This short review covers the use of ionic liquids (ILs) and polymeric ionic liquids (PILs) in solid-phase microextraction (SPME) and dispersive liquid–liquid microextraction (DLLME). 

Over the past decade, ionic liquids (ILs) and polymeric ionic liquids (PILs) have been widely studied and applied in multiple areas of science and engineering. ILs are organic salts with melting points at or below 100 °C, and PILs are polymers synthesized from IL monomers. These "molten salts" are typically composed of an organic cation and an organic or inorganic anion. Given that the cations and anions can be readily interchangeable to form new compounds with different properties, it has been estimated that there can be as many as 10

  possible combinations of ILs (1). ILs and PILs have been previously applied in various extraction methods including single-drop microextraction (SDME), liquid-phase microextraction (LPME), solid-phase microextraction (SPME), dispersive liquid–liquid microextraction (DLLME), hollow fibre-supported liquid membrane extraction (HFSLME) and solid-phase extraction (SPE) (2–7). With the exception of sorbent-based extraction procedures, many of these sample preparation methods involve the use of organic solvents. Compared to traditional organic solvents, ILs can be engineered to exhibit low to negligible volatility, and sometimes lower toxicity, which makes their use in sample preparation highly attractive (3). 

Although ILs and PILs have been applied in many sample preparation methods, this article will focus on the application of these compounds in two rapidly emerging techniques, namely, SPME and DLLME. ILs and PILs exhibit a number of unique properties that make them highly useful extraction media in SPME and DLLME because of their high thermal stability, variable viscosity and negligible vapour pressure. However, the main advantage of using ILs and PILs is their tunable selectivity for specific classes of analytes. By imparting functional groups to the chemical makeup of ILs and PILs, the selectivity of these compounds can be enhanced as a result of favourable analyte-to-IL or -PIL interactions. As a result, higher analyte enrichment factors and extraction efficiencies often can be achieved. In this column instalment, the thermal stability, viscosity and selectivity of ILs and PILs and their relevance to SPME and DLLME will be discussed. An overview of the basic principles of operation for both DLLME and SPME will be covered. Additionally, the current challenges of ILs and PILs in sample preparation will be highlighted and ideas for future research will be discussed. 

Unique Characteristics of Ionic Liquids and Polymeric Ionic Liquids for Applications in DLLME and SPME 

The thermal stability of an IL or PIL is critical for applications in sample preparation that require the use of high temperatures. When SPME is coupled to gas chromatography (SPME-GC), the IL- or PIL-based sorbent materials must be capable of withstanding the high operating temperatures of the GC injector (250–280 °C) during analyte desorption (5). Figure 1 shows the structures and names of common ILs and PILs used in DLLME and SPME. Most ILs or PILs used in SPME and DLLME are composed of imidazolium-, pyrrolidinium- or phosphonium-based cations. Functionalizing the nitrogen atoms of the imidazolium ring with linear aliphatic hydrocarbons can further increase the thermal stability of the resulting IL (8). The anion, however, often plays a more significant role in the thermal stability of ILs and PILs (9). Typically, smaller and less polarizable anions, such as halides, decrease the overall thermal stability because of their susceptibility to undergoing nucleophilic substitution with the alkyl substituents of the IL cation (10). On the other hand, larger anions that exhibit higher electron delocalization, such as bis[(trifluoromethyl)sulphonyl] imide ([NTf

 ), produce compounds with higher thermal stability (8,10,11). 

 Figure 1: Chemical structures of IL and PIL cationâanion pairs previously used in IL- and PIL-based SPME and IL-based DLLME: (a) poly(1-vinyl-3-R-imidazolium), (b) 1-R-3-methylimidazolium, (c) poly(1-R-3-vinylbenzylimidazolium), (d) R-pyridinium, (e) N,N-methyl-R-D-glucaminium, (f) 1-R1-1-R2-pyrrolidinium, (g) bis[(trifluoromethyl)sulphonyl] imide, (h) hexafluorophosphate, (i) tris(pentafluoroethyl)trifluorophosphate, (j) tetrafluoroborate, (k) taurinate, (l) chloride, (m) bromide and (n) iodide.			

 Similar to the thermal stability, the viscosity of an IL or PIL is an important consideration in sample preparation, particularly for SPME and DLLME. The viscosity of ILs is governed largely by their intermolecular interactions, such as hydrogen bonding, van der Waals forces and electrostatic interactions (12). ILs containing halide anions typically exhibit higher viscosities because of their ability to undergo stronger hydrogen bonding and electrostatic interactions with the cations while larger or more asymmetrical anions often possess lower viscosities (11,13,14). Substituting the cationic moiety of the IL with linear or branched aliphatic hydrocarbons will also increase the viscosity as a result of enhanced van der Waals interactions (9,12,14,15). In SPME, it is favourable to use ILs and PILs with relatively high viscosities because the viscosity of ILs tend to decrease at high temperature, such as during thermal desorption in the GC injector at high temperatures. Such thermal desorption may result in the IL coating flowing off the fibre support into the GC injector and jeopardizing both the fibre lifetime and its analytical performance. In addition to producing chromatographic ghost peaks, active sites and chromatographic background noise, IL buildup within the injector may require frequent GC system maintenance. In IL-based DLLME, the viscosity of an IL extraction solvent can be modified to a certain extent by using a disperser solvent, an organic modifier that is miscible in both the sample matrix and the extraction phase. In cases where a disperser solvent is not used, it is necessary to use an IL with a suitable viscosity to allow for homogenous mixing during extraction. 

Variable Selectivity by Structural Tunability

A major advantage to using ILs and PILs as extraction phases in sample preparation is their tunable selectivity for specific analytes. For example, it is beneficial to take advantage of the electrostatic interaction capabilities of ILs in addition to functionalizing them with hydrogen-bond acidic hydroxyl groups to enhance the extraction efficiency of deoxyribonucleic acid (DNA) in DLLME (16). Likewise, the extraction efficiency of analytes containing aromatic moieties, such as polycyclic aromatic hydrocarbons (PAHs), can be enhanced by imparting aromatic character to the PIL-based sorbent coating to increase p-p interactions in SPME. In the same context, engineering PILs with large aliphatic hydrocarbon chains can increase the selectivity of longchained alkyl halides as a result of enhanced dispersion interactions (17). Therefore, to fully exploit ILs and PILs as selective extraction media, the intermolecular interactions between these compounds and the target analytes should be carefully considered.   

ILs and PIL-Based Sorbent Coatings in Solid-Phase Microextraction  

SPME, developed by Pawliszyn and coworkers in the early 1990s, is a popular sorbent extraction technique that combines sampling and sample preparation into one step. Unlike other sorbent-based extraction techniques such as SPE, SPME non-exhaustively preconcentrates the analytes of interest (18,19). Figure 2 illustrates the basic operating principles of SPME. Initially, a fibre support is coated with a thin film of a sorbent coating. During the extraction step, the coating is exposed to a sample solution containing the analytes of interest, either in headspace or direct-immersion mode. Extraction is achieved via adsorption, partitioning of the analytes to the sorbent coating, or both adsorption and partitioning. The analytes are then desorbed from the sorbent coating by exposing the fibre to a GC injector at high temperatures for thermal desorption or to a high performance liquid chromatography (HPLC) injector for solvent desorption (20).  

Figure 2: Schematic of the basic operating principles in SPME: (â) analyte, (â ) sample matrix. 			

