Instrument Considerations in the Transfer of Chromatographic Methods, Part II: System Considerations Thomas E

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Characterization for Biopharmaceutical The Analytical Toolbox The Analytical Toolbox ES39215_LCGC1118_CV1.pgs 11.05.2018 14:54 UBM METHODS INSTRUMENT THE TRANSFER OF CONSIDERATIONS IN CONSIDERATIONS CHROMATOGRAPHIC CHROMATOGRAPHIC www.chromatographyonline.com Volume 36 Number 11 November 2018 36 Number 11 November Volume Understanding Stationary-Phase Selectivity for GC yellow cyan black magenta

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CONTENTS COLUMNS

796 LC TROUBLESHOOTING Mixing and Mixers in Liquid Chromatography—Why, When, and How Much? Part II, Injections Dwight R. Stoll What happens when we inject a sample into the mobile-phase stream? Many Volume 36 Number 11 November 2018 LC practitioners are surprised to learn just how serious the effect of the injected www.chromatographyonline.com sample solvent can be.

802 SAMPLE PREP PERSPECTIVES A Look Back and A Look Forward—An Annual Check-up on the State of Sample Preparation Douglas E. Raynie

We assess the state of the field, first looking back at developments presented INSTRUMENT

Understanding CONSIDERATIONS IN The Analytical Toolbox at conferences this year, reader questions, and the passing of a pioneer in solid- Stationary-Phase THE TRANSFER OF for Biopharmaceutical Selectivity for GC Characterization CHROMATOGRAPHIC phase extraction. Then, we look to the future of sample preparation. METHODS

COVER DESIGN BY 806 GC CONNECTIONS Dan Ward Stationary Phase Selectivity: The Chemistry Behind the Separation Cover image courtesy of Nicholas H. Snow Ioana Davies (Drutu) / Here, we focus on selectivity: its definition, its importance for generating stock.adobe.com separations and resolution; and its role in column polarity.

814 FOCUS ON BIOPHARMACEUTICAL ANALYSIS Analytical Characterization of Biotherapeutic Products, Part II: The Analytical Toolbox DEPARTMENTS Anurag S. Rathore, Ira S. Krull, and Srishti Joshi 794 Peaks of Interest The analytical techniques used for characterizing biotherapeutics have evolved. We review the utility of the traditional tools and discuss the new, orthogonal 836 Products & Resources techniques that are increasingly being used. 837 Ad Index 838 THE ESSENTIALS HPLC Column Maintenance: Tips for Extending HPLC Column Lifetime Follow these tips to protect your columns and extend their useful lifetime.

PEER-REVIEWED ARTICLES 824 Instrument Considerations in the Transfer of Chromatographic Methods, Part II: System Considerations Thomas E. Wheat Scientists executing a method transfer often do not have access to the originating system. Thus, alternative approaches to matching chromatographic results must be considered.

830 Chromatography Fundamentals, Part V: Theoretical Plates: Significance, Properties, and Uses Howard G. Barth The number of theoretical plates forms the basis of chromatographic theory, and is a key parameter used in all modes of chromatography for measuring column efficiency. Fortunately, it’s easy to measure.

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Editorial Advisory Board

• Kevin D. Altria – GlaxoSmithKline, Ware, United Kingdom • Ronald E. Majors – Analytical consultant, West Chester, Pennsylvania • Jared L. Anderson – Iowa State University, Ames, Iowa • Debby Mangelings – Department of Analytical Chemistry and • Daniel W. Armstrong – University of Texas, Arlington, Texas Pharmaceutical Technology, Vrije Universiteit Brussel, Brussels, Belgium • David S. Bell – Restek, Bellefonte, Pennsylvania • R.D. McDowall – McDowall Consulting, Bromley, United Kingdom • Dennis D. Blevins – Agilent Technologies, Wilmington, Delaware • Michael D. McGinley – Phenomenex, Inc., Torrance, California • Zachary S. Breitbach – AbbVie Inc., North Chicago, Illinois • Victoria A. McGuffin – Department of Chemistry, • Deirdre Cabooter – Department of Pharmaceutical and Pharmacological Michigan State University, East Lansing, Michigan Sciences, KU Leuven (University of Leuven), Belgium • Mary Ellen McNally – FMC Agricultural Solutions, Newark, Delaware • Peter Carr – Department of Chemistry, • Imre Molnár – Molnar Research Institute, Berlin, Germany University of Minnesota, Minneapolis, Minnesota • Glenn I. Ouchi – Brego Research, San Jose, California • Jean-Pierre Chervet – Antec Scientific, Zoeterwoude, The Netherlands • Colin Poole – Department of Chemistry, • André de Villiers – Stellenbosch University, Stellenbosch, South Africa Wayne State University, Detroit, Michigan • John W. Dolan – LC Resources, McMinnville, Oregon • Douglas E. Raynie – Department of Chemistry and Biochemistry, • Michael W. Dong – MWD Consulting, Norwalk, Connecticut South Dakota State University, Brookings, South Dakota • Anthony F. Fell – School of Pharmacy, • Fred E. Regnier – Department of Chemistry, Purdue University, University of Bradford, Bradford, United Kingdom West Lafayette, Indiana • Francesco Gasparrini – Dipartimento di Studi di Chimica e Tecnologia • Koen Sandra – Research Institute for Chromatography, Kortrijk, Belgium delle Sostanze Biologicamente Attive, Università “La Sapienza,” Rome, Italy • Pat Sandra – Research Institute for Chromatography, Kortrijk, Belgium • Joseph L. Glajch – Momenta Pharmaceuticals, Cambridge, Massachusetts • Peter Schoenmakers – Department of Chemical Engineering, • Davy Guillarme – University of Geneva, University of Amsterdam, Amsterdam, The Netherlands University of Lausanne, Geneva, Switzerland • Kevin Schug – University of Texas, Arlington, Texas • Richard Hartwick – PharmAssist Analytical Laboratory, Inc., • Dwight Stoll – Gustavus Adolphus College, St. Peter, Minnesota South New Berlin, New York • Michael E. Swartz – Stealth Biotherapeutics, Newton, Massachusetts • Milton T.W. Hearn – Center for Bioprocess Technology, • Caroline West – University of Orléans, France Monash University, Clayton, Victoria, Australia • Thomas Wheat – Chromatographic Consulting, LLC, Hopedale, Massachusetts • Emily Hilder – University of South Australia, Adelaide, Australia • Taylor Zhang – Genentech, South San Francisco, California • John V. Hinshaw – Serveron Corporation, Beaverton, Oregon • Kiyokatsu Jinno – School of Materials Science, CONSULTING EDITORS: Toyohashi University of Technology, Toyohashi, Japan Jason Anspach – Phenomenex, Inc.; David Henderson – Trinity College; • Ira S. Krull – Professor Emeritus, Department of Chemistry and Tom Jupille – LC Resources; Sam Margolis – The National Institute of Chemical Biology, Northeastern University, Boston, Massachusetts Standards and Technology; Joy R. Miksic – Bioanalytical Solutions LLC

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PEAKS of Interest

Shimadzu Celebrates 50th Anniversary CHROMATOGRAPHY Shimadzu has celebrated 50 years of MARKET PROFILE business in Europe, with 300 guests attending an event in Duisburg, Germany Chinese Laboratories to commemorate the anniversary. The Share Views in Survey “Magic Moments Night” took place at For high performance liquid chroma- the Mercator Hall, and featured music, tography (HPLC), ultrahigh-pressure LC show acts, dinner, speeches, greeting (UHPLC), and LC–mass spectrometry notes, and a “Walk of History.” The (LC–MS) technologies, China has con- musical part of the evening was tinued to provide solid growth opportu- Sector distribution of Chinese HPLC and covered by members of the Duisburg nities, as a result of the country’s invest- LC–MS survey respondents (n = 223). Philharmonic Orchestra. The show ments in the pharmaceutical industry, as well as agriculture and food testing. Major act performed by “Physikanten & Co” instrument manufacturers including Agilent, Shimadzu, Thermo Fisher Scientific, combined entertainment and science. and Waters have established a stronghold in the market, and continue to per- Giant vortex rings flew 20–30 m across form well. As the market continues to evolve, Chinese HPLC users are expressing the hall, and, in a rapid sequence of their preferences, gravitating to only a few manufacturers. Some Chinese users experiments, the fascinating aspects of maintain a strong allegiance to indigenous HPLC suppliers. However, they also carbon dioxide were explored. For the recognize that the quality, reliability, and performance from the international HPLC “Walk of History.” Shimadzu collected manufacturers are unrivaled, swaying purchase decisions in favor of global brands. historic advertisements, brochures, and Top-Down Analytics (TDA) recently surveyed over 200 Chinese HPLC, UHPLC, photographs from exhibitions covering and LC–MS users who provided opinions about their instrument and consumables 50 years of corporate history in Europe. suppliers. Approximately 30% of the respondents were from pharmaceutical and Shimadzu’s Japanese-based Super- biotechnology laboratories. Agriculture, food and beverage, and government labo- visory and Executive Board flew in from ratories each represented about a fifth of the responses, and LC users from chemical Japan to attend the party with the Euro- laboratories accounted for about 14% of the survey participants. pean Shimadzu team, as well as Shimad- When asked to compare domestic or Chinese LC manufacturers with global zu’s distributors and subsidiaries. The brands, some respondents were quite candid, indicating that the Chinese brands evening’s program was hosted by Ger- were improving, but still far from catching up to the quality of the international sup- man television journalist Asli Sevindim. pliers. For columns and other consumables, many Chinese laboratories prefer to use local distributors. Waters Opens Food and Water The use of UHPLC continues to increase in popularity, keeping pace with North Center in Singapore American and European trends. The overall market for HPLC, UHPLC, and LC–MS in Waters Corporation (Milford, Massachu- China represents a significant share of the overall market, accounting for about 11% setts) has opened a new International of the worldwide analytical instrument industry. In 2017, TDA estimates there were Food and Water Research Centre (IFWRC) about 7000 combined HPLC and LC–MS installations in China. Growth is expected in Singapore to address the growing chal- to remain quite robust for the next few years, as a result of continued expansion of lenges of food and water security and life science research laboratories and applied markets. safety. The center will be led by a scien- Market size and growth estimates were adopted from TDA’s Industry Data, a tific advisory panel that will identify mean- database of technology market profiles and benchmarks, and the 2018 Instrument ingful, innovative projects by working with Industry Outlook report from independent market research firm Top-Down Analytics academic and industrial leaders. (TDA). Survey data was extracted from TDA’s report, “A Liquid Chromatography Important research areas such as food Survey in China: Chinese Scientists Share Their Preferences.” For more information, fraud discovery, water contamination contact Glenn Cudiamat, general manager, at (888) 953-5655 or glenn.cudiamat@ research, food quality enhancement, and tdaresearch.com. Glenn is a market research expert who has been covering the new ingredient and formulation studies analytical instrumentation industry for more than two decades. will be prioritized as the research center seeks to find solutions to food and water supply challenges around the world. ted with analytical instrumentation from who will work closely with project owners Researchers will gain access to Waters. In addition, the laboratory will throughout implementation. ◾ IFWRC’s state-of-the-art facilities outfit- be staffed with scientists and researchers 7 8 9 4 5 6 DAWN 1 23 HELEOS-II 0 .

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LC TROUBLESHOOTING Mixing and Mixers in Liquid Chromatography– Why, When, and How Much? Part II, Injections

Is a mixer needed between the injector and column in HPLC?

Dwight R. Stoll

n the previous installment of “LC Trou- that has a volume of about 1.5 mL. However, injectors in use in LC today. This is con- Ibleshooting” (1), I reviewed the basic I also think many practitioners of LC are sur- ceptually similar to the way that fluids are working principles behind the two most prised to learn just how serious the effect combined in the case of a low-pressure commonly used LC pump designs in use of the injected sample solvent can be, most mixing pump; that is, volumes of the flu- today: so-called low- and high-pressure commonly when they observe bad results. I ids are introduced to a single flow path in mixing systems. I then discussed why am hopeful that this installment will shed a a serial fashion, or “end to end.” In the using a mobile-phase mixer between the little more light on these issues, and particu- case of a typical sample injection, the fluid convergence point at the pump and larly address the question of whether or not consequence of this is quite striking. If the sample injector is needed in both mixing is needed after the injector. we assume that a 10 μL portion of sam- cases, albeit for different reasons. Finally, ple is injected into a 120 μm (0.005”) i.d. I showed the effect of using mixers with Combining Fluids in LC tube leading to the LC column; by dividing different volumes for different separation Systems—Where and How? the sample volume by the cross-sectional conditions, and discussed some of the Three of the different ways that fluids are area of the tube, we find that the sample advantages and disadvantages associ- brought together in LC systems are illus- could occupy as much as a 90 cm length ated with changing mixer volumes. trated schematically in Figure 1. The first of this tubing, bracketed on both ends This month, I am continuing with the two represent the ways fluids are brought by mobile phase. Given the degree of theme of mixing and mixers, but this time together in either high-pressure or low- physical separation of the middle of the focusing on what happens when we inject pressure mixing pump systems. Although sample plug from the closest mobile a sample into the mobile-phase stream, I discussed the differences between phase fluid (in this case 45 cm), and the particularly in cases where there is a mis- these designs in detail in the previous relatively short time it takes for the sample match between the compositions of the installment of “LC Troubleshooting,” the to reach the column under typical condi- two fluids. This mismatch most commonly differences are actually quite relevant tions (a few seconds), there is absolutely exists as a difference in solvent composi- to the topic of sample injection, and so no way that the two fluids will actually tion (for example, injecting an analyte dis- worth repeating here. In short, the major mix before the sample reaches the col- solved in 100% acetonitrile into a mobile fundamental difference between the two umn. And so, this then leads to the ques- phase of 20/80 acetonitrile/water) between approaches (Figure 1A and 1B) is that, in tion, “Under what circumstances should the sample and mobile phase. However, the first case, the two fluid streams con- a physical mixer be deployed to ensure there certainly are situations where differ- verge in a kind of parallel fashion, so that that the sample mixes with the mobile ences in the pH or buffer composition of the two fluids are always in close contact, phase before reaching the column?” the two fluids can also be very important. whereas in the second case, small packets It is most certainly true that there are of each fluid are introduced into a single Does it Really Matter If the Sample is many analytical situations where the effect fluid path in a kind of serial fashion. Mixed with the Mobile Phase? of the injected solvent is practically negli- The third scenario of Figure 1 illustrates As with many things, the answer here is gible; for example, injecting 1 μL of sam- the way that a sample is introduced into “It depends.” In my thinking about this,

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different injection conditions. Clearly, the first case (A) yields a very nice separation, (a) A Tube i.d. ~170 μm whereas in the second case (B), the five compounds are not well separated and the peak shapes are terrible. The difference here is that, in Case A, the analytes are dis- B solved in a 30/70 acetonitrile/water mixture, and, in Case B, a 70/30 acetonitrile/water (b) mixture. The solvent gradient starts at 50% acetonitrile, and runs to 90% acetonitrile at the end of the gradient. We explain this result by recognizing that, in Case A, the analytes are dissolved in solvent that con- Tube i.d. ~120 μm tains less acetonitrile than the mobile phase itself (50%) at the beginning of the analysis. (c) Under these conditions, the analytes will be For 10 μL injected ~90 cm well retained by the stationary phase, have a low velocity, and are effectively “stuck”at the column inlet. We say that they are FIGURE 1: Idealized representation of the different ways two fl uids converge under “focused” or “compressed”into a narrow different circumstances in LC systems: (A) convergence in a binary high-pressure mix- band, and that this narrow width estab- ing pump, (B) convergence at the outlet of a solvent proportioning valve in a low-pres- lishes a kind of initial condition from which sure mixing pump, and (C) convergence when a sample is injected into a mobile phase stream that will carry the sample to the LC column. the rest of the separation (and subsequent peak broadening) develops. This effect has been known for decades (2), but also continues to be a subject of active research (3,4). 1500 (a) On the other hand, in case B, the sample contains more acetonitrile than the mobile 1000 phase at the starting point of the analysis. Under these conditions, the analytes will 500 be poorly retained, have a high velocity approaching the mobile phase velocity, and 0 are spread out across a large fraction of the 0 0.1 0.2 0.3 0.4 0.5 column bed. This, too, establishes a kind of 1500 (b) initial condition for the analyte bands, but, in contrast to case A, one that involves very broad peaks, from which the separation 1000

Absorbance (mAU,254 nm) cannot recover because the peaks will only get broader as the separation develops. 500 So, to answer the question that heads this section, I would say that, generally 0 0 0.1 0.2 0.3 0.4 0.5 speaking, if the injection conditions favor Time (min) focusing of the analytes at the column inlet, as is the case in Figure 2A, then no actual mixing of the sample and mobile phase is FIGURE 2: Comparison of chromatograms obtained from injection of samples in (A 30/70 needed to obtain good results. Indeed, acetonitrile/water, or (B) 70/30 acetonitrile/water. Conditions: column, 50 mm x 2.1 mm i.d. the chromatogram in Figure 2A is convinc- C18; injection volume, 40 μL; gradient elution from 50-90% acetonitrile from 0–15 s, with water as the aqueous phase; fl ow rate, 2.5 mL/min.; analytes are alkylphenone homologs from ace- ing evidence for this. Of course, there are tophenone to hexanophenone. always exceptions (see the section later about viscous fingering); however, this egories: conditions that favor focusing of the chromatograms obtained when a sim- view should be pretty broadly applicable. the analyte at the column inlet, and those ple mixture of alkylphenones is injected On the other hand, if the injection condi- that do not. This is most effectively under- into a reversed-phase column followed tions are not favorable for focusing, as in stood by way of example. Figure 2 shows by solvent gradient elution, but with two Figure 2B, then this can be a real problem, WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 799

and solving the problem may or may not effects of sample solvent composition we cannot account for, and it possible require a physical mixer, depending on and volume on peaks that we observe that the viscous fingering could explain how the solution is implemented. in real experiments without invoking the some of these differences. Clearly, more effects of more complex processes, such work on this is needed to more fully Dealing with Situations Involving as viscous fingering (for example, we have understand these effects. Unfavorable Solvent Mismatch been able to faithfully predict results like Our work on this topic has yielded In my laboratory, we have studied the those shown in Figure 2 [4]). Neverthe- some experimental results that are effect of the composition and volume of less, there are some differences between instructive here. First, the peaks shown in the injected sample on separation per- simulation and experimental results that Figure 3 were obtained from experiments formance (for example, as in Figure 2) extensively. When I discuss this work with people, the two things I hear most commonly are that: 1) extensive mixing of the sample with the mobile phase is needed to achieve good results as in Figure 2A; and 2), it is physical differences between the sample and mobile phase (for example, viscosity) that are the root cause of poor results (as in Figure 2B). Here, I’d like to discuss a few results that I think shed some light on these issues. Viscous fingering is a physical phenomenon that can develop when a less viscous fluid (for example, acetonitrile) is injected into a more viscous one (for example, water) that flows into a porous medium. In this situation, local flow insta- bilities can develop that produce “fin- gers” of the injected fluid that appear to reach into the adjacent fluid (in the chromatographic context, the mobile phase). This effect has been known in preparative chromatography for some time, but more recently was also visu- ally demonstrated under analytical scale chromatography conditions by Samuels- son, Fornstedt, and coworkers (5). This, and related work, provides compelling evidence that viscous fingering can occur in analytical chromatography columns and probably leads to effects on chromatographic efficiency (that is, plate number, or plate height) that cannot be accounted for using simple plate models of chromatography. In our own work, we have adapted a simple plate model of liquid chromatography that enables us to simulate the effects of sample solvent composition and volume on chromatographic peak shape and efficiency (4,6). To summarize a great deal of work in this direction, I would say that, by using this simple plate model, we can account for a large majority of the 800 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

which will both effectively increase the 700 volume of the injected sample, and add (a) 13 μL gradient delay volume to the system. If 600 neither of these issues is detrimental for 500 the analysis at hand, then adding such a mixer could be a good solution to the 400 problem. In our own work on the sample 300 solvent problem in the context of 2D-LC where analysis time is a precious resource, 200 we have developed an approach referred 100 to as active solvent modulation (ASM) that

