Divide and conquer

As ultra high performance liquid chromatography gains in speed and resolution sample prep becomes ever more important. Vivek Joshi and Estelle Riche let us in on the best practices for sample and mobile phase preparation for UHPLC

Ultra high performance chromatography (UHPLC) offers well-recognised benefits. Improvements in speed and resolution of 30 to 50% over standard HPLC separations are typical. Chromatography runs that would have taken five to 20 minutes just a few years ago now require only a fraction of that time, and the increased resolution allows for better separation of components from a complex mixture. 

However, the greater speed and resolution of UHPLC are putting increased demands on sample and mobile phase preparation to avoid system downtime and ensure quality results. Smaller packing material and tubing diameters make these systems highly susceptible to clogging by impurities. Selection of the optimal sample and mobile phase filtration strategy for UHPLC is critical.

With UHPLC’s extremely low detection limits, attention must also be focused on the quality of the reagents. Ultrapure water is the largest component of the mobile phase, and it plays an essential role for running blanks, diluting samples, mixing buffers, and preparing standards. The water used for UHPLC analyses must be of the highest quality to ensure accurate, reproducible results. 

The speed of UHPLC also puts great demand on the throughput of sample preparation processes. Commonly used sample prep filtration devices such as syringe filters are tedious, slow, and unable to keep pace with the higher throughput of chromatography systems.

In UHPLC, sample clarification and fine particle removal are essential steps in preventing column clogging and system downtime. Some other sample preparation techniques (solid-phase extraction and liquid-liquid extraction) also reduce sample complexity, which leads to higher signal-to-noise ratios and cleaner baselines. Samples must be free of interfering matrix components that can bind the stationary phase in the UHPLC column.

Depending on the type and number of samples, a range of preparation methods can be chosen for UHPLC sample preparation, including sample filtration using 0.2 µm filters; protein precipitation followed by centrifugation or filtration; solid phase extraction; ultrafiltration; or liquid-liquid extraction (Table 1).

 When using sample filtration techniques, it is important to understand that the retention characteristics of syringe filters can vary, and this variance can impact the quality of sample preparation. Figure 1 compares the retention characteristics of syringe filters from two different suppliers. The data show that when comparing nylon and PES membranes, both products show similar particle retention (> 95%). In the case of PTFE and PVDF, membrane filters from Supplier A retain greater than 95% particles, but the membrane filters from Supplier B only retain about 80% of particles. The filters from Supplier B, which allow 20% of the particles to pass through, may cause a column to clog. 

As the throughput of chromatography systems continues to improve, sample preparation techniques struggle to keep pace, becoming more of a bottleneck. Syringe filtration is a very simple sample preparation technique, but serial processing of samples can easily become the slowest and most tedious step in the entire analytical workflow. At the other end of the sample preparation spectrum are robotic systems, which can be expensive and offer too much capacity for labs that handle a few dozen samples per day.

Recent innovations in sample processing such as the Samplicity filtration system (Figure 2) allow multiple samples to be processed in parallel, offering a throughput capacity that is well-aligned with the needs of most labs. The system allows up to eight samples to be simultaneously vacuum filtered directly into LC vials in seconds.

Unlike HPLC where mobile phase filtration requirements aren’t as stringent, typical UHPLC columns have small interstitial spaces, as well as 0.2 µm frits at the end of the columns to retain particles, which can get clogged. Filtering mobile phase components with the optimal membrane filter helps protect UHPLC systems from particulate impurities that may clog and shut down the system. Most UHPLC suppliers recommend the use of filters with a 0.2 µm pore size for preparing the mobile phase. 

In a study conducted by Merck Millipore, three common mobile phase solvents (water, acetonitrile, and methanol) were used for mobile phase preparation and the effect of filtration on back-pressure of the UHPLC system was evaluated (Figure 3). Of the various membrane filters evaluated, hydrophilic polytetrafluoroethylene (PTFE) provided the best filtration performance as indicated by the lowest back-pressure increase in a UHPLC system. Hydrophilic polypropylene (PP) filters were unable to retain particulate impurities present in the solvents as indicated by highest back-pressure gain of all the filters studied. Nylon and hydrophilic polyvinylidine fluoride (PVDF) filters show an intermediate performance in terms of particle retention and subsequent pressure increase.

