SuppleMent to
May 2014 www.chromatographyonline.com
Recent Developments in Sample Preparation
magentablackcyanyellow ES431378_LCESUPP0514_CV1.pgs 04.29.2014 22:32 ADV magentablackcyanyellow ES430543_LCESUPP0514_CV2_FP.pgs 04.29.2014 01:16 ADV Recent Developments in Sample Preparation
5 Introduction Recent Developments in Sample Preparation Ronald E. Majors
6 Analytical Supercritical Fluid Extraction Goes Back to the Future Larry T. Taylor Past, present, and future developments of supercritical fuid extraction (SFE) are outlined. New vendor activity in terms of automation and hyphenation with chromatographic separations is discussed.
10 Recent Advances in Pressurized-Fluid Extraction Aaron Kettle Current advances in commercial pressurized-fuid extraction (PFE) instrumentation are discussed, along with the advantages of performing PFE prior to chromatographic analysis.
15 Microwave-Accelerated Extraction — SW-846 Method 3546 and Beyond Bobbie McManus, Michelle Horn, Steve Smith, Bob Lockerman, and Greg LeBlanc Microwave-accelerated extraction (MAE) is described and evaluated. The latest enhancements to this technology are discussed from a hardware and applications perspective.
22 Green Chemistry Perspectives on Analytical Extractions Victor Essel and Douglas E. Raynie This article introduces some of the concepts behind green chemistry, covering solvent selection and extraction techniques.
26 Superheated Water as an Extraction Solvent in Sample Preparation Roger M. Smith The current status of superheated water extraction is reviewed, and the extraction methods, applications, and problems encountered are discussed.
30 Sample Preparation for Chromatography: How Much Can Be Automated? Edward Pfannkoch The author reviews the automation capabilities available, and some practical considerations to take into account when choosing to automate some or all of the sample preparation and handling steps that must be done before analysis.
www.chromatographyonline.com 3
magentablackcyanyellow ES431108_LCESUPP0514_003.pgs 04.29.2014 20:18 ADV Published by Editorial Advisory Board
Kevin Altria Hans-Gerd Janssen Group Publisher Chief Executive Officer GlaxoSmithKline, Harlow, Essex, UK Van’t Hoff Institute for the Molecular Mike Tessalone Joe Loggia Daniel W. Armstrong Sciences, Amsterdam, The Netherlands [email protected] University of Texas, Arlington, Texas, USA Kiyokatsu Jinno Chief Executive Officer Michael P. Balogh School of Materials Sciences, Toyohasi Editorial Director Fashion Group, Executive Waters Corp., Milford, Massachusetts, USA University of Technology, Japan Laura Bush Vice-President Coral Barbas Huba Kalász Tom Florio Semmelweis University of Medicine, [email protected] Faculty of Pharmacy, University of San Pablo – CEU, Madrid, Spain Budapest, Hungary Executive Vice-President, Editor-in-Chief Brian A. Bidlingmeyer Hian Kee Lee Chief Administrative Officer & National University of Singapore, Alasdair Matheson Agilent Technologies, Wilmington, Chief Financial Officer Delaware, USA Singapore [email protected] Tom Ehardt Günther K. Bonn Wolfgang Lindner Institute of Analytical Chemistry, Managing Editor Institute of Analytical Chemistry and Executive Vice-President Radiochemistry, University of Innsbruck, University of Vienna, Austria Kate Mosford Georgiann DeCenzo Austria Henk Lingeman [email protected] Faculteit der Scheikunde, Free University, Executive Vice-President Peter Carr Department of Chemistry, University Amsterdam, The Netherlands Assistant Editor Chris DeMoulin of Minnesota, Minneapolis, Minnesota, USA Tom Lynch Bethany Degg BP Technology Centre, Pangbourne, UK Executive Vice-President, Jean-Pierre Chervet [email protected] Business Systems Antec Leyden, Zoeterwoude, The Ronald E. Majors Agilent Technologies, Rebecca Evangelou Netherlands Sales Manager Jan H. Christensen Wilmington, Delaware, USA Lindsay Jones Executive Vice-President, Department of Plant and Environmental Phillip Marriot [email protected] Human Resources Sciences, University of Copenhagen, Monash University, School of Chemistry, Victoria, Australia Julie Molleston Copenhagen, Denmark Sales Executive Danilo Corradini David McCalley Liz Mclean Sr Vice-President Istituto di Cromatografia del CNR, Rome, Department of Applied Sciences, [email protected] Tracy Harris Italy University of West of England, Bristol, UK Hernan J. Cortes Robert D. McDowall Vice-President, Legal H.J. Cortes Consulting, McDowall Consulting, Bromley, Kent, UK Subscriber Customer Service Michael Bernstein Midland, Michigan, USA Mary Ellen McNally Visit (chromatographyonline.com) Gert Desmet DuPont Crop Protection,Newark, to request or change a Vice-President, Transport Modelling and Analytical Delaware, USA Media Operations subscription or call our customer Separation Science, Vrije Universiteit, Imre Molnár Francis Heid Brussels, Belgium Molnar Research Institute, Berlin, Germany Service Department on John W. Dolan Luigi Mondello +001 218 740-6877 Vice-President, Treasurer & LC Resources, Walnut Creek, California, Dipartimento Farmaco-chimico, Facoltà Controller USA di Farmacia, Università di Messina, Bridgegate Pavilions, 4A Adele Hartwick Roy Eksteen Messina, Italy Chester Business Park, Sigma-Aldrich/Supelco, Bellefonte, Peter Myers Wrexham Road, Pennsylvania, USA Department of Chemistry, Chester, CH4 9QH CORPORATE OFFICE Anthony F. Fell University of Liverpool, Liverpool, UK Tel. +44 (0)1244 629 300 641 Lexington Ave, 8th Fl. Pharmaceutical Chemistry, Janusz Pawliszyn New York, New York 10022 USA University of Bradford, Bradford, UK Department of Chemistry, University of Fax +44 (0)1244 678 008 Attila Felinger Waterloo, Ontario, Canada Professor of Chemistry, Department of Colin Poole Analytical and Environmental Chemistry, Wayne State University, Detroit, University of Pécs, Pécs, Hungary Michigan, USA Francesco Gasparrini Fred E. Regnier Dipartimento di Studi di Chimica e Department of Biochemistry, Purdue Tecnologia delle Sostanze Biologica- University, West Lafayette, Indiana, USA mente Attive, Università “La Sapienza”, Harald Ritchie Subscibe on-line at Rome, Italy Trajan Scientific and Medical. Milton www.chromatographyonline.com Joseph L. Glajch Keynes, UK Momenta Pharmaceuticals, Cambridge, Pat Sandra Massachusetts, USA Research Institute for Chromatography, Kortrijk, Belgium SUBSCRIPTIONS: LC•GC Europe is free to qualiàed readers in Europe. Jun Haginaka To apply for a free subscription, or to change your name or address, go to www.lcgceurope.com, School of Pharmacy and Pharmaceutical Peter Schoenmakers click on Subscribe, and follow the prompts. Sciences, Mukogawa Women’s Department of Chemical Engineering, To cancel your subscription or to order back issues, please email your request to University, Nishinomiya, Japan Universiteit van Amsterdam, Amsterdam, [email protected], putting LCE in the subject line. Javier Hernández-Borges The Netherlands Please quote your subscription number if you have it. Department of Analytical Chemistry, Robert Shellie MANUSCRIPTS: For manuscript preparation guidelines, visit www.lcgceurope.com or call the Editor, Nutrition and Food Science University of Australian Centre for Research on +44 (0)1244 629 300. All submissions will be handled with reasonable care, but the publisher Laguna, Canary Islands, Spain Separation Science (ACROSS), University assumes no responsibility for safety of artwork, photographs or manuscripts. Every precaution John V. Hinshaw of Tasmania, Hobart, Australia is taken to ensure accuracy, but the publisher cannot accept responsibility for the accuracy of Serveron Corp., Hillsboro, Oregon, USA Yvan Vander Heyden information supplied herein or for any opinion expressed. Tuulia Hyötyläinen Vrije Universiteit Brussel, DIRECT MAIL LIST: Telephone: +44 (0)1244 629 300, Fax: +44 (0)1244 678 008. VVT Technical Research of Finland, Brussels, Belgium Reprints: Reprints of all articles in this issue and past issues of this publication are available Finland (250 minimum). Contact Brian Kolb at Wright’s Media, 2407 Timberloch Place, The Woodlands, TX 77380. Telephone: 877-652-5295 ext. 121. Email: [email protected]. ©2014 Advanstar Communications Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including by photocopy, recording, or information storage and retrieval without permission in writing from the publisher. Authorization to photocopy items for internal/educational or personal use, or the Follow us @ LC_GC ‘Like’ our page LCGC Join the LCGC LinkedIn group internal/educational or personal use of specific clients is granted by Advanstar Communications Inc. for libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA 01923, 978-750-8400 fax 978- The Publishers of LC•GC Europe would like to thank the members of the Editorial Advisory Board for 646-8700 or visit http://www.copyright.com online. For uses beyond those listed their continuing support and expert advice. The high standards and editorial quality associated with above, please direct your written request to Permission Dept. fax 440-756-5255 or 10% Post LC•GC Europe are maintained largely through the tireless efforts of these individuals. email: [email protected]. Consumer Waste LCGC Europe provides troubleshooting information and application solutions on all aspects of separation science so that laboratory-based analytical chemists can enhance their practical knowledge to gain competitive advantage. Our scientific quality and commercial objectivity provide readers with the tools necessary to deal with real-world analysis issues, thereby increasing their efficiency, productivity and value to their employer. 4 Recent Developments in Sample Preparation May 2014
magentablackcyanyellow ES431109_LCESUPP0514_004.pgs 04.29.2014 20:19 ADV Recent Developments in Sample Preparation
Ronald E. Majors, LCGC Sample Preparation Perspectives Column Editor
To keep readers informed of the preparation trends (4) to the last on analytical extractions. Reduced latest developments and trends reader survey (5), the techniques of solvent consumption, safer solvent in sample preparation, LCGC supercritical fluid extraction (SFE), alternatives, and reasonable Europe occasionally runs reader‑ pressurized‑fluid extraction (PFE), energy demands all contribute and expert‑surveys that provide and microwave‑accelerated (or to a greener environment. In that information on overall technology and assisted) extraction (MAE) have all respect, Professor Roger Smith usage patterns. In addition, special shown substantial growth. Of the of the Chemistry Department of issues on popular topics of the time readers surveyed, SFE usage went Loughborough University in the United bring together a host of experts from 1.9% to 3.7%, PFE from 5.8% to Kingdom has a lot of experience in who cover their specific areas of 8.0%, and MAE from 2.6% to 8.0%. All the use of superheated water as an expertise in more depth than might three techniques haven’t surpassed extraction solvent and it is amazing be warranted in a monthly guest more popular techniques such as the dissolution power that this entirely author column. In the past, we have solid‑phase extraction (SPE) and safe solvent can provide for organic assembled special issues on Current solid‑phase microextraction (SPME) compounds when heated well above but have established themselves in the its boiling point. He will provide environmental, food safety, and natural a nice overview of the role of this products markets in particular. To novel extraction solvent in sample bring us up‑to‑date in SFE, Professor preparation. Larry Taylor of the Department of Finally, if sample loads eventually Chemistry, Virginia Tech, Virginia, increase enough, safety concerns USA, provides a good perspective arise, or labour costs become on why this extraction technique, an issue, automation becomes so popular in the 1990s, is coming a necessary part of the sample “back to the future” by providing a preparation portion of the analytical safer, greener, and rapid method for cycle. Most chromatography not only non‑polar compounds but laboratories use autosamplers as the also for polar compounds. Part of the standard form of sample injection answer lies with the introduction of but modern instrumentation permits the latest generation of supercritical automation beyond injection. Edward fluid chromatography (SFC) Pfannkoch from Gerstel USA has a instrumentation. On the PFE front (also good handle on the role of sample Ronald E. Majors referred to as accelerated solvent preparation beyond the autosampler extraction [ASE]), Aaron Kettle from and will discuss the various steps prior Trends and Developments in Sample Thermo Fisher Scientific reviews the to injection that are now automatable, Preparation (1), Sample Preparation latest instrumentation developments freeing up the user to perform less of Solids (2), and Sample Preparation as well as selected applications of routine tasks. of Volatiles (3). Since the last special this rapid extraction technique. For If you are faced with sample issue, some of the sample preparation MAE, Greg LeBlanc and coworkers preparation challenges in your own technologies that were new then from the CEM Corporation provide an laboratory, I hope you find something have advanced further so it is time in‑depth update on greatly improved of interest in this special issue of to re‑visit those technologies to see instrumentation in the field as well as LCGC Europe. what improvements have been made. some novel applications. In addition, we will look at some Since the last special issue, the References newer trends, particularly in the area movement towards green chemistry (1) R.E. Majors, Ed. “Current Trends and Developments in Sample Preparation”, of green sample preparation and has been a driving force in chemistry LCGC 16(5S), S1–S60 (1998). automation. in general and analytical chemistry (2) R.E. Majors, Ed. ”Sample Preparation Technologies for the extraction and sample preparation in particular. of Solids”, LCGC 17(6S), S1–S44 of solid materials have come a Professor Douglas Raynie and his (1999). (3) R.E. Majors, Ed. “Sample Preparation long way since the advent of the graduate student Victor Essel from for Volatile Compounds”, LCGC 17(9S), Soxhlet extractor, particularly in the Department of Chemistry and S1‑S36 (1999). the automation of solid sample Biochemistry, South Dakota State (4) R.E. Majors, LCGC North Am. 31(3), 190–203 (2013). preparation techniques. In comparing University, South Dakota, USA, (5) R.E. Majors, LCGC 20(12), 1098 –1113 this year’s reader survey on sample discuss green chemistry perspectives (2002).
www.chromatographyonline.com 5
magentablackcyanyellow ES431111_LCESUPP0514_005.pgs 04.29.2014 20:19 ADV SFE System Oven Preheat coil BPR Conditioning H2O Extraction Pump Solvent Vessel MeOH Autosampler Pump (AS1) Waste V1 Analytical Supercritical High-pressure CO2 Pump SwitchingValve Unit PDA Detector CO2 UHPLC Switching UHPLC System Valve Unit V2 Separation Column PDA Detector Fluid Extraction Goes Waste Oven Oven 0.2% Formic Acid Pump4 Autosampler Mixing (AS2) Trap Unit Column Acetonitrile Pump5 Back to the Future
Larry T. Taylor, Department of Chemistry, Virginia Tech, Blacksburg, Virginia, USA.
The past and future development of analytical supercritical fuid extraction (SFE) is traced in terms of experimental strategies, applications, vendor support, and timely acceptance of the existing technology. The current state of the art is compared with research activity in the 1980s. New vendor activity, in terms of automation and hyphenation with chromatographic separations, is discussed.
For someone who has between 1980 Purgatory”. He writes: “SFE generates prior to chromatographic analysis. and 2000 (i) published hundreds high interest in surveys, but low sales. Today these vendors are Jasco, Inc., of peer-reviewed manuscripts All the necessary groundwork seems Supercritical Fluid Technologies, Inc., on analytical supercritical fluid to be there, but where are the orders? Waters Corp., Applied Separations, extraction (SFE) for chromatographic Where are the users?” and Taiwan Supercritical Technology. analysis and isolation of additives, Parenthetically, at about this same The problem of long-term neutraceuticals, pollutants, and time, it was reported by experts in analytical SFE exploitation may so on; (ii) co-taught short courses the field that “SFE is over the hump also be in part an education on SFE in Europe, South America, and that it is rapidly developing into issue as evidenced by the failure North America, and Asia; and (iii) the extraction method of choice for of academicians to both learn, guest lectured numerous times on an the 21st century with more and more explore, and apply the technology ACS short course entitled “Sample laboratories around the U.S. and in junior-senior chemistry laboratory Preparation for Chromatography” the world embracing it for sample courses at the university level – organized by Harold McNair, the cleanup and sample preparation” (2). even when new instrumentation current state of SFE does sometimes No doubt this assessment was partly is made available. Furthermore, sound as if we are going back to the based upon the fact that between the perceived remarkable speed future. 1987 and 1989 more than 100 papers and versatility of SFE often tempts A sample preparation survey in were published concerning the use of beginners to look for shortcuts rather 2002 suggested that SFE was being supercritical fluids for extraction (3). than to examine the application used by less than 2% of respondents. Possible explanations for the systematically (4). A hasty approach A similar survey repeated in 2012 perceived current lack of enthusiastic to method development often leads to showed that the use of SFE had only growth among analytical SFE unexpected problems and analytical doubled, despite the obvious overall practitioners are the limited number of SFE is a sophisticated technology advantages of supercritical carbon suppliers that market the technology that is best mastered by adhering to dioxide-based fluids (1). today and the exodus of vital vendors the rigour required when developing The situation now is different from such as Hewlett Packard, Suprex, any analytical method. It should be the 1980s because instrumentation Dionex, ISCO, and Lee Scientific remembered that while there has is more robust and engineering from the field around the turn of the been and continues to be a plethora applications in food, pharmaceuticals, century. A somewhat similar vendor of applications in engineering, SFE bioanalytical, and materials are more exodus occurred slightly earlier in was only developed as an analytical plentiful. On the other hand, analytical time in the field of SFC but, thanks technique in the mid-1980s. To SFE has not changed very much to the efforts of Terry Berger and gain some appreciation for past because there is a lack of support associates, the drought was not as developments (5–10), the reader is from major vendors and newcomers to long-lived, and SFC has survived encouraged to read the numerous the technology are forced to re-learn to “live” again. Hopefully many of reviews that have appeared recently SFE history and protocols that have the developments that benefited as well as in the older literature. been around for years. the SFC community will now foster Look for details concerning the pros To put it in perspective, consider further development of analytical SFE. and cons of: (a) On-line or off-line a report of the 9th Annual Waste Currently, there appears to be a small coupling with a variety of separation Testing and Quality Assurance core of established vendors, as well techniques; (b) dynamic versus static Symposium held in Crystal City, as relatively new vendors, that are extraction protocols; (c) solid-phase Virginia (USA) dated 23 July 1993 by committed to making analytical SFE a or liquid-phase extract trapping; Robert Stevenson, entitled “SFE in viable method for sample preparation (d) modifier addition to the matrix
6 Recent Developments in Sample Preparation May 2014
magentablackcyanyellow ES431105_LCESUPP0514_006.pgs 04.29.2014 20:18 ADV Taylor
could be adversely affected by high Figure 1: Schematic of a SFE–UHPLC system. temperatures and the presence of solvent residuals. SFE using CO is SFE System 2 Oven now an established industrial process Preheat coil BPR for the production of high-value Conditioning H2O Extraction Pump natural products such as hops, Solvent Vessel MeOH Autosampler Pump decaffeinated tea and coffee, herbs (AS1) Waste V1 and spices, medicinal herbs, seeds, and marine oils. Further examples High-pressure CO2 Pump SwitchingValve Unit include extraction processes where PDA Detector an undesirable component is removed CO2 UHPLC Switching from the matrix, such as pesticides UHPLC System Valve Unit V2 Separation from medicinal herbs. In ancillary Column PDA Detector fashion, SFE processing continues Waste Oven to find applications as shown by the Oven 0.2% Formic Acid Pump4 Autosampler variety of lipophilic extracts available Mixing (AS2) Trap as commercial products such as Column Unit polyunsaturated fatty acid esters Acetonitrile Pump5 derived from fish oils, neat and roasted sesame seed oil, cranberry seed-based oils, oils high in n-3 and versus modifier addition to the fluid; The use of SFE in modern process n-6 fatty acid content, and pumpkin (e) adsorbent in the extraction thimble engineering applications was initiated seed extracts coupled with more to retain unwanted compounds such in Germany during the late 1960s. traditional SFE-derived products such as water; (f) experimental strategies These early studies showed that SFE as decaffeinated coffee (11). for extracting analytes from solids, was a viable alternative to distillation The scope of this report is therefore gels, creams, sludges, liquids, and and solvent extraction processes. limited to advances concerning so on; and (g) polar-modified, high Furthermore, it allowed the processing analytical SFE. The goals of this density CO2. of substances whose extraction article are to both describe the current
Touchscreen High End HPLC System SpotPrep II The highest capacity, the highest versatility the smallest footprint!