0 works quite well (7). 0123 The last bit of data I’d like to discuss here actually comes from our work on 700 (b) 40 μL ASM, where an injected sample is mixed 600

Absorbance (mAU, 254 nm) with the mobile phase stream in more of 500 a parallel fashion (that is, like converging streams in Figure 1A) than the more typ- 400 ical serial fashion (as in Figure 1C). Fig- 300 ure 4 shows a comparison of the sample 200 plug profiles observed when the injected 100 sample is brought together with mobile phase in a serial fashion (black traces), or 0 0123 in a parallel fashion (red traces). In these Time (s) cases, the mobile phase was 50/50 acetonitrile/water, and the injected sample was acetonitrile (4A), or 2-propanol (4B), each FIGURE 3: Comparison of sample plug profi les obtained from injections of either 13 or 40 μL of containing 0.1% acetone as a tracer that sample from a conventional fi xed loop injector. The mobile phase was 50/50 acetonitrile water, is observed by UV absorbance detec- pumped at 2.5 mL/min., and the sample was the same solvent spiked with uracil at 10 μg/mL. tion. There are two main points I’d The injector was connected directly to the detector with a short length of 75 μm i.d. tubing. Adapted with permission from ref. (4). like to make about these results. First, here, as in Figure 3, we see that in aimed at understanding the shape of the the sample plug and the surrounding the case of serial sample introduction injected sample plug injected into the mobile phase. I think this is relatively there is little mixing of the sample with second dimension column in two-dimen- easy to understand when we imagine the surrounding mobile phases. On sional liquid chromatography. In this case, what happens inside the system using the other hand, when the sample and the mobile phase was 50/50 acetonitrile/ the illustration in the third case of Fig- mobile phase are brought together in water, and the sample was the same solu- ure 1. The practical consequence of a parallel fashion, the mixing is very tion but spiked with 10 μg/mL of uracil, this, then, is that if we have analytes effective, as indicated by the lowered which is used to trace the concentration dissolved in a sample with a high con- concentration of acetone detected profile by UV absorbance detection. centration of acetonitrile and we inject during introduction of the sample In other words, the uracil is a proxy for this into a mobile phase with a much plug. Note that the profile is wider in other sample solvent components, such lower concentration of acetonitrile, time because the effective volume of as acetonitrile. These experiments are there will be a point where the sample the sample plug is increased as it is done without a column installed, such solvent acts as the mobile phase inside mixed with mobile phase. The differ- that the profile we observe is essentially the column because there is insufficient ences in the extent of dilution of the the sample profile as it would enter the mixing with the surrounding mobile acetone (as indicated by the different LC column. The point I want to empha- phase. This brings us back to the ques- peak heights) are related to the differ- size here is that, even with a relatively tion, “Should we install a mixer between ent viscosities of the two samples. Sec- small injection volume of 13 μL, there is a the injector and the column?” The main ond, there are not obvious differences point in the center of the profile where the problem with installing a simple mixer in the sample plug profiles observed fluid that is detected is essentially pure (think spinning stir-bar) in this context is for the two sample solvents injected, sample. In other words, there is very that effective mixing of the sample will even though their viscosities vary by a little mixing between the center of require a relatively large volume mixer, factor of about seven. WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 801

(2) L.R. Snyder and D.L. Saunders, J.Chromatogr. Sci. 7, 195–208 (1969). doi:10.1093/chromsci/7.4.195. 90 Sample introduced serially (3) S.R. Groskreutz and S.G. Weber, J. Chromatogr. A 1409, 116–124 80 Sample introduced (2015). doi:10.1016/j.chroma.2015.07.038. 70 in parallel (4) D.R. Stoll, R.W. Sajulga, B.N. Voigt, E.J. Larson, L.N. Jeong, and 60 S.C. Rutan, J. Chromatogr. A. 1523, 162–172 (2017). doi:10.1016/j. 50 (a) Sample in acetonitrile chroma.2017.07.041. 40 (5) J. Samuelsson, R.A. Shalliker, and T. Fornstedt, Microchem. J. 30 130, 102–107 (2017). doi:10.1016/j.microc.2016.08.007. 20 (6) L.N. Jeong, R. Sajulga, S.G. Forte, D.R. Stoll and S.C. 10 Rutan, J. Chromatogr. A 1457, 41–49 (2016). doi:10.1016/j. chroma.2016.06.016. 0 051015 20 (7) D.R. Stoll, K. Shoykhet, P. Petersson, and S. Buckenmaier, Anal. 120 Chem. 89, 9260–9267 (2017). doi:10.1021/acs.analchem.7b02046. (b) Sample in PrOH 100 ABOUT THE COLUMN EDITOR

Absorbance (mAU, 220 nm) 80 Dwight R. Stoll is the editor of “LC Troubleshooting.” Stoll is a profes- 60 sor and co-chair of chemistry at Gustavus Adolphus 40 College in St. Peter, Minnesota. His primary research focus is the development of 2D-LC for both targeted 20 and untargeted analyses. He has authored or coauthored more than 50 peer-reviewed publications and three book chapters in 0 separation science and more than 100 conference presentations. 0 5 10 15 20 Time (s) He is also a member of LCGC’s editorial advisory board. Direct correspondence to: [email protected]

FIGURE 4: Comparison of sample plug profi les for injections of either (A) acetonitrile or (B) 2-propanol into a 50/50 acetonitrile/water mobile phase. Both samples contained 0.1% acetone (v/v). The ASM injection loop volume was 40 μL, and the injection valve was connected directly to the detector using a short length of 120 μm i.d. tubing.

Summary When possible, it is desirable to match the sample solvent to the mobile-phase composition used in a LC method, and use an injection volume that is small relative to the volume of the LC column itself to minimize the effects of the injected sample on separation performance. However, there are some situations where this is not possible because of limitations on analyte solubility, or the need to inject large volumes to improve detection sensitivity. In these situations, it is helpful to have a detailed understanding of what happens during the injection process. In situations where the relationship between the properties of the sample solvent and the mobile phase favor analyte focusing, most likely a mixer is not needed between PINNACLE PCX the injection point and the LC column. However, if the situation ( Reliably Sensitive ) does not favor analyte focusing, this can lead to very bad results (see for example, Figure 2B), and in these cases installing a mixer, or Glyphosate Analysis in Food using an alternate means of sample injection may be helpful. The Experts for 30 Years

Acknowledgements www.pickeringlabs.com I want to thank Gustavus Adolphus College student Hayley Lhotka for collecting the data shown in Figure 4.

References CATALYST FOR SUCCESS (1) D.R. Stoll, LCGC North Amer. 36(10),746–751 (2018). 802 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

SAMPLE PREP PERSPECTIVES A Look Back and A Look Forward—An Annual Check-up on the State of Sample Preparation As this is the final “Sample Prep Perspectives” column of the year, it is fitting to assess the state of the field by taking a look back and a look forward. Specifically, we’ll start by addressing a reader’s email, then look at the state of sample preparation at two recent conferences. In the August issue, we looked at the role of blanks (samples lacking the analyte of interest used to determine or track the source of contamination or sample degredation and taken through the analytical process) in understanding the sample analysis process. One reader suggested that it is appropriate to address the total Youden blank, an often overlooked and perhaps the most true blank. The total Youden blank completely eliminates the constant error component arising from any source of bias involved in the measurement. In August 2017, we lost Dr. Patrick McDonald. McDonald was a Research Fellow at Waters Corporation and one of the pioneers in the development of solid- phase extraction. At the Fall National Meeting of the American Chemical Society, a symposium was held in his memory. We’ll review the symposium and McDonald’s contributions to get an up-to-date snapshot of the field of SPE. Finally, at this summer’s ExTech (International Symposium on Extraction Technologies), an expert panel offered their views on the future of sample preparation. A summary of this panel discussion is presented.

Douglas E. Raynie

α ince I took responsibility for this col- of multivariate analysis to data sets from [SA] = K + [ A]CA, where K is the Youden umn, I’ve tried to focus on techniques, chemical determinations (2–6). The cali- blank or “true sample blank,” [α ] is the S A with a balance of theory, applications, and brations and methods presented deter- slope of the calibration curve (or analytical operational aspects. I’ve aimed to provide mine, among other uses, constant errors sensitivity), and CA is the analyte amount a balance that will be of interest to both with proposed calculations to confront (either concentration or weight). Several the bench chemist and the laboratory proportional errors. These methods use a reviews or tutorials, such as those in refer- supervisor, at all levels of education and fractional factorial design to minimize the ences 7–10, provide more detailed treat- background. Occasionally, trends related number of analyses in assaying several ments of the Youden method. to sample preparation are reported. This factors. In single laboratory validations, month, the final installment of the year will the susceptibility of analytical methods to In Memory of Patrick D. McDonald take a slightly different approach. Three small changes in parameters is examined. and Recent Advances in Solid- vignettes are presented, either looking For example, in sample preparation, we Phase Extraction Symposium back at important, but overlooked, devel- have several optimization parameters of One of the pioneers in the development opments, or prognosticating the future. varying importance, as illustrated in Table of solid-phase extraction (SPE), Patrick I (7). This list of optimization parameters D. McDonald, passed in August 2017. Dr. The Total Youden Blank is not exhaustive, but limited to the major McDonald (Figure 1) was hired by Waters In our previous column (1), we discussed influences. Other extraction methods, and and Associates in 1974, initially to develop the role of sample blanks in chemical anal- each step in an analytical procedure, will preparative liquid chromatography (LC) ysis, and provided an overview of various contribute different, and additional, opti- instruments. Later, he was charged to “find types of blanks. One concerned reader mization parameters. Evaluating each of new, faster, more convenient ways to per- sent an email to comment on an often these in a one-at-a-time approach would form traditional sample preparation oper- overlooked, but highly important, type of be prohibitive. When analytical signals are ations (11).” While others had used adsor- blank, the total Youden blank. From the due solely to the presence of analyte, with bents in sample clean-up procedures, his mid-1940s to the early 1970s, W. J. Youden no matrix-based interference, the signal team went from proposing the use of LC presented research on the application from the analyte [SA] can be modeled as technology in a June 1977 internal memo to Photographers, Zugcic Zugcic, Joe Inc. image: Icon Minimize complexity. Magnify your focus.

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The life science business of Merck .*D$'DUPVWDGW*HUPDQ\RSHUDWHVDV MilliporeSigma in the U.S. and Canada. 804 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

TABLE I: Potential factors to be exam- TABLE II: Papers presented at “Recent Advances in Solid Phase Extraction: Symposium in ined in the robustness testing of com- honor of Patrick D. McDonald,” held at the Fall 2018 National ACS Meeting mon sample preparation techniques (from reference 7) Harnessing the Power of Solid-Phase Extraction for Peptide Bioanalysis. M. Lame (Waters)

Sample Solid-Phase Extraction (SPE) in Bioanalytical Method Development for Therapeutic Peptides. K. Lee (Waters) Preparation Factors Technique Development, Validation and Application of a Cation-exchange, Solid-Phase Extraction for the Determination of Nanoparticle-released Drug Concentrations in Plasma. C. Holliman, W. Solid-Phase Sorbent type Song, J. Tweed, Z. Gu (Pfi zer) Extraction Recent Advances in Solid-Phase Extraction for Biological Sam- Sorbent manufacturer ples–Fulfi lling the Promise of SPE. J. Danaceau (Waters) Sorbent mass New Developments in SPME. J. B. Pawliszyn (University of Waterloo) Sample mass Effective Simplifi ed SPE for Modern Multiresidue Analysis: Recent Developments for or volume Pass-through, Dispersive, and Retention/Elution SPE. M.S. Young, K. Tran (Waters) Wash solvent Lipid Selective SPE Materials Simplify Sample Preparation and Improve Results. D. Lucas, B.E. Elution solvent Richter, L. Zhao (Agilent Technologies) Evaporation Variability of Solute-Sorbent Binding Constants in SPE Materials. D.E. Raynie, S. temperature Pandey, S. Subedi, D. Lucas, B.E. Richter (South Dakota State University) Sample pH Porphyrin-based Magnetic Nanocomposites for Effi cient Extraction of Polycyclic Aromatic Hydrocarbons from Water Samples. J. Yu, S. Zhu (China University of Geosciences) Buffer pH Matrix Solid-Phase Sorbent type Dispersion McDonald also edited Waters’s “Sol- Sorbent manufacturer id-Phase Extraction Applications Guide Sorbent mass and Bibliography.” By the sixth edition in Sample pH 1995, over 3000 applications were com- Buffer pH piled. In 1996, he and his team devel- Sonication time oped and patented the Waters Oasis Evaporation HLB copolymer (12). This sorbent is a temperature hydrophilic–lipophilic balance copoly- Wash solvent mer. The water-wettable sorbent retains Elution solvent analytes over a wide polarity range and Sample mass is stable from pH 1 to 14. or volume In memory of McDonald, a symposium on recent advances in SPE was held at the 256th National Meeting of the American shipping the first “Sample Enrichment and Chemistry Society at Boston in August, FIGURE 1: Dr. Patrick D. McDonald (1944- Purification” (SEP–PAK) product in January organized by Tom Walter, a corporate 2017), Research Fellow at Waters and Associ- 1978. SEP–PAK featured a heat-shrinkable fellow at Waters and former associate of ates and inventor of SEP-PAK, Oasis, and other leading advances in SPE. (Photo courtesy polyethylene body, triaxial compression McDonald. The nine invited oral presenta- of Tom Walter, Waters.). technology, and preparative LC C18-silica tions, listed in Table II, can be considered particles. Figure 2 shows the cover of the an assessment of the current state of the (13). The topic was to assess the trends original February 1978 marketing brochure field. In particular, small-scale biological and developments in the chromatogra- for SEP–PAK cartridges. Note that these samples, matrix removal, and magnetic phy sector. Specifically, representatives questions concerning the cost, time, and sorbent particles are all driving present from Biotage (Paul Roberts), CEM (Alicia interferences of sample preparation still res- research in SPE. This column and peer-re- Stell), Eprep (Peter Dawes), Gerstel (Oli- onate today, though it is hoped that sam- viewed analytical chemistry journals review ver Lerch), Phenomenex (Matt Brusius), ple preparation advances have significantly the current state of SPE periodically. and UCT (Danielle Mackowsky) were improved over the decades and the current asked their opinions on emerging sam- situation reflects the simultaneous advances Views on Future Developments ple preparation trends, important recent in analysis techniques! As SEP–PAK car- in Sample Preparation developments, obstacles to continued tridges became accepted and other manu- Last spring, our sister publication fea- sample preparation developments, and facturers developed other approaches, J.T. tured an interview conducted by editors the biggest accomplishments in the past Baker’s term, solid-phase extraction (SPE), of LCGC with panelists from compa- year. While each of the six panelists pro- came to represent the technique. nies that exhibited at Analytica 2018 vided perspectives from their viewpoints, WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 805

