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 to UHPLC Conversion Notes (Column Dimensions, Flow Rate, Injection Volume & System Dispersion)

The use of ultra-high performance liquid chromatography (UHPLC) columns to reduce analysis times and sometimes improve detection limits is a hot topic. UHPLC presents a number of new issues. The incorporation of smaller 1.9 to 3.0 micron particles and smaller frits will raise backpressures and increase system wear and tear. Smaller diameter lines are often used (I.D. of 0.12mm or less) which can increase blockages and clogs if you do not filter your mobile phase and samples through a 0.45 or 0.2 micron filters. Piston seals and valve rotors can wear out early due to the very high pressures, heating and stress imposed on them. You should monitor your HPLC system carefully over time and consider increasing the frequency of preventative maintenance and inspection services as well. However, the smaller particle sizes can provide better resolution in some applications so they are well worth evaluating.

I must answer twenty or so questions each week in the area of UHPLC. The most common questions deal with selection of an UHPLC column and making adjustments to a method for the changes which effect: (1) Column Dimensions; (2) Flow Rate; (3) Injection Volume and (4) System Dispersion. The good news is that some of these questions can be answered with some basic math while others just require a basic understanding of how the system works.

(1) COLUMN DIMENSIONS: Let's start by making things as simple and brief as possible (this is supposed to be a "hint & tip", not a thirty page article). When initially converting from a convention HPLC column (e.g. with 5 micron particles) to an UHPLC column (e.g. with 1.9 to 3 micron particles), initially select a column with the same I.D. and length for the calculation. This way only the particle size changes. *I like to change one variable at a time. If you would like to change the column length to take advantage of some of the increased efficiency (and decrease the pressure!) which results from smaller particles, then please refer to the following equation.

     EQUATION A:  'Lc2' = ('Lc1' * 'p2') / 'p1'

[ 'Lc1' = Length of Column #1 in mm; 'Lc2' = Length of Column #2 in mm; 'p1' = particle size of Column #1 in microns; 'p2' = particle size of Column #2 in microns].
                    
   Example: Column # 1 is a standard HPLC column;  4.6 mm x 250 mm (5u) Column. You want to find out the length of an equivalent column which uses 1.9 micron particles instead of the 5 micron particles.

   'Lc2' = (250 * 1.9) / 5 ; Answer is: 'Lc2' = 95 mm. *So a 10 cm long column would be a good choice here.


(2) FLOW RATE: Flow rate is directly proportional to column diameter and as we saw above in Equation A, the particle size can also affect it too. If you keep the column length and internal diameter the same, then the linear flow will be unchanged with the same particle size. A change to the particle size alone will change the flow rate as follows: 'Fc2' = 'Fc1' x ('p1'/'p2').

A change to a smaller diameter column to compensate for the improved efficiency will require a change to the original flow rate to preserve the linear velocity. Please refer to the following equation.

     EQUATION B:   'Fc2' = ('d2' / 'd1')^2 * 'Fc1'

['Fc1' = Flow Rate of Column #1 in ml/min; 'Fc2' = Flow Rate of Column #2 in ml/min; 'd1' = Column #1 Diameter in mm; 'd2' = Column #2 Diameter in mm].
                     
   Example: Column # 1 is a standard 4.6 mm ID Column. You want to find out what the linear flow rate should be if you use a smaller diameter column (2.1mm in this example).

   'Fc2' = (2.1/4.6)^2 * 1.000 ; Answer is: 'Fc2' = 0.208 ml/min. *A flow rate of 200 ul/min would be fine. 

However, one other factor should be considered. The optimum flow rate for sub 2.5u particles are often about double that of the "normal" linear flow rate used with conventional particles (>2.5u). Evidence for this has been shown through analysis of the van Deemter curve with the tiniest particles showing much flatter curves. Retention (K prime) can often be maintained by combining twice the normal flow rate and speeding up the gradient time by a factor of 2. So a method utilizing std sized particles with a linear flow rate of 0.200 ml/min might benefit from a faster flow rate of 0.400 ml/min and a twice as fast gradient composition change.


(3) INJECTION VOLUME: A change in the column dimension may require a change to the injection volume (note: "volume" and concentration are two different things. If the solution concentration remains the same and you inject less, the on-column sample concentration will also be less). The smaller the internal volume of the column, the smaller the injection volume. To calculate the linear change in volume, please refer to the following equation.

     EQUATION C:   'V2' = 'V1' * {('d2'^2 * 'L2') / ('d1'^2 * 'L1')}

['V1' = Injection Volume #1 in ul; 'V2' = Injection Volume #2 in ul; 'L1' = Column #1 Length in mm; 'L2' = Column #2 Length in mm; 'd1' = Column #1 Diameter in mm; 'd2' = Column #2 Diameter in mm].
                     
   Example: Current injection volume is 10 ul. Column # 1 is a standard 4.6 mm ID x 250 mm Column. You want to find out what the equivalent injection volume should be for a 2.1 mm ID x 150 mm column.

   'V2' = 10 * (2.1^2 * 150) / (4.6^2 * 250) ; Answer is: 'V2' = 1.25 ul.



(4) SYSTEM DISPERSION: When converting HPLC methods to "UHPLC" methods, few parameters effects the results obtained more than the HPLC system's System Dispersion. The volume of liquid that is contained between the injector needle and flow cell (with the column removed or by-passed) is know as the system dispersion volume. This volume is determined by how the specific HPLC is designed and plumbed. On most HPLC systems, it can be easily changed and optimized to fit the specific application desired and only requires that you have a solid understanding of how the HPLC system works. The choice of connection tubing ID and length, how the autoinjector is programmed, its loop size and the detector's flow cell volume all contribute to the system dispersion volume. In the same way that changes to the total column volume can effect the peak shape and resolution, the internal system dispersion volume also contributes to the results. 

With standard sized analytical columns (i.e. 4.6 x 250 mm), the typical HPLC's system volume is so small relative to the volume of the column (e.g. 100 ul system dispersion vs 2900 ul column volume, or 3.5%) that it does not negatively impact the chromatography. However, anytime we utilize a tiny HPLC column whose column volume is a fraction of that found in a standard column (i.e. 100 ul system dispersion vs 2.1 x 50 mm column with 120 ul volume), diffusion and band spreading can quickly become so significant that effective plate numbers are quickly reduced below values found on a standard sized column. As column volume decreases (and approaches the system volume) the total system dispersion volume must also decrease. In general, try and keep the system dispersion volume at or below 10% of the column dead volume. This is most easily accomplished by reducing the number of connections and fittings used, reducing the lengths of all tubing used, using much narrower ID tubing (e.g. 0.12 mm vs 0.17 mm ID), reducing flow cell volume and reducing the flow-through volume in the autoinjector (i.e. loop size, needle seat, etc). The injector is often the largest contributor to the system dispersion so concentrate efforts here (e.g. after injection, switch the loop out of the flow path).