Pittcon is returning to the sunny city of Orlando, the fourth largest airport destination the United States. Florida is rapidly becoming the "innovation hub of the Americas." The state's biotechnology, pharmaceutical and medical device sectors continue to grow, making Florida the epicentre of some of the most exciting research, promising discoveries, and successful commercialization of final products in the world.

will showcase the latest innovations in analytical science and chromatographic technology from nearly 900 leading companies from over 40 countries. 

Orange County Convention Centre, Orlando, Florida, USA.

Approximately 50% of exhibitors use Pittcon as the premier event to launch new products and 20% offer "on-site only" exclusive deals and discounts on equipment and services. 

  which is located in the centre of the  exhibition floor, will present a unique robotic display, exhibitor videos projected on large screen monitors and complimentary copies of leading industry publications.  

This exciting exposition will be complemented by an all inclusive technical programme offering more than 2400 technical presentations, as well as  affordable educational short courses and unique networking opportunities. The advance registration fee of $115 gives conferees unlimited week-long access to the entire technical programme and the exposition floor. 

 includes symposia, contributed oral sessions, awards, workshops, posters and 

 world-renowned speakers will present symposia contributed and oral sessions, workshops, awards and posters covering diverse subject matter. Hundreds of technical sessions will focus on topics relevant to liquid and gas chromatography. Highlights include:  

The Role of Ionic Media in Influencing Electrostatic Interactions in Chromatographic Separations

 by Brian Bidlingmeyer from Agilent Technologies. This presentation starts by reviewing the ways in which control of peak retention is accomplished by using ion-pairing followed by an explanation of the mechanism for ion-pairing. 

Why Consider Ionic Liquids as Mobile Phase Modifiers for Liquid Chromatography?

 by Neil Danielson, Ling Zhou and Matthew Collins, University of Miami, will focus on the use of ionic media in separation science. 

Stable, Microfabricated Thin Layer Chromatography Plates Prepared on Infiltrated, Patterned Carbon Nanotube Forests

 by Matthew Linford, David Jansen, Supriya Kanyal, Cody Cushman, Laurel Peacock, Michael Vail, Andrew Dadson, Richard Vanfleet and Robert Davis will cover lab-on-a-chip/microfluidics, liquid chromatography, nanotechnology and thin layer chromatography. 

The Role of Temperature in HPLC Efficiency and Speed

 by Pat Sandra from the Research Institute for Chromatography, Belgium, will present recent research on optimizing speed and efficiency by using temperature optimization in RP–LC, HILIC and enhanced fluidity LC (EFLC). 

The Judicious Use of pH Gradients Can Double or Triple the Peak Capacity and Resolving Power of High- or Ultra-Pressure Liquid Chromatography

 by Joe Foley and Adam Socia from Drexel University. Discussion will include the benefits of SE–LC with sequential elution modes, theory summarization and applications with an emphasis on an approach using two pH gradients prior to a solvent gradient that can be used to separate weak acids, weak bases and neutral compounds from each other. 

MEMS and Nanotechnology: Taking [micro]GC Beyond Miniaturizing Gas Chromatography

 by Masoud Agah from Virginia Tech, will include discussions on the invention of new µGC columns, semi-packed and multicapillary and demonstrations on the marriage of MEMS and nanotechnology to improve  both stationary phase coating for µGCs and develop new architectures for high-speed, high-performance gas chromatography. 

 by Alastair Lewis, Jacqueline Hamilton, Jaydene Halliday, Richard Lidster and Sam Edwards from the University of York, will present a selection of results from the GC×GC–PID, including assessment of the performance of planar glass columns compared to fused silica, the capabilities of the built-in fluidic modulator and the utility and applicability of a compact photoionisation detector as a means of detection in GC×GC. 

GC-on-a-Chip: Fundamental Considerations of Microcolumn Separation

 by Joshua Whiting, Joseph Simonson and Pete Stevens. This session will focus on several aspects of the theory and modeling of microcolumns for the separation of vapour mixtures, with comparisons to experimental results. Two different applications will be presented: a vacuum outlet system with atmospheric pressure air as a carrier gas using a low aspect ratio column (120×250 µm); and a comprehensive two-dimensional chromatography (GC×GC) system with high-pressure hydrogen as the carrier gas using a high aspect ratio column (30×685 µm).  

, which will be presented by Steven A. Benner, Distinguished Fellow at the Foundation for Applied Molecular Evolution. Pittcon is also pleased to present R. Graham Cooks, Henry B. Hass Distinguished Professor–Analytical Chemistry, Department of Chemistry, Purdue University, who will deliver the Plenary Lecture 

Ambient Ionization and Mini Mass Spectrometers: In situ MS for Everyone

. Mixers following each of the lectures will provide an informal networking venue allowing conferees to continue the discussion.  

Every year Pittcon recognizes and honours scientists who have made outstanding contributions to analytical chemistry and applied spectroscopy. This year 

, University of Texas at Arlington, will receive the prestigious 

The Chromatography Forum of Delaware Valley 

for his contributions to separation sciences which have been both esoteric and practical. He is credited with the development of electrodialytic suppressors, eluent generators and post column reagent introduction devices. His contributions to the use of ion chromatography in environmental analysis have been particularly important, from the discovery of the ubiquitous presence of perchlorate in mother's milk to near real-time gas/aerosol composition analyzers.  

Sessions associated with this award presentation include T

he Evolution of Electrodialytic Devices in Ion Chromatography

Recent Progress in the Synthesis of Anion Exchange Condensation Polymers

Development of Ionic Liquids for Chemical and Biochemical Analysis

Recent Advances in the Charge Detector Concept for Ion Chromatography

session will be webcast and available for post-event online viewing. 

 professor of analytical chemistry in the Department of Chemistry at The University of Toledo, will receive the 

ACS Division of Analytical Chemistry Award 

Young Investigators in Separation Science

. 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. This award recognizes outstanding contributions in the field of separation science by a young chemist within 10 years of their highest degree. Sessions included in this award presentation include 

Exploiting the Versatility of Ionic Liquids and Polymeric Ionic Liquids in Separation Science

Separations as Intermolecular Interaction Amplifiers

Cyclofructans: The Newest Chiral Macrocycle

Combining Sensors with Separations for Enhanced Selectivity

For those who are new to the field of chromatography or for veterans looking to enhance professional development, Pittcon provides an affordable 

covering more than 60 topics which range from beginner to advanced levels, in half-, one- or two-day courses. There are approximately 55 courses offered in various topics of chromatography. Some of the new courses for this year include 

Advances in Countercurrent Chromatography and Related Techniques

Fundamentals and Practical Application of Size-exclusion Chromatography

How To Launch 100% of Liquid Samples Into ESI Mass Spectrometers and More About the Nanoliter Regime

mpurities in Pharmaceuticals - A Survey Course

Physical Chemistry of Macromolecules, Part I - Basic Principles

 will help iOS and Android device users to organize agendas, get information about technical sessions and short courses, search exhibitor listings and find local restaurants and entertainment. On 

 more than 18,000 attendees in industry, academia and government from over 90 countries will converge at 

making it the perfect event to get connected to science, discovery and colleagues — while enjoying the sights and sounds of the great city of Orlando. For complete up-to-date event details visit 

Orange County Convention Centre, Orlando, Florida, USA 

 Follow Pittcon on Twitter, Facebook, LinkedIn, Google +

Articles

A look at what's on offer at this year's Pittcon.