In addition to offering the lowest back pressure increase, hydrophilic PTFE filters also show the best chemical compatibility for various solvents and mobile phase modifiers commonly used in UHPLC (Table 2).

It is also critical to use high quality buffer salts when preparing the mobile phase for UHPLC. Yet even with use of high quality salts, the presence of insoluble impurities can clog the UHPLC column. It is therefore important to filter mobile phase components prior to their use.

Few factors affect high-performance liquid chromatography analyses more than contaminants in the water used for the mobile phase. While poor water quality is one of the easiest problems to fix, it is one of the least-understood factors in an analytical laboratory.  Reports indicate that 70 to 80% of chromatography performance issues are ultimately attributed to water quality in eluents, samples, and standards1.

Water contaminants that affect HPLC also impact UHPLC, but to a more significant extent in some cases.  With detection limits down to part-per-trillion levels, the water used for running blanks, diluting samples, mixing buffers, and preparing standards must be of the highest quality. 
Water used for UHPLC should be free of particulates, organic contaminants, bacterial contamination and ionic impurities.

Particulates
The presence of particulates in water can have a significant impact on UHPLC, due to its lower interstitial void volumes and decreased column diameters. Although UHPLC columns still operate at flow rates typical of conventional HPLC, the columns are more susceptible to premature plugging by particulates compared to HPLC columns.

Organics
Organic contamination of ultrapure water may affect chromatographic separations in a number of ways. Organic molecules may accumulate at the head of the column and subsequently elute as contaminant or ghost peaks. If the level of organic contamination is very high, the contaminants may act like a new stationary phase, causing shifts in retention time and peak tailing. Accumulation of organic material in the column may also lead to back pressure increases. This would ultimately shorten the column lifetime. In addition, the presence of organic contaminants in the water used to prepare the eluents may lead to a loss in resolution and reduced sensitivity of the analytical method. It is therefore critical to accurately monitor the level of organics in water used for UHPLC via an on-line TOC monitor.

Bacteria
Bacteria may plug column and frits and release organic by-products which can, in turn, contribute to the effects seen with organic contamination.

Ionic Impurities
Modification of the ionic strength of the eluent may affect some separations. If the ionic contaminant is UV-absorbing (e.g., nitrates, nitrites), it will elute as a peak and make data analysis difficult. If a mass spectrometer (MS) is used as a detector, formation of adducts other than the protonated one (e.g. Na+, K+ adducts) can also impact data analysis.

Water purification systems combine several purification technologies to ensure that these important water contaminants are efficiently removed, and the highest water purity level is reached. A good pre-treatment system combines reverse osmosis and electrodeionisation technologies (such as in the Elix system). Water is further purified with a polishing unit (e.g. a Milli-Q system) combining UV photo-oxidation, ion exchange resins, activated carbon, as well as microfiltration at the point-of-use.

The storage of ultrapure water in plastic containers can also impact UHPLC as the containers may introduce leachables into the water. Glass containers are preferred over plastic, as they do not leach as many organics, however they can introduce ions into the water. Storing ultrapure water can also encourage the proliferation of bacteria. Therefore, freshly purified ultrapure water should be preferred2.

The benefits of UHPLC are clear: higher speed, improved resolution, and increased throughput. In order to fully achieve these benefits, however, processes used for sample and mobile phase preparation must meet higher standards that those used for HPLC applications. The use of the most appropriate filtration materials and sample prep technologies, as well as the highest quality water, all contribute to maximizing the effectiveness of UHPLC. 

References:
1. Mabic S., Regnault C., Krol J.  The misunderstood laboratory solvent: reagent water for HPLC. LCGC North America 23(1):74-82 (2005).
2. Tarun M., Monferran C., Devaux C., Mabic S. Improving chromatographic performance by using freshly delivered ultrapure water in the mobile phase. LCGC „The Peak“, June, 7-14 (2009)

Author:
Vivek Joshi, PhD, Senior Scientist, Bioscience Business Unit and Estelle Riche, Senior Scientist, Lab Water Business Field, Merck Millipore

Contact: 
e: vivek.joshi@merckgroup.com