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 Mobile Phase Composition and LC-MS Electrospray Voltage

I am often asked about the importance of selecting and optimizing the LC-MS Electrospray Ionization Interface (ESI) voltage. To better understand why it is necessary to do so and how it effects the results obtained, let us review some key facts about ESI first.

• While a gas sheathed flow of volatile mobile phase is directed into the MS source, a strong positive or negative electric field (KV) is applied across the MS inlet. The effluent is atomized and evaporated to form ions (voltage polarity determines positive/negative mode).
• Too high of a capillary voltage may produce electrical arcing resulting in damage to the system (e.g. PEEK needle may melt, burn and/or clog).
• Too low of a capillary voltage and ion evaporation will not occur.
• The voltage needed to produce efficient desolvation and ion evaporation are directly related to the sheath gas flow rate, the mobile phase composition and the flow rate.

What Can You Do To Insure Finding A Suitable ESI Capillary Voltage?

1. High quality HPLC methods which utilize fully volatile mobile phases and first retain, hold, then elute all samples are needed to generate LC-MS or LC/MS-MS methods. Optimize the HPLC column type, dimensions, MS compatible mobile phase composition and flow rate before optimizing the MS settings. If you have enough sample available, use an infusion method (continuous flow injection) to establish the initial MS settings needed to detect the sample before continuing with the LC/MS method development optimization. Infusion (with a syringe pump) provides the needed time to makes changes, observe how they change the signal for fastest optimization.
2. The HPLC mobile phase and any dissolved additives or buffers used for LC/MS analysis must be of high purity and fully volatile.
3. Make sure your sample is fully dissolved in the mobile phase and filtered (0.22 u filter) before injecting into the system.
4. Basic samples can be protonated to form [M+H]+ clusters in acidic mobile phases.
5. Acidic samples can be deprotonated to form [M-H]- clusters in basic mobile phases.
6. The electrospray ionization (ESI) process used in LC/MS or LC/MS-MS analysis is affected by the surface tension of the HPLC mobile phase used. Water has a higher surface tension than most organic solvents (i.e. Methanol, Acetonitrile, Ethanol, IPA). Using conventional flow rates with highly aqueous mobile phases requires a higher initial voltage for ion evaporation to occur. IOW: Mobile phase mixtures high in water content will require a higher capillary voltage.
7. Higher organic solvent content usually leads to better atomization / droplet formation and require less capillary voltage to maintain.
8. Lower HPLC flow rates usually lead to better atomization / droplet formation and require less capillary voltage to maintain.
9. To optimize the ESI capillary voltage it is necessary to carry out experiments trying different voltages and monitoring the signal (S/N of a standard or sample) to find the best voltage which results in good signal quality and low noise. This process requires experience to know which settings are likely to enhance the signal and a great deal of skill operating the Mass Spectrometer.

Optionally, ESI signal output may be enhanced using: Adducts or changing the solution chemistry with other mobile phase additives.