Gas Chromatography–Mass Spectrometry of Organochlorine Pesticides in Environmental Waters Claire A

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www.shimadzu.eu/coupling-powers CONTENTS

August | 2019 Volume 32 Number 8

COVER STORY 396 Development of Fully Automated Bubble-in-Drop Microextraction–Gas Chromatography–Mass Spectrometry of Organochlorine Pesticides in Environmental Waters Claire A. Taylor, Hui Min Lee, and Hian Kee Lee This article describes the intentional incorporation of an air bubble into a solvent droplet for fully automated bubble-in-drop microextraction–gas chromatography–mass spectrometric (BID-GC–MS) analysis of selected organochlorine pesticides as model analytes.

FEATURES (HPLC) modules (pump, autosampler, UV detector, and chromatography data system) to be published 420 Sizing Up Size-Exclusion Chromatography in 2019. This instalment provides an overview Alasdair Matheson for analytical-scale HPLC pumps, including their André Striegel spoke to LCGC Europe about requirements, modern designs, operating principles, innovations in size-exclusion chromatography (SEC) in trends, and best practices for trouble-free operation. polymer analysis, including the benefits of hyphenating Recommendations for selection and guidelines for SEC with on-line multi-angle static light scattering operating and troubleshooting are provided. (MALS) and differential refractometry (DRI) detection, and the role of quintuple detection. DEPARTMENTS 424 Company Profiles COLUMNS A selection of profiles on leading separation science 405 LC TROUBLESHOOTING companies. Pass the Salt: Evolution of Coupling Ion-Exchange 454 Products Separations and Mass Spectrometry Dwight R. Stoll 456 Events At the recent HPLC meeting in Milan, Dwight Image credit: Denis Gladkiy/stock.adobe.com Stoll noted several topics of interest to the “LC Troubleshooting” readers. Here, he focuses on the first: MS-friendly ion-exchange separations. 410 PERSPECTIVES IN MODERN HPLC Modern HPLC Pumps: Perspectives, Principles, and Practices Konstantin Shoykhet, Ken Broeckhoven, and Michael W. Dong This instalment is the first of a series of four white papers on high performance liquid chromatography

394 LCGC Europe August 2019 EDITORIAL ADVISORY BOARD

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Please note that Wright’s Media is the only authorized company that we’ve partnered with for Multimedia Healthcare materials. © 2019 Multimedia Healthcare LLC (UK) all rights reserved. No part of the publication may be reproduced in any material form (including photocopying or storing it in any medium by electronic means and whether or not transiently or incidentally to some other use of this publication) without the written permission of the copyright owner except The Publishers of LCGC Europe would like to thank the members of the Editorial Advisory in accordance with the provisions of the Copyright Designs & Patents Act (UK) Board for their continuing support and expert advice. The high standards and editorial 1988 or under the terms of the license issued by the Copyright License Agency’s quality associated with LCGC Europe are maintained largely through the tireless efforts 90 Tottenham Court Road, London W1P 0LP, UK. Applications for the copyright of these individuals. LCGC Europe provides troubleshooting information and application owner’s permission to reproduce any part of this publication outside of the solutions on all aspects of separation science so that laboratory-based analytical chemists Copyright Designs & Patents Act (UK) 1988 provisions, should be forwarded in can enhance their practical knowledge to gain competitive advantage. Our scientific quality writing to Permission Dept. fax +1 732-647-1104 or email: jfrommer@mmhgroup. 10% Post and commercial objectivity provide readers with the tools necessary to deal with real-world com. Warning: the doing of an unauthorized act in relation to a copyright work Consumer analysis issues, thereby increasing their efficiency, productivity and value to their employer. may result in both a civil claim for damages and criminal prosecution Waste www.chromatographyonline.com 395 LEE ET AL.

PEER REVIEW Development of Fully Automated Bubble-in-Drop Microextraction– Gas Chromatography– Mass Spectrometry of Organochlorine Pesticides in Environmental Waters

Claire A. Taylor, Hui Min Lee, and Hian Kee Lee, Department of Chemistry, National University of Singapore, Singapore

In this study, the intentional incorporation of an air bubble into a solvent droplet is proposed for fully automated bubble-in-drop microextraction–gas chromatography–mass spectrometric (BID-GC–MS) analysis of selected organochlorine pesticides as model analytes. By using a commercial autosampler coupled to a GC–MS system, all experiments could be performed sequentially and automatically, without any human intervention. By applying orthogonal array design (OAD), optimization of the extraction procedure was performed with a minimum number of experiments. The automated procedures presented the advantage of high precision (5.8–7.7%) and good figures of merit. Using the optimized BID parameters, enrichment factors were determined to be between 23 and 326 for α–hexachlorocyclohexane, dichlorodiphenyldichloroethane, and dieldrin. Limits of detection ranging from 1.74 μg/L to 2.51 μg/L were obtained. Fully automated BID-GC–MS was successfully applied to the analysis of tap water and reservoir water samples.

Over the years, interest in developing fast, convenient, and sensitive analytical methods has been on the rise. Much of the effort has been on the development of sample preparation procedures, a critical stage in an analytical method (1). Such a step is required for extraction and, of course, analyte enrichment. Another reason for sample preparation is clean-up (or matrix minimization, if not matrix elimination). One of the oldest preconcentration and matrix isolation techniques in analytical chemistry is liquid–liquid extraction (LLE). Conventional KEY POINTS LLE uses large amounts (several hundred millilitres) of extractant solvents, • A fully automated BID-GC– which are expensive, and often hazardous. The procedure is time-consuming MS method has been and tedious. Moreover, appreciable amounts of waste solvents are generated. In developed and evaluated. short, LLE is not a very environmentally friendly procedure. In attempts to lessen • This is the first application of OAD- the ecological impact of LLE, there have been many studies emphasizing optimized fully automated BID. the miniaturization of LLE, specifically to reduce solvent use. Liquid–liquid • The method was demonstrated microextraction (LLME) is the miniaturized alternative to LLE and was introduced to be viable for quantitative by Murray in 1979 (2); the procedure was conducted in a flask with a capillary determination of OCPs in real tube and a side arm. Subsequently, Jeannot and Cantwell reported solvent environmental water samples. microextraction in which a low microlitre volume amount of solvent (compared to several hundred microlitres in LLME) was used for extraction (3); this eventually

396 LCGC Europe August 2019 [email protected] | + 41 41 925-6200

PERFORMANCE EQUAL TO OR BETTER THAN GC DETECTORS WITH CONVENTIONAL RADIOACTIVE SOURCES.

The VICITM pulsed discharge detectors (PDD) utilize a stable, low-powered, pulsed DC discharge in helium as the ionization source.

Two Modes:

• Electron capture mode as a selective detector monitoring high electron CHƂPKV[EQORQWPFUUWEJCUHTGQPUEJNQTKPCVGFRGUVKEKFGUCPFQVJGT halogen compounds.

• Helium photoionization mode, as a universal, non-destructive, high UGPUKVKXKV[FGVGEVQT

6JG8+%+/QFGN&2&&KUFGUKIPGFHQTRNWICPFRNC[KPUVCNNCVKQPQPVJG popular Agilent 6890 and 7890, and is optimized for trace level work in the JGNKWORJQVQKQPK\CVKQPOQFG6JG&WVKNK\GUVJGGNGEVTQPKEUCPFRQYGTUWRRN[ of the host GC.

• Universal detector • Large linear range • Mass sensitive • 'CUKN[TGVTQƂVUQPVQGZKUVKPIKPUVTWOGPVCVKQP • 5VCDNG PQPƂPKEM[PQVUGPUKVKXGVQGZVGTPCNEJCPIGU NQYOCKPVGPCPEG LEE ET AL.