FAST, RELIABLE, CONVENIENT External Detector Option HPLC PURIFICATION
® www.gilson.com/spotprep
www.chromatographyonline.com 7
magentablackcyanyellow ES431107_LCESUPP0514_007.pgs 04.29.2014 20:19 ADV Taylor
state of the art in relation to new Based upon the control software PIC Solution, Inc. has recently instrumentation and to address unique chosen and the application-specific reported an useful technique to extraction strategies, theories, and component options used, the system improve the quality of preparative applications. In addition, interesting offers the ability to do everything from separations called “injection by hyphenations of analytical SFE with sophisticated studies to routine scale extraction” as a substitute for chromatography and spectroscopy up. Complementary techniques such traditional mixed stream injection that have been reported during the as counter current chromatography, and modifier stream injection (14,15). past three years, as well as new aids particle formation (for example, RESS Often one is able to load significantly for achieving successful extraction and SAS), phase monitoring, and more sample using this technique with CO2 will be considered. reaction engineering options are because of better solubility and less available. peak distortion. In addition, the vendor Vendor Activity Two years earlier Jasco, notes that the procedure avoids many The development of second Inc. introduced a supercritical of the problems associated with more generation SFC instrumentation fluid extraction coupled to conventional injection techniques. In by several major vendors over the ultrahigh-pressure liquid particular sample precipitation, which past four to five years introducing chromatography (SFE–UHPLC) is a problem often met in preparative improvements to mobile phase system (Figure 1). The vendor SFC, is eliminated by dissolution of delivery, reproducible modifier noted that hyphenating multiple the sample in the mobile phase. The introduction, and greater analytical methods in an analysis system technique is also useful for purification sensitivity appears to have facilitated has many advantages such as of samples with insoluble impurities analogous selective advances in decreasing the analysis time, or that require extraction from a matrix SFE. While these developments reducing cost, simplifying sample before separation. are encouraging, they will not take pre-treatment, and increasing away, for example, the critical need sensitivity and selectivity. In addition Hyphenation of SFE for quantitative trapping that must to hyphenation of SFE with SFC, gas Historically, the development of occur when modified fluids are used chromatography (GC) and HPLC analytical SFE has been associated to extract polar analytes that have have been reported in the open with a form of chromatography. marginal solubility in pure CO2. literature (12). Hyphenation of SFE The research of Stahl in Germany Furthermore, a greater understanding and UHPLC by Jasco, Inc. has only combined SFE with thin layer of matrix effects and the availability been reported in a vendor application chromatography (16). In 1989, of universal SFE methodologies that note. The on-line SFE–UHPLC Anderson and colleagues reviewed are not specifically matrix and analyte employs valve-switching techniques. some of the theoretical considerations sensitive and dependent is also Piperine in peppers was extracted surrounding the use of SFE as the desired. effectively in the SFE system and means of introducing samples into Waters Corp. introduced at Pittcon then trapped and concentrated chromatographic systems (17). The 2013 an accelerated analytical on the trap column that was then data presented indicated that SFE supercritical fluid extraction system connected in the high-pressure could be fine-tuned into a selective that provides unattended operation switching valve unit section (13). sample preparation or injection for method development, multiple After the extraction, a conditioning method for chromatography. The samples, and multiple collection solvent was delivered from the practice of analytical SFE is divided vessels. The unit utilizes ChromScope conditioning pump to remove the between “off-line” and “on-line” software to achieve better selectivity gaseous CO2 in the trap column. methods. Such definitions refer to and specificity with no organic solvent The extract was then automatically the mechanism of conducting the exposure and no disposal cost. The introduced into the UHPLC system extraction. The on-line methods are system accommodates 10 sample and subjected to rapid separation usually combinations of SFE with vessels and 12 collection vessels and photodiode array detection with ancillary techniques such as GC, SFC, that can be the same or different in high sensitivity. The overall system is and HPLC. Off-line SFE offers more vessels that range in volume from reported to offer very high speed and flexibility with respect to extracting 5-mL to 15-mL. The key parameters extremely sensitive analysis without different sample sizes and types, of CO2 pressure, vessel temperature, complicated pre-treatment. RSD for as well as in the choice of the final percent co-solvent, and use of retention time was listed as 0.36% analytical method. The bottom line dynamic and static conditions, as well and for peak area was 1.91%. Overall is that the selection of an analytical as collecting extracts at individual piperine recoveries were 96.0%. SFE method should be based on user criteria can all be programmed Several other vendors are quite the problem faced. The design of and run unattended. The unit may active and supportive of advances in the interface is a crucial aspect in use up to six different co-solvents to SFE. Applied Separations, Inc. offers a developing a hyphenated technique develop new methods for multiple four vessel simultaneous oven-based involving the direct coupling of SFE samples while enhancing the extraction system with modifier to a destructive or non-destructive extraction process. addition capability. Temperatures up analytical detector. A second product line is to 240 °C, pressures up to 680 bar, More current studies concerning pre-assembled and cart-mounted that and flow rates up to 400 mL/min can hyphenation continue to be published varies in size from 100 mL to 2 × 5 L. be accommodated. in the open literature. A direct aqueous
8 Recent Developments in Sample Preparation May 2014
black ES431110_LCESUPP0514_008.pgs 04.29.2014 20:18 ADV Taylor
SFE system designed to extract from shrimp (27); (f) cannabinoids from (13) M. Saito et al., American Laboratory water samples contained in vials marihuana (28); (g) abuse drugs in website, posted 1 February 2011 (http://www.jascoinc.com/blog/chroma- has been coupled with a reversed- biological media (29); and (h) benign tography-applications/blog/2013/02/2/). phase LC–MS–MS system using a resolution of trans-1,2-cyclohexanediol (14) M. Shaimi, “Direct Sample Injection single 10-port valve (18). An SFE trap by two-step SFE (30). Technique for Preparative Supercritical Fluid Chromatography: Solventless system using a C1 stationary phase In summary, there is not only interest Injection,” presented at PREP 2001, connected to a C18 analytical HPLC in SFE as an analytical tool but also Washington, DC., USA. column enabled the SFE–LC–MS–MS for process development. It is clear (15) K. Gahm, “On-Line Coupling of analysis of three polyether ionophore from the recent literature that SFE Supercritical Fluid Extraction with Supercritical Fluid Chromatography for antibiotics in water. In another study, for processing commands a greater Chiral Separation of Racemic Mixture a new hyphenated technique for interest at this time in a variety of areas Containing Insoluble Impurities”, the extraction and determination of such as food science, natural products, presented at SFC 2013, Boston, MA, USA. isoflavones in sea and freshwater pharmaceuticals, bioprocessing (31), (16) E. Stahl, K.W. Quirin, and D. Gerard, algae and cyanobacteria with and environmental science. Advances Dense Gases for Extraction and Refining, sonication sample pretreatment has in analytical SFE appear to have waned (Springer-Verlag, Berlin, Germany, 1988). been developed (19). A third reported during the past decade as vendor (17) M.R. Andersen, J.T. Swanson, N.E. study concerns the development commitment to the technology has Porter, and B.E. Richter, J. Chromatogr. of an analytical strategy based on been sporadic. Yet, mathematical Sci. 27(7), 371–377 (1989). rapid extraction techniques coupled modelling appears to be on the (18) E.D. Ramsey, A.T. Rees, G. Wei, J.Y. Liu, and X.H. Wu, J. Chromatogr. A 1217, to comprehensive bi-dimensional rise (32). Improved instrumentation 3348–3356 (2010). gas chromatography (GC×GC) for for analytical applications such (19) B. Klejdus, L. Lojkova, M. Plaza, the characterization of the volatile as automation, coupling to M. Snoblova, and D. Sterbova, J. Chromatogr. A 1217, 7956–7965 (2010). fraction of tobaccos. The high peak chromatographic and spectroscopic (20) J. Vial, D. Thiébaut, P. Sassiat, M.S. capacity of GC×GC allowed global techniques, and sample throughput Beldean-Galea, M.J. Gomez Ramos, extraction techniques that do not would benefit the technology. The G. Cognon, S. Mallipattu, B. Teillet, and focus on restricted chemical families time seems right for analytical SFE M. Bouzige, J. Chromatogr. Sci. 48(4), 267–273 (2010). to be considered (20). Finally, a novel, to expand on the gains made over (21) F. De Andres, M. Zougagh, G. sensitive, and selective method for the past 25 years as a rapid and Castaneda, and A. Rios, Electrophoresis the separation and quantification efficient sample preparation tool. The 31(13), 2165–2173 (2010). (22) A. Rios, M. Zougagh, and F. De Andres, of a group of nonpolar heterocyclic technological developments first made Bioanalysis 2(1), 9–25 (2010). amines in commercial meat samples in fast liquid chromatography and now (23) R.M.A. Domingues, M.M.R. de Melo, has been developed (21). The method supercritical fluid chromatography will E.L.G. Oliveira, C.P. Neto, A.J.D. Silvestre, and C.M. Silva, J. Supercrit. is based on the combination of a no doubt bring greater emphasis and Fluids 74, 105 –114 (2013). SFE procedure followed by analysis applications to analytical SFE. (24) M. Banchero, G.Pellegrino, and L. of the extracted plug by capillary Manna, J. Food Eng. 115(3), 292–297 electrophoresis under fluorimetric References (2012). (1) R.E. Majors, LCGC (2002, 2012). (25) J. Fojtova, L. Lojkova, and V. Kuban, detection. (2) C.L. Phelps, N.G. Smart, and C.M. Wai, Cent. Eur. J. Chem. 8, 409–418 (2010). J. Chem. Ed. 73(12), 1163–1168 (1996). (26) B.A. Domadia and N.R. Vaghela, J. Applications of SFE (3) M.S. Ray, Sep. Sci. Tech. 29, 2203 Chem. Pharm. Res. 5(4), 188–191 (1994). (2013). The bioanalytical applications of (4) W. Pipkin, LCGC 10, 15 (1992). (27) W.L. Liu, R.J. Lee, and M.R. Lee, Food supercritical fluid techniques such as (5) J.W. King and M.L. Hopper, J. AOAC Chem. 121(3), 797–802 (2010). SFE are of increasing interest. The International 75(3), 375–378 (1992). (28) J. Omar, M. Olivares, M. Alzaga, and N. main role of this technique is in the (6) J.M. Levy, R.M. Ravey, and R. Panella, Etxebarria, J. Sep. Sci. 36(8), LCGC 15, 570 (1998). 1397–13404 (2013). sample preparation and separation of (7) M.Herrero, J.A. Mendiola, A. Cifuentes, (29) D.J.E. Mirson and O.E. Roses, Int. J. biologically active compounds such and E. Ibanez, J. Chromatogr. A 1217, Environment and Health 4(1), 18–39 as drugs and their metabolites, as well 2495–2511 (2010). (2010). (8) M.D. Luque de Castro, M. Valcarcel, (30) E. Szekely, G. Bansaghi, P. Thorey, P. as endogenous compounds (22). The M.T. Tena, Analytical Supercritical Fluid Molnar, J. Madaarasz, L. Vida, and B. selection of the operating conditions Extraction (Springer-Verlag, New York, Simandi, Ind. Eng. Chem. Res. 49(19), depends on the specific compound USA, 1994). 9349–9354 (2010). (31) O. Catchpole, S. Tallon, P. Dyer, F. or compound family to be extracted. (9) L.T. Taylor, Supercritical Fluid Extraction (John Wiley & Son, Inc, New York, USA, Montanes, T. Moreno, E. Vagi, W. Molecular weight and polarity have 1996). Eltringham, and J. Billakanti, Amer. J. to be taken on a case-by-case basis. (10) Analytical Supercritical Chromatography Biochem. Biotech. 8(4), 263–287 (2012). Some recently published applications and Extraction, M.L. Lee and K.E. (32) L.G. Oliverira, A.J.D. Silvestre, and C.M. Markides, Eds (Chromatography Silva, Chem. Eng. Res. Design 89(7), have been selected to demonstrate Conferences, Inc, Provo, UT, USA, 1990). 1104–1117 (2011). the great variety of applications and (11) J.W. King and J.E. France, Basic (33) B. Honarvar, S.A. Sajadian, M. Khorram, to indicate the diversity of journals Principles of Analytical Supercritical and A. Samimi, J. Chem. Eng. 30(1), Fluid Extraction, in Analysis with 159–166 (2013). in which SFE is presented: (a) SFE Supercritical Fluids: Extraction and of triterpenic acids from Eucalyptus Chromatography, B. Wenclawiak, Ed Larry T. Taylor is Emeritus Professor globulus bark (23); (b) acrylamide from (Springer, Berlin, Germany, 1992). of Chemistry at Virginia Tech, coffee (24); (c) monoterpenes from (12) Hyphenated Techniques in Supercritical Blacksburg, Virginia, USA. Please Fluid Chromatography and Extraction, coniferous needles (25); (d) lycopene K. Jinno Ed (Elsevier, Amsterdam, the direct correspondence to ltaylor@ from tomatoes (26); (e) amphenicols Netherlands, 1992). vt.edu
www.chromatographyonline.com 9
magentablackyellow ES431106_LCESUPP0514_009.pgs 04.29.2014 20:19 ADV Solvent Solvent Solvent
Mixing Valve Pump
Relief Valve Recent Advances in
Oven Extraction Cell Pressurized‑Fluid Static Value Extraction Collection Bottle Aaron Kettle, Thermo Scientific, Sunnyvale, California, USA.
Pressurized‑fuid extraction (PFE) is an automated extraction technique that uses elevated temperature and pressure to increase the rate and efficiency of the extraction process. PFE reduces the amount of time and solvent required, while signifcantly improving laboratory throughput relative to traditional extraction techniques such as Soxhlet. This article discusses current advances in commercial PFE instrumentation and shows how these systems can beneft scientists in analytical laboratories who wish to perform automated extraction.