ration of core-shell magnetic materials needs to the push of new technologies will drive advances in magnetic-particle– in search of meaningful applications. based separations. Psillakis, on the other However, education toward a more com- hand, offered the view that advanced, plete understanding of solubility and or smart, materials are not necessary for phase equilibria will guide all analysts in development of sample preparation. For coming up with the sample preparation example, she has demonstrated success approaches of the future. in the manipulation of vacuum conditions to provide more efficient extractions and References mentioned the salting-out effect in driving (1) D.E. Raynie, LCGC North Am. 36(8), chemical separations. Neither of these 494–497 (2018). manipulations (reduced pressure and salt- (2) W.J. Youden, Anal. Chem. 19, 946–950 (1947). ing out) can compete with the effect of (3) W.J. Youden, Biometrics 3, 61 (1947). temperature in driving separations. This FIGURE 2: Questions asked on the cover of (4) W.J. Youden, Mater. Res. Stand. 1, led other members of the panel, nota- 268–271 (1961). a February 1978 brochure for Waters’s SEP- bly Psillakis, Pawliszyn, and me, to stress PAK cartridges (from reference 11). (5) W.J. Youden, Statistical Techniques for that what is missing, and limiting future Collaborative Tests (Association of Offi- common threads centered on develop- innovations, is understanding of the fun- cial Analytical Chemists, Washington, DC, 1967). ment of faster and higher throughput damentals of extraction. For example, at (6) W.J. Youden and E.H. Steiner, Statistical techniques, automation, and accommo- its heart, all extractions involve manipula- Manual of AOAC (Association of Official dation of smaller samples. Given that tions of phase contact, solubility, diffusion, Analytical Chemists, Washington, DC, this interview is freely available on-line and phase separation in a thermodynam- 1975). (http://www.chromatographyonline.com/ ically consistent manner. Knowledge of (7) E. Karageorgou and V. Samanidou, J. Chromatogr. A 1353, 131–139 (2014). trends-and-developments), the reader is the chemistry of analytical systems and (8) R.C.C. Castells and M.A. Castillo, Anal. invited to peruse this article. the impact of various manipulations (such Chim. Acta 423, 179–185 (2000). Meanwhile, at about the same time, the as vacuum or temperature) can influence (9) A.R. Mauri, M. Llobat, and D. Adria, Anal. 20th International Symposium on Advances many technologies. Pawliszyn mentioned Chim. Acta 426, 135–146 (2001). in Extraction Technology (ExTech) in Ames, the integration of the individual steps in an (10) A.G. Gonzalez and M.A. Herrador, TrAc Iowa (June 19–22) took place. During analysis scheme, modeling, optimization Trend Anal. Chem. 26, 227–238 (2007). one feature, conference organizer Jared of mass transfer, and direct coupling to (11) P.D. McDonald, “James Waters and his Liquid Chromatography People: Anderson from Iowa State University mass spectrometry as needs for the future A Personal Perspective,” http://www. assembled a panel that included technical development of sample preparation. waters.com/webassets/cms/library/docs/ experts from sample preparation vendors Pawliszyn noted the lack of indus- wa62008.pdf. and academia. I was honored to join the trial chemists at the ExTech conference, (12) E.S. Bouvier, R.E. Meirowitz, and P.D. McDonald, U.S. Patent 5,976,367 (1996). panel, which also consisted of Veronica though it is noted that the infrequent (13) LC/GC Editors, The Column, 14, 2-19 Pino Estevez (Universidad de La Laguna, appearance of ExTech symposia in (2018). Spain), Elia Psillakis (Technical University North America (the previous ExTech in of Crete), Janusz Pawliszyn (University of the United States was in the Black Hills Waterloo), Bruce Richter (Agilent Technol- of South Dakota nearly ten years ago, ABOUT THE AUTHORS ogies), Dan Cardin (Entech), and Jason in 2009) and the plethora of specialized Douglas E. Raynie Herrington (Restek). An interesting mix of meetings can contribute to lower atten- “Sample Prep Perspec- views, with some significant convergence, dance. He observed a conflict between tives” editor Douglas E. was noted. Based on the panel discussion, industrial adoption of new separation Raynie is a Department Head and Associate Pro- new developments in the near term were technology with early inventions. Sim- fessor at South Dakota State Univer- discussed. These include approaches for ilarly, claims that regulatory methods sity. His research interests include matrix removal (which differ from sample generally are developed by academics green chemistry, alternative solvents, enrichment); field-based extractions and and technology manufacturers, due to sample preparation, high-resolution chromatography, and bioprocessing in other approaches to take the extraction budgetary and workload issues in lab- supercritical fluids. He earned his PhD to the sample; and even one-size-fits-all oratories associated with government in 1990 at Brigham Young University approaches to the extraction of com- agencies, may also minimize industrial under the direction of Milton L. Lee. plex samples. Pino Estevez and Richter adoption of new sample preparation Raynie is a member of LCGC’s editorial advisory board. Direct correspon- discussed how separation scientists can technologies. Hence, the drivers for dence about this column via e-mail to learn from materials science. For exam- sample preparation development have [email protected] ple, Pino Estevez opined that the prepa- shifted from being the pull of industry 806 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

GC CONNECTIONS Stationary Phase Selectivity: The Chemistry Behind the Separation

“The column is the heart of the separation.” Perhaps more accurately, the column is where the chemistry that generates a separation happens. For chemists and non-chemists alike, the chemistry that drives the utility of a column to solve a separation problem can be complex and confusing. Selectivity describes the ability of a column to effect a separation. This installment of “GC Connections” focuses on selectivity, its definition, and its importance for generating separations and resolution. We will also see how selectivity is the concept that underlies the idea of column polarity. We begin by asking two simple questions about common observations, then extend these observations into a capillary GC column, and conclude with an introduction to methods for evaluating the quality, selectivity, and polarity of a stationary phase or column.

Nicholas H. Snow

hy does a puddle evaporate? On the windy day, the wind carries water evaporates as an azeotrope. Rubbing alco- WThe next time it rains, observe molecules away, so the air above the pud- hol evaporates faster than water, due to the puddles of water on the sidewalk or dle does not approach saturation and the its higher liquid-vapor partition coefficient, street after the storm is over. As we know, water evaporates more quickly. In both thus its higher vapor pressure. the water evaporates at the air tempera- cases, the system (surface, puddle and Taking this further into chromatogra- ture, say 25 oC, yet the boiling point of air above it) is driving toward equilibrium, phy, all separations are also governed by water is 100 oC, and the water still evapo- saturation of the air above the puddle. A a similar phase equilibrium. Equation 3 rates. This is due to the vapor pressure of puddle evaporates faster on a windy day. represents the partitioning of an analyte water. Water will continue to evaporate Why does rubbing alcohol evaporate (A) from the mobile phase, the phase in until the air above the puddle becomes faster than water? Try a simple experi- which it enters the column after the injec- saturated (100% relative humidity). This ment. Rub a small amount of water on your tion, into the stationary phase: relationship can be expressed chemically arm. As we know, when the water evapo- by the following equations: rates, energy transfers from your arm to the ⇔ [A(sp)] A (mp) A (sp) Kc = water, evaporating the water and making [A(mp)] [3] H O(l)H O(g) K =P your arm feel cool. Try again with rubbing 2 2 p H2O [1] alcohol. The alcohol evaporates faster, and Kc is the partition coefficient for the pro- The vapor pressure of the water is repre- your arm feels cooler. This difference arises cess of sorption from the mobile phase sented as P , and K is the pressure-based from differences in the heat of vaporization into the stationary phase. A(mp) refers to H2O p equilibrium constant for the evaporation and vapor pressure of water and rubbing an analyte dissolved in or moving in the process. Figure 1 shows a stylized puddle. alcohol. This is an example of selectivity. mobile phase, and A(sp) refers to an ana-

Figure 1A shows some water molecules, The ability of the water (H2O) or alco- lyte dissolved in or sorbed on a station- represented by dots, evaporating on a calm hol (Alc) to evaporate is governed by ary phase. A higher Kc indicates stron- (no wind) day. Figure 1B shows the same simple chemical equations: ger analyte attraction to the stationary puddle, but with the wind blowing. In which phase, leading to longer retention times. situation does the water evaporate faster? H2O(l) H2O(g) Kc = [H2O(g)] Figure 2 shows this relationship applied [2] On the calm day, there is no wind to carry Alc(l) Alc(g) Kc = [Alc(g)] to a column with flowing mobile phase. the evaporated water molecules away, so Note how this figure looks very much like the air above the puddle becomes more In this case, the rubbing alcohol mix- the puddle with the wind blowing shown in

saturated and evaporation slows down. ture is considered a single substance, as it Figure 1B. A column in gas chromatography Photographers, Zugcic Zugcic, Joe Inc. image: Icon ADDITIONAL 15% DISCOUNT

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at the chromatogram in Figure 3, the resolution may be calculated as the difference between the two retention times divided by the average of the two peak widths:

2(tR(2)–tR(1)) Rs= [6] W1+W2

(a) (b)

If Rs is greater than 1.5, the peaks are FIGURE 1: A) Water evaporating from a puddle in still air. Evaporated water molecules baseline separated. To understand the role are represented as dots. B) Water evaporating from a puddle with blowing wind. In still of selectivity in resolution, the fundamental air, the water stays above the puddle. In wind, the water is carried away. principles behind resolution in chromatography can be considered using the following equation:

k α-1 √N R = [7] s α 1+k 4

Resolution is obtained from three basic principles: • retention factor (k), which is a measure FIGURE 2: Analytes partitioning into the stationary phase in a capillary column. Below of how long the analyte is in the col- the normal boiling point most of the molecules (represented here as dots) will partition umn. Too small k (< 2), and there is into the stationary phase. The few dots in the mobile phase are moved along the column by the fl owing mobile phase. not enough contact with the stationary phase for the most effective chromatog- behaves in a manner very similar to a pud- books on GC (1–3). Ultimately, selectivity raphy. Too large k (> 10), and there is a dle on a windy day, with the analyte being comes from thermodynamics. From Gen- diminishing return with longer time. analogous to the puddle, the stationary eral Chemistry, the Gibbs Equation relates • theoretical plates (N), the measure of phase being analogous to the surface and the partition coefficient to the standard free column efficiency. The more theoretical the mobile phase being analogous to the energy (4). plates, the better the resolution. wind. Like the rubbing alcohol versus water • selectivity (α), the separating power of 6 o example, if the puddle were a mixture of G = -RT lnKc [4] the stationary phase based on differ- two liquids, they would evaporate at differences in the strength of intermolecu- ent rates, based on their vapor pressures. If two analytes are present, as seen in Fig- lar interactions between the stationary In the column, if there are two analytes ure 3, this equation becomes: phase and analyte molecules. they will move at different rates, based on Figure 4 shows two chromatograms with -∆(∆G°) K2 K2 the difference in their partition coefficients ∆(∆G°) = –RT In and α = = e RT equal selectivity. In Figure 4A, an example K K and the resulting vapor pressures above the 1 1 from HPLC, the column has 18,285 theoret- surface. For two analytes to be separated [5] ical plates and in Figure 4B, in an example on a column, the difference in partition A more detailed description of the ther- from capillary GC, the column has 120,000 coefficient gives rise to selectivity, which modynamic relationships involved in gas theoretical plates. With k being equal, a gives rise to the separation. chromatography can be found elsewhere dramatic effect on resolution is seen. Note How is selectivity determined and (5). Selectivity comes from the difference in that, while the peak maxima are equally where does it come from? Figure 3 shows free energy change for the partitioning of spaced in the two chromatograms, the the calculation of selectivity from a chro- the analyte(s) from the mobile phase into peaks are broader in Figure 4A, reducing matogram, with the relevant equations. It the stationary phase, as seen in Equation 3. the resolution. In classical packed column is simply the ratio of the adjusted retention We have now seen the fundamental and GC and in HPLC, with lower N, adjusting times (t’R, the difference between the reten- thermodynamic basis of selectivity. Next, we the selectivity is critical to obtaining nearly tion time, tR and the gas hold up time tM) of discuss the impact of selectivity on the sep- all separations. In today’s capillary GC, two peaks in the chromatogram. It is also aration and resolution. selectivity, with high N, is less important the ratio of the retention factors (k) and the How does selectivity impact the sepa- but still must be considered. partition coefficients (Kc). Detailed descrip- ration and resolution? The first goal of any The lower the separation efficiency (low tions of the basic theory behind these rela- chromatographic method development is N), the higher the selectivity needed to tionships can be found in most basic text- to optimize the resolution. Looking again achieve separation. In most applications, 808 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

methods for evaluating the selectivity of stationary phases, and to a better understanding of the term “polar,” which is often used to describe stationary phases in GC. Most of the development of these methods occurred in the early days of GC, when packed columns, with much lower efficiency than capillary columns, were predominant. What does “polar” really mean in GC? Stationary phases are often described using terms such as “polar”, “non-polar”, “mod- erately polar” and the like. These terms are often used very broadly, and can easily generate confusion and misunderstanding. The first problem with descriptions such as “polar” is that they require context. As an example of context, think about other simple terms: “hot” and “cold”. In the FIGURE 3: Determination of selectivity from chromatogram with defi ning equations. environs of New York City, in the winter, a Chromatogram adapted from ChromAcademy (www.chromacademy.com, accessed day with a high temperature of 65 oF would September 2018). be considered unusually hot, while in summer, unusually cold. When describing the weather, the meaning of “hot” and “cold” TABLE I: Components of the Grob Test Mixture and their column performance and depends greatly on the environment. The chemistry measures. meaning of “polarity” requires similar con- Component Interaction(s)/Performance Tested text. In describing columns, any discussion Decane, undecane Column effi ciency of “polarity” must be based on specific

Fatty acid methyl esters C10, C11, C12 Column effi ciency intermolecular interactions between the 1-octanol Hydrogen bonding, presence of silanol groups stationary phase and the analytes. The Nonanal Aldehyde adsorption; non-hydrogen bonding strength of these interactions determines the free energy required for the analytes 2,6-dimethyl phenol Acid-base interactions to partition into the stationary phase, and 2,6-dimethyl aniline Acid-base interactions therefore determines the selectivity of the 2-ethyl hexanoic acid Irreversible adsorption stationary phase and, along with the col- Dicyclohexyl amine Irreversible adsorption umn dimensions, ultimately the separating ability of the column. Over the years, numerous tests have TABLE II: List of test probes and interactions for the McReynolds test mixture been developed to evaluate the intermo- Test Probe Interaction(s) lecular interactions that occur in gas chro- Benzene Pi-pi, aromatic and olefi nic hydrocarbons matographic columns. These interactions Ethanol or n-butanol Hydrogen bonding for alcohols, nitriles include the same ones we learned about 2-butanone or 2-pentanone Proton acceptor – ketones, ethers, aldehydes, esters in school: dipole-dipole, dipole-induced dipole, van der Waal forces, acid-base, Nitromethane or nitropropane Dipole-dipole interactions electrostatic interactions, and hydrogen Pyridine Strong proton acceptor – acid character of column bonding. These classical studies generally involve injecting test mixtures containing selectivity has a critical impact on sep- peaks. If the selectivity calculated by the a mix of compounds, each selected to aration and resolution. As columns and equations shown in Figure 3 is one (1.00), probe a specific interaction. These tests, instruments become more efficient (more then the two analytes are not separated and many other aspects of column evalu- theoretical plates, N), the need for high at all and cannot be separated no matter ation, are discussed in great detail in the selectivity lessens. In Figure 4A, a selectiv- how efficient the column and instrument. textbook by Barry and Grob (6). ity of 1.04, which could be a nearly 1 min Fortunately, this is not often the case. Hav- The Grob test mix, developed in the late difference in retention time in a 20 min sep- ing seen the basics of the thermodynamic 1970s is the most commonly used mixture aration, is not sufficient to fully resolve the background of selectivity, we now turn to for testing the interactions and quality of WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 809

log (t’R)u – log (t’R)x I =100 +100x [8] α log (t’R)x+1 – log (t’R)x Rs = 1.3, = 1.04, N = 18285 (Peak*)

(a) α = 1.04 The subscript u refers to the analyte. The subscript x refers to the number of carbon atoms in the normal alkane eluting immediately prior to the analyte. The subscript x+1 refers to the number of carbon atoms in the normal alkane eluting immediately following the analyte. For example, an analyte eluting between hexane α = 1.04 (C6) and heptane (C7) might have a Kovats Retention Index of 650. (b) An example calculation is shown in Figure 6. In theory, the Kovats α Rs = 3.0, = 1.04, Retention Index is linearly related to the free energy change for N = 120,000 (Peak*) the sorption process into the stationary phase, as described in Equation 5, and it accounts for differences in the column dimensions. The Kovats Retention Index of an analyte is a constant for a given stationary phase and temperature and is independent of FIGURE 4: Comparison of chromatograms with α = 1.04, con- column length, internal diameter and film thickness. stant k and differing N. Adapted from ChromAcademy (www. In the 1960s, based on Kovats Retention Indexes, two systems chromacademy.com, accessed September 2018). for assessing stationary phase polarity, or really the strength of capillary columns (7,8). Every time you purchase a column, you are a stationary phase for separating various classes of compounds provided with a column test mixture chromatogram that either uses were developed by Rorhschneider and McReynolds (10,11). the Grob test mix, or is based on the principles described in the These are used in column evaluation and are most commonly original papers. Figure 5 shows a typical chromatogram of a column termed McReynolds Constants. The experiment is simple. A test test mix run on a commercially available column, with the test mix components identified. There are several quality tests resulting from this analysis, including reactivity and sensitivity to several intermolecular interactions, column efficiency, and overall separating power. The most important selectivity calculation is the spacing of the fatty acid methyl ester peaks. This spacing must be even as each meth- ylene unit (-CH2-) is added, indicating proper selectivity. The resolution of adjacent peaks, which also results from selectivity is tested, as is the retention time and peak shape of each component, which is determined by the specific intermolecular interactions between that component and the column wall or stationary phase. Table I lists the Grob test mix components with the specific intermolecular interactions they probe. Comparing Table I to the chromatogram shown in Figure 5, basic performance tests such as the spacing and shape of the hydrocarbon and fatty acid peaks indicate a properly installed generally well-performing column. The acceptable shapes of the alcohol, phenol, aldehyde, and aniline indicate a column that should perform well for weaker acids and bases. The very poor shapes of the hexanoic acid and dicyclohexylamine indicate that this column is likely to adsorb stronger acids and bases. The Grob test mix thus provides an excellent snapshot for the quality and performance of a column. !∀ Most column manufacturers use their own variation of the Grob test mix to demonstrate column quality and performance. # In order to discuss most measures of intermolecular interactions with proper context, retention time data must be cor- rected to account for differences in column dimensions, including length, inside diameter, and stationary phase film thickness. The classical means for this is calculating the Kovats Retention Index for each analyte using Equation 8 (9): 810 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

mixture is injected under isothermal con- stationary phases are given in Table III a column may have a high constant for one ditions, and the Kovats Retention Index of (12,13). Table III also includes Mondello test probe, but a lower constant for another, each analyte is determined and compared polarity numbers, described below. indicating different degrees of “polarity” to the retention index of that test analyte McReynolds constants can be used to depending on the analyte. McReynolds on a standard non-polar stationary phase. estimate the ability of a stationary phase constants provide the necessary context The McReynolds constant for that analyte to separate analytes of various compound for describing whether a stationary phase on that column at the given temperature classes. For example, a high McReynolds is “polar”. In general, as the McReynolds is the difference between the measured constant for benzene indicates a stationary constants increase, the stationary phase retention index and the standard reten- phase with strong pi-pi interactions and may be described as more polar. tion index. A list of the five most com- ability to separate aromatic analytes. If the This discussion leads to the final ques- mon test probes with the interactions McReynolds constants are high for all of the tion: How polar is my column? This they were proposed to probe is given in test probes, the stationary phase may be topic has seen renewed interest with Table II and some constants for common accurately described as “polar”. Note that the advent of ionic liquids as stationary phases for capillary GC in the mid- 89 2000s (14,15). An ionic liquid is a molten 11 organic salt that is in the liquid phase at or near room temperature. As stationary phases, ionic liquids are considered highly polar, plus they have low vapor 3 1 2 5 pressure and do not decompose at the 7 temperatures normally employed in GC. They seem to extend the polarity range 4 of stationary phases available in capillary GC. An interesting difference between ionic liquid stationary phases and most 10 traditional capillary GC stationary phases

6 is that the ionic liquids are liquid salts, whereas the traditional stationary phases are liquid polymers. At a molecular level, 8 10 12 14 16 18 20 22 24 Retention Time (min) the actual partitioning process may be different for ionic liquid columns than for FIGURE 5: Chromatogram of a column test mixture. 1) decane, 2) 1-octanol, 3) undec- traditional polymeric columns. This pos- ane, 4) 2,6-dimethyl phenol, 5) nonanal, 6) 2-ethyl hexanoic acid, 7) 2,6-dimethyl aniline, 8) methyl decanoate, 9) methyl undecanoate, 10) dicyclohexylamine, 11) methyl laurate. sibility merits further research. column: 5% phenyl polydimenthyl siloxane, 30 m x 0.25 mm x 0.25 μm. Temperature In 2011, while evaluating the new ionic Program: 70 oC/1 min, 5 oC/min to 250 oC. liquid stationary phases, Mondello devel-

TABLE III: McReynolds constants and polarity numbers of selected stationary phases. Data from references 12, 13 and 16.