1 × 12 TABLE 1: Assignment of factors and level settings for experimental runs in the orthogonal array (OA)16 (4 2 ) matrix (water samples at a concentration of 0.05 μg/mL were considered)

Column No. Extraction 12345678910111213 Solvent

AB(AXB)1 (AXB) 2 (AXB) 3 C(AXC)1 (AXC) 2 (AXC) 3 BXC D E # 1 Toluene 0.2 μL 1.0 μL 10 min 0% # 2 Cyclohexane 0.8 μL 1.5 μL 20 min 10% # 3Isooctane # 4Dichloromethane # A = Extracting solvent; B = bubble size; C = extraction solvent volume; D = extraction time; E = salt concentration (m/v) (ionic strength); # = dummy factors; AXB = interaction between extracting solvent and bubble size; AXC = interaction between extracting solvent and extracting solvent volume; BXC = interaction between bubble size and extracting solvent volume became known as single-drop microextraction (SDME). In (HCH), dichlorodiphenyldichloroethane (DDD), and dieldrin 1997, He and Lee reported a concept to perform LLME in (DID) as model analytes in water samples, was developed a simple apparatus or device (in this case, the commonly and evaluated. Because optimization of microextraction used microsyringe), which served as a microseparatory procedures, including LPME, involves the consideration funnel for extraction, before the extract was injected, using of several parameters, as well as the interactions amongst the same device, into a gas chromatograph for analysis them, orthogonal array design (OAD) (9–11) was applied (4). The technique was termed liquid-phase microextraction to streamline the process, and to reduce the number (LPME), which is now a generic term to represent all of experiments needed to arrive at the set of the most solvent-based microextraction procedures. This LPME favourable extraction conditions (see, for example, a approach successfully paved the way for its full automation, recent OAD application for automated dispersive liquid– with the use of a commercial autosampler and software (5). liquid microextraction [12]). The present preliminary study SDME is useful in trace analysis, but the method has constitutes the first application of OAD-optimized fully notable shortfalls. Droplet instability is always a potential automated BID, seamlessly integrated with GC–MS analysis. problem. Additionally, volatilization of the organic solvent, especially at relatively high ambient temperatures, may Experimental cause the formation of bubbles in the droplet, which is Materials and Reagents: HCH, DDD, and DID, dissolved undesirable. However, Williams and associates purposely in methanol, at concentrations of 10 μg/mL, were incorporated an air bubble within the droplet in SDME, purchased from SPEX. All the extraction solvents used with interesting results (6). The enrichment factors of the were of HPLC-grade. Cyclohexane was purchased from experiment with a bubble in the droplet were reportedly Emsure. Isooctane and toluene were obtained from higher than those of the same experiment with no bubble. Tedia. Dichloromethane was purchased from MSR (P.P. This was due to the higher surface area of the solvent in Chemicals). Analytical-grade acetone and HPLC-grade contact with the sample, in comparison to the solvent drop methanol were also obtained from MSR. Sodium chloride without a bubble, resulting in greater extraction efficiency. was obtained from GCE. Nanopure water was produced Although SDME and its bubble-in-drop variant are simple using an Elga Purelab Option-Q water purification system. to use and operate, it would be beneficial if the entire Aqueous working solutions were prepared daily by procedure could be automated, as has been reported (7,8). spiking of the stock solution into ultrapure water. This has the great implication that human manipulation, Automated BID Procedure: All the following steps, including and therefore error, are kept to an absolute minimum when rinsing of syringe, introduction of extraction solvent into the dealing with such small (μL) volumes of extraction solvent. microsyringe, incorporation of an air bubble, and withdrawal In the present work, a fully automated bubble-in-drop (BID) of the final extract for injection into the GC injector, were microextraction approach coupled to gas chromatography– performed automatically on a CTC Analytics CombiPAL mass spectrometry (GC–MS) for the analysis of three autosampler and its Cycle Composer software. Instructions organochlorine pesticides (OCPs), α–hexachlorocyclohexane to handle the automated operations were written using the

398 LCGC Europe August 2019 LEE ET AL.

1 × 12 TABLE 2: Orthogonal array (OA)16 (4 2 ) matrix with experimental results

Trial Factors and Interactions Response No. AB(AXB)1 (AXB)2 (AXB)3 C (AXC)1 (AXC)2 (AXC)3 BXC D E # HCH DID DDD Sum 1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 10 9 48 2 1 1 1 1 1 2 2 2 2 2 2 2 2 41 10 8 60 3 1 2 2 2 2 1 1 1 1 2 2 2 2 39 12 9 60 412 2 2 2 2 2 2 2 111144 18 17 78 5 2 1 1 2 2 1 1 2 2 1 1 2 2 22 8 7 37 621 1 2 2 2 2 1 1 221141 16 15 72 7 2 2 2 1 1 1 1 2 2 2 2 1 1 27 11 11 49 822 2 1 1 2 2 1 1 112239 15 13 68 9 3 1 2 1 1 1 2 1 2 1 2 1 2 29 11 11 52 10 3 1 2 1 1 2 1 2 1 2 1 2 1 32 11 10 54 11 3 2 1 2 2 1 2 1 2 2 1 2 1 15 5 5 25 12 3 2 1 2 2 2 1 2 1 1 2 1 2 27 9 9 45 13 4 1 2 2 2 1 2 2 1 1 2 2 1 2 1 2 5 14 4 1 2 2 2 2 1 1 2 2 1 1 2 5 2 2 8 15 4 2 1 1 1 1 2 2 1 2 1 1 2 3 1 2 6 16 4 2 1 1 1 2 1 1 2 1 2 2 1 3 2 2 7 r1 61 42 37 43 43 35 38 42 45 42 40 45 42 r2 56 42 47 41 41 49 46 42 40 42 44 39 42 r3 44 r4 7 d540 102 2 148 0 5 0 460 Refer to Table 1 footnotes and to text explanation related to Table 2 macro-editor of the software. To perform BID, the software GC–MS Analysis: Analysis was carried out using a was programmed so that 2 μL of the organic solvent was Shimadzu QP2010 GC–MS system and a DB-5MS fused withdrawn into a 10-μL microsyringe, followed by withdrawal silica capillary column (30 m × 0.25-mm, 0.25-μm) from of 1 μL of air into the solvent droplet. The needle of the J&W Scientific. High purity (99.999%) helium was used as microsyringe was then set to penetrate 29 mm into a 2-mL the carrier gas at a flow rate of 1.67 mL/min. The injector GC vial with a polytetrafluoroethyelene septum containing temperature was kept at 280 °C, and operated in the an aqueous solution (1.0 mL) of the OCPs. A penetration splitless mode. The MS system was operated in the electron depth of 29 mm ensured that the needle tip was below impact ionization mode, and the interface temperature the surface of the aqueous solution. The solvent (and was set at 280 °C. The GC temperature program was as bubble) were slowly exposed to the aqueous solution to follows: initial temperature 80 °C, held for 1 min; increased form a droplet suspended at the tip of the needle, until all by 20 °C/min to 240 ºC, held for 4 min. A mass range of the solvent had been pushed out from the microsyringe m/z 170–250 was scanned to obtain the fragmentation barrel. After a 35-min extraction time, the solvent droplet spectra of the analytes, and selective ion monitoring (SIM) was retracted back into the microsyringe barrel (together mode was performed for quantification. Detection was with the air bubble, which provided the added benefit of achieved in SIM mode, with a solvent delay time of 8 min. a clearly visible boundary between the organic solvent OAD Optimization and Investigation of Interactions and the aqueous solution), and then injected into the GC Between Variables (6–10): Five main parameters, including injector port. The microsyringe was rinsed with acetone the effect of salt addition (salting-out effect), type of by repeatedly drawing and dispensing it (3×) into a waste extraction solvent, volume of air bubble, volume of extraction reservoir, with a final rinsing (2×) with the extraction solvent solvent, and length of extraction time, were considered for to prevent carryover, before the next BID experiment. OAD. Because the type of extraction solvent is a qualitative www.chromatographyonline.com 399 LEE ET AL.