Connecting with Chromatography at Pittcon 2012

A mysterious autosampler problem is solved.

We recently experienced some mysterious and sporadic liquid chromatography (LC) results using an ultrahigh-pressure LC (UHPLC) system. This incident took us a week of intensive investigation involving many hypotheses and numerous blind alleys until we found the root cause. We believe this issue has not been previously reported and could easily be missed by many LC users (1–4). Here is our story. 

Our problems began when we performed potency and impurity assays of two newly developed clinical drug products (capsules and tablets). We followed a validated 42-min procedure that had been used successfully for two years and modified it into a faster and equivalent 15-min UHPLC method (5). The sample preparation procedure in the modified method uses 20 mM ammonium formate, pH 3.7, as the extraction solvent. We used an Agilent model 1290 UHPLC (Santa Clara, California, USA) with 3-μL injections and performed many area-percent analyses without any issues. However, this time the potency assay failed system suitability tests because the response factors for duplicate standard (calibrator) preparations differed by ~20%. Curiously, peak area repeatability from the same vial was excellent (<0.3% RSD), indicating that the autosampler was not malfunctioning. Duplicate standard preparations were then made by two analysts. Highly variable results and high responses for the calibrator were observed, which led to artificially low potency values of the drug products. We decided to launch a more systematic investigation.  

We started our investigation by interviewing the primary analyst and checking all the calculations for the method. We noted higher than expected responses from some calibrator vials but not others — even though all calibrator solutions (~0.5 mg/mL) should have provided nearly identical responses. It seemed quite inconceivable that we obtained responses from two duplicate calibrator preparations that varied as much as 20–30%.  

Peak areas in chromatograms of drug product extracts typically were quite consistent. Next, we scrutinized each analyst's weighing techniques and the quantitative transfer procedure of the weighed powder to the volumetric flask. We verified the analytical balance calibration and visually checked the homogeneity of the reference standard. At that point, all practices appeared to be in order, and we found no clues for the disparate responses. Next, we turned our attention to the existing sample preparation procedure and designed an experiment using stronger extraction solvents. For quicker problem diagnosis, we switched to a 2-min fast LC, nonstability indicating method (Waters XBridge C18 column [Milford, Massachusetts], isocratic mobile phase consisting of 25% acetonitrile in 0.1% trifluoroacetic acid, 1-mL/min flow rate, 2-μL injection) (6). The results of the extraction study are summarized in Table 1, which shows encouraging results in study 1 with all three extraction solvents (ammonium formate, 0.1 N hydrochloric acid and 20% acetonitrile in 0.1 N hydrochloric acid) yielding the expected potency of ~100%. However, reinjecting the very same sample vials on a different model 1290 UHPLC system yielded data with 30% higher peak areas for the standards and low potency results of ~74–78% (study 2, Table 1). A repeat of the same sample set in study 3 using the 15-min UHPLC method on the second system also yielded the low potency results, but at ~8–11% higher potency than those in study 2 (fast LC). A review of historical response factors showed higher responses for some of the calibrator solutions as the key issue. The high calibrator responses were consistent with an extra injection volume of ~0.6 μL; that is, 30% for 2 μL, 20% for 3 μL, and 6% for 10 μL. We were puzzled with these consistent observations that could be caused by an extra 0.6 μL that was injected at some times, but not at others.  

Table 1: Drug product potency assay results with different extraction solvents. 

We ruled out instrumental problems because the same observations were made with two UHPLC systems — both well maintained and almost brand new. We considered other hypotheses such as software issues or corrupted sample sequences, contamination of the sampling needle with surfactant from the drug product excipients, overfilling the sample vials, and over-tightening of the septa that might cause pressurization of the vials (3,4). We designed experiments to test each hypothesis using the 2-min isocratic method, which allowed us to watch each injection closely and check the resulting peak area quickly. Nevertheless, none of these hypotheses could be verified. Finally, during close scrutiny of one of the "offending" calibrator vials, we saw a "shimmering" reflection caused by an extra layer of liquid adhering to the neck of the vial (Figure 1). This was a "eureka" moment and we knew we had found the root cause.  

Figure 1: A picture and a drawing of an HPLC vial containing an extra layer of liquid near the neck of vial. This extra layer of liquid is easily formed by inverting the filled vial and is physically stable. 

As it turns out, our analyst is very conscientious and often shakes or inverts LC vials to homogenize sample extracts before loading sample trays. Because of the high surface tension of the aqueous buffer, an extra layer of solution adhering to the septum or around the inside of the neck of the vial is formed. This layer of liquid somehow causes an extra ~0.6 μL to be injected. Drug product extracts have surfactants from the formulation which reduce surface tension and tend not to form this extra layer. As a result, the calibrator solution injections are larger, leading to biased results for the samples. To verify this explanation, we conducted an experiment on two calibrator vials without and with the extra liquid layer. Figure 2 shows that while both responses are linear and have the same slope, the 

-intercepts are 0.00 μL and -0.65 μL respectively, indicating a bias of 0.65 μL from the vial with the extra layer.  

Figure 2: Two response curves obtained by injecting the same standard solution from two vials: (a) without and (b) with the extra liquid layer. 

Why Doesn't this Problem Occur More Often?

The next question was obvious: Why hasn't this problem been observed previously or reported in the literature? The extra layer appears to form easily using all types of LC sample vials. We subsequently tried to reproduce this problem with other HPLC systems in our laboratory (Waters Alliance, Waters Acquity UPLC and Agilent 1200) without any success. The Waters Alliance system has a side-port sampling needle and the Waters Acquity UPLC system has a double-needle design; those systems do not appear to be susceptible to over-volume injection from the extra layer. However, the Agilent 1200 autosampler appears to have a similar injection mechanism to the 1290, so why wouldn't it be affected?  

Response from Agilent indicated that the 1290 UHPLC autosampler uses an injection technique that pulls an air gap of ~0.6 μL into the tip of the sampling needle inside the vial near the septum before the needle leaves the vial. The use of an air gap, sometimes referred to as the "leading bubble technique" (7), has long been known (8) as a way to decrease band-broadening and dispersion during sample injection. Some Agilent autosamplers use this approach to increase the precision for samples of small volumes. In the present case, instead of an air gap, the needle will contain ~0.6 μL more sample if a plug of liquid is trapped in the vial neck after vial inversion. According to Agilent Technologies, a new firmware revision is under evaluation that will allow reproducible injections of very small volumes without this issue. 

