Translator for HPLC HINTS and TIPS for Chromatographers

Sunday, May 1, 2011

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

Column Dead Volume, Column Dead Time or 'Column Void Volume' (the preferred name) 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 column volume changes for different column dimensions and different column supports.

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 sample retention for a long enough time period on the column. How do you know how long is long enough? Start by estimating the Column Void Volume (use our table or calculate it), and then, calculate the K prime value for your sample. The K prime for each peak should be at least 1.5 (>2.0 is the accepted standard) for the method to be useful and selective. *A more accurate answer will be found by measuring the void volume of your column (read on).

Knowing the Column Void Volume and the Flow Rate allows you to calculate the Column Void Time (which is the most useful value). Determining  the column void time or T0 ("Tee Zero" as we call it), is necessary to find other important chromatography values such as: the Resolution, Separation Factor and Capacity Factor (K1) in a chromatography separation. Ideally, it is measured by injecting a sample which is unretained by the column & mobile phase (passes right through the column support with little to no interaction). It may also be easily estimated for most spherical, bare or coated silica supports if you know a few physical specifications of the column and media used. You should first estimate it, then measure it (the values should be close, +/- 15%). Note: A practical "tip". You can also estimate T0 by noting when the small injector valve pressure peak ('blip') appears on the baseline. It results from the pressure change which occurs from switching the injection valve from the "load" to "inject" positions.

Here is short list of typical HPLC column dimensions and their associated estimated 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  50                                                                  0.12
                         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  50                                                                  0.58
                         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). Estimating 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).


  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?

    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.

  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?

    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).

  3. If your sample(s) elute at or near the Column Void Volume, then no chromatography has taken place and no HPLC method has been developed. Please do not be confused if your calculated Column Void Volume time is slightly longer than your measured time. The "calculated" value is an ESTIMATE only. That means it will be close, but you need to measure it to find the actual value. The estimate provides you with an initial value to use when developing the method. You can quickly tell if the peak is retained or not.

    Measurement of Column Void Volume: In addition to injecting an unretained compound as described in the article, you may also want to try injecting a small volume of mobile phase or solvent (no sample) and monitoring the UV/VIS detector at low wavelength (~ 220 nm) for a 'blip' to appear. You should see a small peak appear at or near the T zero time caused by the HPLC injector valve cycling during the injection. The disturbance in back pressure will result in a small peak which is easily visible and provides for a mechanical marker of the void time.

  4. When we inject our 3 samples, all of them come out right around the Tzero time, but we can see peaks (1 with a shoulder and 2 peaks). Our flow rate is 1 ml. 4.6 * 250 mm. 1st one at 2.75 min, next one 2.82 and third at 3.06 minutes. It is a validated method from our other lab. I asked the lab about what there Tzero was and they did not know what it was. I asked them if there k primes were greater than 2 and they did not know. How could they validate method and did they make a mistake?

    1. Yes, they made an error. The "method" developed does not demonstrate that it is specific for the sample(s) and does not show good selectivity for the sample(s) analyzed. As presented, it is INVALID and ignores basic HPLC fundamentals.

      If we estimate the Column Void Time for your method (~ 2.9 min), then we find it is very close to your first peak (2.75 min). We can assume that your first sample peak is the actual Column Void Time (T zero). We would asign it a K prime value of 0 (zero), since it is not retained on the column. This would mean that your 2nd peak has a K prime of ~ 0.03 and the third peak a K prime of ~ 0.11. An acceptable method guideline is that all peaks should have K prime values of > 1.5 (or equal to or greater than 2, for most regulatory methods). The method fails to show proper retention of the compounds so any purity statements associated with this method may be unreliable or inaccurate.

      Training in basic chromatography fundamentals is needed to develop a new method which selectively retains the compounds.

  5. What value (instead of 0.7) would be most suitable to estimate a void volume for a column filled with sub 2 μm fully porous particles?
    Thank you!

    1. They should be similar to other fully porous silica particles. We have been measuring between 0.65 and 0.70 for the latest sub 2.5u supports so those values may serve as estimates.

      To be sure, you should always measure it using an unretained standard to find out the actual value. Having an estimate helps you to develop the method (and also to quickly invalidate poor quality methods, which are more common than you may think).

      As a secondary estimate we often run the same method with mobile phase as the sample (or even a tiny amount of organic solvent). Set your detector (UV/VIS) to monitor at a low wavelength and low scale. Watch for a small baseline "blip" (small peak which goes up and down) near the estimated time. When the injector valve switches position, a disturbance in the back pressure occurs and this will be seen on the detector as a blip. You can use this as a secondary check.

  6. Great post, I want to measure the actual column void volume of my specific HPLC column with a compound which is unretained by the column. For Biopartitioning Micellar Chromatography (BMC) applications. I would like to use Thiourea to measure column dead time, and then, find K prime (retention factor). how can I prepare thiourea to be used in HPLC system to measure T0 ?

    1. The injection solution of thiourea should be prepared at a concentration which may be easily detected (~ 241 nm) on your HPLC detector and which will be within the lower end of your detector's dynamic range (On scale. Perhaps between 0.05 and 0.20 Au). Keep the injection volume small (or the same volume which is appropriate for your column and conditions). Try a few different concentrations to find the one that works best. *The mobile phase you inject it into may effect the result (run under mostly organic conditions and also, the same conditions as you use for your actual method).