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 Column PORE SIZE (or Pore Diameter) and Retention Time

Think of your typical porous bare silica support as a big sponge full of holes. All of those holes (pores) are where the sample will migrate through before emerging out the other side. With conventional chromatography supports, most of the interaction takes place inside the particle, not on the surface. The size and number of these openings relate to retention time. Besides particle size (particle diameter), pore size is one of the most important characteristics of silica based chromatography supports.


The pore size or pore diameter is often expressed in Angstroms (i.e. 80 A = 8 nm). The degree of porosity relates to the hydrodynamic volume of your sample and is inversely related to the surface area of the support. The larger the surface area of the support (smaller pore size), the longer the possible retention of the sample. For small drug molecule samples under 1,000 daltons (an estimate only) we often use high surface area supports with small pore sizes between 60 and 150 Angstroms (~ 200 to 500 square meters per gram). These provide high retention characteristics useful in separating apart many small compounds in one analysis run. For larger molecules (i.e. peptides and proteins), we employ supports with larger pore sizes (~300 Angstroms). Particles with small pores have larger surface areas which can provide more interaction with the sample. Note: Pore size is often determined using the BET Nitrogen adsorption/desorption equation. Due to endcapping of the support (e.g. C8 or C18), the actual value obtained is often 20-30% less than the original value.

When comparing bare silica columns or trying to identify similar conventional columns for use in a method, pore size must be considered. Manufacturer's publish the pore size in Angstroms (*sometimes in nm) for their different supports. Choosing columns with similar pore sizes is just one of many parameters needed to provide similar retention characteristics. 

Introduction to Size Exclusion Chromatography (SEC HPLC)

Size Exclusion Chromatography. Often known as “SEC”.

Other names used to describe SEC:

• Gel Filtration Chromatography or “GFC” is a commonly used phrase when you are separating biological molecules in aqueous (or sometimes organic containing mobile phases). It is often described as a gentle form of chromatography leaving the protein or sample intact (*Proteins are one of the most common molecules separated using this technique, but if needed intact, must be kept away from denaturing agents). 
• Gel Permeation Chromatography or "GPC" usually refers to the separation of polymers using an organic solvent, but water soluble polymers are also applicable too.

Basic Principle: Used to separate molecules based on their molecular size in solution (as the primary mode of separation). The pore size and interstitial volume of a packed column must be determined to find out which molecules it excludes. Small molecules which are smaller than the pore size will enter the particles and spend more time navigating the channels within than larger molecules which will be excluded from entering the particles and exit quickly. It is extremely important to measure this so you know what the actual column volume is AND what the exclusion limit is. Manufacturer’s often report these exclusion limits via calibration tables for linear standards such as dextran or polystyrene though some provide data using globular standards which provides more accurate data when running many proteins or peptides. Please keep in mind that the actual confirmation (hydrodynamic volume) of the compound in the mobile phase may be different than what any of these standards are so the best column to choose may be one with a different pore size than suggested (this is why it is so important to test your compound on actual columns). Determine the actual exclusion volume running actual samples. They should elute at the Tzero point (column void volume).

Support Types: Available supports are most commonly based on either silica gel or polymeric materials (e.g. DVB). Their properties and chemical compatibility may vary so be sure to document which back-pressure ranges, pH, flow rates, temperature and/or solvents are safe to use with them.

Technique: Improved resolution often results from chaining columns together, in-series, with the same pore size. Additionally, a broader range (size) of molecules can often be separated using multiple columns with differing pore volumes together, in-series (very common in GPC applications). Single "Mixed Pore" columns are also available from many manufacturers which allow a wide range of molecular weights to be screened, though often at reduced resolution. It is important to make sure that there is no interaction between the stationary phase used and the solute employed to transport the sample. This will insure that the only mechanism being used is size exclusion.

Misc. Method Development Notes: 

(1) As the primary mode of chromatography is based on "size", achieving acceptable K prime values for retention are not applicable in this mode. K prime is NOT applicable to ion exchange or SEC modes. You must achieve retention past the initial pore exclusion point to demonstrate that the compound(s) are interacting with the pores of the phase. Measure the actual column volume to determine Tzero (this is very important). Inject an unretained compound to confirm and record the pore exclusion limit with a suitable high Mw standard.

(2) For silica based supports, strong salt buffers are often employed. You must insure proper miscibility of the sample and mobile phase at all times. Be sure and flush the system of all buffers at the end of each day. This is critical and not an optional step if you want to maintain the chromatography hardware. Salt crystals can be corrosive to the steel used in these system and may result in damage to the pump, injector and other components if not flushed out. Use a flushing solution that is similar to your mobile phase, but without the buffer. If you see any salt crystals forming on the instrument, then you have not been flushing the system down properly, or often enough. Salt should never be visible on the outside of the instrument. 

(3) Method development using buffered mobile phase solutions may employ several key variables to achieve good results. After selecting the correct column(s) use a linear flow rate and systematically adjust: (a) the molarity of the buffer salt used (e.g. 10 mM, 50 mM, 100 mM, 0.5M ...); (b) the pH of the solution (acidic, neutral, basic); (c) the temperature of the column to achieve satisfactory resolution. Note: Selecting the best column is the single most important aspect of success. If you select a column that is poorly suited to the separation, a great deal of time and money will be spent on the method development with poor results. Start with the most suitable column(s).