This article demonstrates the application of UPLC to peptide mapping in a series of experiments. Most of the instruments and
chemicals were from Waters with three exceptions: acetonitrile (Optima Grade) was from Fisher, TFA was from Pierce, and formic
acid was from EMD. Exact conditions are listed in Table 1.
The ACQUITY C18 chemistry is based on a bridged ethyl hybrid base particle, specifically designed for operation at higher
pressure. It has an average pore diameter of 130 Å a pore volume of 0.7 mL/g, and a surface area of 185 m2/g. Its diameter is 1.7 μm.
PLATE HEIGHT AND VELOCITY
The chromatographic benefits of UPLC are largely derived from reduced band-broadening that is, in turn, a consequence of reduced
diffusion distances in small particles. This process is quantitatively described in the van Deemter equation that relates
height equivalent of a theoretical plate (H) to linear velocity. Figure 1 graphs this relationship for a peptide of 1,500 Da on 3.5 μm and on 1.7 μm packings. The minimum
in the curve corresponds to the maximum efficiency, and greatest resolving power, for each particle size.
Figure 1. Van Deemter Plot for 1,500 Da peptide. The equation has the form, H = A + (B/u) + Cu, where H = Height equivalent
of a theoretical plate (cm), u = average linear velocity (cm/s), and A, B, C are constants.
At linear velocities or flow rates above and below the optimum, resolving power declines. The smaller particles have higher
resolving power at a higher linear velocity. In Figure 1, the 3.5 μm particles have a minimum plate height of 8.11 μm at a
linear velocity of 0.17 mm/s. In contrast, a minimum plate height of 3.94 μm is observed at 0.33 mm/s with the 1.77 μm particles.
In practical terms, these suggest that the small particles used in UPLC could increase the resolving power in a peptide mapping
experiment, and should simultaneously reduce the separation time because the optimum is achieved at a higher linear velocity.
For the 3.5 μm particle, the optimum linear velocity corresponds to a flow rate of about 24 μL/min on a 2.1 mm i.d. column
or about 5.5 μL/min on a 1 mm column. In practice, such flow rates would never be used for a peptide map because the separation
times would be far too long. It is common practice to operate at higher flow rates, typically about 250 μL/min and 60 μL/min
on 2.1 and 1 mm columns, respectively. A linear velocity of about 1.7 mm/s corresponds to a plate height of about 21 μm. This
2.6-fold loss of resolution with a 10-fold increase in separation speed becomes an accepted compromise in the analytical community.
For 1.7 μm particles, however, the plate height at 250 μL/min only increases to 6.45 μm, a 1.6-fold sacrifice from the optimum.
This analysis suggests several ways to approach improving peptide maps using UPLC. First, columns with the smaller particles
(1.7 μm) will improve both resolution and sensitivity by reducing diffusion-related band broadening. Second, reducing the
plate height is consistent with obtaining the same or better resolution with shorter columns and higher flow rates. Third,
the compromise between separation time and resolution is more favorable with the smaller particles.