The use of ILs as sorbent coatings for headspace SPME was first investigated by Liu and coworkers in 2005 (21). Table 1 provides a brief summary of all studies discussed in this article. The 1-octyl-3-methylimidazolium hexafluorophosphate ([C8MIM][PF6]) IL was exploited in the extraction of benzene, toluene, ethylbenzene and xylenes (BTEX) in paints. The IL-based coatings provided lower limits of detection (LODs) for the BTEX analytes compared to a commercial poly(dimethylsiloxane) (PDMS) coating, demonstrating its high selectivity. Nevertheless, the IL-coated fibre was not reusable and had to be recoated following every extraction or desorption step as a result of a decrease in the viscosity of the IL and it flowing off from the fibre (21).  

Table 1: Summary of studies for IL- and PIL-based SPME and IL-based DLLME			

Following this initial study, different approaches were reported to increase the amount of IL loaded on the fibre as well as to stabilize it on the fibre. A perspective of the different sorbent coating techniques used in IL- and PIL-based SPME is shown in Table 2. A Nafion (DuPont) membrane was applied to pretreat the fibre surface before loading various ILs possessing the [TfO]

  anion on a bare silica fibre (22). This method not only allowed for an even IL coating but also enabled a large volume of these ILs to be stabilized on the fibre via electrostatic interaction with the Nafion membrane (22). A wet-chemical etching method using ammonium hydrogen difluoride was developed by Huang and coworkers to increase the surface area of a bare fused silica before IL loading (23). A larger volume of the 1-butyl-3-methylimidazolium hexafluorophosphate ([C

]) IL was loaded using the etched fibre compared to a non-etched bare fibre for the extraction PAHs in which a fivefold increase in extraction efficiency was observed (23). Amini and coworkers chemically bonded an organosilane-derivatized IL, namely, 1-methyl-3-(3-trimethoxysilyl propyl) imidazolium bis[(trifluoromethyl)sulphonyl]imide ([MTPIM][NTf

]), to a bare silica fibre. The chemically immobilized coating provided improved mechanical and thermal stability in addition to being reusable up to 16 extraction or desorption steps (24).  

Table 1: (continued): Summary of studies for IL/PIL-based SPME and IL-based DLLME			

In an effort to further extend the lifetime and reusability of the sorbent coating, our group exploited PILs as selective SPME sorbent coatings (25). Compared to ILs, PILs possess higher thermal stability and are less prone to flowing when subjected to high desorption temperatures. Three PILs, namely, poly(1-vinyl-3-hexylimidazolium bis[(trifluoromethyl)sulphonyl]imide) (poly([ViC

])), poly(1-vinyl-3-dodecylimidazolium bis[(trifluoromethyl)sulphonyl]imide) (poly([ViC

])) and poly(1-vinyl-3-hexadecylimidazolium bis[(trifluoromethyl)sulfonyl]imide) (poly([ViC

])), were applied as PIL-based coatings for the selective extraction of fatty acid methyl esters (FAMEs).  

Table 2: Summary of coating and fiber modification methods in IL- and PIL-based SPME			

Outstanding fibre lifetimes of approximately 150 extractions were observed when using these PILcoated fibres (25). Subsequent investigations focused on improving the coating-to-analyte selectivity by imparting various substituents to the PIL backbone. For example, aromatic moieties were imparted to the poly((1-4-vinylbenzyl)-3-hexadecylimidazolium bis[(trifluoromethyl)sulphonyl]imide) (poly([VBC

])) PIL to produce enhanced selectivity toward PAHs by p-p interactions, illustrated in Figure 3 (17,26,27). As shown, the non-functionalized poly([ViC

]) PIL resulted in a significant increase in the mass of PAHs extracted compared to a commercial PDMS coating. Exploiting the benzy-lfunctionalized poly([VBC

]) PIL provided even higher extraction efficiencies, especially for benzo[

]anthracene and chrysene (26). The selectivity of hydrogen-bond acidic analytes, such as volatile fatty acids (VFAs), alcohols, aldehydes and amines, was significantly improved by incorporating a hydrogen-bond basic chloride anion to the PIL-based sorbent coating (17,28–31). Favourable selectivity was obtained in the extraction of long-chained aliphatic alkyl halides by using a glucaminiumbased PIL comprising a hydrogen-bond acidic carbohydrate moiety and linear aliphatic hydrocarbon chains capable of undergoing dispersive-type interactions (17). The poly([ViC

IM][taurinate]) PIL was used to extract CO

 (32,33). High selectivity was achieved as a result of the ability of the amine group within the anion to undergo complexation with CO

 to form a carbamate salt. The selectivity of this PIL was further demonstrated in the extraction of CO

 in a simulated flue gas atmosphere (32,33).   

Figure 3: Comparison of mass extracted using three studied fibres: (â ) 7 Âµm PDMS, (â ) 12 Âµm poly([ViC16IM][NTf2]) (â ) and 12 Âµm poly([VBC16IM][NTf2]). The extractions were performed for 30 min with a stir rate of 800 rpm at 22 Â°C. The concentration of the analytes was: 6 Âµg/L of naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene and fluoranthene; 3 Âµg/L of pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene and benzo[k]fluoranthene. Adapted from reference 26.			

Recent efforts have explored fibre surface modification and sorbent loading techniques to improve the overall mechanical, thermal and chemical stability of PIL-based SPME sorbent coatings. An IL–polyaniline (IL–PANI) composite coating, namely, 1-butyl-3-methylimidazolium tetrafluoroborate–PANI ([C

]–PANI), was coated on a fibre support via electrochemical deposition. This coating exhibited good resistance to organic solvents in addition to high extraction efficiency of benzene derivatives compared to a neat PANI coating and a commercial PDMS coating (34). The [BF

  anion of this composite was also exchanged to [PF

  in an effort to increase the thermal stability while allowing the fibre to be reused up to 250 times (35). Sol-gel chemistry was used to chemically bond silica-PIL composite coatings to the fibre support (36–38). Sorbent coatings synthesized by this technique exhibited high thermal stability in addition to possessing acceptable tolerance to large pH ranges (36,37) and good selectivity toward analytes such as phenolic environmental estrogens (PEEs), phthalate esters (PAEs), aromatic amines, fatty acids and alcohols (38). To further improve the overall stability and robustness of the sorbent coating, dicationic IL crosslinkers were added to monomeric IL monomers to form a crosslinked PIL by azobisisobutyronitrile (AIBN)initiated free radical polymerization (30). Our group further examined a quicker approach to produce crosslinked PILs through a solvent-free "on-fibre" polymerization technique by UV-initiated free radical polymerization (39). This technique combined surface modification methods, such as etching and derivatization, with the UV-initiated copolymerization of dicationic IL crosslinkers and monocationic IL monomers consisting of halide anions. This enabled the directimmersion SPME of polar analytes, an impossible task for analogous non-crosslinked PILs containing the same halide anions because of the miscibility of these PILs in aqueous sample matrices. The crosslinked PIL-based coatings were reusable up to 90 times in simple and complex water matrices including river and well water (39). 

