Reflecting on the Influence of the Current State of Sample Preparation on GC, Part 2: Techniques

Recently, we examined major trends in sample preparation and how they relate to gas chromatography (GC), based on a 2023 user survey by LCGC International. In Part 2 of LCGC’s survey of sample preparation users, the prevalence of sample preparation techniques, from the most common, such as weighing, to the most complex, such as online extractions, was discussed. In this installment, we examine trends in the use of sample preparation techniques through the lens of instrumental analysis by gas chromatography (GC) and GC–mass spectrometry (GC–MS). We see that, although common and classical techniques, such as filtration and centrifugation, are still generally the most widely used, newer techniques that allow automation, simplification of analysis steps, and green analysis are gaining traction.

High resolution and sensitivity instrumental techniques, including gas and liquid chromatography (LC), are unable to perform as expected without effective sample preparation. As seen in a recent survey conducted by LCGC International and discussed by Raynie, there are myriad techniques available for sample preparation in conjunction with gas chromatography (GC) (1). This survey is a follow-up to previous surveys, and looking at all of them provides interesting perspectives about the evolution of sampling and sample preparation for chromatography (2). Sample preparation techniques for GC range in complexity from simple dilutions and “neat” liquid injections to complex online automated extraction systems. Not surprisingly, the simpler and more fundamental chemical techniques are reported by users are being used the most often. Although the survey includes all areas of chromatography and sample preparation, we can see trends and interests that directly relate to GC.

Users were surveyed first about the instrumental techniques they use. In 2023, GC holds its place along with high performance liquid chromatography (HPLC) dominating the users’ responses. HPLC users are now almost evenly split between traditional HPLC and ultrahigh-pressure liquid chromatography (UHPLC). In GC, a new response category for GC with headspace sampling was added in 2023, and a similar number of respondents reported using GC with headspace sampling as GC alone and more than gas chromatography–mass spectrometry (GC–MS). The most recent (and now classical) literature discussing the basics of headspace sampling with GC is the text by Kolb and Ettre, published nearly 20 years ago (3). With so many laboratories performing headspace analysis, managers should consider additional training, and instrument vendors should increase support and knowledge sharing in this area.

Sample Mass and Volume

The data presented on the volume and mass of collected samples and samples following sample preparation show interesting trends. The survey showed that most chromatographers’ initial samples have volumes between 0.5–20 mL of liquid or gas samples or initial mass of 0.055 g for solid samples. The roughly identical initial volume of liquid or gas samples is not as surprising as it may seem. Initial liquid samples of up to 20 mL are likely to undergo additional sample preparation, such as solid-phase extraction (SPE) or liquid–liquid extraction (LLE), prior to injection, resulting in a lower final sample volume prior to injection. The survey data reflects this as the majority of all liquid samples are prepared to a final volume of 1–2 mL prior to injection.

Gas samples have a much larger volume for the same mass as liquid samples. So, in GC, gas samples are generally directly injected using a sample valve or syringe that accommodates the larger volume. Gas samples have a much larger volume for the same mass as liquid samples. So, in GC, gas samples are generally directly injected using a sample valve or syringe that accommodates the larger volume.

Interestingly, for all three initial sample phases, the volume or mass used is generally convenient for the user and is reminiscent of sample volumes used over the decades of my own career. Final sample volumes, most of which are in the 1–2 mL range for liquids, match closely with the classical 2 mL auto-injector vials used for decades in most autosamplers. In GC, there is much room for reducing final sample volumes prior to injection, as the typical injected volume of a liquid sample is only 1 µL. The vast majority of all final samples in the vial for gas chromatography are eventually disposed as waste. Finally, these initial and final sample volumes are easy for users to handle with standard glassware and equipment. With the increasing interest in sustainability and green chemistry, we can expect initial and final sample volumes to decrease over time, but this may require some rethinking of glassware, sample handling, and methods to limit the expected increases in experimental uncertainty that come with smaller sample volumes (4).

Techniques

Turning to the techniques used by chromatographers, we see that classical glassware handling and wet chemical techniques continue to outpace instrumental and automated techniques, in addition to those that require phase changes or alter the chemical nature of the analyte. Raynie notes that the order of the techniques may have shifted, with biological techniques, such as centrifugation and sample cooling, now occupying spots near the top of the most commonly used techniques. Table I shows the techniques discussed in the survey, separated into classical or wet chemical and phase change, and they are listed in the popularity order presented in the survey, with the most popular at the top of the list. With the exception of pressurized fluid extraction, which somehow shows up at second on the overall list, the classical techniques that involve manipulating or transferring a sample rather than a phase change or an instrument are generally more popular; techniques that were introduced more recently or those requiring instrumentation were less popular.

TABLE I: Popularity of sample preparation techniques separated by classical versus instrumental. Listed in order from most to least popular.

Classical Techniques

Looking more closely at Table I, some interesting trends are seen, with the observation that sample preparation methods for all chromatographic techniques are included, not just those primarily used with GC. As Raynie observes, centrifugation and cooling, which are more commonly used with biological sample analysis, usually with LC-related techniques, were the most popular classical methods in the survey. These are followed by filtration, dilution, concentration, internal standard addition, and weighing, a set of techniques used in most chromatographic methods across the entire field.

