HPLC Column Support Pore VOLUME

If an HPLC column had no packing material inside it, then the volume of liquid contained in the cylinder could be calculated using the formula for the volume of a cylinder as follows: 

      Volume of Cylinder = Pi * r² * L;     

          [where Volume is in ul; Pi = 3.14; r = column radius (mm) and L= column length (mm)]

  Example: Using the above formula, a 4.6 mm x 250 mm column would have an empty volume of 4,155 ul (~ 4.16 mls).

For most chromatography applications we pack the column with a high surface area porous media. Often this is a silica based support. This support media fills the empty space inside the column reducing the total volume accessible by a liquid (or to the samples). If the media used was not porous, it would fill most of the space (depends on size and shape of media). Most commonly used chromatography supports are porous and leave about 70% (0.7) of the original volume available to the mobile phase and sample [Pore Volume = Surface Area (m²/g) x Pore Diameter  (Å) / 40,000]. Based on this information, we use a value of 0.7 as the average pore volume for a packed chromatography column (some supports will have pore volumes which are larger or smaller than this value. The manufacturer will often measure it and provide the value on their published specification sheet).


Using a typical 4.6mm x 250mm column we found the total volume to be 4,155 ul (4.16 mLs). If we now multiply this empty column volume by 0.7 (note: use 0.7 or 70% for columns with fully porous particles and 0.55 or 55% for superficially porous particles) we obtain 2,908ul total volume (2.9 mLs). This is the estimated volume of the fully packed column. This value is very important as it provides an estimate of what the column dead volume will be so we can calculate the 'T' zero time of an unretained analyte. This estimate will depend on the column dimensions, using our HPLC method (be sure and take into account the measured flow rate to determine the column "dead time"). This is one of the very first calculations you make when starting or modifying an HPLC method and is critical information to know at all stages of method development. All chromatographers should know how to estimate this value before using an HPLC system. *You should confirm this estimate by injecting an unretained sample onto the column and measure the retention volume, then compare the two values. The measured value is the most important number (the one we use for calculations), but the estimate should be close (+/- 15%). The estimate is still useful for troubelshooting and method development as when combined with K prime, it provides a quick measure if chromatography has occurred (retention).

For more information on the importance of knowing the HPLC Column Dead Time, please refer to this article link. 

Notes: The measured support pore Diameter (SIZE) is important for determining if the sample will have access to the inside of the support (e.g. A support with a pore size of 80Å will be too small for most large peptides or proteins, but a support that is 300Å will allow access to many, not all, larger molecules). A support with too small a pore diameter will not allow the sample to access the high surface area inside the support. Instead, the sample will be unretained and pass by it eluting at the column's void volume. This is the basis of SEC or GPC analysis where we use columns with different pore sizes to "filter" samples based on size. Large pores for large Mw samples and small pores for low Mw samples. A general rule is use 300Å or larger pores for samples with Mw > 10,000 and 80Å to 150Å for smaller samples.

More info on pore volume can be found at this article link: https://hplctips.blogspot.com/2014/12/hplc-column-pore-volume-or-pore.html

HPLC Capillary Tubing Connection Volumes:

The length and internal diameter of the HPLC interconnecting tubing used in your system really does matter. The total volume contained in the tubing can dilute your sample or separated peaks. This can effectively undue the work of separating the peak(s) on a column. Extra volume in the tubing can also have the effect of increasing the gradient delay factor for your method (the greater the volume of the tubing from the pump head to the column inlet, the greater the delay in the solvent mixture arriving at the column). In general, keep the the total delay volume as low as possible. This is accomplished by connecting the various modules together using the shortest lengths of tubing possible. For systems which use standard sized HPLC columns (e.g. I.D.'s of 3.0 to 4.6mm and lengths from 100mm to 300mm) the tubing internal diameter should be 0.17mm (0.007"). For systems which use very short, mini or micro bore sized HPLC columns (e.g. I.D.'s of 1.0 to 2.1 mm and lengths from 50mm to 250mm) the tubing internal diameter should be 0.12mm (0.005"). Looked at another way, if the total column volume is less than 750 ul, consider using the smaller internal diameter tubing (0.17mm) to reduce band broadening. 

Here are some tubing volumes to help you evaluate the effect changing the I.D. or length has on the tubing that you use.

I.D. (mm)	I.D. (inches)		ul / cm	ul / inch
0.12	0.005		0.127	0.323
0.17	0.007		0.249	0.632
0.25	0.010		0.507	1.288
0.51	0.020		2.026	5.146
1.02	0.040		8.103	20.581

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

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 Volume (ul) = (d^2 *Pi * L * 0.7) / 4 ;

•  Column Void Volume Equation for SUPERFICIALLY Porous Supports (e.g. Fused-Core, Core-Shell etc):

Column Volume (ul) = (d^2 *Pi * L * 0.5) / 4 .

   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).