1 × 12 TABLE 3: Analysis of variance (ANOVA) table for experimental responses in the orthogonal array (OA)16 (4 2 ) matrix SS d.f. MS F SS' PC(%) Extracting solvent (A) 7366.63 3 2455.54 5777.18 7365.36 82.05 Bubble size (B) 0.39 1 0.39 0.91 0.00 Extraction 1765.47 749.97 8.35 750.40 1 750.40 solvent volume (C) Extraction time (D) 39.88 1 39.88 93.83 39.46 0.44 Salt concentration (E) 113.03 1 113.03 265.92 112.60 1.25 (AXB) 386.94 3 128.98 303.45 385.67 4.30 (AXC) 316.19 3 105.40 247.97 314.92 3.51 (BXC) 2.92 1 2.92 6.86 2.49 0.03 Pooled error (#) 0.4250 1 0.43 6.34 0.07 Total 8976.80 15.00 8549.19 100.00 SS = sum of squares; d.f. = degree of freedom; MS = mean of squares; F = critical values; SS’ = purified sum of squares; PC(%) = percentage contribution

TABLE 4: Assignment of factors and level settings for optimization of bubble-in-drop (BID) microextraction experiments by orthogonal 13 array (OA)27 (3 ) matrix; water samples at a concentration of 0.05 μg/mL were considered Column No. Level 1 2345678910111213

AB(AXB)1 (AXB)2 C(AXC)1 (AXC)2 D ##E ## 1 Toluene 0.8 μL 1.5 μL 25 min 0% 2 Cyclohexane 1.0 μL 2.5 μL 30 min 3% 3 Isooctane 1.2 μL 3.5 μL 35 min 6% A = extract; B = bubble size; C = extracting solvent volume; D = extraction time; E = salt concentration (m/v); # = dummy factors; AXB = interaction between extracting solvent and bubble size; AXC = interaction between extracting sovent and extracting solvent volume; BXC = interaction between bubble size and extracting solvent volume factor, a four-level setting was considered. Additionally, The responses (enrichment factors) from the array were the four remaining factors were quantitative factors, hence calculated, and are tabulated in Table 2. Data analysis two-level settings were considered. Therefore, a mixed was carried out according to the equations and methods 1 × 12 level OAD with an OA16 (4 2 ) matrix was chosen for the given for data analysis strategy employing analysis of optimization process because it allowed the effect of the five variance (ANOVA) and percentage contribution techniques main parameters and possible interactions to be considered (10). The average response of each factor was calculated simultaneously, with the least number of experiments. (rx) for each of the chosen levels. ANOVA results are 1 × 12 By choosing a mixed-level OA16 (4 2 ) matrix, the shown in Table 3. The results showed that the choice of main variables together with their interactions can be organic acceptor solvent (A) was the most significant simultaneously examined by 16 experimental trials. The variable followed by the extraction solvent time (C). For main variables included the choice of organic extraction the first time, it was shown that the interaction between solvent, bubble size, extraction solvent volume, extraction the type of extracting solvent and bubble size (A × B) time, and salt concentration of the sample solution. Three and that between the type of extracting solvent and the selected interactions were also investigated. Being extraction solvent volume (A × C) were also significant. a qualitative variable, the choice of organic acceptor The effects of the bubble size (B), extraction time (D), solvent was considered at a four-level setting, whereas salt concentration (E), and the interaction between bubble the rest of the quantitative variables were set at two-level size and extraction solvent volume (B × C) were negligible. settings, as shown in Table 1. As one column was However, the main variable bubble size (factor B) had to be assigned as a dummy factor, no repetition for the same considered at a higher level array because the interaction experimental trial was necessary because the error (A × B) was shown to be important. The percentage variance could be calculated from the dummy factor (10). contribution coming from the error (uncontrollable or unknown

400 LCGC Europe August 2019 LEE ET AL.

13 TABLE 5: Orthogonal array (OA)27 (3 ) matrix with experimental results

Trial Column No. Response No. 12345678910111213HCHDEDDDDSum 11111111111111114 27 21 163 21111222222222152 28 22 202 31111333333333163 25 21 208 41222111222333106 30 28 163 51222222333111155 22 30 207 61222333111222107 23 20 150 71333111333222114 23 19 157 81333222111333102 26 23 151 91333333222111110 22 26 158 10212312312312351 13 13 77 11212323123123154 18 17 90 12212331231231243 14 14 70 13223112323131244 16 15 74 14223123131212353 28 39 120 15223131212323152 16 18 86 16231212331223148 12 10 70 17231223112331243 14 12 69 18231231223112354 10 14 79 19313213213213257 12 12 82 20313221321321364 16 16 96 21313232132132179 11 10 100 22321313221332182 20 19 121 23321321332113293 21 25 139 24321332113221363 16 16 95 25332113232121367 16 16 99 26332121313232168 20 20 108 27332132121313255 16 27 98

r1 173 121 127 129 112 118 117 109 116 116 125 115 122

r2 82 128 118 113 132 120 122 120 122 119 116 124 116

r3 104 110 114 118 116 122 108 130 122 125 118 121 121

d 91181316204142169996 Note: HCH = α–hexachlorocyclohexane; DID = dieldrin; DDD = dichlorodiphenyldichloroethane factors), which was computed from the dummy factor, was solvent to use. However, these are preliminary results as low as 0.07%. This implies that there are no other essential and further investigation is deemed necessary. factors or interactions which are left out in the initial stage According to the initial results, a three-level 13 of the array design. Hence, the rest of the interactions OA27 (3 ) array (Table 4) was chosen for a more between the factors could be disregarded in this comprehensive optimization of the variables study. The optimal extraction conditions are shown affecting the fully automated BID procedure. as the highest average response for each factor. For Experimental results of the three-level array are example, for factor A, r1 displays an average response presented in Table 5. Although toluene gave the best of 61 (underlined in Table 2, second column). This response in the mixed-level array, the best three of the means that toluene is most likely the optimal extraction four organic solvents were selected for the three-level www.chromatographyonline.com 401 LEE ET AL.

13 TABLE 6: Analysis of variance (ANOVA) table for experimental responses in the orthogonal array (OA)27 (3 ) matrix Source SS d.f. MS F SS' PC(%) Extracting solvent (A) 41072.98 2 20536.49 138.48*** 40776.38 79.81 Bubble size (B) 1572.05 2 786.03 5.30** 1275.46 2.50 Extraction solvent 6.53** 1641.31 3.21 1937.90 2 968.95 volume (C) Extraction time (D) 1998.94 2 999.47 6.74** 1702.35 3.33 Salt concentration (E)351.542 175.77 1.19 54.95 0.11 (AXB) 2045.64 4 511.41 3.45* 1452.45 2.84 (AXC) 923.92 4 230.98 1.56 330.73 0.65 Pooled Error (#) 1186.3779 8 148.30 3855.73 7.55 Total 51089.35 26.00 51089.35 100.00

SS = sum of squares; d.f. = degree of freedom; MS = mean of squares; F = critical values; SS’ = purified sum of squares; PC(%) = percentage contribution. *** F(2,8) = 18.49 at P < 0.001; **F (2,8) = 4.46 at P < 0.05; * F (4,8) = 2.81 at P < 0.1

be decided based on the interactions. This can TABLE 7: Three-by-three table for the analysis of (A × B) be evaluated by considering a three-by-three table interaction for each interaction, which had been described in Extraction Solvent Type Bubble Size detail for a similar situation (9). The computed results A A A 1 2 3 for (A × B) interactions are listed in Table 7.

B1 122 108 86 It can be seen from Table 7 that the combination of A1 B 193 88 120 2 (toluene) and B2 (1.0 μL) gives the best enrichment factor.