Pressurized-fluid extraction (PFE) is a Inc. (FMS) (the pressurized liquid under the same programmed method technique performed to extract solid extraction [PLE] system); and Büchi conditions. The total extraction time or semi-solid samples using organic (the SpeedExtractor system). Each is usually less than 20 min and the solvents. Elevated temperatures (up system offers automation capabilities amount of solvent used is approximately to 200 °C) are used to increase the for the analytical laboratory to reduce 1.5 times the volume of the sample cell kinetics of the extraction process while the amount of time spent on sample (for example, 15 mL for a 10-mL cell). applying high pressures (for example, preparation. These systems use elevated Extracts are delivered to the collection 1500 psi) to maintain the organic solvents temperature and pressure to improve vessels through a filter inserted at the in the liquid state. PFE is unique in that extraction efficiency and productivity bottom of the cell, and in many cases extractions are performed rapidly with compared with traditional extraction do not need any additional preparation reduced solvent use, compared with techniques such as Soxhlet. A summary prior to analysis. The schematic shown traditional extraction techniques. For of each system along with significant in Figure 1 demonstrates the operating example, PFE can reduce the extraction features is provided in this article. principles of this system. time down to 20 min per sample versus The features of the ASE method are hours using Soxhlet and reduce solvent Accelerated Solvent Extraction summarized in Table 2. The ASE system consumption to 30 mL per sample. The (ASE) is capable of performing in-line clean physiochemical processes influenced by Accelerated solvent extraction (ASE) up through use of adsorbents to the PFE are described in Table 1. was first introduced at Pittcon in extraction cell. These absorbents are PFE instrumentation follows a common 1995 by Dionex (now part of Thermo layered at the bottom of the cell prior pathway to produce extracts. An Fisher Scientific). ASE increases to sample introduction and selectively extraction cell containing the sample is extraction efficiency by using elevated remove interfering compounds during loaded into an oven and a pump transfers temperatures of up to 200 °C with the extraction. For example, adding extracting solvent into the cell from a a fixed pressure of 1500 psi using activated alumina oxide eliminates the reservoir. The cell is then pressurized both stainless steel and zirconium co-extraction of lipids when extracting and heated to a preset temperature. extraction cells (1–100 mL). These organic compounds. Several different The temperature and pressure in the latter cells are especially resistant to absorbents can be used and the correct extraction cells rises above ambient low concentrations of mineral acids selection is documented in reference levels and the hot solvent enhances the and strong bases. Currently, two ASE 1. Once the extraction is complete, the extraction rate of the analytes from the instruments are available from Thermo collection vial can be interfaced with a matrix. PFE systems are designed so that Fisher: The ASE 150 Accelerated Rocket Evaporator (Genevac Ltd.) for solvent will flow through the extraction Solvent Extraction instrument, a automated sample concentration and cell and be collected into a bottle or tube single-cell system for the extraction of evaporation. This method significantly at the end of the flow path. Once the solid and semisolid samples; and the improves the sample preparation extraction is complete, the extraction cell multiple cell ASE 350 instrument. The process by eliminating the need for is purged with nitrogen gas to remove ASE 350 instrument can process up to off-line clean-up steps and by enabling residual solvent and the collected extract 24 samples in a batch and store up to 24 the direct transfer of samples from the is ready for concentration and analysis. extraction methods. Extraction methods system to an automated evaporator. PFE systems are currently manufactured can be preprogrammed and multiple With the use of in-cell adsorbents and by three vendors: Thermo Scientific (the extraction methods run in a single the Rocket Evaporator, ASE is able to accelerated solvent extraction [ASE] batch, providing sequence control. combine sample filtration, cleanup, and system); Fluid Management Systems, Each sample is processed sequentially evaporation into one workflow.
10 Recent Developments in Sample Preparation May 2014
magentablackcyanyellow ES431127_LCESUPP0514_010.pgs 04.29.2014 20:20 ADV Kettle
Pressurized‑Liquid Extraction instrument with in-line cleanup is shown This configuration helps to automate the (PLE) in Figure 2. sample preparation process and improve Pressurized-liquid extraction (PLE) The PLE instrument can be interfaced productivity prior to analysis. Table 3 lists is widely used in persistent organic with both in-line clean columns and an the key features for the FMS PLE system. pollutants (POPs) extraction from evaporator that combine the extraction, environmental matrices such as soil, and cleanup, and concentration procedures Parallel Sample Extraction from biological matrices such as tissue. into one step. In-line cleanup of extracts The Büchi Labortechnik AG The FMS PLE instrument makes use of is accomplished with the use of in-cell SpeedExtractor is a parallel extraction elevated temperature and pressure to packing materials (for example, silica or instrument that can process up to increase the rate and efficiency of the carbon), and by interfacing with columns six samples simultaneously. For six extraction process, producing extracts in that are packed with adsorbents (carbon, samples, a total of 20–40 min is required as little as 20 min. Up to six samples can silica, and alumina). A flow-through for extraction, and 5–60 mL of solvent is be extracted simultaneously using the design is used to move solvent downward required per sample, depending on the systems parallel extraction mechanism. through a heated and pressurized application. Stainless steel extraction The system is modular, can be expanded extraction cell and through the packing from one to six channels, and uses material that is located at the bottom of stainless steel extraction cells (5–250-mL) the cell. An additional column clean-up Figure 1: Accelerated solvent with end caps that contain Teflon filters. module can be added to the output extraction (ASE) system schematic. DMS-6000 Editor software is used to of the extraction cell for cleaning the control the instrument, store multiple samples prior to analysis. In addition, extraction methods, and plot temperature PLE instruments can be interfaced Solvent Solvent Solvent and pressure data for each channel directly with a concentrator to automate Mixing used. The operating principles of the PLE the entire sample preparation workflow. Valve Pump
Table 1: Physiochemical processes influenced by pressurized-fluid extraction (PFE). Relief Valve Parameter Effect on the Extraction Process Temperature Elevated temperature increases analyte diffusion from the matrix and improves analyte solubility in the extraction solvent. Oven Extraction Cell Pressure Increased pressure enables liquid solvents to be used at high temperature. Analyte solubility Increases as temperature increases to improve extraction efficiency (for example, solubility of anthracene increases 13-fold in DCM [50 oC to 150 oC]). Static Value Solvent viscosity Decreases as temperature increases. Improves solvent migration through the matrix to increase extraction efficiency. Collection Bottle Solvent surface Decreases as temperature increases. Allows solvents to better coat the tension matrix and helps improve analyte diffusion.
Table 2: Summary of Thermo Scientific accelerated solvent extraction (ASE) features. Feature Description Use Static extraction cycles Extraction cells filled with solvent are held at elevated Maximizes analyte diffusion from the matrix while temperature and pressure without a continuous flow of reducing the amount of solvent required for the solvent. extraction. In-line filtration Disposable filters (cellulose or fibre glass). Automatically removes solid particulates from the extract. In-line cleanup Adsorbents (for example, silica, activated alumina). Automatically removes interfering molecules (for example, lipids) from the extract. Rocket evaporator Centrifugal system that evaporates up to 18 accelerated Automates the evaporation process for high-throughput solvent extractions. laboratories. pH-hardened pathways Zirconium extraction cells. Allows the extraction of acid or alkaline pretreated samples (for example, total fat from foods). Integrated solvent Internal proportioning valve that allows solvent mixing Automatically mixes up to three solvents for complex controller with user-defined ratios. extractions. Solvent saver modes Programmable modes in the extraction method that Reduces the amount of solvent required to 5 mL per further reduce the amount of solvent required for the sample. extraction.
Chromeleon 7.2 Software to standardize control of the instruments Controls the ASE 350 instrument and chromatographic chromatography data involved in the analytical workflow. systems involved in the workflow. system software control Sequence processing Increases flexibility by allowing multiple methods to be Multiple extraction methods can be run on the same control run in a single batch. sample or series of samples within a single batch.
www.chromatographyonline.com 11
magentablackcyanyellow ES431124_LCESUPP0514_011.pgs 04.29.2014 20:19 ADV Kettle
cells (10–120-mL) are added to a Figure 2: FMS PLE system schematic. pressurized heating block for operation up to 200 °C and 2000 psi. Extraction cells are automatically sealed once entered into the oven permitting the use of up to four solvents. Software control is NITOGEN PRESSURE PRESSURE available to record method parameters PC VALVE GAUGE CONTROL and run preprogrammed extraction methods. The operating principles of the HEATER BLOCKS SpeedExtractor instrument are shown in CHANNEL Figure 3. SELECTION VALVE COOLING Similar to ASE and PLE FAN OUTPUT instruments, the SpeedExtractor VALVE instrument offers a parallel PUMP evaporation option that further automates the sample preparation NITROGEN workflow. Once the extraction PLE EXTRACTION CELL is complete, bottles containing the extracts can be placed into the Multivapor evaporator for concentration. Up to six samples can be simultaneously concentrated SOLVENTS COLLECTOR CONTROL SIGNAL improving sample throughput CONTROL LINE SOLVENT/GASFLOW prior to analysis. Features of the SpeedExtractor are listed in Table 4.
Table 3: Summary of FMS pressurized-liquid extraction (PLE) instrument features. Feature Description Use In-line filtration Extraction cell end caps contain Teflon filters. Automatically removes solid particulates from the extract. In-line cleanup Adsorbents added to extraction cell and in-line Automatically removes interfering molecules (for columns. example, lipids) from the extract. In-line concentration Evaporator that processes up to six samples Automates the evaporation process for high-throughput simultaneously. laboratories. Modular design One to six channels are available to meet changing Additional channels can be added to the system as throughput needs. laboratory workload increases. Parallel extraction Processes multiple samples simultaneously. Improves throughput by extracting up to six samples in 20 min. DMS-6000 Editor software Software that controls the extraction process and Enables multiple extraction methods to be run and control allowing the laboratory to edit extraction methods and plots temperature and pressure in real time for each monitor and store extraction data. channel. Solvent selection valve Valve that permits the use of up to five solvents. Enables the use of a different solvent for each extraction channel.
Table 4: Summary of Büchi SpeedExtractor instrument features. Feature Description Use Sealed extraction cells Extraction cell caps are automatically sealed when Cell caps do not need to be manually tightened as part placed in the oven. of the sample preparation. Multivapor concentrator Parallel concentration system that processes up to six Samples can be added to the concentrator immediately extracts. following extraction and concentrated simultaneously. Parallel extraction Processes multiple samples simultaneously. Improves throughput by extracting up to six samples in 20 min. SpeedExtractorRecord Software that controls and records method parameters Method parameters and external intervention are software from the extraction process. recorded for reporting. Multiple collection 60-, 150-, 220-, or 240-mL collection bottles. Flat-bottom vials that are compatible with the Multivapor bottle sizes concentrator. Integrated solvent mixer Two or four-port mixer for multiple extraction solvents. Solvent mixtures can be prepared to handle complex extractions.
12 Recent Developments in Sample Preparation May 2014
magentablackcyanyellow ES431125_LCESUPP0514_012.pgs 04.29.2014 20:20 ADV Kettle
Table 5: Representative applications using pressurized-fluid extraction (PFE). Feature Description Use Total fat in food Total fat (bound and unbound) extraction from foods. Enables compounds to be extracted from acid/base pretreated food matrices. Acid pretreated biomass Use of zirconium extraction cells to extract water and Enables extraction of sugars from acid treated biomass ethanol soluble components from acid pretreated to expand the versatility of PFE. biomass. Persistent organic PAHs, PCBs, brominated flame-retardants, dioxins/ Rapid extraction of a broad class of compounds from pollutants furans, pesticides from soil, sediment, and tissue. environmental matrices. Extraction times as low as 20 min and 30 mL of solvent per sample. Plasticizers in polymers Extraction and determination of plasticizers in polymeric Important step in assuring the quality of the end material for manufacturing quality control. product prior to release. PFE can reduce extraction time to 12 min per sample vs. 6 h with Soxhlet and typically requires 30 mL of solvent per sample. Active ingredients in Extraction and determination of active ingredients Verifies label claim and ensures quality control for pharmaceuticals and from herbal supplements (for example, St. Johns Wort, production. Reduces extraction time to as low as natural products goldenseal root). 14 min per sample with 15 mL of solvent used. Pesticides and herbicides Extraction of pesticides and herbicides from multiple Helps ensure food safety by allowing rapid extraction in foods different types of food including fruits and vegetables. of multiple pesticides from food products. Compounds can be extracted in as little as 12 min per sample using 15 mL of solvent. PFE can work with high water content, highly pigmented, and high fat content samples.
extraction time and approximately 30 mL Figure 3: Büchi SpeedExtractor instrument schematic. of solvent per sample to extract myriad compounds for analysis. Solvent Reservoirs Examples of key PFE applications include POPs and dioxin extraction from a diverse range of matrices. In one study, the performance of ASE was compared with Soxhlet for the extraction of pesticides, PCBs, and dioxins (polychlorinated dibenzodioxins [PCDDs] and polychlorinated dibenzofurans Extraction Chambers [PCDFs]) in soil, river sediment, and other solid matrices (2). The extraction efficiency was measured by the average percent recovery for six samples and Collection Vessels reproducibility was measured by % relative standard deviation (% RSD). Tables 6–8 show the results of the ASE extraction as a proportion (%) of Soxhlet. This example illustrates the effectiveness of the ASE technique in obtaining recoveries of analytes equivalent to the Table 6: Average recovery of pesticides from three soil types. Soxhlet approach. Pesticide Average Recovery* Average RSD (%) Another study used a Büchi Heptachlor 88.0 Matrix ASE Soxhlet SpeedExtractor instrument and an ASE instrument for dioxin and furan extraction Aldrin 94.9 Clay 5 9.7 from soil (3). The extraction efficiency Gamma chlordane 99.5 Loam 7.8 6.2 of both instruments was compared and Alpha chlordane 102.2 Sand 12 10.1 it was demonstrated that both could meet the recovery requirements of Dieldrin 101.2 *% of Soxhlet, n = 6 EPA Method 3454A (4). The extraction Endrin 97.2 efficiency was measured through the p,p'-DDT 74.9 mean quantitated amount by performing gas chromatography coupled with high New Applications Using applications across a wide spectrum of resolution mass spectrometry (GC– Pressurized‑Fluid Extraction industries including the environmental, HRMS). Table 4 lists the comparison PFE is versatile and can be applied to food, chemical, petrochemical, and of both instruments for several PCDDs numerous solid matrices, as shown in pharmaceutical industries. Most and PCDFs and demonstrates that the Table 5. PFE has been used for new applications now require only 20 min of SpeedExtractor Model E-916 instrument
www.chromatographyonline.com 13
magentablackcyanyellow ES431123_LCESUPP0514_013.pgs 04.29.2014 20:20 ADV Kettle
delivers extractions equivalent to those improved sample preparation throughput References obtained with the ASE. by combining what were once separate (1) Thermo Scientific Technical Note 210 [Available from: http://www.dionex.com/ procedures. Over the last 10 years the en-us/webdocs/57304-TN210-ASE-In- Summary continuing of these combined techniques Line-Interferences-Removal-05Oct2012- The principle of pressurized-fluid has improved the productivity of sample TN70242_E.pdf]. extraction has not changed significantly preparation and reduced bottlenecks in (2) Thermo Scientific Application Note 352 [Available from: http://www.dionex.com/ over the last 10 years; however, the level sample preparation. With the advances en-us/webdocs/40970-AN352-ASE- of automation provided by this technique made in these techniques, laboratories Persistent-Organic-Pollutants-23Jun2011- has expanded greatly. The biggest are now able to able to remove analytes LPN1676-03.pdf]. (3) Büchi Application Note 012/2009 [Available advance in PFE instrumentation has been from the original matrix and place them from: http://www.buchi.com/fileadmin/ the addition of in-line cleanup techniques into a gas chromatography (GC) or liquid user_upload/01_products/10_extraction/ and direct transfer of samples to chromatography (LC) autosampler vial for pdf/SN%20012-2009%20Dioxin_Furan%20 concentrators. The advance has markedly analysis with minimal analyst intervention. Soil_p%20Version%20B.pdf]. (4) EPA Method 3454A: Pressurized Fluid Extraction (PFE), January 1998 [Available from: http://www.epa.gov/sam/pdfs/EPA- Table 7: Average polychlorinated biphenyls (PCB) recovery from river sediment 3545a.pdf]. (SRM 1939). PCB Congener Average Recovery* RSD (%) Aaron Kettle is the Product Manager for Dionex ASE systems at Thermo Fisher PCB 101 89.2 3.7 Scientific. Aaron has been with Thermo PCB 153 62.3 4.1 Fisher Scientific for five years through PCB 138 122.1 2.3 legacy Dionex and has held both sales PCB 180 111.5 5.9 and marketing positions. Previously, he was a commissioned officer in the U.S. *% of Soxhlet, n = 6 Navy and served as a technical director and biochemist for the Department of Table 8: Total polychlorinated dibenzo-p-dioxins. Defense Forensic Laboratory. Aaron holds a Master of Science in Toxicology Sample Matrix Soxhlet (ng/kg) ASE (ng/kg) from the University of Michigan’s School Chimney brick 8040 8170 of Public Health (Michigan, USA) and a Urban dust 1110 1159 Master of Business Administration from Fly ash 93,000 107,9 0 0 The Lake Forest Graduate School of Management (Lake Forest, Illinois, USA). Hamilton Harbor sediment 4283 4119 Direct correspondance to: aaron.kettle@ Parrots Bay sediment 2836 2444 thermofisher.com
Table 9: Mean values in pg/g and % relative standard deviation (%RSD) for dioxins and furans. Buchi SpeedExtractor Thermo Fisher Acelerated Solvent Extraction Content (pg/g) (n = 3) %RSD Content (pg/g) (n = 2) %RSD 2378-TCDF 154 17 158 11 2378-TCDD 7 29 7 5 12378-PeCDF 504 2 439 6 23478-PeCDF 370 15 325 18 12378-PeCDD 47 4 51 5 123478-HxCDF 657 9 557 9 123678-HxCDF 311 14 265 15 234678-HxCDF 276 21 208 2 123789-HxCDF 190 10 201 1 123478-HxCDD 35 14 32 12 123678-HxCDD 58 5 54 3 123789-HxCDD 33 9 39 3 1234678-HpCDF 2807 11 2068 13 1234789-HpCDF 529 5 484 15 1234678-HpCDD 672 5 519 7 OCDF 2006 2 1191 23 OCDD 2003 10 1509 17 WHO-TEQ 476 6 424 4
14 Recent Developments in Sample Preparation May 2014
magentablackyellow ES431126_LCESUPP0514_014.pgs 04.29.2014 20:20 ADV Plug Cap Seal Microwave-Accelerated Extraction — SW-846 Liner Method 3546 and Beyond
Bobbie McManus, Michelle Horn, Steve Smith, Bob Lockerman, and Greg LeBlanc, CEM Corporation, Matthews, North Carolina, USA.
