J U LY 2 0 1 7 IMPROVING SAMPLE PREPARATION IN HPLC Sponsored by Presented in partnership ® SAMPLE PREP OVERVIEW 485F US Highway One South, Suite 210, © 2017 UBM. All rights reserved. No part of this Iselin, NJ 08830 publication may be reproduced or transmitted in any (732) 596-0276 form or by any means, electronic or mechanical including by photocopy, recording, or information storage PUBLISHING & SALES and retrieval without permission in writing from the publisher. Authorization to photocopy items for internal/ Michael J. Tessalone educational or personal use, or the internal/educational Vice President/Group Publisher or personal use of specific clients is granted by UBM for [email protected] libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA Edward Fantuzzi 01923, 978-750-8400 fax 978-646-8700 or visit http:// Publisher www.copyright.com online. 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Megan L’Heureux Managing Editor, LCGC North America UBM Americas (www.ubmlifesciences.com) is a leading Stephen A. Brown worldwide media company providing integrated Group Technical Editor, LCGC North America marketing solutions for the Fashion, Life Sciences and Powersports industries. UBM Americas serves business Cindy Delonas professionals and consumers in these industries with its Associate Editor, LCGC North America portfolio of 91 events, 67 publications and directories, 150 electronic publications and Web sites, as well as Alasdair Matheson educational and direct marketing products and services. Editor-in-Chief, LCGC Europe Market leading brands and a commitment to delivering Kate Mosford innovative, quality products and services enables UBM Americas to “Connect Our Customers with Theirs.” Managing Editor, LCGC Europe UBM Americas has approximately 1000 employees and Lewis Botcherby currently operates from multiple offices in North America Assistant Editor, LCGC Europe and Europe. INTRODUCTION hile sample preparation is a routine task for chromatography labs, it cannot be taken for granted. Sample prep may well be one of the most important ways of ensuring the selectivity, sensitivity, and Wreproducibility of an analytical method. In Improving Sample Preparation in HPLC (sponsored by GE Healthcare, and presented in partnership with LCGC), experts explain how labs can ensure their sample prep techniques are supporting the success of even the most advanced chromatographic methods. First, Nicolas Snow, PhD, a professor of chemistry and biochemistry at Seton Hall University, provides an overview of how some basic factors—like lab glassware choice and solvent mixing—can all have a major effect on the reproducibility of a separation method. Snow also reviews the fundamentals of some of the most common sample preparation techniques, such as liquid–liquid extraction, solid-phase extraction, solid-phase microextraction, and QuEChERS, and the importance of keeping equilibrium and kinetics in mind. Next, “Sample Prep Perspectives” columnist Douglas E. Raynie interprets the results of a recent LCGC North America readership survey about the use of sample preparation techniques. He examines both short-term trends (especially in the use of automation and newer instrumental extraction techniques) and long-term trends spanning over a decade. In a separate piece, Raynie and Ronald E. Majors explore high-throughput sample preparation as a possible technique for accelerating sample preparation time. They suggest that the best approach may be a combination of methods for achieving high-throughput sample preparation. Last, Dwight Stoll, editor of LCGC’s LC Troubleshooting column, discusses some best practices in instrument set-up related to filters and filtering, two important parts of any liquid chromatography system. As this LCGC eBook demonstrates, a well-thought out sample preparation technique is a lab’s first step in an ensuring an accurate and reliable chromatography method—and proper planning for this stage is critical. TOC Improving Sample Table of contents Preparation in HPLC Sample Prep Overview Sample Preparation: The Forgotten Dimension in HPLC Nicholas H. Snow 5 Sample Prep Trends Trends in Sample Preparation Douglas E. Raynie 13 High-Throughput Sample Prep Exploring the Possibilities of High-Throughput Sample Preparation Douglas E. Raynie and Ronald E. Majors 30 Filtration Filters and Filtration in Liquid Chromatography: What To Do Dwight Stoll 37 SAMPLE PREPARATION: ThE FORGOTTEN DIMENsiON IN HPLC Nicholas H. Snow Although often forgotten, sample preparation is the most important factor in achieving reproducibility from even the most advanced chromatographic methods. Overview Sample preparation techniques are often thought of as basic chemistry, but they may be the most important aspects of running analytical methods. In this article, learn about some ways that sample preparation can affect the selectivity, sensitivity, and reproducibility of an analysis. Classic Sample Prep Techniques While sample preparation is a routine task, it can be a critical factor that affects a method’s accuracy and reproducibility. Reviewing the basics of sample prep, it is vital that: 1. Analysts collect samples according to standard operating procedures. 2. Solvents and glassware are chosen according to the method’s needs. TUDIO 3. Samples, reagents, and chemicals are S handled safely. 4. Sample preparation techniques are optimized using the ideas of equilibrium and kinetics. SHUTTERSTOCK.COM/LOOKER_ 5 | July 2017 | LCGC SAMPLE PREP SAMPLE PREP HIGH-ThROUGHPUT FILTRATION OVERVIEW TRENds SAMPLE PREP Red = 2015 Blue = 2013 Figure 1: Popularity of sample preparation techniques. Source: D.E. Raynie, LCGC North America, 34(3), 174–188 (2016). 5. Samples are kept as clean as a form that is ready to be injected into an possible. instrument can require numerous steps, Sample preparation requires precise each with its own potential for introducing handling and manipulation of samples variance to the method. Gravitmetric steps from the time they enter the lab until the often have the fewest errors associated vial is placed on the tray. One should with them, assuming the balances are always think about which steps of the properly maintained and calibrated, and sample handling process could enable proper operating procedures are used. hidden errors to creep into the method. Dilutions, on the other hand, are often a Several techniques are used in neglected source of variance. sample preparation, ranging from well- Glassware choice. It is common established procedures like weighing, to do simple dilutions in volumetric dilution, and filtration, to relatively glassware. It is imperative to use good new technologies like QuEChERS. The techniques and avoid bad practices results from two recent surveys about that can negatively affect quantitative the popularity of sample preparation analysis. For instance, one may be techniques are shown in Figure 1. Older introducing precision and accuracy methods are by far the most common. errors into the method by using smaller The process of turning a raw sample into glassware, which is chosen for smaller 6 | July 2017 | LCGC SAMPLE PREP SAMPLE PREP HIGH-ThROUGHPUT FILTRATION OVERVIEW TRENds SAMPLE PREP samples. Larger flasks for larger samples variance that blindly picking a stopper offer better precision, at least on a from the drawer could result in a poor fit. percentage basis. In addition, when If a leak occurs during the inversion and using nonaqueous solvents, analysts mixing step, some solvent will be lost and may also see some small errors in the concentration will be thrown off. working with volumetric glassware One way to check to see if a leak is because they were calibrated for water. present is to insert the lid and see if any Class A glassware has a known ground glass is visible. If there is, switch to tolerance that should be considered, another lid until no ground glass can be and is higher for smaller volumes. For seen. example, a 250-mL volumetric flask has a Last, glassware has a finite lifespan. The tolerance of 0.048%, while a 10-mL flask volume of a heavily used flask will drift is 0.20%. Performing serial dilutions is a over time. If the etched labeling on the good way to save on the use of solvent, side of the glassware is no longer legible, but introduces extra steps into the it might be time to consider replacing it.