Technology Forum: Sample Preparation

Each month in our Technology Forum we will feature a discussion between industry experts on various trends and issues in the chromatography field.

This month’s Technology Forum looks at the topic of Sample Preparation and the trends and issues surrounding it. Joining us for this discussion are Kary Staples, with Gilson, Inc., Mike Kopczynski, with ENS Nanotechnology, and Ron Majors, LCGC Columnist.

What trends do you see emerging in sample preparation?

Kary Staples: The demand for automation of sample preparation continues to grow in almost every market ranging from food to pharmaceutical to environmental, forensics and much more. The demand for automation of solid phase extraction continues to grow as well as scientists time is more valuable than ever. There seems to be a growing trend in higher throughput devices in both number of samples processed as well as volume of sample depending on market segment. Many scientists are looking for processing more samples at a time or parallel processing to speed up the extraction or clean up for further analysis. Positive Pressure Solid Phase extraction seems to also be growing as the method of choice for SPE with many instrument manufacturers coming out with positive pressure devices. A part of this change has been for better reproducibility that this method allows for.

Mike Kopczynski:As Sr. Director of R&D Applications at ENS Nanotechnology, my interest and knowledge is primarily in the area of novel nanotechnology applications for chiral separation for small molecules. Until recently, most of the developments in sample prep have been focused on simply improving the same techniques available for years by making them faster or cheaper. On the screening side, the trend continues to be the automation and acceleration of processes to increase throughput. At the bench top, we are seeing new types of selectivities being added to the current variety of products, such as chiral enrichment, where we see the growth going forward. The time is ripe for a major advance that enables chiral separation and permits much faster, more efficient progress from discovery to semi-prep and prep stages.

Ron Majors: One of the more obvious trends has been the increasing use of smaller sample sizes. With the advent of the widespread use of more sensitive and selective analytical techniques such as LC/MS-MS, a smaller amount of sample is required for analysis, down into the microgram to nanogram masses. This observation is especially true in proteomics research where a single cell can be analyzed for hundreds and thousands of proteins. Indeed, in this field, looking for a needle in a haystack is becoming an everyday occurrence. Even in the analysis of drugs in biological fluids, the increased use of 96-well plates, pipette tip SPE, and similar low volume sample preparation devices is evident. Also, by using some of the newer sample preparation techniques such as stir-bar sorbent extraction (SBSE) or solid-phase microextraction (SPME), analysts can now measure lower concentrations of analytes in these smaller samples.

Another trend that is evident is the work being done to make sample preparation faster. Most of the modern chromatographic techniques have the capability for high speed separations. For example, HPLC with short, sub-two micron column generates chromatograms in the minute range. Entire GC chromatograms of complex samples sometimes only require a few minutes. Many of the currently used sample preparation techniques are much slower and have created a bottleneck which slows down overall productivity. Unfortunately, automation of some of these sample preparation techniques has not kept up with the analysis, nor is even integrated. Full laboratory robotics is a thing of the past and the trend seems to have evolved to dedicated workstations. I noticed that in putting together my Pittcon 2007 articles for LCGC No. America this year, quite a few SPE workstations have been introduced.

There has been a trend to make sample preparation safer and more environmentally friendly. Fortunately, as sample sizes decrease, the amount of required solvent decreases proportionally. So the analyst not only saves on solvent cost but also on disposal costs. Some of the sample preparation techniques are even solvent-less such as SPME or matrix-assisted sorbent (MASE) extraction. The use of subcritical water extraction is an area that deserves more attention.

In your opinion, what is the future of sample preparation?

>Kary Staples: Solid Phase Extraction continues to grow as the method of choice as it lends itself to automation much easier than other methodology. The future of this field is to minimize the scientists time needs for the basic and mundane processes. SPE is a critical and crucial step in the process but is many times perceived as a more basic step. Scientists time is critical and by allowing them to focus on the science through automation rather than spending their time doing extractions by hand, will allow them to be more effective in their job, while increasing throughput and ensuring reproducibility.