Microwave-accelerated extraction (MAE) was evaluated by the authors of this review in 1999 (1). Since this time, the United States Environmental Protection Agency (USEPA) has promoted the use of SW-846 Method 3546 using MAE for the extraction of organic compounds from solid matrices. Applications for this technique have increased and MAE equipment has been streamlined to dramatically increase throughput and ease of use. This article reviews the latest enhancements to this technology from a hardware and applications perspective.
In 1999, microwave-accelerated sample extraction methods. The • ASTM 5765 – 05 (2010) is the extraction (MAE) was a relatively matrix is either aqueous, solid, an Standard Practice for Solvent new technique for solvent extraction, air sampling train, or non-aqueous Extraction of Total Petroleum and approvals were only obtained soluble. Analytes are characterized Hydrocarbons from Soils and when the technique became as either nonvolatile or semivolatile Sediments Using Closed Vessel mainstream. The first approval organic compounds. All samples Microwave Heating (4). The soil was from the state of California analyzed for non-volatile or or sediment sample is extracted under its California Environmental semi-volatile organic compounds with acetone or hexane in a sealed Technology Certification programme require a solvent extraction step, with microwave transparent vessel for the extraction of semivolatile the exception of non-aqueous solvent using microwave heating to an organic compounds in soil, soluble samples. internal temperature of 150 °C, sediment, and sludge (2). As the Microwave extraction (Method 3546) producing an extract suitable for technique substantially reduces was formally included in SW-846 in analysis by gas chromatography sample extraction time and solvent Final Update IV of the Third Edition (GC) or gravimetric techniques. consumption, the sample turnaround of the manual in 2008 (3). Microwave • ASTM D6010-12 is the Standard time for data generation improved extraction is the process of heating Practice for Closed Vessel significantly. The certification was solid sample–solvent mixtures in a Microwave Solvent Extraction of intended to encourage use of sealed (closed) vessel with microwave Organic Compounds from Solid the technique where data quality energy under temperature-controlled Matrices. The soil, sediment, sludge, objectives could be met by its use. conditions. The temperature is or waste sample is extracted in an The United States Environmental elevated significantly above the acetone-hexane mixture at 115 °C Protection Agency’s (USEPA’s) Test atmospheric boiling point of the producing an extract suitable for Methods for Evaluating Solid Waste solvent, accelerating extraction while analysis of semi-volatile or volatile (SW-846) provides a comprehensive giving performance comparable to organic compounds by GC or gas source of information on sampling, the standard Soxhlet method. Solvent chromatography–mass spectrometry sample preparation, analysis, and consumption is only 25–50 mL per (GC–MS) (5). This standard practice reporting for compliance with the sample. reduces sample preparation time, Resource Conservation and Recovery The American Society for solvent consumption, and operating Act (RCRA). SW-846 outlines test Testing and Materials (ASTM) is an costs. procedures used to characterize international standards organization • ASTM D7210-13 is the Standard solid waste in accordance with that publishes voluntary consensus Practice for Extraction of Additives 40 CFR Part 261, Identification and technical standards, including test in Polyolefin Plastics. This Listing of Hazardous Waste. The methods that define how a method is provides guidelines for extracting sample preparation and analytical performed and the accuracy of the phenolic antioxidants, phosphite procedures (or determinative steps) result. Test results may be used to antioxidants, UV stabilizers, are categorized by the analyte, which assess compliance with a Standard antistatic agents, and slip additives can be inorganic or organic. Specification. The ASTM has three from milled polyolefin plastics (6). The sample matrix and analytes test methods that incorporate the It now includes an MAE technique define the SW-846 3500 series MAE technique: for subsequent analysis of the
www.chromatographyonline.com 15
magentablackcyanyellow ES431148_LCESUPP0514_015.pgs 04.29.2014 20:21 ADV McManus et al.
Table 1: Approved microwave accelerated extraction techniques. Governing Body Method Sample Solvent Analytes United States SW 846 - 3546 Soils, sediments, and Acetone:Hexane Semivolatile organic compounds, Environmental sludges organophosphorus pesticides, organochlorine Protection Agency pesticides, chlorinated herbicides, phenoxyacid herbicides, substituted phenols, PCBs, and PCDDs or PCDFs American Society of D 5765 - 05(2010) Soils and sediments Acetone:Hexane Total petroleum hydrocarbons Testing and Materials American Society of D 6010 - 12 Soil, sediments, Acetone:Hexane Semi-volatile and volatile organic compounds Testing and Materials sludges, or waste samples American Society of D 7210 - 13 Polyolefins Sample Antioxidants, UV stabilizers, slip, and anti-static Testing and Materials dependent agents Consumer Product CPSC- Plastics (children's Acetone:Hexane Phthalates Safety Commission CH-C1001-09.3 toys or childcare articles)
energy to separate compounds Figure 1: Representation of a batch (multimode) microwave extraction system. from the sample to the solvent. To Fume The extraction is most commonly Hood performed in a sealed vessel under temperature-controlled conditions. This provides a Multimode significant temperature elevation above the atmospheric boiling point Direct Microwave Cavity of the solvent and accelerates the Temperature extraction process. Sensor Batch Microwave Systems: The Solvent components of the batch style Detector microwave system remain the same as when reviewed in 1999 (1) in terms of microwave energy generation, application of microwave energy to the sample load, safety concerns, sample stirring, and indirect heating. However, there have been significant enhancements in terms of its Air Flow “intelligence”, vessel technology, and temperature control. This review IR focuses attention on these areas of the Temperature batch microwave system for solvent Sensors extraction applications (see Figure 1). Microwave Magnetic Recent systems have a level Field Stirring of “intelligence” not present in earlier versions. The latest systems possess libraries of predefined methods loaded into software with extract by chromatographic children’s toys and childcare articles the capability to count the number techniques. was the result of standards passed of samples and recognize the in the Consumer Product Safety vessel type loaded — this enables Phthalates are mainly used as Improvement Act Section 108. The test “one-touch” operation. A method is plasticizers to increase flexibility method uses a microwave-extraction selected by the user based on the and durability, but they are linked to technique based upon EPA Method sample matrix. The sample is then health issues and are being phased 3546 as an acceptable method of weighed into the vessel, solvent is out of use. The Consumer Product extraction. added, and the vessel is sealed. Safety Commission (CPSC) issued The turntable with the vessels is Test Method: CPSC-CH-C1001-09.3, Advances In Hardware And then placed into the system cavity. a Standard Operating Procedure for Instrumentation On pressing the start button, the Determination of Phthalates in April MAE involves heating solid sample– system can then determine the 2010 (7). Phthalate content analysis in solvent mixtures with microwave vessel type and number of vessels,
16 Recent Developments in Sample Preparation May 2014
magentablackcyanyellow ES431147_LCESUPP0514_016.pgs 04.29.2014 20:21 ADV McManus et al.
components — a vessel body, a Figure 2: Microwave extraction vessel components and assembly. plug seal, and a cap — simplifying assembly. Figure 2 represents an example of this design. Plug Cap With the reduction in the number Seal of components and size, up to 41 samples can be processed for each batch using 75-mL vessel assemblies. When processing larger scale samples, 100-mL vessel assemblies are available that have a throughput of 24 samples for each batch. Both vessel sizes have a built-in pressure relief mechanism for safety purposes. If the pressure inside the vessel exceeds its Liner operating limitations, the vessel will automatically vent to relieve the internal pressure. The 100-mL vessel assemblies can be used with an optional disposable glass liner inserted inside the vessel liner. This component insertion eliminates the need to clean the vessel liner in between runs, eliminating issues associated with carry over contamination. Table 2 compares modern features of currently available batch-type MAE systems on the market. Figure 3: Graphical representation of temperature versus vessel using “all vessel” Previous generation systems temperature control. used a temperature sensor inserted into one of the vessel assemblies 200 containing a sample with solvent to create a feedback control loop regulating microwave input power to maintain target temperature. This 150 assumed equivalent absorbtion for all samples in the system. This temperature technology has since 100 been replaced with a floor-mounted temperature sensor that measures the temperature of all samples as
Temperature (ºC) Temperature they rotate inside the system. The 50 system then takes an average of all the sample temperatures measured to regulate the microwave input power for the temperature control. 0 The enhancement improves control 5 10 15 20 25 30 35 40 over the whole extraction process Vessel Position and removes the need for an operator to insert a temperature probe into one of the sample subsequently applying the correct Past generation vessels vessel assemblies. Figure 3 shows level of microwave energy to meet were assemblies containing six vessel-to-vessel temperature the method-defined temperature for components that were rather variation and is typical for all vessel its hold time to complete extraction. cumbersome, and only 12–14 temperature controlled systems. This minimizes operator error as it samples could be loaded into the The discussion up until this point automatically defines the input power system at one time. To properly has related to batch style microwave levels needed for different numbers seal vessels, a torque wrench systems or systems that extract of samples in the system and the was required. The latest vessel a batch of samples at one time. conditions for the extraction. technology now only has three These systems are well-suited to the
www.chromatographyonline.com 17
magentablackcyanyellow ES431143_LCESUPP0514_017.pgs 04.29.2014 20:20 ADV McManus et al.
Table 2: Batch, closed vessel microwave accelerated extraction (MAE) system comparison.
Supplier Anton Paar CEM Corporation Milestone PerkinElmer
Model Multiwave Pro MARS 6 Ethos EX Titan MPS
Feature
Power output (Watts) 1500 1800 1200 1500
Direct, in-vessel temperature control Yes (Gas bulb) Yes (Fibreoptic) Yes (Fibreoptic) No
Contactless, all vessel temperature control Yes (Measures only) Yes Yes Yes
Direct pressure control Yes Yes Yes No (Indirect optical technique)
Stirring Yes Yes Yes No
Time to parameter software control No Yes Yes No
Vessel recognition and counting capability No Yes No No
User interface Integrated touchscreen Integrated touchscreen External Integrated touchscreen touchscreen
Viewing window Yes Yes Yes No
Exhaust solvent sensor Yes Yes Yes No
Number of vessels or batches 16 40/24 41/24 16
Volume of vessel (mL) 100 75/100 70/75 75
Vessel liner material of construction Teflon Teflon/optional glass Teflon/optional Teflon glass
Indirect heating Silicon carbide inserts Carboflon Weflon No
Instrument safety approval (ETL/UL/NRTL) Yes Yes Yes No
needs of laboratories processing Sample pressure is measured using or the number of vessels, that can environmental samples. a sensor in the component that be held for automated processing Sequential Closed Vessel MAE “seals” the vessel into the microwave is variable — most commonly 48 or Systems: Microwave systems system cavity. The component 60 of 10-mL size vessels and 24 for that process samples sequentially measures the deflection of the vessel 80-mL size vessels. Table 3 provides have recently been introduced for cap as pressure is generated during a comparison of the important extraction applications. The systems the irradiation cycles causing the features of the sequential MAE consist of a microwave component cap to move outward. The system systems on the market. with a relatively small “focused” stirs the sample solvent mixture The potential benefits of a cavity, and an automated vessel in the vessel using a magnetic sequential versus a batch style handling component that feeds and component located under the microwave system are: removes vessels containing sample cavity floor and a magnetic spinbar • The option to use unique extracting to and from the microwave system placed in the extraction vessel. At conditions for each sample. cavity. the end of the irradiation cycle, the • Selection of precise temperature When samples are introduced sample is automatically cooled using control for every sample. into the microwave system cavity, compressed air, rapidly decreasing • Different sample types can be vessels are sealed so samples can the temperature and pressure of processed within a “rack” of achieve elevated temperatures and the vessel. The “cool down” time samples. pressures during the microwave is reduced resulting in turnaround • Samples can be processed with irradiation cycle. The maximum times as low as five min per sample. different extracting solvents within operating conditions for vessels are Figure 4 depicts a typical design of a a “rack” of samples. up to 300 °C and 450 psi (28 bar) — sequential style MAE system. • A sample can be immediately easily meeting conditions needed for Vessels used with sequential style removed after completion for post extraction applications. systems are available in sizes from extraction handling. Temperature is monitored and 5–80 mL with a working volume • Small sample sizes/solvent measured within each vessel using range of 2–50 mL respectively. The volumes can be processed (where an infrared sensor mounted in the vessel material is normally glass, feasible) with the 10-mL vessel. cavity. Temperature data is used to with quartz as an option. Caps have regulate the microwave input power a Teflon sealing surface between This sequential approach provides and control the temperature in the the cap and vessel lip to minimize more flexibility than with a batch-style same way as the batch systems. contamination issues. The rack size, system. Both systems apply microwave
18 Recent Developments in Sample Preparation May 2014
magentablackcyanyellow ES431145_LCESUPP0514_018.pgs 04.29.2014 20:20 ADV McManus et al.
to determine MeHg+, EtHg+, and Figure 4: Representation of a focused (single-mode) microwave extraction system. Hg2+ in sediment samples. The limits of detection were 0.013 ug/L, Pressure Transducer 0.022 ug/L, and 0.011 ug/L for mercury species using the microwave technique. The technique was validated by applying it to two certified reference materials — IAEA-405 and ERM-CC580 — that gave good agreement with the Reaction High Density certified value. Vessel Microwave Field Antioxidants: There are potential health advantages to diets rich in Waveguide antioxidant compounds that include lowering the risk of cardiovascular disease and certain cancers. In 2011, Mathur and co-workers investigated IR Single Mode MAE versus traditional solvent Temperature Microwave extraction techniques for screening Sensors Cavity plant extracts for antioxidant activity (9). The MAE technique was Magnetic optimized on 20 g of plant samples Stirring using methanol as the extracting solvent at 80 °C with a 20 min hold time. Extracts were screened for in energy to sample–solvent mixtures at method. Key benefits are enhanced vitro antioxidant activity. The results elevated temperature and pressure yield, quicker isolation times, and an indicated that extracts prepared by under controlled conditions with environmentally friendly approach. the MAE technique showed potent sample stirring to accelerate the antioxidant activity relative to the extraction process. For laboratory New Applications Areas traditional method of extraction. scientists, the sample variety, The introductory section of this Active Pharmaceutical throughput, and objectives will define review discussed approvals Ingredients: The extraction of which system is better suited for their granted for the MAE technique. active pharmaceutical ingredients needs. For a technique to be approved (APIs) from solid dosage forms is Solvent-Free MAE System: for a specific application, it must critical to validate concentrations Milestone recently introduced a have records documenting its within formulations. Brannegan system capable of performing performance for a wide-variety of published a chapter titled “Extraction solvent-free microwave extraction matrices. The primary use of MAE is Techniques Leveraging Elevated following a collaboration with the in environment-related applications. Temperature and Pressure” Université d’Avignon et des Pays However, scientists typically explore within Sample Preparation of de Vaucluse (Avignon, France). other applications for existing Pharmaceutical Dosage Forms: The system uses a technique of technologies once they have been Challenges and Strategies (10). The microwave hydrodiffusion (a type proven to work. This section looks application of MAE can facilitate of steam distillation) followed by at other applications for the MAE rapid troubleshooting of low potency gravity separation to produce technique. results and help identify the source essential oils in a concentrate form Mercury Speciation: The total of these issues. MAE can be from raw aromatic plant materials. mercury concentration in a sample performed in parallel, samples can The technique is performed at does not provide an accurate picture be stirred during the extraction step, atmospheric conditions and does for “cause–effect” relationships. The and rapid heating of the sample not use any solvent or added distribution of mercury species can is provided, therefore providing water. Internal heating of the water provide an improved understanding solubilization and extraction of within the plant material causes of data relationships leading to more APIs from the solid dosage forms. cell disruption and release of the accurate conclusions. In 2013, Leng The chapter provides case studies extract (water and essential oils). and co-workers published an article documenting the benefits of MAE to The extract is then gravity fed into titled “Speciation Analysis of Mercury troubleshoot low potency results. a collection system underneath in Sediments by HPLC Following RoHS/WEEE: The adoption of the microwave system to separate Microwave-Assisted Extraction” the “Restriction of Hazardous the essential oil from the water. (7). The authors proposed the Substances (RoHS) and Waste from Samples are processed one at a combination of microwave-assisted Electrical and Electronic Equipment time in a 1- to 2-L proprietary glass extraction of samples with HPLC– (WEEE) Directives” by the European vessel using a microwave power VGAFS determination. It provided Union (EU) created a problem. There and time- or temperature-based a sufficiently low detection limit were no reliable, cost-effective
www.chromatographyonline.com 19
magentablackcyanyellow ES431153_LCESUPP0514_019.pgs 04.29.2014 20:22 ADV McManus et al.
Table 3: Sequential, closed vessel microwave accelerate extraction system comparison.
Supplier Anton Paar Biotage CEM
Model Monowave 300 with MAS 24 Initiator Robot 60 Discover SPX
Feature
Power output (Watts) 850 400 300
Infrared temperature control Yes Yes Yes
Direct pressure control Yes Yes Yes
Stirring Yes Yes Yes
Post reaction cooling Yes Yes Yes
Time to parameter software control Yes No Yes
User interface Integrated touchscreen Integrated touchscreen External controller
Volume of vessel (mL) 4, 10, or 30 5, 10, and 20 10, 35, and 80
Vessel material of construction Glass/Quartz Glass Glass/quartz with optional Teflon liners
Autosampler capacity 24 60/30 72/36/24 (Number of vessels)
Indirect heating Silicon carbide inserts No Carboflon
Instrument safety approval (ETL/UL/NRTL) Yes Yes Yes
methods of testing for the presence aromatic plants. The traditional of sample types and analytes. This is of high-mass unit additives such as approaches to extract the essential especially true for those compounds polybrominated biphenyls (PBBs) oils use a chemical process that might degrade based upon and polybrominated diphenyl ethers of steam distillation or solvent temperature variability in a batch (PBDEs) in polymer samples. NSL extraction, or a mechanical process process. Analytical resolved the problem by using a cold press technique. Current research being performed developing an MAE technique for Vian and associates developed on sequential systems includes the extraction of PBB and PBDE from a microwave-assisted technique extraction of fatty acid methyl polymers and subsequent analysis for the extraction of essential oils esters (FAMEs) in a wide variety by GC–MS (11). The technique without the use of added solvents or of food samples, as well as amino provided savings in time and cost water — microwave hydrodiffusion acid hydrolysis for adulteration (labour, solvent cost, and disposal) and gravity, a new technique for testing in products — such as while providing 99% recovery of the extraction of essential oils (13). skimmed milk and infant formula. additives from sample sizes of only The microwave extracted essential Work is also being conducted to 0.5 g. oils were produced with yields and test the possibility of performing Food Safety: Dicyandiamide is a aromatic profiles similar to standard simultaneous hydrolysis and white crystalline compound primarily methods but in one-sixth of the time. extraction of total fats in foods. used for the production of melamine. With the increasing levels of food The detection of dicyandiamide Future safety and fraud concerns, it is in milk-based products can be Over the past decade, MAE has likely that this technique will expand indicative of melamine, responsible become a mainstream sample significantly in the food testing arena for kidney stones. Shen and preparation method, because of in the coming years. Other areas coworkers developed a MAE advances in vessel technology, of research include the extraction method in combination with liquid software control, and throughput. of AZO dyes from textiles that have chromatography tandem mass The remaining obstacle for the been banned in Europe since 2003, spectrometry (LC–MS–MS) for the batch-style approach is sample– as well as metabolite extractions in determination of dicyandiamide solvent separation after the the biological field of study. residue in infant formula (12). extraction step and before the The extraction was performed in analytical step. The majority of future Summary 5% formic acid with recovery in application areas are likely to be Over the past 14 years, MAE the range of 83–96% at levels of focused on developing flexibility of has become well-accepted for 0.25 mg/kg. sequential systems. The ability to environmental testing. Regulatory Essential Oils: Essential oils control conditions for each sample approval by the EPA, ASTM, CPSCs, are highly concentrated, volatile extraction will enable scientists to and others validate the technique oils that can be extracted from rapidly develop methods for a variety as an analytical method. The
20 Recent Developments in Sample Preparation May 2014
magentablackyellow ES431152_LCESUPP0514_020.pgs 04.29.2014 20:22 ADV McManus et al.
combination of high-throughput, (6) Standard Practice for Extraction of served as Product Manager for both the solvent reduction, and ease of use Additives in Polyolefin Plastics, DOI: Analytical and Process areas where she 10.1520/D7210-13 (http://www.astm. make it an excellent alternative org/Standards/D7210.htm). participated in several AOAC studies and to other extraction techniques. (7) Standard Operating Procedure for approvals for new technology. Advances in temperature Determination of Phthalates, (http:// Michelle M. Horn is a scientific writer www.cpsc.gov/PageFiles/110957/ measurement, software control, CPSC-CH-C1001-09.3.pdf) (2010). for CEM Corporation. She writes and vessel technology, and automation (8) Geng Leng, Li Feng, Shao-Bo Li, edits a variety of content in the areas of have allowed expansion of the Ping Yang, and De-Zhong Dan, LCGC chemistry, biochemistry, and peptide Europe 26(5), 250–258 (2013). technique into the regulatory synthesis. (9) A. Mathur, D. Mathur, G. Prasad and environmental arena, a trend that V.K. Dua, Asian Journal of Biochemical Steve Smith is illustrator extraordinaire should continue. and Pharmaceutical Research 1(2), for CEM Corporation. He provides 410–418 (2011). marketing communication support for (10) Beverly Nickerson, Ed. Sample References Preparation of Pharmaceutical Dosage their laboratory grade microwave system (1) G. LeBlanc, LCGC 17(6S), S30–S37 (1999). Forms Challenges and Strategies for product offerings. (2) California Regulatory Notice Register, Sample Preparation and Extraction, (2011). Bob Lockerman is the Analytical (11) A. Kovalenko, and B. Bacher, Register 2000, December 15, 2000, Product Line Manager for CEM Volume 50-Z, pages 2136 with Microwave Extraction Provides More Evaluation Report Reliable Analysis of High Mass Unit Corporation. He oversees the (3) Federal Register, Vol. 73, No. 2, Additives, (http://www.nslanalytical. microwave-based, solvent extraction Thursday, 3 January 2008, Notices, com/sites/default/themes/custom/nsl/ pdf/Microwave%20Digestion.pdf) product offerings. pages 486-489. (4) Standard Practice for Solvent Extraction (12) Y. Shen, C. Han, X. Zhou, and X. Chen, Greg LeBlanc is a New Business . of Total Petroleum Hydrocarbons from J. Dairy Sci DOI: 10.3168/jds.2013- Development Manager with CEM 6881, (2013). Soils and Sediments Using Closed Corporation. He has over 28 years of Vessel Microwave Heating, DOI: (13) M. Vian, X. Fernandez, F. Visinoni, and experience in the use of laboratory 10.1520/D5765-05R10, (http://www. F. Chemat, J. Chromatogr. A 1190, astm.org/Standards/D5765.htm). 14–17 (2008). microwave systems for sample (5) Standard Practice for Closed Vessel preparation as a front end for Microwave Solvent Extraction of Organic Bobbie McManus is the Director of chromatographic and spectroscopic Compounds from Solid Matrices, DOI: 10.1520/D6010-12, (http://www.astm. North American Sales at CEM Corporation. analysis. Direct correspondence to: Greg. org/Standards/D6010.htm). During her 25 year career at CEM, she has [email protected]
Introducing the LCGC EUROPE APP for your iPhone or iPad
Designed for both the iPhone and iPad, LCGC Europe’s new app may be found on iTunes, under the Business category, and downloaded for free. Exploring the latest news and trends for chromatographers has never been easier. Download it for free today at http://www.chromatographyonline.com/LCEApp
www.chromatographyonline.com 21
magentablackcyanyellow ES431151_LCESUPP0514_021.pgs 04.29.2014 20:21 ADV Methodology Simpler,generic Less selective • Direct injection methodology Minimal sample cleanup • Filtration • Centrifugation • Dilute and shoot • Sonication • Lyophilization • Protein precipitation • Distillation • Dialysis and ultra+ltration • Liquid–solid extraction and pressurized-,uid Green Chemistry extraction • Soxhlet extraction • Solid-phase microextraction • Supported liquid extraction • Liquid–liquid extraction • Solid-phase extraction • QuEChERS • Turbulent ,ow chromatography Perspectives on • Derivatization • Column switching and heart cutting • Immunoaf+nity sorbents • Molecularly imprinted polymers More complicated Greater selectivity methodology Optimal sample cleanup Analytical Extractions
Victor Essel and Douglas E. Raynie, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, South Dakota, USA.
The growing interest in green chemistry requires fresh perspectives on analytical extractions. Reduced solvent consumption, alternative safer solvents, and reasonable energy demands must be balanced with traditional analytical considerations such as extraction yield and selectively. This article introduces some of the concepts behind green chemistry, and discusses green solvent selection and extraction techniques. An overview of alternatives to conventional solvents, new green solvents, ionic liquids, and other solvent options are also described.
The history of modern analytical chemical process rather than after miniaturization. Miniaturization is extractions mirrors the development it has been performed. Despite advantageous when used properly, of green chemistry. For nearly a the fact that modern extraction but it seems that many analysts century Soxhlet extraction, combined technologies and green chemistry embrace miniaturization because with shake-flask methods, was the are contemporaries, the two fields they can, rather than following standard method for the isolation of have advanced independently of appropriate sampling theory. analytes from solid samples, while each other. The green advantages of Miniaturization may reduce solvent multiple liquid–liquid extractions newer extraction methods are widely use and waste generation, but it (LLEs) using separatory funnels promoted, but they are rarely placed does not address, for example, was the method of choice for liquid in the context of the green chemistry solvent toxicity. samples. In the mid-1980s, new principles. R.E. Majors recently reported on forms of analytical extractions were Green chemistry in the developments in an article entitled developed and popularized — such chromatography laboratory was ‘“Just Enough” Sample Preparation: as supercritical fluid extraction recently reviewed and this article A Proven Trend in Sample Analysis’ (SFE), pressurized-fluid extraction described ways to save on solvent (3). Following the examples of (PFE), solid-phase extraction (SPE), consumption, alternatives to using “just-in-time” inventory control in solid-phase microextraction (SPME), acetonitrile in liquid chromatography total quality management systems, microwave-assisted extraction (LC), and how to assess the “just-enough” sample preparation (MAE), single-drop microextraction “greenness” of analytical methods balances analytical considerations (SDME), and ultrasonic extraction. (2). These topics will not be repeated such as selectivity and quantitative These new extraction techniques in this review. needs with labour, time, and had benefits such as faster times, solvent intensiveness. Important lower cost for each extraction, Green Chemistry in the “just-in-time” methods are presented improved yield and reproducibility, Planning Stages in Figure 1. This approach reiterates and lower solvent volumes. Lower A somewhat tongue in cheek, the importance of understanding the solvent volumes provide significant though serious, adage is that “the purpose of a given analysis: environmental advantages. ‘greenest’ analytical procedure is “Minimizing the number of sample Extraction solvents generally provide that which you do not perform”— but handling steps in any analytical the bulk of the waste encountered in well-executed analytical methods technique is desirable since the more any analytical method and often have can inherently be green. Following times the sample is transferred, the health and safety concerns, such as correct sampling theory (as outlined greater the chance of analyte loss toxicity or flammability. in several undergraduate textbooks), (or modification), thereby resulting In parallel with these the minimum number of samples in poor analytical precision and developments, the concept of and minimum sample size is desired. accuracy,” — R.E. Majors (3). green chemistry emerged. This Samples collected should also Similarly, the appropriate choice culminated in the statement of the support the problem-solving efforts of the subsequent analytical 12 principles of green chemistry in behind the analysis. characterization technique can 1998, which are shown in Table 1 (1). One disturbing trend of many drive sample preparation and the The goals of green chemistry are to modern extraction technologies, overall “greenness” of an analytical address environmental, health, and that runs counter to the planning procedure. For example: Desorption safety concerns when planning a of an analysis, is the trend toward electrospray ionization (DESI) and
22 Recent Developments in Sample Preparation May 2014
magentablackcyanyellow ES431149_LCESUPP0514_022.pgs 04.29.2014 20:21 ADV Essel and Raynie
Table 1: Principles of green chemistry. Adapted from reference (1). Table 2: Isolation of divalent cations from water with EDDS (7). 1. Prevention It is better to prevent waste than to treat or clean up waste after it has been created. Cation Recovery with EDDS relative 2. Atom Economy to Recovery with EDTA Synthetic methods should be designed to maximize the incorporation of all materials used (%RSD) in the process into the final product. Ca2+ 102.3% (0.32) 3. Less Hazardous Chemical Syntheses Mg2+ 96.7% (0.62) Wherever practicable, synthetic methods should be designed to use and generate substances that possess little or no toxicity to human health and the environment. Mn2+ 115.3% (0.44)
4. Designing Safer Chemicals Zn2+ 101.1% (0.19) Chemical products should be designed to affect their desired function while minimizing their toxicity. Pb2+ 110.0% (1.11) 5. Safer Solvents and Auxiliaries The use of auxiliary substances (for example solvents, separation agents) should be made Here, we present a brief overview of unnecessary wherever possible and innocuous when used. analytical extraction technologies 6. Design for Energy Efficiency with green attributes. Energy requirements of chemical processes should be recognized for their environmental Micro-Liquid–Liquid Extraction: and economic impacts and should be minimized. If possible, synthetic methods should be In a review of classical extraction conducted at ambient temperature and pressure. procedures, Murray reported an 7. Use of Renewable Feedstocks approach for extracting large liquid A raw material or feedstock should be renewable rather than depleting whenever sample volumes with a minimal technically and economically practicable. amount of solvent, mixing 980 mL 8. Reduce Derivatives of aqueous sample and 200 µL of Unnecessary derivatization (use of blocking groups, protection/deprotection, temporary hydrocarbon (9). The addition of modification of physical/chemical processes) should be minimized or avoided if possible, water through a side-arm forces because such steps require additional reagents and can generate waste. the lighter organic phase into a 9. Catalysis capillary tube where it is sampled Catalytic reagents (as selective as possible) are superior to stoichiometric reagents. with a syringe. With three extractions 10. Design for Degradation recoveries greater than 90% are Chemical products should be designed so that at the end of their function they break down obtained. Of course, a highly into innocuous degradation products and do not persist in the environment. favourable solute partition coefficient 11. Real-time Analysis for Pollution Prevention and complete water immiscibility of Analytical methodologies need to be further developed to allow for real-time, in-process the extracting solvent are vital to the monitoring and control prior to the formation of hazardous substances. success of this approach. 12.Inherently Safer Chemistry for Accident Prevention Single-Drop (10) and Suspended Substances and the form of a substance used in a chemical process should be chosen to Microdroplet (11): These related minimize the potential for chemical accidents, including releases, explosions, and fires. techniques also rely on favourable partition coefficients. However, the single-drop approach may also be direct analysis in real time (DART) of organics for subsequent used in the headspace mode. Once approaches to mass spectrometry chromatographic analysis, these optimized, these techniques can (MS) eliminate or minimize the need results demonstrate the application provide good analytical results. for sample preparation (4,5,6). In of green chemistry principles across Sorptive-Based Extractions: both approaches, the sample (often a broad spectrum of chemical Since its introduction in the 1970s, in solid form) is bombarded by analysis. SPE has become almost routine in energetic species to create ions of many laboratories, especially in the interest. Further development and Green Sample Preparation pharmaceutical industry. In addition maturation of these techniques (and Methods to the extraction selectivity offered those that they have inspired), will As emphasized, nearly all of the by the stationary phase, substantial contribute to the overall “greenness” extraction technologies developed solvent savings may be made. of the analytical endeavour. within the past twenty years tend to Refinements and developments The use of the biodegradable have green advantages, regardless of other extraction technologies chelant ethylene diamine disuccinate of whether that was the initial intent. based on sorbents provide even (EDDS) has been investigated as a SFE, MAE, accelerated-solvent greater green chemistry advantages. substitute for the common ethylene extraction (ASE), and the use of Among the most established diaminetetraacetic acid (EDTA) in superheated water for analytical sorptive methods that apply these analytical applications (7). Table 2 extractions are all discussed advantages are the use of molecular displays the quantitative results for elsewhere in this supplement. recognition sorbents (12), SPME (13), the isolation of aqueous divalent Table 3 shows a comparison of the stir-bar sorptive extraction (SBSE) cations. Although this review green attributes of these, and similar, (14), and supported liquid–liquid focuses on the sample preparation sample preparation methods (8). extraction (15).
www.chromatographyonline.com 23
magentablackyellow ES431146_LCESUPP0514_023.pgs 04.29.2014 20:21 ADV Essel and Raynie
Membrane Methods: Supported Extraction Solvent green chemistry principle. There are liquid membranes (16) and Considerations two approaches to addressing green microdialysis also consume small Regardless of extraction technique, chemistry concerns from the solvent volumes of extracting solvents for one key green attribute is solvent perspective — minimization of specialized applications. use, as directly addressed in the fifth solvent use and the use of “greener” solvents. With the exception of Figure 1: Summary of just-in-time sample preparation methodologies and their superheated water, each of the attributes (3). extraction techniques discussed in this supplement approaches this Methodology Simpler,generic Less selective by reducing solvent use. However, • Direct injection methodology Minimal sample cleanup solvent selection considerations are • Filtration • Centrifugation also important. • Dilute and shoot Solvent Selection: In addition to • Sonication the physical-chemical properties • Lyophilization • Protein precipitation of solvents, cumulative energy • Distillation demand, toxicity, flash point, and • Dialysis and ultra+ltration • Liquid–solid extraction and pressurized-,uid similar characteristics should be extraction addressed. For example, hexane is • Soxhlet extraction widely used as a nonpolar organic • Solid-phase microextraction • Supported liquid extraction extraction solvent; yet it is the most • Liquid–liquid extraction neurotoxic of the n-alkanes (17) • Solid-phase extraction and hexane–acetone mixtures have • QuEChERS • Turbulent ,ow chromatography even greater toxicological effects. • Derivatization Heptane is preferable as a nonpolar • Column switching and heart cutting • Immunoaf+nity sorbents solvent. Many major pharmaceutical • Molecularly imprinted polymers companies have led efforts to More complicated Greater selectivity replace solvents. For example: Pfizer methodology Optimal sample cleanup recommends the substitution of ethyl acetate, methyl-t-butyl ether, toluene,
Table 3: Comparison of greenness issues of typical sample pre-treatment techniques used for trace analysis C. Adapted and reproduced with permission from Elsevier (8).
Treatment Method “Green Issues” Time Energy Safety Solvents
Inorganic
Dry ashing 6-8 h High consumption Very safe Use of small volumes of diluted acids to solubilize the ash
Wet digestion (open Several High consumption Risk of explosion and spills Use of large volumes of concentrated vessel) hours mineral acids
Microwave-assisted <1 h Moderate Risk of explosion, safety at the Use of small volumes of concentrated digestion (closed consumption expense of expensive instrumental mineral acids vessel) equipments
Organic
Soxhlet 6–24 h High consumption Exposure risk to organic vapours 150–500 mL of organic solvents
Microwave-assisted 10–30 Moderate Potential explosion risk with closed 10–40 mL of organic solvents extraction min consumption vessels
Supercritical fluid 10–60 Moderate Very safe (high pressure and 2–5 mL (solid trap); supercritical CO2 used extraction min consumption temperatures) as extractant fluid
Accelerated solvent 10–20 Moderate Very safe (high pressure and 10–40 mL of organic solvents extraction min consumption temperatures)
Organic and inorganic
Ultrasound extraction <1 h Moderate Very safe, extractions performed Use of moderate volumes of concentrated with a bath sonicator consumption at atmospheric pressure and room mineral acids and organic solvents for temperature inorganic and organic analysis, respectively
Ultrasound extraction <1 h Moderate Very safe, extractions performed Use of small volumes of diluted mineral with an ultrasonic probe consumption at atmospheric pressure and room acids and organic solvents for inorganic and temperature organic analysis, respectively
24 Recent Developments in Sample Preparation May 2014
magentablackcyanyellow ES431144_LCESUPP0514_024.pgs 04.29.2014 20:20 ADV Essel and Raynie
or 2-methyltetrahydrofuran for studies indicated that these DES fall (16) J.A. Jonsson, Comp. Anal. Chem. 37, dichloromethane during extractions. into the category of polar hydrogen 503–530 (2002). (17) Agency for Toxic Substances and These industrial efforts have led to bond-donating solvents because of their Disease Registry (July 1999). N the development of several methods high ET(30) or ET values. In addition, “Toxicological Profile for n-Hexane”. to characterize “greenness” of the effect of temperature and water Atlanta, GA: U.S. Department Of Health And Human Services. solvents (18,19,20) and solvent content on polarity and Kamlet–Taft (18) C.S. Slater and M. Savelski, J. Environ. guides. These reports also include parameters of DESs was studied. Sci. Health A 42, 1595–1605 (2007). summaries of computer-aided (19) R. Gani, C. Jimenez-Gonzalez, and solvent selection and property tools. Summary D.J.C. Constable, Comput. Chem. Eng. 29, 1661–1676 (2005). Green Solvents: Several natural Modern sample preparation (20) C. Jimenez-Gonzalez, A.D. Curzons, product-derived organic solvents technologies grew up alongside, D.J. Constable, and V.L. Cunningham, are being explored for their green but separate from, green chemistry. J. Clean Technol. Environ. Policy 7, 42–50 (2005). attributes. Jessop has called However, the environmental aspects (21) P.G. Jessop, D.A. Jessop, D.Fu, and L. for the continued investigation featured in these technologies Phan, Green Chem. 14(5), 1245–1259 into new solvents (21). Among parallel the 12 principles of green (2012). (22) K. Faure, E. Bouju, P. Suchet, and A. emerging green solvents of chemistry. Recent developments Berthod, Anal. Chem. 85(9), interest are d-limonene (22), ethyl have tended to focus on reducing 4644–4650 (2013). lactate (23), γ-valerolactone (24), solvent usage in chemical (23) S. Aparicio and R. Alcalde, Green 2-methyltetrahydrofuran (25), and extractions. Over the next decade Chem. 11(1), 65–78 (2009). (24) I.T. Horvath, H. Mehdi, V. Fabos, L. cyclopentyl methyl ether (26). or so, advances based on research Boda, and L.T. Mika, Green Chem. Ionic Liquids and Deep Eutectic into green solvents, ionic liquids, and 10(2), 238–242 (2008). Solvents: The use of ionic liquids deep eutectic solvents, as well as (25) D.F. Aycock, Org. Process Res. Dev. 11(1), 156–159 (2007). to extract solvents was recently solvent cumulative energy demand (26) K. Watanabe, N. Yamagiwa, and Y. reviewed (27). Emphasis was placed and chemical toxicity will drive the Torisawa, Org. Prcess Res. Dev. 11(2), on single-drop microextraction, green chemistry attributes of current 251–258 (2007). SPME, dispersive liquid–liquid and future sample preparation (27) T.D. Ho, H. Yu, W.T.S. Cole, and J.L. Anderson, LCGC 31(2), 92–107 microextraction (DLLME), hollow technologies. (2013). fibre-supported liquid membrane (28) Y. Dai, G.J. Witkamp, R. Verpoorte, References extraction, and SPE. While ionic and Y.H. Choi, Anal. Chem. 85(13), (1) P.T. Anastas and J.C. Warner, Green 6272–6278 (2013). liquids may be tuned for the Chemistry: Theory and Practice (Oxford (29) Y. Dai, J. van Spronsen, G. Witkamp, R. application, have negligible University Press, New York, USA, Verpoote, and Y. Choi, Anal. Chim. Acta vapour pressure, and possess 1998). 766, 61–68 (2013). (30) M. Francisco, A. van den Bruinhorst, high thermal stability, their high (2) R.E. Majors and D.E. Raynie, LCGC North America 29(2), 118–135 (2011). and M. C. Kroon, Green Chem. 14(8), viscosity and toxicity (especially (3) R.E. Majors, LCGC North America 2153–2157 (2012). of methylimidazolium ionic 30(12), 1024–1031 (2012). (31) Y.H. Choi, J. van Spronsen, Y. Dai, M. liquids) render their widespread (4) Z. Takats, J.M. Wiseman, B. Gologan, Verberne, F. Hollmann, I.W.C.E. Arends, and R.G. Cooks, Science 306(5695), G.-J. Witkamp, and R. Verpoorte. Plant use somewhat limited. It will be 471–473 (2004). Physiol. 156(4), 1701–1705 (2011). interesting to see what developments (5) H. Chen, N.N. Talaty, Z.Takats, and (32) G. Degam and D.E. Raynie, Chimica occur over the next decade enabling R.G. Cooks, Anal. Chem. 77(21), Oggi (submitted). utility of these intriguing solvents. 6915–6827 (2005). (6) R.B. Cody, J.A. Laramee, and H.D. On the other hand, deep eutectic Durst, Anal. Chem. 77(8), 2297–2302 Victor Essel works as a Postdoctoral solvents (DESs) — formed by the (2005). Research Fellow in the Department of interaction of, for example, hydrogen (7) D.E. Raynie, G. Degam, B.A. Anderson, Chemistry and Biochemistry at South and K. Odegaard, “Greener Chelating bond donors with quaternary ammonium Agents for Analytical Titrations,” Dakota State University. His field compounds — offer several unique presented at the 14th Green Chemistry of interest is in analytical chemistry advantages compared with their ionic and Engineering Conference, and green chemistry, with specialty Washington, DC., USA, (2010). liquid cousins. DESs can be an order (8) C. Bendicho, I. De La Calle, F. Pena, M. in the pretreatment of biomass for of magnitude, or more, less viscous, Costas, N. Cabaleiro, and I. Lavialla, biofuel production, chromatographic inexpensive, and nontoxic. Natural DES, Trends Anal. Chem. 31, 50–60 (2012). separations, and analytical method made from sugars, water, and small (9) D.A.J. Murray, J. Chromatogr. A. 177(1), development and validation. 135–140 (1979). acids have been reported as extraction (10) F. F. Cantwell and M. Losier, Comp. Douglas Raynie is an Associate media (28,29,30,31). Their high Anal. Chem. 37, 297–340 (2002). Research Professor at South Dakota solubilization power for both polar and (11) L. Yangcheng, L. Quan, L. State University. His research interests Guangsheng, and D. Youyuan, Anal. nonpolar compounds was demonstrated Chim. Acta 566(2), 259–264 (2006). include green chemistry, alternative in the isolation of coumaroyls and (12) H. Kataoka, Trends Anal. Chem. 22(4), solvents, sample preparation, high other safflower compounds, non-water 232–244 (2003). resolution chromatography, and (13) C.L. Arthur, L.M. Killam, S. Motlagh, soluble bioactive natural products, M. Lim, D.W. Potter, and J. Pawliszyn, bioprocessing in supercritical fluids. gluten, starch, DNA, and quercetin. The Environ. Sci. Technol. 26(5), 979–983 He earned his Ph.D. in 1990 at properties of DES comprised of choline, (1992). Brigham Young University under chloride, or acetylcholine chloride (14) E. Baltussen, P. Sandra, F. David, and the direction of Milton L. Lee. Direct C.A. Cramers, J. Microcol. Sep. 11(10), with urea or glycerol have also been 737–747 (1999). correspondence to: Douglas.Raynie@ intensively studied (32). Solvatochromic (15) R. E. Majors, LCGC 24(2), 118 (2006). SDSTATE.EDU
www.chromatographyonline.com 25
magentablackyellow ES431150_LCESUPP0514_025.pgs 04.29.2014 20:21 ADV (a)
400 baicalin baicalein 200 Superheated Water as
0 10.978 8.043 0 5 10 15 20 25 30 Time (min) (b) an Extraction 500 Solvent baicalin 10.987 baicalein
250
0 0 5 10 15 20 25 30 in Sample Preparation Time (min)
Roger M. Smith, Department of Chemistry, Loughborough University, Loughborough, UK.
Pressurized high temperature or superheated water is a green extraction solvent used in food, environmental, and traditional medicine studies for the extraction of non-polar and polar analytes including essential oils and spices, agrochemicals, pharmaceuticals, and petrochemicals. The technique can be used on both the analytical and preparative scale to give a clean, solvent-free product for chromatographic analysis. This article reviews the current status of superheated water extraction, discussing extraction methods, applications, and, briefy, problems encountered with this approach.
Water at high temperature is often as an extraction solvent, as a (GC) oven can be used that will forgotten as a potential extraction chromatographic eluent, and as a have good temperature control up to solvent for sample preparation, yet solvent for organic synthesis. The 300 °C. An empty column or similar it can be used in a wide range of recent interest in sample preparation configuration can act as the extraction applications, including the extraction and extraction was aroused in the vessel. Extraction can either be of polycyclic aromatic hydrocarbons 1990s by Hawthorne and co‑workers performed in a static or dynamic (PAHs) from soil and nutraceuticals who used superheated water as an mode. In the latter case, a system as from plant materials, for both extraction solvent in environmental simple as a capillary or narrow‑bore non‑polar and polar analytes. Unlike studies for the extraction of PAHs from tube can provide sufficient restriction many other solvents, water is readily soils (1) and compared it to alternative and back pressure to maintain the available in high purity at minimal extraction methods (2). They found water in the liquid phase in the cost, with minimal disposal costs and that for non‑polar analytes the change oven. The solvation power of high a negligible environmental impact. Its in extraction power with temperature temperature water is effectively potential as a solvent is the result of it’s can be dramatic. For example, the independent of the pressure, so unique properties when heated — the solubility of anthracene, chrysene, an accurate control is usually not polarity changes from a polar solvent and perylene in water increased needed. Some recent extractions at room temperature with a specific by 20,000‑fold over the range have used commercial accelerated permittivity of ε = 80 to ε = 30 at 25–200 °C. solvent extraction systems but these 220 °C in a pressurized system, In later studies by a number of are limited to 200 °C. which is comparable to many organic groups a wide range of analytes and After the extraction, the eluent solvents at room temperature, such matrices have been examined and water is cooled to room temperature as methanol ε = 33. Mechanically these have been presents in reviews and if sufficiently concentrated can it is easy to handle because only by Smith in 2002 (3); by Kronholm, be analyzed directly either by high low pressures (15 bar at 200 °C) Hartonen, and Riekkola in 2007 (4); performance liquid chromatography are required to maintain the liquid and most recently in 2010 by Teo and (HPLC) or gas chromatography (GC). state. Unlike most organic solvents, co‑workers (5) and Smith (6). The extraction can also be linked it presents no danger from ignition directly to HPLC or superheated or toxicity, and, although it is more Extraction Methods water chromatography, for example, volatile than ionic liquids, its vapours In common with other solvents, to determine herbicides in compost are environmentally benign. superheated water is most suitable (7). In that study, a single stream of Hot water is not often thought of as for the extraction of solid samples water steam performed a sequential a typical extraction solvent but it is such as soils, environmental solids, extraction, fractionation, and used every day at 100 °C to extract and dried plant material but can be chromatographic separation by using tea leaves and coffee beans. When difficult to apply to tissues, impervious temperature control. we raise the temperature further, the food samples, or clinical samples. For less concentrated samples, polarity decreases and extraction The equipment (shown in Figure 1) analytes can be extracted from power increases. From 100 °C up to is fairly simple, consisting of a water the polar aqueous phase and around 300 °C under low pressures supply, pump, an oven containing concentrated before an assay it is referred to as either superheated an extraction vessel, and post‑oven using either a small volume of water, subcritical water, or pressurized restrictor to maintain the pressure. organic solvent, or a solid‑phase hot water and has found application Typically, an old gas chromatograph microextraction (SPME) fibre,
26 Recent Developments in Sample Preparation May 2014
magentablackcyanyellow ES431179_LCESUPP0514_026.pgs 04.29.2014 20:22 ADV Smith
Figure 1: Superheated water extraction system. Applications The wide scope of superheated water extraction is shown in the range of environmental, food, and Pump herbal extracts that are listed in the many examples given in the earlier reviews (3–6). Some recent examples Oven 100–300 °C from the areas of environmental and petrochemical samples and from Restrictor or the rapidly growing area of food and Back pressure regulator plant materials illustrate the possible roles for the technique. These studies include non‑polar Extraction vessel samples, such as aliphatic hydrocarbons from petroleum source Water rocks (9) and hydrocarbons from Extract Huadian oil shale in China (10), and more polar PAH metabolites and pesticides (Table 1). Other Table 1: Recent examples of the use of superheated water extraction for environmental studies have included environmental analysis and related topics. the determination of parabens from Analyte Matrix Conditions Ref house dust (14) and personal care Aliphatic hydrocarbons Petroleum source rocks 250 °C 9 products in marine sediments. In Hydrocarbons Oil shale 260 °C 10 a related study, superheated water has been used to release DDE and PAHs Sediments 100–200 °C 11 related compounds, which had been Pesticides Soil and sediment 120–180 °C 12 trapped and concentrated on a Neonicotinoid insecticides Eels 120 °C 13 nanospun fibre (17). Parabens House dust 80 °C 14 This ability to extract non‑ polar analytes has also led to the Hydroxylated metabolites of PAHs Sediment samples 150 °C 15 use of superheated water in soil Perfumes, personal care products, and Marine sediments 200 °C 16 remediation as it does not markedly pharmaceuticals disrupt the soil structure unlike some alternative techniques. For Figure 2: Comparison of high performance liquid chromatography (HPLC) example, in recent studies Islam chromatograms obtained for baicalin and baicalein in Scutellariae radix by: and co‑workers removed PAHs (18) (a) Soxhlet extraction; and (b) pressurized hot water extraction. Adapted and and lubricating oils (19) from soils; reproduced with permission from Elsevier (36). and superheated water has been evaluated for the remediation of (a) pesticide‑contaminated soil (20). 400 The main growth in recent years baicalein baicalin in superheated water extraction has been for the extraction of 200 plant materials to yield natural products, such as essential oils or nutraceuticals (Table 2). Although
0 10.978 8.043 similar to other methods that 0 5 10 15 20 25 30 Time (min) can be slower, such as steam (b) distillation or solvent extraction, the proportions of polar and non‑polar 500 10.987 compounds can differ, altering baicalin baicalein the quality of the extracts. In a 250 recent study by two‑dimensional gas chromatography (2D–GC), the essential oil from Rosa damascena 0 Mill obtained by superheated water 0 5 10 15 20 25 30 Time (min) extraction was compared with steam distillation and direct thermal desorption (27). With the growth adsorbent disc, stir‑bar, hollow Post extraction derivatization or of interest in the identification of fibre, or a solid‑phase extraction modification can also be performed traditional Chinese medicines and (SPE) cartridge before assay. in the aqueous solution (8). related products, it has become
www.chromatographyonline.com 27
magentablackcyanyellow ES431182_LCESUPP0514_027.pgs 04.29.2014 20:22 ADV Smith
Conclusion Table 2: Recent examples of the application of superheated water to the extraction Superheated water extraction of plant materials. between 100 °C and 250 °C can Analyte Matrix Conditions Reference provide an environmentally friendly Terpenoids Zingiber cassumunar 140 °C 21 method of extraction for both polar and non‑polar analytes from a wide Bioactive species Chrysanthemum 140 °C 22 range of matrices, more rapidly than Essential oil Origanum micranthum 100–175 °C 23 Soxhlet methods, and without the problems of flammable and often Essential oils Camomile Matricaria 100, 125, 150, and 24 Chamomilla L. 175 °C toxic organic solvents.