B3 153 75 101 Therefore, based on the OAD experiments, the optimized extraction conditions were 1.0 μL of air incorporated array to study the significant interactions (A × B) and with 2.5 μL of toluene as the extraction solvent, an (B × C) more thoroughly. Table 6 shows the ANOVA extraction time of 35 min, and with no salt added. results for the three-level array. Similarly, the error in It should be noted that, since the OAD strategy the ANOVA table was pooled from the dummy factors. required multiple experiments, it provided the perfect The effects of the variables on the response function opportunity in this work to demonstrate the automation of can be evaluated by both their significance (F ratio) a BID-GC–MS sequence over many cycles, without any and their percentage contribution (PC%). The F-test operator intervention, to generate the optimization data. indicated that the organic extraction solvent (A) was statistically significant at P < 0.001. Bubble size (B), Results and Discussion extraction solvent volume (C), and extraction time (D) OAD Optimization Results: According to the results were statistically significant at P < 0.05. The results from the mixed-level OAD, the type of extraction were further confirmed by the analysis of PC%, which solvent was the most significant variable, followed by indicates how important each variable or interaction volume of the solvent, and the ionic strength (added is amongst the rest of the considered factors (11). The salt) of the sample solution. It was also shown that results pertaining to the importance of the variables and the interaction between the type of extraction solvent interactions were in accord with those of the mixed-level and bubble size and the interaction between the array. A low error of 7.55% was also obtained. type of solvent and the solvent volume were also 1 × 12 13 From the analysis of the mixed-level OA16 (4 2 ) significant. A three-level OA27 (3 ) array was then 13 × and three-level OA27 (3 ) arrays, interactions (A B) chosen to study the effects of these significant variables were statistically significant, as discussed in detail and interactions at more specific levels for a more previously. In this case, because the interaction comprehensive optimization of automated BID. (A × B) contained a non-significant variable (B), the The responses (enrichment factors, taken to optimum level for the non-significant factor must represent BID efficiency) from the array were

402 LCGC Europe August 2019 LEE ET AL.

calculated according to the following equation: to 0 μL), were carried out. The EFs obtained are shown in Table 8. The EFs for HCH, DID, and DDD were 232,

Enrichment factor (EF) = Ca,final / Cs,initial [1] 19, and 28, respectively, compared to 326, 23, and 39 achieved using BID-SDME. Thus, it is possible that the

where Ca,final is the final concentration of increase in the extraction efficiencies for OCPs were analyte in the extract phase, and Cs,initial is the related to the increase in interfacial area of the organic initial analyte concentration in the sample. drop, given that all other factors were kept the same. Based on the OAD experiments, the final optimized extraction conditions were 1.0 μL of air incorporated with 2.5 μL of toluene as the extraction solvent, an extraction time of 35 min, and with no salt added. BID-GC–MS Performance: Evaluation of the method’s linearity, sensitivity, and precision was carried out under the optimized conditions, and the results are shown in Table 8. The analytes exhibited good linearity, with superior coefficients of determination (r2) of between 0.9987 and 0.9991. The precision of the method was determined by performing six consecutive fully automated extractions from aqueous samples spiked with 0.05 μg/mL of each OCP. The relative standard deviations (RSDs) of the analyte responses obtained were between 5.8% and 7.7%. The enrichment factors (EFs) obtained for the three OCPs were between 23 and 326, which demonstrate effective enrichment of the target analytes. Limits of detection (LODs), calculated at a signal-to-noise (S/N) ratio of 3, ranged between 1.74 and 2.51 μg/L. Comparison of SDME with BID: To support the hypothesis that the deliberate inclusion of air bubble in the microdroplet could lead to the increase in extraction efficiency, replicate SDME experiments of 0.05 μg/mL sample solution, using the same conditions and parameters (with the exception of bubble size, which was changed

www.chromatographyonline.com 403 LEE ET AL.

TABLE 8: Performance of BID-GC–MS, and comparison of enrichment factors (EFs) of SDME-GC–MS and BID-GC–MS SDME-GC–MS BID-GC–MS OCP EF EF RSD (%) Linearity Coefficient of LOD (n = 6) range (μg/L) determination (r2) (μg/L) HCH 232 326 5.8 8.28–80 0.9987 2.49 DID 19 23 7.7 8.39–80 0.9988 2.51 DDD 28 39 5.6 4.63–80 0.9991 1.74 OCP = organochlorine pesticides; HCH = α–hexachlorocyclohexane; DID = dieldrin; DDD = dichlorodiphenyldichloroethane

TABLE 9: Analysis of spiked tap water and reservoir water samples Conclusion Tap Water Reservoir Water Fully automated bubble-in-drop microextraction was set up in this work to address the shortcomings and inconvenience Spiked to 50 μg/L Linearity LOD OCP concentration range (μg/L) (μg/L) of previously reported manual approaches of this procedure. Recovery (%) RSD (%) Recovery (%) RSD (%) With the simplicity of operations and handling enabled by (n = 5) (n = 5) automation, including the implementation of orthogonal HCH 83 5.5 76 4.3 array design optimization, the sample preparation procedure could be carried out easily, and sequential extractions could DID 92 5.3 75 5.9 be performed conveniently using a commercially available DDD 96 4.3 69 10.0 autosampler. There was less error than that associated OCP = organochlorine pesticides; HCH = α–hexachlorocyclohexane; DID = dieldrin; DDD = dichlorodiphenyldichloroethane with manual injection, and significant reduction in labour intensiveness, allowing good analytical performance of the Real Sample Analysis: The applicability of the proposed fully procedure for the determination of several representative automated method to a real environmental aqueous matrices organochlorine pesticides. However, this is only a preliminary was evaluated using tap water and reservoir water samples. study, and more work is needed to improve analytical The unprocessed water samples were first extracted under performance, particularly in terms of the limits of detection. the optimized conditions, and then analyzed. No target analytes were detected, or their concentrations were below References the LODs. This is not surprising because these OCPs have 1) M. Smith, J. Chromatogr. A 1000, 3–27 (2003). 177 not been used in Singapore since the 1970s. Nevertheless, 2) D.A.J. Murray, J. Chromatogr. A , 135–140 (1979). 3) M.A. Jeannot and F.F. Cantwell, Anal. Chem. 68, 2236–2240 (1996). each OCP was spiked at 50 μg/L concentrations into 4) Y. He and H.K. Lee, Anal. Chem. 69, 4634–4640 (1997). the samples, and the latter extracted and analyzed 5) J. Lee and H.K. Lee, Anal. Chem. 83, 6856–6861 (2011). using the method developed. The relative recoveries 6) D.B.G. Williams, M.J. George, R. Meyer, and L. (RRs) (defined as the ratio of the concentration of analytes Marjanovic, Anal. Chem. 83, 6713–6716 (2011). from spiked real sample extracts to that from spiked 7) D.C. Wood, J.M. Miller, and I. Christ, LCGC Asia Pacific 7, 8–11 (2004). 88 ultrapure water extracts) were calculated to evaluate 8) L. Guo, N. Binte Nawi, and H.K. Lee, Anal. Chem. , 8409–8414 (2016). 9) W.G. Lan, M.K. Wong, N. Chen, and Y.M. Sin, matrix effects. Results of the RRs and RSDs of spiked tap Analyst 119, 1659–1667 (1994). water and reservoir water samples (Table 9) demonstrated 10) W.G. Lan, K.K. Chee, M.K. Wong, H.K. Lee, and that satisfactory RRs (69–96%) were attained; the RSDs Y.M. Sin, Analyst 120, 281–287 (1995). ranged from 4.3% to 10.0%, which are considered very 11) W.G. Lan, M.K. Wong, N. Chen, and Y.M. Sin, 120 good (the lowest RR and highest RSD values were for the Analyst , 1115–1124 (1995). 12) L. Guo and H.K. Lee, Anal. Chem. 86, 3743–3749 (2014). relatively more complex reservoir samples, which is not surprising). The generally good results can be attributed to the full automation of the procedure; overall, the data Claire A. Taylor, Hui Min Lee, and Hian Kee Lee showed that the matrices had only minor effects on the are with the Department of Chemistry at the National method. The fully automated BID-GC–MS approach was University of Singapore. Direct correspondence demonstrated to be viable for quantitative determination to H.K. Lee at: [email protected] of OCPs in real environmental water samples.

404 LCGC Europe August 2019 LC TROUBLESHOOTING

Pass the Salt: Evolution of Coupling Ion-Exchange Separations and Mass Spectrometry

Dwight R. Stoll, LC Troubleshooting Editor

Direct coupling of ion-exchange separations to mass spectrometric (MS) detection is increasingly being used for analyses of molecules ranging from organic acids to proteins. These approaches leverage both the exquisite selectivity of the ion-exchange mode for charge-based separation, and the tremendous power of mass spectrometry for identification of unknowns and trace-level quantitation.

The 48th annual International Symposium Introduction to the question is that ion-exchange on High-Performance Liquid Phase Historically, the coupling of ion-exchange separations were developed long Separations and Related Techniques, separations and MS has not been so before electrospray MS was developed. more commonly known simply as “The straightforward because the high levels There are still good chromatographic HPLC meeting”, was held in Milan, Italy, of nonvolatile salts that are typically used reasons to use particular nonvolatile a few weeks ago (16–20 June). Upon with many ion-exchange methods simply salts over volatile ones in certain preparing for the conference I thought cannot be used with mass spectrometers. situations. However, in the early days of about listening to the presentations this However, for some applications there ion-exchange separations mobile phase year with an ear towards ideas that could is tremendous incentive to couple additives were chosen on the basis of be helpful for people developing and ion-exchange and MS because of the attributes other than volatility, such as troubleshooting liquid chromatography incredibly rich qualitative information transparency to optical detectors. For (LC) methods. As usual, I walked MS detection can provide. Whereas the example, two of the most commonly used away from the conference with many selectivity of ion-exchange separations mobile phase additives for ion-exchange research ideas, but also ideas for two can be very powerful (for example, one separations—sodium phosphate and topics I’d like to address in this column, can easily separate 150 kDa mAbs that sodium chloride—are highly transparent this month and in September. The first differ only by a single charged amino to UV light, and thus low wavelengths topic is focused on the evolution of the acid), if analytes are detected by a around 200 nm can be used to increase coupling of ion-exchange separations nonspecific detector (most commonly, detection sensitivity when these additives with mass spectrometric (MS) detection. UV absorbance), one is frequently left are used. Furthermore, one of the other During the HPLC meeting, there was wondering, ”What is that peak?” Currently, attractive attributes of phosphate salts an entire session dedicated to this this type of question is most commonly is that phosphoric acid is a triprotic acid specific topic, with six oral presentations answered by collecting fractions of what with pKa values at about 2.1, 7.2, and covering aspects ranging from stationary is eluted from the ion-exchange column 12.3. The multiple pKa values and their phase development to development of and re-analyzing them using a more spread across a wide pH range means MS-compatible ion-exchange separations MS-friendly separation technique such that this single mobile phase additive for molecules ranging from metabolites as reversed-phase LC. This process can be used to effectively buffer the to intact monoclonal antibodies (mAbs). is very tedious and time-consuming, mobile phase pH across large pH ranges. Clearly, there is a lot of interest in this of course, and so the idea of directly Although phosphate cannot be used to area at the moment, and given that coupling the ion-exchange separation provide buffering capacity over the entire coupling ion-exchange separations to to MS detection is attractive. range, it is frequently complemented MS represents a significant change One might reasonably ask why with other (nonvolatile) additives such as from the way ion-exchange has been nonvolatile salts are used so often when citrate, which have pKa values that fill the practiced historically, we all have a they are known to be incompatible gap between 2.1 and 7.2 for phosphate. lot to learn as we move forwards. with MS detection. The short answer Cocktails of such complementary www.chromatographyonline.com 405 LC TROUBLESHOOTING

nucleotides and sugar phosphates, FIGURE 1: Cation-exchange chromatograms of intact mAbs. Mobile phase A: 10 mM where the results of ion-exchange MS ammonium acetate + 10 mM acetic acid; mobile phase B: 50 mM ammonium acetate + 50 mM ammonium carbonate; gradient: 10–70% B in 8 min; flow rate: separations complement those from 0.3 mL/min; column: 50 mm × 2 mm weak cation exchanger. Peaks: eculizumab other methods, including reversed-phase (1), panitumumab (2), reslizumab (3), pembrolizumab (4), atezolizumab (5), and hydrophilic interaction liquid adalimumab (6), and rituximab (7). Reprinted with permission from D. Guillarme (8). chromatography (HILIC) separations coupled with MS detection (2,3). At 7 the HPLC meeting in Milan, the last presentation in the session on coupling p/ = 9.4 ion-exchange and MS by Hvinden 6 described the use of cation-exchange separations for metabolomics (4). p/ = 8.9 5 Direct Coupling of Ion-Exchange

p/ = 8.6 and MS Using Mobile Phase pH 4 Gradients and Volatile Buffers Currently, the most active area p/ = 7.6 of research on the coupling of 3

Signal (AU) ion-exchange and MS is focused on separations of biologics such as mAbs. p/ = 7.1 Indeed, four of the six presentations in 2 the session on coupling ion-exchange and MS at the HPLC meeting in p/ = 6.8 Milan were focused on this topic in 1 particular. Whereas small organic ions can be eluted from ion-exchange p/ = 6.1 012345678 stationary phases with modest mobile Retention time (min) phase concentrations of counterion (for example, 100-mM hydroxide in reference 3), larger molecules, such as additives can be prepared for use The development of suppressor proteins, can be more difficult to elute at as so-called “universal buffers”. technology for ion-exchange separations a fixed pH if they are multiply charged in Unfortunately, there are no obvious (1) has had a tremendous impact on solution. For example, in our own work alternatives to additives like phosphate several application areas. Whereas we have found that mAbs can be eluted and citrate that are both volatile enough much of the early research on this from conventional cation-exchange to be used in high concentration with MS, technology was focused on the analysis phases using volatile buffers such as and provide high buffer capacity across of inorganic ions using conductivity ammonium acetate (pH 6) that would a wide pH range. Nevertheless, the detection, more recent research has be suitable for coupling to MS, but at attractiveness of coupling ion-exchange enabled high performance separations of rather high concentrations on the order and MS directly for some applications organic ions coupled with MS detection. of 0.2 M (5). While such mobile phases has prompted several groups to In the case of coupling ion-exchange may be used with electrospray MS investigate conditions that are viable with MS, the suppressor is important for a short time, prolonged use under for such separations. In the following because it enables inline removal of these conditions is not recommended. sections, I provide examples of a few nonvolatile inorganic components of the The high level of salt required for elution different lines of research in this direction. mobile phase (for example, potassium of proteins has motivated a number of ions) prior to MS detection. This type of groups to explore the potential to elute Direct Coupling of Ion-Exchange separation is increasingly being used in proteins from ion-exchange phases using and MS Using Mobile the metabolomics field for quantification gradients in mobile phase pH, rather than Phase Salt Gradients of anionic metabolites, including mobile phase salt concentration. The

406 LCGC Europe August 2019 LC TROUBLESHOOTING

central principles that control retention is straightforward, actually executing for separations of biomolecules. On and elution in this case are pretty this in practice, with buffers that are the other hand, MS-friendly additives, straightforward. Consider the behaviour MS-compatible, is where the difficulty such as ammonium ion and formic of a protein as an example. One of the lies. As discussed above, one of the acid, are only monoprotic acids, with important characteristics of a protein virtues of phosphate as a buffering pKa values that are far apart, and this is its isoelectric point (pI)—the pH at mobile phase additive is that phosphoric limits their effectiveness when used which the net charge on the molecule acid is a triprotic acid. This enables in a pH gradient scenario. In spite of in solution is zero. This does not mean effective buffering of the mobile phase these challenges, in very recent work it is uncharged; rather, it means that at over much of the pH range that is useful several groups have demonstrated the pI, the number of negative charges on the protein (from deprotonated carboxylate groups, for example) is equal to the number of positive charges on the protein (from protonated amino groups, for example). At pH levels well below the pI the protein will carry a net positive charge and be retained by a stationary phase designed for BioLC Innovations... cation-exchange separations, and at pH levels well above the pI the same protein ...with Incredible Reproducibility! will be unretained. Moreover, changes to the protein that alter the pI, such as the addition or deletion of amino acids with ionizable side groups, or a change to an amino acid such as deamidation, will in turn influence retention in an ion-exchange separation. So, the strategy to separate two molecules having different pI values using a pH gradient and a cation-exchange stationary phase would be to first inject the sample into the column running with Proteins a mobile phase buffered well below the Antibodies pI of the analytes, with relatively low salt Oligonucleotides concentration, so that they are retained. This could be something like 25-mM Peptides ammonium acetate, with the pH adjusted to 5 with acetic acid. Then, using a • SEC for high resolved MAbs second buffer, such as ammonium • RP-C4-Widepore with superior stability carbonate adjusted to pH 10, a gradient of the two buffers is carried out such • IEX for high recovery that the pH of the mobile phase mixture inside the column slowly increases • HIC with exceptional efﬁciency from 5 to 10 over the course of the separation. The protein with the lowest pI should elute first, and the protein with the highest pI should be eluted last. Discover more at www.ymc.de While the main principle of separating ionizable analytes using a pH gradient www.chromatographyonline.com 407 LC TROUBLESHOOTING

ion-exchange mobile phase itself FIGURE 2: (a) LC×LC–MS separation of IdeS digested trastuzumab, with more compatible with MS detection by cation-exchange and reversed-phase separations in the first and second dimensions, respectively. Panels (b) and (c) show deconvoluted mass spectra obtained in real replacing the commonly used nonvolatile time following the second dimension separation. Adapted from reference 5. mobile phase additives with volatile ones. A very different approach to obtain MS (a) information about analytes separated 80 by ion-exchange in real time is to use

