5 June 2014 Volume 10 Issue 10 Cover Story 2 Sense and Sensitivity: The Role of Separation Science in Sensory Evaluation Kate Mosford of The Column spoke to Giorgia Purcaro of the Department of Food Science at the University of Udine, Italy, and Chiara Cordero of the Department of Drug Science and Technology at the University of Turin, Italy, about the sensory evaluation of olive oil and the benef ts of a comprehensive approach. Features 16 Principles of Detection and Characterization of Branching in HPLC GC MS SSPEynthetic IRand Natural by MALS Stepan Podzimek, SYNPO and University of Pardubice powered by This article provides insight into the basic principles of the MALS technique, and how it can be applied to the detection and characterization of branching in polymers. 20 Polyolefin Characterization by High Temperature Adsorption Liquid : A Review of Solvent Gradient (SGIC) aEluennd Thte Crmhemistral Gradiyent Interaction Chromatography (TGIC) B. Monrabal, Char G The use of high temperature adsorption liqruadienid chromatography for the characterization of polyolef ns is detailed. t Elution 25 Using Multi-Detection GPC/SEC to Determine Impact of Sterilization on Medical-Grade Polymers Bassem Sabagh1 and Angel Valero-Navarro2, 1Malvern Instruments, 2nanoMyP Multi-detection GPC/SEC is described and new data showing how the technique is LIVE being used to determine the impact of sterilization on medical grade poly(L-lactide) is presented. on the web Regulars 10 News Optimization Text to come, text to come, text to come, text to come, text to come, text to come, text to come, text to come, text to come, text to come, text to come. Column 12 Tips & Tricks GPC/SEC: Selection The Art of Analyzing High Molar Mass Samples HPLC Method Daniela Held, PSS Polymer Standards Service GmbH GPC/SEC can be performed for samples that range from a few hundred to several million daltons in molar mass. It can often require more skill to analyze high molar mass samples in the molar mass range of several million dalton. DevelopmenHeadline_ hetadline_ This article explains more. F30ind CHROMac outademy more @ Subhead_ subhead_ subhead_ subhead Update on what’s new on the professional site for chromatographers. 31 Training Courses and Events Starts July 2014 www.CHROM33 Staff academy.com

magentablackcyanyellow ES447539_LCTC060514_cvtp1.pgs 05.29.2014 18:21 ADV 5 June 2014 Volume 10 Issue 10 Cover Story 2 Sense and Sensitivity: The Role of Separation Science in Sensory Evaluation Kate Mosford of The Column spoke to Giorgia Purcaro of the Department of Food Science at the University of Udine, Italy, and Chiara Cordero of the Department of Drug Science and Technology at the University of Turin, Italy, about the sensory evaluation of olive oil and the benef ts of a comprehensive approach. Features 16 Principles of Detection and Characterization of Branching in Synthetic and Natural Polymers by MALS Stepan Podzimek, SYNPO and University of Pardubice This article provides insight into the basic principles of the MALS technique, and how it can be applied to the detection and characterization of branching in polymers. 20 Polyolefin Characterization by High Temperature Adsorption Liquid Chromatography: A Review of Solvent Gradient (SGIC) and Thermal Gradient Interaction Chromatography (TGIC) B. Monrabal, Polymer Char The use of high temperature adsorption liquid chromatography for the characterization of polyolef ns is detailed. 25 Using Multi-Detection GPC/SEC to Determine Impact of Sterilization on Medical-Grade Polymers Bassem Sabagh1 and Angel Valero-Navarro2, 1Malvern Instruments, 2nanoMyP Multi-detection GPC/SEC is described and new data showing how the technique is being used to determine the impact of sterilization on medical grade poly(L-lactide) is presented. Regulars 10 News Red wine could help to protect against dental cavities, HPLC–MS demonstrated as a clinical tool to monitor patients taking anti-hypertension drugs, and the New Orleans HPLC 2014 poster award winners are all featured in this issue. 12 Tips & Tricks GPC/SEC: The Art of Analyzing High Molar Mass Samples Daniela Held, PSS Polymer Standards Service GmbH GPC/SEC can be performed for samples that range from a few hundred to several million daltons in molar mass. It can often require more skill to analyze high molar mass samples in the molar mass range of several million dalton. Sense and Sensitivity This article explains more. 30 CHROMacademy The sensory evaluation of olive oil Update on what’s new on the professional site for chromatographers. 31 Training Courses and Events 33 Staff

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Kate Mosford of The Column spoke to Giorgia Purcaro of the Department of Food Science at the University of Udine, Italy, and Chiara Cordero of the Department of Drug Science and Technology at the University of Turin, Italy, about the sensory evaluation of olive oil and the benef ts of a comprehensive approach.

Q: Olive oil is classif ed by law according therefore been subjected to frauds by the to both the chemical composition and addition of oils of lower quality and price. the sensory evaluation. Is olive oil the Since the f rst European Regulation only food product analyzed by sensory citing olive oil, which was enacted in 1966 evaluation? (Regulation no. 136/1966)1 and directed to Giorgia Purcaro: Sensory evaluation of market regulation on fats and oils within foods has been widely applied in industry , both chemical and sensory evaluations to study consumer acceptance, which have been used for assessment of purity and inf uences business decisions and guides quality of olive oil. Although the def nitions of product development to get closer to sensory quality were initially “blurry” (“perfect a benchmark product. In academia the f avour”, “good f avour”, and “off-f avour”), correlation between sensory perception they were elucidated in detail in the European and chemical composition and texture has Regulation no. 2568, which was enacted in always been an important topic of study 1991. In fact, this regulation introduced — in food science. Sensory quality has been along with a long list of chemical analyses used as an additional requirement in several with an accurate description of the analytical food certif cations, such as PDO (product methods — a detailed explanation of the designation of origin). Sensory evaluation procedure to be performed in the training of has never been used as a legal parameter panellists, how to carry out the panel session, to def ne any kind of food products and the attributes to be used to evaluate the before olive oil. Olive oil has been a very sensory quality of olive oil.2 important product of the Mediterranean basin since ancient times, and it has always Q: What does the sensory evaluation been recognized, from both a quality and involve and what are the drawbacks? a commercial viewpoint, as high value oil GP: Sensory evaluation is fundamental

Photo Credit: Ursula Alter/Getty Images compared to other vegetables oils. It has in classifying olive oil within the specif c

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commercial class, namely extra virgin, virgin, since panellists are usually employed in other and lampante olive oil [Reg. CEE 2568/91, Reg jobs; (b) each session is very tiring, consequently CE 796/02; Reg CE 640/08].2–4 only a limited number of samples (maximum For example, extra virgin olive (EVO) oil, of six) can be analyzed for each session; apart from respecting all the required chemical (c) low reproducibility has been observed parameters, has to present a fruity aroma among different panels, mainly as a result of and no defects (muddy, fusty, rancid, musty, uneven training, which is related also to the vinegary, or metallic) that have originated diff cult availability of reference standards — in during olive or oil storage. Described in particular when presenting only one defect at a detail by both the European Regulation CEE time. Therefore, an objective sensory evaluation 2568/912 (and following modif cations) and is costly and time-consuming.8 by several International Olive Oil Council (IOC) documents (COI/T.20/Doc. No 15/Rev. 6; Q: What analytical techniques have been COI/T.20/Doc. No 13; COI/T.20/Doc. No 4),5–7 used to analyze olive oil previously and Is the foundation of your molecular characterization based on measurements without SEC-MALS? the sensory analysis is based on the judgement what are the benef ts of a comprehensive Scientists shouldn’t have to guess the molecular weights of their samples. But that’s pretty much what they do when they don’t of a panel of assessors, composed of a approach? use Wyatt’s SEC-MALS solutions to calculate molecular weights. Only Wyatt’s industry-leading DAWN® multi-angle light scattering (MALS) instruments make absolute molar mass determinations—without column calibration or reference minimum of eight testers (generally 12) headed GP: To study the volatile prof le of olive oil, the standards. When you connect a DAWN to your SEC/GPC system, you’ll determine absolute molar masses completely by a panel leader, who has to motivate the most suitable technique is gas chromatography independent of elution times. judges, process the data, interpret results, and (GC), usually coupled with a mass spectrometer Since 1982, we’ve been committed to solving the toughest macromolecular characterization problems for our customers. Every day, thousands of leading scientists throughout the world depend on our instruments. And more than 9,300 peer- draft the f nal report. (MS) detector for identif cation purposes. review articles have been published, all of which attest to the versatility and superiority of our unique MALS technology.

The sensory assessment (both tasting and A few studies have reported the use of an Free training and PhD-level customer support come with your purchase, as well as a remarkable, three-day, all-expenses-paid smelling) is performed according to codif ed olfactometric detector to describe the odour Light Scattering University® (LSU) training course in Santa Barbara (US customers only). Our personalized, friendly and accessible customer support staff includes 18+ PhD scientists dedicated to sharing their expertise in chromatography, polymer rules, in a specif c tasting room, and using a perception generated by specif c compounds.9 chemistry, protein science, biochemistry and, of course, light scattering. 7 specif c vocabulary (COI/T.20/Doc. No 4). The Several sampling techniques have been used, There’s no mystery in determining absolute molecular weights—just a superior solution. And panel has to be continuously trained and the mainly static,10 dynamic headspace (HS), and by choosing Wyatt Technology you’ll get measurements that are solidly grounded in science, not based on a house of cards. performance constantly evaluated, usually using purge-and-trap techniques9,11 and more reference samples provided by the IOC, to recently solid-phase microextraction (SPME).12,13 verify the reliability of the results and harmonize The latter technique represents an easy-to-use

the perception criteria. However, despite a pre-concentrating method, alternative to the DAWN® HELEOS® Multi- Optilab® T-rEX™ 'iijerential re- Mobius® Electrophoretic mobility Eclipse™ &olXmn-Iree ıeld-IJow DynaPro® Plate Reader Auto- angle light scattering for fractometer for on-line concentra- from proteins to nanoparticles. fractionation of macromolecules mated dynamic light scattering for characterizing macromolecular tions. Unparalleled measurement Uniquely sensitive and versatile and nanoparticles in solution. proteins and nanoparticles, in-situ rigid codif cation of all the aspects of sensory dynamic HS that provides a representative mass, size and conformation. and temperature ranges. for all types of solvents. One system covers all your in standard micro-well plates. No other MALS detector comes separation needs. Now with a camera! analysis, several drawbacks can be pinpointed: picture of the sample volatiles as a function of close in range or sensitivity. ©2013 Wyatt Technology. DAWN, Optilab, DynaPro and ViscoStar are registered trademarks, and the Eclipse is a trademark of Wyatt Technology Corporation. (a) Organization of a panel session is not easy analyte volatility and relative polymer aff nity.