Another useful observation is that this extra layer of liquid can be formed only for sample-solvent strengths less than 20% acetonitrile or 25% methanol. Liquids with higher organic-solvent concentrations have lower surface tension, which will not allow the extra layer to form. As an alternative to inversion, vortexing sample vials provides any necessary mixing but lessens the chance of forming this layer because of the rapid vibration of the vortexing action. 

We learned several valuable lessons from this investigation and troubleshooting episode.  

Don't invert LC vials before loading them into the sample tray. Most sample solutions are homogenous, and further agitation should not be required. In cases where agitation may be helpful (for example, frozen samples, emulsions or suspensions), use vortexing and check afterward to confirm the absence of any extra layer of liquid. 

Use stronger extraction solvents (if possible) to ensure robust and quantitative extractions from all sample matrices. Our revised drug product method now uses a stronger extraction solvent of 20% acetonitrile in 0.1 N hydrochloric acid and a larger injection volume of more-diluted sample solutions (5 μL for UHPLC or 20 μL for HPLC). The increased organic reduces the solution surface tension, thus minimizing the chance of forming the extra liquid layer in the vial neck; the larger injection volume reduces the percent error. 

Effective LC troubleshooting may include looking for common symptoms and verifying corrective actions (for example, instrument, column, baseline, pressure, data and sample preparation) using a step-by-step procedure to isolate and diagnose the cause (1–4). A good strategy is to propose hypotheses and then conduct experiments to verify or eliminate each potential causative factor. The use of short run times can speed up such problem diagnosis.

Although toll-free technical service hotlines and service engineers typically are helpful in troubleshooting, there may be circumstances where personal vigilance and keen observations are required by analysts. Here, keeping our eyes open and focused on the key issue provided us with the solution to this mystery of sporadic potency results.

The authors thank Jack Pellett, Derrick Yazzie, Tania Ng, Max Zhong, Kelly Zhang and Nik Chetwyn from Genentech (San Francisco, California, USA); Tom Waeghe of Mac-Mod (Chadds Ford, Pennsylvania, USA); Tom Jupille and John Dolan from LC Resources (Walnut Creek, California, USA); and Christian Gotenfels of Agilent Technologies for their helpful discussions and suggestions. 

is a research assistant at Genentech, Small Molecule Analytical Chemistry and QC, in South San Francisco, California, USA.  

is senior scientist responsible for multiple early phase drug development projects and new analytical technologies at Genentech, Small Molecule Analytical Chemistry and QC, in South San Francisco, California, USA. He is also a member of 

 is vice president of LC Resources, Walnut Creek, California, USA. He is also a member of

 editorial advisory board. Direct correspondence about this column should go to "LC Troubleshooting", 

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

 (Humana Press, Clifton, New Jersey, 1989).  

HPLC Maintenance and Troubleshooting Guide, in Modern HPLC for Practicing Scientists

 (Wiley-Interscience, Hoboken, New Jersey, 2006) Chapter 10. 

Handbook of Pharmaceutical Analysis by HPLC

, S. Ahuja and M.W. Dong, Eds. (Elsevier/Academic Press, Amsterdam, 2005) Chapter 9.   

LC Troubleshooting

A Case of Sporadic LC Assay Results

  Food samples cover a wide range of physical matrix types and the range of potential chemical contaminants are equally diverse. Some contaminants, such as banned substances, require analysis at extremely low levels to 'demonstrate' that they are absent and the method of analysis must enable determination at trace and ultra-trace levels. The method of analysis is usually chromatographic and to have any practical validity, good sample extraction and preparation are vitally important to enable separation of the contaminant from the complex matrix and reduce the potential impact of any matrix effects. A variety of sample extraction techniques are available to the analyst and the method of choice will depend on the matrix type and analyte(s) of interest. The move towards microextraction techniques or analysis with little or no sample preparation aims to provide a more "environmentally friendly" approach by using less solvents, but must be used with caution for a complex, naturally non-homogeneous matrix, such as food. Examples of sample preparation techniques used for analysis of trace-level contaminants in foodstuffs are outlined and the advantages and disadvantages of each are assessed.

Food contaminants cover a diverse range of compounds from a number of potential origins. They can be present at trace and ultra-trace levels and therefore identification and quantification in foods can present a challenge to the analytical chemist. Many known contaminants are at levels of toxicological concern, or a maximum level permitted by legislation, but for banned substances the analytical method must be capable of measuring as low as is reasonably practicable to demonstrate absence. Moreover, for unknown contaminants, such as those responsible for taints and off-flavours, the method must also provide unequivocal identification of the causative compound(s).  

As a result of the variety of analytes that can be regarded as 'food contaminants', including pesticides, packaging contaminants, environmental contaminants, processing contaminants, mycotoxins and veterinary drugs, several methods of extraction are generally required. The choice of sample preparation techniques used will depend on the contaminant(s) of interest, the food matrix and the final analytical determination/instrumentation to be used (1). The requirements of the analysis must always be considered and the method used should be fit for purpose — in terms of identification of unknown analytes, accuracy required (qualitative or quantitative) and sensitivity if working to known limits. 

Typical steps in an analytical method include sampling/homogenization, extraction, clean-up, and concentration before the final analysis. A simplified schematic showing the options for both solid and liquid samples is shown in Figure 1.  

Figure 1: A simplified schematic showing the sample preparation options for both solid and liquid samples. 

For the majority of food contaminants, the final analysis is performed using a chromatographic technique, combined with an appropriate detector. The increased use of mass spectrometric detectors has enabled selectivity and sensitivity to be optimized at the detection stage and has led to the development of methods with minimal sample preparation. Such approaches provide rapid methods and can cover a wide range of analytes, with crude extractions. It should be noted, however, that good sample extraction and preparation are still important, and a 'cleaner' extract will improve chromatography, reduce instrument maintenance and, in particular for liquid chromatography mass spectrometry (LC–MS), reduce the potential impact of matrix effects. 

Food can encompass a number of matrix types, and may include different biological matrices, such as meat, fish, grain, seeds and plant material, as well as dry powders, fats and liquids. The complexity of food products means that there is the potential for many interferences with the analytes of interest and that different techniques may be required depending on the matrix. When looking for trace-level contaminants, large concentrations of matrix components can cause problems, such as ethanol for alcoholic drinks. 

Foodstuffs are naturally non-homogenous and this presents a challenge given that a lot of modern techniques have been developed with smaller sample sizes in mind. It is essential to ensure that any sample taken for analysis is representative of the bulk matrix. A large sample size may be needed to obtain the necessary sensitivity, although in some cases, such as a complaint from a customer, this may not be possible. When analysing for trace and ultra-trace levels of known contaminants, increased method selectivity (that reduces potential matrix interferences) can help provide the sensitivity required to determine the analytes of interest. However, for the determination of unknown contaminants, analysis becomes more of a challenge as selective extraction may not be possible. In this instance, a more investigative approach is required.  

Where an analyte is known (or suspected to be) volatile then headspace analysis can be used. This technique has the advantage of being suitable for all matrix types and uses little or no solvents. The headspace of a sample is injected directly into a gas chromatograph connected to a suitable detector and it is therefore a clean technique and avoids the non-volatile components entering the chromatographic system. Direct static headspace involves the incubation of a sample (usually with heating and shaking) for a defined period of time (ideally until equilibrium is reached). The amount of analyte in the headspace is directly proportional to the amount in the sample. This technique suits volatile and semi-volatile analytes because temperatures and incubation times can be adjusted accordingly and salt can also be added to encourage analytes into the vapour phase. However, as only a proportion of the vapour phase (headspace) is injected, direct headspace provides only limited sensitivity. A summary of headspace GC analysis for the determination of food contaminants was published in 1984 (2) and it has been reported more recently to determine 1,3-dichloropropanol in soy sauce (3) and furan in food stuffs (4). 

The distribution of an analyte between the sample and gaseous phase will vary with the matrix and for quantitative work, matrix-matched standards or the use of the method of standard additions is recommended. An internal standard (IS) of similar volatility, or preferably isotopically labelled, can also be used. There are several approaches to increasing the sensitivity in headspace extraction. One approach is to use multiple headspace extraction (MHSE) and perform sequential headspace analysis from the same vial. This can provide exhaustive extraction of an analyte and theoretical amounts can be calculated from a limited number of consecutive extractions.  

Another approach to enable a more exhaustive extraction of the sample is to use dynamic headspace or gas sampling techniques. This enables enrichment of volatile analytes in a cold trap or on an inert support by continually flushing the headspace of the sample. Thermal desorption enables direct transfer of the analytes into the GC instrument following extraction.  

Traditional dynamic headspace (purge and trap) GC is rarely used for trace contaminant analysis in foods. This is largely a result of the lack of selectivity that it offers as, although large concentration factors are possible, any interferences present are also concentrated and therefore increases in sensitivity can be limited. However, the use of more selective absorbent materials as traps may help overcome this and additional cryo-focusing steps help to minimize losses before injection. Because of the flows needed on some systems to desorb the trap, a split is required, leading to additional potential losses in sensitivity. Applications using commercially available needle-trap systems are increasing, as illustrated in a recent review of needle trap and related techniques (5). It has been reported for analysis of food volatiles (6), although food applications are limited and, as with all miniaturized techniques, limitations in sample size have to be considered to ensure that any sample is representative.  

Sorptive extraction techniques are based on the extraction of analytes from the matrix into a non-miscible extracting phase, which can be coated on a solid support. Sorption can include extraction onto active sites on the surface (adsorption) or partition of the analytes into the extracting phase (absorption), or a combination of both. The main advantages of sorptive extraction compared to some other extraction techniques are the reduction in solvent use, the combination of sampling and extraction into one step and the high enrichment factors that can be achieved.  

As with headspace analysis, sorptive extraction techniques are generally equilibrium based and do not provide an exhaustive extraction. However, although the maximum sensitivity is achieved at equilibrium, extractions can be performed for a defined period of time, providing conditions are kept consistent. As with headspace, the use of ISs, matrix-matched standards or the method of standard additions is recommended for accurate quantitation. Some techniques enable pre-doping of the extraction phase with standards and, in some cases, kinetic calibration regimes have been reported to improve quantitation (7). 

Solid-phase microextraction (SPME) is a commonly used sorptive extraction technique. It can be used to sample liquids or solutions directly, or to analyse the headspace above a sample. For food contaminant analysis direct immersion SPME is rarely used because of issues with fouling of the fibre or interference from a high concentration of matrix components. Headspace SPME sampling enables analysis of almost any matrix for volatile and semi-volatile compounds. The fibres are available with different extraction coatings and the process can be automated using a modified microsyringe. Following extraction, the fibre is desorbed thermally directly into a GC, or can be eluted with solvent for subsequent GC or LC analysis.  

Fibre coatings can be pure liquid polymers [such as polydimethylsiloxane (PDMS) or polyacrylate (PA)] or mixed films containing liquid polymers and solid particles [such as carboxen-PDMS]. The choice of phase will depend on the properties of the analyte (if known). A mixed fibre, such as divinylbenzene(DVB)/carboxen-PDMS, enables extraction of a wide range of analytes and is often used for volatile screening methods. PDMS is also a commonly used phase because it has generic selectivity for many types of non-polar analytes, but for more polar analytes, polyacrylate and carbowax-divinylbenzene (CW-DVB) are more suitable. Alternative coating materials are also available that provide extraction for specific analytes, including affinity coatings and chiral coatings for optically active analytes. It should be noted that when using headspace SPME, because of the partitions involved (as illustrated in Figure 2), more volatile compounds may be lost from the fibre because they will favour the vapour phase at increased temperature. This can be a useful tool for selective analysis, as the fibre will favour lower volatility compounds than direct headspace. Cold fibre SPME, (CF–SPME), enables the fibre to be cooled while the sample is heated, and is thus reported to increase extraction efficiencies. Derivatization can be used to maximize extraction efficiencies for some analytes, either prior to extraction or on-fibre.  

Figure 2: Partitions in HS-SPME between sample matrix, vapour and fibre. 

SPME offers an increased sensitivity compared with direct headspace because of the high enrichment factors that can be achieved. Moreover, as a result of the different selectivity, HS–SPME is most commonly used to obtain  volatile/semi-volatile profiles, particularly in flavour analysis. Applications for the determination of trace contaminants in foods include determination of furan in baby food (8), phthalates in wine (9) and ham sausage (10) and PAHs in tea (11).  

However, one of the drawbacks of the technique can be the limited volume of stationary phase that can be bound to the fibre.  

To overcome the limited extraction capacity of SPME fibres, stir bar sorptive extraction was developed. This technique uses a glass stir bar, which can be coated with a larger volume of stationary/extracting phase than a SPME fibre. The 'Twister' bars are coated with a bonded adsorbent layer of PDMS and, following extraction, can be thermally desorbed directly into a GC or eluted with solvent for subsequent analysis by GC or LC. Unlike SPME fibres, twisters are more often used for direct sampling, but can also be used for headspace sampling to provide additional selectivity and enrichment. Because of the non-polar nature of the PDMS, this extraction technique works best for analytes with octanol-water distribution coefficients between 100,000 and 100 (log K

 between 2 and 5). As the technique can be used for liquid or semi-solid samples, it is suitable for many food types and can also be used following an initial extraction. To enable extraction of the more polar analytes, the matrix can be modified or derivatization can be used. Currently, alternative coatings are being developed to offer different selectivity. Long extraction times may be required for exhaustive extraction and generally this technique is considered an equilibrium-based extraction and therefore optimization of conditions is required for accurate quantitation. The use of matrix matched standards or ISs are recommended. For food contaminant analysis, to date, applications are mainly for non-fatty food matrices and non-polar analytes, although some multi-analyte methods have been reported (12, 13). 

For solid samples, the efficiency of extraction depends on penetration of the sample by the solvent, therefore an initial homogenization step, such as drying and grinding, is often used. Following this there are a number of extraction techniques available. 

One technique which enables homogenization and extraction in one step for solid or liquid samples is matrix solid-phase dispersion (MSPD). With this approach, the sample is mixed with a sorbent, such as C18-bonded silica or hydromatrix, and the analytes eluted with solvent. The porous structure provided by the matrix aids penetration by the extraction solvent and can also have some functionality which can retain unwanted matrix components, such as fats/lipids. This technique is particularly useful for samples with a high proportion of fats, such as animal tissue samples, where drying can be problematic and is commonly used in pesticide analysis (14–18). A review of MSPD for extraction of contaminants from foodstuffs was published in 2007 (19). 

A number of techniques that have been used for extraction of contaminants from solid samples are outlined below. 

Solvent Extraction Techniques For Solid Samples

Soxhlet extraction has been widely used for the extraction of thermally stable analytes from foodstuffs, such as polyaromatic hydrocarbons (PAHS) and polychlorinated biphenyls (PCBS) (20,21). It provides an exhaustive extraction by the continuous cycling of solvent through the heated sample matrix, with collection of analytes into the hot solvent. Automated systems enable several simultaneous extractions to be performed. To improve extraction efficiencies, soxhlet can be combined with microwave or ultrasonic extraction. A related technique: pressurized liquid extraction (PLE) – uses elevated pressures, which enables a more rapid extraction because the solvent remains in the condensed state. Alternative names of PLE include accelerated solvent extraction (ASE), high-pressure solvent extraction (HPSE), pressurized hot solvent extraction (PHSE) and subcritical solvent extraction (SSE).  

If water is used as an extraction solvent it can be known as super-heated water extraction (SHWE), subcritical water extraction (SWE), hot water extraction (HWE), pressurized hot water extraction (PHWE) or high temperature water extraction (HTWE). As a result of the changes in polarity of water at high temperature, this can have additional advantages. The use of a supercritical fluid (SFE) (such as carbon dioxide) as an extraction solvent provides another green alternative and enables the easy removal of the 'solvent' following extraction. 

The selectivity of these solvent extraction techniques can be improved by the addition of sorbents to the extraction cells or by modifying the extraction solvent. Microwaves and ultrasonic extraction can be employed to enhance extraction efficiencies. For microwave-assisted extraction (MAE), at least some polar solvent needs to be used, and as the sample is heated it is only suitable for thermally stable analytes. Most of these extraction techniques are not suitable for volatile analytes. A wider choice of extraction solvent can be used for ultrasonic solvent extraction (USE) and the technique needs no specialized equipment so is relatively inexpensive. However, although multiple extractions can be performed simultaneously, it is not easily automated.  

Using these techniques for the analysis of food contaminants, means that further steps following extraction are invariably required to enable analyte enrichment and/or concentration. Reported applications for food contaminant analysis include PLE for determination of Bisphenol A diglycidyl ether (22), ochratoxin A (23) and various persistent organic pollutants (24), SHWE for selected pesticides from fruit and vegetables (25), SFE for pesticides (26,27) and PAHs (28). The use of USE for pesticides (29,30), glycyrrhizic acid from liquorice root (31) and quinolones in pig muscle has been reported (32). Determination of contaminants in food and soil has been reviewed by Tadeo 

Complete extraction solutions are becoming more common and enable on-line cleanup; for example, PLE systems linked to a solid phase extraction (SPE) cartridge or disk to allow for selective clean-up and enrichment directly following extraction.  

For solid samples, following the extraction step, the extracts can then be treated as liquid samples and further extraction/cleanup procedures can be used prior to analysis.  

Extraction Techniques: Liquid Samples or Solid Sample Extracts 

For liquid samples, or solutions obtained following an initial extraction, analytes can be partitioned directly into an immiscible extracting phase. For non-homogeneous samples this can be preceded by a dissolution or homogenization step and possibly filtration.  

Direct partitioning liquid-liquid extraction (LLE) enables an analyte to be selected based on its solubility or affinity for the immiscible extracting phase compared to the sample matrix. Traditional methods use a separating funnel, and after mixing the phases are allowed to separate and the phase containing the analyte is retained (and invariably concentrated). The selectivity of the technique is through choice of extraction solvent. To improve efficiency of extraction, matrix modification such as addition of salt or change in pH can be used and, if necessary, centrifugation to ensure a distinct phase barrier is achieved.  

As the extraction solvent can be chosen to suit the analyte properties, direct liquid extraction can be used for the majority of food contaminants, although with the complexity of some food matrices, the challenge can be the preferential extraction of analytes versus unwanted matrix components. For trace contaminants in foods a further concentration step and possibly cleanup (such as solid phase extraction) is generally required and this is one of the major disadvantages of traditional liquid-liquid extraction. Where a food contaminant is unknown, for example, in the case of taint analysis or investigations into customer complaints or suspected adulteration, isolation from food matrix components can be a challenge. For this analysis and also for multi-class or multi-residue methods, a compromise in the choice of solvent is generally made to give optimum extraction for the most number of compounds. 

To enable analytes to be exhaustively extracted from the samples, multiple extractions are often required and therefore large volumes of solvents may be needed. As a result miniaturized liquid extraction techniques have now been developed. 

Liquid-phase microextraction (LPME) involves the partition of analytes between the bulk aqueous phase and a very small volume of extraction solvent. In single-drop microextraction (SDME), a single drop of organic solvent is suspended at the tip of a needle in the sample solution. This extraction can be performed in static or dynamic mode, or the drop can be protected by a hollow fibre. This technique can also be used for headspace analysis to enhance selectivity for volatile analytes (34). The developments and applications of SDME were the subject of a review in 2010 (35). 

The choice of extraction solvent is limited because it has to have sufficient viscosity to avoid it dropping off the needle, and in headspace mode the solvent must have a boiling point high enough to avoid evaporation. The use of an IS is recommended for accurate quantitation. There are only a few reported methods that have been developed for food contaminant analysis, including ochratoxin in wine (36), herbicides in milk (37) and organophosphorus pesticides in orange juice (38). A related approach known as dispersive liquid-liquid microextraction (DLLME), mainly used for analysis of water, uses a mixture of two organic solvents (extractant and dispersant). 

This technique has recently been reported for multiresidue determination in fruit juices (39) and fungicides in wine (40). It has also been combined with USE for extraction of pesticides from tomatoes (41). 

Continuous Liquid Extraction/Distillation (SDE and SAFE)

Examples of a wide variety of sample preparation techniques for the analysis of trace-level contaminants in food and the advantages and disadvantages of each are discussed in this article.

Sample Preparation for Food Contaminant Analysis

 in Anaheim, California, USA. This year's conference continues the tradition of visiting celebrated cities. Although the HPLC Symposium has previously visited the West Coast of the United States, it has never been held in Southern California. Thus, 

 Anaheim marks the symposium's Southern California debut. While some may consider this city a suburb of Los Angeles, Anaheim is an easy-to-reach world-class destination in its own right, welcoming 40 million visitors each year. It is well known for its theme parks, two professional sports teams and proximity to world-famous Southern California beaches. The conference venue is conveniently located in the heart of the Anaheim resort district, offering excellent meeting and exhibition space together with unrivaled amenities and services.  

 conference provides a superb venue for learning the latest advances in liquid phase separations, interacting with colleagues and renewing old friendships. The conference features plenary and keynote lectures, short courses and workshops to get the attendees acquainted with current separation techniques and technologies. Among the many topics to be discussed are sessions on the newest developments in chromatographic column technologies including monolithic columns, small and core-shell particles, fundamentals of chromatographic separation methods, high temperature, high-speed and high-resolution separations, HPLC in systems biology, microfluidic and nanofluidic systems, as well as new instrumentation and detection schemes. 

Applications in pharmaceutical, biopharmaceutical, environmental and food safety analysis in life sciences and polymer characterization will also be featured. The wide-ranging vendor exhibition will include a plethora of companies showing their latest innovations in instrumentation for chromatographic separation and related research. Conference proceedings will be published in regular issues of the 

 and also collected for an internet-only special issue. All presenters at the symposium are invited to contribute.  

The multiplicity of components at the HPLC symposia make it a valuable resource for analytical chemists, biochemists, molecular biologists, as well as those interested in the separation sciences seeking practical solutions to their problems. In addition to the extensive technical programme full of lectures and poster presentations, the symposium addresses the diverse challenges faced by today's scientists in half- and full-day short courses. These courses are designed for attendees to gain an insight into the latest advances in liquid phase separations – and this year one short course is bringing it back to basics by advising how to prepare perfect manuscripts, attractive PowerPoint presentations and award-winning posters. The tutorial sessions and educational lectures are popular among students, postdocs and newcomers and provide opportunities to broaden your understanding of specific elements of chromatographic science and technology. A novelty introduced in this year's programme is a panel discussion entitled 

What are Today's Grand Challenges in Chromatography?

The social programme is always a favourite among attendees. This year it includes opening and closing receptions, and the symposium dinner at the Disney California Adventure Park and World of Colour. 

Before or after the conference there are many excellent choices to extend the visit in the city or in the nearby scenic areas. 

For more detailed information, visit our website at 

 Dubai International Convention and Exhibition Centre's Sheikh Saeed Halls, Dubai, UAE 

14th International Trade Fair of Analytical, Measurement and Control Technology EuroLab 2012

 MT Polska Centre, 95 Makowska St, Warsaw, Poland 

 New Munich Trade Fair Centre, Munich, Germany 

 Silesian School of Management, Katowice, Poland 

Institute of Environmental Engineering of Polish Academy of Science 

36th International Symposium on Capillary Chromatography (Riva 2012) and 9th GC

Luigi Mondello, Chromaleont, University of Messina, Italy 

Departments

Event News

In gas chromatography selectivity is commonly thought to be about exclusivity of the column stationary phases and sometimes extended to the detector. This instalment of "Column Watch" explains how to incorporate selectivity throughout the sample analysis cycle, from the sampling analysis cycle through data analysis and report generation.

Many samples to be analysed using gas chromatography (GC) are complex. There may be many analytes of interest to be separated, possibly at very low concentrations. Some of the analytes may be difficult compounds, meaning they may be thermally labile, active or of too high molecular weight for analysis by GC. There also may be a high loading of matrix and the presence of matrix interferents. From sample collection to data analysis, there are many steps in the process. Of course, the final step is the reporting of the required information about a sample. To get reproducible and accurate results, it is important to think about and understand the sample first, and then to identify the steps to select the best techniques for the analysis. 

In chromatography, when selectivity or being selective is mentioned, first thoughts are always directed toward the analytical column's stationary phase, especially in GC where this parameter has the biggest effect on the separation of analytes. In GC, the mobile phase, a gas, has little interaction with analytes, the matrix or the stationary phase, and mainly serves to transport compounds down the column. However, each of the steps identified in the collection, analysis, and reporting of a sample can have selectivity applied to it. Figure 1 identifies some of these key steps. Although some of the steps involved in selectivity enhancement are often the same for gas and liquid chromatography, I will focus this discussion on considerations in GC.  

Figure 1: Steps in sample collection, preparation and analysis. 

The first and most important step in any separation is to determine how to collect a representative sample, enough for the analysis. One must consider the nature of the sample (gas, liquid, solid or something in-between); the analytes (volatility, stability); and the location of the sample. It may be relatively easy to collect and return some samples to the laboratory for analysis, while in other cases, sample changes (such as oxidation, chemical breakdown or chemisorption) can occur during the transport or storage process. It may be the case that the analysis needs to be performed 

Selectivity can be considered in this first step — to selectively collect only the portion of the sample of interest with as little matrix as possible. Selectivity can be based on volatility, where only analytes within a certain volatility range are sampled, or on the nature of the analyte, where only target analytes with certain characteristics such as polarity are collected. In a case where there is a high level of matrix interferents, a technique may be selected to avoid these; for example, a reversed-phase solid-phase extraction (SPE) sorbent may be used to concentrate organics and to avoid trapping water. 

One technique for selectively sampling on site is thermal desorption; the thermal desorption tube packing material is selected to retain the target analytes and not the matrix. Solid-phase microextraction (SPME) and stir-bar sorptive extraction (SBSE) are other techniques where the phase can be selected for the analytes and not the matrix, and can be used on site. 

The aims of sample preparation can be to make the analytes of interest amenable to GC; to concentrate the analytes; and to remove matrix (if possible). Selectivity can also be considered at this step, to extract only the analytes and not the matrix. Again, selectivity can be based on volatility or the nature of the analytes.  

For example, when using SPE, analysts can choose a phase that is selective for either the analytes or the matrix, by thinking about the chemistry of the analytes and selecting a sorbent stationary phase that provides the best interaction to enable extraction of the analytes from the sample. Likewise, in SPME, SBSE and liquid–liquid extraction (LLE), the solvent can be selective. Techniques like headspace and purge-and-trap sampling use volatility to selectively extract the analytes from the sample. Whereas parameters like temperature are important to ensure the best extraction efficiency of target analytes, their effect on the coextraction of matrix components must also be considered. With dynamic headspace and purge-and-trap sampling, the selectivity of the trap can also be chosen to reduce the trapping of matrix coextractives.  

Unfortunately, during sample preparation there is often the potential for the loss of analytes and the concentration of unwanted matrix. Given that there are many steps in the analysis of the sample where we can be selective for one over the other, it is important not to lose analytes at this step for the purpose of removing matrix; the latter can be examined again later on in the process. One important factor to consider in sample preparation is that the more individual steps used to isolate or concentrate the analytes of interest, the bigger the chance for analyte loss.  

Although not as popular as it once was, chemical derivatization of analytes (or perhaps the matrix) can be used to achieve additional selectivity. By adding new functional groups or eliminating "old" functional groups, chromatographic selectivity changes can be dramatic and may sometimes allow analytes to be completely resolved from interferences. Derivatization can enhance selectivity by introducing functional groups that allow the use of a selective detector (as discussed later). In addition, derivatization can be used to enhance volatility such that nonvolatile high-molecular-weight compounds that ordinarily couldn't be passed through a GC column can be separated by GC. The downside to analyte derivatization is that it adds an extra step, sometimes lengthy, to the overall analysis. 

Steps 1–3 may be automated or manual, but with step 4 we are now entering the instrumentation, and most modern GC instruments utilize automation exclusively. 

The aim of sample introduction is to repeatably and reproducibly introduce a representative portion of the sample onto the analytical column in a narrow sample band with no chemical change. Usually, the analytes are dissolved into a liquid solvent that requires evaporation in the GC inlet; this liquid expands into a large vapour volume that then must be squeezed into a narrow column. Analytes already in the vapour phase have to be quickly flushed onto the column to ensure a narrow sample band and hence produce sharp peaks. Sample introduction is often the trickiest part of the process, with problems like mass discrimination, activity and breakdown frequently occurring in the inlet.  

However, discrimination doesn't have to be all bad; selective discrimination based on Grob-type principles uses the volatility of the sample components to selectively transfer analytes into the analytical column from the GC inlet. There are two main areas where this principle can be applied. At the front-end of the chromatogram, it can be used to eliminate major components. An example of this is large volume injection (LVI), where the solvent is evaporated out of the split exit before it is closed and the concentrated analytes are transferred onto the column. The principle can also can be applied to the back-end of the chromatogram, to leave nonvolatile material within the inlet liner, protecting both the column and detector as well as reducing run times. 

Frequently, the temperature of a hot GC inlet is not optimized, leading to unnecessary problems for sample analysis results and maintenance of the instrument. When selecting the inlet temperature, the default seems to be 250 °C, which may be too hot or too cold for certain analyses. Frequently, a temperature is chosen that is too high, because the analyst considered the boiling points of the components rather than thinking about their volatility. Figure 2 shows a plot of the literature boiling point of a hydrocarbon mixture against the effective boiling point — the temperature at which 100% of the analyte is vapourized and transferred onto the column. To vapourize the analytes, the inlet temperature is important, but so is the flow through the liner, which is dependent on column flow and split flow. As Figure 2 shows, the inlet temperature for this particular inlet type, an Atas Optic 2 (Atas GL, Eindhoven, The Netherlands) and liner flow, only needs to be a maximum of half of the boiling point for total transfer of the analyte onto the column.  

Figure 2: Literature boiling point vs. effective boiling point. 

Figure 3 shows an example, for a hydrocarbon mixture, of profiling the inlet temperature required to transfer components out of the inlet while retaining others within the inlet liner. For example, at an inlet temperature of only 80 °C, the C18 hydrocarbon and those above are retained within the inlet liner, while 80% of C16 is vapourized and swept out and 100% of C14 and below are vapourized. To vapourize 100% of C18, an inlet temperature of 130 °C is required, but this is much lower than its boiling point of around 320 °C.  

Figure 3: C14âC36 hydrocarbon vaporization efficiency plotted as a function of temperature. 

As mentioned, this information is very useful when considering selectivity within the sample introduction step of the analysis. Here, selectivity is based on the volatility of the analytes; the control of the inlet temperature can be used to selectively transfer or exclude sample components from the analytical column. Figure 4 shows the discrimination envelope, where again for this inlet the temperature can be selected to either transfer an analyte or retain it within the inlet liner, with knowledge of the literature boiling point.  

Figure 4: Temperature discrimination envelope: upper curve = the total transfer of analyte; lower curve = total exclusion of analyte. 

For example, the least volatile analyte of interest in a sample has a literature boiling point of around 350 °C, which means the inlet temperature must be at least 140 °C (upper curve) for total transfer from the inlet onto the column. However, a large matrix peak with a literature boiling point of around 430 °C is preferred to be retained within the liner rather than being transferred onto the column, where it will potentially damage the stationary phase, dirty the detector and lengthen the run time to elute it from the column. An inlet temperature of 170 °C (lower curve) or higher would mean transfer of this component onto the column. Therefore, an inlet temperature of at least 140 °C would mean all of the compounds of interest would be transferred to the column, but the higher the inlet temperature, especially around 170 °C, would also mean transfer of less-volatile matrix components, an undesirable result. 

Selectivity within the inlet can also be used for the concentration of analytes with LVI. The volume of solvent injected into a GC inlet is limited by its vapour volume; if the volume is too large, the capacity of the liner can be exceeded, causing contamination of the inlet, septum purge, split and even the carrier gas inlet lines. Transferring too large a volume of solvent onto the column can result in distorted peaks because of the long solvent band. An LVI approach with a programmable temperature vapourizer (PTV) enables the evaporation of the majority of the solvent before the analytes are vapourized and transferred onto the column. The inlet temperature is chosen to enable the evaporation of the solvent while retaining the analytes within the inlet liner; usually this is 30 °C below the boiling point of the solvent. The discrimination envelope can be used here, but this time in reverse. The solvent boiling point is put into the upper curve for total transfer out of the split exit, although a slightly lower temperature is preferable to increase the solvent evaporation time so that it is more controlled. The boiling point of the most volatile analyte of interest is put into the lower curve. This temperature shouldn't be exceeded; otherwise loss out of the split exit can occur. This procedure can be used to eliminate any major component at the front end of the chromatogram, not just solvents. 

Figure 5 shows the power of selectivity within the GC inlet to selectively take a volatility cut of a petrochemical sample and transfer it onto the column for separation while flushing the unwanted parts out of the split exit. The upper trace (Figure 5a) shows the entire sample transferred and separated on the column. If the less-volatile components were not of interest, as in the example above, up to 100% of C20 can be transferred and separated on the column, as shown in Figure 5b, which shows the front end of the chromatogram. If only the less volatile components were of interest, only 100% of C40 and above can be transferred to the column, as shown in Figure 5c. Figure 5d shows a volatility cut, where only 100% of C30 is transferred with the minimum of more- and less-volatile analytes. In all of these traces, the unwanted components are flushed out of the split exit rather than being retained in the liner. Hence, traces of unwanted components are seen, the amount depending on the split ratio. Figure 5b can be achieved with any GC inlet type, selecting the correct inlet temperature to get 100% transfer of all analytes of interest; retaining unwanted less-volatile matrix components in the inlet liner is important for reproducibility of the target compounds while minimizing column and detector maintenance and GC run times. This approach also has the advantage of vapourizing the sample at the lowest possible temperature, hence causing fewer problems for thermally labile analytes. Figures 5c and 5d can only be achieved in cases where the inlet temperature can be varied during the sample introduction, with inlets like PTVs.  

Figure 5: GC analysis of a petrochemical sample: (a) entire sample transferred and separated; (b) transfer of only 100% of C20 and earlier; (c) transfer of only 100% of C40 and later; (d) transfer of only 100% of C30. 

The separation of analytes and any transferred matrix on the analytical column is the main area where selectivity is considered in the GC analysis of a sample. By thinking about selectivity in steps 1–4, and finding ways to collect, extract and transfer only the analytes of interest and not matrix, we can have fewer peaks to separate in the fifth step. In addition, less instrument and column maintenance may be required.  

When selecting the GC column stationary phase, phrases such as "like separates like" or "polar separates polar" are useful. Considering the chemical composition of the whole sample — both analytes and matrix — is important to separate them all from each other. Volatility as well as the functional groups present must also be considered when choosing the stationary phase, because very polar columns are often not able to reach the temperatures required to elute high-molecular-weight analytes. Stationary phase film thickness, column length and internal diameter should be just enough to separate the components while minimizing band broadening and run times. 

Sometimes, however, even if an analyst is very selective in the choice of analytical column as well as in steps 1–4, there just aren't enough theoretical plates for acceptable separation of the large number of analytes and matrix peaks transferred onto the column. Or, the components of the sample might have mixed characteristics in terms of polarity to make selection of the proper stationary phase difficult, resulting in good separation of some compound classes but not others. Introducing further selectivity approaches can help with these problems.  

 Heart-cutting transfers a "cut" of the remaining coeluted peaks from the separation on one stationary phase onto a secondary column containing a different, more selective stationary phase. Several cuts can be performed across a chromatogram. 

Comprehensive two-dimensional gas chromatography (GC

 This method transfers many smaller cuts, usually 1–10 s long, onto a second column with a different stationary phase throughout the whole analysis; therefore every peak is separated on two different stationary phases. This separation enhancement is achieved by having the separation on the second column take no longer than the cut from the first column; for example, if the cuts from the first column are 4 s long, the separation on the second column takes no longer than 4 s. 

 Another method of introducing more selectivity is to hyphenate a different chromatographic technique to GC. For example, the sample might be first analysed by liquid chromatography (LC), where selectivity in the mobile and stationary phases are very different, followed by analysis using GC. This is sometimes referred to as LC–GC. 

There are more than 20 detectors for GC on the market, many using quite different chemical or physical principles. When choosing a detector for a GC method, the aim is to select one that has adequate sensitivity with a linear response over the concentration range, and is stable, reproducible and gives the required information to confirm the presence or absence of the target analytes, along with a response for quantitation if required. By introducing selectivity in this detection step, we can improve sensitivity or make it much easier to observe the target analyte among coeluting analytes and matrix components. 

Certain detectors respond only to certain compound classes; for example, an electron-capture detector (ECD) only responds to analytes capable of capturing an electron. Other detectors only respond to certain atoms; for example, a sulfur chemiluminescence detector is used for sulfur-containing molecules, and a nitrogen–phosphorus detector is used for nitrogen- or phosphorus-containing molecules. A mass selective detector adds another dimension of information and selectivity by providing a full mass spectrum, where a single ion or ions can be chosen that are unique to that molecule but not the coeluted peaks; the accurate mass of a molecule; or an MS–MS product ion spectrum. A detailed discussion of GC detectors is beyond the scope of this paper. It suffices to say that detection selectivity can be another addition to our "spectrum" of selectivity enhancements. 

Unfortunately, data analysis sometimes takes just as long if not longer than the whole sample collection, preparation and analysis. Properly understanding the problem and the questions to be answered is the starting point. Then, introducing selectivity at each of the steps discussed above to obtain high quality data showing the analytes of interest with as little interference as possible helps reduce the data analysis time as well as improve the quality of the results. Unfortunately, even after carefully optimizing each of the previous steps to be as selective as possible, there can still be some coelutions between analytes and matrix that cause problems, especially if very low-concentration analytes are being analyzed in dirty or complex samples.  

If the sample has been analyzed with a detector that generates another dimension of data, more selectivity can be introduced at this step, too. With full scan data from a mass spectrometer, ions can be selected for qualitative and quantitative analysis that are unique to that analyte, which means the ions are not present in coeluted peaks, ion ratios show little variation and integration is much easier baseline-to-baseline, reducing manual data analysis time. 

Failing that, advanced techniques like deconvolution automatically unknit coeluted mass spectra for confirmation or identification of an analyte. The deconvoluted ions can then be used for accurate quantitation. Statistical analyses of the data using chemometric techniques like principal component analysis (PCA) are also helpful when analysing complex data sets. PCA can assist with method development, the identification of outliers, determining patterns of association and variability, applying confidence intervals to determine the reproducibility of data and determining mass spectral match coefficients. Even simple analyses can benefit from this automated approach. 

Yet however good these advanced data analysis techniques are, they will always work best on high quality data, generated from a fully optimized sample collection, preparation and analysis method where the techniques are selected to fit the analysis. 

Like all parts of a method, optimization of the data analysis step takes time but is crucial to getting accurate results with as little manual input as possible, resulting in less data analysis time overall. 

Before determining which techniques to use at each step of the analysis, here are some factors that should be considered:  

The sample phase. Is it a gas, a liquid, a solid or something in between?

The analytes, including their volatility, nature and concentration. Are very low detection limits needed? Are they thermally labile, active or high-molecular-weight compounds?

The matrix (volatility and nature). What proportion of the total sample is the matrix?

The sample location. Can a representative portion be taken back to the laboratory easily without the sample changing? Is sampling 

 needed? Does the analysis need to be performed on site?

Column Watch

This article looks at selectivity throughout the whole sample analysis cycle from the sampling stage, through sample preparation, sample introduction, analyte separation, analyte detection, data analysis and report generation.

LCGC Europe-02-01-2012

Connecting with Chromatography at Pittcon 2012

A Case of Sporadic LC Assay Results

Sample Preparation for Food Contaminant Analysis

Event News

It's All About Selectivity

How Raw Are Your Data — 2012?