Column Hold-up Volume, Column Dead Time or 'Column Void Volume' (the preferred name) are all different terms we apply to find the internal volume of a packed column (divided by the flow rate and usually expressed in minutes for the Column Void Time). You must know what this value is BEFORE starting to run an HPLC method or perform liquid chromatography. The value for column void volume changes for different column dimensions and different column support types (e.g. fully porous, superficially porous etc) .
Are you peaks or samples eluting at or near the column void volume? If so, for most modes of chromatography, this implies that no chromatography has taken place and no HPLC method has been developed (SEC/GPC separate based on hydrodynamic volume, so elution at or near the column volume means the sample(s) were excluded from the column). Individuals with little to no chromatography training or experience often make this mistake and create methods which show poor retention. Make sure your methods are designed to retain each sample for a long enough time period on the column (K prime). How do you know how long is long enough? Start by estimating the Column Void Volume (use our table or calculate it for an estimate) 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 most regulatory authorities) for the method to be useful and selective. *A more accurate value of column void volume will be found by measuring the void volume of your column (please read on).
Knowing the Column Void Volume and the Flow Rate used allows you to calculate the Column Void Time (which is the most useful initial 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 (K prime aka: "K1") in a chromatography separation. Ideally, it is measured by injecting a sample which is unretained by the column & mobile phase (it passes right through the column support with little to no interaction). It may also be easily estimated for most fully porous, 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 two 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. Use a low UV wavelength to observe this deflection on the baseline.
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
- Column Void Volume Equation for Std Sized, FULLY Porous Supports:
- Column Void Volume Equation for SUPERFICIALLY Porous Supports (e.g. Fused-Core, Core-Shell etc):
Note: Column Diameter & Length are in mm. Volumes are estimates (always measure to find the actual value).
[Note: All you need is the column's length and ID to estimate it. For most fully porous supports, use a 'Pore Volume' value of 0.70 in the above equation. This is the most commonly measures pore volume found for non-encapped, fully porous spherical bare silica support (please check with the manufacturer for the actual value of your support). For superficially porous supports, use a value of 0.50. 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).
We use this table all of the time in our lab! Thanks.
ReplyDeleteQuestion - 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?
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).
DeleteThe 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.
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?
ReplyDeleteIf 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).
DeleteIf 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.
ReplyDeleteMeasurement 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.
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?
ReplyDeleteYes, 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.
DeleteIf 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.
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?
ReplyDeleteThank you!
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.
DeleteTo 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.
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 ?
ReplyDeleteThe 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).
DeleteIn the void volume calculation what is the role of the divisor 4?
ReplyDeleteBasic math really, but the version that I have shown in the article uses the measured column DIAMETER, not radius, for convenience.
DeleteTo estimate void volume of a packed HPLC column we use the standard formula to find the volume of an empty cylinder plus factor in the contribution of the packing material inside (pore volume). Because HPLC columns are sold based on their Diameter and Length, we use their diameter (not radius) to simplify the equation. *That is why the denominator (4) is used. You can of course use a similar formula to calculate the volume of a cylinder (i.e. Pi x r2 x L) x Pore volume to obtain the same result (no '4' needed). Give it a try and you will see that you get the same answer.
Understood about the convenience of using diameter.
DeleteYes, same answer either way! It is now crystal clear. Thanks!
Why did we use .0.7 value to calculate column volume
ReplyDeleteThe column is not an empty cylinder, but a cylinder filled with porous particles. Those particles occupy volume (space) inside the tube so the actual HPLC column volume must be less than the theoretical volume for an empty cylinder. Different particle types will occupy different volumes. *We need to adjust for this fact in our calculation. A value of 70% (0.70) is consistent with many commercial HPLC supports. In fact, it may be a bit more or less than this estimate. That is why we only use this value as an ESTIMATE and MUST measure it for ALL methods. Once known, we can correctly calculate K prime and other important values to determine if our proposed method really is selective for the sample under analysis.
DeleteCan you suggest me where can I find this formula in literature?
ReplyDeleteYou have found it right here! [Letter, W.S. "Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero)"; https://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html ].
Delete*HPLC Column Volume is based on the volume of a cylinder. The formula can be found in any basic math textbook. The HPLC column packing material contributes to the internal volume (decreases it). The particle 'porosity' values come from the manufacturers. They vary, and so the formula used. We use 0.7 as a generic value, but with modern supports could 0.5 to 0.7. *The formula is designed to provide an ESTIMATE only. Estimates are useful when first developing a method or when reviewing a method to see if it meets basic accepted criteria. To provide a basic level of scientific validity, you must estimate and MEASURE the column volume to solve for T zero for ALL samples and ALL methods.