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Many studies14–16 have been performed highlighted as an important issue from the to unravel the complex mixture of olive oil analytical viewpoint.19 volatiles and to understand their relationship With this perspective, the use of with quality. Aldehydes, ketones, alcohols, comprehensive GC (GC×GC) coupled with esters, hydrocarbons, furans, lactones, and MS, in combination with HS sampling and others still unknown are responsible for the performed with different techniques, and whole aroma of olive oil. However, their implemented with advanced data elaboration exact composition and relative prof le may tools, can be a valuable and more informative be affected by several parameters, therefore analytical approach. In fact, GC×GC is a complicating the identif cation of target relatively novel technique,20 which improves compounds. Cultivar, geographic region, selectivity, using different column stationary fruit ripeness, processing method, and phases in each dimension, resolution storage may signif cantly affect the volatile (theoretically corresponding to the product composition.17 All of these variables affect of the peak capacity of each column), and the intensity and quality of the green and provides a useful support for identif cation fruity perception, while the presence of of “unknowns” thanks to the formation of defects is mainly a result of inappropriate chemically-similar compound patterns when a manufacturing practices. homologous series of compounds are present. A few studies9,18 have focused on the Furthermore, depending on the kind of determination of target compounds related interface (modulator) used between the to standardized defects using standards two columns, the comprehensive system provided by the IOC, and mainly applying can provide an important increase in the one-variable-each-time approach. sensitivity, eventually allowing the detection However, despite an extensive knowledge of informative volatiles present at low of the volatile prof le of olive oil, any concentrations, but with a low odour recognized analytical procedure has not threshold. replaced sensory evaluation yet. Several Moreover, the selection of a comprehensive aspects to explain this situation have been set of samples (extra virgin, virgin, and recently discussed, and the need to improve lampante oil) to be analyzed together the analytical determination, along with allows a wider set of informative data to be the pre-concentration procedure, both in collected, and requires suitable chemometrics terms of selectivity and resolution, has been to derive useful information.

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Q: How easy is comprehensive GC to basic theory and experience in different implement in practice? branches of the GC f eld are of great help for GP: The introduction of the comprehensive rapid and effective GC×GC optimization.23,24 GC×GC system in 1991 by Liu and Philips20 However, a bigger effort in method has been one of the greatest advancements optimization is largely rewarded by the towards increasing separation power in advantages obtained from such a technique. GC applications. The separation process In fact, the introduction of GC×GC has is performed on two columns of different not only allowed a better understanding of selectivity (with almost orthogonal complex matrices, but it has also given a discrimination principles) connected in new insight into the so-called “well-known” series and interfaced by a modulator, which samples. represents the heart of the system. Since However, since the authors are aware that the introduction of this technique, different such a powerful technique is not widespread kinds of modulators have been developed yet, the main goal of our work in def ning over the years, which can be classif ed the blueprint of extra virgin olive oil will be to on the basis of the specif c operational identify reliable target compounds through characteristics in thermal and pneumatic the support of advanced analytical techniques. modulators.21 The setting of a GC×GC Development of selective sample preparation system is a rather simple task. Essentially, techniques will follow, which can be used it can be achieved using any commercially- routinely in conventional, more widespread, available GC instrument by installing a and affordable GC techniques. modulator between two different columns. In our recent work,22 one of the two Q: You mention using different and involved developed its own complementary sampling techniques to lab-made cryogenic (CO2) modulator, which obtain comprehensive results. Can you performed comparably to the commercial comment on this further? one used in the other . Chiara Cordero: Sampling is a key step of any Although hardware conf guration is quite analytical method, and when the application straightforward, method optimization can be deals with the prof ling of volatiles and high a cumbersome issue and is probably the main volatiles it is also challenging. In particular, factor inhibiting the widespread use of GC×GC. in the sensory quality assessment of olive oil Indeed, strong knowledge of chromatographic the sampling should provide a consistent and

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meaningful picture of all sensory-informative extraction from the HS (orthonasal) and from analytes, including chemicals present at trace the liquid sample that will be introduced in the and ultra-trace level. oral cavity (retronasal). The results obtained have With this perspective, we have adopted been really straightforward and, in our opinion, a well-established miniaturized sampling represent a bridge between high-throughput technique (that is, SPME applied to the HS screenings with a complete and almost extraction of volatiles) that enables an ad comprehensive prof ling of volatiles related to hoc tuning of the extraction selectivity by the aroma perception. In addition, by tuning modifying physico-chemical characteristics of the extraction capability of sampling towards a the extractants and of the sampling conditions wider range of polarities and volatilities, most Thinking Forward. (time, temperature, and volume or mass of of the sensory descriptors of the product can the extraction phase). SPME is also f exible in be monitored and subsequently related with terms of extraction eff ciency and capability, corresponding odour perceptions. In such a GPC/SEC offering different commercial solutions that context, the information potential of each Theory or practice? combine single or combinations of extraction analysis increases and an almost complete polymers whose mechanisms are based on mild sensory prof le can be objectively delineated. interactions that limit artifact formations. Last If there is PSS are world leaders in macromolecular characteri- but not least, the possibility of full integration Q: What are your next steps in olive oil zation and have the expertise to help you with your and automation of the extraction process, analysis? Do you see the comprehensive one thing analysis requirements. We offer a range of products and services from instruction courses and training thru therefore including sample preparation as an approach totally replacing sensory contract analysis, consulting, method development additional dimension in the analytical platform, evaluation? we can do, and qualifi cation services all the way up to supplying turnkey GPC/SEC and LC/2D systems. is another key aspect to take into consideration, GP, CC: Our study, although not conclusive, All this comes with the personal and direct support especially in view of a routine quality control. has emphasized the analytical advantages it’s both. of our dedicated team of innovative and pioneering However, we have recently experienced of GC×GC–MS in terms of sensitivity, specialists. Is there any better way of achieving your analysis goals? the advantages of a “truly” complementary reproducibility (evaluated by cross-validation sampling on milk aroma prof ling by collecting between laboratories and instrumental setup), Driving GPC/SEC forward analytes from HS and in-solution sampling of and information potential, and proposes a 25 milk samples. The sampling design has been productive investigation strategy for a Phone + 49 6131 962390 inspired by the pathways aroma compounds reliable quality assessment of EVO and VO oils. You‘ll fi nd the ideal GPC/SEC solution at PSS under: www.pss-polymer.com follow to reach the regio olfactoria: orthonasal The results obtained to date will be [email protected] and retronasal. The characterization of volatiles implemented by extending the number of should be consequently run through their representative samples to include in the

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classif cation model and by approaching the sample constituents and related attributes true quantitation of key targets to obtain (physicochemical properties, concentration reference blueprints of defects to adopt as in-the-matrix) together with their sensory More Flexibility – decision makers. properties (odour quality, odour threshold [OT], More Information In this context, the investigation of odour activity value [OAV]).27,28 alternative high concentration capacity HS The multidimensionality of the approach sampling techniques, (that is, headspace represents a key aspect in this perspective, sorptive extraction – HSSE with different polar enabling some of the limits of conventional or apolar extraction polymers) will be decisive platforms and methods to be overcome as a future perspective, which improves by applying orthogonal discrimination in f ngerprinting sensitivity and enables reliable multiple analytical dimensions and combining: quantitation of key-odourants (by multiple (a) the separation of analytes based on headspace extraction [MHE]).25 volatility, polarity, partition coeff cient, and In our vision, a robust, reliable, and sensitive solubility; (b) identity assessment, provided method able to provide an objective picture of by mass spectroscopy; (c) quantitation (true key-odourants distribution would be a valuable concentration and relative abundance); and tool to support, or even replace, sensory (d) odour activity characterization when evaluation. olfactometric detection is included. Thermal Desorption, Pyrolysis Applications include studies on cocoa and and more – in one system! Q: Do you think this approach to chocolate aroma characterization, roasted Thermal Extraction / Thermal The modular GERSTEL systems extract significantly more Desorption (TDS & TDU) information from your sample: analyzing olive oil could be useful in coffee volatiles distribution, quality assessment Multiple techniques on one GC/MS system Automated Pyrolysis Efficient, flexible automation other applications? of hazelnuts by aroma and technological (PYRO) Easy and convenient operation CC: Actually, methods and approaches adopted markers distribution, and wine quality by Compatible with all standard GC/MS systems Thermal Extraction of in our investigation of olive oil chemical aroma prof ling and origin authentication. liquids in µ-vials (ATEX) Application support at your service blueprint are included in the analytical tools of Thousands of users in leading companies world-wide Dynamic Headspace (DHS) rely on our Thermal Desorption Solutions. sensomics.26 This intriguing discipline focuses Q: How important are the “-omics” in and Headspace What can we do for you? analytical efforts on revealing sensorially-active food analysis? SPME and Multi Fiber compounds to link the chemical composition GP, CC: The role of academia has to be Exchanger (MFX)

with food sensory quality (f avour). The oriented towards advanced, innovative, MAESTRO PrepAhead productivity investigation is extended to all possible stimuli and novel techniques within the f eld of of the multimodal perception (aroma, taste, the modern “–omics” disciplines, such as www.gerstelus.com www.gerstel.com texture etc.) by comprehensively treating foodomics, f avoromics, and sensomics in food

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f elds. Such techniques are extremely powerful References and they allow a huge amount of data that 1. Reg. CEE n. 136/66 Off. J. Eur. Comm. L172 (1966) requires advanced chemometrics to extrapolate 3025. the useful information to be collected. It may 2. Reg. CE n. 2568/91 Off. J. Eur. Comm. L347 (1991) seem a rather complicated task and far from 69. everyday practice, because, as is usually the 3. Reg. CE n. 796/02 Off. J. Eur. Comm. L128 (2002) 8. case, the solution to a problem is simple and 4. Reg. CE n. 340/08 Off. J. Eur. Comm. L178 rather linear. However, when working with (20 08) 11. complex matrices such as foods, one has to 5. International Olive Council (IOC). Document isolate information deriving from informative COI/T.20/Doc. No 15/Rev.6. Madrid, , 2013. analytes (such as compounds characterizing 6. International Olive Council (IOC). Document botanical origin, technological process, or COI/T.20/Doc. No 13. Madrid, Spain, 1996. sensory quality) from interferences to get close 7. International Olive Council (IOC). Document to a truly comprehensive practical solution that COI/T.20/Doc. No 4. Madrid, Spain, 1987. is able to act as “decision maker”. 8. D.L. García-González and R. Aparicio, Grasas y A comprehensive approach is fundamental Aceites 53(1), 96–114 (2002). to have a complete overview of the sample to 9. M.T. Morales, G. Luna, and R. Aparicio, Food Chem. be studied and to provide a reliable indication 91, 293–301 (2005). on the variables to focus on to solve any kind 10. I. Marcos, J.L. Pérez, M.E. Fernández, C. García, of problem. We are aware of the industrial and B. Moreno, J Chromatogr A. 945, 221–230 needs for easy and straightforward solutions (2002). to practical problems; we think that exploiting 11. A. Kanavouras, A. Kiritsakis, and R.J. Hernández, comprehensive approaches and extending the Food Chem. 90, 69–79 (2005). analytical investigation to the whole chemical 12. J.-F. Cavalli, X. Fernández, L. Lizzani-Cuvelier, and pattern would increase knowledge of the A.-M. Loiseau, J Agric Food Chem. 51, effects of processing, storage, fermentation, 7709–7716 (2003). and biotransformation on the overall quality of a 13. S. Vichi, A.I. Castellone, L. Pizzale, L.S. Conte, S. food product. Buxaderas, and E. López-Tamames, J Chromatogr A. In this context, academia and industry would 983, 19–23 (2003). work synergically towards a common objective 14. C.M. Kalua, M.S. Allen, D.R. Bedgood Jr, A.G. and promote a truly technological transfer of Bishop, P.D. Prenzler, and K. Robards, Food Chem. knowledge and methods. 100, 273–286 (2007)