F(ab’)2 variants online two-dimensional chromatography 60 (2D-LC). The virtue of this approach D (s) 2 40 is that existing ion-exchange methods Fc/2 (or others, such as size-exclusion 20 reversed-phase [SEC]) can be used as they are, where high mobile phase concentrations of 10 20 30 40 50 1D (min) nonvolatile additives may be required to cation-exchange improve peak shape (9). Briefly, in online (b) 25000 F(ab’) – 97,630 Da 2D-LC, fractions of effluent from the first 20000 2 dimension column are collected in loops 15000 or traps and transferred (injected) into 10000 a second dimension column for further 5000

MS Signal (Counts) separation. In a simple case the second 0 dimension separation can be a short, fast 97400 97500 97600 97700 97800 97900 98000 Deconvoluted Mass (Da) desalting step using reversed-phase or SEC conditions, for example, where the 6000 (c) Fc/2 + H3N4F1 role of the second separation is to simply Fc/2 + H4N4F1 4000 separate nonvolatile components of the

2000 first dimension effluent from the analytes of interest. The second separation can

MS Signal (Counts) 0 25200 25250 25300 25350 25400 25450 25500 also be much more powerful, of course, Deconvoluted Mass (Da) and provide additional separation of analytes that were not resolved by the first separation. An example of such a the potential for direct coupling of ammonium carbonate (unadjusted separation from our own work is shown cation-exchange separations to MS pH ~9.0). Note that a gradient made with in Figure 2, where a salt gradient of detection using volatile mobile phase these buffers will increase in both pH and ammonium acetate at pH 6 was used additives and pH gradients (6–8). Much salt concentration over the course of the to elute mAbs from a first dimension of this work to date has been focused gradient. Figure 1 shows representative cation-exchange column, followed by on separations of mAbs. Farsang, chromatograms from their work, where a second dimension reversed-phase Fekete, and coworkers compared the the retention of the mAbs is highly separation running with 0.1% formic performance of mAb separations by correlated with pI, as expected. The acid as a mobile phase additive, prior cation-exchange with pH gradients using ability to couple these chromatographic to MS detection (5). In this case the mobile phases prepared with different conditions—as a generic starting second dimension separation not only types of MS-compatible additives (8). point in method development—with separates the high concentration of They report that the following single set of the identification power of mass ammonium acetate from the protein buffers enable separations of mAbs that spectrometry is indeed very powerful. analytes of interest, but also separates span a range of pIs, with good retention, subunit variants of the mAb that were not peak shape, and selectivity. Buffer A: Indirect Coupling of Ion- separated by cation-exchange. Readers 10 mM ammonium acetate + 10 mM Exchange and MS Using 2D-LC interested in learning more about 2D-LC acetic acid (unadjusted pH ~4.7); Buffer In the preceding two sections, I described are referred to recent review articles that B: 50 mM ammonium acetate, 50 mM recent research aimed at making the describe the state of the art (10,11).

408 LCGC Europe August 2019 LC TROUBLESHOOTING

Chem. 88, 11799–11803 (2016). doi:10.1021/acs. 519–531 (2017). doi:10.1021/acs. Closing Thoughts analchem.6b03435. analchem.6b03506. Each year I attend the HPLC meeting, 4) I.C. Hvinden, J. Walsby-Tickle, S. Liu, C.J. Scho⇒ eld, 11) B.W.J. Pirok, D. Stoll R., and P.J. Schoenmakers, I walk away with a lot of excitement and J.S.O. McCullagh, Developing Cation Exchange Anal. Chem. 91, 240–263 (2019). doi:10.1021/acs. about the innovative and powerful Chromatography Coupled to Mass Spectrometry analchem.8b04841. for Metabolomics Applications, (2019). https:// things researchers are doing with www.hplc2019-milan.org/ (accessed 10 July liquid chromatography, which is 2019). Dwight R. Stoll is the editor of “LC sometimes perceived as a mature 5) M. Sorensen, D.C. Harmes, D.R. Stoll, G.O. Troubleshooting”. Stoll is a professor technique. In fact, there are many Staples, S. Fekete, D. Guillarme, and A. Beck, and co-chair of chemistry at exciting developments each year, and MAbs 8, 1224–1234 (2016). doi:10.1080/194208 Gustavus Adolphus College in St. the evolution of coupling ion-exchange 62.2016.1203497. Peter, Minnesota, USA. His primary 6) Y. Yan, A.P. Liu, S. Wang, T.J. Daly, and N. Li, Anal. separations directly to MS detection research focus is on the development Chem. 90, 13013–13020 (2018). doi:10.1021/acs. is one development that should be analchem.8b03773. of 2D-LC for both targeted and interesting to follow in the years to come. 7) F. Füssl, K. Cook, K. Scheɞ er, A. Farrell, S. untargeted analyses. He has Mittermayr, and J. Bones, Anal. Chem. 90, authored or coauthored more than 50 References 4669–4676 (2018). doi:10.1021/acs. peer-reviewed publications and three analchem.7b05241. 1) H. Small, T.S. Stevens, and W.C. Bauman, Anal. book chapters in separation science 47 8) E. Farsang, A. Mursier, K. Horvath, O. Colas, A. Chem. , 1801–1809 (1975). doi:10.1021/ and more than 100 conference ac60361a017. Beck, D. Guillarme, and S. Fekete, LCGC Europe 37, 34–38 (2019). presentations. He is also a member 2) H.F.N. Kvitvang, K.A. Kristiansen, and P. Bruheim, J. Chromatogr. A 1370, 70–79 (2014). 9) P. Petersson, K. Haselmann, and S. of LCGC’s editorial advisory 1468 doi:10.1016/j.chroma.2014.10.029. Buckenmaier, J. Chromatogr. A , 95–101 board. Direct correspondence to: (2016). doi:10.1016/j.chroma.2016.09.023. 3) C. Petucci, A. Zelenin, J.A. Culver, M. Gabriel, K. [email protected] 89 Kirkbride, T.T. Christison, and S.J. Gardell, Anal. 10) D.R. Stoll and P.W. Carr, Anal. Chem. ,

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www.chromatographyonline.com 409 PERSPECTIVES IN MODERN HPLC

Modern HPLC Pumps: Perspectives, Principles, and Practices

Konstantin Shoykhet1, Ken Broeckhoven2, and Michael W. Dong3, 1Agilent Technologies, Waldbronn, Germany, 2Vrije Universiteit Brussel, Brussels, Belgium, 3Perspectives in Modern HPLC Editor

This instalment is the first of a series of four white papers on high performance liquid chromatography (HPLC) modules (pump, autosampler, UV detector, and chromatography data system) to be published in 2019. This instalment provides an overview for analytical-scale HPLC pumps, including their requirements, modern designs, operating principles, trends, and best practices for trouble-free operation.