Polarity Stationary Phase Name X’ Y’ Z’ U’ S’ Total Number Squalane 0 0 0 0 0 0 0

Polydimethyl siloxane (DB-1, SPB-1, ZB-1, Rtx-1, etc.) 16 55 44 65 42 222 5

5% Phenyl polydimethyl siloxane (DB-5, SPB-5, ZB-5, Rtx-5, etc.) 33 72 66 99 67 337 8 50% phenyl polydimethyl siloxane (DB-17, SPB-50, Rtx-50, ZB-50, etc.) 119 158 162 243 202 884 20 Polyethylene glycol (WAX) 322 536 368 572 510 2,308 52 1,12-di(tripropylphosphonium) dodecane bis(triﬂuoro- 338 505 549 649 583 2,624 59 methanesulfonyl)amide (SLB-IL-59) 1,9-di(3-vinylimidazolium)nonane bis(trifl uoromethanesulfonyl)imide (SLB-IL-100) 602 853 884 1017 1081 4,437 100 X’ = benzene Y’ = n-butanol Z’ = 2-pentanone U’ = nitropropane S’ = pyridine WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 811

(9) E. Kovats, Helv. Chim. Acta 41, 1915–1932 (1958). (10) L. Rohrschneider, J. Chromatogr. 22, 6–22 (1966). (11) W.O. McReynolds, J. Chromatogr. Sci. 8, 685–691 (1970). (12) “Technical Bulletin 880: The Retention Index System in Gas Chromatography: McReynolds Constants”, Sigma-Aldrich, https://www.sigmaaldrich.com/Graphics/ Supelco/objects/7800/7741.pdf (Accessed September, 2018). (13) E.F. Barry and R.L. Grob, Columns for Gas Chromatography Performance and Selection (John Wiley and Sons, New York, 2007), pp. 32–44. FIGURE 6: Chromatogram and calculation of Kovats retention index. Adapted from (14) C. Yao and J.L. Anderson, J. Chromatogr. ChromAcademy (www.chromacademy.com, accessed September 2018). A 1216 1658–1712 (2009). (15) “Introduction to Ionic Liquid Columns” oped a straightforward overall column Conclusions https://www.sigmaaldrich.com/content/ dam/sigma-aldrich/docs/Supelco/ polarity scale, based on McReynolds The ability of a stationary phase to per- Posters/1/ionic_liquid_gc_columns.pdf constants (16). These values for some sta- form a separation is based on selectivity, (Accessed September, 2018). tionary phases are included in Table III. To the difference in the strength of intermo- (16) C. Ragonese, D. Sciarrone, P.Q. Tranchida, determine the polarity number, the five lecular interaction between each analyte P. Dugo, G. Dugo, and L. Mondello, Anal. Chem. 83 7947–7954 (2011). McReynolds constants are totaled, and and the stationary phase. Selectivity derives then the ratio of this total to the total for a from the partitioning process for the ana- highly polar ionic liquid column (SLB-IL100) lytes between the mobile and stationary ABOUT THE AUTHOR is expressed with the SLB-IL100 column phases and it plays a key role in the abil- Nicholas H. Snow having the value 100. Squalane is the ity to achieve desired resolution. Column is the Founding Endowed standard non-polar stationary phase for quality and polarity are also determined by Professor in the Depart- McReynolds constant determination (all the intermolecular interactions that occur ment of Chemistry and Biochemistry at Seton Hall McReynolds constants and polarity num- between compound classes of interest and University. He is also the university’s ber are zero), but in practice, it is rarely the stationary phase. Methods including the Director of Research and Adjunct Pro- used with capillary columns. The classical Grob test mix, McReynolds constants and fessor of Medical Science. During his non-polar phases, polydimethyl siloxane polarity numbers provide the tools to ana- 30 years as a chromatographer, he has published more than 60 refereed arti- and 5% phenyl polydimethyl siloxane lyze these interactions and to better under- cles and book chapters and has given have very low polarity numbers, so they stand the chemistry behind the separation. more than 200 presentations and short are accurately described as non-polar. courses. He is interested in the funda- 50% phenyl polydimethyl siloxane sta- References mentals and applications of separation science, especially gas chromatography, tionary phase, long considered moder- (1) H.M. McNair and J.M. Miller, Basic Gas sampling, and sample preparation for ately polar shows a polarity number of Chromatography, (John Wiley and Sons, New York, 2nd ed., 2009). chemical analysis. His research group is 20, while polyethylene glycol phase, tradi- very active, with ongoing projects using (2) C.M. Poole, Ed., Gas Chromatography GC, GC–MS, two-dimensional GC, and tionally considered among the most polar (Elsevier, Amsterdam, 2012). extraction methods including head- capillary column stationary phases, only (3) R.L. Grob, Ed., Modern Practice of Gas space, liquid–liquid extraction, and sol- reaches a polarity number of 52. Chromatography (John Wiley and Sons, id-phase microextraction. Broad descriptions of polarity and New York, 4th ed., 2004). polarity numbers are useful but with cau- (4) N. Tro, Chemistry: A Molecular Approach, (Pearson, New York, 4th Ed., 2016) Chapter tion. In Table III, the n-butanol McReyn- 18. ABOUT THE COLUMN EDITOR olds constant is higher for polyethylene (5) N.H. Snow, J. Chem. Educ. 73(7), 592–597 glycol then for SLB-IL-59, yet the over- (1996). John V. Hinshaw “GC Connections” editor all polarity number is lower. SLB-IL-59 is (6) E.F. Barry and R.L. Grob, Columns for John V. Hinshaw is a Senior Gas Chromatography Performance and an overall more polar stationary phase, Scientist at Serveron Cor- Selection (John Wiley and Sons, New York, poration in Beaverton, yet polyethylene glycol is likely to more 2007). strongly retain polar alcohols. Results such Oregon, and a member of LCGC’s (7) K. Grob, Jr., G. Grob, and K.J. Grob, Chro- editorial advisory board. Direct corre- as this and the advent of ionic liquid col- matogr. 156, 1–20 (1978). spondence about this column to the umns are leading to new thinking about (8) K. Grob, Jr., G. Grob, and K.J. Grob, Chro- author via e-mail: [email protected] column polarity and how it is described. matogr. 219, 13–20 (1981). Experience the New Proﬁciency Testing Portal Ensuring trust and conﬁdence through accurate results every time!

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The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. W Tools for Analytical Tools Analytical for There isanever-increasing interest inattain- (2), are listedinTable I.This review focuses Anurag S.Rathore, IraS.Krull, andSrishtiJoshi attributes. ofthese analysis for used arebeing that practices evolving and current the present wewill issue, this In toabiotherapeutic. arepertinent that attributes quality various the wediscussed series, ofthis part first the In II: Part Products The Analytical Toolbox Biotherapeutic of Characterization Analytical World HealthOrganization (WHO)guide- Electrophoresis (PAGE) oneofthemost Cost-effectiveness andrelative easeof tion tools and covers the new generation of tion toolsandcoversthenewgenerationof the molecule(3–5).Attributestobecovered terization. Thesetoolsare carefully chosen terizing theseattributeshavealsoevolved. the analyticaltechniquesusedforcharac- Characterization analytical hardware thatisincreasingly being analysis. Inthecaseofbiotherapeutic on theutilityoftraditionalcharacteriza- cal, andbiologicallyrelevant characteristicsof orthogonal, high-resolution toolsforcharac- exhibited bybiopharmaceuticalproducts, it estimate the size and isoelectric point of estimate thesize andisoelectricpointof characterization, PAGE istypicallyused to commonly usedtechniquesfor protein in biotherapeutic characterization, as per in biotherapeuticcharacterization,asper ing ahigherstructuralresolution ofthese lines toevaluatequality, safetyandefficacy has become the norm to use a multitude of has becomethenormtouseamultitudeof used orthogonallytothetraditionaltoolbox. use havemadePoly-Acrylamide Gel products (1,2). In view of the complexity products (1,2).Inviewofthecomplexity biotherapeutics andsubsequentbiosimilars, structural, physicochemical,immunochemi- such thattheplatformcoversallcritical 814 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 attached to structural attributes of attached tostructuralattributesof ith increasing importancebeing ANALYSIS BIOPHARMACEUTICAL ON FOCUS (2D-PAGE). Iso-ElectricFocusing(IEF),which for 2D-PAGE andprovides higherresolution, for resolving complexmoleculessuchas trophoresis (2D-DIGE).Proteins are directly tric point is also a commonly used platform tric pointisalsoacommonlyusedplatform the molecule. It serves both as a detecting the molecule.Itservesbothasadetecting and post-translational modifications. (6). and post-translationalmodifications.(6). a denaturingseconddimensionresolution aration from otherproteins isattainedina are scanned using an instrument capable are scanned usinganinstrumentcapable accurate quantificationofspots. Thegels as wellresolving technique,andrelies evaluation ofcharge heterogeneity, stability example, 2D-PAGE has alsobeenshownto only onsize.UsingTrastuzumab asan either inthefirstorseconddimension dye bindscovalentlyto or inreduced form(SDS-PAGE). Acombi- of detectingdifferent CyDyeindependently. of lysineresidues inproteins, allowingfor on theproperty ofcharged moleculesto in theirnativeform(non-denaturingPAGE) when compared with 2-D separation based when compared with2-Dseparation based labelled withfluorescent dyes(CyDyes), resolves moleculesbasedontheiriso-elec- non-denaturing firstdimensionfollowedby monoclonal antibodies(mAbs),where sep- nation ofthetwocanalsobeemployed resolved on a polyacrylamide matrix, either resolved onapolyacrylamidematrix,either migrate inanelectricfield.Proteins are platform hasbeenof2D-Difference GelElec- be a quick and easy method for qualitative be aquickandeasymethodforqualitative pooled andseparatedona2D-PAGE. The NOVEMBER 2018 An interesting recent introduction inthe2D

ε -amino groups IonExchange(IEX)Chromatography • 2D-DIGE can further be coupled with mass 2D-DIGE canfurtherbecoupledwithmass (pH gradient)are routinely usedtoascer- for identification on specific gel bands (first for identificationonspecificgelbands(first Liquid Chromatography (HPLC)acorner- tility. Gelmatrixcanbeeasilymodifiedto tain theisoelectricpoint.Moreover, PAGE achieve a specific resolution. Gradient gels achieve aspecificresolution. Gradientgels and robustness have made High Performace and robustness havemadeHighPerformace and related variants and impurities, which and related variantsand impurities, which alleled selectivitybetweenabiotherapeutic and thestationaryphase.Thisyieldsunpar- allows theanalysttofine-tunetypeof dimension) orspots(second(8,9). can becoupledwithMassSpectrometry cochemical properties. Some ofthecom- otherwise mayhavenearidenticalphysi- chemistry andchoiceofmobilephase interactions allowed between the analyte interactions allowedbetweentheanalyte monly usedmodalitiesofHPLCinclude: nucleic acids,orsmallmoleculesincom- ubiquitously usedforanalysisofproteins, by using a liquid mobile phase and a solid by usingaliquidmobilephaseandsolid plex mixtures. Typical separation isachieved spectrometry forprotein identification(7). stone of biopharmaceutical analysis. It is stone ofbiopharmaceuticalanalysis.Itis stationary phase.Diversityinsolidphase A majoradvantageofPAGE isitsversa- High resolution, varied choice of phase High resolution, variedchoiceofphase their charge. Thestrength ofbinding on theirtotalcharge. Itenablesthe is determined by theaffinity ofthe is usedtoseparatemolecules based separation ofmoleculesbased on WWW.CHROMATOGRAPHYONLINE.COM

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TABLE I: Techniques used for analytical characterization as mentioned in WHO and ICH Q6b guidelines. CD - Circular Dichroism, DSC - Dynamic Light Scattering, FTIR - Fourier Transform Infra-Red Spectrometry, LC - Liquid Chromatography, MS - Mass Spec- trometry, NMR - Nuclear Magnetic Resonance, Cryo-EM - Cryo Electron Microscopy, IEX - Ion Exchange Chromatography, CE - Cap- illary Electrophoresis, IEF - Isoelectric Focusing, RP-HPLC - Reverse Phase High Performance Liquid Chromatography, SEC - Size Exclusion Chromatography, UV - Ultraviolet, MALS - Multi Angle Light Scattering, FFF - Field Flow Fractionation, AUC - Analytical Ultracentrifugation, SDS-PAGE - Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis, TEM - Transmission Electron Micros- copy, ELISA- Enzyme Linked Immunosorbent Assay, SPR - Surface Plasmon Resonance, ITC - Isothermal Titration Calorimetry, BLI - Bio-Layer Interferometry.

Type Attributes WHO (2013) ICH Q6b (1999) OTHERS Amino-acid sequence, Molecular weight, extinc- tion-coeffi cient, disulfi de linkage, N-terminal HPLC (SEC, RP, Phys- methionine, signal/leader sequence, N-/C-termi- IEX, Affi nity), MS, iochemical Primary nal modifi cations, C-terminal processing, N-ter- HPLC, MS (ESI, MAL- SEC, SDS-PAGE, MALS, character- structure minal pyroglutamate, deamidation, oxidation, DI-TOF), MS/MS IEF, UV-VIS DSC ization isomerization, fragmentation, disulfi de bond spectroscopy, mismatch, N-/O-linked oligosaccharide, glyco- Western Blot, CE sylation, aggregation, C-terminal lysine presence HPLC, Electropho- Glycan Glycan content, glycan structure, glycan pattern, resis, MS, MS/MS, CE-MS structure glycosylation site, glycan charge pattern UV-FLD, CE, IEF X-ray crystallography, NMR, CD, FTIR, Fluo- Cryo-EM, rescence, DSC, proton Higher Order secondary structure, tertiary struc- Raman nuclear magnetic CD, NMR Structure ture, quarternary structure spectros- resonance (IH-NMR), copy Hydrogen-Deuteri- um exchange MS Animal-based ADCC, CDC, Apopto- Biological biological assays, Effector function, complement bind- sis assay, Fc-y receptor SPR, BLI, character- cell culture-based ing and activation, potency binding, Neonatal Fc ITC ization assays, biochem- receptor binding ical assays Immu- Binding as- nochemical Product Affi nity, avidity, immunoreactivity, epitope char- SPR, BLI, Cell-based assays say, western character- related acterization, glycosylation/ PEGylation profi le ITC blot, ELISA ization HPLC, Electrophore- HPLC, SEC- Impurities, Fragmentation, amino acid modifi cation, sis, MS, CE, SEC, FFF, HPLC, SDS-PAGE, MALS, contam- Higher molecular weight species, particles AUC, Fluorescence, peptide mapping, SEC, MFI inants Light scattering CE, MS, CD

proteins to the “Ion-Exchanger” linked similar in size but differ in charge, using based on their hydrophobicity and to the resin (stationary phase). Cat- cation exchange HPLC (11). their charge. It is a relatively gentle ionic exchangers possessing negative • Size Exclusion Chromatography (SEC) separation technique as the chosen charge bind positively charged entities, enables separation of molecules based conditions are minimally denaturing whereas anionic exchangers bind neg- on their size. The stationary phase con- and as a result do not significantly atively charged entities. Furthermore, sists of a gel containing beads of a affect the biological activity of the ion-exchangers can be weak or strong specific pore distribution. The choice protein. Traditionally used as a pol- depending upon the range of pH within of pore distribution allows the user to ishing step in monoclonal antibody which they can sustain their charge. This achieve separation of species in the purification, it has also been used influences the range and type (strong/ desired range of size. In the case of to characterize drug distribution in weak) of binding that can be achieved biotherapeutics, a popular application antibody-drug conjugates (ADCs) by (10). Elution is typically achieved by either is the resolution of size based hetero- exploiting the hydrophobicity of the altering the pH of the mobile phase or geneities, especially aggregates (12). conjugated small molecule (13). increasing the ionic concentration of the Co-eluting host cell proteins can also • Reverse Phase (RP) Chromatography mobile phase (salt gradient IEX). A com- be resolved using SEC (9). exploits reversible adsorption of bio- monly used application involves sep- • Hydrophobic Interaction Chroma- molecules based on their hydrophobic- aration of charged variants, which are tography (HIC) separates molecules ity under conditions where the stationary WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 817

