

Step 1: Reduce Column Length
Simply shortening the HPLC column reduces separation time; however, resolution will also be reduced due to a decrease in the plate number (available stationary phase surface). In general column length is directly proportional to retention time, column efficiency, and backpressure. Reduction of column length is acceptable as long as column efficiency remains sufficient for separation; therefore, when decreasing the column length as a means to decreasing analysis time the efficiency of the peaks must be considered and improved if necessary. 



Step 2: Reduce column I.D., decrease flow (maintain constant linear velocity)
Achieving increased linear velocity can be achieved in several ways, however the combination of eluent flow rate and column internal diameter ultimately define the achievable linear velocity with any particular column.
Where any of the numerators increase, flow (F) or column length (L) for instance, a resultant increase in back pressure will be observed. Where any of the denominators decrease, column radius (r) or particle size (d_{p}) for instance, an increase in back pressure is also observed. Note that the radius and particle size functions are raised to the second power, thus, causing their impact on back pressure to be much more dramatic. Larger diameter HPLC columns require higher flow rates; therefore, larger volumes of mobile phase will be used. Changing from a 4.6 mm (1) to a 3.2 mm (2) ID column can reduce the flow rate and solvent volume required to reach the same optimum linear velocity without increasing the run time. The new flow rate can be calculated using Equation 5.
When column internal diameter is decreased an increase in sensitivity (23 fold) can be expected when injecting the same analyte mass. This is due to there being an increased analyte concentration in the mobile phase. 



Step 3: Reduce particle size and increase flow rate  


By reducing the particle size diameter, not only is the efficiency of the column increased (plate height (H) is reduced), but the optimum linear velocity at which minimum plate height achieved is also increased. More efficient peaks can be achieved at elevated flow rates, leading to a reduction in analysis time accompanied, potentially, by an increase in resolution. Shorter diffusion paths and, hence, increased mass transfer kinetics (C term from the Van Deemter equation) are one reason behind this reduction in plate height. Figure 6 and 7 show the relationship between particle size, flow rate (linear velocity), and plate height for columns packed with various silica particle diameters. 



It may seem that the smallest diameter particles will be the obvious choice for high efficiency separations; however, this benefit does come at a substantial price, that of increased back pressure. The pressure increase is inversely proportional to the square of the particle diameter as shown in Equation 6. Note the increase in backpressure when decreasing particle size in Figure 7. It can be seen that reducing particle size when using traditional porous silica can result in high back pressures which may not be tolerated by traditional HPLC instruments. The use of superficially porous materials allows the use of particle sizes which produce lower back pressures (i.e. 2.62.7 µm) while still increasing efficiency and allowing the use of increased flow rates, which ultimately results in faster analyses. This is due to the increased mass transfer kinetics associated with superficially porous particles (C term of the van Deemter equation) for larger molecules (biomolecules) and an increase in packing efficiency and reduction in multiple path lengths through the column (A term of the van Deemter equation) for small molecules, which is demonstrated by the very flat van Deemter curves produced by this packing material type (Figure 8). 
