encapped, fully porous spherical bare silica support (please check with the manufacturer for the actual value of your support). Calculating the value will often get you close to the measured value, but due to the unique chemistries used to prepare supports, it is only an approximation].

Always measure the actual void volume 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 "Column 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 includes the volume from the pump (or gradient valve) to the column head.

A more detailed version of this table with other common HPLC Column Sizes and Tubing Volumes for capillary lines are available at the following links (Link #1) or (Link #2).

4 comments:

  1. We use this table all of the time in our lab! Thanks.

    Question - What about the newer columns which have the core shell particles inside. Do these have the same volume as the regular particles inside the column?

    ReplyDelete
    Replies
    1. Great question! The most commonly used HPLC supports are known as FULLY porous supports and we often use a value of 0.70 for their pore volume (as noted above).

      The more recently introduced SUPERFICIALLY porous supports (e.g. Fused-Core, Core-Shell) particles have less volume, so a value of 0.50 is often used.

      Best ways to determine it? (1) Measure it using an unretained standard to find out the actual value. (2) Contact the manufacturer of the specific support (particle) to obtain the most accurate value to use. The values supplied here are good estimates only.

      Delete
  2. I have the problem of my sample coming out at 1.7 min with a flow rate of 0.7ml/min. and my void volume is 1.75mL so therefore my sample comes before my void volume of 2.5 min, it is 100% not sample carry over, any suggestions?

    ReplyDelete
    Replies
    1. If you have measured the column void volume and flow rate correctly, then the problem that you have observed could be caused by: (1) Sample Overload (don't exceed an injection volume of more than 4% of the column's void volume) or (2) perhaps "channeling" (this is when a very narrow internal channel forms down the inside of the length of the column allowing liquid to shoot through. When channeling occurs, the column must be disposed of (or repacked).

      Delete