Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, January 30, 2016

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 of 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 superficially porous and leave about 70% 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 we obtain 2,908ul total volume (2.9 mls). This is the estimated volume of the packed column. This value is very important as it provides an estimate of what the column dead volume (aka: "column dead time") will be so we can calculate the 'T' zero time of an unretained analyte using our HPLC method (be sure and take into account the measured flow rate). 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. *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 valuable number, but the estimate should be close. 

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

Notes: The measured 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 proteins, but a support that is 300Å will allow access to many 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. 

More info on pore volume can be found at this article link: