This month we complete a list of questions about gases that began in a previous instalment (1).

The range of questions about gases in gas chromatography (GC) is wide and complex. Even barring direct questions about hydrogen as a carrier gas, my attempts to address at least most of the core questions has already taken two instalments of 

. I hope this month's additional topics will suffice, but I also encourage readers to correspond with any other questions they may have on these or any other GC-related subjects. 

More than one reader commented that in the previous column (1), no explicit precaution was given regarding the use of polyfluorocarbon tape or sealant with swaged fittings. The use of any tape or sealant is clearly proscribed for swaged fittings as well as for cylinder compression fittings. A better statement would be that polyfluorocarbon tape specifically sold for high-purity gas distribution is the only type of tube or fitting sealant that can be used for gas chromatography, and the only place that it is appropriate for use is on pipe-thread fittings where it functions as a sealant and prevents gas from flowing around the pipe threads themselves. This applies not only to ¼- or ⅛-in. (6- or 3-mm) tubing and associated fittings but also to larger sizes such as sometimes found in manifolded gas distribution systems (see below for more). Thanks to those readers for helping to clarify this issue. 

More Questions That Should Be Asked Frequently 

Here are the remaining questions on my list regarding gases and gas delivery for GC. As before, these questions do not address anything about the gases after they reach the GC system. Therein lie even more questions that also should be asked frequently. 

What Types of Filters Should Gas Chromatographers Use?: 

Recommendations for filter types vary for different instrument manufacturers as well as for different consumables suppliers and producers. Filter requirements are driven by the types of injection, column and detection technology in use. In mixed situations, higher purity requirements override less stringent ones, so always use a filtering scheme that is appropriate for the component that requires the highest purity level. Table 1 is an expanded version of a table included in the previous instalment (1) that gives a filter and gas selection matrix for commonly used inlets, columns and detectors. Be sure to choose filters that are rated for the desired gas purity level and devices in use.  

Table 1: Filter and gas selection matrix.			

For example, if using a split–splitless inlet with a wide-bore porous-layer open-tubular (PLOT) column and thermal conductivity detection (TCD), the TCD makeup gas — which has a less strict purity requirement — would be subsumed to the carrier gas because of its higher purity specification. A separate makeup gas supply is not required with TCD; makeup gas can be pulled from the higher-purity carrier-gas supply. An electron-capture detector with a split–splitless inlet, capillary columns, and hydrogen carrier gas would require at least 99.9995% carrier gas and multiple high-purity carrier-gas filters, plus a separate supply of 99.9995% impurity-free nitrogen or argon plus 5% methane ionization gas with its own set of high-purity gas filters.  

In general, most chromatographers will simply install the highest level of filtration and use the highest gas purities that apply to all in-use or anticipated combinations of inlet, column and detector on a particular instrument population. 

When Should I Change or Replace My Gas Filters?: 

Gas filters for GC either include an end-of-life indicator or they are specified to purify a set gas volume. The indicating types are more efficient, in the sense that they don't need to be changed until they indicate the approach of exhaustion. A non-indicating filter that expires after a number of tanks of gas must be changed at that point, whether truly exhausted or not. A non-indicating filter also creates the extra burden of noting for each filter how many tanks of gas have gone through it — assuming, of course, that it will purify more than one tank. Non-indicating filters will usually include a specification for just how contaminated the incoming gas can be to meet the filter lifetime specification. However, feeding less-contaminated gas — as printed on the gas tank analysis — cannot be assumed to yield a longer filter lifetime. The filter is supposed to remove extra contamination that may be entrained into the gas and not improve on the purity of the gas that is in the tank. 

How Do I Leak-Check the Gas Delivery System?: 

I use three methods to check a gas delivery system for leaks. First, determine if there are any gross leaks by performing a pressure-drop test. Pressurize the gas lines and operate at normal flows at the instrument for a few minutes. Then turn off the flows at the instrument and mark the position of the high-pressure gauge needle by attaching a sticky-note to the gauge face with its edge aligned to the needle. Next, turn off the high-pressure tank valve. The high-pressure gauge should remain steady. If it does not then there is a serious leak somewhere. If the gauge appears steady, then wait 5 min. Now, while watching the high-pressure gauge needle in relation to the sticky note, turn the high-pressure valve back on. The gauge needle should remain steady. If it moves upward slightly then there is a low-level leak somewhere. 

Even if no apparent leak is indicated by the pressure-drop gauge test, use an electronic leak detector as a second way to verify critical gas lines such as carrier or make-up gas, as well as any hydrogen lines. The pressure-drop test will usually suffice for air lines, as an electronic leak detector does not work for air. If a leak is indicated for an air line, use a few drops of pure liquid water — not soap solution — to try and find it. If you are really concerned, it is possible to pressure the line with helium for the purposes of a leak-check with the electronic meter. 

Should I Use Gas Tanks or a Gas Generator?:

 This question is closely related to the adoption of hydrogen carrier gas. So many have asked about it, however, that I'm including some responses here.  

Gas generators provide a high level of convenience over cylinders, but at a higher initial cost; they are an excellent way to replace gas tanks in laboratories that consume them frequently. How often is "frequently"? That depends on the trade-offs between the cost of the individual tanks taken against the cost and maintenance of a gas generator over its useful lifetime. The advantages of gas generators also depend strongly on the real safety and convenience advantages gained from having fewer heavy, high-pressure cylinders to manage. In some situations this advantage alone makes changing to gas generators an easy decision. The following examples are based on full retail prices for gas generators taken from one chromatography supplier's website in November 2012, and while the numbers are certainly representative, it would be crucial to work with a supplier to arrive at realistic figures for a specific laboratory. 

Suppose a 49-L tank of high-purity helium costs $400 in quantity under a supplier contract — if you can find some, of course. A GC carrier-grade hydrogen generator with 500-mL/min capacity might cost $10,000–$15,000. Each full helium cylinder of this size contains around 8 m

  of gas, which would last for about 11 days at a 500-mL/min delivery rate to two or three GC systems with split–splitless inlets. In one year, this laboratory would consume around 32 cylinders of carrier gas for these GC systems, at a cost of $12,800, plus any extra demurrage for keeping spare cylinders on-hand. A more accurate return on investment (ROI) calculation can be obtained by considering the cost of the funds to purchase the generator and the tax advantages its depreciation would produce, but it's clear from the above quick estimate that the hydrogen gas generator saves its cost in helium cylinders within a year or so. Of course, this assumes that the laboratory can replace expensive helium carrier gas with hydrogen within the scope of their operating procedures and methodology. There is a cost of changing to hydrogen because of translating and validating methods, but in most cases this is not too difficult. The good news is that lots of support is available for this one-time activity. 

What about a zero-grade air generator — really an air purifier — for flame ionization detectors? This device actually consists of two components: a zero-air purifier and an air compressor to supply it. Together, they might cost around $4000 for a 1-L/min system that could supply two flame ionization detectors. Zero-grade air tanks might cost about $75 a piece, and such a tank would last for about a week at 800 mL/min. So, in a year the cost of the 52 air tanks consumed would be around $3900. Again, ignoring the finer points of an ROI calculation, it is clear that replacing air tanks with an air generator for flame ionization detection (FID) consumption would pay for itself in about one year.  

Gas generators do have recurring costs for filter replacements, but this is minor compared to the savings that will result over a 5-year service period for split–splitless carrier gas and FID air generators, not to mention the labour savings and safety gains from not having to haul nearly 100 tanks into and out of the laboratory in a years' time. 

When might it not be a good idea to install a gas generator? Consider replacing only the hydrogen tanks used for FID while not switching to hydrogen carrier gas. Around 45 mL/min hydrogen is needed for each flame ionization detector, so the time for a single detector to consume a full hydrogen tank would be about 125 days: that's just three cylinders per year. A hydrogen cylinder for FID use is less than $100, or about $300 per year per detector. By comparison, a smaller capacity 165-mL/min hydrogen generator, which is suitable for three flame ionization detectors, costs about $8000. The three detectors would consume a total of nine cylinders in a year, at around $900 cost, so the ROI period is something on the order of 10 years. 

Clearly, the payback on an FID hydrogen-only configuration is not as attractive as the initial gain when moving directly to hydrogen carrier-gas generation from helium cylinders. What about going to hydrogen but without a generator — on cylinders only? Hydrogen cylinders to replace the 32 carrier-gas cylinders in the first example above would cost around $3200, so the net gain in a year compared to using helium tanks would be about $9600. That's close to the cost of a hydrogen generator, so it makes good sense to put the first year and some month's of this saving directly into a hydrogen generator, and save the intermediate transition from helium to hydrogen cylinders for an initial test exercise in method translation, if there are any questions about the switch over. 

What About Tanks Kept Outdoors for Gas Distribution Into the Lab?:

 Many laboratories use an outdoor shed to hold both in-use and spare gas tanks. This is an excellent solution to concerns over housing compressed gas tanks in the laboratory, and this arrangement liberates some laboratory space otherwise occupied by numerous tanks. However, such a setup comes with extra requirements. 

First of all, outdoor tanks are subject to extreme temperature swings both daily and as seasons change. Daily temperature fluctuations can drive the internal tank pressure up and down by about 5–7%, while pressure changes could swing as much as two to three times more annually in areas that experience large seasonal temperature changes.  

The combination of widely fluctuating tank pressure and temperature affects pressure regulator output levels. Data from outdoor single-stage regulators with outlet pressure monitors that experienced ambient temperatures from -15 °C to +40 °C over the course of six months showed an inverse outlet pressure variation from 86 psig (593 kPa) to 78 psig (538 kPa) in one case, about ±5%. A similar effect was noted for all the observed regulators (2). 

A tank heating blanket is another solution for temperature fluctuations at the tank, but usually this is used only on gas-mixture tanks in which a component might liquefy or stratify at cold temperatures. Dual-stage regulators help maintain a more constant output pressure, too, but tank heaters or dual-stage regulators are not necessary for outdoor GC gas tanks, as explained below. 

Tank regulators and associated valves and fittings outdoors can be subject to high levels of dust, moisture and chemical pollution. Brass regulators and conventional pressure gauges — even with nickel flashing on the outer surfaces — can undergo significant degradation in aggressive environments such as near sea water, in a desert location or in proximity to a chemical plant. Unsealed regulators and gauges are subject to dust and insect intrusion. Any of these can cause the regulator mechanisms to exhibit bias, stick or even fail. 

Beyond regular inspection and maintenance operations to keep regulators in good operating condition, I have encountered a few solutions to the above environmental problems. Installation of outdoor-rated stainless steel regulators and sealed stainless steel gauges is a simple solution, but such devices can be difficult to acquire. A sealed regulator enclosure, with a short flexible high-pressure hose that connects to the gas tank, effectively isolates the regulator from long-term exposure to environmental pollutants. Enclosures specifically designed for this service will have an external safety vent in addition to the inlet and outlet connections. I have also seen sites where the tanks and regulators were located in an enclosed heated gas shed. It is also possible to route high-pressure hoses and piping through outdoor walls to a protected location, but this arrangement requires special materials and skills to properly contain the associated high pressures coming directly from the gas tanks. 

The pressure fluctuations in the output of a single-stage outdoors pressure regulator are large enough to cause some supply pressure problems at the gas chromatograph: Why not use a dual-stage regulator? A better solution consists of two single-stage regulators, with one at the tank (outdoors) followed by in-line regulators between the incoming gas line and the backs of the instruments. Set the tank regulator to 150–200 psig (1–1.4 MPa) and set the in-line regulators to deliver 80–100 psig (500–700 kPa) or as required by the instruments.  

One feature that is invaluable in outdoor installations is a lock-nut knob-free arrangement for the outlet pressure settings on all regulators. This prevents the inevitable knob-twiddling from no doubt well-intentioned persons. I personally witnessed one such situation where the twiddler said, "Hey, more pressure is better, right?" Another time, someone turned the pressure knob all the way clockwise in a mistaken attempt to turn it off; the result was a broken regulator. 

New, cleaned ¼-in. (6-mm ) o.d. copper tubing is a good choice for plumbing between the tank area and the laboratory, if the tubing will not be flexed or disturbed much during its service lifetime. (The metric diameters given here are the commonly available sizes, not the exact equivalent of the fractional sizes.) Larger diameter ⅜-inch (10-mm) tubing is appropriate for runs longer than 50 ft (15 m). Stainless steel tubing is better, but it is also more expensive and difficult to cut and bend. For either tubing material, it's a good idea to connect 6-ft (2-m) lengths of convoluted stainless steel–core hose with a stainless braided outer covering between the tank regulators and the start of the tubing that runs to the laboratory. The flexible links will prevent repeated stress and eventual failure of rigid tubing because of normal tank changes when connected directly to the pressure regulators.  

Remember to include a valve and purge-tee arrangement close to the regulator. The purge tee will speed up tank changes by accommodating purging of only the pressure regulator and short tubing in front of the tee. Everyone purges the regulator upon tank changes, don't they? And yes, this is another knob for the twiddlers. Install a lock-out style valve with a padlock if you are seriously concerned. 

For high-flow gases such as carrier gas with split injection or air for flame-type detectors, where the total gas flow delivery is greater than a few litres per minute, consider using a tank regulator with a higher flow rating and perhaps even two tanks with an automatic or manual switch over manifold. 

Include a pressure gauge, shut-off valve and another purge-tee arrangement where the incoming gas supply tubing enters the laboratory from the outdoor tanks; this will facilitate purging the long tubing runs from the tanks when necessary. Connect the incoming tubing to a series of tee fittings to distribute the gases to in-line regulators in parallel. Install suitable pressure gauges at the outlets of the in-line regulators if the connected gas chromatographs cannot display incoming gas-line pressures. Additional shut-off or isolation valves at each in-line regulator or, even better, at each instrument will make it easier to install or remove instruments when necessary. 

A single large in-line filter is unnecessary if the gas delivery system is well designed, clean and leak-tight. In fact, a low-grade high-capacity filter might introduce more contamination than it removes at first. However, be sure to include appropriate filters after each in-line regulator, or position individual filters or filter banks close to the back of each instrument. 

These two instalments have covered a range of issues about carrier and detector gases, gas distribution, gas generators, filter selection, regulators, tubing, fittings and connections. Chromatographers should pay careful attention to these areas when setting up or expanding a laboratory, when installing instruments, and of course during routine use. Readers are encouraged to send their comments and additional questions about these or any other GC-related topics to 

 is a senior research scientist at BPL Global Ltd., Oregon, USA, and is a member of 

 editorial advisory board. Direct correspondence about this column should be addressed to "GC Connections", 

, 4A Bridgegate Pavillion, Chester Business Park, Wrexham Road, Chester, CH4 9QH, UK, or email the editor-in-chief, Alasdair Matheson, at 

(2) J.V. Hinshaw, personal observations.  

Articles

GC Connections

In part 2 of this series, we complete a list of questions and answers about gases for GC.

Q&A Gases: Part 2

Pittcon will be held in Philadelphia for the first time this year. Philadelphia is the centre of one of the top life science clusters in the USA, bringing together the pharmaceutical, biotechnology, medical devices, R&D and a wide range of other supporting industries. 

 offers a unique opportunity to get a hands-on look at the latest innovations in analytical science and chromatographic technology from leading companies from over 30 countries. 

Pennsylvania Convention Center in Philadelphia, Pennsylvania

Approximately 50% of exhibitors use Pittcon as the venue to launch new products and 20% of companies offer exclusive on-site discounts on equipment. Several specialty areas will be open on the floor including: New Exhibitor, Life Science, and Laboratory Information Management Systems (LIMS) areas and international pavilions for Japan and Germany. Attendees will get the chance to participate in live demonstrations and product seminars. Technical personnel will be available to talk about challenges faced by laboratory managers and offer solutions to improve efficiency. 

This dynamic exposition will be complimented by a diverse all-inclusive technical programme offering more than 2,000 technical presentations, as well as affordable short training courses and unique networking opportunities.  

As members of the scientific community from over 90 countries converge on Pittcon 2013, networking will become a primary activity among participants. The advance registration fee of $130 before 18 February ($260 after) gives conferees unlimited access to the exposition floor and technical programme.  

World-renowned speakers will present symposia, contributed and oral sessions, workshops, awards and posters covering a wide range of subjects. This year's programmme will include the Wallace H. Coulter Plenary lecture, 

"Exameter Objects to Nanometer Ones and Back Again,

" by Nobel Laureate, Sir Harry Kroto, Francis Eppes Professor of Chemistry at Florida State University and the Wallace H. Coulter Keynote lecture: 

"How The Higgs Boson Saved Us From A Cold, Dark, Lifeless Universe,"

 by R. Michael Barnett from Lawrence Berkeley National Laboratory & Coordinator ATLAS Experiment. Other highlights include: 					

ACS ANYL SCSC - Supercritical Fluid Chromatography

 by David Pinkston, Kellogg, and Larry T Taylor, Virgina Tech, will discuss how SFC (supercritical fluid chromatography) is finding a new place in trace analysis and validated methods.

Adaptation of Platform Analytical Technologies in Pharmaceutical Development 

organized by Michael W. Dong from Genentech, will outline the concepts, benefits and applications of platform technologies in pharmaceutical development.

Applications of Two-Dimensional High Performance Liquid Chromatography,

 organized by Dwight R. Stoll, Gustavus Adolphus College and Peter W. Carr from the University of Minnesota, will highlight recent work of leading experts in a range of applications, with updates on recent theoretical work. Participants in this session can expect to walk away with a good sense for the state-of-the-art of 2D LC, in terms of the scope of the technique, best practices and applications where an immediate impact can be realized.

Microfluidics for Detection of Circulating Tumour Cells, 

organized by Hugh Fan from the University of Florida, will present different perspectives from oncologists and technologists.

Advanced Mass Spectrometry for Food Safety and Cosmetics, 

organized by Perry G. Wang, US FDA, will focus on the application of advanced mass spectrometry techniques, such as infusion nano-electrospray high-resolution mass spectrometry, Orbitrap technique and tandem mass spectrometry (MS–MS) on food safety and cosmetics. A new sample preparation technique — "QuEChERS Sample Preparation Approach" for pesticides in foods will be introduced as well.

New Ionization Approaches in Mass Spectrometry: Molecular Imaging,

 organized by Sarah Trimpin, Wayne State University and Charles N. McEwen, University of the Sciences, will discuss new promising ionization approaches to molecular imaging. 

Structure Elucidation in Native Mass Spectrometry,

 organized by Evan R. Williams, University of California-Berkeley, will talk about new strategies to obtain structural information about macromolecules and macromolecule complexes, including chemical foot-printing, ion mobility spectrometry and tandem mass spectrometry and the application of these methods to interesting problems in structural biology.

Environmental Applications of High Resolution Mass Spectrometry, 

organized by Earl Michael Thurman, University of Colorado and Imma Ferrer, University of Colorado, will introduce the latest advances in mass spectrometry such as the limits of high resolution and accurate mass, as well as the latest advances in mass spectrometry instrumentation. Topics such as the study of pesticide degradates and pharmaceuticals in wastewater, fracking (removing natural gas from shales), the application of proteomics to environmental studies of contamination in water supplies, and natural pesticides from around the world will be addressed using Orbitrap and other high resolution methods. 

Every year, Pittcon recognizes and honours scientists who have made outstanding contributions to analytical chemistry and spectroscopy. This year, The Society for Electroanalytical Chemistry (SEAC) — 

 assistant professor of chemistry at the University of Washington, Washington, USA. Research in the Zhang group is focused on the understanding of fundamental and applied aspects of electrochemistry and bioanalytical chemistry using nanoscale materials. 

 for Analytical Science will be given to 

the Grace-Rupley Professor of Chemistry at Notre Dame University, Indiana, USA. The Royal Society of Chemistry has awarded the prize to Dovichi for pioneering development of ultrasensitive separations. Dovichi developed the first separations at zepto- and yoctomole levels and capillary electrophoresis-based DNA sequencing. 

The ACS Division Of Analytical Chemistry Award For 

Young Investigators in Separation Science 

, Shimadzu Distinguished Professor of Analytical Chemistry and Biochemistry at The University of Texas at Arlington, Texas, USA. Schug was selected for his outstanding contributions to separation science in the ten years since the completion of his PhD. He was previously awarded the international Emerging Leader in Chromatography Award sponsored by 

The Chromatography Forum Of The Delaware Valley 

, senior investigator of clinical investigation at the National Institute on Ageing, Maryland, USA. Wainer has published over 300 scientific papers, and has received a number of awards including the A.J.P. Martin Medal presented by the Chromatographic Society for contributions to the development of chromatographic science. Wainer's research focuses on the effect of the disease state on drug metabolism, the development of chromatographic techniques for drug discovery and the development of bioanalytical methods for the determination of drugs within biological matrices. 

For the recent graduate new to the field of chromatography, or for the seasoned professional interested in enhancing professional development, Pittcon offers an affordable Short Course Programme offering more than 50 courses in topics relevant to liquid and gas chromatography. These courses, taught by leading experts in their field, range from beginner to advanced levels, in half-, one- or two-day sessions. Some of the new titles for 2013 include 

Advances in Countercurrent Chromatography and Related Techniques; Gas Chromatography/Infrared Spectroscopy; Introduction to LC–MS for Chromatographers and Beginners; Advanced Topics in Capillary Gas Chromatography 

 Getting More from Your GC Supercritical Fluid Chromatography in the Pharmaceutical Industry.

will help iOS and Android device users organize schedules, technical sessions, short courses, search exhibitor listings and find local restaurants and entertainment. This free app is a valuable resource tool to use before, during, and after the event. In addition, 

 will be running a dedicated Twitter feed throughout the event keeping you up-to-date on highlights and news from the conference. Follow us: @LC_GC to keep up with the latest developments. 

 Pennsylvania Convention Center, Philadelphia, Pennsylvania, USA 			

Feature

A preview of what Pittcon will offer chromatographers in Philadelphia this year.

Pittcon 2013 – What's in Store for Chromatographers

This month's instalment surveys traditional and modern approaches for extracting and determining additives, contaminants and low-molecular-weight components that may be present in synthetic polymers or polymeric materials. After sample workup, components are typically analysed by chromatographic methods. This information is needed for product certification to comply with governmental safety and health regulations. 

High-purity polymers are typically required for special applications such as implants or prosthetics, pharmaceuticals, electronic devices and polymer reference standards. Most polymers of commercial interest, however, contain low-molecular-weight additives that are intentionally added to extend the material's serviceable lifetime or alter its properties and performance. There are also nonpolymeric compounds, oligomers and low-molecular-weight components found in the polymer that originate from the polymerization process itself. Lastly, polymeric material can become contaminated during production, handling, storage or transportation.   

Component levels and their identification are needed for product certification, meeting end-use performance criteria and for ensuring product stability and safety. (Please note that the term "component" is used throughout this survey as a general label for additives, residual solvents, unreacted monomers, oligomers, chain fragments, low-molecular-weight polymerization by-products or reaction side-products or contaminants.) Because extractables and contaminants may pose health risks, especially if the polymer is processed into film, packaging or parts that contact food, pharmaceuticals, viable organisms and biologicals, accurate and precise analytical data are required. 

To comply with governmental safety, health and transportation regulations, it is necessary for agencies and companies to monitor, control and certify product quality. This month's 

 instalment surveys approaches for extracting and concentrating polymer additives and side products that may be present in synthetic polymers or polymeric materials for subsequent chromatographic analysis. 

The sample preparation process is an important part of the overall analysis and is often the rate-determining and error-prone part of the analytical cycle. Nonporous solid materials such as polymers represent a special sample preparation challenge as many of the additives are located deep within the matrix itself and must be released in a quantitative manner. 

Sources of Components Potentially Present in Polymeric Materials

 Most commercial polymers and polymeric materials contain intentionally added components, many of which are small molecules. Additives are essentially used to extend the lifetime of materials by inhibiting polymer ageing and deterioration. Additives are also used to alter the chemical or physical properties of polymeric materials, whereas others act as processing aids. A list of their uses and functions is given in Table 1.  

Table 1: Categories of polymer additives and their functions*.			

Additives are usually applied so that they are uniformly distributed throughout the bulk of a polymer, especially at high processing temperatures. Processing aids that help to release formed parts from molds, and finishes applied to fibres to withstand high-speed spinning and winding, are examples of components that are applied to the surfaces of materials.   

If the solubility parameter, which is related to its polarity of an additive, is greater than the polymer's solubility parameter, additives will tend to "bloom" or migrate toward the polymer's surface with time, decreasing it's effectiveness, and causing some surface cloudiness. The degree of blooming depends on the additive's concentration, molecular weight, ambient temperature, the article's size and shape and the solubility and diffusion coefficient of the additive in the polymer. 

Components Formed During Polymerization: 

Polymers may also contain a host of other components left over or formed from the polymerization process, such as residual solvents and surfactants, unreacted monomers or comonomers, linear and cyclic oligomers, and decomposition products of catalysts, initiators or chain-transfer agents. After the polymerization, polar components may tend to migrate or bloom to the surface.  

It is important to realize that with certain types of polymerizations such as random or block copolymerizations, the polymeric material formed may be compositionally heterogeneous, containing polymers that have different chemical compositions or architectures such as branched and cross-linked structures, unreacted blocks or branches, or copolymers enriched in one or the other comonomer. Because of their relatively high molecular weights and unusual compositions, their separation and analyses are indeed challenging (and will not be covered in this column). To complicate matters, most polymers have a molecular weight distribution that can range from oligomers to high-molecular-weight polymers, depending on the polymerization mechanism.  

Polymers in any physical form can become contaminated during or after polymerization, processing or fabrication. Usually contamination is first realized by the appearance of an unknown peak during chromatographic analysis of a polymer extract. Finding the source of contamination and determining the contaminant's chemical structure, especially at low levels, are indeed daunting and time-consuming tasks. Furthermore, contamination can be quite expensive, especially because of cleanup and disposal. Contamination is usually a surface phenomenon unless it occurs during a polymerization or processing operation such as extrusion. 

Typical potential sources and occurrences of polymer contamination are as follows: 					

within the polymerization reactor, holding tanks, feed lines and associated process equipment if any unit was not thoroughly flushed during changeovers and in-between production runs,

during transportation from plant, fabricator, then to customer's site using trucks, tanks or railcars not properly cleaned before use, or 

from improper storage in opened or exposed containers. 

Because of the huge negative impact that arises from product contamination, it would be highly desirable for chemical and polymer manufacturers to consider setting up additional rapid-response high performance liquid chromatography (HPLC) or gas chromatography (GC) screening facilities at critical points within the plant and nearby loading docks. 

Particle Size Reduction of Polymeric Materials

Polymer samples submitted to the analytical laboratory can be of many different physical shapes and sizes. Physical forms can range from pellets, fibres and irregular pieces, to fabricated finished products — just about any form imaginable. Polymeric materials can be soft (for example, polybutadiene elastomers) to extremely hard (such as polycarbonate pellets) with varying degrees of porosity, surface area and other physical and chemical attributes. Before extraction and analysis, the polymeric material itself must be changed to a suitable form for further treatment. It is usually desirable to render solid samples into a finely divided state. 

Statistical methods of sampling must be used to obtain meaningful data. Furthermore, samples being compared should have the same average particle size and distribution. To ensure good laboratory practice and to obtain statistically reliable analytical data, it is desirable to comminute and combine samples that have the same production dates and lot numbers. This approach also helps to smooth over discordant data points. When finely divided samples are combined, sample uniformity and homogeneity are ensured, allowing more representative subsampling with greater precision and accuracy. As examined in greater detail in the next section, small-particle-size samples dissolve faster and have larger extraction recoveries because of their greater surface area. 

Popular laboratory methods for reducing the size of polymers are listed alphabetically in Table 2. Size reduction of a polymer should be carried out at low temperature, preferably well below its glass-transition temperature (

) to prevent softening and sticking of the sample. Many of the devices listed in Table 2 can keep the sample chamber cooled with liquid nitrogen, which is sufficiently cold to maintain the temperature below the 

 of most polymers while pulverizing. The low temperature will also keep samples brittle so that they can easily be pulverized. Since most particle-size reduction techniques will generate a significant quantity of heat, cooling the sample will also prevent possible degradation of additives or the polymer itself, which could lead to spurious results.  

Table 2: Recommended methods for reducing particle size of polymeric materials.			

The choice of procedure or instrument in Table 2 is dictated by a number of practical considerations: 					

noise level abatement needed for most of these instruments;

location availability of device, that is, analytical laboratory or plant site;

sample hardness or degree of softness that can be accommodated;

particle size range and size distribution that can be generated;

maximum size and volume of part that can be processed;

maximum number of samples that can be accommodated;

maximum throughput that can be accommodated;

continuous or batch-wise sample processing;

and sample contamination risk of a given instrument.

Most size-reduction techniques are performed in a dry state, but for certain polymers that tend to agglomerate during grinding, wet or slurry grinding should be considered. However, if a wet or slurry reduction is performed in water or another liquid, precautions must be taken to ensure that particle components are not extracted from the polymer matrix during wet grinding, and that no contamination occurs. 

Because all comminutive methods involve some sort of abrasion, sample contamination by abrasion particles should not be a concern, unless trace metals are being determined. Good laboratory practices must be strictly followed to prevent contamination of all areas exposed to the polymer sample. This can be done by thoroughly cleaning tubes and magnetic pulverizing devices after each use.  

Details on commercially available instrumentation for particle-size reduction are beyond the scope of this article. Nonetheless, readers are referred to references on the subject (3–5) as well as manufacturer's literature. Companies active in this area include Retsch Technology, Spex SamplePrep, Fritsch and Buehler, among others. 

Extracting Components from Polymer Matrices 

Removing and determining components of interest in polymers can often be quite demanding. In most instances, components must be separated or removed from the polymer matrix before being analysed to prevent polymer interference. There are three basic approaches to removing and measuring these components: 					

Traditional extraction method using a thermodynamically good solvent for the components and a thermodynamically poor solvent for the polymer; temperature is used as the main variable

Modern extraction method as in the first approach, but using pressure as an added variable

Complete dissolution of the polymer, thereby releasing components; choice of solvent and temperature are variables here.

Tables 3 and 4 provide an overview of traditional and modern techniques, respectively, for component extraction from polymers. The next sections discuss some of these techniques in more detail.  

Table 3: Traditional methods of sample extraction of polymeric materials.			

Components found in polymer samples can be either adsorbed on the polymer surface, as in the case of surface contamination, or absorbed within the bulk of the sample, as with additives. Adsorbed components can be either extracted or washed from the surface with an appropriate solvent and analysed chromatographically. Absorbed components can be extracted from a finely divided polymer with an appropriate solvent. Alternatively, although not an "extraction" technique, the polymer sample can be completely dissolved to release absorbed components from the matrix into solution.  

Table 4: Modern extraction methods for polymer samples.			

When the polymer matrix is dissolved on the addition of a good solvent, component molecules become solvated along with the polymer chains. Thus, in principle, the use of a good solvent to dissolve the polymer matrix will also solvate all released components. The analytical challenge is to find the appropriate conditions (that is, solvent and temperature), to dissolve the polymer matrix, and desorb and solvate component molecules. Perhaps the best way of dissolving a polymer is to use a solvent that has a solubility parameter that is numerically close to that of the polymer. For the reader's convenience, Table 5 lists solubility parameters of commonly used solvents, and Table 6 is a compilation of estimated solubility parameters of polymers. The role of temperature is discussed in the following section.  

Table 5: Solvent solubility parameters*.			

Figure 1 illustrates conceptualized plots of the concentration of extracted additives against particle size of the sample for two cases: adsorbed and absorbed components. If additives are applied only to the surface of the polymer, as in the case of finishes or contaminants, a horizontal line is obtained as shown by Figure 1, line 1a. It is quite obvious that the amount of extracted or recovered component does not depend on particle size; the same results would be obtained whether the extract is from a simple polymer wash or from a comminuted sample.  

Table 6: Estimated polymer solubility parameters*.			

If, however, components are absorbed throughout the polymer bulk phase, as with polymerized-generated components or the anti-ageing additives listed in Table 1, then the concentration of extracted components will depend on surface area or particle size, as seen in Figure 1, curve 1b. As the particle size of the sample is reduced, the amount of recovered component will increase asymptotically, reach a maximum at point A and remain level as the particle size is further reduced to its zero particle size limit at point B. In essence, point B is the concentration of the component as determined by complete dissolution of the polymer in the solvent; that is, at infinitely small particle size (point C). The segment between points A and B represents the finite depth that the extracting solvent penetrates.  

Sample Preparation Perspectives

This instalment focuses on approaches and techniques for extracting and concentrating small molecules of interest present in polymeric materials.

Approaches for Extracting and Determining Additives, Contaminants and Low-Molecular-Weight By-Products in Synthetic Polymers

29th International Symposium on MicroScale Bioseparations (MSB 2013) 

 The conference will build on the rich history of separation science and discuss a wide spectrum of current microscale bioseparation techniques. The 2013 conference is the 25th year of the series and was previously held in locations throughout North America, Europe and Asia. The University of Virginia, founded in 1819 by Thomas Jefferson, is a historic academic venue and ideal for the event, according to the organisers. 

The symposium was first held in 1989 in Boston, Massachusetts, USA, with the format of the symposium continuing to evolve. The organizers of 

 report that they will introduce new and substantial changes to the programme format. Registration is currently limited to 375 participants to encourage scientific exchange between delegates and preserve the academic atmosphere of the conference. About 70% of the presentation programme will be built built around blind peer-reviewed presentation abstracts. A layer of confidentiality has been added to protect unpublished work, allowing comfortable and open discussion in forums arranged following the presentations.  

Among the many topics to be discussed in key note, plenary lectures and presentations include 

biologics; clinical pharmacogenomics; carbohydrates; nucleic acids; food and health; proteomics; affinity capillary electrophoresis; glycomics; hyphenated techniques; metabolomics; micro- and nano-scale sample preparation; integrated microsystems; forensics; microfluidic controls; nanoparticle characterisation; novel detection; late breaking developments and advanced instrumentation. 

 programme will also include a number of short half- and full-day training workshops and courses, including a laboratory-based course on paper-based analytical devices. 

programme is a complimentary Science Café scheduled daily, to provide refreshment during discussions of advances in commercial MicroScale Bioseparation products, alongside presentations by sponsors presenting new technologies.  

will be the recognition of the achievements of analytical scientists, represented by a series of award ceremonies and presentations. The Arnold O. Beckman Medal and Award which is presented annually at the North American and European MSB conferences respectively will be presented to a nominated scientist for lifetime contributions in the field of electrodriven separation techniques.  

A symposium of poster presentations will be held to promote the exchange of scientific ideas at the cutting edge of separation science. A poster award competition will be held to reward the top three presentations. The selected authors will have the opportunity to present an oral presentation during the symposium finale, a must-see attraction of the conference. 

To encourage student participation and stimulate discussion between delegates, 

 will provide a limited number of travel grants for students and post-docs presenting at the conference; research students from industry and academia are encouraged to apply. 

The conference co-chair Jeff Chapman, director of marketing, Beckman Coulter Life Sciences looks "forward to a deeply engaging, scientifically rewarding gathering of the microscale separation community" and reports that "attendees at this year's meeting will find much to absorb, and be hosted by a wonderful campus and community." 

29th Symposium on Microscale Preparations

				University of Virginia, Charlottesville, Virginia, USA 			

				Pennsylvania Convention Centre, Philadelphia, USA 			

 The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy 			

Institute of Environmental Engineering of Polish Academy of Science 			

www.ipis.zabrze.pl/en/conferences/65-planowane-ipi-pan

				Amsterdam RAI Conference Centre, Amsterdam, The Netherlands 			

20th International Symposium on Electro- and Liquid-Phase Separation Techniques, ITP2013

				Puerto de la Cruz, Tenerife, Canary Islands, Spain 			

Event News

The 29th International Symposium on MicroScale Bioseparations (MSB 2013) will be held 10–14 March 2013, at the University of Virginia in Charlottesville, Virginia, USA. The conference will build on the rich history of separation science and discuss a wide spectrum of current microscale bioseparation techniques.

MicroScale Bioseparations (MSB) 2013

An excerpt from LCGC's e-learning tutorial on solid-phase extraction at CHROMacademy.com

Solid-phase extraction (SPE) is a sample cleanup and preconcentration technique used to reduce sample and chromatographic complexity or increase analytical sensitivity. 

Often called "digital chromatography," the technique uses silica or polymeric stationary phase particles that are often functionalized with various ligands to offer a particular selectivity to compounds partitioning between the liquid mobile phase and solid stationary phase surface. There are many different sorbent chemistries available, including most of the popular high performance liquid chromatography (HPLC) phases, ion exchangers (both weak and strong) and "mixed-mode" phases using both hydrophobic and ionic ligands. Polymers are used as mixed-mode phases and have found widespread use as "generic" phases in which a fixed protocol of solvents can be used across a wide variety of application types.  

The sorbent materials are often packed into syringe barrel tubes or well-plate devices and protocol solvents are passed through the packed beds using gravity, vacuum or positive pressure of an inert gas. The amount of sorbent is chosen to reflect the capacity required to retain the analytes of interest, although usually in SPE the mass of all sample components is considered in this calculation and not just the analyte of interest. Silica phases typically have a capacity of around 5% (w/w) by bed mass and polymers around 20% (w/w). 

The majority of SPE methods — or protocols as they are often called — have six discrete stages. 

typically involves changing a liquid sample pH, dissolving a solid sample in a suitable solvent or altering the sample buffer strength to ensure maximum retention by the chosen sorbent. 

 uses a solvent (often methanol) to activate the bonded phase on the sorbent surface and acts to avoid dewetting effects.   

 prepares the sorbent surface to accept the sample, ensures optimum analyte recovery and minimizes "breakthrough" — this might include adjusting the pH of the solvent within the sorbent bed, altering the ionic strength or changing the type of buffer used. The adjustment of the solvent pH may also be used to control the degree of ionization for ligands containing ionogenic functional groups. 

 Figure 1: Steps in solid-phase extraction.   

 is usually performed in a similar solvent to that used during the equilibration step, and careful control of solvent flow rate is important to ensure optimum analyte retention, especially where ionic (point to point) interactions are concerned. Flows rates of 1 mL/min are typical.   

 steps require careful design of the protocol solvent and the eluotropic strength, ionic strength and pH may all be adjusted to ensure that the maximum amount of potentially interfering species are removed while leaving the analyte in as tight a band as possible on the sorbent surface. The specificity of the protocol can be optimized, if required, by using ligands that undergo ion-exchange interactions where the options for chemically differentiating between analyte and interferents include pH, ionic strength and the nature of the counterion used, thus creating cleaner final extracts. More than one washing step may be used if necessary. Method developers commonly use wash solvents that are eluotropically weaker than necessary for fear of analyte loss. 

 protocol solvents are designed to recover the maximum amount of analyte from the stationary phase while leaving any remaining potential interferents behind, and, once again, percentage of organic modifier, pH, ionic strength and the nature of the counterion are available to maximize analyte recovery. Depending upon the nature of the wash and elution solvents and the mode of analyte retention, a soak step may be used in which the elution solvent is allowed to remain stationary for a fixed period of time, ensuring good penetration into the pores of the stationary phase, minimizing dewetting phenomena and allowing time for the analyte to equilibrate into the elution solvent. Again, a common mistake at this stage is the use of protocol solvents that are "too strong" and elute interfering compounds that could otherwise be left on the sorbent for fear of low analyte recovery. Both wash and elution stages should be carefully optimized to maximize selectivity of the process. 

The volume of elution solvent can be altered by changing eluotropic strength to achieve a concentration effect in which the analyte concentration increases with the ratio of sample volume to elution volume. If required, the elution solvent may be evaporated under an inert gas and reconstituted in a solvent more suited to the analytical technique or in a lower volume, again achieving an increase in analyte concentration. This evaporation or reconstitution step provides extra complexity and a possible reduction in precision and should be carefully considered. 

The Essentials

Solid-Phase Extraction

Many edible oils contain natural antioxidants that protect the oil from lipid oxidation. Knowledge on the identity of these oxidation inhibitors is crucial for finding natural ways to protect healthy unsaturated fats and oils from turning rancid. In this article a new assay that allows rapid quantification of the lipid oxidationinhibiting potential of pure compounds or liquid chromatographic (LC) fractions is described. The assay uses accelerated ageing of docosahexaenoic acid ethyl ester (DHA-EE) to quantify the extent of oxidation and oxidation inhibition. In the assay fast gas chromatography (GC) is used to monitor the disappearance of the DHA-EE and the appearance of volatile oxidation products. The method is fast and allows multiple fractions to be tested in parallel. 

A good flavour and taste, a pleasant texture and good nutritional characteristics are important criteria for consumers to appreciate and purchase specific food products. As far as edible oils and fats are concerned, the fatty acids present determine much of the flavour and the health aspects of the food. From a health perspective, consumption of saturated fatty acids (SAFAs) should be minimized, and mono- and poly-unsaturated fatty acids should preferably be consumed. However, in terms of food processing and shelf life, these unsaturated fatty acids are less attractive because they are much more susceptible to oxidation and off-flavour formation. The addition of antioxidants increases product stability. For this reason there is great interest in natural antioxidants that inhibit oxidation and can prevent off-flavour formation for products rich in unsaturated fatty acids. 