Essential oils Thymus vulgaris L. 125 °C 25 References Essential oils Cinnamon bark and leaves 200 °C 26 (1) S.B. Hawthorne, Y. Yang, and D.J. Miller, Anal. Chem. 66(18), 2912–2920 Essential oil Rosa damascena Mill 150 °C 27 (1994). Phenolic and flavouring Cinnamon bark 150, 200 °C 28 (2) S.B. Hawthorne, C.B. Grabanski, E.
compounds Martin, and D.J. Miller, J. Chromatogr. A 892(1–2), 421–433 (2000). Phenolic compounds Pomegranate 100–220 °C 29 (3) R.M. Smith, J. Chromatogr. A 975(1), 31–46 (2002). Curcumin Turmeric 197 °C 30 (4) J. Kronholm, K. Hartonen, and M.L. Riekkola, Trends Anal. Chem. 26(5), Alkaloids Goldenseal 140 °C 31 396–412 (2007). (5) C.C. Teo, S.N. Tan, J.W. Yong, C.S. Alkaloids Sophora flavescens Ait 79–190 °C 32 Hew, and E.S. Ong, J. Chromatogr. A 1217(16), 2484–2494 (2010). Anthraquinones Roots of Morinda citrifolia 150–200 °C 33 (6) R.M. Smith, Superheated Water Bioactive compounds Gastrodia elata Blume 60–120 °C 34 Extraction in : Handbook of Sample Preparation (Wiley, New York, USA, Flavanoids Various plants 150–170 °C 35 2010) pp. 181. (7) R. Tajuddin and R.M. Smith, J. Chromatogr. A 1084(1–2), 194–200 increasingly important to identify extract oxytetracycline, tetracycline, (2005). (8) A.M. Carro, P. Gonzalez, and R.A. the composition and sources of and chloramphenicol antibiotics Lorenzo, J. Chromatogr. A 1296, the constituent plant materials. from animal feeds for HPLC analysis 214–225 (2013). Superheated water extraction was (42). In another study Murakami (9) A. Akinlua and R.M. Smith, Energy found by Ong and co‑workers (36) and co‑workers used superheated Fuels 23(12), 6020–6025 (2009). (10) S. Deng, Z. Wang, Q. Gu, F. Meng, J. to give comparable characteristic water up to 225 °C to rapidly extract Li, and H. Wang, Fuel Proc. Tech. 92(5), biomarkers to conventional Soxhlet fluoxetine‑hydrochloride from 1062 –1067 (2011). extraction (Figure 2). capsules for HPLC analysis (43). (11) V. Fernández‑González, E. Concha‑Graña, S. Muniategui‑ As well as analytical sample Superheated water can also be used Lorenzo, P. López‑Mahía and D. preparation for chromatographic for inorganic analysis and Morado Prada‑Rodríguez, J. Chromatogr. A analysis, many of these applications Piñeiro and co‑workers determined 1196, 65–72 (2008). have been used in large‑scale arsenic and arsenic species in (12) O. Chienthavorn and P. Su‑in, Anal. Bioanal. Chem. 385(1), 83–89 (2006). extraction processes of materials, scalp hair by using superheated (13) Z. Xiao, Y. Yang, Y. Li, X. Fan, and S. such as onion skins (37), to provide water extraction followed by Ding, Anal. Chim. Acta 777, 32–40 potential commercial products. liquid chromatography coupled to (2013). (14) N. Ramírez, R.M. Marcé, and F. Borrull, These applications often include inductively coupled plasma mass J. Chromatogr. A 1218, 6226–62231 HPLC or GC assays and have been spectrometry (LC–ICP–MS) (44). (2012). reviewed by Mustafa and Turner (15) X. Wang, L. Lin, T. Luan, L. Yang, and N.F. Tam, Anal. Chim. Acta 753, 57–63 (38), Chemet and co‑workers (39), Problem Areas (2012). Mukhopadhyay and Panja (40), One concern that is always present (16) S. Morales‑Muñoz, J.L. Luque‑García, and Shi and co‑workers (41). One when high temperatures are used is M.J. Ramos, A. Fernández‑Alba, and advantage is that the absence of an the potential degradation of analytes, M.D. Luque de Castro, Anal. Chim. Acta 552(1–2), 50–59 (2005). organic solvent means that potential but there are few reported examples. (17) D. Adeyemi, J. Mokgadi, J. Darkwa, C. issues associated with solvent Although some degradation of Anyakora, G. Ukpo, C. Turner, and N. residues are avoided, particularly hydrastine and berberine was Torto, Chromatographia 73(9–10), 1015–1020 (2011). important if the product is intended observed by Mokgadi and coworkers (18) M.N. Islam, Y.T. Jo, and J.H. Park, for human consumption. (31) during the extraction of J. Ind. Eng. Chem. 8(5), 1689–1693 Because superheated water Goldenseal at 160 °C and 180 °C, (2012). extraction is generally unsuitable for this was not an issue at 140 °C. (19) M.N. Islam, Y.T. Jo, and J.H. Park, J. Ind. Eng. Chem. (2013) (Available clinical tissues or aqueous samples, Leppänen and co‑workers reported on‑line: http://dx.doi.org/10.1016/j. it has not been widely applied in that at 240 °C, cellulose in Norway jiec.2013.07.040). the pharmaceutical area. However, spruce samples could break down (20) M.N. Islam, Y.T. Jo, S.K. Jung, and J.H. Park, Water, Air, Soil Pollut. 244, Wang and co‑workers found that and be extracted (45) but not at 1652 (2013) (DOI: 10.1007/s11270‑013‑ water at 100 °C could be used to lower temperatures. 1652‑8).
28 Recent Developments in Sample Preparation May 2014
magentablackcyanyellow ES431181_LCESUPP0514_028.pgs 04.29.2014 20:22 ADV Smith
(21) O. Chienthavorn, T. Poonsukcharoen, Bioanal. Chem. 401(9), 2977–2985 Chem. Eng. 51(4), 311–324 (2010). and T. Pathrakorn, Sep. Sci. Technol. (2011). (41) J. Shi, S.J. Xue, Y. Ma, Y. Jiang, X. 46(4), 616 –624 (2011). (31) J. Mokgadi, C. Turner, and N. Torto, Ye, and D. Yu, in Green Technologies (22) F. Liu, E.S. Ong, and S.F.Y. Li, Food Amer. J. Anal. Chem. 4, 398–403 in Food Production and Processing, Chem. 141(3), 1807–1811 (2013). (2013). J.I. Boye and Y. Arcand, Eds. (Food (23) F. Gogus, M.Z. Ozel, and A.C. Lewis , J. (32) H. Wang, Y. Lu, J. Chen., J. Li, and S. Engineering Series, Springer, New York, Chromatogr. Sci. 43(2), 87–91 (2005). Liu, J. Pharm. Biomed. Anal. 58, 146 USA, 2012) pp. 273–294. (24) M. Khajenoori, A.H. Asl, and H.N. (2012). (42) L. Wang, H. Yang, C. Zhang, Y. Mo, and Bidgoli, IJE Trans. B. Appl. 26(5), 489– (33) B. Pongnaravane, M. Goto, M. Sasaki, X. Lu, Anal. Chim. Acta 619(1), 54–58 494 (2013). T. Anekpankul, P. Pavasant, and A. (2008). (25) A.L. Dawidowicz, E. Rado, and D. Shotipruk, J. Supercritic. Fluids, 37, (43) J.N. Murakami, K.B. Thurbide, Wilanowska, J. Sep. Sci. 32(17), 390–396 (2006). G. Lambertus, and E. Jensen, J. 3034–3042 (2009). (34) C.C. Teo, S.N. Tan, J.W.H. Yong, C.S. Chromatogr. A 1250, 80–84 (2012). (26) B. Jayawardena and R.M. Smith, Hew, and E.S. Ong, J. Chromatogr. A (44) A. Morado Piñeiro, J. Moreda‑Piñeiro, Phytochem. Anal. 21(5), 470–472 1182(1), 34–40 (2008). E. Alonso‑Rodríguez, P.López‑Mahía, (2010). (35) M.J. Ko, C.I. Cheigh, and M.S. Chung, S.Muniategui‑Lorenzo, and D. (27) M.Z. Özel, F. Gög˘ üş , and A.C. Lewis, Food Chem. 143, 147–155 (2014). Prada‑Rodríguez, Talanta 105, 422–428 Anal. Chim. Acta 566(2), 172–177 (36) E.S. Ong, J.S.H. Cheong, and D. Goh, (2013). (2006). J. Chromatogr. A 1112(1–2), 92–102 (45) K. Leppänen, P. Spetz, A. Pranovich, K. (28) P. Khuwijitjaru, N. Sayputikasikorn, (2006). Hartonen, V. Kitunen, and H. Ilvesniemi, S. Samuhasaneetoo, P. Penroj, P. (37) M.J. Ko, C.I. Cheigh, S.W. Cho, and Wood Sci. Technol. 45(2), 223–236 Siriwongwilaichat, and S. Adachi, J. M.S. Chung, J. Food Eng. 102(4), (2011). Oleo Sci. 61(6), 349–355 (2012). 327–333 (2011). (29) L. He, X. Zhang, H. Xu, C. Xu, F. Yuan, (38) A. Mustafa and C. Turner, Anal. Chim Roger M. Smith is a Professor of Ž. Knez, Z. Novak, and Y. Gao, Food Acta 703(1), 8–18 (2011). Chemistry at Loughborough Univerisity, Bioprod. Process. 90(2), 215–223 (39) F. Chemat, M.A. Vian, and G. Cravotto, Loughborough, UK. Please direct (2012). Int. J. Mol. Sci. 13(7), 8615–8627 (30) M.A. Euterpio, C. Cavaliere, A.L. (2012). correspondence to: R.M.Smith@lboro. Capriotti, and C. Crescenzi, Anal. (40) M. Mukhopadhyay and P. Panja, Ind. ac.uk
LCGC ONLINE
Selected highlights of recent digital content from LCGC Europe and LCGC North America:
CONNECT WITH LCGC LCGC BLOG How do you like to stay updated with the What Is the Optimal Training to Provide to Students latest troubleshooting tips and technical Interested in a Career in Industry? advice? If you are “social” you can follow Analytical chemists entering the work force are not us on Twitter (@LC_GC), join our LinkedIn always ready to face the group, or “like” our page on Facebook. challenges of private industry. Alternatively, if you prefer e‑mail, you can Kevin Schug discusses how best subscribe to our weekly e‑newsletter here: to prepare students for the next goo.gl/R8SLP2 stage of their careers.Available from: goo.gl/JIqnTS
PRACTICAL ADVICE TOPICAL INTERVIEW SFC Column Selection Green Foodomics A column classification system for SFC was developed Is “green foodomics” another buzzword or a new to help with the process of method development. This direction in food analysis? Kate Mosford of The Column article demonstrates how the spoke to Professor Elena Ibañez system can be used to select of the Institute of Food Science the appropriate column when Research (CIAL), Madrid, Spain, developing a new SFC method. to find out more. Full interview Article available here: goo.gl/ available at: goo.gl/B4zeux FBMGxg
www.chromatographyonline.com 29
magentablackcyanyellow ES431180_LCESUPP0514_029.pgs 04.29.2014 20:22 ADV 0Sample Preparation for Chromatography: How Much Can Be Automated?
Edward Pfannkoch, Gerstel Inc., Maryland, USA.
Modern autosamplers and workstations possess a range of capabilities, in addition to simple liquid handling, that allow automation of sample preparation steps traditionally performed manually. Scientists in many laboratories are considering automating labour-intensive sample processing steps — such as weighing, fltration, dilution, solid-phase extraction, evaporation, and reconstitution. Most quantitative methods require preparation of calibration standards that can be as time-consuming as preparing the samples. This article reviews the automation capabilities available and some practical considerations to take into account when choosing to automate some or all of the sample preparation and handling steps that must be done before analysis.
Those of us who experienced the communication between instruments current automation capabilities? What are early days of high performance liquid to allow automation devices to the next developments on the horizon? chromatography (HPLC) may remember communicate with any vendor’s manual injections and strip‑chart chromatography platforms. Defning Sample Preparation recorders on chromatographic Opinions differed regarding the One of the most formidable challenges instruments. In the mid‑1980s rapid importance of integrated (serial) sample to standardization and automation in development of autosamplers, processing versus batch processing, the analytical laboratory is the broad integrators, and personal computers and the need for flexible (user‑defined) range of sample types. Samples can (data systems) greatly simplified sample robotic systems that provide a set be gaseous, liquid, solid, or a mix preparation and contributed to the of “tools” that could be customized of phases (for example, biological development of modern automated versus specific systems designed to tissues). Some sample types lend chromatographic instrumentation. provide automated defined solutions. themselves to relatively straightforward Fifteen years later, in 1995, an article It was stated that developments in collection of homogeneous samples published in LCGC North America (1) detection technology, particularly in — for example water, some liquids, provided insights from experts regarding the area of liquid chromatography– and air samples. Other solid sample sample preparation for chemical mass spectrometry (LC–MS), might types, such as whole fruits and analysis, and predictions regarding reduce the need for extensive sample vegetables or production batches of where developments would head over preparation. pharmaceuticals, require physical the next decade. Increasing sample A later article (1998) (2) on sample homogenation of larger representative loads, the need for higher productivity preparation for LC–MS–MS emphasized samples before reducing the sample in the face of decreasing analyst skill automation of three common procedures size to a more easily manipulated levels, and increasing issues with worker — dilute‑and‑shoot, solid‑phase volume. This article will address only the safety, regulatory constraints, and extraction (SPE), and liquid–liquid automation of processing steps for small, information management were all listed. extraction — as well as post‑injection homogeneous, or homogenized samples. Sound familiar? techniques — column switching and Samples are often intact prior to Noteworthy were consensus on‑line SPE— to manage the sample sample preparation, but there are a opinions on the need for rugged and matrix. The authors also recognized the few examples of sample collection reliable automation systems that could emerging need for “massively parallel” devices that perform partial sample be automated. It was agreed that sample preparation and analysis driven preparation as part of the process of future trends would include sample by modern drug discovery practices collecting the sample. Air sampling onto miniaturization and reduced solvent such as combinatorial chemistry. adsorbent tubes concentrates analytes usage. Also, experts agreed that This review addresses the following and simplifies transport. Similarly, pressure from industry programmes questions: What driving forces are solid‑phase microextraction (SPME) and government agencies could stimulating the continued development (3) and stir‑bar sorptive extraction play a significant role in stimulating of the automation of sample preparation (SBSE) (4) concentrate analytes onto development. Another major theme for gas chromatography (GC) and HPLC extraction phases on fibres or stir was the need for better, more universal today? Where do we stand regarding bars, respectively, that can stabilize
30 Recent Developments in Sample Preparation May 2014
magentablackcyanyellow ES431194_LCESUPP0514_030.pgs 04.29.2014 20:23 ADV Pfannkoch
new Fast Fit Fibre Assemblies Figure 1: xyz‑robotic autosampler configured for automated solid‑phase extraction (developed jointly with Chromline srl (SPE) before high performance liquid chromatography (HPLC) injection: a) sample Prato) designed for easier field sampling vial storage, b) standards, c) incubation and agitation, d) SPE, e) eluate storage, and and offer barcode labelling and stable f) liquid chromatography (LC) instrument valve. transport after sampling. Modules are available that offer three preconditioned SPME fibres, or alternatively a fibre can be picked from a tray (preloaded in the field) and automatically desorbed into the GC inlet. Thermal introduction techniques for GC (thermal desorption and thermal extraction) also provide some automated sample preparation capabilities. Thermal desorption is the process of heating a sampling device containing a sorbent medium, such as an air sampling adsorbent tube or phase‑coated stir bar, on which analytes have been trapped and concentrated. Thermal extraction involves directly heating a solid sample in a tube under a gas stream to extract and concentrate the analytes and facilitate transport. an autosampler can perform large the volatiles on an integral secondary Although typically performed manually, volume injection (LVI) eliminating manual trap (available from Markes International, these methods simplify downstream sample concentration or evaporation (5). Frontier, Perkin Elmer, or CDS Analytical) sampling and analysis, and can be Likewise, autosamplers for headspace or in a cryogenically cooled inlet automated with relative ease. injection techniques — whether (Gerstel) from which they are transferred dedicated units (the Agilent 7694E or directly to the GC column. Direct thermal Automating Sample Introduction the PerkinElmer TurboMatrix) or flexible extraction requires small sample sizes Virtually all manufacturers of syringe‑based instruments such as (5–100 mg) of ideally finely divided chromatographic instrumentation those based on the CTC PAL platform powder or thin films, but can eliminate provide some type of automation for (CTC Analytics) — already transport, sample preparation steps such as liquid sample introduction into their systems thermostat, and reproducibly transfer extraction and filtration. Accessories and users expect this capability as a samples from the vial headspace are available (from Gerstel and CDS standard offering. Numerous companies into the GC inlet eliminating manual Analytical) to automate gas switching also manufacture devices for sample handling steps and interference from during thermal extraction or pyrolysis (to introduction that can be interfaced nonvolatile sample matrices. More allow heating in the presence of air for with a variety of chromatographic specialized samplers and accessories oxidation studies or addition of reagents platforms. Basic engineering decisions from suppliers such as Dani, Tekmar, to perform thermochemolysis (Gerstel, made about the core design of these EST Analytical, OI Analytical, Gerstel, Frontier), for example to study lignin systems often defines whether they and others can automate dynamic structure (6). are dedicated or flexible; compact or headspace sampling techniques. Dedicated accessory instruments are expandable; push‑button‑intuitive; or SPME is another automatable GC available for automating some types of user‑programmable. It is not possible injection technique that includes sample cleanup and extraction as part to review all available instrumentation sample preparation. A number of SPME of HPLC injection. Figure 1 shows a for sample preparation here. Instead, innovations have been automated diagram of an autosampler platform with features of representative commercially including: accessories for automated SPE using available instruments are described. • Pre‑ or post‑extraction derivatization: a disposable cartridge format. On‑line Given that upgrades and new designs The autosampler inserts the SPME SPE is available in a variety of formats are constantly released, this review fibre into a vial containing reagent and is used for rapid preconcentration will provide a snapshot in time of this either before or after exposure to the and cleanup of samples. Valco continuously evolving field. sample. Instruments lists a number of valving Modern autosamplers used for • Hot injection and trapping (HIT): The configurations, with formats ranging from sample injection already include some SPME fibre is inserted into a thermal regenerated and reused SPE cartridges sample preparation. Liquid injections, desorption unit mounted above a configured as part of the sample loop used almost universally for injections cold inlet that allows refocusing of the to multivalve column switching systems into LC and GC instruments, may analytes improving peak shapes for such as those offered by Cohesive not normally be considered sample early eluting compounds. Technologies (Thermo Fisher Scientific). preparation. However, when coupled Alternatively, systems are available with a programmed temperature New multi‑fibre exchange modules for from Spark Holland and Gerstel that vaporizing (PTV) inlet in a GC system, the CTC‑type sampler use Supelco’s automatically exchange disposable
www.chromatographyonline.com 31
magentablackcyanyellow ES431196_LCESUPP0514_031.pgs 04.29.2014 20:23 ADV Pfannkoch