Mike Kopczynski:While there have been many notable advances in the area of biomolecule analysis, particularly preparing for LC-MS for biomarkers and metabolomics, the small molecule field is overdue for similar innovative tools which add another dimension of selectivity while preparing a sample for further analysis. For instance, the emphasis on single enantiomer leads in the pharmaceutical industry calls for new products to provide chiral enrichment early in discovery. Our own ENS Chiral SPE cartridges are at the front of this trend for the synthetic chemist.

Ron Majors: The drivers in laboratory analysis will also drive the need for better, faster, more efficient sample preparation. For example, every company wants to get to market faster and thus laboratory productivity is increasingly important. With fewer skilled chemists available and sample loads increasing, everybody wants to do more with less. This has a direct impact on sample preparation which is often viewed upon as the slowest part of the analytical cycle. Ideally, no sample preparation would be the goal of every laboratory but this goal is unrealistic. However, one of the initiatives in the pharmaceutical industry is process analytical technology (PAT) where measurements will be made on the production line without the need to collect a sample and run it back to the analytical laboratory. Such approaches will cut down on the need for off-line analysis and will eliminate the need for sample preparation. But for most work, some sample preparation will still be required. The way to improve productivity and improve analytical results will be some form of automation, probably by a careful investigation of the workflow to see which of the sample preparation steps are the slowest and in most need of automation that can keep up with the analytical instrument. Another area driven by validation and chain of custody concerns is the need to track a sample through the sample preparation-analysis cycle. Bar codes were never that useful since in sample preparation, multiple containers are used to transfer samples. There is some interesting work to use nanoparticles as pseudo-internal standards that can be followed through the entire process.

Improvements in sample prep devices will aid in cutting down on the number of sample preparation steps. For example, in my 2002 survey on sample preparation, the average number of steps in a typical sample preparation protocol was three. Each additional step leads to more time required and increases the potential error. Very selective SPE phases such as molecular imprinted polymers (MIPs) and affinity phases can allow the analyst to minimize the number of sample preparation steps down to one or two. Selective detection will also minimize sample preparation steps. If the detector can pick out one or two analytes based on elemental composition, mass selectivity, or other chemical/physical attribute, then the need to first isolate the analyte from the matrix may be eliminated.

As mention earlier, the development of sample preparation techniques that use less or no organic solvent will cut costs, improve safety, and speed up the whole process. The larger the amount of solvent used, there is more chance to introduce undesired impurities. If one evaporates a large volume of solvent to concentrated analytes, any solvent impurity will also be concentrated and may cause additional interferences in analysis or require additional sample prep steps to eliminate these impurities. Matrix-solid phase dispersion (MSPD) and dispersive SPE (dSPE) are techniques that use no or a minimal amount of organic solvent yet provided excellent extraction efficiencies for solid- and semi-solid samples. The use of subcritical water seems to be an interesting inexpensive and effective extraction solvent. When water is superheated above its boiling point in a closed vessel, it develops organic-like solvent power, resembling ethyl acetate or even methanol. Extracted analytes can easily be concentrated by passing the cooled extractant through a reverse-phase SPE column. More work is needed to show the versatility of this environmentally friendly solvent.

What one recent development in the area of sample preparation would you say is the most important?

Kary Staples: I don’t believe there has been one specific development but in general more phases are being developed and applied to Solid Phase Extraction Cartridges by the various Column vendors and many are developing specific sample preparation assays on SPE cartridges that are packaged in kits which is helping open the doors to more and more automations of this process. This even includes assays that were never thought to be SPE processes such as Chiral separations, etc.

Mike Kopczynski:New base materials that enhance stability or extend solvent or pH range have the potential to rapidly expand sample prep into areas that were deemed too difficult in the past. The application of nanotechnology to create completely novel structural frameworks can add functionality and transcend the limitations of the traditional materials and are destined to change the direction of sample prep in the future.

Ron Majors: With the increasing role of proteomics in investigating of new drug targets and biomarkers of disease, there is hope that this field of human research can help in improving the health of millions. However, most of these proteins that are indicative of disease states are buried deep in the proteome in minute concentrations. The tiny amounts of protein are masked by the massive amounts of high abundance plasma proteins, such as human serum albumin and IgG, so that they cannot be sufficiently uncovered. So I would say that the development of high abundance protein depletion sample preparation columns is a step forward in helping biochemists and molecular biologist to uncover the trace proteins that could be identified and characterized to help in the ongoing battle to defeat cancer and other life threatening illnesses.