Ionic Liquids in Dispersive Liquid–Liquid MIcroextraction

Dispersive liquid–liquid microextraction (DLLME) is a rapidly maturing solvent extraction technique for sample preparation. Several features that make this approach attractive include its ease of operation, high throughput and the capability to achieve high analyte enrichment factors. Introduced by Rezaee and coworkers in 2006, DLLME is a technique that preconcentrates analytes from an aqueous phase to an extraction phase in the presence of a disperser solvent (40). Figure 4 illustrates the basic operating procedures of DLLME. During extraction, a hydrophobic extraction solvent, typically dissolved in a disperser solvent, is injected into an aqueous sample solution. As noted earlier, the disperser solvent is an organic modifier, typically acetone or methanol, that is miscible in both the sample matrix and the extraction phase. This solvent facilitates the formation of extraction solvent microdroplets in the aqueous sample solution. Because of the large surface area of the microdroplets, analytes are quickly extracted, often with high extraction efficiency. The extraction solvent is recovered by centrifugation and separated from the aqueous matrix. The extraction phase is then injected to a chromatographic system for analysis. 

Figure 4: Schematic of the basic operating principles in DLLME. (â) Analyte, (â ) aqueous matrix, (â ) extraction and disperser solvents.			

Zhou and coworkers first applied ILs as extraction phases in DLLME (41). The 1-hexyl-3-methylimidazolium hexafluorophosphate ([C

]) IL was used to extract pyrethroid pesticides from water. Unlike traditional DLLME, the solubility of the IL was manipulated in the aqueous sample by increasing the temperature of the system. After dissolution of the IL in the aqueous phase, the temperature was decreased to allow for the formation of a turbid solution followed by analysis of the extraction phase. Baghdadi and Shemirani (42) expanded the application of IL-based DLLME for the determination of metal ions using [C

] ILs as extraction phases. Triton X-114 (Dow Chemical Company) was added to the extraction phase as an antisticking agent to minimize the amount of IL residue remaining on the wall of the sample tube after centrifugation. As a result, the overall absorbance of select metal ions increased and IL carryover from the sample tubes was minimized. Ultrasound-assisted IL-based DLLME was used to minimize the use of high temperatures during extraction. Using this technique, dispersion and homogenization of the IL was achieved within 5 min and excellent enrichment of aromatic amines was observed (43). The extraction of analytes from aqueous solutions containing high ionic strength in IL-based DLLME can result in poorer extraction efficiency because the IL may dissolve in the ion-rich aqueous matrix (44). This was circumvented by exploiting the common ion effect. By adding NaPF

 to the aqueous sample matrix containing high NaNO

 or NaCl content, the miscibility of the [C

] IL was decreased and enhanced extraction efficiency was achieved (44). 

 IL-based DLLME was developed by our group to increase the extraction efficiency of analytes and also simplify the extraction steps of traditional IL-based DLLME (45). Figure 5 illustrates the procedure for 

IL-based DLLME. The watermiscible 1-butyl-3-methylimidazolium chloride ([C

MIM][Cl]) IL was fully dissolved in an aqueous sample solution. An ionexchange reagent, namely LiNTf

 , which immediately resulted in the formation of a turbid solution of the waterimmiscible [C

] IL phase was subjected to HPLC analysis. This approach provided significant advantages over IL-based DLLME and traditional DLLME as it does not require the manipulation of temperature (heating or cooling) and also avoids the use of organic solvents and the need for a disperser solvent. Additionally, because the original extraction phase is dissolved completely in water before the formation of a turbid solution, the microdroplets formed using this method are often smaller, which produced higher enrichment factors for aromatic compounds (45).  

Figure 5: Schematic of the basic operating principles in DLLME. (
•) Analyte, (â ) aqueous matrix, (â ) extraction and disperser solvents.			

Figure 6 compares the enrichment factors of aromatic analytes by 

IL-based DLLME and IL-based DLLME using two different disperser solvents. The former method provided the highest enrichment factors for all analytes (45). The selectivity of ILs for the extraction of emerging contaminants was explored using IL-based DLLME, and the results were compared to 

 IL-based DLLME (46). It was observed that the 1-(6-aminohexyl)-1-methylpyrrolidinium tris(pentafluoroethyl)trifluorophosphate ([HNH

MPL][FAP]) IL exhibited high selectivity for tertiary amines, and higher enrichment factors were obtained for polar acidic analytes using the [C

] IL (46). A glucaminium-based IL was applied as a selective extraction solvent in the 

 IL-based DLLME of boron species in water (47). The preconcentration of boron species into the IL phase was made possible by taking advantage of the selective cis diol functionality within the carbohydrate moiety of the cation (47).  

Figure 6: Comparison of the enrichment factors of 11 analytes using the IL-based DLLME method and the in situ IL-based DLLME method. (â) In situ IL-based DLLME using [C4MIM][Cl] + LiNTf2, (â ) Neat [C4MIM][NTf2], (â²) [C4MIM][NTf2] + 0.5 mL acetone, (X) [C4MIM][NTf2] + 0.5 mL methanol. Analytes: 1 = nitrobenzene, 2 = Î±,Î±,Î±,6-tetrafluoro-m-toluidine, 3 = 3-tert-butylphenol, 4 = ethyl benzoate, 5 = 2-nitronaphthalene, 6 = naphthalene, 7 = biphenyl, 8 = acenaphthene, 9 = fluorene, 10 = phenanthrene, 11 = anthracene. In situ IL-based DLLME conditions: [C4MIM][Cl] volume: 35 ÂµL; LiNTf2 solution volume: 434 ÂµL (0.2 g/mL); sample volume: 10 mL; sedimented phase volume: ~12 ÂµL; injection volume: 8 ÂµL; extraction time: 0.5 min; centrifuge time: 5 min. IL-based DLLME conditions: extraction solvent: [C4MIM] [NTf2]; disperser solvent: acetoneâmethanol; extraction solvent volume: 70 ÂµL; sample volume: 10 mL; sedimented phase volume: ~12 ÂµL; injection volume: 8 ÂµL; extraction time: 0.5 min; centrifuge time: 5 min. Adapted from reference 45. 			

Further studies revealed that the glucaminium-based IL can be recovered and reused after the 

 metathesis reaction and extraction by adding 0.1 M hydrochloric acid to the IL phase to displace the complexed boron species (48). Very recently, our group expanded the application of 

 IL-based DLLME to biological samples, namely the extraction of DNA from an aqueous sample solution (16). In an effort to increase selectivity and extraction efficiency, ILs were functionalized with hydroxyl groups as well as aliphatic akyl chains. It was observed that the 1-(1,2-dihydroxypropyl)3-hexadecylimidazolium bromide ([C

POHIM][Br]) IL exhibited high extraction efficiency for DNA presumably as a result of hydrogenbonding capabilities and dispersive-type interactions. Data from 

 P nuclear magnetic resonance (NMR) spectroscopy suggests that the extraction of DNA is also strongly affected by electrostatic and p-p interactions with the IL (16). 

Current Challenges and Future Directions  

Because of their tunable structures, ILs and PILs can be readily engineered to exhibit desirable physicochemical properties, providing them distinct advantages over traditional organic solvents for applications in sample preparation. Functionalized ILs and PILs have expanded the applicability of SPME toward more diverse classes of analytes while providing the high selectivity and sensitivity needed for trace-level analyses. Novel fibre modification techniques and sorbent loading and immobilization methods have improved the overall stability of IL- and PIL-based SPME sorbent coatings, allowing for directimmersion SPME in complex water samples. Analogous to IL- and PIL-based SPME, the use of ILs in DLLME has also been gaining momentum. As a result of the high enrichment factors achieved using functionalized ILs, IL-based DLLME and 

 IL-based DLLME have provided alternative pathways for the preconcentration and analysis of various analytes, including biological samples.    

