We investigated the influence of volumetric flowrate on peptide separation performance with 2.1 mm i.d. columns. A standard
peptide mixture was separated on a UPLC column run at 100 μL/min and at 300 μL/min (Figure 2). Flowrate is the only variable
because the gradient change per column volume is the same, ensuring that the chromatographic selectivity is constant. We ran
a 75 min gradient at 100 μL/min and a 25 min gradient at 300 μL/min. Figure 2 shows a comparison of peak volumes, calculated
by multiplying the flow rate by peak width at the base. Peak volumes at 100 μL/min average about half the volumes at higher
Figure 2. Influence of flow rate in UPLC peptide mapping with a 2.1 mm column. The peaks are normalized versus the tallest
peak, and detected by MS.
We expected peptide peak volumes, a measure of resolution and sensitivity, to be optimum at 100 μL/min. Most peptide maps
done with 2.1 mm HPLC columns are run higher than 100 μL/min. The higher flow rates represent a compromise between resolution
and run time. Other users of HPLC accept this compromise. Another justification are instrumental limits in reproducibly pumping
liquid at flow rates less than 150 μL/min with accurate and precise gradients.
However, the ACQUITY UPLC instrument performs extremely well at a flow rate of 100 μL/min in gradient mode. Figure 3 demonstrates
this performance by the overlay of six gradient runs of a peptide standard, with the average and standard deviations of retention
time listed for each peak.
Figure 3. UPLC peptide mapping retention time reproducibility is good at 100μL/min. Mean and standard deviation are given
for each normalized peak.
In some cases it would be desirable to reduce the run time of a peptide map. HPLC maps often require cycle times of 3 to 5
hours to separate all the peptides within the digest, especially for large proteins like antibodies. While faster peptide
maps are desirable, it is critically important not to compromise resolution to ensure that the test results provide the same
level of information.
The van Deemter equation predicts that plate height will be two to four fold less with 1.7 μm particles than with 3.5 μm particles.
We expect to see the same resolving power with a shorter column.
A test demonstrates how UPLC can resolve the same number of peaks in a peptide map as HPLC but in less time. The separation
of an enolase digest was done on a 50 mm long UPLC column with a 20 min gradient and on a 100 mm long HPLC column with a 40
min gradient; both with flow rates of 100 μL/minute. Figure 4 shows these chromatograms. The UPLC separation shows a comparable
number of peaks and a similar overall elution pattern as the HPLC separation, but in half the time.
Figure 4. Using a shorter UPLC column to improve peptide-mapping throughput. Top plot is UPLC. Bottom plot is HPLC. The peaks
are normalized versus the tallest peak.