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15. F. Angerosa, Eur. J. Lipid Sci. Technol. 104, Giorgia Purcaro obtained (HRGC–FID, HRGC–MS, HPLC–UV, HPLC– year 1996–97. Since 2001 she has worked 639–660 (2002). her Masters degree in food FLD), as well as advanced multidimensional as assistant professor of food chemistry at 16. G. Luna, M.T. Morales, and R. Aparicio, Food Chem. science and technology in and comprehensive chromatography ones the Dipartimento di Scienza e Tecnologia 98, 243–252 (2006). 2002 with “summa cum (GC×GC, LC–GC, LC×GC) to apply to food del Farmac o and Faculty of Pharmacy at the 17. F. Angerosa, M. Servili, R. Selvaggini, A. Taticchi, laude” and she gained analysis. University of Turin. S. Esposito, and G. Montedoro, J. Chromatogr. A her PhD in food chemistry Her work on the capability of a quadrupole In 2008, she was awarded the Leslie S. 1054, 17 –31 (2004). in 2008, both at the MS for GC×GC detection was awarded with Ettre Award for the young scientist who 18. G. Procida, A. Giomo, A. Cichelli, and L.S. Conte, J. University of Udine (Italy) the prestigious Leslie S. Ettre Award in 2010 presented the most interesting original Sci. Food Agric. 85, 2175–2183 (2005). under the supervision of Professor L. Conte. for the young scientist who presented the research in capillary gas chromatography with 19. R. Aparicio, M.T. Morales, and D.L. García-González, In 2006, she was awarded the scholarship most interesting original research in capillary an emphasis on environmental and food safety Eur. J. Lipid Sci. Technol. 114, for international mobility within the Regional gas chromatography with an emphasis on during the 32nd International Symposium 1114 –1125 (2012). Operative Programme, Objective 3- European environmental and food safety during the on Capillary Chromatography (ISCC) and 5th 20. Z. Liu and J.B. Phillips, J. Chromatogr. Sci. 29, Social Fund 2000–2006, axe D, Measure D4 34th International Symposium of Capillary GC×GC Symposium. The study focused on 227–231 (1991). “Improvement of the human resources in Chromatography and 7th GC×GC Symposium the application of GC×GC/qMS for group-type 21. P.Q. Tranchida, G. Purcaro, P. Dugo, and L. the research and technological development in Riva del Garda (Italy). and f ngerprint analysis approaches in the Mondello, Trends Anal Chem. 30(9), 1437–1461 f eld”, and she spent six months at the RMIT Chiara Cordero received characterization of roasted food matrices. (2011). University in Melbourne (Australia) under the her Masters degree in Her research activities are focused on the 22. G. Purcaro, C. Cordero, E. Liberto, C. Bicchi, and L.S. supervision of Professor P.J. Marriott, working on pharmaceutical chemistry development of innovative and advanced Conte, Journal of Chromatography A. 1334, 101–11 the development of rapid analytical techniques and technology in 1998 instrumental conf gurations for GC×GC (2014). based on hyphenated chromatography methods with “summa cum coupled to MS applied to quali-quantitative 23. M. Adahchour, J. Beens, and U.A.Th. Brinkman, (GC×GC) for food safety assessment. From laude” at the University characterization of complex food samples J.Chromatogr. A 1186, 67–108 (2008). 2008 to 2011 she worked in the laboratories of Turin (Italy), supervised (roasted coffee, roasted hazelnuts, herbal 24. H.J. Cortes, B. Winniford, J. Luong, and M. Pursch, of Professor L. Mondello at the University by Professor Carlo extracts, and essential oils), also through J.Sep. Sci. 32, 883–904 (2009). of Messina (Italy), where she developed Bicchi. Thanks to her curriculum studiorum the application of “omics” approaches 25. C. Cordero, C. Cagliero, E. Liberto, L. Nicolotti, P. comprehensive chromatography methods she was awarded with the Silver Medal (multidimensionality in sample preparation Rubiolo, B. Sgorbini, and C. Bicchi, J. Chromat. A. (GC×GC, LC×GC) with different interface and (Medaglia d’argento dell’Università di Torino) and separation) to molecular sensory science, 1318, 1–11 (2013). detection systems for the investigation of natural as best graduate student in pharmaceutical followed by advanced data elaboration 26. J. Kief , G. Pollner, and P. Schieberle, J. Agric. Food complex food matrices. Since 2012 she has chemistry and technology for the academic approaches. Chem. 61(22), 5226–5235 (2013). worked as assistant professor at the Department 27. J. Charve, C. Chen, A.D. Hegeman, and G.A. of Food Science at the University of Udine. E-mail: [email protected]; [email protected] Reineccius, Flav. Fragr. J. 26, 429–440 (2011). Her research interests include development Website: http://www.uniud.it; http://www.unito.it 28. W. Grosch, Chem Senses 26, 533–545 (2001). of conventional chromatography techniques

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magentablackcyanyellow ES447494_LCTC060514_009.pgs 05.29.2014 18:17 ADV 10 magenta black cyan yellow www.bis.gov.uk/rgf For more information visit: output is exported. company estimate that around 95% of its America, China, , and Korea. The around the world in Europe, North around people, 340 and has subsidiaries Malvern in the UK,the company employs in Headquartered manufacturing. and that are used in research, development, systems characterization biophysical and fyears. ve of 47 new jobs in the UK over the next that the funding will lead to the creation estimate Malvern employment. sustainable sector investment to create growth and programmes that are also raising private that was set up to support projects and (RGF). The RGF is acompetitive fund government’s Regional Growth Fund secured £3 million support from the UK Malvern Instruments (Malvern, has UK) Government Secures £3 million from UK Malvern Instruments Malvern is amanufacturer of materials 20 2 Monrabal PurcaroQ&A: and Cordero 25 Journal of Agricultural and Chemistry. Food cavities, according to research published in the Red wine could help to protect against oral human microbiota, particular, in microbiota. gut on the interactions of wine polyphenols and had greatly contributed to the knowledge Column The of the study, M. Victoria Moreno-Arribas, told antimicrobial properties of wine. Lead author This prompted the current investigation into the growth of different Streptococcus spp. and grape phenolic could extracts inhibit the worldwide. affect between 60% and 90% of the population the most prevalent oral diseases in humans and cavities, periodontal disease, and tooth loss are of bacteria are diff very cult to remove; dental formation (dental plaque). communities These stick for long periods of time, leading to biof lm the teeth —which allow microorganisms to because it contains nonshedding surfaces — in the sampled red wine. found compounds phenolic to analyze MS–MS) (UHPLC–ESI– ionization electrospray with together chromatography spectrometry mass tandem The team used ultrahigh-pressure liquid Recent research has suggested that wine The mouth is aunique area of the body Sabagh & Valero-Navarro & Sabagh 10 0 : “In the last 5years, our group News Red Wine Health Benefts strains. strains. 1

30 against against with grape seed extract were also effective benef ofts red wine. The solutions spiked health the about knowledge existing and an oral bioflm model ( growth of some pathogenic species in concentrationsmoderate inhibited the passage.”digestive through oraland transformation products polyphenols, as well as for the study of their identif wine of characterization and cation demonstrated to be apowerful tool for the detection to LC systemsESI–MS–MS has been UHPLC–ESI–MS–MS was used: “Coupling of extract. Moreno-Arribas explained why (as acontrol), wine extract, and grape seed red wine, dealcoholized red wine, water biof lm model. The biof lms were dipped into ( Gram-negative Gram-positive ( bacteria responsible for dental diseases, both study.” this wine is rich in polyphenols, we decided to tackle that is behind the wine consumption and that Considering that the oral cavity is the f barrierrst The team concluded that red wine at The team grew avariety of cultures of CHROMacademy S. oralisS. 12 12 F. nucleatum, oralis, S. and 2 ). This confrmed the Tips &Tricks: GPC/SEC A. oris,A. mutans, S. oralis S. F. nucleatum, V. dispar F. nucleatum 31 A. oris A.

Training Events & ), as a . This This . ) and and ) ES447481_LCTC060514_010.pgs 05.29.2014 18:16 ADV 1. I. Muñoz-González, I. Thomas Thurnheer, Begoña 1. Reference inter-individual differences.” K.M. — or adhered to oral surfaces, as well as intra/ considering bacteria present in the saliva and/ studies in ex vivo and in vivo conditions, also model. In the step, next we will perform human pathogenic oral bacteria in abiof lm and grape extract rich in polyphenols to inhibit ability of moderate consumption of red wine this is the f study rst that has demonstrated the care products to prevent dental cavities. of anatural ingredient in the formulation of oral result in particular could lead to the development Moreno-Arribas said: “To our knowledge, 16 4731–4737 (2014).4731–4737 Journal of Agricultural and Chemistry Food Bartolomé, and M.Victoria Moreno-Arribas, Podzimek 33 Staff