As interests within the field of sample preparation are expanding toward the analysis of biological, environmental and pharmaceutical samples, concerns over biofouling and reduction in sensitivity can arise in both SPME and DLLME. Thus, it is imperative to develop novel ILs and PILs that can both exhibit high analyte selectivity and resist the potentially damaging components of the sample matrix. Further investigations in sorbent loading methods, coatingtosupport immobilization and using sorbentprotective membranes are possible means to improve matrix resistance of IL- and PIL-based SPME sorbent coatings. Varying extraction and agitation conditions while exploring the use of various functionalized ILs may reduce matrix interference in IL-based DLLME. 

For ILs and PILs to be more widely adopted in the analytical chemistry community, their toxicity and environmental effects need to be further investigated. An existing criticism sometimes heard regarding the use of ILs and PILs in sample preparation is their high cost of production compared to many organic solvents. However, because the amount of IL or PIL required is often significantly smaller as a result of their designed high selectivity, the total cost of production can be the same or lower than when using other solvents. The synthesis of highly pure ILs can be challenging and may require purification to minimize the presence of low-level by-products and impurities. Signal interference can be a problem in sample preparation and analysis because some of these impurities may contribute to background UV–vis absorption or fluorescence emission. To ensure high IL purity, analytical laboratories can synthesize ILs in smaller scales while also taking advantage of chromatographic purification techniques. Although careful consideration is needed when synthesizing ILs in bulk, it may be necessary to explore alternative synthetic routes that can provide high yields with minimal purification steps. Given the enormous flexibility offered by ILs and PILs in sample preparation, their use will continue to grow tremendously and it is anticipated that they will soon replace organic solvents in niche applications. 

received his BS degree in science from Aquinas College (Michigan, USA) in 2009. He currently attends the University of Toledo as a PhD student in Dr Jared L. Anderson's group. His research involves examining the selectivity of various functionalized polymeric ionic liquid–based SPME sorbent coatings. 

 graduated from China Agricultural University and received a BS degree in chemistry in 2010. Currently, she is working as a PhD student in Dr Jared L. Anderson's group. Her research involves the analysis of water by using polymeric ionic liquid-hydrogels as sorbent coatings in SPME. 

 is a junior undergraduate student at the University of Toledo pursuing a double major in Chemistry and Applied Mathematics with a minor in Physics. Will currently works in Dr Anderson's research group focusing on the development of novel SPME coatings using PILs. 

 obtained his BS degree in chemistry at South Dakota State University in 2000 and his PhD in analytical chemistry at Iowa State University in 2005. He is a Professor of Chemistry at The University of Toledo and a member of 

 editorial advisory board. His research interests include the synthesis of new classes of ionic liquids and materials derived from polymeric ionic liquids, the use of ionic liquids as catalytic solvents, and the use of ionic liquids in all aspects of separation science including analytical extractions, purification and chromatography. Direct correspondence to: 

 is the editor of "Sample Prep Perspectives" and a senior scientist in the columns and supplies division at Agilent Technologies in Wilmington, Delaware, USA. He is also a member of 

 editorial advisory board. Direct correspondence about this column should go to 

 editor-in-chief, Alasdair Matheson, at Advanstar Communications, 4A Bridgegate Pavilion, Chester Business Park, Wrexham Road, Chester, CH4 9QH, UK, or e-mail 

(3) R. Liu, J. Liu, Y. Yin, X. Hu and G. Jiang, 

(4) L. Vidal, M.L. Riekkola and A. Canals, 

(5) T.D. Ho, A.J. Canestraro and J.L. Anderson, 

(7) N. Fontanals, F. Borrull and R.M. Marcé, 

Articles

Columns

This short review covers the use of ionic liquids (ILs) and polymeric ionic liquids (PILs) in solid-phase microextraction (SPME) and dispersive microextraction (DLLME)

Ionic Liquids and Their Applications in Sample Preparation

A panel of experts discuss the latest trends in sample preparation.

This article was written as part of a special series to commemorate 30 years since the launch of 

This is certainly an accomplishment that we in the separations community have enjoyed and benefited from, and we wish many more happy years of success. This article is intended to be a follow-up of a sample preparation special report published in 1995 by Ron Majors, entitled "Trends in Sample Preparation and Automation — What the Experts Are Saying" (1). Now, we have asked a new panel of experts similar questions on sample preparation. This is a compilation of what these experts think about the current practice of sample preparation, and where they predict it will be 10 years from now. Just for fun, comparisons to the predictions of the expert panel in 1995 have been made where possible.   

Sample preparation remains the single most challenging aspect of chemical analysis. Chromatographic instrumentation, including column technology, instrument reliability, durability, capabilities and most certainly detector technology, continues to advance and move the business of chromatography forward at a steady pace and frequently with an incremental leap. Sample preparation still relies on scientists' basic chemical knowledge of the analytes being measured; the techniques and methods they have to measure them; and the experience to couple this knowledge effectively. Fortunately, advances in chromatography and chromatographic detection have reduced some of the need for complex and tedious sample preparation. But, as our experts reveal, challenges still remain. 

How valuable are automated sample preparation techniques, such as supercritical fluid extraction (SFE), microwave extraction, automated solvent extraction (ASE) and solid-phase extraction (SPE)?

In 1995, the overall general opinion of the earlier panel was that sample preparation was a manual technology, considered "low-tech" and assigned to the least trained staff. Now, that is changing. "Sample preparation stations are becoming more and more powerful, and aside from extraction and enrichment they offer features of standard addition, derivatization and so forth," says Pat Sandra from the University of Ghent. "The application of automated sample preparation units with injection (that is, on-line systems) is presently rather limited but will become more and more important in the future."

Other experts concur that for automated sample preparation to realize its goals of being commercially advantageous, efficient and "green," samples need to be run in parallel batches, similar to how sample preparation is done using manual labour. If sequential sample handling is the automated mode of operation, it should be very rapid and subsequently integrated with parallel analysis of final extracts to expedite the whole process. All the panelists agree that simple solvent based extractions can be faster and easier for a few samples and sometimes using SPE or SFE just to concentrate, purify or desalt samples in preparation for further mass spectrometry (MS) analysis is the simplest procedure to follow. 

Are preset sample preparation methodologies, in kit form, such as QuEChERS (quick, easy, cheap, effective, rugged and safe), decreasing method development time and making sample preparation a more routine, predictable laboratory operation?

These kits are recommended highly by our panelists as excellent tools for routine sample analysis at facilities that measure hundreds of analytes in large numbers of samples. Sergio Nanita, a residue analytical chemist from DuPont Crop Protection, states that the kits "allow laboratory personnel to become very familiar with the analytical steps, thus reducing analyst-to-analyst variability and errors that can occur when changing operations between multiple methods." Better expected precision is an added advantage of the kits.  

Analysts should ensure that the kits for matrices and target analytes have passed validation studies. In addition, users should be mindful that the kits contain preweighed materials designed for a specific concentration range of the analytes themselves. Karyn Usher, an Associate Professor in the Department of Chemistry at West Chester University, reminds analysts that if the kits do not meet the exact requirements of sample concentration levels, custom kits can be requested from most suppliers or the content of the kit can be modified to meet specific sample requirements, for example, by adding more sorbent or more salt. Since the technology of QuEChERS was introduced in 2003 (2), additional kits for removal of proteins and phospholipids from serum have also been marketed (3). In 1995, these advanced sample preparation techniques were not available. 

Will advantages in detection limits in MS, when used with massive dilution of complex samples and single ion monitoring (SIM), eliminate the need for sample preparation altogether? Are there particular sample types where this just doesn't work?

