New sample preparation requirements for Ultra-High Performance Liquid Chromatography

New sample preparation requirements for Ultra-High Performance Liquid Chromatography
Tony Lewtas, Vivek Joshi and Marcy Engelstein

Introduction to UHPLC technology

Significant improvements in separation times and efficiencies can be achieved by using next generation High Performance Liquid Chromatography (HPLC) technologies such as Ultra-High Performance Liquid Chromatography (UHPLC or UPLC™). To maximize the benefits of UHPLC technology, a few, simple modifications to sample preparation procedures are required. A brief explanation of the technology is useful in understanding why these changes are necessary.

Chromatographers have long known that sub-2 µm particles packed in short columns can deliver reduced sample run times while maintaining good separation efficiency. However, an undesirable side effect of these columns is system back-pressure that far exceeds the limits of traditional HPLC systems. To achieve the benefits of this new column technology, manufacturers have developed instruments capable of running at pressures much higher (up to 15,000 psi) than standard HPLC systems. This new instrument/column combination is known as UHPLC.

The main advantage of UHPLC comes from the sub-2 micron particles leading to improved separation efficiency as predicted by the Van-Deemter equation. The columns packed with smaller particles can be run at higher flow rates without compromising separation efficiency, peak capacity and resolution. Resolution improvements of up to 50% over standard HPLC separations are routine. The advantages that a UHPLC system can bring to an analytical lab include a general increase in productivity, reduced method development time, more data from smaller samples and decreased solvent usage and disposal costs.

Rethinking sample preparation procedures, UHPLC Challenges

Most methods developed on traditional HPLC systems can be transferred to a UHPLC system without much difficulty. However, UHPLC, with its smaller particle sizes, lower interstitial void volumes, decreased column diameters and higher flow rates, presents scientists with new challenges:

• Ruggedness, accuracy and reproducibility of the system
• Low sample loading capacity of columns
• High instrumentation costs
• Shorter column life for UHPLC columns compared with HPLC
• Increased attention to sample and mobile phase preparation

Clean samples and running buffers are the key to addressing some of the challenges associated with UHPLC technology. To minimize system failure and maximize system performance, instrument manufacturers recommend using ultra-pure water and filtering mobile phase components through 0.2 µm membrane filters. Poor water quality and unfiltered buffer salts result in particulates in the mobile phase. Particles can cause increased back-pressure, column clogging and eventual system shut down.

For the same reason, meticulous attention should be given to sample preparation. Even though the volume of sample injected on the system is small (2-10 µL) compared to the solvent volume (300-1000 µL/min), sample preparation techniques utilizing 0.2 µm filters, which help remove particulate impurities from the sample, should be practical.

Summary

Using general lab filtration techniques is an easy way to achieve the highest levels of UHPLC system performance. Membrane filtration devices are available in a variety of formats and materials, including pressure-driven syringe filters, for preparing up to 10 mL of sample, and disposable plastic or reusable glass vacuum-driven vessels for filtering liters of solvents and buffers. Simple filtration provides a fast and economical means of preparing samples and mobile phases for optimal UHPLC results. A precut disc filter that costs about $1.50 is a far better place to collect damaging particulates in mobile phases than a high priced column critical to the operation of sensitive and expensive UHPLC equipment.