62 ,

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HPLC 2014 Agilent Technologies Monitoring High Blood Pressure Best Poster Award Winners The 41st International Symposium on High Treatment Performance Liquid Phase Separations and Related Techniques took place from 11–15 May A new study in the journal Heart suggests that high performance liquid chromatography tandem mass spectrometry (HPLC–MS–MS) can be 2014 in New Orleans, USA. One of the highlights performed as a standard screening tool to identify patients who do not take the anti-hypertensive medications as prescribed by their doctor.1 The of the conference was the poster presentation authors suggest that up to one in four patients is misdiagnosed as suffering with “resistant hypertension”, when in fact the patient has not been sessions, which were entered into a competition taking their prescribed medications as advised. for the Agilent Technologies Best Poster Co-author, Pankaj Gupta, Consultant Metabolic Medicine/Chemical Pathologist at the University of Leicester Hospital, told The Column that the study Award. The winning candidates are as follows: was inspired four years ago when a young patient was referred as being diagnosed with resistant high blood pressure. He said: “Maciej Tomaszewski Stephen Groskreutz from the University of and I had a discussion about the patient after his clinic visit and we were pondering on how we would conf rm his adherence. I was aware of this Pittsburgh (Pennsylvania, USA) won the technique [HPLC] and I conf rmed in our laboratory that with a bit of modif cation we could test for the anti-hypertensive drugs. We were able to f rst place prize with his poster entitled conf rm the absence of all drugs in the patient. The patient admitted to poor adherence to medications when we discussed the results with him. This “Modelling Temperature Assisted Solute subsequently led to the audit — the results of which have been recently published in Heart.” Furthermore, Gupta said that non-adherence can occur in Focusing in Capillary High Performance up to 50% of patients diagnosed with resistant hypertension, and is estimated to cost the UK health economy £4 billion annually. Liquid Chromatography”. The team spot sampled urine from 208 patients with long-standing high blood pressure who had been referred to a specialist hypertension Dina Lakayan from VU University Amsterdam clinic in Leeds, UK. HPLC–MS–MS was performed on the samples to detect the presence of a range of drugs known to be used in the treatment (Amsterdam, The ) came in at of hypertension. One in four patients were not taking their prescribed medication and one in 10 samples did not contain any anti-hypertension second place with a poster entitled medication. The average number of drugs in samples was also lower than expected. Gupta said: “We are the f rst laboratory in the UK and the “Hyphenation of Surface Plasmon Resonance second in the world to set this method up. It detects 40 antihypertensive drugs and requires investment in a specialist and sensitive piece of kit. Optical Sensing to Size Exclusion Chromato- More importantly we are very fortunate to have the very best of expertise in our laboratory graphy for Analysis of Antibody Preparations”. to detect drugs in urine and therefore setting up this method was something we were very In joint third place were Alexander capable of doing.” Siegle from the University of Heidelberg, The results suggest that HPLC–MS–MS could be used as a tool to screen for non-adherence to (Heidelberg, ) with a poster entitled prescribed drug courses, however, the sample size is small and further investigations will need to be “Hadamard Transform Multiplexing LC: carried out in the future. Gupta told The Column: “Since the presentation of our results, there Boosting Through-put and Sensitivity of HPLC” has been a lot of interest nationally and we are receiving samples for routine testing from hypertension and Suresh Kumar from Brigham Young clinics in the UK. There has also been interest expressed by other researchers. We are excited about this University (Utah, USA) with a poster entitled study and have applied for funding for larger projects. I think this test can lead to a paradigm shift in “An Integrated Microf uidic-based System for the way hypertension and other chronic cardiovascular diseases such as diabetes and heart failure Complete Analysis of Preterm Birth Biomarkers”. are managed.” — B.D. For more information please visit: Reference

www.agilent.com 1. M. Tomaszewski, C. White, P. Patel, et al., Heart (First Published Online: 2 April 2014) doi: 10.1136/heartjnl-2013-305. Photo Credit: Henry Steadman/Getty Images

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magentablackcyanyellow ES447482_LCTC060514_011.pgs 05.29.2014 18:16 ADV Tips & Tricks GPC/SEC: The Art of Analyzing High Molar Mass Samples

Daniela Held, PSS Polymer Standards Service GmbH, Mainz, Germany.

Gel permeation/size-exclusion chromatography (GPC/SEC) can be performed for samples that range from a few hundred to several million daltons in molar mass. Analysis of low molar mass samples is relatively straightforward; however, it requires more skill to analyze high molar mass samples in the molar mass range of several million dalton.

The analysis of macromolecules requires if the dissolution process takes several patience, especially when analyzing high days or weeks. Keeping the dissolution molar mass samples. The dissolution time container in a dark environment is also required by macromolecules is signif cant often recommended. If needed, the and can take up to several weeks in a worst dissolution container can be gently swirled case scenario. Dissolution time is dependent to homogenize the solution. on a number of parameters such as sample Lower concentrations of high molar mass and solvent, molar mass, polydispersity, samples should be used than otherwise chain chemistry, crystallinity, composition, recommended when analyzing low and stereochemistry. As a rule of thumb, the molar mass samples. High molar mass higher the molar masses and narrower the molecules need space to occupy their distributions the more time that is required. hydrodynamic volume without interference It is not easy to speed up the polymer from other chains. Table 1 summarizes dissolution process. The use of ultrasonic recommended sample concentrations devices is not recommended because it based on molar mass, but please note that is likely to result in degradation of the these recommendations are for samples sample, and for ultrahigh molar mass with narrow molar mass distributions. samples even the use of a magnetic For samples with a high polydispersity stir bar might result in chain scission. (broad ) higher Therefore, the only option is to wait until concentrations are possible. the sample is dissolved into single isolated The effect of sample concentration on

Photo Credit: Ryan McVay/Getty Images chains. Stabilized solvent should be used elution volume increases with molar mass.

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Figure 1: Overlay of chromatograms of two polystyrene reference materials for three different concentrations. While there is only a minor effect for the 5000 Da sample, the elution volume and even the peak shape of the 500,000 Da sample is highly affected. FRESH CHOICE Polystyrene Molecular weight for proteins Flow rate = 1 mL/min and polymers by SEC-MALS Inj. Vol. = 20 µL Column = SDV 5-µm 10e5 Å, 8 x 300 mm 20 Angles More MALS detector angles = More accuracy. 2 Clicks From data to results - It’s so easy to get precise, repeatable results. Mw = 500,000 Mw = 5000 1 Call 10 mg/mL For Worldwide service and support by Detector Signal (V) the global light scattering experts.

5 mg/mL

1 mg/mL

Elution Volume (mL)

MORE ANGLES MORE DATA MORE CHOICE Figure 1 shows the elution volume and peak position of a sample will be compared to The amazing new Viscotek SEC-MALS 20 detector is the shape for reference materials with molar that of a calibration standard. The change in new choice from Malvern for molecular weight and size of proteins and polymers. With 20 angles, the revolutionary masses of 5000 Da and 500,000 Da. There peak shape (indicating the lack of separation) Xertical ƃow cell with radial oRtics RrodWces the best M#.5 is a very small change seen for the low molar affects all kinds of calibrations, even those data obtainable anywhere. Make the fresh choice now! mass sample, compared to a signif cant with on-line viscometers, light scattering change in the elution volume and the peak detectors, or triple detection systems. shape of the high molar mass reference Filtering sample solutions through a CLICK HERE standard. The change in elution volume is membrane filter is usually recommended TO FIND OUT MORE AT

especially problematic when the GPC/SEC for solutions containing gels or particles, www.malvern.com/morechoice Material relationships system is calibrated with narrow distributed but care must be taken for high molar molar mass standards where the peak mass samples. It is advisable to adjust

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Figure 2: Overlay of chromatograms for a high molar mass sample of 3.8 million Da obtained at 0.5 mL/min f ow rate (red trace) and 0.25 mL/min f ow rate (green trace). Reducing the f ow rate to 0.1 mL/min can further improve the results.

Flow rate 0.25 mL/min

Flow rate 0.50 mL/min Detector Response

Elution Volume (mL)

the pore size of the filter to avoid sample • Larger particle sizes are generally used for degradation, and if at all possible, filtration higher molecular weights to avoid shear of high molar mass sample solutions should degradation of the samples. be avoided completely. For samples that are several million Dalton Chromatographic Conditions in molar mass, additional experimental The following rules apply for all samples that aspects need to be considered. The most are to be analyzed with GPC/SEC: important step is to adapt the flow rate • The separation of higher molar masses of the system, because polymers show requires separation columns with larger significant peak broadening or unexpected pores. peak shapes when run at too high flow

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Figure 3: Slice concentration (measured using an RI) and on-line determined molar mass An on-line light scattering detector a reduction of the draw-speed of the (MALLS) for a run with a too high f ow rate (0.5 mL/min). is a good tool to identify unsuitable autosampler is recommended. This chromatographic conditions. These detectors will increase the reproducibility of the are often required to overcome the lack of injections. ultrahigh molar mass calibration standards, and so many setups already include such a Good Practices for the Analysis of device. High Molar Masses Figure 3 shows the multi-angle light • Be patient and give enough time for scattering results for the high molar mass dissolution. sample at a flow rate of 0.5 mL/min. The • Use low concentrations and, if required, measured molar mass stays nearly constant increase the injection volume. Slice concentration Molar Mass (MALLS) for a wide elution volume range. This is • If possible, avoid sample f ltration. If this is the result of inefficient separation because not possible use adequate f lter pore sizes. unsuitable chromatographic conditions • Use low flow rates (0.25–0.1 mL/min) have been applied. and reduce the autosampler draw- Other practical considerations to make speed.

Elution Volume (mL) when analyzing high molar masses are • Use columns with a large particle detection limit and sample viscosity. The size and large pores. In the case S/N ratio for concentration detectors may of suspicious shoulders in the Table 1: Recommended sample concentrations with respect to sample molar mass. be too low, because low concentrations chromatogram check the exclusion limit are applied. However, instead of increasing of the columns. Molar Mass Range Concentration the sample concentration, it is better 100–10,000 2 mg/mL (0.2%) to increase the injection volume for the Daniela Held studied polymer chemistry 10,000–1,000,000 1–2 mg/mL (0.1–0.2%) injection because this will also increase the in Mainz, Germany, and works in the PSS > 1,000,000 0.5 mg/mL or less (0.05%) overall injected mass and therefore improve software and instrument department. the signals. Please note that this is not She is also responsible for education and rates as a result of their extremely low the long polymer chains. Figure 2 shows recommended for lower molar masses. In customer training. diffusion coefficients. Reducing the flow an overlay of a high molar mass sample those cases it is better to increase rate to 0.25 mL/min or even less will result measured at 0.5 mL/min and at 0.25 mL/ the concentration and to inject a small in more realistic peak shapes for high min. The curve for 0.50 mL/min is shifted volume. E-mail: [email protected] molar mass samples. Another factor to be to artificially high elution volumes, and If the sample viscosity is high because Website: www.pss-polymer.com considered is shear-induced stretching of again the peak shape changes. of the presence of high molar masses,

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magentablackcyanyellow ES447502_LCTC060514_015.pgs 05.29.2014 18:18 ADV Principles of Detection and Characterization of Branching in Synthetic and Natural Polymers by MALS

Stepan Podzimek, SYNPO, Pardubice, Czech Republic, and University of Pardubice, Institute of Chemistry and Technology of Macromolecular Materials, Pardubice, Czech Republic.

Branching is an important structural parameter of many synthetic and natural polymers. It can inf uence the mechanical and thermodynamic properties of polymers, and also affect the viscosity and rheological behaviour of polymer solutions and melts. Quantitative data about branching topology is therefore vital to understanding polymerization processes and the development of novel polymer-based materials with enhanced properties. Multi-angle light scattering (MALS) is one analytical technique that can be performed to identify branching in macromolecules. This article provides insight into the basic principles of this technique, and how it can be applied to the detection and characterization of branching.

Branching is widely recognized as relevant each size fraction individually to obtain a behaviour. AF4–MALS is therefore ideal same molar mass (M). R and M are both to synthetic polymers, but has more complete and accurate distribution. for the separation of large and highly determined independently of MALS. A recently become relevant to natural The most common method of separating branched macromolecules. MALS provides differential refractive index (dRI) detector polymers. For example, hyaluronic acid, polymers in solution is gel permeation or the required quantitative information about is used for measuring concentration. The an important biopolymer with numerous size-exclusion chromatography (GPC/SEC). branching topology. branching ratio (g) is directly related to medical and pharmaceutical applications, SEC–MALS is a well-established technique the number of branch units in randomly was believed to have a linear structure until for the absolute characterization of typical The Theory Behind Branching branched polymers or to the number of multi-angle light scattering (MALS) analysis polymers; however, large and highly The development of quantitative branching arms in star-branched polymers.2 In general, proved otherwise.1 branched polymers can exhibit abnormal analysis began in 1949 when Zimm and g ≤ 1 where the equality sign stands for Full characterization of branching requires conformation plots in SEC.5 An alternative Stockmayer2 introduced the theoretically linear polymers. Lower values of g tend to the coupling of a separation device to method is asymmetrical f ow f eld-f ow derived “branching ratio” (g): correspond to higher degrees of branching. separate molecules of varying size over a fractionation (AF4) coupled with MALS. AF4 R2 For example: g ≈ 0.1–0.2 indicates a highly = branched period of time; and an analytical detector does not require the diffusion of molecules g 2 [1] branched structure. Rlinear to determine molecular properties such as in and out of a porous solid phase, and is M Ten years after the def nition of g by Zimm molar mass, size, or branching ratio. This therefore not subject to the “anchoring” R2 is the mean square radius of branched and Stockmayer, Zimm and Kilb3 introduced

coupling allows the detector to characterize mechanism that leads to abnormal elution and linear macromolecules having the an alternative branching ratio based on Photo Credit: Naoko Suguta/Getty Images

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Figure 1: Analysis of linear (blue) and branched (red) poly(lactic acid). (a): Mark-Houwink obtained by performing the following Suitability of Ionic plots, exhibiting slopes of 0.56 and 0.31 for linear and branched molecules, respectively. methods: (b): Molar mass versus elution volume overlaid with RI chromatograms. • Liquid GC Columns for Radius method: Calculate g from the Various Applications 35 conformation plot (log–log plot of R versus (a) (b) 30 M) using equation 1, MALS, and dRI 25 LIVE WEBCAST: 5 20 10 detectors. Thursday, June 12, 2014 at 8:00 am PDT/ 10:00 am CDT/ 11:00 am EDT 15 • Viscosity method: Calculate g’ from the Register free at Mark-Houwink plot (log–log plot of [η] http://www.chromatographyonline.com/suitability 10 104 versus M) using equation 2, MALS, dRI, EVENT OVERVIEW: The next great platform in GC stationary phase technology is here: and dVI. the ionic liquids. Several inherent features result in chromato- Molar Mass (g/mol) graphic benefts over polysiloxane polymer and Intrinsic Viscosity (mL/g) 5 • Mass method: Calculate g from the plot glycol stationary phases. Some of our recent work has progressed 103 of molar mass versus elution volume towards specifc uses in chemical, petroleum, food and beverage, 103 104 105 12 13 14 15 16 17 agriculture, favor and fragrance, environmental, biofuel, and clini- cal industries. Discussed will be: Molar Mass (g/mol) Volume (mL) using equation 4, MALS, and dRI, plus n Our process for evaluating selectivity measurement of a linear counterpart under n Our naming system, based on the McReynolds method n Chromatographic performance, using multiple intrinsic viscosity: to determining root mean square (RMS) the same SEC conditions as those used for example chromatograms radii above ~10 nm (corresponding to a branched sample Key Learning Objectives: []η branched 5 1. Physical and chemical makeup of an ionic liquid GC stationary g‘ = [2] molar mass of ≈ 10 g/mol for typical phase. []η linear 2. Features and benefts of ionic liquid stationary phases compared M polymers). Alternatively, either intrinsic The radius method is the simplest to to polysiloxane polymer and polyethylene glycol GC stationary phases. viscosity or SEC elution volume can be used implement, but for polymers smaller than 3. Suitability for several applications, including industrial solvents, impurities in feedstocks, downstream where [η] is the intrinsic viscosity of branched as a size parameter. The former is used in ~10 nm in radius, the viscosity or mass petrochemical products, benzene in gasoline, PAHs, omega 3 and omega 6 FAMEs, cis/trans FAME isomers, FAME profle of biodiesel, and linear polymer molecules having the same equation 2, whereas the latter appears in method is required. profle of essential oils, and PCBs. molar mass. [η] and M are determined using a the approach of Yu and Rollings: 4 Who Should Attend: n GC and GC-MS method developers and analysts, lab managers, + MALS, dRI, and a differential viscometry (dVI) l a Case Studies and lab auditors. e detector for intrinsic viscosity. The relationship M Polyester based on lactic acid (PLA) is a Presenters: = linear between g’ and g is described via the g [4] biocompatible and biodegradable polymer Mr. Leonard M. Sidisky M R&D Manager (Gas Separations), branched V so-called “draining parameter” (e): that can be used as a drug delivery Sigma-Aldrich/Supelco, Bellefonte, PA, USA M is the molar mass of linear and branched material. Its ability to swell, degrade, Moderator: ‘ = e [3] g g Kristen Moore molecules eluting at the same elution and release an active compound can be Webcast Manager The parameter e is expected to vary in the volume, V, and a is the exponent of the controlled by the degree of branching. LCGC range of 0.5–1.5, but a typical value is e ≈ 0.7. Mark-Houwink equation for a linear The release of an active compound can Presented by Sponsored by MALS can measure molar masses from polymer. M is determined by MALS + dRI. be controlled by altering the degree of below 1 kDa up to ~1 GDa, but is limited In summary, the branching ratio may be branching to alter the rate of swelling and For questions, contact Kristen Moore at [email protected]

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Figure 2: (a): Conformation plots of linear (blue) and branched (red) polystyrene. (b): The an Agilent 1100 HPLC instrument corresponding plot of branching ratio versus molar mass. (c): The number of branch units (Agilent Technologies). Tetrahydrofuran From TLC to Flash per molecule plotted versus molar mass. The plot of branch units per molecule versus molar was the solvent for both the SEC and to Prep-HPLC An all-in-one solution, the mass is overlaid with the cumulative molar mass distribution (red), and the 3rd order f t to AF4 analysis. experimental data points (magenta). The slopes of the conformation plots of linear and NEW BUCHI PrepChrom C-700 The characterization of branching for LIVE WEBCAST: branched polymer are 0.59 and 0.48, respectively. Wednesday, June 18, 2014 at 8:00 am PDT/ small PLA molecules is depicted in Figure 1, 11:00 am EDT/ 4:00 pm BST/ 5:00 pm CEST 60 (a) (b) 1.0 which compares Mark-Houwink plots Register free at www.chromatographyonline.com/prepchrom 50 and plots of molar mass versus elution EVENT OVERVIEW: 40 What if there were a way to combine fash chromatography 0.8 volume of linear and branched molecules. and preparative HPLC into one solution that took up a quarter of the space? Learn about how you can go from TLC to purifed 30 Both indicate the presence of branched product without multiple instruments and without being an 0.6 expert in chromatography. A test application will show how molecules and may be used to calculate g one product can give you the results you need quickly and 20 by means of equations 2–4. Figure 2 shows easily. Branching Ratio

RMS Radius (nm) 0.4 Key Learning Objectives: conformation plots of linear and branched n Learn about your options to purify compounds and reaction polystyrene. Branching is shown by the mixtures. 10 0.2 n Learn how several techniques involved in preparative 105 106 105 106 measured slopes of 0.59 and 0.48. These chromatography can be combined into one solution. n See interesting applications optimized from TLC to fash and Molar Mass (g/mol) Molar Mass (g/mol) can be compared with the two limiting from fash to HPLC fractions. (c) 8 1.0 theoretical values: 0.58 for linear polymers If you are a scientist involved in the following felds: n Medicinal chemistry in thermodynamically good solvents, and n Organic synthesis 0.8 n Natural product research 6 0.33 for compact spheres that can be n Polymer chemists n Nutraceutical research 0.6 considered “inf nitely branched”. …and you’re struggling with difcult separations, 4 The conformation data transform to plots this webinar will address your needs. 0.4 of molar mass dependency of the branching Presenters: Jason Wagner 2 ratio, and the number of branch units per Product Manager for Chromatography 0.2 BUCHI Corporation, New Castle, Delaware

molecule, shown in Figure 2. Overlaying the Dr. Birke Gotz Branch Units per Molecule

Cumulative Weight Fraction Global Product Manager for Preparative Chromatography 0 0.0 branching units plot with the cumulative BUCHI Labortechnik, Flawil, Switzerland 4 5 6 10 10 10 distribution of molar mass facilitates Moderator: Molar Mass (g/mol) Kristen Moore quantitative evaluation of branching. Multimedia Producer LCGC Figure 2 shows that ≈ 28% of molecules Sponsored by Presented by degradation of the polymer. an Optilab T-rEX refractive index detector, with molar masses below ≈ 60,000 g/mol Method: The data presented in this study and an Eclipse A4F system, and processed do not contain branch units. Notably,

were obtained with a DAWN HELEOS MALS with Astra 6 software, all from Wyatt SEC–MALS is capable of detecting just a For questions, contact Kristen Moore at [email protected] photometer, a ViscoStar on-line viscometer, Technology. SEC was performed with single branch until in polymer chains.

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For such polymers, AF4 has proven to References Figure 3: Conformation plots of cellulose tricarbanilate, a branched polymer exhibiting 6 “anchoring”, determined by SEC–MALS (red) and AF4–MALS (blue). provide better results. 1. S. Podzimek, M. Hermannova, H. Bilerova, Z. A comparison of conformation plots Bezakova, and V. Velebny, J. Appl. Polymer Sci. obtained by SEC–MALS and AF4–MALS 116, 3013 (2010). 100 for cellulose tricarbanilate is depicted in 2. B.H. Zimm and W.H. Stockmayer, J. Chem. Figure 3. The separation by AF4 is not Phys. 17, 1301 (1949). affected by the anchoring of branched 3. B.H. Zimm and R.W. Kilb, J. Polym. Sci. 37, 19 molecules and the upswing is completely (1959). eliminated. 4. L.P. Yu and J.E. Rollings, J. Appl. Polym. Sci. 33, 1909 (1987). Conclusion 5. S. Podzimek, T. Vlcek, and C. Johann, J. Appl. The demand for an absolute technique that Polym. Sci. 81, 1588 (2001).

RMS Radius (nm) can provide robust and reliable polymer 6. S. Podzimek: Light Scattering, Size Exclusion characterization has led to recognition of the Chromatography and Asymmetric Flow powerful capabilities of MALS. This study Field Flow Fractionation, Wiley, 2011. DOI: 10 shows that the most direct and fundamentally [978-0470386170]. 105 106 107 correct technique for characterizing branching Molar Mass (g/mol) in polymers is the MALS-based radius Stepan Podzimek heads the Department method, though it is limited to molecules of Analytical and Physical Chemistry The SEC–MALS radius method may MALS measures the weight-average molar with RMS radii > 10 nm. Smaller branched at SYNPO, a Czech R&D company fail for some large, highly branched mass (Mw) but the z-average RMS radius polymers can be characterized by adding conducting contract research in synthetic polymers because of limitations of the (Rz). As long as elution fractions are a differential viscometer to a SEC–MALS polymers and related materials. He SEC separation mechanism, where the reasonably monodisperse, the weightings system for Mark-Houwink plots, or MALS- also holds a professorial position at the branches are temporarily “anchored” in of Rz and Mw are nearly identical, but based determination of the relation between Institute of Chemistry and Technology of the pores of SEC column packing.5 These with increased polydispersity they the molar mass and elution volume. When Macromolecular Materials at the University polymers then elute abnormally at a diverge. The combination of polydisperse, separation is adversely impacted by the of Pardubice, Czech Republic, and is a retention time that corresponds to a much abnormal SEC elution with disparate anchoring of branched macromolecules in scientific consultant for Wyatt Technology smaller hydrodynamic volume than actually weightings of R and M by MALS results the pores of SEC column packing, AF4–MALS Corporation. presented by the molecule.5 As a result, in upswings on the conformation plots offers great separation and yields correct fractions at large elution volumes become at the low end of the molar mass axis conformation plots and branching ratios. E-mail: [email protected] highly polydisperse containing both very (horizontal) and consequently incorrect AF4–MALS is suitable for all types Website: www.synpo.cz small and very large branched species. values of g.5 of polymers.

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magentablackcyanyellow ES447509_LCTC060514_019.pgs 05.29.2014 18:19 ADV Polyolef n Characterization by High Temperature Adsorption Liquid Chromatography: A Review of Solvent Gradient (SGIC) and Thermal Gradient Interaction Chromatography (TGIC)

B. Monrabal, Polymer Char, Valencia, Spain.

This article provides an overview of solvent gradient interaction chromatography (SGIC) and thermal gradient interaction chromatography (TGIC) for polyolef n characterization.

Polyolef n resins account for more than half of (TREF),1 crystallization analysis fractionation the world’s plastics demand and, in spite of (CRYSTAF),2 and crystallization elution their simple chemistry (carbon and hydrogen fractionation (CEF).3 All of these techniques atoms), they can be made in a variety of separate the polymer molecules according complex microstructures that demand to crystallizability and provide a predictable extensive characterization. Most polyolef ns separation of the polymer fractions depending are copolymers of ethylene or propylene on the presence of branches, irregularities, with the incorporation of other alpha olef n or tacticity differences. In recent years new co-monomers that result in short chain copolymers of lower crystallinity have been branches, which may or may not be uniformly developed, extending the polyolef n products distributed between molecules. The analysis into the elastomers region. Consequently, of the co-monomer distribution in the chain, new characterization techniques also have also referred to as short chain branching to be developed to properly characterize (SCBD) or chemical composition distribution those more amorphous resins. The most (CCD), is in many cases the most fundamental signif cant contribution has been the use of microstructure feature in the industrial resins. high temperature liquid chromatography in The CCD is typically measured by adsorption mode on graphitized carbon.4 The

Photo Credit: Alto Classic/Getty Images established crystallization techniques: use of this adsorbent in the characterization temperature rising elution fractionation of polyolef ns has been intensively investigated

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Figure 1: Analysis of different ethylene copolymers by SGIC on a graphitized carbon column. Adapted and reprinted with permission from Analytical and Bioanalytical Chemistry 399(4), T. Macko, R. Brüli, R.G. Alamo, F.J. Stadler, and S. Losi, Separation of short-chain branched polyolef ns by high-temperature gradient adsorption liquid chromatography, 1547–1556. Features of Different Mass Analyzers © 2011 Springer (7). Applied to Multi-Allergen Screening E/C2 [47-49] E/C4 [47-49] ON-DEMAND WEBCAST 14.0 E/C6 [47-49] (Fig. 4) Register free at http://www.chromatographyonline.com/Multi_Allergen E/C6 [49] (Fig. 5) E/C6 [50-53] (Fig. 6) EVENT OVERVIEW: Presenters: 13.5 E/C8 [54] Mass spectrometry can provide some advantages over more tradi- E/MP [54] tional methods of allergen detection like ELISA and PCR, including Linda Monaci Institute of Sciences E/MP [55, 56] quantitation and reduced risk of false positives and false nega- of Food Production tives. Features provided by the latest technology of mass analyz- CNR-ISPA (Bari, Italy) ers have pushed in the direction of developing multi-target and 13.0 sensitive MS-based methods, provided that certain requirements are met for the fnal protein identifcation. Moderator: In this webinar, a comparison of two mass spectrometry-based Laura Bush Editorial Director Elution volume (mL) approaches for multiplex detection of residual allergens in food 12.5 LCGC matrices will be discussed. As it will be presented, the Thermo Scientifc Exactive Orbitrap mass spectrometer enabled untar- geted, data independent acquisition at the highest resolving Who Should Attend: 12.0 power, generating extracted ion chromatograms based on the 0 2 4 6 8 10 12 14 16 18 20 22 accurate masses of the selected marker ions along with their n Laboratory managers and technical respective fragments. As an alternative, the use of the Thermo specialists working in food safety analysis Content of alkene (mol %) Scientifc LTQ Velos linear ion trap mass spectrometer allowed high n Analytical professionals from regulatory speed data dependent MS/MS acquisition to search for proper and ofcial control authorities focused on peptide markers and transitions to be further implemented in an allergen research in recent years by many researchers using has been extensively investigated by SRM method. n Analytical and R&D scientists interested 6 in new developments in accurate mass a solvent gradient approach which has Professor Pasch’s group at the DKI (now spectrometry in the feld of protein and Key Learning Objectives: become known as solvent gradient interaction Fraunhofer Institute) in Germany. High peptide detection n Discover how mass spectrometry helps to overcome chromatography (SGIC), or a temperature temperature is needed for the dissolution traditional challenges of immunoassay-based methods Sponsored by n Learn how to improve your lab throughput and save costs by gradient approach, known as thermal gradient of the resin and the non-functionality of performing routine screening for food allergens interaction chromatography (TGIC).5 the polymer molecules make it diff cult to n Learn how to improve confdence in your data by eliminating false positives and negatives use LC in interaction mode for polyolef n Presented by Solvent Gradient Interaction analysis. Chromatography (SGIC) An important turning point came For questions, contact Kristen Moore at [email protected] In the last decade the application of liquid with the f nding of a graphitized carbon chromatography (LC) to analyze polyolef ns column by Macko and Pasch4 that could

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Figure 2: TGIC analysis of a series of octene copolymers on a graphitized carbon column and calibration curve. Adapted and reprinted with permission from Macromolecules 44(8), R. Cong, W. DeGroot, A. Parrott, W. Yau, L. Hazlitt, R. Brown, M. Miller, and Z. Zhou, A new technique for characterizing comonomer distribution in polyolef ns: High-temperature thermal gradient interaction chromatography (HT–TGIC), 3062–3072. © 2011 American Chemical Society (5).

0.33

EO-1 0.0 mol% octene 0.28 EO-2 1.33 mol% octene EO-3 3.99 mol% octene 0.23 EO-4 8.52 mol% octene 160 EO-5 13.88 mol% octene 140 0.18 EO-6 19.0 mol% octene y=-3.0704x + 149.8 2 EO-7 21.7 mol% octene 120 R = 0.9905 C) 0.13 EO-8 32.5 mol% octene o 100

IR-4 Output EO-9 50.7 mol% octene 80 0.08 60 of TGIC ( 40 0.03 Peak temperature Peak temperature 20 -0.02 0 -5 15 35 55 75 95 115 135 155 0 10 20 30 40 50 60 Ocetene mol% Elution temperature (oC)

perform the separation of polyethylene and obtained between the co-monomer mole could handle the various percentage incorporated and the elution tacticity conf gurations using a gradient volume. These were similar results to that of decanol-trichlorobenzene in a very obtained by the crystallization techniques, short analysis time. The most signif cant but were capable of extending the analysis work came later, with the separation to the elastomers region. of ethylene-copolymers by the level of Solvent gradient interaction co-monomer incorporation,7 again using a chromatography (SGIC) can be used to graphitized carbon column, and resulting analyze copolymers from 0% to 100% of in a linear calibration as shown in Figure 1. co-monomer incorporation, which is not Similar results were obtained by Miller et al.8 possible with crystallization techniques. using the same carbon column. The presence SGIC has been used in the separation of branches in the ethylene-copolymers of ethylene-propylene copolymers9 and reduced their surface area of interaction the analysis of ethylene propylene diene with graphite, and a linear correlation was monomer (EPDM) resins.10 A drawback of the

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Figure 3: TGIC analysis of a series of octene copolymers on different adsorbents and of synthetic polymers by molar mass on other layered adsorbents was proposed by comparison with a TREF column. Paper presented by B. Monrabal at the Macromolecule a reversed-phase column.16 A different Monrabal.18 This is based on weak van der Symposium (2013). approach was suggested by Cong et al.5 Waals forces interaction of the non-polar when they used temperature gradient polyolef n with an atomic level f at surface 160 TGIC and TREF corrected To chromatography with a graphitized carbon (ALFS) adsorbent. The adsorption strength 140 TGIC 20/2/0.5 TREF 0.5/1/0.5 column that resulted in the separation of depends on the available surface area of o-DCB 120 polyolef ns by composition as shown in the molecule in contact with the adsorbent, Figure 2. This technique is known as thermal and the chemical structure of graphene 100 C)

o Graphites gradient interaction chromatography (TGIC). should not be as important for interaction

80 WS2 The separation obtained was similar to that with the polymer as the ALFS itself. This was BN Absorption MoS2 seen in SGIC, but in this case there was the conf rmed by using other types of layered

Peak T ( 60 advantage of the isocratic conditions, which packing materials like molybdenum sulphide, 40 allowed the use of a linear IR detector, as boron nitride, and tungsten sulphide, which

20 TREF well as an in-line viscometer or LS molar mass provided the same organized separation detection. order as the graphitized carbon column in 0 0 2 4 6 8 10 12 14 16 The comparison of TGIC and TREF for spite of their different surface chemistry and Ocetene mol% a series of ethylene-octene copolymers polarity. This is shown in Figure 3 for a series has been reported,17 demonstrating that of ethylene octene copolymers. resolution with TREF is slightly better than The only difference among ALFS adsorbents technique is the current lack of appropriate composition interdependence could also be with TGIC for medium density resins. TREF is the strength of adsorption, but all of them detectors. analyzed.11 or other crystallization techniques are result in linear calibration curves with the To overcome detector limitations, the SGIC 2D has been utilized for the also more appropriate for industrial LLDPE same slope placed above the TREF (dissolution combination of SGIC with gel permeation/ characterization of ethylene-propylene materials. However, TGIC does not suffer after crystallization) calibration line. size-exclusion chromatography (GPC/SEC) in copolymers, EPDM resins,13 high impact from co-crystallization effects and it covers Polypropylene (PP) homopolymer is a second dimension (SGIC 2D) was proposed polypropylene,14 and ethylene octene a broader copolymer range down to the semicrystalline, and because it has so many by Roy et al.11 using a gradient of decanol copolymers, alongside the addition of light elastomer region, which crystallization methyl branches that restrict adsorption, or ethylene-glycol mono-butyl-ether and scattering (LS) and viscosity detectors.15 techniques cannot reach. TGIC’s broad range it does crystallize before it adsorbs in ALFS trichlorobenzene (TCB) on a graphitized of separation down to 50% octene mol5 is adsorbents; therefore, when analyzing it by carbon column. A similar approach was also Thermal Gradient Interaction smaller than that of SGIC, which covers the TGIC it elutes at the TREF temperature.18 The followed by Ginsburg et al.12 The GPC/SEC Chromatography (TGIC) full range of co-monomer incorporation. separation of ethylene-propylene copolymers second dimension used infrared (IR) detection Temperature gradient chromatography has A separation mechanism for interaction results in a U-shape calibration line where the and, besides the convenience, the molar mass been shown to be of value in the separation chromatography on graphitized carbon and ethylene-rich copolymers are separated by

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adsorption (TGIC), while the propylene-rich for both approaches and it is related to the References V.B.F. Mathot, and H. Pasch, Macromolecules 45, copolymers are separated by crystallization spatial microstructure of the polymer chain, 1. L. Wild and T. Ryle, Polym. Prepr. (Am. Chem. 2025−2034 (2012). (TREF).18,19 which def nes the surface area available for Soc., Div. Polym. Chem.). Div. 18, 182 (1977). 15. D. Lee, M.D. Miller, M.D. Meunier, J.W. Lyons, Other than the peculiar behaviour of PP interaction with an ALFS. 2. B. Monrabal, J. Appl. Polym. Sci. 52, 491 (1994). J.M. Bonner, R.J. Pell, and C. Li Pi Shan Huang, J. resins, TGIC is a very practical technique to SGIC can analyze the whole range of 3. B. Monrabal, J. Sancho-Tello, N. Mayo, and L. Chromatogr. A. 1218, 7173 (2011). analyze polyolef n elastomer resins. It has ethylene-copolymers using the broad range Romero, Macromol. Symp. 257, 71 (2007). 16. T. Chang, H.C. Lee, W. Lee, S. Park, and C. Ko, also been used for the analysis of olef n block of solvent strength available in the approach. 4. T. Macko and H. Pasch, Macromolecules 42, Macromol. Chem. Phys. 200(10), 2188 (1999). copolymers.20 This is potentially the most important 6063 (2009). 17. B. Monrabal, N. Mayo, and R. Cong, Macromol. attribute of the technique, together with 5. R. Cong, W. DeGroot, A. Parrott, W. Yau, Symp. 12, 115-129 (2012). Conclusions ability to obtain narrow peaks with short L. Hazlitt, R. Brown, M. Miller, and Z. Zhou, 18. B. Monrabal, E. Lopez, and L. Romero, Crystallization techniques appear to be analysis time. However, SGIC is currently Macromolecules 44(8), 3062–3072 (2011). Macromol. Symp. 330, 9–21 (2013). the preferred approach for the analysis limited to the high temperature ELSD 6. H. Pasch, Polym. Chem. 4, 2628–2650 (2013). 19. B. Monrabal and L. Romero, Macromol. Chem of semicrystalline industrial polyolef ns detectors that have been discontinued 7. T. Macko, R. Brüll, R.G. Alamo, F.J. Stadler, and and Phys. (2014) , accepted for publication. down to approximately 20% co-monomer commercially. S. Losi, Anal. Bioanal. Chem. 399(4), 1547–1556 20. R. Cong, Proceedings of International Conference incorporation. Above this co-monomer SGIC 2D overcomes the solvent gradient (2011). on Polyolef ns Characterization, Houston, USA, level the crystallinity is so neglible that detection diff culties by adding an isocratic 8. M.D. Miller, A.W. deGroot, J.W. Lyons, F.A. Van 2012. crystallization cannot be used as a separation separation step by molar mass, which also Damme, and B.L. Winniford, J. Appl. Polym. Sci. method. takes care of the solvent removal. SGIC 123, 1238–1244 (2012). Benjamin Monrabal is the R&D director at The development of high temperature 2D isocratic elution allows for infrared and 9. T. Macko, R. Brüll, Y. Wang, B. Coto, and I. Polymer Char, Valencia, Spain. He received interaction chromatography for the analysis optional viscosity and/or LS detection. Suarez, J. Appl. Polym. Sci. 122, 3211 (2011). his degree in chemical engineering from of polyolef n copolymers comes at a time TGIC is performed on a simple TREF or 10. R. Chitta, T. Macko, R. Brüll, G. Van Doremaele, the Instituto Químico de Sarriá in Barcelona, when new elastomer resins appeared on the CEF instrument with isocratic elution that and L.C. Heinz, J. Polym. Sci., Part A: Polym. Spain; and his PhD in polymer chemistry market that cannot be analyzed by standard allows the use of infrared and molar mass Chem. 49, 1840 (2011). from Virginia Polytechnic Institute, USA. crystallization techniques. The most signif cant detectors. It requires two temperature 11. A. Roy, D.M. Miller, D.M. Meunier, W. DeGroot, He is the inventor of the CRYSTAF and CEF development is the use of adsorbents cycles (adsorption and desorption) in a W.L. Winniford, F.A. Van Damme, R.J. Pell, and technologies, and has been responsible for the such as graphitized carbon that separate similar way to crystallization techniques. J.W. Lyons, Macromolecules 43, 3710–3720 design of new instrumentation for polyolef ns ethylene copolymers by the level of branches Ethylene-copolymers can be analyzed down (2010). characterization. He is currently developing incorporated. Chromatographic separation to approximately a 50% co-monomer 12. A. Ginsburg, T. Macko, V. Dolle, and R. Brüll, J. methods for interaction chromatography. with these adsorbents can be performed content; the limit is given by the temperature Chromatogr. A 217, 6867 (2010). using a solvent gradient or a thermal gradient required to adsorb the most amorphous 13. A. Ginsburg, T. Macko, V. Dolle, and R. Brüll, Eur. E-mail: [email protected] approach. The organized separation order in copolymer, as well as the solvent strength and Polym. J. 47, 319–329 (2011). Website: www.polymerchar.com the series of ethylene-copolymers is the same freezing point. 14. S. Cheruthazhekatt, T.F.J. Pijpers, G.W. Harding,

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magentablackcyanyellow ES447521_LCTC060514_024.pgs 05.29.2014 18:21 ADV Using Multi-Detection GPC/SEC to Determine Impact of Sterilization on Medical-Grade Polymers

Bassem Sabagh1 and Angel Valero-Navarro2, 1Malvern Instruments, Malvern, UK, 2nanoMyP, Armilla, Granada, Spain.

Sterilization is a core part of the medical-grade polymer formulation process. However, high-energy sterilization techniques can have a destructive inf uence on core polymer features inducing chain scission, bond cleavage, and changes in molecular weight (Mw). For high-end polymers with engineered functionality, such as novel medical materials for timed drug release, the inf uence of sterilization must be carefully monitored because changes to polymer structure can substantially alter performance. Traditional single detection gel permeation/size-exclusion chromatography (GPC/SEC) is limited in its application for novel material analysis because there are not the appropriate reference standards needed and it does not give the advanced structural analysis required. Multi-detection GPC/SEC overcomes these issues by combining light scattering, refractive index (RI), and viscometry detection to determine absolute Mw and detailed structural information. This article gives an introduction to multi-detection GPC/SEC and presents new data showing how the technique is being used to determine the impact of sterilization on medical grade poly(L-lactide).

Polymer-based controlled drug release systems a given specif cation by precisely engineering the level needed to induce polymer bond and macromolecules. The f rst stage of the are used to deliver discrete therapeutic drug performance-def ning polymer characteristics breakage and chain scission, which means analysis involves the separation of a dissolved dosages to patients over days, weeks, and — including molecular weight (Mw), molecular that sterilization can directly affect molecular sample on the basis of size using a column even months following a single injection. weight distribution, and structural features such weight characteristics. Appropriate polymer containing microporous packing material. These systems avoid the need for repeated as branching — during the formulation process. characterization techniques provide the The second step involves detection of eluting administration, and so decrease patient It is vital to monitor molecular weight detailed insight required to quantify these compounds, and this determines the amount discomfort and save on healthcare resources. parameters throughout the preparatory stages changes. of information generated. Certain polymer materials can be manipulated leading up to administration, particularly Traditional GPC/SEC techniques incorporate to dissolve at selective rates in vivo to ensure following sterilization. Energy applied by Multi-Detection GPC/SEC a single refractive index (RI) detector that that a precise quantity of drug is released at processes such as gamma irradiation and GPC/SEC enables the determination of determines the concentration of samples in

the required time. Such behaviour is tailored to electron collision sterilization often exceed molecular weight for polymers, proteins, each size fraction and produces a relative Malo/Getty Miguel Credit: ImagesPhoto

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Figure 1: (a) Smaller, isotropic scattering molecules scatter light evenly in all directions while E-Book Educational Series (b) anisotropic scatterers scatter light at different angles at different intensities. Advances in GC-MS (a) : Isotropic scattering Analysis of Pesticides

Advances in GC-MS Analysis of Pesticides Constructive interference In this new e-book, authors from the US Department (b) : Anisotropic scattering of Agriculture, academia, and a European Union Reference Laboratory discuss advances in the Scattered light analysis of pesticide residues o at 0 using gas chromatography– mass spectrometry (GC–MS) methods. Destructive interference A format for ARTICLES INCLUDE DISCUSSIONS OF: everyone – How to improve detection limits and reduce matrix interferences molecular weight distribution via a calibration Modern GPC/SEC instruments often This new e-book is to extend the range of pesticides that can be analyzed with GC curve. It relies on external reference standards incorporate additional detectors alongside available for the iPad The advantages of negative chemical ionization (NCI) instead of electron ionization (ESI) for calibration because an RI detector, in RI to increase the information f ow from or for a standard computer web browser Using tandem mass spectrometry in SRM isolation, is unable to measure absolute each experiment. For example, triple mode to improve selectivity molecular weight. In addition, single detection detection GPC/SEC also includes a light DOWNLOAD PDF Lowering detection limits through large-volume injection (LVI) using cold on-column injection or a programmable-temperature vaporizer GPC/SEC offers no information about scattering detector and a viscometer. A TODAY! The benefits of using an accurate-mass database the material structural characteristics. For light-scattering detector uses the direct for screening pesticides by GC-TOF-MS custom-engineered polymers without easily relationship between the angle and A comparison of qualitative and quantitative results obtained using GC –TOF-MS and GC-triple-quadrupole MS assessable reference standards, and where intensity at which molecules scatter light, SPONSORED BY Sample preparation using QuEChERS with acetonitrile structure strongly inf uences functionality, and the molecular mass to calculate extraction and dispersive solid-phase extraction (dSPE) single detection GPC/SEC cannot provide the absolute molecular weight data without An evaluation and comparison of three dSPE sorbent combinations, The world leader in serving science comparing cleanup efficiency and contaminant recovery data needed to support development. any requirement for column calibration. A

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Figure 2: Molecular weight distribution overlay of PuraSorb PLDL 7028A before and after Figure 3: A Mark-Houwink plot for treated vs. untreated PuraSorb PLDL 7028A samples sterilization treatment. Green = before treatment. Orange = gamma irradiation. Purple shows that the degree of branching within the higher molecular weight region is changed = electron collision. by sterilization.