According to Steve Lehotay, lead scientist at the US Department of Agriculture's Agricultural Research Service Eastern Regional Research Centre, dilute-and-shoot sample preparation methods for many analytes from both simple sample types and complex applications are available using mass spectrometers with exceptional detection limits. However, some analytes do not ionize well and cannot be diluted enough and still meet the detection limit needs of the desired analysis. With current instrumentation, the experts agree that there is a constant trade-off between the ionization capabilities of the molecules and how much dilution can actually be used without losing accurate and precise quantitation. This goes hand in hand with ion suppression and ion enhancement, which need to be evaluated on an analyte-toanalyte basis. Removal of high-molecular-weight material with simple sample preparation, using an automated liner-exchange system and backflush options on MS equipment were recommended by Sandra if sample preparation is reduced or omitted. Agreement among the experts was not obtained about the benefits vs. cost of mass spectrometers to achieve these sample preparation reductions. Generally, academics viewed the most sensitive mass spectrometers as having a "high capital cost" needing a significant up-front investment and a specially trained and expensive operator. Conversely, their industrial counterparts stated that even if a less expensive technology — for example high performance liquid chromatography (HPLC) with UV detection — may be the "logical" choice, using the highly specific mass spectrometer pays for the costly instrument over time because the overall procedures are faster, with shorter sample preparation times and chromatographic runs of only a few minutes. To achieve this investment in state-of-the-art technology that yields improved efficiency and can translate into savings in all laboratories, cost improvement trends in spectrometers with improved ionization capabilities and detector sensitivity must continue. Undoubtedly, they will.   

Are there specific sample types where SPE disk and cartridge technologies coexist, or does each have specific applications where it excels?

SPE, whether disk or cartridge, really shines for use with dirty matrices, such as urine (cartridges) and large sample volumes, such as in water analysis (disks). These are classical applications, which Nick Snow, Professor of Analytical chemistry and Separation Science as well as the Director of the Centre for Academic Industry Partnership at Seton Hall University, views as the "bread and butter" for SPE. He reasons that cartridges have high capacity so analytes will not easily be lost or hidden by interferences, while disks make it easy to pass large volumes of sample through quickly. Both disks and cartridges are helpful in enriching or concentrating solutes from a matrix while also eliminating some interferences. Matrix solid-phase adsorption techniques (MSPD), such as QuEChERS, provide the purification step, but not the enrichment step. In 1995, the question asked of the panel was if both SPE disks and cartridges would survive in the future, and the answer was that they would likely coexist. The answer is the same 12 years later: Disks are best suited for concentrating large volumes such as environmental water samples and cartridges are more suited to a wider variety of cleanup applications.   

Are specific detectors, such as mass spectrometors, eliminating or greatly reducing the need or use of sample preparation in your laboratory today?

Amgen's Peter Grandsard, who leads the company's Discovery Analytical Sciences Group, comments that they are on a quest to perform "top-down" MS of large, heterogeneous molecules — that is, biologics and full antibodies. The "top-down" technique determines the intact molecular weight of each and every protein identified by peptide mass fingerprinting and peptide mapping. If Amgen's goal is achieved, the need for sample preparation currently associated with "middle-down" or "bottom-up" MS analysis will be greatly reduced. In "bottom-up" or "middle-down" MS, peptides are determined in given proteins, and online databases are used to identify the most likely structures (3). So, the quest to eliminate sample preparation and achieve a better answer remains in some specific fields. In general, as Usher representatively reports, using SIM mode in either GC–MS or LC–MS, most sample types still require some sample preparation, but this type of detection eliminates the need for more complex sample preparation methodologies that would otherwise be required. Laboratories need to prepare samples to the extent that they are clean enough to not foul the instrument and enable an accurate and reliable result to be obtained. W. Jeffrey Hurst, a member of the 1995 panel and a scientist at Hershey Foods Technical Centre in Hershey, Pennsylvania, USA, predicted the start of this trend with a statement that ideally analysts should perform no sample preparation, thus eliminating the use of solvents and reducing potential errors. 

What do you consider the best tool in your current toolbox of sample preparation methodologies?

Snow states that solid-phase microextraction (SPME), using both direct immersion and static headspace extraction, along with classical liquid–liquid extraction techniques, is frequently the best place to start in his laboratory. Lehotay concurs; he relies on extraction by shaking with solvents, liquid–liquid portioning and dispersive-SPE cleanup with the options of a variety of sorbents. QuEChERS, cartridge-based SPE and gel permeation chromatography (GPC) are preferred for more extensive cleanup. Usher sums it up for all three laboratories, stating that the most useful for a number of different samples is SPE. "Method development time was often longer than for some of the other techniques, but we were typically able to get the desired result," she said. These views are consistent with the 1995 panel, which asserted that automated or on-line analysis would mimic traditional analysis and that until new analytical techniques were developed, sample preparation would continue to be performed as necessary. 

If you had a crystal ball, where would you predict that sample preparation would be in 5–10 years?

Sample preparation, and even chromatography, will become less critical in MS- and nuclear magnetic resonance (NMR)-based methods in the next 10 years, predicts Grandsard. So it is dependent on the evolution and adoption of highend MS and NMR in the biologics arena. He does maintain that sample preparation techniques may be needed more in prototypical, biophysical methodologies such as cryogenic transmission electron microscopy (cryoTEM) and atomic force microscopy (AFM) that will likely have greater ongoing use in the analysis of biologics. For ultratrace-level analysis, DuPont's Nanita suggests that the survival of chromatography and MS for years to come will depend on rigorous methods using SPE, robotics, preconcentration steps and more-sophisticated instrumentation to achieve the most selective and sensitive chemical analysis. He feels that sensitivity improvements in MS will continue as will research on fast methods based solely on MS, making the techniques more rugged, placing them in laboratories around the world for routine sample analysis and replacing some of the classical extraction methodologies currently in chemists' toolboxes. Sandra simply states that application-dedicated sample preparation stations (off-line) will be increasingly developed, as he feels they are much more robust than on-line systems. 

These were not the same futuristic viewpoints of the expert panel 17 years ago. However, upon review, most of their predictions have come true. Examine the list below and see which predictions you believe they hit the mark on.   					

Greater integration of sample preparation and analysis with information feedback, automated record keeping and greater use of hyphenated techniques. 

Increased standardization, automation and modularization.

Techniques that use less organic solvent.

Less sample preparation because of improvements in selective spectroscopic measurements.

In summary, everything is expected to get smaller, labonachip is expected to be more widespread, kits are expected to be more broadly applied to a wider variety of sample applications, automation will increase and sample preparation will, in some cases, be eliminated because of more selective detectors. The panel predicted many of the same things with a few unexpected changes because of cautiously evolving technology. 

 is a Technical Fellow at the DuPont Crop Protection, Newark, Delaware, USA. 

(2) M. Anastassiades, S.J. Lehotay, D. Stajnbaher and F.J. Schenck, 

(3) Wako Diagnostics, Clinical Diagnostic Reagents, Richmond, Virginia, Catalog No. 433-36201. 

(4) I.S. Krull, A.S. Rathore and S. Kreimer, 

A panel of experts discuss the latest trends in sample preparaion.

Sample Preparation: The State of the Art

Elevated-temperature separations offer significant benefits compared to those performed at ambient temperature. This article discusses twelve of these benefits and then describes some practical considerations for implementing elevated-temperature liquid chromatography.

