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. 

Pressure Drop Across an HPLC / UHPLC Column

Many of you prefer tables of data over equations that you must work out. So, instead of providing you with another equation, I have done some basic measurements for you to provide a general overview of how particle size (porous) effects System backpressure.

For simplicity, let us start with a few parameters. Pore Volume = 0.70; Linear Velocity = 1.44 mm/sec; Solvent Viscosity = 0.89 cP at 25C (Water). 

Pore Volume and Flow Resistivity will vary by column type. Obviously the back pressure will be higher with more viscous solvents (e.g. EtOH is 1.20 cP) and lower with less viscous solvents (e.g. ACN is 0.34cP). A Table of HPLC Solvent Viscosity values can be found here [ http://www.hplctools.com/lcsolvent.htm ]. Linear flow rates have been used for all column I.D.'s to better illustrate the relationship between column dimensions and flow rate. If you double the flow rate, then the pressure will approximately double as well. 

Note that when run at traditional linear velocities, most 2.5u particles are within the maximum pressure limits of most HPLC systems (under 400 bars). Only the newer sub 2.0 micron particles used in long columns exceed the 400 bar limit. The higher maximum pressure limits of many UHPLC systems allow the use of higher flow rates with these particles. Naturally, you should optimize both column efficiency and system dwell volume when developing any UHPLC method. Failure to optimize the dwell volume (and minimize all volumes) may result in very poor chromatography separations. Meeting any/all backpressure requirements to run a method does not translate to success in sample analysis. Successful ultra-fast separations require ultra-low system dwell volumes, higher sampling rates and usually smaller flow cell volumes.

HPLC Column I.D. (mm)	Particle Size (u)	Column Length (mm)	Flow Rate (mL/min)	Observed System Back Pressure (Bars)
4.6	5	250	1.000	89
4.6	5	150	1.000	54
4.6	5	100	1.000	36
4.6	5	50	1.000	18
4.6	3.5	250	1.000	182
4.6	3.5	150	1.000	109
4.6	3.5	100	1.000	73
4.6	3.5	50	1.000	36
4.6	2.5	250	1.000	357
4.6	2.5	150	1.000	214
4.6	2.5	100	1.000	143
4.6	2.5	50	1.000	71
4.6	1.9	250	1.000	618
4.6	1.9	150	1.000	371
4.6	1.9	100	1.000	247
4.6	1.9	50	1.000	124
3.0	5	250	0.430	90
3.0	5	150	0.430	54
3.0	5	100	0.430	36
3.0	5	50	0.430	18
3.0	3.5	250	0.430	184
3.0	3.5	150	0.430	110
3.0	3.5	100	0.430	74
3.0	3.5	50	0.430	37
3.0	2.5	250	0.430	361
3.0	2.5	150	0.430	217
3.0	2.5	100	0.430	144
3.0	2.5	50	0.430	72
3.0	1.9	250	0.430	625
3.0	1.9	150	0.430	375
3.0	1.9	100	0.430	250
3.0	1.9	50	0.430	125
2.1	5	250	0.210	90
2.1	5	150	0.210	54
2.1	5	100	0.210	36
2.1	5	50	0.210	18
2.1	3.5	250	0.210	184
2.1	3.5	150	0.210	110
2.1	3.5	100	0.210	73
2.1	3.5	50	0.210	37
2.1	2.5	250	0.210	360
2.1	2.5	150	0.210	216
2.1	2.5	100	0.210	144
2.1	2.5	50	0.210	72
2.1	1.9	250	0.210	623
2.1	1.9	150	0.210	374
2.1	1.9	100	0.210	249
2.1	1.9	50	0.210	125

* The results obtained in this table from are from one of our HPLC systems and reflects the total system backpressure (what the pressure gauge reads), with the column inline. Your results may vary due to differences in HPLC system used, flow path, tubing ID, column choice and mobile phase selected.