Speed Up HPLC Analysis Time Using Higher than "Normal" Flow Rates with SMALLER Particles

Column efficiency (as described by Van Deemter) in HPLC is largely a function of dispersion, column particle size and the flow rate of the mobile phase.After a column has been selected, the Flow rate should be optimized for all methods (start with the nominal linear velocity). Once the optimum flow rate range is achieved, little to no advantage in analysis time or solvent savings is found by increasing it (as column efficiency normally decreases at higher flow rates).

From a practical point of view, columns packed with porous 3 to 5 micron diameter supports show only small differences in efficiency as the flow rate is varied above the initial, optimum level (linear velocity). Running at too low a flow rate serves no purpose, increases dispersion/diffusion and delays the peaks from eluting off the column in a timely manner. Higher rates often decrease column efficiency. Once the flow rate has been set within the 'optimized zone', it no longer becomes a variable in HPLC method development. 

Many ~ 3 micron supports do demonstrate some ability to maintain optimum efficiency at slightly higher flow rates (e.g. with linear velocities > 1 mm/second), but significant advantages in using higher flow rates to save time and solvent are not obvious unless the particle size is reduced further. 

With the much smaller diameter ~ 2 micron particles, column efficiency can be further optimized using higher than "normal" flow rates on standard columns. Columns packed with these smaller porous particles show optimized flow rates at much higher linear velocities (e.g. 2x normal or ~ 2 mm/second for standard analytical sized columns, but experiment using 2 to 5x the normal linear velocity to compare results). 

• For example: If your method currently runs at 1.000 mL/min, you may be able to run the same method at 2.000 mL/min OR if your method currently runs at 0.200 mL/min, you may be able to run the same method at 0.400 mL/min or higher using one of the 2.5 or smaller particles. 

This increased efficiency coupled with proper optimization of the HPLC's flow path to reduce dispersion, allows for a doubling of the flow rate without a loss of efficiency (or loss of resolution). Depending on the scaling used, a two-fold savings in analysis time over conventional methods using larger particles may be observed. There may be a corresponding increase in system back-pressure too (* if only the particle size is changed and the column dimensions are unchanged). *Some of this can be countered using proper scaling of the column dimensions too). 

NOTE: Do Not Optimize HPLC Methods for "Pressure". This goes against basic chromatography fundamentals. Back Pressure is a result of pushing mobile phase through the tubing and column and is not a method development tool or variable. As mobile phase composition changes, so does the pressure. Flow rates should be stable. Work within a pressure range that is high enough to permit the pump(s) to function properly, but below the point in which frictional heating interferes with the method.

Optimization of method resolution, overall analysis time and solvent usage should be considered. The increased efficiency gained from the smaller particle size supports also allows for scaling down the column dimensions (i.e. length, ID or both) too, though a trade-off between overall column efficiency vs. analysis time and/or too high a back-pressure must be addressed to optimize the method and meet the application goals.

Summary: HPLC analytical column flow rate is often ignored in method development (* esp after it has been adjusted to the initial optimum, often 1.0 mL/min for a 4.6 mm ID column), but IF you are using porous HPLC particles that are smaller than 3.5 micron diameter, please be sure to investigate if you should re-optimize the flow rate used in your method / application so you can take advantage of any increases in column efficiency and/or scaling. As with ALL applications using these very small particles, pre-optimization of the HPLC flow path is often needed to achieve many of the available benefits.

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 Peak Tailing - Some of the Most Common Reasons For it

Three easy ways to minimize chromatography peak tailing:

(1) Tailing often results from using “Type – A” HPLC silica. Type-A silica often contains more acidic silanol groups and metal impurities than Type-B. To improve peak shape, use modern “Type – B” silicas which are of higher overall purity, have less metal contamination and feature minimal silanol ionization under higher pH conditions.

(2) Minimize ionic interactions and utilize a buffer or ion pairing agent (e.g. TFA 0.02%) in your mobile phase. Select a buffer that is at least 2.0 pH units away from your sample's pKa and use the smallest concentration or amount that gets the job done. For LC/MS or MS/MS applications, remember to only use volatile buffers and avoid the use of ion pairing agents unless absolutely necessary (and if used, use at the lowest possible concentration to avoid source contamination).

(3) Always use a freshly washed and equilibrated column. Is the column fouled or the inlet frit dirty? If the head of the column is fouled from sample overloading or from a failure to wash off strongly retained compounds from many runs (much more common problem), then your peak shape and reproducibility will suffer. Incorporate a washing step in between your analysis methods which utilizes a solvent which is stronger (in concentration) than your mobile phase to wash off any strongly retained material after each run. For example, if you normally end a method with an 80% concentration of ACN, utilize a separate wash method which has 95% ACN in it. Allow enough wash time for this work.

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.