phase is more hydrophobic than the ing it a cost-effective technique. CE coupled shows much promise in monitoring batch to mobile phase. It is quite similar to HIC with nano-ESI-MS has been shown to be batch variation in manufacturing as well as in in principle, however, the RPC medium particularly useful for N-Glycan analysis of biocomparability exercise. is much more hydrophobic than in HIC monoclonal antibodies as well as for detec- X-ray crystallography is considered the and elution is achieved by the use of tion of charge variants (15). The availability gold standard for protein structural stud- non-polar, organic solvents. This makes of various CE modes such as capillary zone ies. It works on the principle of X-ray dif- it a very desirable technique for cou- electrophoresis, capillary gel electrophore- fraction. The angle and the intensity of the pling with MS for peptide mapping and sis, capillary isoelectric focusing and micel- diffracted beam from a crystal are used to other comparability studies. Coupled lar electrokinetic chromatography allows construct a 3-dimensional image of the with mass spectrometry, the reverse for characterization of different attributes molecular structure. However, applica- phase has been extensively used in pri- such as intact mass, reduced mass, charge tion of this technique for characterization mary structure characterization of bio- variants, as well as glycosylation pattern. CE and biocomparability is challenging as the therapeutics as well as in comparability coupled with MS is increasingly being used protein of interest has to be purified and studies of biosimilars (14). as a complementary platform to traditional crystallized, which may not be possible for High Performance Capillary Electropho- LC-MS for biotherapeutic characterization biotherapeutics due to the presence of resis (CE) is another technique increasingly (16). Innovative integration of CE with MS, inherent heterogeneity in the form of sev- being applied in conjunction with MS for such as in ZipChip has reduced the analysis eral post-translational modifications (PTMs). charge variant characterization, isoelectric time to under three minutes and bypassed Although the data obtained is a direct mea- focusing and biosimilarity assessment. Mol- issues of individual component integra- surement of the crystal structure, the tech- ecules are separated across a fine capillary tion and capillary damage due to handling. nique itself is too cumbersome to be used with the internal diameter as small as 50 μM Demonstrated applications include glyco- as a routine analytical technique for biosim- via application of high voltage (~30 kV). This sylation profiling of mAb directly from cell ilar characterization and comparability (18). miniaturized format requires minimal sample culture with sample volume requirement of Nuclear Magnetic Resonance (NMR) with flow rates in the range of nL/min, mak- under fifty microliters (17). The technique exploits the magnetic properties of specific 818 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

tallography which requires time-resolved TTABLE II: Publications per technique since 2010 for biotherapeutics (Google Schol- ar) LC - Liquid Chromatography, CE - Capillary Electrophoresis, TOF - Time of fl ight, experiments to capture structural changes Q-TOF- Quadruple-time of fl ight, MALDI - Matrix Assisted Laser Desorption ioniza-- due to biochemical reactions (19, 20) tion, TRIPLE-QUAD - Triple Quadrupole, CD - Circular Dichroism, DSC - Dynamic Light Transmission Electron Microscopy (TEM) Scattering, FTIR - Fourier Transform Infra-Red Spectrometry, NMR - Nuclear Magnetic is a microscopy technique in which a beam Resonance, HDX-MS - Hydrogen/Deuterium exchange-Mass spectrometry, CRYO-TEM of electrons is transmitted through a spec- - Cryo-Transmission Electron Microscopy, TEM - Transmission Electron Microscopy, FFFF - Field Flow Fractionation, SPR - Surface Plasmon Resonance, ITC - Isothermal Titration imen to form an image. Due to the much Calorimetry. The publication data were generated using Google Scholar for papers smaller wavelength of electrons as com- from 2010-2018 (excluding citations and patents). Search word combination used was pared to light, the resolution of the image is “technique name” “biopharmaceutical” and “technique name” “biopharmaceutical”” greater by orders of magnitude with details and “monoclonal antibody”. up to atomic level, and hence TEM finds Tool Category use in the characterization of biotherapeu- 2010-2017 Biopharmaceuticals Monoclonal antibodies tic aggregates (21). Recent developments HPLC 7440 1600 in achieving greater resolution, especially in Cryo-TEM, have enabled researchers to CE 1880 1040 observe protein complexes such as mAbs HPCE 109 44 in their native formulation (without crys- ESI-TOF 2140 703 tallization) (22). The technique is currently Q-TOF 559 441 underutilized in the field of biotherapeutics MALDI-TOF 2630 835 but has significant potential to grow. ORBITRAP 882 603 Circular Dichroism (CD) Spectroscopy TRIPLE-QUAD 39 251 is based on the difference observed in the absorption of left and right-handed circularly HDX-MS 406 665 polarized light of a molecule in the presence CD 1750 781 of light absorbing chiral groups. This prop- DSC 1670 847 erty of biomolecules is frequently utilized in FTIR 1720 362 the examination of the secondary structure DLS 4330 1080 of the proteins and is employed for assessing NMR 3550 796 HOS comparability. Using thermal denatur- CRYO-TEM 144 24 ation, information about molecule stability TEM 3760 650 as well as folding and unfolding mechanisms can be elucidated. An application of CD FFF 550 307 in the aggregate characterization of mAbs SPR 2060 997 has also been demonstrated (23, 24). ITC 505 156 Fourier Transform Infrared Spectros- copy (FTIR) is a spectroscopic technique atomic nuclei. Although a gold standard for for deflection by the surrounding electron that monitors the characteristic infrared protein structural studies, its routine applica- clouds, NMR is based on the absorption of absorption of molecules and translates it tion in the biotherapeutic industry has been electromagnetic radiation in the radio-fre- into structural information. Similar to CD, it limited due to several factors, including the quency (RF) range. In NMR, proteins are gives structural information about the sec- large size of protein biopharmaceuticals, the analysed in solution and the final image is ondary structure of the protein, mainly the relatively low sensitivity of the NMR signals, a compilation of a number of low energy alpha helices and beta sheet. It serves as an and the low natural abundance of active states of the protein in different orientations orthogonal technique to CD in characteriza- nuclei. However, in instances where biother- as compared to single instance images of tion and biocomparability studies (25). apeutics of small molecular size are being X-ray crystallography. Both the techniques Although both CD and FTIR can provide characterized, NMR might be utilized to gain require high concentration, homogenous information with regards to components in-depth HOS information (18). sample preparations. With respect to proof the secondary structure of a molecule, It should be noted that although both tein size, NMR is more limiting. Proteins with it is only through higher resolution visual- X-ray crystallography and NMR provide a size larger than 40 kDa exhibit a slower ization techniques such as CryoTEM, X-ray structural details at a near-atomic level, the molecular tumbling in solution leading to crystallography and NMR that the spe- techniques differ in their principle, informa- spectral overlap and peak broadening. In cific arrangement of these components in tion and bottlenecks. X-ray crystallography terms of information obtained, NMR can space can be unravelled. requires the formation of the uniform crystal unravel information on structural dynamics Dynamic Light Scattering (DLS) is com- lattice and utilizes very high energy X-rays and flexibility more readily than x-ray crys- monly used to determine the size distribu- WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 819

tion profile of small particles in biothera- aggregate analysis because it causes physi- typically used in line with a resolving tech- peutic formulations. The particle size profile cal separation of molecular species of differ- nique such as SEC to determine the size of is determined by measuring the random ent mass or shape. This is a matrix-free tech- the different molecular species as they pass changes in the intensity of light scattered nique as no column or gel matrix is required through the MALS detector. It adds a quanti- from the sample solution. It is often used in for size fractionation to occur (31). AUC-Sed- tative measure of analysis to techniques like conjunction with SEC and analytical ultracen- imentation Velocity (AUC-SV) is often used SEC. As the sample passes through the laser trifugation for aggregate studies. An interest- to quantify high molecular weight species beam, light is scattered at multiple angles ing application of High-throughput platform present in biopharmaceuticals (32). and the detector collects this data to approx- in DLS (HT-DLS) has been in screening stud- Multiple Angle Light Scattering (MALS) is imate the sample size (33). It has been used ies to quantify viscosity of mAb formulations (26). Using automated HT-DLS, effects of buffer conditions and temperature on aggregate formation have also been reported (27) Raman Spectroscopy is used to observe ® vibrational, rotational, and other low-fre- TSKgel SuperMultipore quency modes in a system, generally applied GPC/SEC columns to study the secondary structure of proteins by studying the Amide I band between 1600 New multiple pore size columns and 1700 cm−1. An advantage that Raman spectroscopy offers over other secondary for extended linear separation range structure characterizing techniques is its Individual particles with broad pore size distribution low susceptibility to water interference as it 108 TSKgel SuperMultiporeHZ-N detects scattered light and water is inefficient 7 Multiple Pore Sizes 10 TSKgel SuperMultiporeHZ-M TSKgel SuperMultiporeHZ-H at scattering in this part of the spectrum (28). 106 5 In a recent study, Raman Optical Activity SuperMultipore columns 10 104

3 (ROA) was evaluated as a means to detect Log molar mass 10 102 early thermal instability in mAb samples kept Pure packings with multi-pore size distribution 101 (TSKgel SuperMultiporeHZ column) 1.5 2.5 3.5 4.5 5.5 6.5 at 50 °C for a month. Significant structural Retention time (minutes) changes could be observed at one week of 100 stress. This provides an advantage over SEC 80 TSKgel SuperMultipore HZ-M • Each particle has a linear calibration curve – in monitoring aggregation as ROA would 60 no inﬂ ection point provide information about subtle differences 40 • No chromatogram distortion in tertiary structure whereas Raman/ROA 20 for more accurate molecular spectra can elucidate on changes at the 0 weight distributions Competitive mixed bed column secondary structure level (29). Another inter- -20 6 8 10 12 14 16 18 20 esting recent application of Raman spectroscopy has been in drug identification, specifically in the identification of monoclonal 90 antibodies by exploiting subtle differences Semi-micro columns Conventional columns ×4 70 in vibrational modes of the antibodies (30). • Small particle size (3 to 6 μm) It would be interesting to see if this applica- and smaller column dimensions 50 (4.6 mm ID × 15 cm L) tion can be further modified to distinguish 30 Detector response (mV) between biosimilars and innovator product. • Higher throughput, higher 10 resolution, reduced solvent TSKgel SuperMultiporeHZ-N ×4 Analytical Ultracentrifugation (AUC) is -10 consumption 10 20 30 40 a versatile tool for quantitative analysis of Retention time (minutes) macromolecules in solution. It employs the Accurate results every time! principle of centrifugal acceleration to separate particles based on their size and mass. Contact us: 800-366-4875, option #4 Two types of hydrodynamic analyses, namely Tosoh Bioscience and TSKgel and are registered trademarks of Tosoh Corporation. sedimentation velocity and sedimentation equilibrium, are able to make distinctions in formulation components based on shape www.tosohbioscience.com and mass or mass alone, respectively. Sed- imentation velocity is a mode of choice for 820 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

to monitor the formation of soluble, HMWS used to induce fluorescence. Fluorescence is teases make it possible to map disulphide so as to better quantify and model non-na- routinely used in thermal stability studies of links present and the different glycans tive aggregation kinetics in α chymotrypsino- biotherapeutics, especially monoclonal anti- attached to the protein (18,40). gen (34). Composition-Gradient Multi-Angle bodies (38). At best, it is an indirect measure Another useful application of MS, when Light Scattering (CG-MALS), a variation on of changes in a protein’s tertiary structure and coupled with Hydrogen/Deuterium MALS, employs a series of unfractionated does not provide information about position exchange (HDX) is in elucidating protein samples of different composition or concen- and the specific nature of these changes. conformational dynamics and protein inter- tration in order to characterize a wide range Micro-Flow Imaging (MFI) is an up and actions. In the biopharmaceutical industry, of macromolecular interactions. CG-MALS, a coming technique that combines digital HDX-MS has established itself in the analysis complementary technique to AUC and DLS, microscopy with microfluidics to capture of protein-small molecule interactions, char- has been used to characterize self-associa- and quantify sub-visible particles in the acterization of bio-therapeutics/biosimilars, tion in model mAb molecule (35). Although range of 1 to 300 μm in a solution. It can and epitope mapping of biotherapeutics. not yet commonly used, CG-MALS could provide information about particle size, The technique relies on isotope labelling to be used orthogonally to Surface Plasmon concentration and morphology (39). How- probe the rate at which protein backbone Resonance (SPR) to study receptor bind- ever, rather than protein characterization, it amide hydrogens undergo exchange. MS ing kinetics in biotherapeutics. Unlike SPR, is more suited for profiling and classifying is then used to monitor the mass-shift as a CG-MALS would not require binding of the particulate size in a given formulation. result of incorporation of deuterium in the the receptor to any chip and hence would Mass Spectrometry (MS) is a powerful protein. The rate of exchange provides infor- be a truly label-free technique to quantify and data-intensive technique for deter- mation regarding the conformational mobil- interactions, similar to Isothermal Titration mining protein mass (intact, fragmented ity, hydrogen bonding strength, and solvent Calorimetry (ITC). Although the latter is and reduced), sequence, and for probing accessibility in protein structure (41,42) more suited to study the thermodynamic and quantifying protein modifications. Differential Scanning Calorimetry (DSC) parameters of an interaction. MS involves ionization of the sample is a versatile technique that measures the Field Flow Fractionation (FFF) is a unique fragments followed by their separation quantity of heat radiated or absorbed by separation technique used for analysis of based on their mass to charge ratio (m/z). the sample on the basis of a temperature aggregates. Samples are pumped into a By accelerating the ionized particles and difference between the sample and the narrow tube perpendicular to the flow and subjecting them to an electric or magnetic reference material. It is used to determine separation occurs due to the difference field, the ions get deflected depending on equilibrium thermodynamic stability and in mobility of the species in the mixture the mass and charge that they carry. Ions folding mechanism of proteins and finds under the field applied. Depending upon are detected by an electron multiplier and routine use in thermal stability characteriza- the properties of the species in the sample the results are displayed as a spectrum of tion of biotherapeutics (43). mixture, different flows such as electrical, the relative abundance of different ions magnetic, thermal-gradient, gravitational based on the m/z ratio. Tools for Functional Characterization: or centrifugal can be applied. FFF serves Analytical capability of an MS platform is Enzyme-linked Immunosorbent Assay as a complementary technique to DLS and defined by the kind of ionization source and (ELISA) is a popular diagnostic technique MALS for determining the presence of the type of mass analyser being used. Exam- used to assess the immunogenicity of a ther- sub-micron particles in the sample (36). ples of ionization sources include fast atom apeutic product. In a typical assay, a ligand, Fluorescence spectrometry is based on bombardment (FAB), chemical ionization typically an antigen, is non-specifically or spe- the principle that certain molecules called (CI), atmospheric-pressure chemical ioniza- cifically bound to the polystyrene well of a 96 fluorophores emit light upon excitation by tion (APCI), electrospray ionization (ESI), and well microtiter plate. Enzyme-linked antibod- an external source such as an incandescent matrix-assisted laser desorption/ionization ies are used for colorimetric detection of a lamp or a laser which produces a spectrum. (MALDI). Examples of mass analyzers include positive interaction upon addition of the This technique is helpful in exploring HOS Time-of-flight (TOF), quadrupole mass filter, enzyme substrate. ELISA has several appli- of a protein (to an extent), mainly the tertiary and ion-traps. Some of the popular combi- cations in biotherapeutic characterization. A structure via the intrinsic fluorescence of the nations of the two are ESI-TOF, MALDI-TOF common application of this assay format is in protein. Changes in the local environment and ESI-Q-TOF (Table II). Host Cell Protein analysis where polyclonal of tryptophan, the strongest intrinsic fluo- MS is usually coupled with LC or CE as antibodies raised to the host cell are used rophore, are reflected in the emission spec- the first dimension of separation. Charac- to detect the presence of HCPs in the sam- tra of the molecule and are a measure of terization of complex molecules such as ple (44). However, with high specificity tech- change in the protein tertiary structure (37). monoclonal antibodies requires mapping niques such as MS (detection) and Surface In cases where a biomolecule does not have of the different fragmentation patterns in plasmon resonance (interaction) becom- an intrinsic fluorophore, extrinsic fluorophore MS such as Electron Transfer Dissociation ing affordable and routine, ELISA can only dyes such as Thioflavin-T (ThT) or 1-anili- (ETD) and Collision-Induced Dissociation be used for a precursor technique for quick no-8-naphthale-nesulfonate (ANS) can be (CID) along with the use of different pro- estimation with limited confidence prior to WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 821

employing tools with much higher specificity. reflected from two surfaces: a layer of Summary Surface Plasmon Resonance (SPR) immobilized protein on the biosensor tip With the increase in complexity of the bio- (interaction) is a label-free technique and an internal reference layer. Changes therapeutics undergoing manufacturing, the that allows for real-time detection of occurring in the number of molecules need for in-depth analytical characterization biomolecular interactions. The SPR phe- bound to the biosensor tip is reflected as a has also increased. As protein molecules, nomenon occurs when polarized light shift in the interference pattern. Similar to even minute changes in the structure of the strikes an electrically conducting surface SPR, binding kinetics can be determined biotherapeutic can confer altered functional- at the interface between two media. In by this technique. Because of its robust- ity, sometimes leading to immunogenic reac- response, plasmons are produced, which ness and ease of implementation, BLI is tions in the patients. In view of our depen- are electron charged density waves. gaining application as a complementary dency for production of these molecules These waves reduce the intensity of technique to SPR for studying and com- on biological machinery, the formation of reflected light at a specific angle known paring biotherapeutic binding kinetics (44). numerous altered conformations of the mol- as the resonance angle, in proportion to Isothermal Titration Calorimetry (ITC) is a ecule is unavoidable. However, significant the mass on a sensor surface. SPR allows physical technique used to determine the advancements have been made in analytical for real-time monitoring of both associ- thermodynamic parameters of interactions methodology increasing our ability to char- ation and dissociation of an interaction, in a solution. It measures the heat released acterize a biotherapeutic. This is highlighted generating reproducible kinetic data. or absorbed by mixing the two interactants by the increase in the number of technique Due to its sensitivity, ease of use and via titration. It is widely considered an abso- rich publications on analysis of biotherapeu- automated data analysis, it is widely used lute and direct measurement of interaction. tics since 2010 and the introduction of the to study the ligand-receptor kinetics of Thermodynamic parameters of an interac- US-FDA BPCI Act 2009 (Table II). monoclonal antibodies (14) tion can be assessed via this technique and Our understanding of the relevance of the Bio-Layer Interferometry (BLI) is a label- it is fast gaining importance as an orthog- different measured attributes with respect to free, optical analytical technique that ana- onal technique to SPR for ligand binding the safety and efficacy of a biotherapeutic lyzes the interference pattern of white light studies in biocomparability (45). has been improving with time and experi-

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ence. Some recently evolving techniques (10) J. R. Auclair, A. S. Rathore Chitra, and I. S. (32) R. Esfandiary et al., J. Pharm. Sci. 102(9), Krull, LCGC North Am. 36(1), 26–36 (2018). 3089–3099 (2013). such as CG-MALS for macromolecular (11) S.K. Singh, G. Narula, and A.S. Rathore, (33) J. Liu, T. Eris, C. Li, S. Cao, and S. Kuhns, Bio- interactions and intrinsic Förster resonance Electrophoresis 37(17–18), 2338–2346 (2016). Drugs 30(4), 321–38 (2016). energy transfer (iFRET) for in vivo target pro- (12) A. Singla, R. Bansal, V. Joshi, and A.S. (34) P. Garidel, M. Hegyi, S. Bassarab, and M. tein detection are yet to find their place in Rathore, AAPS J. 18(3), 689–702 (2016). Weichel, Biotechnol. J. 3(9–10), 1201–1211 the biotherapeutic characterization toolbox (13) A. Wakankar, Y. Chen, Y. Gokarn, and F.S. (2008). while other well-established techniques such Jacobson, MAbs 3(2), 164–175 (2011). (35) M. Weichel, S. Bassarab, and P. Garidel, Bioprocess Int. (5), 42–52 (2008). at . technological advancements would be the (16) M. Han, B. M. Rock, J. T. Pearson, Y. Wang, (36) M.K. Joubert, Q. Luo, Y. Nashed-Samuel, increasing throughput of techniques such and D. A. Rock, Therapeutic Monoclonal J. Wypych, and L.O. Narhi, J. Biol. Chem. as LC by parallelization (9). Short analysis Antibody Intact Mass Analysis by Capillary 286(28), 25118–33 (2011). Electrophoresis–Mass Spectrometryin Cap- time and micro- and nanoplatforms with ill. Electrophor. Spectrom., 13–34 (Springer (37) A. Guttman, LCGC North Am. 30(5), 412– minimal sample consumption would help International Publishing, Cham, 2016). 421 (2012). cut down the cost of analysis associated doi:10.1007/978-3-319-46240-0_3. (38) C.M. Johnson, Arch. Biochem. Biophys. 531(1–2), 100–109 (2013). with limited and expensive samples such as (17) S.A. Berkowitz, J.R. Engen, J.R. Mazzeo, and G.B. Jones, Nat. Rev. Drug Discov. 11(7), (39) D.G. Bracewell, R. Francis, and C.M. Smales, monoclonal antibodies. 527–540 (2012). Biotechnol. Bioeng. 112(9), 1727–1737 (2015). Despite these advancements, we are yet (18) S.A. Berkowitz, Analytical Characterizationin (40) U. Sinha-Datta, S. Khan, and D. Wadga- to reach a point where a product such as a Biosimilar Drug Prod. Dev., 15–82 (2017). onkar, Biosimilars 5, 83–91 (2015). complex biotherapeutic, can be completely doi:10.1201/9781315119878-3. (41) C.A. Challener, BioPharm Int. 28(1) (2015). fingerprinted in a manner similar to a phar- (19) B. Carragher, A. Schneemann, J.J. Sung, (42) T.K. Dam, M. Torres, C.F. Brewer, and A. S.K. Mulligan, J.A. Speir, K. On, and C.S. maceutical (small molecule) product. More- Casadevall, J. Biol. Chem. 283(46), 31366– Potter, Microsc. Microanal. 21(33), 2014– 70 (2008). over, each technique comes with its unique 2015 (2015). (43) M.G. Petroff, H. Bao, J.P. Welsh, M. van limitations and pitfalls. This ensures that the (20) J. T. Yang, C.-S. C. Wu, and H. M. Martinez, Beuningen-de Vaan, J.M. Pollard, J.D. topic of analytical characterization of bio- Methods Enzymol. 130, 208–269 (1986). Roush, S. Kandula, P. Machielsen, N. Tugcu, doi:10.1016/0076-6879(86)30013-2. therapeutics will continue to be an area of and T. O. Linden, Biotechnol. Bioeng. 113(6), (21) V. Joshi, T. Shivach, N. Yadav, and A.S. 1273–1283 (2016). research in the time to come. Rathore, Anal. Chem. 86(23), 11606–11613 (44) V. Kamat and A. Rafique, Anal Biochem. (2014). 536, 16-31 (2017) References (22) L.R. Tsuruta, M. Lopes dos Santos, and A.M. (45) S. Perspicace, A.C. Rufer, R. Thoma, F. (1) ICH Expert Working Group, Specif. Test Moro, Biotechnol. Prog. 31(5), 1139–1149 Muller, M. Hennig, S. Ceccarelli, T. Schulz- Proced. Accept. Critreia Biotechnol. Prod. (2015). Gasch and J. Seelig, FEBS Open Bio. 3, (March), 1–20 (1999). (23) F. He, G. W. Becker, J. R. Litowski, O. L. 204-211 (2013) (2) WHO, Guidelines on the Quality, Safety and Narhi, D. N. Brems, V. I. Razinkov, Anal. Bio- Efficacy of Biotherapeutic Protein Products chem. 399(1), 141–143 (2010). ABOUT THE AUTHORS Prepared by Recombinant DNA Technology, (24) L. Aileen, A. Seneviratne, G. Ratnaswamy, WHO Technical Report Series 814, 91 (2013) and J. Park, Pharm. Technol. 38(10), 32–39 Anurag S. Rathore (3) L.A. Bui, S. Hurst, G.L. Finch, B. Ingram, I.A. (2015). is a professor in the Depart- Jacobs, C.F. Kirchhoff, C.K. Ng, and A.M. (25) R.J. Falconer, D. Jackson-Matthews, and ment of Chemical Engineer- Ryan, Drug Discov. Today 20(S1), 3–15 (2015). S.M. Mahler, J. Chem. Technol. Biotechnol. ing at the Indian Institute of (4) A.S. Rathore, Trends Biotechnol. 27(12), 86(7), 915–922 (2011). Technology in Delhi, India. 698–705 (2009). (26) G. Thiagarajan, E. Widjaja, J.H. Heo, J.K. (5) A. AL-Sabbagh, E. Olech, J.E. McClellan, Cheung, B. Wabuyele, X. Mou, and M. Sha- Ira S. Krull and C.F. Kirchhoff, Semin. Arthritis Rheum. meem, J. Raman Spectrosc. 46(6), 531–536 is a Professor Emeritus with 45(5), S11–S18 (2016). (2015). the Department of Chemis- (6) D. Nebija, C. Noe, E. Urban, and B. Lach- (27) S.K. Paidi, S. Soumik, R. Strouse, J.B. try and Chemical Biology at mann, Int. J. Mol. Sci. 15(12), 6399–6411 McGivney, C. Larkin, I. Barman, Anal. Chem. Northeastern University in (2014). 88(8), 4361–4368 (2016). Boston, Massachusetts, and a member (7) R. Diez, M. Herbstreith, C. Osorio, and O. (28) T. Arakawa, J.S. Philo, D. Ejima, K. Tsumoto, of LCGC’s editorial advisory board. Alzate, 2-D Fluorescence Difference Gel and F. Arisaka, Bioprocess Int. 4, 42–43 Electrophoresis (DIGE) in Neuroproteomics (2006). Srishti Joshi Neuroproteomics (CRC Press/Taylor & (29) L. Wafer, M. Kloczewiak, and Y. Luo, AAPS J. is a post-doctoral research Francis, 2010). at . (30) A. Oliva, M. Llabrés, and J.B. Fariña, J. and Bioprocessing Lab, (8) C. Reichel and M. Thevis, Bioanalysis 5(5), Pharm. Biomed. Anal. 25(5–6), 833–841 under the tutelage of Pro- 587–602 (2013). (2001). fessor Anurag. S. Rathore, Department (9) S. Fekete, D. Guillarme, P. Sandra, and K. (31) Y. Li, W. F. Weiss, and C. J. Roberts, J. of Chemical Engineering, Indian Insti- Sandra, Anal. Chem. 88(1), 480–507 (2016). Pharm. Sci. 98(11), 3997–4016 (2009). tute of Technology, Delhi. The Visible Difference In Laboratory Science Expositions

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Instrument Considerations in the Transfer of Chromatographic Methods, Part II: System Considerations

The process of transferring chromatographic methods between users and laboratories is often complicated and time consuming. This is the second installment of a three-part consideration of this common activity. In part 1, the focus was on the method itself. In this second part, the techniques and concerns about characterizing the systems in use in the originating laboratory and the new facility will be described. Given that the scientists executing the transfer often do not have free access to the originating system, alternative approaches to matching chromatographic results will be considered.

Thomas E. Wheat

ransferring chromatographic meth- here. It is, however, very often impossible These guidelines, often mentioned as Tods among users and laborato- to maintain that consistency. The instru- “<621>”, specify changes to the method ries is a common and important activity ments used in various laboratories are that may be implemented without revali- that has often proven more difficult than often different models or brands, and it dating the method. The chapter has been expected. This is the second installment is not usually financially sensible to pur- summarized in many places, but the original of a three-part discussion focused on the chase chromatography instruments for document should always be consulted. We most rigorous method transfer leading each specific new method to be imple- will allude to specific items in these guide- to the duplication of results of the estab- mented. Furthermore, the usable life- lines in the context of specific challenges in lished methods. The first installment (1) time of a method is often much longer method transfer. It should be emphasized described the aspects of the method than that of an instrument. Duplicating that many laboratories follow these limits that specifically affect the transfer of the instruments, therefore, may not be pos- and practices, but they are not universal method. In the future, the final part of this sible to begin and execute a method regulations. They are absolute require- series will consider the details of aligning transfer. We must, then, consider the ments only for the compendial methods of individual instrument modules. In this part, differences among instruments that can the USP. we address the chromatographic systems affect method transfer. The transfer of a and how they may be characterized and method from one instrument to another The Fluid Path compared for use in the transfer. It is gen- may require some adjustment of the There are important factors associated erally assumed that modern instrumenta- method. Many laboratories adhere to with the fluid path in general; the pump- tion delivers the volumes, temperatures, the guidelines found in Chapter 621 of ing or solvent delivery system; the sample and so on that are programmed in the the current United States Pharmaco- introduction or injector system, and the control software. That is generally true for peia. The currently applicable chapter detector. Each component will be consid- well-maintained systems. There are, how- specifically states: ered for its potential impact on the method ever, subtle differences in the exact deliv- in terms of altered retention time as it may ery and conditioning of flow that can have Adjustments to the specified chro- affect peak identification and resolution; significant effects on the chromatographic matographic system may be nec- altered chromatographic selectivity as it results. Such details occur in all the system essary in order to meet system suit- may affect resolution and quantification; modules. We discuss here the characteriza- ability requirements. Adjustments to and peak shape as it may affect resolution tion of the systems to identify these opera- chromatographic systems performed and sensitivity. tional differences. in order to comply with system suit- The fluid path of the instrument includes ability requirements are not to be all the tubing, and other elements where General Considerations made in order to compensate for col- liquid moves through the system. To con- The chromatographic instrument itself is umn failure or system malfunctions. sider the impact on transfer, we must dis- often the largest contributor to inconsis- Adjustments are permitted only when tinguish between segments that transport tencies in the transfer of methods. The . . . adjustments or column change the sample and segments that are only common principle applied for all other yields a chromatogram that meets all exposed to the mobile phase. We gen- considerations, “Use exactly what was used the system suitability requirements erally assume that the fluid is unaltered in the originator’s laboratory,” is desirable specified in the official procedure (2). during this transport, but this may not be WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 825

from the column. Many standard methods have peak volumes near or above 50 μL. 100.00 100% t μ G The effect of a 10 L tubing volume will be relatively small. Even for the most modern 80.00 ultra-high performance liquid chromatography (UHPLC) methods, with peak volumes 60.00 near 10 μL, 0.004 inch (~102 μm) contributes 50% about 2 μL per foot, and 0.0025 inch (63.5 μ μ 40.00 m) is less than 1 L per foot. Gradient (%) When implementing the above tubing t 1 t D t – – G considerations, many scientists overlook 20.00 1/2 2 the back pressure that can arise simply V = t D G F from flow through tubing. At the midpoint 0.00 of a water–methanol gradient, at 1 mL/ 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 min, the resistance to flow through 1 m of Time (min) a 0.0025 inch (~63.5 μm) tubing generates t 1/2 (2) a back pressure greater than 5000 psi. For this reason, the tubing used to assemble a FIGURE 1: The measurement of the volume of a chromatographic system is based on system should be no smaller than required running a gradient after removing the column. Mobile-phase A = water. Mobile-phase B = for minimizing dispersion. water with 10 mg/mL caffeine. Detection wavelength = 273 nm. The strong solvent is spiked with a UV absorbing marker (in this case, caffeine). Mobile-phase gradient: hold at 0% B for Pumps 5 min, then 0–100% B in 20 min, 100% B for 5 min, 100–0% B in 5 min; fl ow rate: 1 mL/min. (Reprinted with permission from reference 2). The pumping, or solvent delivery system, has received the most attention of any absolutely true. While the instrument may on both the diameter and length of the instrument module in terms of its effect on create trace chemical changes in the mobile tubing. It is important here to distinguish method transfer. Isocratic separations are phase, such as metal leaching, this seldom between parts of the flow path used for reasonably simple because modern sys- complicates method transfer, since mate- mobile-phase transport only and the parts tems reliably deliver the specified flow rate rials of construction are consistent across downstream from the injector where the from a reservoir of preblended solvent, models and manufacturers. The worrisome sample is in the flow path. As a general with the preparation constraints discussed effects are physical. In addition, the tubing rule, the upstream parts of the system in part I of the series. Gradients are much dimensions create some timing differences. can contribute some mixing to blend the more complicated to duplicate between The time to transit the fluid path is often mobile phase. However, as discussed systems. We assume that the pump is of great concern to laboratory scientists above, the volumes and the residence delivering liquid flow at the programmed using chromatographs and transferring time are so small that there is no significant rate, and that the percentage composi- methods; these scientists frequently com- contribution to mixing. There is, of course, tion accurately corresponds to the pro- ment that tubing must be shortened and substantial solvent blending in all systems, grammed value. We also expect that any modules placed closer together. In fact, and we will discuss that below in the con- gradient follows the intended profile and such changes are almost imperceptible. As text of the pumping system. that the specified composition reaches the a point of reference, a 3 m piece of 0.005 Dispersion in the fluid path downstream column at the intended time. All modern inch (~127 μm) tubing has a volume of from the injector has an impact on both chromatographic systems closely approach about 12.7 μL. So the transit time at 1.00 peak shape and resolution. This has been these assumptions, but there are always mL/min is only 0.01 min or 0.75 s. Even dou- considered in detail in other investigations deviations from the ideal. The differences bling the tubing diameter only increases the (3). The largest contribution to dispersion in the deviations between different brands time to 3 sec. Larger differences are typically during sample transport originates with the or models of pumps create complications allowed for flow rate or retention time vari- tubing diameter, with length as a smaller for method transfer. The differences can ability in typical system suitability specifica- contributor. It is, therefore, important during affect retention time and selectivity. It is tions. So, there is little reason to focus on method transfer to ensure that the tubing less well recognized that the differences tubing length as a factor in time offset. used for connecting the modules of the tar- can also affect column regeneration and The tubing in the system also contrib- get system are the smallest possible diam- reequilibration. Most scientists consider utes some mixing or dispersion, primar- eter and the shortest length. In following the system volume to be the major source ily because of laminar flow differences this guideline, however, it is not necessary of the deviations so method transfer strate- between the tubing wall and the center. to take extreme measures. Consider the gies are often based on equalizing the The magnitude of this effect is dependent expected volume of the peaks as they elute system volume differences. Silicon Wafer Technology Makes LC Analysis for Proteomics More powerful and Effective, A Q&A A User’s Experience

s the field of proteomics grows more complex, traditional separation techniques like nano-liquid chromatography (LC) have a harder time extracting critical information A ™ efficiently. PharmaFluidics’ μPAC , a breakthrough in column design, helps bring LC analysis up to speed. LCGC recently sat down with Geert Van Raemdonck, global field support expert at PharmaFluidics, to discuss this topic. After using the μPAC™ at the Geert Van Raemdonck University of Antwerp, Van Raemdonck was convinced about the benefits of micro-pillar Global Field Support Expert array column technology. His enthusiasm resulted in him joining the team of PharmaFluidics. PharmaFluidics LCGC discussed with Van Raemdonck the unique properties of the μPAC™ columns and got a user’s-eye view of what it’s like to work with them in proteomics applications. Van Raemdonck also shared his ideas about how future products could meet even more advanced analytical needs.

LCGC: What are the biggest differences between the μPAC™ columns and conventional nano-LC columns? Van Raemdonck: The biggest difference of the column is the way the backbone of the stationary phase is manufactured. μPAC™ incorporates a chip made of a silicon wafer, and freestanding pillars are etched out of the wafer, which results in a perfectly ordered structure. This leads to a high separation performance because there is almost no peak dispersion. Sharp peaks also lead to higher sensitivity, so that small amounts of molecules can be detected more easily. In addition, back-pressure is significantly lower as compared to conventional columns, which allows you to operate your column at a broader flow range, going from about 300 nL up to 1 μL per minute. And last, since the μPAC™ column is etched out of a silicon wafer, there is no batch-to- batch variation, as every column that is produced is exactly the same.

