HPLC Column Dimensions. HPLC columns are manufactured in a variety of different internal diameter and length combinations, as well as having an assortment of particle sizes. HPLC column dimensions will affect the efficiency sensitivity, and speed of an analysis. The choice of column dimensions will depend on the chromatographic application; analytical, semipreparative, preparative, number of analytes in the mixture etc. However, the dimensions can also be altered to improve chromatography by achieving more efficient, sensitive, and faster analyses. The primary column dimensions of particle size, length, and internal diameter will be discussed and their effect on chromatographic separations demonstrated, providing some practical insight into how to select the best column dimensions for a particular application. 



Particle Size The particle size of the support material is of primary importance when selecting a stationary phase. 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 the minimum plate height is 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 2 shows 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 1. 

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.
Particle size particularly affects the efficiency term of the fundamental/Purnell resolution equation (Equation 2). Efficiency is inversely proportional to particle size (Equation 3); hence, a decrease in particle size will result in an increase in efficiency. This increase in efficiency will allow the use of shorter columns and/or faster flow rates which will ultimately result in faster analyses without loss of resolution, as is demonstrated in Figure 3. Note the increase in system back pressure caused by reducing the particle size. 



Particle Size Distribution 



Length Efficiency is directly proportional to column length (Equation 3). Increasing column length will increase efficiency. Doubling column length increases resolution but only by a factor of 1.4. Short column lengths (3050 mm) will give short run times and low backpressures and are ideal for gradient analyses. Longer columns (250300 mm) will give greater resolution but with longer analysis times and at a greater cost. 

The separation of four paraben compounds detailed in Figure 5 demonstrates the effect of column length. As can be seen decreasing the column length reduces analysis time, however, resolution is also decreased.  




Internal Diameter 

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 4. 

Where: 