In part I of this series we discuss the various advantages and practical considerations of performing elevated temperature liquid chromatography (LC) separations. In part II, we will continue our discussion of this topic, exploring some basic thermodynamic relationships, including the derivation and use of the van 't Hoff equation. Finally, in part III we will present a more detailed discussion of the van 't Hoff equation, exploring its usefulness and relevance using various examples from the literature. 

Elevated-temperature separations offer many benefits, which are discussed below. 

Lower Column Back Pressures and Faster Separations

: Because solvent viscosity is a strong function of temperature, elevated temperatures decrease mobile-phase viscosities, which leads to lower column back pressures and faster separations. The dependence of viscosity on temperature is exponential and is given by the following equation: 

 are coefficients that are specific to a solvent, and 

 is the temperature in kelvins (1). Accordingly, noticeable reductions in viscosity, and therefore back pressure, are observed in systems running even at relatively low temperatures. For example, the viscosity of water decreases from 0.890 centipoise (cP) to 0.547 cP and acetonitrile decreases from 0.369 cP to 0.284 cP at 25 °C and 50 °C, respectively (2). This advantage only increases with increasing temperature: A 20-fold improvement in analysis time can be realized when working at 150–200 °C (3–5). 

: With the reduction in mobile-phase viscosity with increasing temperature, less toxic and "greener," but inherently more viscous, organic modifiers can be contemplated, such as ethanol. Separation of 18 fat- and water-soluble UV filters for cosmetic formulations was achieved using ethanol as an organic modifier under gradient conditions at 45 °C (6). Ethanol and water were also used to separate various phthalates at 35 °C (7). 

Temperature Can Exceed Solvent Boiling Point

: Column temperatures exceeding the normal boiling points of the typical solvents used in LC can be used in elevatedtemperature LC because a relatively inexpensive back-pressure regulator can be installed at the end of the system, such as after the UV detector, to suppress boiling of the mobile phase in the column. The typical pressure range for a back-pressure regulator is 20–70 bar. 

The increase in solvent boiling point with external pressure follows from the integrated Clausius-Clapeyron equation (8): 

* is the vapour pressure of the liquid at a temperature of 

 is the vapour pressure at the temperature 

 is the enthalpy of vaporization of the liquid, and 

 for various solvents of importance in chromatography are given in Table 1. Because a liquid boils when its vapour pressure exceeds the pressure acting on it, it is clear that the column pressure must be kept above the vapour pressure of the mobile phase to prevent it from boiling. A backpressure regulator of approximately 30 bar is usually sufficient to keep typical mobile phases from boiling (9).  

Table 1: Enthalpy of vaporization (ÎvapH) for various solvents (2). Note that enthalpies are generally fairly constant with temperature. 

Water Behaves More Like an Organic Solvent:

 As the temperature increases, the static permittivity of water decreases, so water behaves more and more like an organic solvent. Thus, at elevated temperatures, water is increasingly able to dissolve nonpolar species (10), and separations using more aqueous mobile phases can mimic those using more organic modifiers at lower temperatures. For neutral compounds on silica- and zirconica-based columns, an increase of 4–5 °C affects retention like a 1% increase in organic modifier (11–13). More specifically, a temperature increase of 3.75 °C has a similar elution strength change as a 1% increase in methanol (14), and a 5 °C increase in temperature was comparable to a 1% increase in acetonitrile (11). Figure 1 shows graphically how heated water can mimic the elution strengths (dielectric constants) of water–methanol or water–acetonitrile mixtures. For example, to replace a separation that uses 40:60 water–methanol (v/v) with a pure aqueous mobile phase the chromatographer would operate near 130 °C. 

Figure 1: Change in polarity (dielectric constant) of water with changing temperature at 100 and 600 bar (bottom x-axis) (15), compared to the effect of mixing an organic modifier into water at 25 Â°C and ambient pressure (top x-axis). Labels: * = 100 bar (water), 
• = 600 bar (water), â = methanol, â  = ethanol, â² = 2-propanol, ÃÂ =Â tetrahydrofuran, -Â =Â acetonitrile (16,17). Lines are guides to the eye. 			

In some applications pure aqueous mobile phases have been used at temperatures exceeding 100 °C. This method has been called "superheated water chromatography," "subcritical water chromatography" or "chromatography in very hot water" (13). Figure 2 shows the separation of several steroids at various temperatures using a superheated water mobile phase (18). The separation performed at 120 °C was far from ideal. As the temperature was increased to 140 °C and then 185 °C, the analysis time was reduced tremendously: from 30 min to 18 min to 5.5 min. Notice that the degree of tailing decreased with increasing temperature, which can be attributed to a decrease in the number and effectiveness of secondary interactions. 

Figure 2: Isocratic and isothermal separation of a mixture containing four steroids on a polybutadiene-coated zirconium dioxide column (150 mm Ã 4.6 mm, 3-Âµm dp, 300-Ã ZirChrom-PBD). Chromatographic conditions: temperature: 120, 140 and 185 Â°C; flow rate: 1 mL/min; mobile phase: deionized water; detection: UV at 200 nm. Peaks: 1 = estriol, 2 = androstadiendione, 3 = dehydroepiandrosterone, 4Â =Â estrone. Adapted from reference 19 with permission. 			

The van Deemter equation sheds light on some of the benefits of elevated-temperature LC: 

 is the height equivalent of a theoretical plate; 

 is the eddy diffusion coefficient, which depends on the quality of the column packing and the size of the particles in the packed bed, but is otherwise unaffected by temperature; 

 is the linear velocity of the mobile phase. A simplistic breakdown of the 

 terms shows that both depend on the analyte diffusion coefficient, 

 is strongly temperature dependent (as is η in equation 1 (1), and depends on 

 decreases. As a result, an increase in temperature will increase the degree to which longitudinal diffusion occurs (the 

 term), unfavourably raising plate heights and broadening bands. However, elevated temperatures lead to more efficient solute and mass transfer between the mobile and stationary phases, and diffusion is accelerated within the mobile phase itself (20,21), which reduces resistance to mass transfer of the analyte (the 

 term), and lowers plate height and is favourable. Two important consequences of these effects are that as temperature increases, the minimum in the van Deemter equation (

 (21,22) and van Deemter curves flatten (see Figure 3). Thus, elevated temperatures allow for faster separations with little or no loss of efficiency. Note that in general, 

 stays roughly constant with increasing temperature, so efficiencies do not change significantly with changing temperature as long as the linear velocity of the separation is near the optimum. 

Figure 3: Plots of the reduced plate height (the plate height, H, divided by the particle diameter, dp) vs. the reduced velocity (linear velocity, u, multiplied by dp, divided by the diffusion coefficient for the analyte) scaled to Dm,25 (the diffusion coefficient of the analyte at 25 Â°C) with the temperature as the parameter. Conditions: totally porous particles, rapid sorption kinetics, Dm,25 = 6 Ã 10-7 and dp = 3 Âµm. Plate height vs. linear velocity at various temperatures. Figure adapted from reference 23. The same trends are observed in plots of H vs. u (22). As temperature increases: the minimum value of H stays roughly constant, B increases and C decreases, which leads to the expected flattening of the van Deemter curve. (As noted, in the classical expression for the van Deemter equation, the A term does not depend on temperature [21]. However, there is at least some suggestion that the higher analyte diffusion coefficients at elevated temperatures reduce the effects of multiple paths for analytes in a column and may lead to some improvement in A [24]). 			