LCGC: For which applications are these μPAC™ columns best suited? Van Raemdonck: Special applications seen in the field of proteomics are analyses of samples with low amounts of peptides—for instance, the analysis of biopsies or protein extracts from a tissue. But, there’s also the opportunity for the analysis of complex samples that require longer gradients, as for biomarker discovery. The technology is also suitable for chemically labeled samples like iTRAQ or TMT, where the gradients applied for separation often last four hours and longer. Additional applications are in pharmaceutical fields, like in the detection of small subtle differences in biopharmaceuticals, and biosimilars, like for antibody production and quality AN EVALUATION OF USING μPAC™ COLUMNS FOR PROTEOMIC APPLICATIONS

control. In metabolomics, it might also be helpful to use Van Raemdonck: I used the μPACTM for the first time when I was μPAC™ columns because of the low concentrations of mol- working at the University of Antwerp. The installation was done ecules, and also in lipidomics experiments, where a high perfectly by the team of PharmaFluidics. One thing that you resolution is required. have to keep in mind is that you have to adapt your methods to the internal volume of the 200-cm column. This is about 9 μL, LCGC: Are there any drawbacks to these types of so it’s important that at the end of your gradient, you provide columns compared with classical columns? enough equilibration time to equilibrate your column before you Van Raemdonck: In my opinion, there are no specific draw- inject your next sample. If you desire, you can also increase your backs related to the μPAC™ columns. However, I think it’s flow rate (due to the lower back-pressure) in order to reduce important to keep a few things in mind. this equilibration time. First of all, the back-pressure of the μPAC™ should always stay below “Since the μPAC™ column LCGC: Why is grounding the 350 bar, or 5,000 PSI, to prevent column so important? any damage to the freestanding is etched out of a silicon Van Raemdonck: The column is pillars that form the separation made out of a single silicon wafer bed of the column. This maximum that’s semi-conductive. So, when the back-pressure is lower than for clas- wafer, there is no batch- column is not grounded and when a sical nano-LC columns, but it also high voltage is applied to the spray lowers the shear force of the LC to-batch variation, as every emitter that is used to transfer your instrumentation. molecules to your mass spectrometer, Secondly, it’s important to ground column that is produced is the current can actually reach the the column in case of applying a high column and this will influence the voltage to the spray emitter of the retention of the column. mass spectrometer. This prevents exactly the same.” So, this will result in some charging any charging effects since the effects that will retain your tryptic column is made of a semi-conductive silicon chip. peptides, which will result in significantly broader peaks. And Finally, the price of a μPAC™ column is higher than this discrepancy can be really big—you’ll see it immediately most commercially available columns. However, since the when a column is not grounded; you’ll be having very broad lifetime of a μPAC™ column outperforms any conventional peaks. Unfortunately, adequate grounding is often a step nano-LC column, the price per injection will be at least that’s forgotten. equal or even lower. LCGC: For which proteomics applications do you see LCGC: What product improvements would you like to the biggest potential for the μPACTM technology? see in the future from a user’s point of view? Van Raemdonck: On the one hand, there are the small Van Raemdonck: I think an introduction of trapping columns sample sizes that I already mentioned—like the pro- would be very useful since this would reduce the loading time tein or peptide extractions from tissues and biopsies. and also offer higher flexibility in the sample volumes that can Furthermore, there are also the top-down proteomics be injected into the column. applications with the C4 or C8 coating. But, there are also Further, it would be interesting to have the addition of opportunities for protein–protein interactions and host-cell some products to our current portfolio—like, for instance, protein identifications since there will be very tiny amounts those with bigger pore sizes in combination with other coat- of compounds detected. ings, like reverse-phase C8 or C4, which could be applied There is also a benefit to using the μPAC™ technology for in peptidomics and top-down applications. targeted applications like scheduled parallel reaction moni- And finally, if it would be possible to integrate the grounding toring, since the elution profiles can be set really narrow. mechanism, that would also be very handy. So, you can include much more of the compounds that you want to validate in a single run. And further, it could also be LCGC: Did you encounter any issues during the instal- very interesting to use the very stable retention times of the lation and first use of the column? μPAC™ columns for data-independent analysis. 828 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

System Volume The measurement of dwell volume does Mixers The system volume, also called dwell vol- not ensure perfect replication of the gradi- The differences among mixers can have ume, void volume, or delay volume, is the ent to be transferred. When the gradient three effects on chromatography. First, amount of liquid from the point where two, trace generated during the measurement with a smaller system volume, the leading or more, solvents meet and the point where is examined in detail, it is always observed edge of the gradient reaches the column the blended solvents reach the head of the that the offset time or volume is not con- and causes peaks that elute very early in column. Two systems with different dwell stant throughout the gradient profile. the gradient to be shifted to an earlier volumes, delivering the same gradient, will This variation is a consequence of sev- retention time. Second, because the entire give different retention times. It has long eral factors. Systems generally create the mixer volume must be flushed with the been recognized that characterizing the programmed percentages by blending final conditions of the gradient before that volume of a system is the key first step for volumes of each solvent. There is, how- strong solvent is delivered to the column, maintaining constant retention time in a ever, some inaccuracy associated with the the column may not be regenerated on method transfer. There are many published nonadditive effects associated with pairs one system as compared to the other. This and freely circulated procedures for mea- of solvents. In addition, all instruments can lead to a drift in retention times over a suring system dwell volume (4,5). The gen- make some adjustment for compressibil- series of injections. Third, on return to ini- eral approach is to prepare two batches of ity changes. The much larger source of tial, the entire mixer volume must again be the same solvent, one of which is spiked changes in the gradient profile is the mix- flushed before re-equilibration can begin. with a UV-absorbing marker, such as uracil, ing that is incorporated in all systems. Gra- Again, the two systems may differ in the acetone, propyl paraben, or caffeine. The dient systems must include a volume for equilibration to initial conditions for the column is replaced with a short length of mixing of the proportioned solvents. Many next injection. In this case, the two systems small inner diameter (i.d.) tubing. A gradi- different designs have been used for mix- may prove reproducible but different from ent is run from 100% A to 100% B over 20 ers, but they all must meet the two criteria one another. These three problems have min, followed by a return to 100% A over 5 of stable, reproducible retention times and different symptoms, and solutions, from a min. A hold, typically 5 min, is incorporated ripple-free baselines. The ripples reflect simple mismatch in measurement of dwell at 100% B, and at the return to 100% A. The solvent concentration inhomogeneities volume. Strategies for correcting system gradient is observed as the UV absorbance that may alter retention and will certainly volume differences will be discussed in trace at the wavelength appropriate for the complicate quantitative integration of the part III. selected marker. The volume of the system peaks. The alternative mixer designs all is best measured at the midpoint of the gra- are intended to smooth these ripples by Gradient Mixing dient. The difference in time between the making the solvent changes uniform with There is one additional difference among programmed midpoint and the observed time. To achieve this, the mixer must have chromatographic systems that must be midpoint is multiplied by the flow rate to a common volume that is large enough to considered. Pumps are usually classi- obtain the system dwell volume. Although span the period of the ripple. This com- fied as multi-pump gradient, high-pres- it is sometimes suggested that the volume mon volume, however, also distorts the sure mixing systems or as single-pump be measured with an instantaneous step programmed profile of the gradient. When gradient, low-pressure mixing systems. from A to B or, alternatively, at the first lift- a change in composition is initiated, that The high-pressure mixing systems cre- off of the gradient trace, the midpoint is change is intended to be uniformly dis- ate specific compositions by varying the much preferred as reflecting a steady state tributed throughout the mixer volume. But flow from two or more pumps to gen- transfer through the mixing volume of the that change, now much smaller because erate the desired solvent percentages. system, as discussed below. Although sys- it is diluted by the mixer volume, begins The low-pressure mixing approach uses tem volume values typically are published to emerge from the mixer immediately. In a solenoid valve where each port opens by instrument manufacturers, these val- other words, it reaches the head of the col- for a percentage of the valve cycle time ues are seldom ideal for method transfer umn in much less than the physical volume corresponding to the programmed com- experiments because the measurement of the system. Over the course of the gra- position. This series of solvent aliquots is procedures are not consistent from one dient, this phenomenon becomes less carried to the pump through a single tub- manufacturer to another. It is, therefore, significant as the series of small composi- ing piece. Some blending of the solvent necessary to measure the particular sys- tion increments settles into a steady state segments occurs in the transport tubing tems in use. For most purposes, the best change. This is the basis for recommend- and then in the pump heads as the mobile method is the one recommended by the ing that the system volume be measured phase is brought to the pressure and flow manufacturer of the system, but the overrid- at the gradient midpoint. Measurement for the method. Both system designs can ing consideration is that the same method at the start of the rise in the gradient work very well in terms of compositional be used to measure the volume of both monitoring trace gives a much smaller accuracy and precision. The system vol- the original and the target system. A typical volume than the physical volume, or the ume is usually somewhat smaller with a useful procedure is shown in Figure 1 (5). steady state condition. multi-pump gradient system, whereas the WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 829

single pump system can provide more to evaluate the shape of the gradient ence in retention time multiplied by the convenience as described in part I in the on various systems (6). This tool can be flow rate gives the difference in system context of solvent preparation. implemented from an open-source web volume between the two systems. This There is an additional characteristic asso- tool: http://www.measureyourgradient. trial can then be repeated after apply- ciated with low-pressure mixing systems org/index.php. For this approach, the ing the strategies discussed in part III that is not generally recognized. The fluid intent is to recreate the identical gradient to match the volumes between the two in the transfer tubing from the proportion- on the two systems in question. The gra- systems. After a few iterations of this ing valve enters the pump head as a block dient table entries will be different, but empirical adjustment, the chromatogram corresponding to the delivery volume of the the solvent composition delivered to the should be close to meeting specification. pump head. That series of solvent segments column will be identical. The “Measure If there are still discrepancies, apply a is mixed, more or less completely, into a sin- Your Gradient” tool uses a very exactly correction to the target system that mini- gle packet at the composition specified at defined mixture of test compounds along mizes all the differences in time. that time. That blended volume of solvent with a specified column, mobile phase, is then delivered into the flow path towards and method. This test is run on both sys- Conclusions the column inlet. Rather than the smoothly tems and the retention times are entered We have considered the contribution blended continuous gradient that we envi- into the software. The gradient is calcu- of system characteristics to transferring sion, the pump transmits a series of small lated to give a specific array of retention chromatographic methods. The possible steps that are somewhat smoothed at the times. Because this approach provides a differences between the originating and edges of the packets. This flow and com- multi-point calibration that addresses the the target systems can alter resolution position pattern is further complicated by a several physical factors discussed above by changing the characteristics of sol- mechanical characteristic of pumps. When that make gradient transfer imperfect, vent delivery and by altering dispersion the piston, or plunger, expels the liquid it provides a very good adjustment for of the separated peaks as they migrate from the head, it does not dispense all the method transfer. It is, however, limited through the system. Consideration must liquid, because there is space between the if the information is not available about also be given to the resistance to flow surface of the piston and the walls of the both systems in the method transfer. within the system tubing and the blend- pump head. This unswept liquid can be a This problem arises when implementing ing of solvents. In our final part of this substantial fraction of the total volume of methods that are even a few years old three-part series, we will consider the the pump head, on the order of 40% of where the systems may no longer exist. modules that comprise each system and its physical volume. That residual volume Even in the case where current instru- the ways that they can be aligned for mixes with the incoming mobile phase of ment systems were used to develop the consistent results. the next segment of solvent aliquots. The method, those instruments may not be result of this sequence of events is that the available to the laboratory responsible References mobile phase is diluted with the composi- for the transfer. This same obstacle can (1) T.E. Wheat, LCGC North Am. 36(9), tion of the previous pump cycle, which was interfere with the conventional measure- 693–696 (2018). also similarly diluted. This contributes ment strategies discussed above. When (2) General Chapter <621> “Chromatogra- phy” in United States Pharmacopeia 40 to the larger system volume associated the observed system volume is to be National Formulary 35 (USP 40-NF 35, with single-pump systems, and, more used to match the gradients delivered United States Pharmacopeial Convention, importantly, adds to the required time to by two systems, the same measurement Rockville, Maryland, 2017), pp. 508-520. reach the regeneration step in a gradient protocol must be used on both systems. (3) F. Gritti, T. McDonald, and M. Gilar, J. Chrom. A 1420, 54-65 (2015). method and also to complete the return That may simply not be possible. (4) J. W. Dolan, LCGC Europe, 19(6), 336-343 to initial conditions for re-equilibrating for An alternative to measuring the exact (2006). the next injection. system volume can be suggested for (5) P. Hong and P.R. McConville; Waters transfer of the separation method. The Corporation White Paper; 720005723EN; Tools for Instrument originator method (the method being ©2016 Waters Corporation (2016). Calibration in Method Transfer duplicated), should provide system suit- (6) M.H. Magee, J.C. Manulik, B.B. Barnes, D. Abate-Pella, J.T. Hewitt, P.G. Boswell; J. The physical factors that directly affect ability criteria specifying the retention Chrom. A 1369, 73-82 (2014). the shape of the gradient in a given time for each sample component that system have elicited interest in alterna- is to be measured. Use the established tives to the common and simple ways method on the new system. This initial Thomas E. Wheat to estimate and use the system volume. experiment may not meet the specifi- is a principal scientist with Chromato- One interesting alternative is the use of cations, but the major analyte peak will graphic Consulting, LLC in Hopedale, Massachusetts. Direct correspon- marker compounds to calibrate the sys- be recognizable. Compare the retention dence to: chromatographic.consult- tem. A more general tool, called “Mea- time of this peak on the new system with [email protected]. sure Your Gradient,” has been developed the time in the specifications. The differ- 830 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

Chromatography Fundamentals, Part V: Theoretical Plates: Significance, Properties, and Uses

This month’s “Chromatography Fundamentals” is a continuation of the development of the theoretical plate concept, with emphasis on its significance, properties, and uses as applied to liquid chromatography.

Howard G. Barth

lates are generated during the elu- varies during the distillation process. transfers are completed. The amount of Ption of solutes through a chromato- At each stage, equilibrium is reached solute that is equilibrated between the graphic column and contain a wealth between the vapor and liquid phases, two phases depends on the distribution of information about the separation and separation occurs between high- coefficient of the solute, K, and relative process, mainly peak dispersion. It is an and low-vapor-pressure components. volumes of the two phases (1,3). easily measured quantity used to probe The number of theoretical plates is Distribution profiles of solutes are column properties. proportional to column length, and constructed manually by plotting solute We had previously shown in part IV of depends upon column design. It concentration, c, against the number of this series (1) that the plate concept orig- should be emphasized that distillation transfers, r, or tube location. If we assume inated from fractional distillation and was is not a chromatographic process, but a Gaussian distribution (see Figure 1), the later used to interpret results obtained a countercurrent process of rising vapor number of theoretical plates of a CCD from countercurrent distribution (CCD) in contact with descending distillate run can be approximated using extractions. At about the same time that droplets. CCD was being developed, A. J. P. Mar- To compare distillation efficiencies N ∝ rK [2] tin (1910-2002) and R. L. M. Synge (1914- among columns of different lengths, col- 1996) formulated a theory of chromatog- umn length, L, is divided by plates, N, to with a standard deviation defined by raphy using the theoretical plate concept give the height equivalent of a theoretical σ = (2,3). These researchers went on to win the plate, HETP, or simply plate height H: √rpq [3] Nobel Prize in Chemistry in 1952 for the invention of partition chromatography. H = L/N [1] where p and q are the respective sol- In this article, we will focus on the ute fractions in the two immiscible development of theoretical plates and Although distillation is a complex pro- phases at equilibrium. (By convention, peak broadening, and arrive at relation- cess, each plate can be viewed as a vir- p is the solute fraction in the upper ships that are applicable to liquid chro- tual platform on which vapor-liquid equi- compartment or mobile phase and q matographic (LC) separations. The more librium occurs. is the solute fraction in the lower com- important relationships from distillation partment or stationary phase). We can and CCD will once again be reviewed for CCD Extractions now see that the chromatographic the benefit of the reader. A cleverly designed automated CCD theory, including peak broadening, is extraction apparatus, comprised of beginning to take shape. Background Information multiple two-compartment transfer Fractional Distillation tubes, was invented by Craig and Post Martin and Synge’s The concept of theoretical plates (2). Craig tubes consist of upper and Contributions to LC Theories was first introduced with respect to lower compartments, filled respectively Martin and Synge sought to improve fractional distillation, which required with two immiscible solvents (1,2). The upon CCD extractions with a paradigm an accounting of the separation first tube contains sample dissolved in shift by inventing partition chromatog- efficiency of different lengths and either the upper or lower immiscible raphy, a technique that uses a liquid designed distillation columns (1). It was liquid phase. After each extraction, stationary phase. Their experiments con- assumed that the distillation process the upper phases are simultaneously sisted of using LC columns packed with occurs in stages along the length of a transferred to adjacent tubes, contents an adsorbent coated with an immiscible distillation column, the location of which mixed, and the process repeated until all liquid phase. (In addition, paper chroma- WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 831

8. After equilibrium, solute is carried by the mobile phase to the next theoretical plate and the process repeated until components emerge from the column with characteristic retention times and peak widths, as described by a Gaussian distribution. Martin and Synge published two experimental methods for determining the num- Inﬂection Point ber of theoretical plates of an LC peak (4). 2

W h Method 1 1/2 A Gaussian distribution was used to describe solute concentration as a func- 0.607 h tion of retention time t, where tr is peak 0.5 h maximum, σ is the standard deviation,

Injection and x = t − tr:

c = (1/√ 2π)e −1/2 (x2/σ2) W = 4 [4]

t , V , or d r r Based on this equation, the following relationship was derived: FIGURE 1: Gaussian chromatographic peak indicating different methods of measuring theoretical plates. Refer to Table I and equations 6, 12 to 15. N = 2π (h2l2/A2) [5]

tography was also introduced by these Nobel laureates.) They made the following assumptions, SPEED UP YOUR PESTICIDE RESIDUE ANALYSIS which are universally valid for chromato- ™ ® T FAST EN ED T graphic techniques (4,5): WITH SILIA FaPEx CARTRIDGES A

P Y

G T O 1. Separation is uniform throughout a E L C H N O chromatographic column. 2. A column can be divided into equal Unique on the market lengths, stages, or segments. 3. Within each stage, there is sufficient Up to 120 X faster time for equilibrium to be reached, than existing methods as solutes partition between mobile and stationary phases. Simple 4. Solutes are sufficiently dilute so that their retention characteristics, i.e., Wide spectrum thermodynamic properties, are inde- Cost effective pendent of one another. 5. When applied to LC (or GC), each stage approximates one theoretical plate. 6. The number of theoretical plates generated by a solute can be calcu- Fast™ ® ■ www.silicycle.com/fapex lated by representing each peak as a Learn more about Silia FaPEx : Visit: Gaussian distribution. ■ Contact us: [email protected] 7. Each theoretical plate is considered to be a discrete site (a nano-size separa- www.silicycle.com tory funnel, if you wish), in which sol- CERTIFIÉE ISO 9001:2015 CERTIFIED utes distribute between two phases. 832 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

TABLE I: Summary of different representations of calculating theoretical plates from LC (t )2 (t )2 N = r = 16 r [12] data. Refer to Figure 1. (w /4)2 (w )2 t t Eqn Relationship Applicability