The high performance liquid a mechanism like a circular is determined by the timings of the chromatography (HPLC) pump, often cam or screw drive to transform opening of the solenoid valves. called a solvent delivery system, rotations into linear motion. • Dwell volume: The volume in the provides a precise flow of mobile • Pump head: A metal body in HPLC system from the point of phases of a specified composition to which one or multiple cylinders mixing of two or more solvents the column. The historical evolution of or chambers are milled or drilled to the head of the column. Dwell HPLC pumps and their characteristics to accommodate the piston(s). volumes translate into gradient delay has been reviewed in many • Check valve: A device that allows times, which become important textbooks (1–3), and journal articles liquid flow in one direction (typically for high-throughput analysis and (4–6). Their performance, pressure using a passive gravity-based low-flow gradient applications. ratings, and reliability have increased ball-and-seat design). It prevents • Mixer: Mixers are required to dramatically in the last two decades to any backflow and pressure drops, ensure adequate mixing of blended accommodate the demands from the such as those that occur during the mobile phases. The volume of the use of narrow columns packed with piston’s recharge cycle. Some check mixer should be large enough for small particles in ultrahigh-pressure valves are active or electronically applications sensitive to composition liquid chromatography (UHPLC) actuated (examples include fluctuations, such as UV-detection (5–6). In this instalment, we provide an Agilent 1100, 1200, and 1260). with UV-absorbing additives in overview of modern HPLC pumps by • High-pressure pump unit: A subunit the mobile phases. Mixer volumes describing the design and operating of an HPLC pump delivering a range from a few μL to 2 mL in principles of key components, and the high-pressure flow for a single or analytical HPLC systems. best practices in pump operation. mixed solvent. Connecting the outlets • Pulse damper: An elastic chamber of two pump units makes a binary located in the high-pressure flow Glossary of Key Terms pump. A pump unit may be a core of path of the pump that functions Let’s start by defining the key an isocratic pump, or a quaternary to reduce residual flow pulsations terms related to HPLC pumps: pump by attaching a four-port from the reciprocating pump • Piston: A rod made of inert materials proportioning valve at the inlet side. unit(s). like sapphire, ceramics, steel, or • Proportioning valve: A block with • Degasser: An in-line device to alloy. The piston is reciprocating typically four solenoid valves, each reduce the gas content of the within a piston chamber or connected to a separate solvent line solvent before entering the pump cylinder in the pump head. to allow blending of a mixed mobile unit. The most common design • Drive: A mechanical unit that phase from up to four solvents using uses tubes of semi-permeable actuates a piston’s back-and-forth a single pump unit. The opening of membrane located inside an motion. The drive train is typically the solenoid valves is electrically evacuated chamber to eliminate powered by a motor, and includes driven, and the solvent mixing ratio dissolved oxygen and nitrogen.

410 LCGC Europe August 2019 HILICON

iHILIC® -Fusion advances HILIC separations in UHPLC and HPLC

iHILIC® -Fusion iHILIC® -Fusion(+) iHILIC® -Fusion(P) Silica based Silica based Polymer based

Charge modulated hydroxyethyl amide/amide HILIC columns Three complementary selectivities for separation of polar compounds Excellent durability and ultra-low bleeding for LC-MS Versatile columns for ”Omics” studies and other applications iHILIC®® -Fusion and iHILIC -Fusion(+): pH 2-8; 1.8, 3.5, and 5 μm iHILIC® -Fusion(P): pH 1-10; 5 μm

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2.0E+05

0.0E+00 0123456789101112131415 Time (min)

Constant Flow Pumps: Constant flow TABLE 1: Requirements, desirable characteristics, and key components of the analytical HPLC pump rate pumps have been the standard approach since the debut of the Requirements first commercial HPLC system in the • Provides constant and pulse-free flow of common mobile phases to HPLC columns 1960s. This is important because under high pressures most chromatograms use detector • HPLC (0.1–10 mL/min, 6000 psi or 400 bar) signals plotted against the x-axis • UHPLC (0.01 to 2–5 mL/min, >15,000 psi or 1000 bar, up to 22000 psi or in time (as minutes) to yield peak 1500 bar), intermediary UHPLC (9–12,000 psi or 600–800 bar) retention times for analyte identification. • Micro LC (0.001 to 0.1 mL/min) Though peak retention volume (VR) • Has a flow path consisting of metals and other materials or polymers compatible is the more inherent property in the with most common HPLC solvents chromatographic theory for solute • Allows blending of two or more solvents with accurate compositional control for retention, time has established itself isocratic or gradient operation as the preferred x-axis coordinate in • Precise operation with long-term reliability; control by a controller or chromatography because it can be chromatography data system (CDS) measured more easily and precisely Desirable characteristics than volume. Since VR is equal to • Dimensions compatible with other HPLC modules (stackable to minimize footprint) retention time (tr) multiplied by flow • Easy to operate and maintain (front-panel accessibility of key components) rate (F), F must remain constant • Low dwell volumes to minimize gradient delay times throughout the chromatographic run for proper evaluation and quantitation of • Biocompatibility: flow path made of titanium, tantalum, PEEK, or other salt and corrosion-resistant materials chromatographic peak area versus time. Although the delivery of a constant Key components flow under isocratic conditions and low • Drive mechanism: motor with a drive train for a reciprocating piston pressures can be easily accomplished • Mobile phase selection, conditioning, and control: filter, solvent selection valve, by a direct displacement pumping proportioning valve, prime–purge valve, mixer, degasser mechanism, it is considerably • Other components or optional accessories: pressure transducer, pulse dampener, more difficult to deliver a variety piston-seal wash, leak detector of mixed solvents with changing composition at high pressures Requirements, Desirable instrumental development (3,4). The accurately. Solvents have different Characteristics, and following are brief highlights of the coefficients of compressibility and Key Components development of different types of thermal expansion coefficients, which Table 1 summarizes the requirements, pumps, leading to the dual-piston should be compensated for to obtain desirable characteristics, and key renditions commonly in use today. reproducible HPLC separations components of a modern HPLC Constant Pressure Pumps: Perhaps across a variety of HPLC platforms. pump, followed by a discussion the simplest way to pump liquids into Syringe Pumps: Many early HPLC of historical perspectives, recent a column is to pressurize the mobile pumps in the 1960s and 1970s developments, and design rationale, phase reservoir with a gas, such as were syringe pumps (such as the plus operating mechanisms of key nitrogen. The fundamental problem Varian 8500 Dual Syringe Pump components. Our goal is to increase with this simple constant pressure System in 1975). The syringe pump the understanding of the modern approach is that the flow rate will be is ideally suited for the generation HPLC pump by laboratory scientists, dependent on the flow resistance of the of a perfectly constant flow without thus allowing the implementation column. As pressure drop changes, the pulsation against a constant load. of better operating practices. flow rate would be barely controllable, The gradient can be formed by using leading to unpredictable retention two or three syringe pumps. The big Brief Historical Perspectives times. Also, the gas solubility in the disadvantages are the cumbersome There are numerous historical solvents and the control for accurate screw-driven syringe mechanisms accounts of HPLC and its blending can be fundamental issues. (fill volumes of 250–1000 mL) needed

412 LCGC Europe August 2019 PERSPECTIVES IN MODERN HPLC

to provide common analytical flow FIGURE 1: Schematic diagrams: (a) single-piston reciprocating pump driven by rates for each chromatographic a motor with a circular cam; (b) dual-piston in-parallel format with two pistons run, and the painfully slow syringe operating 180 degrees out of phase; (c) dual-piston in-series format with a primary retraction process before each sample (first) piston and a secondary (accumulator) piston, shown in the primary stroke injection. Today, syringe pumps are phase. Note: the primary piston provides the flow to fill up the secondary cylinder rarely used, except for micro or nano with a retreating secondary piston, and at the same time the system is flowing. HPLC instruments (Eksigent nanoLC 400), and portable units (Axcend Single-Piston Reciprocating Pump Dual-Piston In-Parallel Dual-Piston In-Series Focus LC) where the smaller flow (a) (b) (c) rates allow for more compact pump Pump Head designs, or post-column derivatization. Piston Seal Outlet Single-Piston Reciprocating Pumps: Check Valve The single-piston reciprocating pump Cylinder was the mainstay in HPLC solvent Piston Ruby Inlet Motorized Ball Check Valve delivery using piston mechanisms Cam Sapphire in its early days. Its strengths Seat are compactness, low-cost, and continuous delivery, while the key weakness is the pulsed flow caused by the piston recharge cycle. FIGURE 2: Schematic diagrams: (a) high-pressure mixing quaternary pump; Figure 1(a) shows a schematic (b) low-pressure mixing binary pump. of the simple reciprocating pump using a single-piston mechanism. High-Pressure Mixing Pump (b) Pressure Low-Pressure Transducer Filter Here, a motorized cam drives a Mixing Pump