6 5 1.0 4 0.8 3 0.6 2 0.4 WF / dLog MW 0.2

1

0.0 Intrinsic Viscosity (dL/g)

100 200 1000 2000 104 2x104 105 2x105 106 2x106 107 Molecular Weight (Da) 105 2x105 3x105 4x105 5x105 6x105 7x105 8x105 9x105 106 2014-01-30_18;14;29_PuraSorb_PLDL_7028A_03-0000.vdx : WF / dLog MW 2014-01-31_01;48;58_PuraSorb_PLDL_7028A_g_03-0000.vdx : WF / dLog MW Molecular Weight (Da) 2014-01-30_21;03;58_PuraSorb_PLDL_7028A_e_02_0000.vdx : WF / dLog MW 2014-01-30_16;19;02_PuraSorb_PLDL_7028A_01-0000.vdx : Intrinsic Viscosity (dL/g) 2014-01-30_21;03;58_PuraSorb_PLDL_7028A_e_02-0000.vdx : Intrinsic Viscosity (dL/g) 2014-01-30_23;26;28_PuraSorb_PLDL_7028A_g_01_0000.vdx : Intrinsic Viscosity (dL/g)

viscometer determines intrinsic viscosity (IV) to investigate the structural characteristics from measurements of the pressure drop of a polymer by using a Mark-Houwink Case Study: Using Multi-Detection to f exibility required for timed-release systems. across a capillary bridge as a function of (MH) plot. An MH plot is a log–log Monitor Structural Changes Following Method: Three commercially available sample solution f ow rate. IV is an important graph of intrinsic viscosity against Sterilization PLA copolymers, two from Corbian Purac molecular characteristic directly related to molecular weight distribution and its y A study was performed to investigate the (Purasorb PDLG 7502A and Purasorb PLDL the density of the macromolecules and is intercept, gradient, and overall shape inf uence of high-energy sterilization on three 7028A [PDL]) and one from PolySciTech essential in determining accurate structural yield information. For example, a material medical-grade, commercially available block (PolyVivo-AK24) were analyzed using a information. with a high degree of branching is likely and co-block polylactic acid (PLA)-based triple detection GPC/SEC system (Viscotek These additional detectors give valuable to have a shallower slope than one with polymers. These materials are biocompatible TDAmax, Malvern Instruments), prior to and information when used individually but a more linear structure. The ability of in vivo and dissolve within the body at a following either electron collision sterilization also work in tandem to maximize analytical multi-detection GPC/SEC to deliver this rate determined by molecular weight and (e) or gamma irradiation (g).Both kinds of insight. For example, absolute molecular information makes it a valuable technique structural features of the polymer. Molecular samples (gamma and electron sterilized) weight data derived from light scattering for medical polymer analysis weight and structure can be controlled by were exposed to an absorbed radiation level can be used alongside IV measurements and development. varying reaction conditions to deliver the of 25 kGy (25000 Grays) for a few seconds.

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Light Scattering Explained — LALS, RALS, and MALS

When a molecule in solution is irradiated with a beam of light, it scatters light with an intensity that is directly related to its molecular weight, a relationship described by the Rayleigh equation. Light-scattering detectors determine molecular weight from measurements of light scattering intensity using the Rayleigh equation. Three types of detector are currently available that are DOWNLOAD suited to different applications: Low-angle light scattering (LALS), right-angle light scattering LCGC E-Book Educational (RALS), and multi-angle light scattering (MALS). Series: Advances in The Rayleigh equation is simplif ed if scattering intensity is measured at 0º to the incident Ion Chromatography beam. However, measuring at this angle is impractical because the scattered light signal is indistinguishable from the incident beam. Small molecules (<10–15 nm radius) scatter light This exclusive e-book assembles the top experts in ion chromatography from across the world to provide isotropically so intensity can be measured at any angle to the incident beam. RALS uses a single state-of-the-art views and reviews of developments PDF and advances in this powerful separation technique. detector to measure scattered light at 90º and is well-suited to smaller molecules such as proteins. For larger molecules (>10–15 nm radius), the intensity of scattered light is a function of the KEY TOPICS INCLUDE: È angle at which it is measured. LALS detectors measure at an angle as close to the incident beam Developments in ion-exchange columns È Advances in detection for IC as is physically possible — 7º — to minimize the effect of anisotropic scattering. In contrast, Today È Best practices in MALS detectors measure as many as 20 angles and apply extrapolation techniques to determine sample preparation intensity at 0º. Generally speaking, LALS detectors provide the most accurate measurements of È Tools for simulation and optimization of IC separations the molecular weight of larger molecules; however, MALS can also provide radius of gyration È High-speed and high- data, which can be very useful for certain polymer applications. resolution methods È Perspectives from Hamish Small, the inventor of ion chromatography, on the development of the technique The GPC/SEC system consisted of a light IV is intrinsic viscosity (dL/g), and Rh is + A format for everyone – scattering detector (LALS RALS), viscometer, hydrodynamic radius (nm). This e-book is available for and RI detector. Samples were dissolved Results: One initial observation from the the iPad or for a standard computer web browser in chloroform and gently agitated to fully results was that the reported Rh values span dissolve samples and ensure homogeneity the boundary of isotropic and anisotropic SPONSORED BY prior to injection onto the system. Table 1 scattering (see box “Light Scattering summarizes the results obtained for each Explained”), a transition which occurs at sample where Mw is the average molecular around 10–15 nm radius. In this instance weight (Da), Mn is number average molecular successful analysis of the samples was weight (Da), Mw/Mn is polydispersity, therefore enabled by the coupling of both

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Table 1: Molecular weight, intrinsic viscosity, and molecular size data for PLA polymer samples However, the plots do not exhibit such index, and viscometer detection to provide measured using triple detection GPC/SEC before and after electron collision sterilization (e) or a pronounced difference in gradient in the sophisticated polymer and macromolecular gamma irradiation (g). low molecular weight region or vary in characterization beyond what is achievable Sample Mw (Da) Mn (Da) Mw/Mn IV (dL/g) Rh (nm) terms of their point of intercept, indicating with conventional single detection techniques. PolyVivo-AK24 2676 1040 2.6 0.047 1.2 that the specif c monomeric density (and The results presented here illustrate PolyVivo-AK24 (e) 2771 2278 1.2 0.044 1.2 hence the structure of the repeating unit) how multi-detection GPC/SEC enables and the uniformity of low molecular units is quantif cation of the destructive inf uence PolyVivo-AK24 (g) 2749 1789 1.5 0.043 1.2 unaffected by sterilization. One interpretation of sterilization on medical-grade polymers of this data is that the degradation process and supports the development of effective PuraSorb PDLG 7502A 8468 2417 3.5 0.115 2.4 has broken down the polyuniformity of the preparation methods. PuraSorb PDLG 7502A (e) 7731 3838 2 0.114 2.3 polymer into smaller fragments, increasing PuraSorb PDLG 7502A (g) 9614 8059 1.2 0.125 2.6 internal cross-branching without affecting the Bassem Sabagh is a product technical monomeric structure. These changes, along specialist at Malvern Instruments, UK, and with the striking decrease in Mw, suggest that is an expert on the application of GPC/ PuraSorb PLDL 7028A 271,687 62,069 4.4 1.372 17 high energy gamma irradiation and electron SEC for the characterization of natural and PuraSorb PLDL 7028A (e) 89,205 27,30 0 3.3 0.635 9.1 collision sterilization disrupt the PLDL structure synthetic polymers and macromolecules. PuraSorb PLDL 7028A (g) 80,225 34,531 2.4 0.586 8.5 and may impact performance. Although it He has been at Malvern Instruments since is hard to directly correlate how sterilization 2008 supporting customers in their use of RALS and LALS detectors, which allows the polymer structure and may potentially impact will inf uence drug release, these results company products. analysis of relatively small and large particles product performance. clearly suggest that the PolyVivo-AK24 and Dr. Angel Valero-Navarro is a founder within the same experiment. A Mark-Houwink plot (Figure 3) for the Purasorb PDLG 7502A, which are unaffected and COO (Chief Operating Off cer) of the Of the three polymer samples, only PLDL untreated PLDL (blue) and for each treated by sterilization, may be more suitable for this technology-based company Nanomateriales underwent substantial physical changes PLDL sample (red and green) illustrates how application. y Polimeros (nanoMyP) in Armilla, Spain. following sterilization. The Mw signif cantly the structural characteristics of the polymer Angel’s research interests lie predominantly decreased following both sterilization have been altered by sterilization. The plots for Conclusion in the areas of synthetic polymer chemistry treatments, a change that can be clearly seen the treated PLDL samples differ clearly from Materials with built-in functionality help and materials science, with special emphasis from the shift to the left in the molecular the untreated sample, particularly within the to deliver closely controlled drug delivery on the design and the synthesis of functional weight distribution plot (Figure 2). The high Mw region. Here the slope is noticeably performance but their development relies polymeric nanomaterials. calculated polydispersity is appreciably lower shallower for the treated samples, suggesting on smart analytical techniques that offer the for the treated samples. This marked change that these polymers are denser. Increases insight required to understand and shape E-mail: [email protected] in physical characteristics suggests that in density are associated with a less linear polymer performance. Multi-detection Website: www.malvern.com sterilization has a destructive inf uence on the structure and a greater degree of branching. GPC/SEC combines light-scattering, refractive

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