What obstacles do you think stand in the way of sample preparation development?

Kary Staples: Developing an instrument capable of extremely high throughput that is reliable, reasonably priced, and that doesn’t limit the application to only a microplate format.

Mike Kopczynski:Two issues have constrained growth of sample prep. First, while some of the new methods have produced real advances to the field, they have inherent restrictions. The marketplace is looking for predictable methods that are reproducible with many sample types. This had led to long development times to work out the kinks. Second, the materials on which traditional sample prep products are based have solvent. pH, and temperature limitations that have restricted expansion of sample prep into new areas.

Ron Majors: I would say the biggest obstacle in sample preparation development is its stature in the analytical laboratory. Sample preparation has always been viewed as a manual, tedious technology that was oftentimes given to the lower skilled workers to perform. It has been relatively unattractive from an academic viewpoint and unrecognized as a field of analytical chemist. Hence, sample preparation technology gets very little recognition and study in the universities. In addition, most analysts consider themselves as above sample preparation as well and view their job as running sophisticated instruments, evaluating and implementing new analytical technologies and generating and interpreting data. Even the major analytical instrument companies have not devoted a lot of development effort in sample preparation preferring instead to generate more sophisticated measurement devices; even today many of the sample prep instrument companies are smaller companies that provide dedicated machines to perform one or two sample preparation tasks. One outcome of these developments is that many of the standalone sample preparation instruments are not well integrated into the analytical instrument since software, connectivity, and the hardware are not always mutually compatible. Another area that had exciting beginnings was robotic automation but again early attempts to integrate robots and analytical instruments were carried out by chemists and not engineers so that there were many failed attempts to integrate the two. Standardized communication protocols and non-standardized sample containers were other obstacles to the acceptance of full laboratory automation.

How difficult is it to find sample preparation technology that is safe, fast, environmentally friendly, and cost efficient?

Kary Staples: In general, there seems to be a lack of rugged automation instrumentation available. Most equipment on the market tends to be very expensive and has had a history of not being very reliable especially for sample preparation and specifically SPE. Gilson is one of few companies currently that has focused on solving this dilemma and offers this type of SPE instrumentation.

Mike Kopczynski:While there are new sample prep products and methods that meet these criteria, they are not widely applicable and tend to be focused on specialty areas. For general use, sample prep users continue to have to strike a balance between these needs that suits their own situations.

Ron Majors: As stated earlier, the ideal sample preparation technique would be no sample preparation at all. But, in a non-ideal world, samples come in a variety of matrices that don’t fare well in an analytical instrument. So sample preparation technologists have been looking for a technique that meets all of your criteria. Supercritical fluid extraction had a flurry of development in the early-to-mid 1990s. With the use of the environmentally friendly, non-toxic, solvent-power tunable carbon dioxide as an extractant, everybody thought that the ideal sample preparation technique had been found. Unfortunately, strong analyte-matrix interaction problems hampered SFE and this universal sample preparation technique was put on the back-burner. It does find limited use in extractions of fats from food and a few more applications but the high volume extraction of solid environmental samples and Environmental Protection Agency approval as a universal method never came to be. Accelerated solvent extraction that took its place was found to be too powerful and non-selective but has found more acceptance in environmental sample prep. Hopefully, this new sample prep technology will also be easily automated something that MSPD, dSPE, and SBSE don’t have going for them.

It will be extremely difficult to find a universal sample preparation technique that can deal with the wide variety of analyte-matrix combinations that analysts encounter. Biological samples such as body fluids and tissue have many water-soluble substances while polymer and environmental matrices often need organic extractants due to the nonpolar nature of encountered analytes. Inorganic matrices require an entirely different set of sample extraction protocols. Gaseous samples such as air or refinery gas effluents require different sample preparation approaches. So, although feasible to find an ideal sample prep technique to attack one analyte-matrix combination, it will probably not be useful for another combination. So, the search for the perfect extraction technique goes on.