Table 1: Typical features of software available for sample preparation. a dilutor module from CTC Analytics was used to automate ASTM standard General Features method D6584‑07 for biodiesel (10). Controls multiple instruments from one computer The widely practiced dilute‑and‑shoot Interfaces with multiple chromatography data systems technique used in LC–MS–MS to Compatible with LIMS reduce interference and ion suppression can also be automated by using Logfiles for traceability autosamplers capable of handling both 21 CFR Part 11 capable larger diluent and smaller microlitre User‑friendly, Microsoft Windows or icon based programming volumes. Simplified command lists based on hardware configuration Automated manipulation of the sample is not the only benefit provided On‑line help menus and prompts by these flexible prepstations. Integrated testing and debugging of program steps The additional liquid‑handling Automated error notification capability has been routinely used Graphical display of sample processing schedule for serial dilution and automated Running methods and sequences that can be edited preparation of calibration standards for liquid, headspace, and SPME Multiple programmable inputs and outputs available injection methods (11,12). This is High‑throughput capabilities (prepare ahead, parallel preparation) particularly beneficial to automating matrix‑matched standard preparation SPE cartridges placed in‑line between method, or GC autosamplers performing — blank sample matrix samples are the injection valve and the analytical LVI, may be able to use larger syringes processed at the same time as test column. to add relevant volumes of internal samples, before being spiked with The need for rapid and widespread standards, derivatizing reagents, or standards. For example, automated screening of blood samples has sample modifiers (salts, acids, or extraction and analysis of pesticides stimulated the development of methods bases) as part of the automated sample using the QuEChERS (quick, easy, for collecting dried blood spots (DBS) processing steps. cheap, effective, rugged, safe) on filter paper cards. This technology approach using matrix‑matched was reviewed recently (7) and offers Versatile Prepstations standards improved quantification (13). less invasive, cost‑effective sample In the past 10 years robotic xyz The proliferation of flexible xyz collection, and small sampling volumes. samplers capable of GC or HPLC robotic autosamplers created the Also, when dry, samples are more injection have become the industry need for software control that was stable and pose less risk to analysts standard, although lower‑cost user‑programmable so that customized handling the samples compared to dedicated autosamplers are still quite processing steps could be created liquid samples. Currently, the technique common. The evolution of robotic by the analyst. Firmware and software is mostly manual or semi‑automated by samplers has been driven by the provides the initial framework and punching out blood spots from the DBS addition of specialized hardware tools for customization of autosampler cards. However, new instrumentation modules, and the development of actions. For example, CycleComposer that extracts the spots on the card for versatile, user‑friendly software. One software (CTC Analytics) provides further workup and analysis has been of the first steps in this development detailed control features enabling the introduced. It will be of interest to see if was to mount two robotic samplers analyst to create customized actions, major regulatory bodies will endorse this one above the other (referred to as a but requires an understanding of the new form of blood sample handing. “twin” or “dual‑rail” configuration). CTC underlying macro language. Instrument Analytics then more recently created manufacturers subsequently provided Expanding Autosampler a longer autosampler platform (PAL‑xt) drivers or other integrated control of Capabilities to accommodate more accessories these samplers within their proprietary Samplers configured for headspace and, soon after, integrated two injection chromatographic platforms, but most of or SPME injection are typically units into a single rail xyz sampler. Two the embedded control is for standard preprogrammed with standard injection different syringe types can be mounted injection modes, with relatively little cycle options with few additional simultaneously, allowing a wider flexibility. automation options. However, the range of sample processing options. There is clearly a need for capabilites of modern autosamplers for This configuration has been used to user‑friendly, flexible software liquid injection can often be expanded. automate a variety of headspace GC control of customizable autosampler Samplers designed for GC injection methods with liquid internal standard platforms that can communicate typically use a single small (5–10‑µL) or reagent addition including blood effectively with a variety of different syringe limiting the volume range alcohol (8), and cyanide in drinking chromatographic platforms such as that can be manipulated for other water (9). ChemStation (Agilent Technologies), reagents, but they may still be able to Alternatively, specialized liquid MassHunter (Agilent Technologies), automatically add internal standards to delivery modules are available that Analyst (AB Sciex), Xcalibur (Thermo sample vials. Samplers injecting into can deliver millilitre volumes of a Fisher Scientific), Millenium (Waters HPLC valves using the loop overfill dilution solvent. A configuration using Corp.), and ChromaTOF (Leco). Third
32 Recent Developments in Sample Preparation May 2014
magentablackyellow ES431195_LCESUPP0514_032.pgs 04.29.2014 20:23 ADV Pfannkoch
Table 2: Sample preparation hardware modules available for several platforms. The newest autosampler platform from CTC Analytics is known as the PAL Mixing (orbital, stirring, high speed vortex) RTC, or Robotic Tool Change system. In Headspace (static, dynamic, purge‑and‑trap, full evaporation) addition to automating standard sample SPME fibre exchange introduction capabilities — liquid, Heating (conventional, microwave) headspace, and SPME — the RTC can automatically exchange tools (in this Cooling (Peltier, circulator) instance, syringe types) enabling a SPE (standard cartridge, ITSP microcartridge, 96‑well plate) single injection unit system to carry out dSPE (vial format, disposable pipette extraction) multiple functions without being limited Thermal desorption or extraction (SBSE, adsorbent tube, solids, pyrolysis) by one single syringe type. As new Evaporation or solvent exchange autosampler platforms enter the market new software control will be needed, but Filtration this will take time. Barcode reading A number of sample preparation Ultrasonication steps required immediately before Weighing sample injection can now be automated, allowing “just in time” sample Centrifugation preparation. For this to be efficient, sample preparation steps must be party instrument distributors focused liquid to be withdrawn from the lower completed during the chromatographic on front‑end sample automation may layer. One alternative approach is separation of the previous sample, to be able to meet this need. Typical membrane‑assisted solvent extraction prevent automated sample preparation software features currently available (MASE) that uses a small polypropylene from becoming a bottleneck and limit on some of these instruments are bag filled with the extraction solvent throughput. listed in Table 1. For example, suspended in the vial (15). Dense Some software (Gerstel Maestro) is Maestro software (Gerstel) provides a solvents can be used because the bag capable of automatically overlapping or user‑friendly Microsoft Windows‑based holds the liquid in a position grouping sample preparation steps to programming interface to control the easily accessible by the autosampler maximize throughput without the need CTC autosampler platform. Standard syringe. for cumbersome analyst programming. injection modes are programmed Evaporation and solvent exchange For example, automated saponification with on‑screen help and prompts for are common procedures to dissolve and esterification for analysis of fatty recommended parameter ranges. A list analytes into a solution compatible acid methyl esters (FAMES) and pain of typical sample preparation modules with the HPLC eluent. Single‑ and management drug screening in urine from a variety of vendors is provided in multi‑position evaporation modules are have been automated (16,17). Table 2. available on some instruments allowing full automation through serial‑ or Standalone Workstations Steps To Automate semi‑batch processing. A number of high‑throughput analysis Highlighted here are a few simple Filtration is often the final step methods are being developed based steps that can be automated and when manually processing samples on fast GC, HPLC, or UHPLC where that are likely to become much more for injection, particularly before the analytical runtime is too short to commonly integrated into routine HPLC or ultrahigh‑pressure liquid allow efficient coupling of sample automated operations. Coupling sample chromatography (UHPLC), to prevent preparation steps. For example, high preparation with GC analysis was blocking the narrow orifices in valves throughput LC–MS–MS analyses may recently reviewed in depth (14). and connection tubing. Particulates be completed in a couple of minutes. Liquid–liquid extraction is often used may already be present in samples or Automated sample preparation before injection, and when large sample may form after experimental steps — in supporting these types of analyses have volumes are not needed, in‑vial liquid– this instance, pH adjustment, solvent therefore been taken off‑line. liquid extraction may become more addition, various digestion procedures, Off‑line automation allows interfacing common. Mixing devices are available or even the simple dilute‑and‑shoot with equipment or processing steps to achieve efficient extraction that can approach. In‑vial devices are available that are currently incompatible with the achieve orbital mixing speeds of 750 (Whatman, Thomson) that allow manual chromatographic system. Application to 3000 RPM. When smaller (2‑, 4‑, or filtration of the sample as it is placed examples include high‑speed 10‑mL) vials are used the extracting into the vial. An accessory is available centrifugation, or weight verification organic solvent may be more or less from Leap Technologies that allows after liquid transfer using a balance. dense than the aqueous phase, and the the CTC autosampler to perform the Weighing solids and powders typically layer to be injected onto the instrument filtration with these devices. Filtration requires more sophisticated robotic can be selected by adjusting the using conventional syringe filters can modules. Sometimes the sample load penetration depth of the autosampler also be automated, although care must for one analysis type may not justify syringe needle. When larger (20‑, 40‑, be taken to ensure particulates do not the expenditure for automation, but a or 100‑mL) vessels and dense solvents contaminate the autosampler syringe or single workstation platform automating are required, the sampler must allow needle. several sample preparation procedures,
www.chromatographyonline.com 33
magentablackyellow ES431193_LCESUPP0514_033.pgs 04.29.2014 20:23 ADV Pfannkoch
potentially “feeding” several different the AutoMate‑Q‑40. The automated (4) E. Baltussen, P. Sandra, F. David, and C.A. analytical instruments, may fit into a system adds solvents, internal Cramers, J. Microcol. Sep. 11, 737–747 (1999). laboratory workflow. standards, and solid salts, performing (5) J. Staniewski and J.A. Rijks, J. Chromatogr. The Agilent 7696A Workbench is all mixing and centrifugation steps. A 623, 105–113 (1992). an example of a standalone sample portion of the upper acetonitrile layer (6) T.R. Filley, R.D. Minard, and P.G. Hatcher, Organic Geochemistry 30(7), 607‑621 preparation platform designed to is removed automatically and added (1999). provide flexible liquid handling and to a tube containing dispersive SPE (7) J. Déglon, A. Thomas, P. Mangin, and some additional features including sorbent that is mixed and centrifuged. C. Staub, Anal. Bioanal. Chem. 402(8), heating, cooling, mixing, and weighing. A portion of the cleaned‑up extract is 2485–98 (2012). (8) C.L. Morris‑Kukowski, E. Jagerdeo, J.E. The system is based on handling then transferred to a final sample tube Schaff, and M.A. LeBeau, Journal of small volumes in 2‑mL vials reducing from which it can be placed into a Chromatography B 850, 230–235 (2007). solvent use and waste generation; sample vial for GC–MS analysis. (9) Method ME355.01, Revision 1.0 “Determination of Cyanide in Drinking but may require modification and Water by GC/MS Head‑space Analysis” revalidation of the analytical method Conclusion May 26, 2009. [Available at https://www. to accommodate the reduced sample The key themes cited by the experts nemi.gov or from James Eaton, H & E size. The Agilent Workbench has been Testing Laboratory, 221 State Street, almost two decades ago have largely Augusta, ME, USA, 04333]. used to miniaturize and automate been realized in modern instrumental (10) J. Stuff and J. Whitecavage, Gerstel liquid–liquid extractions of drugs in sample preparation methods. We now Application Note 1/2010 (2010). plasma, perform serial dilutions to use smaller samples and less organic (11) K. Summerhill and J. Angove, Anatune Chromatography Technical Note No AS51 prepare standards for calibration, and solvent and handle a wider variety of [Available from: http://www.anatune.co.uk/ fatty acid methyl ester (FAME) analysis tasks automatically with instrumentation resources/as51]. in biodiesel (18,19). providing better traceability and (12) J. Angove and K. Summerhill Anatune Chromatography Technical Note No AS45 Gilson offers a number of systems communicating more universally with [Available from: http://www.anatune.co.uk/ for off‑line liquid handling and SPE. The different vendor’s platforms. In the resources/as45]. Gilson GX‑281, for example, is based areas where the experts envisioned (13) V. Settle, F. Foster, P. Roberts, P. Stone, J. on a syringeless pump design and can parallel paths, we have seen parallel Stevens, J. Wong, and K. Zhang, Gerstel Application Note 4/2010 (2010) [Available function as a standalone workstation or development, with the growth of both from: http://www.gerstelus.com/applica‑ can be coupled to an HPLC instrument serial “just in time” processing and tions_category.php?id=65]. by incorporating a proprietary injection batch type “workstation” automation (14) H. Lord and E. Pfannkoch, Comprehensive Sampling and Sample Preparation, J. valve in the configuration. It is designed systems, as well as the development of Pawliszyn Ed. (Elsevier Academic Press, to handle samples in a variety of test both dedicated analyzers and flexible, 2012). tubes or scintillation vials, offering customizable platforms to meet quickly (15) B. Hauser, P. Popp, and E. Kleine‑Benne, J. of Chromatogr A 963, 27–36 (2002). automated valve control to access evolving needs. (16) J. Stuff and J. Whitecavage, Gerstel up to five solvent reservoirs and six One clear challenge is to develop Application Note 3/2013 (2013) [Available rinse solvents, and all flowpaths are flexible software tools that can keep from: http://www.gerstelus.com/applica‑ constructed of biocompatible materials. up with changing computer operating tions_category.php?id=93]. (17) O. Cabrices, F. Foster, J. Stuff, E. With additional accessories, tubes can systems and chromatography platforms. Pfannkoch, and W. Brewer, Gerstel be moved to a compatible balance There is a need for software to take Application Note 1/2012 (2012) [Available option. Other models such as the GX‑274 advantage of the hardware capabilities from: http://www.gerstelus.com/applica‑ tions_category.php?id=88]. allow automated positive pressure elution available, combining them in unique (18) A. Macherone and J. McCurry, SPE in 1‑, 3‑, and 6‑mL cartridge formats. ways to address emerging issues in the “Automating Sample Preparation for the Finally, there is a class of dedicated analytical laboratory. GC Analysis of Biodiesel using Method robotic systems designed for either Pressure to increase capacity to EN14105:2011,” presented at the ASMS Conference on Mass Spectrometry and complete automation of a specific screen more samples faster and more Allied Topics, Minneapolis, Minnesota, procedure or for high‑throughput, accurately continues from a number of USA, 2013. parallel processing of small sample areas (such as pharmaceutical, food (19) S. Salman, S. Palaniswamy, and S. Babu “Extraction of Drugs in Plasma by volumes, often in a 96‑ or 384‑well and beverage, medical, and biological). Automated Liquid‑Liquid Extraction For plate format. Tecan, Tomtec, and High‑throughput and fast analyses Downstream Analysis,” presented at the Zinsser NA provide a range of robotic are likely to require more development ASMS Conference on Mass Sprectrometry and Allied Topics, Minneapolis, Minnesota, systems for liquid handling. Dedicated, in standalone workstations that can USA, 2013. robotic workstations are available prepare samples off‑line. Automation from PerkinElmer (Janus workstation), is unlikely to replace all manual sample Edward Pfannkoch is the Director of Agilent, and others. Teledyne Tekmar preparation in the laboratory, but is likely Technology Development, North America has developed the AutoMate‑Q40 to become an increasingly common part for Gerstel Inc. He has over 25 years for complete automation of the of modern methods. experience in chromatography method QuEChERS extraction for pesticides development, automation, and sample from fruit, vegetables, and other References preparation. He has authored three book foodstuffs. With this system, (1) Ronald E. Majors, LCGC 13(9), 742–749 chapters, numerous scientific publications, homogenized sample is prepared (1995). and dozens of corporate technical reports and appropriate amounts of sample (2) J. Henion, E. Brewer, and G. Rule, Anal. regarding sample preparation, automation, Chem. 70(19), 650A–656A (1998). (typically 15 g) in 50‑mL conical (3) R. Belardi and J. Pawliszyn, J. Water Pollut. and analysis. Direct correspondence to: centrifuge tubes are loaded onto Res. J. Can. 24, 179–191 (1989). [email protected]
34 Recent Developments in Sample Preparation May 2014
magentablackyellow ES431192_LCESUPP0514_034.pgs 04.29.2014 20:23 ADV HPLC GC MS SPE IR
powered by
The next course is: HPLC Method LIVE Development on the web July 2014
• Instructor led training courses - delivered via four 90 minute live web sessions • Key concepts highlighted using real life methods and chromatograms • Troubleshoot problems with hardware and separations • Understand your methods and instrument variables • How to set and optimize critical parameters • Understand instrument maintenance
To fnd out more contact: Glen Murry on +1 732 - 346 - 3056 | e-mail: [email protected]
www.chromacademy.com/2014
magentablackcyanyellow ES430544_LCESUPP0514_CV3_FP.pgs 04.29.2014 01:16 ADV Perfect fit!
We bring the pieces together Whatever your sample preparation requirements, we help bring the pieces together for the perfect solution to meet your GC/MS and LC/MS needs:
Automated Sample Prep Standards & Dilution Series SPE and Online SPE (SPExos) Dynamic Headspace (DHS), HS & SPME Twister & Thermal Desorption/Extraction Intelligent PrepAhead productivity Application support at your service
What can we do for you?
www.gerstel.com
magentablackcyanyellow ES430542_LCESUPP0514_CV4_FP.pgs 04.29.2014 01:16 ADV