Oxidation of lipids is a very complex process that is not fully understood. The common consensus is that lipid oxidation proceeds through radical initiation, formation of non-volatile peroxidized- and hydroperoxidized triacylglycerides and eventually causes the cleavage and loss of volatile aldehydes, ketones and other species (1,2). This complex mixture of volatile compounds gives an aged oil the typical rancid smell.  

Antioxidants are capable of preventing or slowing down lipid oxidation. A series of reviews describing the chemistry of antioxidants (3,4) and natural sources of antioxidants (5) has been published. Important natural antioxidants are extracts of rosemary (6), sage (7) and green tea (8). Once an extract has shown to possess oxidation-inhibiting properties it is important to find out which particular compound in the extract is responsible for this beneficial effect. This question is typically answered through the use of so-called activityguided fractionation approaches (9). The mixture is fractionated using a chromatographic separation system and the activity of the fractions obtained measured. Active fractions are fractionated again using a different separation mechanism and fraction-activity is measured. This process is repeated until a sufficiently simple aliquot is obtained in which the active compounds can be identified using chromatography and/or spectroscopy. The above cycle describes the use of off-line activity testing. On-line systems that use automated set-ups consisting of a chromatographic separation system connected to a continuous 'activity detector' have also been described (10,11). Such systems have also been applied to detect natural antioxidants and radical scavengers (12,13). 

Key factors in the activity-guided fractionation cycle described above are the separation power of the fractionation method, alongside the sensitivity and nature of the assay. The ideal fractionation method would provide large numbers of very different fractions, whereas the ideal assay would be fast and sensitive. In addition to this, and probably even more important, is that the assay should truly mimic the lipid oxidation as it would occur in a food product rich in poly-unsaturated fatty acids (PUFAs). This last requirement is difficult to meet: 'normal' lipid oxidation can take months to occur.  

The ideal assay to assess the lipid oxidation-inhibiting-strength of a compound would be the lipid oxidation reaction itself. Performing the reaction at an elevated temperature and using fast and highly sensitive methods to monitor the disappearance of PUFAs and the simultaneous appearance of volatile products could be a route to achieve this. Gas chromatography (GC) would be the technique of choice because it can detect a very wide range of compounds ranging from the high-molecularweight starting material to the small, volatile products. The flame ionization detector (FID) would provide the universality needed to see all products at a good sensitivity. One analytical difficulty with this route is that fast GC analysis of the high-molecular weight triglycerides is difficult. By working with free PUFAs rather than PUFAglycerol tri-esters, fast GC quantification of the decreasing PUFA level is feasible, while in the same run volatile products that have formed can also be quantified. The use of a methyl- or ethylester of the PUFA as the starting material would simplify the analysis further. Since esters of PUFA with small alcohols are not believed to react differently from PUFA triacylglycerides, this simplification is unlikely to affect the reliability of the assay. 

In this article we will describe a novel, GC-based assay for rapid testing of the lipid-oxidation inhibition of compounds and mixtures. The assay uses an accelerated lipid oxidation experiment with fast GC monitoring of both the disappearance of a PUFA-ester and the simultaneous appearance of volatile products. To allow identification of actives in a complex edible oil, this GC-based assay is used in combination with preparative liquid chromatography as a fractionation method. The optimization of the two systems will be discussed, focusing particularly on their respective requirements and their influence on operation and setup of the complete assay. The combined system is applied to the identification of natural antioxidants in a maize oil sample.  

Preparative Normal Phase Liquid Chromatography: 

Fractionations of edible oil samples were performed on a preparative HPLC system (Shimadzu Europe, Duisburg, Germany) consisting of two LC 8A pumps, a DGU14A degasser, SLC 10A controller and SIL 10A autosampler. The column was a semi-preparative Alltima Silica column of 150 mm length and 10 mm i.d. packed with 5 µm particles from Alltech (Deerfield, Illinois, USA). Gradient elution was applied with a mobile phase of 

-hexane (A)/methyl tertiarybutyl ether (MTBE, B) starting with 4% B for 5 min followed by a linear increase to 45% B in 65 min, back to 4% B in 1 min and a reconditioning time of 19 min (total run time = 90 min). The flow rate was 5 mL/min. Detection was performed with a UV detector (Shimadzu SPD-20A) at a wavelength of 210 nm and data was recorded with a PerkinElmer data acquisition system (PerkinElmer, Norwalk, Connecticut, USA). In the preparative separations, 50 mg edible oil dissolved in 500 µL 

-hexane was injected into the HPLC system. The edible oil studied was an unprocessed maize oil obtained from a Unilever spreads factory. Fractions from the preparative separations were collected for activity testing. Initially, a fraction width of 1 min was applied to roughly detect the region of interest. In a second, more targeted fractionation, fraction width was 30 s. All fractions were collected in 25 mL vials. From each vial 1 mL was used in the lipid oxidation assay. The remainder was kept for later GC–MS identification.  

Many edible oils contain natural antioxidants that protect the oil from lipid oxidation, Knowledge on the identity of these oxidation inhibitors is crucial to find natural ways to protect healthy, unsaturated fats and oils from turning rancid. This article describes a new assay that allows rapid quantification of the lipid oxidation inhibting potenital of pure compounds or liquid chromatography (LC) fractions.

LCGC Europe-01-01-2013

Q&A Gases: Part 2

Pittcon 2013 – What's in Store for Chromatographers

Approaches for Extracting and Determining Additives, Contaminants and Low-Molecular-Weight By-Products in Synthetic Polymers

MicroScale Bioseparations (MSB) 2013

Solid-Phase Extraction

Detection and Identification of Natural Antioxidants in Edible Oils using LC fractionation with Off-line Effect-Based Detection

Making the Most of a Gradient Scouting Run

Vol 26 No 1 LCGC Europe January 2013 Regular Issue PDF