An empirical demonstration that higher flow rates do not strongly affect the quality of a separation at elevated temperature (185 °C) is shown in Figure 4. At 1 mL/min, four steroids were separated in <5.5 min (19). Under identical conditions, the same separation was then performed in <1.5 min using a flow rate of 5 mL/min (18). Even at this greatly increased flow rate, the steroids were still baseline separated. 

Figure 4: Isocratic and isothermal separation of a mixture containing four steroids on a polybutadiene-coated zirconium dioxide column (150 mm Ã 4.6 mm, 3-Âµm dp, 300-Ã ZirChrom-PBD) at flow rates of (a) 5.0 mL/min and (b) 1.0 mL/min. Chromatographic conditions: temperature: 185 Â°C; mobile phase: deionized water; detection: UV at 200 nm. Peaks: 1 = estriol, 2Â =Â androstadiendione, 3 = dehydroepiandrosterone, 4 = estrone. Adapted from references 18 and 19 with permission. 			

: There are a number of additional advantages when using elevated temperature separations. These are described below. 

Temperature is an instrumental variable, not a chemical variable, so with proper equipment it is often easier to change the temperature of a separation than to explore a chemical change in the mobile or stationary phase. 

Changes in analyte selectivity with temperature can sometimes be substantial, and reversal of elution order of certain analytes with increasing temperature may be observed (25). For example, while a decrease in retention times of analytes is usually found with increasing temperature in reversed-phase LC, McCalley (26) demonstrated an increase in retention times of various bases with increasing temperature. This increase in retention could be attributed to a temperature dependency of the p

Elevated-temperature LC raises the possibility of performing retention modelling over a range of temperatures — that is, predicting and optimizing the retentions of analytes with input from relatively few experiments (27–32). Because plots of ln 

 are often linear, and because elevated-temperature LC is increasingly practiced, this advantage is becoming particularly important (33).  

The option of direct extraction of thermodynamic data through van 't Hoff plots, which are plots of ln 

 is the temperature in kelvins. This information can serve both to characterize a column and to allow its comparison to others (34,35).  

In general, thermostating a column at a temperature above room temperature is a very good idea to avoid the daily fluctuations in temperature that take place in most laboratories and that can change analyte retention. Analyte retention typically changes by approximately 2% for each 1 °C change, so an intraday or day-to-day change in laboratory temperature of ±5 °C or more, which can occur in many laboratories, can substantially change analyte retentions (20,36–38).  

The equipment needed for elevated-temperature separations is readily available; relatively inexpensive column ovens are commercially available and provide temperature control and the necessary precolumn heating and postcolumn cooling of the mobile phase (36,39, 40–42). Many high performance liquid chromatography (HPLC) and ultrahigh-pressure liquid chromatography (UHPLC) systems sold today come with column ovens.  

Analyte decomposition in elevated-temperature LC is not generally an issue, because most analytes possess good thermal stability, even at relatively high temperatures, during their short dwell times in a column (3). However, in the case that a thermally labile compound may be part of a separation, or that a reaction may be possible between the mobile phase and an analyte, the questions presented by Thompson and colleagues (3) may help chromatographers decide whether elevated-temperature chromatography is desirable: Does working at elevated temperature reduce the dependability of the analytical calibration curve? Does elevated temperature chromatography induce a significant intercept in the calibration curve? Does temperature significantly diminish the sensitivity? If present, do on-column reactions distort peak shape? Does elevated-temperature chromatography introduce new peaks that interfere with the resolution and quantitation of the analyte or impurities? Pursuant to an impurity analysis, does a high-temperature mobile phase cause chemical reactions that alter the concentration or produce products that would interfere with quantitation of the impurity? If the answer to these questions is no, then elevated-temperature chromatography should be a viable option. 

Practical Considerations for Elevated-Temperature LC

To realize the advantages of elevated-temperature HPLC, the mobile-phase flow rate must increase and the column oven must be equipped with an efficient, low-volume means of properly adjusting the mobile-phase temperature (4). That is, the mobile phase needs to be preheated before entering the chromatographic column to avoid a thermal mismatch between the column and the incoming mobile phase. If a thermal mismatch exists, then band broadening of the peak may result, producing less-than-optimal chromatography. This thermal mismatch should be kept to less than 5 °C (3). Many systems come equipped with column preheaters that can be thermostated independently of the column oven. Figure 5 shows a diagram of a typical elevated-temperature apparatus. As with most systems, the solvents are mixed after the pumps and before the injector. The incoming solvent is preheated. The column is thermostated at a particular temperature via a column oven, and the effluent is cooled before it enters the detector. As discussed earlier, the column is maintained under a certain pressure via a back-pressure regulator that is placed in-line after the detector and before waste. 

Figure 5: Typical apparatus for elevated-temperature LC with a preheater, which can be temperature controlled independently of the column oven, effluent cooler and back-pressure regulator. 			

Figure 6 demonstrates how band broadening may occur if the mobile phase is not properly preheated. In Figure 6(a) the temperature of the incoming mobile phase matches the temperature of the column. When the temperatures of the mobile phase and column match, peak distortion resulting from thermal gradients is avoided. In Figure 6(b) the incoming mobile phase is cooler than the column, which leads to radial thermal gradients — that is, the centre of the column will be cooler than the outer portion of the column. This type of thermal mismatch between the column and mobile phase will result in broader peaks and less than optimal chromatography. In Figure 6(c) frictional heating is present. Frictional heating becomes a problem at high pressures because of friction between the mobile phase and the stationary phase or support. Frictional heating may lead to axial (along the length of the column) and radial thermal gradients. Axial thermal gradients also result in band broadening and a loss of efficiency. Figure 6(d) shows that one may compensate for frictional heating by introducing the mobile phase at a reduced temperature. 

Figure 6: Band broadening because of thermal effects: (a) ideal case, no thermal effects; (b) effect of incoming mobile phase that is at a lower temperature than the column; (c) effect of frictional heating; (d) combined effects of cold incoming mobile phase and frictional heating. An oven temperature of 70 Â°C is assumed. Adapted from reference 43. 			

A chromatographic demonstration of the thermal mismatch diagrammed in Figure 6 is shown in Figure 7. Figure 7(a) shows an ideal chromatogram obtained by preheating the mobile phase to the temperature of the column. Figure 7(b) shows a chromatogram with the incoming mobile phase cooler than the column. This thermal mismatch produces two effects: the retention times of peaks 3–7 are changed when compared to the chromatogram in Figure 7(a) (they are longer because the temperature of the mobile phase is lower), and peaks 6 and 7 are broader and distorted, a result that is attributed to thermal gradients in the column (43,44). Figure 7(c) also shows a thermal mismatch between the entering mobile phase and the column, but in this case these temperatures were adjusted to produce the same retention times as in the ideal chromatogram shown in Figure 7(a). However, peaks 3, 6 and 7 are still distorted, and peaks 6 and 7 are noticeably split. These less-than-desirable effects are again attributed to axial and thermal gradients in the column. 

Figure 7: Effect of the inlet solvent temperature on separation. Flow rate is 2.0 mL/min, incoming and oven temperatures shown in figure. Adapted from reference 43. 			

As a final practical consideration, one must be aware of the temperature limits of any columns that might be contemplated for use in elevated temperature chromatography. Many stationary phases or supports, especially those based on silica, degrade quickly at elevated temperatures. These effects are exacerbated at extremes of pH. However, there are also some materials based on silica that exhibit excellent stability even at temperatures above 100 °C (45). 

are with the Department of Chemistry and Biochemistry at Brigham Young University in Provo, Utah, USA. Direct correspondence to: 

is with the Institut für Energie- und Umwelttechnik e. V. in Duisburg, Germany. 

is with Selerity Technologies Inc., in Salt Lake City, Utah, USA.  

