The small particle sizes used in UHPLC greatly increase the theoretical plates and, thus, the separation power of the column.
Small particle sizes also increase peak capacity, sensitivity, and resolution, which allows a much smaller column to accomplish
the same separation as a much larger HPLC column. In addition, the linear velocity of the UHPLC column leads to much shorter
chromatographic run times for similar separations. The smaller column and particle size also maintains this high linear velocity
at flow rates lower than those in HPLC. Figure 2 consists of an HPLC and a UHPLC chromatogram for the same drug product. Figure
2 demonstrates what increased peak capacity brings to a chromatographic separation by moving from a 5-μm particle to a 1.7-μm
particle. The HPLC method has an 80-min run time at a flow rate of 1 mL/min. The UHPLC method has an 11-min run time at a
flow rate of 0.5 mL/min. The UHPLC chromatogram shows more peaks because of its better sensitivity. These parameters provide
a more than 90% reduction in mobile phase consumption and waste generation.
Figure 2: High performance liquid chromatogram and ultra high performance chromatogram for the same drug product.
UHPLC also can be advantageous in the method development phase. During the preformulation phase of product development, UHPLC
decreases the time needed to develop a method adequate to test research batches. Five columns can be screened in four different
mobile phases in less than 8 h. The entire method development process is considerably shorter, and the sensitivity of UHPLC
is greater than that of HPLC, so the detection limit for impurities and degradants is lower. Method validation and routine
testing can be completed in less time than for HLPC. For example, an HPLC method that has an 80-min run time would take more
than 13 h to complete the analysis for one sample. The same analysis on UHPLC has an 11-min run time, and it would be completed
in less than 1 h. Therefore, the turnaround time for experimental sample analysis is significantly shorter and allows the
formulator to adjust the development strategies without interruption.
Even though the maximum pressure for the UHPLC system is 15,000 psi, columns age faster if they are run routinely at pressures
greater than 12,000 psi. The column cost per analysis of UHPLC is about the same as that for HPLC. In addition, the internal
diameters of the connection tubing are very small (0.02–0.004 in.) and are less forgiving than those of traditional HPLC systems.
Therefore, highly purified reagents and solvents are recommended as well as better filtration of the samples and mobile phases.
Currently, many more vendors manufacture column chemistries for HPLC than for UHPLC. However, the number of companies offering
sub-2 μm columns is growing. To date, at least four vendors offer UHPLC systems and the number of column chemistries is increasing.
The advantages of introducing UHPLC to a contract laboratory are a decrease in sample turnaround time for both manufacturing
and product development, the use of less organic solvents, and a reduction in generated waste. Saving time and lowering cost
without sacrificing quality can give a contract laboratory an advantage over its competitors while providing better service
to its customers. It appears that UHPLC is here to stay.
Allison A. Aldridge is an R&D analytical manager at Mikart Inc., 1750 Chattahoochee, Atlanta, GA 30318, tel. 404.351.4510, firstname.lastname@example.org
What would you do differently? Submit your comments about this paper in the space below.
1. A.D. Jerkovich, J.S. Mellors, and J.W. Jorgenson, "The Use of Micrometer-Sized Particles in Ultrahigh Pressure Liquid
Chromatography," LC/GC North America
21 (7), 60–61 (2003).
2. Michael E. Swartz, PhD, Waters Corporation, "Ultraperformance Liquid Chromatography (UHPLC): An Introduction," Separation Science Redefined (May 2005),
3. Eric S. Grumbach et al., Waters Corporation, "Developing Columns for UHPLC: Design Considerations and Recent Developments,"
Separation Science Redefined (May 2005),
4. UHPLC: New Boundaries for the Chromatography Laboratory (Waters Corporation, Milford, MA, 2004).
5. M.W. Dong, "Ultrahigh-Pressure LC in Pharmaceutical Analysis: Performance and Practical Issues," LC/GC North America
25 (7), 31-35 (2007).