(t )2 which is identical to equation 6. This clas- 6 N = 16 r Commonly used plate equation with units of time. 2 sical equation not only allowed separa- (wt) tion efficiency to be compared among (V )2 columns, but also was key for optimiz- 12 N = 16 r Use when retention volume is required, e.g., SEC. 2 ing LC experimental parameters, as (wv) described below. (d )2 14 N = 16 r Use with planar or open-column chromatography. 2 Theoretical Plate Representations (wd) Retention and peak width in equation 2 6 can be adjusted to accommodate the (tr) Use for asymmetric, skewed, or partially 15 N = 5.54 2 resolved peaks; w is measured at 0.5 x ht. types of measurements being made. (w1/2) Thus these values can be expressed in 2 terms of volume, by multiplying each (tr ) Alternative to eqn 14: w is measured between 16 N = 4 2 peak infl ection points at 0.607 x peak height. term by flow rate, F: (w2σ) t r, retention time. w σ 2 t = 4 t, peak width in units of time. (V ) V r r, retention volume. N =16 [13] w σ (w )2 v =4 v, peak width in units of volume. v d r, distance of peak maximum. w σ d =4 d, peak width in units of length. w σ ½ = 2.35 t, peak width at one-half peak height in time units. w σ σ For researchers who still rely on strip- 2σ =2 t, peak width at 2 in time units. chart recorders, or for estimating plates from TLC or open columns (3), the follow- σ ∝ ∝ Here l is the distance from the point of = √rpq √ n √N [7b] ing equation can be used: injection to the peak maximum, h is peak height, and A is peak area. As previously shown (1), after r trans- (d )2 Equation 5 can now be converted to fers, the CCD tube that contains the max- r [14] N = 16 2 the iconic plate equation, where l is set imum concentration of solute, μ, is given (wd) equal to retention time, tr, and area and by height are transformed into peak width in where dr is either the peak maximum units of time (4–11): μ = rp [8] distance on the strip chart or the migra- tion distance of a solute band or TLC Since μ ∝ t and from equation 2 we know spot, and w is the baseline width. (t )2 r d N = 16 r [6] that r ∝ N, equation 8 can be recast as Equations 5, 6, 13, and 14 are similar, (w )2 t if not identical; however, plate calcula- ∝ tr N [9] tions from planar chromatography and Provided that the peak is a Gaussian open-column LC are estimates, since distribution, equations 5 and 6 are equiv- Taking the ratio of equations 9 and 7b, solute zones are usually not Gaussian. alent. we obtain (Before online computers, peak widths were calculated manually by drawing

Method 2 tr N tangents to the two slopes of a peak, as σ = [10] The second approach used was to √N seen in Figure 1.) compare LC to CCD extractions. Since Occasionally, baseline widths may be r is equal to one transfer in a CCD Squaring numerator and denominator, problematic to measure reliably because analysis, or by definition, r = n, equation of noise, drift, peak asymmetry, or the 3 can be written as presence of partially resolved peaks. A 2 (t ) 2 r N [11] common procedure to avoid these diffi- σ = ∝ √ n σ2 ==N √ rpq [7a] N culties is to measure peak widths higher up the peak, as illustrated in Figure 1. We know that n is a single plate, i.e., Letting time be the unit of measurement, Thus, at one-half peak height, equation σ n = N, thus equation 7a, becomes wt=4 t, we obtain the expected result, 15 is used: WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 833

2 σ2 TABLE II: Factors infl uencing theoretical plates, N=tr / (eqn 11). Relative Influence Terms Most Item LC Variables on Plate Numbers Affected

1. Mobile phase composition medium tr A breakthrough in Stationary phase 2. medium t composition r sample automation 3. Gradient profi le strong t , σ2 r and concentration σ2 4. Column temperature strong tr, 5. Injection volume medium/strong σ2 for GC–MS 6. Injection amount weak σ2 σ2 7. Flow rate weak/medium tr, 8. Column i.d. weak σ2 σ2 9. Column length strong tr, 10. Column confi guration weak/medium σ2 11. Particle size very strong σ2 12. Particle shape medium σ2

13. Pore size weak tr Particle size/ shape 14. medium σ2 uniformity

15. Packing purity weak/medium tr Dead volume of inter- connecting tubing: 16. injector-column very strong σ2 column-column column-detector

(t )2 established most notably with injection N = 5.54 r 2 volume, flow rate, column length, (w1/2) [15] particle size, and dead volume. Except We can move still higher up the peak for column length, which is given in the ® and measure width between inflection next section, these relationships will be Centri points, i.e., 2σ, as depicted in Figure 1, covered in subsequent tutorials. and equation 16: Multi-technique sample Column Length pre-concentration and injection Column length, L, is an integral part of platform, delivering enhanced (t )2 r analytical sensitivity and higher N = 4 [16] plate theory, since t ∝ L. Furthermore, (w )2 r 2σ the number of transfers, r, with respect throughput. to CCD, is analogous to column length. Since equations 6, 15, and 16 may Therefore, from equation 3, σ = √rpq, we ɵ SPME & SPME–trap give different results depending on peak obtain σ ∝ √L and equation 11 becomes: ɵ Headspace & HS–trap shape and overlapping peaks, measurements should be consistent for a given N = L2/σ2 ∝ L2/L∝ L [17] ɵ HiSorb sorptive extraction analysis. These equations are summa- ɵ Thermal desorption rized in Table I. Equation 17 indicates that sufficiently long columns can be used to generate Theoretical Plate Properties enough plates to pull apart neighboring Find out more Table II lists LC experimental peaks. However, there are three critical chem.markes.com/Centri parameters and their qualitative effects limitations: back pressure, analysis time, on the number of theoretical plates, and detectability. Column pressure and as shown in the last column. In spite analysis time are both directly propor- of the complexity of some of these tional to length, and an acceptable col- NEW relationships, correlations have been umn length can be readily predicted from 834 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

Resolution can be related to column TABLE III: Application of theoretical plates for selecting and maintaining high column performance. length by considering the behavior of peaks during elution. As two adjacent Application Procedure Comments peaks travel through a column, peak max- Column Choose column with Procedure for calculating required N is ima move apart, according to selection required N. described by resolution equation.b Column N should be within range of list- Compare N with listed value. Δx ∝ L [20] installation ed value, if not, reinstall column. Column Compare N vs. mL/min plots Select column with shallowest slope. comparisons among potential columns.a Peak dispersion or width, however, also increases, but not as fast. The rela- Column Change column when plate loss reaches Plot N vs. wks of usage tionship between peak width and column stability 10-20% or if Rs becomes unacceptably low. a. A more meaningful plot is H vs. linear velocity, see Chromatography Fundamentals, Part VIII. length becomes apparent by rearranging b. Chromatography Fundamentals, Part VIII of this series. equation 18:

σ = (LH)½ [21] TABLE IV: Using theoretical plates to optimize experimental LC conditions. Experimental Since H is constant for a given column, Data Analysis Optimum Value Variables Plot N vs. μL: plates will be- Select injection volume within σ ∝ ½ Injection L [22] gin to decrease with increas- region giving maximum plates, Volume ing injection volume. maintaining adequate s/n. the resolution equation now can be Plot N vs. mg/mL: plates will Select concentration within Sample Con- begin to decrease with increas- region giving maximum plates, expressed as centration ing injection concentration. maintaining adequate s/n. σ ∝ ½ ∝ ½ Select fl ow rate to achieve Rs=∆x/4 L/L L [23] Plot N vs. mL/min: plates will slowly Flow Rate maximum N maintaining decrease with increasing fl ow rate. adequate analysis time. Based on this relationship, resolution Select temperature at max Column Plot N vs. °C: plates will in- will increase by only 40%, each time col- allowable value without sac- Temperature crease with temperature. umn length is doubled. Furthermore, if rifi cing safety or resolution. we wanted to double resolution, column length would have to be increased by a factor of four. The resolution equation will a single measurement using any conve- Equation 1 can also be expressed dif- be examined in more detail in a subse- nient column length. ferently by letting N = L2/σ2: quent article of this series. As column length increases, peaks broaden with a corresponding decreased H = L/N = Lσ2/L2 = σ2/L [18] Theoretical Plates: Range detector response, defined by equation and Limitations 4. Decreased detector response, however, Plate height now becomes peak vari- It is instructive to estimate the minimum will also affect signal-to-noise ratio, limiting ance per unit column length, a rela- plate count of low-resolution chromato- detection of trace or minor components. tionship used for studying column and graphic methods, like paper, thin-layer, or packing properties that govern peak open-column chromatography. We can Plate Height broadening, as discussed in part VI. assume a worst-case scenario in which An essential relationship derived a solute tails from the point of applica- from theoretical plates is plate height or Chromatographic Resolution tion to its final resting place. If we let x height equivalent of a theoretical plate, Another critical relationship is the reso- be the distance from sample application

HETP (see equation 1). Martin and Synge lution, Rs, between adjacent peaks, to the middle of a sample spot (splotch) referred to it as plate “thickness”, in or tailed band, then σ ≈ ½x. The approxi- σ 6 σ 2 σ2 which the “thicker” or wider the plate, the Rs = (x2-x1)/4 = x/4 [19} mate plate count is N = x / ≈ 4. Indeed, lower the separation efficiency. They pro- the typical plate count of open columns posed that plate thickness corresponded where ∆x is the distance between two seldom exceed 102. Even with these to the depth of the stationary phase and peak maxima, and σ is the average stan- extraordinarily low plate counts, by mod- reasoned correctly that column efficiency dard deviation of two adjacent peaks, ern standards, these methods were highly could be improved by decreasing this which we assume are Gaussian. A value praised for their separation abilities. layer or increasing the rate of transfer of of ≥ 1.5 is required for baseline resolution Modern, high efficiency columns, solute molecules during partitioning. of two peaks of equal areas. by comparison, can typically range WWW.CHROMATOGRAPHYONLINE.COM NOVEMBER 2018 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 835

between 104 to less than 105 plates per such as its thickness and homogeneity, When column length is normalized column, depending, of course, on col- and the diffusion coefficients of solutes with respect to plate number, we obtain umn length. In theory, there is no upper within the stationary phase. In this a new parameter, the plate height, which plate limit, since we can always con- section, only unretained solutes are is a measure of the amount of peak struct longer columns. considered for column evaluation, since broadening that occurs when solute trav- Suppliers sometimes advertise col- they will produce higher plate counts els through a defined length of column. umns based of plates per meter, which than retained components, thus are Solute emerges as a Gaussian distribu- can be misleading since actual column more sensitive probes. tion carrying information regarding sol- lengths are significantly shorter. In order Most LC peaks have profiles that devi- ute and column characteristics, as well as to compare columns, it is best to choose ate from Gaussian distributions and are experimental conditions. columns based on actual plates per col- not strictly symmetrical; consequently, Plate number and height are used umn length; for column comparisons, there will be errors associated with base- to develop and optimize chromato- plate height is preferred, which is inde- line width measurements. In view of this graphic methods, details of which will pendent of column length. imprecision, only two significant figures be presented in subsequent articles Recall that LC plate theory was devel- are justified for reporting plate counts. of this series on the fundamentals of oped by assuming Gaussian peak distri- A summary of theoretical plate chromatography. butions: for non-Gaussian peaks, such as applications is outlined below and in Our next tutorial will review the prop- skewed or tailed shapes, the plate con- Tables III and IV. erties of the Gaussian distribution with cept is no longer applicable, and plate emphasis on peak variance and its effect counts are usually underestimated, unless Getting Started with Plates on chromatographic resolution. peak width is measured at 50% or 60.7% Based on preliminary analytical runs, of peak height (equations 15 and 16). the required number of plates to References Furthermore, plate theory is also based effect a separation are estimated; this (1) H.G. Barth, LCGC North Am. 36(8), 532–535 (2018). on the assumption that chromatographic procedure will be described in part VII. (2) E.W. Berg, Physical and Chemical conditions remain constant throughout With this value, an appropriate column Methods of Separation (McGraw-Hill, NY, 1963). the analysis and that equilibrium has of required plates can be selected. (3) H.G. Barth, LCGC North Am. 36(3), 200- been reached. After a new column is installed, the 203 (2018). When gradient elution or temperature plate number is checked against the sup- (4) R J. Magee, Selected Readings in Chromatography (Pergamon Press, programming is applied, equilibrium plier’s value to ensure that the column Oxford, UK, 1970). may not be attained. In addition, reten- was correctly plumbed into the LC sys- (5) A.J.P. Martin and R.L.M. Synge, Biochem. J., 35, 1359 (1941). tion times are dictated by the gradient tem. (6) R.L. Grob, Chpt. 2, In: R.L. Grob, profile. As a result, strongly retained Experimental parameters, such as Ed., Modern Practice of Gas peaks can remain stationary during injection volume, solute concentration, Chromatography, 2nd ed. (Wiley- Interscience, New York, 1985), pp. part of the gradient and elute at con- flow rate, and column temperature, are 49–114. siderably long times with compressed then optimized for maximum plate count. (7) S.G. Weber and P.W. Carr, Chpt. 1, In: P.R. Brown and R.A. Hartwick, Eds., High peak widths, producing artificially high Column performance typically will dete- Performance Liquid Chromatography plate counts. Under these conditions, riorate with time, as a result of buildup of (Wiley-Interscience, New York, 1989), pp. 1–115. the physical significance of theoretical particulates or contaminants, disruption (8) J.C. Giddings, Unified Separation plates is lost. of packing uniformity, dissolution of silica Theory (Wiley-Interscience, New York, packing, loss of bonded phase, or surface 1991). (9) B.L. Karger, L.R. Snyder, and C. Horvath, Theoretical Plates: Applications oxidation. Column fidelity is ascertained An Introduction to Separation Science Theoretical plate measurements of by monitoring separation attributes, such (Wiley-Interscience, New York, 1973). (10) P.A. Bristow, Liquid Chromatography in unretained solutes are typically used as plate count, solute retention time, and Practice (Handforth, Cheshire, UK, 1976). in the development and maintenance resolution on a routine basis. (11) L.R. Snyder, J.J. Kirkland, and J.W. of LC methods, as described below Dolan, Introduction to Modern Liquid Chromatography (Wiley, New York, 3rd and summarized in Tables III and Conclusions ed., 2010). IV. Unretained solutes reflect the The number of theoretical plates, which uniformity of the packed bed, forms the basis of chromatographic packing particle size, mobile phase theory, is a key parameter used in hydrodynamics, solute diffusion all modes of chromatography for Howard G. Barth coefficient in the mobile phase, and measuring column efficiency. It is is with Analytical Chemistry Consul- tants, Ltd. in Wilmington, Delaware. extracolumn effects. In addition to simply the ratio of retention squared Direct correspondence to: these effects, retained solutes also to peak variance, a relationship first [email protected] probe stationary phase characteristics, realized by Martin and Synge. 836 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM PRODUCTS & RESOURCES Thin film solid-phase microextraction Reference materials Gerstel’s thin-film solid-phase microex- LGC Standards’ reference materials traction device is a 20 x 4 mm carbon are designed for food, beverage, and mesh sheet impregnated with sorptive environmental analysis. 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Parker Balston, Inc...... 815 Wyatt Technology Corporation ...... 795 838 LCGC NORTH AMERICA VOLUME 36 NUMBER 11 NOVEMBER 2018 WWW.CHROMATOGRAPHYONLINE.COM

Excerpts from LCGC’s professional THE ESSENTIALS development platform, CHROMacademy.com HPLC Column Maintenance: Tips for Extending HPLC Column Lifetime

hile the relative cost of HPLC opposite direction, leading to the same size; 0.45 μm for traditional columns and Wcolumns has been reduced chromatographic symptoms. 0.2 μm for UHPLC columns is typical. over time, extending column lifetime If the sample matrix or diluent is likely to remains an important consideration If columns have dried out, initiate harm the sorbent (due to pH, for exam- for most laboratories. The following the flow very slowly (0.1 mL/min/min) ple) or is particularly chemically dirty or tips should help to protect your col- using an eluent containing at least intractable, a guard column may be used, umns and significantly extend the use- 50% acetonitrile. and the phase should be matched with ful lifetime of most phases. If a “standard” (non “aq” or non-polar that of the analytical column. Take great embedded phase type) reversed-phase col- care when selecting the dimensions of the Always read the manufacturer’s litera- umn is suspected of phase collapse (short- guard column and connecting to the ana- ture with respect to the recommended ening retention times, poor efficiency) due lytical column to ensure that the efficiency pressure, eluent pH and temperature to use with 100% aqueous mobile phases, of the separation is not compromised. operating ranges for the column, and the column should be reactivated at high stick to these ranges. flow with 100% acetonitrile at 60 oC (take If column or frit contamination is sus- Note that higher operating temperatures care to not precipitate any solid buffers from pected due to peak splitting or loss of often go hand in hand with reduced pH the eluent remaining within the column). In efficiency, it is possible to reverse the operating ranges. At low pH, the main both of these cases, between 50 and 100 direction of the column for back flush- symptom of column degradation is typ- column volumes may be required to prop- ing purposes, and the column washing ically loss of efficiency (peak broaden- erly re-equilibrate or re-activate the phase. procedure mentioned above is a good ing) and at high pH, peak tailing and an “recipe” for this purpose. increase in column back pressure. Columns should be properly washed One should uncouple the column from after each use. the detector to avoid fouling, and note Avoid mechanical shock of the column A recommended washing routine may be: that reversal for flushing should only be bed, such as dropping the column, and • Current content to 90% acetonitrile at used as a matter of last resort, and that ramp the pressure or flow slowly (1 mL/ 10% organic per 2 column volumes and the original efficiency of the column may min/min is ideal) each time eluent flow hold for 10 column volumes (again take not be achieved. is initiated. care to avoid precipitation of solid buffers Most modern HPLC equipment is capa- by ramping the acetonitrile concentra- Remember that column volume may ble of achieving this flow or pressure tion slowly, as recommended here). be estimated using π x r2 x L x 0.6 ramp automatically through secondary • 10% acetonitrile per 2 column volumes to (the approximate interstitial poros- instrument settings. Bed voiding due 50:50 acetonitrile:water and hold for 10 ity of silica used for HPLC column to pressure shock often manifests itself column volumes. packing materials). via split or very badly tailing peaks. Col- • Remove from the system, end cap and So, for a 150 x 4.6 mm column, this would umns may be reversed for analysis if a store. End capping of the column is very approximate to: replacement is not readily available; important to avoid the phase drying out. however, the efficiency of the column One might use an older HPLC system as 3.142 x (2.3)2 x 150 x 0.6 = 1.496 μL or ~1.5 is likely to reduce much more quickly a column “wash station,” which can save mL as the bed will ultimately slump in the significant amounts of operating time on “live” instruments. These tips will help extend your column life- MORE ONLINE: times. FIND THIS, AND OTHER WEBCASTS, AT If samples are likely to contain partic- www.CHROMacademy.com/Essentials ulate matter, choose a good quality (free until December 20). inline filter with the appropriate mesh FREE CHROMACADEMY MEMBERSHIP

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