(1)  S. Giegold, T. Teutenberg, J. Tuerk, T. Kiffmeyer and B. Wenclawiak, 

D.R. Lide, Ed. (CRC Press, Boca Raton, Florida, 2004). 

Feature

Benefits of elevated-temperature seperations in LC are discusssed.

Elevated Temperatures in Liquid Chromatography, Part I: Benefits and Practical Considerations

"MS: The Practical Art" Editor, Kate Yu, spoke to Fred McLafferty about his pioneering career in mass spectrometry (MS).

Kate Yu: What brought you into the field of mass spectrometry (MS)? 

A crazy coincidence! After a PhD at Cornell University, (Ithaca, New York, USA) and a postdoc at the University of Iowa, (Iowa, USA), I arrived at the Dow Chemical Co., (Midland, Michigan, USA), in 1950 for an interview in their organic chemistry research laboratory. However, Dow also interviewed me for their spectroscopy laboratory in the MS group of Vic Caldecourt and two instrument operators. Vic had made the MS analyses so popular within the company that Dow wanted a chemist for the increased sample load while he concentrated on maintaining and improving the cranky instrumentation, at which he was terrific. No one, not even me, understood why I took the job with absolutely no prior knowledge (1).  

Bendix time-of-flight mass spectrometer at the Dow Chemical Company with Roland Gohlke (foreground) and Fred McLafferty. Published with permission of Fred McLafferty. 			

KY: You were in World War II and were awarded the Combat Infantryman Badge. What were the most remarkable experiences you remember during the war?

I went on active duty in April 1943, just after finishing a BS in chemistry and mathematics at the University of Nebraska (Nebraska, USA). A month before the war ended in Europe, our 2nd Battalion, 253rd Infantry, captured a major remaining German ammunition depot in a fierce battle against an elite SS unit that promised Hitler "no retreat". The battalion members were awarded a Presidential Unit Citation. During this action platoon sergeant John R. Crews of my rifle company was awarded the Congressional Medal of Honor for personally saving part of another company that was trapped. I was wounded earlier the same day. 

KY: Why did you come back to the field of chemistry after the war?

I was offered a battlefield commission from sergeant to officer rank, so I had a "career choice", but I doubt that such a career would allow the "fun participation" that I am having at my present age.  

Time-of-flight mass spectra (oscillograph display) typical of Dow’s early GCâMS capabilities. (a): Mass spectrum of methane (CH4), peaks at m/z = 12â17 (16 tallest). (b): Mass spectrum of acetone (C3H6O, MW 58), peaks at 13â59 (contains impurity peaks, for example, H2O). Published with permission of Fred McLafferty. 			

KY: Looking back at your career, who was the most influential person for you? 

Professor Franklin A. Long, who was at the Cornell University Chemistry Department from 1937 to 1999, was a very special scientific role model and friend during my graduate work from 1947 to 1949, and after I joined the faculty in 1968. He was a member of the National Academy of Sciences, Assistant Director of the US Arms Control and Disarmament Agency, and on the Science Advisory Committees for three US Presidents. But by far the most important person in my life for 65 years has been my wonderful wife: Elizabeth "Tibby" Curley McLafferty.  

KY: Tell us about your experiences at Dow Chemical. How did you develop the GC–MS instrument?

Dow's spectroscopy laboratory was one of the best in "modern" high information methods including direct reading atomic emission spectroscopy (AES), X-ray diffraction (XRD), infrared (IR), nuclear magnetic resonance spectroscopy (NMR) and MS. Dow was also unusually supportive of any new MS applications and related research. At a 1954 Gordon Research Conference, friend Steve DalNogare of duPont and H.N. Wilson from Imperial Chemistry Industries (ICI) (Manchester, UK), told me about gas chromatography (GC) with full details on how to build one. Soon after, Roland Gohlke, who worked with me, developed an improved version to solve problems in Dow laboratories and plants. For example, Roland put the column, a 25-ft copper tubing coil, in a 2L Dewar for better temperature control and often used the detergent "Tide" as a column packing.  

Fred McLafferty (far left); Roman Zubarev, Professor of Medical Proteomics in the Department of Medical Biochemistry and Biophysics at the Karolinska Institutet (Stockholm, Sweden). Roman also directs the institute’s large LCâMS proteomics facility; Dr Gary Valaskovic, co-founder and CEO of New Objective (Woburn, Massachusetts, USA); Neil Kelleher, Professor in Chemistry, Molecular Biosciences and the Feinberg School of Medicine of Northwestern University (Chicago, Illinois, USA). Neil also directs the large LCâMS proteomics facility in Chicago. The photo was taken by Dr Susan Weintraub at the ASMS meeting (Philadelphia, USA) June 2009. Published with permission of Fred McLafferty.			

In addition, my friends Bill Wiley and Dan Harrington at nearby Bendix Research Labs were just commercializing their time-of-flight (TOF) MS instrument (10K spectra/s), and were happy to let us try coupling our GC to the TOF in February 1956. It was very exciting to see on the output oscilloscope the rise and fall of unit-resolution spectra of acetone, benzene and toluene, the first organic compounds run on their TOF instrument (2).  

KY: How did you discover the famous McLafferty Rearrangement and what sort of impact did it have on mass spectrometry?

The infrared capabilities of Dow's spectroscopy laboratory were world-class, helped by the spectroscopy team's extensive efforts to collect a large spectral database of organic compounds. Consequently, during any spare MS instrument time we had, we continued this to build up an MS spectral database using the extensive sample collection available, giving us the mass spectra of many compound classes. This made it possible by 1956 to show (3) a general correlation of novel hydrogen rearrangements, although individual cases had previously been reported.  

At that time Dow sent me to the Boston area to set up a corporate laboratory for basic research. Although for my personal research I had no MS instrument, the structural diversity of our MS database was already unique, allowing extensive details of single and double hydrogen atom rearrangements to be established (4). This helped to show the key role of odd-electron ions in MS ion dissociation mechanisms and convince organic chemists that mass spectra were based on real chemistry. 

KY: Why did you go back to university while having a fantastic career in industry at the time?

The success of Dow's new basic research laboratory depended on selling its research capabilities to the academic community, as well as to meet Dow objectives. New hires found a novel research opportunity with industrial level support. We did recruit great people, including later Nobel Laureate George Olah. But in trying to sell my industrial research to academe, they sold me on academia. In 1964 I went to Purdue University (West Lafayette, USA), as Professor of Chemistry.  

MS: The Practical Art Editor Kate Yu interviews Fred McLafferty about his pioneering career in mass spectrometry

LCGC Europe-02-01-2013

Ionic Liquids and Their Applications in Sample Preparation

Sample Preparation: The State of the Art

Elevated Temperatures in Liquid Chromatography, Part I: Benefits and Practical Considerations

Man of the Masses

Troubleshooting GC Gas Supply, Sample Preparation, and Inlets

The Importance of the Sample Matrix

How Much Value Is There in a Software Operational Qualification?

Event News: HPLC2013

Determination of Pesticides in Red Wine by QuEChERS Extraction, Rapid Mini-Cartridge Cleanup and LC–MS–MS Detection

Vol 26 No 2 LCGC Europe February 2013 Regular Issue PDF