The top half of the classical group of methods in Table I reminds us that the fundamental chemical techniques are still performed the most. Although these may seem simplest as well, they are often rushed, and their impact on overall method performance is underestimated. Mistakes or experimental uncertainty in these techniques can have an outsized impact on method performance, and are one of the first places I look when consulting on method optimization, especially if reproducibility is not satisfactory. These are areas in which it is very common to see analyst-to-analyst variability in technique and performance. In the research community, these techniques are often considered "mature," "fundamental," or "basic," so there is often little emphasis in ensuring correct technique in teaching and training.

Instruments and Phase Changes

Looking at the more instrumental and phase change-related techniques in Table I, we surprisingly see pressurized fluid extraction (PFE) at the top of the list. PFE is usually used to transfer organic contaminants from solid matrices, such as soil, into liquid solvents, using high pressure and often heating (5). The high pressure and temperature force the solvent into small pores in the solid, increasing the surface area exposed to the extraction, and therefore the recovery. High pressure and temperature also make these extractions relatively fast, on the order of 30 min, and the instrumentation is usually automated, with the capability to extract samples in batches. PFE is often considered "green," as using elevated temperature and pressure can allow better extraction kinetics and performance with lower solvent volume and more benign solvents.

Again, among the top five techniques we now see two, column chromatography and protein precipitation, that are more used for purification rather than traditional chromatographic analysis. This is further evidence that the analytical chemistry landscape is changing, with reduced emphasis on small molecule analysis and more on large molecules and biologics, either as a sample matrix or as the analytes themselves.

Classical liquid-liquid extraction (LLE), solid-phase extraction (SPE), and static headspace extraction (SHE) highlight the next several entries. These are all staple sample preparation techniques for GC, and each depends on a phase equilibrium, very similar to chromatography itself, with LLE involving analyte transfer between two immiscible liquid phases, SPE between a liquid and solid phase, and back to a liquid phase, and SHE between a liquid or solid phase and the vapor phase.

Although GC has been automated for decades through autosamplers, the most popular sample preparation techniques are still analyst-intensive and hands-on. There have been many recent advances in automation, instrument operation, and control, yet we still inject using similar syringes to those used fifty years ago, and sample preparation is still generally based on the same techniques in place for decades. Interestingly, as instruments and autosamplers have become more reproducible, this has placed more emphasis on reproducible sample preparation.

As we proceed down the list of phase transfer techniques, we see several that are more instrumental and automated. Over the decades, the route to fully automated sample preparation for GC has been a bumpy one. Automation requires a significant up-front investment, and may not immediately return benefits in reproducibility over manual methods. Furthermore, operation of the automated system itself requires care, maintenance, and troubleshooting, often by a specially trained operator.

Sample preparation methods are becoming more complex and being performed in large batches, as seen in the surveys over time. While there is significant variability, users report about four steps in a typical sample preparation and sample batches commonly ranging over 50 samples. Thinking further about this and the most popular techniques, we see that a typical sample batch can easily include over 200 sample preparation steps, not including standards, blanks, and quality control samples. Again, this points to an ever-increasing need for skilled analysts performing fundamental tasks and processes in analytical laboratories.

Finally, the survey addresses users’ perceptions about the needs and future of sample preparation. Not surprisingly, the most common desires were for more green and miniaturized methods. This may prove challenging, as, while the survey indicates satisfaction with the analytical performance of current methods, the most popular current methods, such as LLE and SPE, may not lend themselves easily to miniaturizing or “greening” while maintaining performance including detection limits, reproducibility, ruggedness, and simplicity.

Summary

Recently, LCGC International has surveyed subscribers on their use and perceptions of sample preparation for chromatography. While GC and LC share roughly equal proportions of the respondents, we see the biggest increases in use of techniques related to biological analysis, most likely for LC. Classical chemistry techniques, including dilution, weighing, pipetting, glassware handling, SPE, and LLC, continue to dominate chromatographic methods, with further automation and miniaturization on the horizon. Sample preparation continues to be analyst intensive, requiring training and practice.

References

(1) Raynie, D. E. Trends in Sample Preparation, Part II: Sample Considerations and Techniques. LCGC International 2024, 1 (3), 12–21. DOI: 10.56530/lcgc.int.mn3284n6

(2) Raynie, D. E. Trends in Sample Preparation. LCGC North Am. 2016, 34 (3), 174–188.

(3) Kolb, B.; Ettre, L. S. Static Headspace-Gas Chromatography: Theory and Practice, 2nd Ed. John Wiley and Sons, 2006.

(4) Snow, N. H. Is the Solution Dilution? Hidden Uncertainty in Gas Chromatography (GC) Methods, LCGC North Am. 2022, 40 (7), 304–308. DOI: 10.56530/lcgc.na.re9187d2

(5) Barp, L.; Višnjevec, A. M.; Moret, S. Pressurized Liquid Extraction: A Powerful Tool to Implement Extraction and Purification of Food Contaminants. Foods 2023, 12, 2017. DOI: 10.3390/foods12102017

ABOUT THE AUTHOR

Nicholas H. Snow is the Founding Endowed Professor in the Department of Chemistry and Biochemistry at Seton Hall University, and an Adjunct Professor of Medical Science. During his 30 years as a chromatographer, he has published more than 70 refereed articles and book chapters and has given more than 200 presentations and short courses. He is interested in the fundamentals and applications of separation science, especially gas chromatography, sampling, and sample preparation for chemical analysis. His research group is very active, with ongoing projects using GC, GC–MS, two-dimensional GC, and extraction methods including headspace, liquid–liquid extraction, and solid-phase microextraction. Direct correspondence to: Nicholas.Snow@shu.edu