Solvent out piston within a chamber or cylinder (a) High Pressure in the pump head to deliver a solvent Pump Mixer through a set of inlet and outlet check Pump Channel A Pump Channel B valves. The typical piston volume was ~100 μL for most HPLC pumps. Because only the inward piston stroke delivers the liquid, a pulse damper Solvents or other means is needed to reduce A-D flow fluctuations. All components Proportioning in the fluidic path of the pumps Valve are made from inert materials (for example, stainless steel pump heads, ruby balls with sapphire seats in Dual-Piston Pumps: Dual-piston flow with significantly less pulsation check valves, sapphire or ceramic pumps are the most popular (Figure 1[b]), such as the Perkin-Elmer pistons, or fluorocarbon pump seals). mechanisms in use today, and are Series 3 in the late 1970s. The same This popular low-cost reciprocating available as dual-piston in-parallel can be accomplished by a single pump design (Milton Roy Mini-pump or dual-piston in-series formats. A motor with a gear system that drives and Altex110A in the 1970s) has detailed explanation of these two two pump heads, as in the compact undergone numerous enhancements formats can be found elsewhere (7), Waters M6000 pump (1972). The to improve performance. but we provide our overview below. M6000 quickly became the gold Nevertheless, the deficiencies Dual-Piston In-Parallel Pumps: standard in HPLC pumps at that time. of the single-piston mechanism Dual-piston in-parallel pumps often The dual-piston in-parallel design quickly led to the development of the use a single motor with a cam has become less common in the dual-piston pump, which became the driving two pistons (180 degrees analytical pumps market after the dominant design starting in the 1980s. out of phase) delivering a combined popularization of the dual-piston www.chromatographyonline.com 413 PERSPECTIVES IN MODERN HPLC

and the design of the mixer can be TABLE 2: Modern trends and best practices in mobile phase selection in reversed-phase chromatography tuned to have the ideal compromise between homogeneity of the blended Overall trends solvents blend and dwell volume. • Simpler binary mobile phases and linear gradients Binary pumps generally cost more • Increased use of MS-compatible mobile phases than their quaternary counterparts, • Reduced use of buffers and other additives but have the advantage of lower

Selection of mobile phase A (MPA) and mobile phase B (MPB) dwell volumes (2). Solvent selection valves are used to enable selection • MPA: Increased use of MS-compatible buffers at lower concentrations, balanced-absorbance, high-pH mobile phases, reduced use of nonvolatile of additional different solvents for ion-pairing reagents unattended method or application • Use of 0.1% phosphoric acid (pH 2.2) for UV detection <210 nm instead of switching. Typically, one solvent phosphate buffer; elimination of triethylamine as a masking agent from up to three solvent reservoirs • MPB: Preferred use of acetonitrile for better separation efficiency, and low-UV (Thermo Vanquish) can be selected sensitivity for each line, though only binary Best practice in mobile phase preparation and operating conditions solvent mixtures can be delivered at • Preference for simpler mobile phase preparation and avoidance of dipping pH one time. Most early UHPLC systems electrodes in MPA during pH adjustments use binary pumps to reduce dwell volumes and for better compositional in-series format. Nevertheless, it was popular HPLC and UHPLC systems accuracy performance (6,8). revived in some UHPLC pumps, such (such as Agilent’s 1100, 1200, 1260 Low-Pressure Mixing Pumps: as the Shimadzu AD models and the with single motor design, Agilent 1290 Spectra-Physics was likely the Thermo Vanquish pumps (2014). with independent piston drives, and company that first introduced a Dual-Piston In-Series Pumps: The Waters Alliance and Acquity UPLC low-pressure mixing system (ternary) dual-piston in-series mechanism instruments with independent piston in the late 1970s (the SP8100). A (Figure 1[c]) has two pistons that can drives). This format is popular in quaternary HPLC system using a be driven by one or two separate low-pressure mixing quaternary pumps. dual-piston in-series pump was independent motors. The first piston High-Pressure and Low-Pressure introduced by PerkinElmer (Series 4, (often called the primary piston) is Mixing Systems: Pumps are 1983). Today, all quaternary pumps used to deliver a single or mixed categorized as high-pressure use a low-pressure mixing design, solvent to the second piston (the mixing (mostly binary pumps) where a single pump unit draws mobile high-pressure piston, sometimes called and low-pressure mixing systems phases from a four-port proportioning the secondary piston or accumulator). (all quaternary pumps) based on valve, as shown in Figure 2(b). The The first piston can have the same or the way two or more solvents are pump’s microprocessor controls a slightly larger displacement volume mixed in preblending isocratic the solvent composition of each than the second piston. The refinement or gradient operation. intake piston stroke by the timed of the movement of the two pistons High-Pressure Mixing Pumps: In opening of each solvent port. during the piston-switching phase, high-pressure mixing systems, two The first piston of a dual-piston when the solvent is passed from the (and occasionally three) separate in-series pump unit withdraws the first chamber into the second piston high-pressure pump units are used solvents to be mixed from the separate chamber, is particularly challenging to to mix solvents at high pressures lines under low pressure, while the allow compensations of compressibility downstream of the pump units second piston pushes an accurate and thermal expansion by elaborate (Figure 2[a]). A coordinated flow flow rate under high pressure into the piston control algorithms and pressure rate control of each pump is used system. Typically, solvent blending sensorics. This configuration is to generate different isocratic occurs inside the first piston cylinder, capable of delivering mobile phases blending or gradient profiles. A though a mixer can be installed right with highly accurate compositional mixer is commonly used to ensure after the proportioning valve in some control and less pulsation. This adequate mixing of the solvents designs (such as the Agilent 1290 configuration was adopted by most for most applications. The volume quaternary pump). All the internal

414 LCGC Europe August 2019 PERSPECTIVES IN MODERN HPLC

The Design of a Modern FIGURE 3: Schematic diagram of key components and flow path of a high speed UHPLC UHPLC Pump: A Case Study binary pump. To illustrate the application of concepts and modern pump technologies, we Binary Pump Schematic Jet Weaver use the Agilent 1290 Infinity II High Mixer Pressure Speed Pump, a binary high-pressure Sensor mixing pump (19,000 psi, 1300 bar),

Inline as an example of design rationale Purge FIlter Valve and performance characteristics (9). Channel A Channel B Figure 3 shows a schematic diagram of the various key components of this pump. Although the design strategy of each manufacturer may vary To Waste Outlet Check Valve significantly, the goal is to provide Degasser a cost-effective system that delivers accurate pulse-free flow rates in a range to support analytical-scale isocratic and gradient analysis. Seal Wash To Waste Pump Solvent Inlet This particular pump has a solvent Selection Check Valve Valve selection valve to allow the selection of two solvents for each pump channel (A1, A2, B1, B2). Like most modern pumps, a solvent vacuum degasser components in the fluidic path (both had a single channel low-pressure (1.5 mL per channel) is built into the cylinders with their dead volumes, metering pump with a proportioning system, to prevent outgassing of air pulse damper if present, purge valve, valve connected to its inlet. The bubbles in the system. The binary pump in-line filter, and pressure transducer) desired composition was generated by has two dual-piston in-series channels; contribute to the internal liquid hold-up the proportioning valve, and the flow each has two pump heads and pistons or dwell volumes (2,8). However, the from the metering pump was provided driven by separate drive mechanisms. major advantages of low-pressure to a membrane booster pump, which Each solvent identity can be named in mixing pumps are the inherent pressurized it and delivered it to the the HPLC method, allowing the system simplicity, and lower cost, because only system at up to 400 bar. The HP1090 to perform compensation of solvent one high-pressure pump unit is needed. DR5 had a set of three metering compressibility, based on an internal The low-pressure mixing pumps, which delivered the desired or user-loaded solvent description quaternary pump has become the composition by continuous parallel table. Although the ZrO2-based ceramic de facto standard pump for many metering of up to three solvents. The piston has a 100 μL-volume, the laboratories in method development composite flow was then boosted by variable stroke feature allows the use and routine analysis because it the high-pressure membrane booster of smaller stroke volumes (5–100 μL) can automatically blend up to four pump. The booster pump operated to improve low flow performance solvents. In comparison with the at 10 Hz. Thus, the residual flow and the use of smaller mixers. binary pump, the quaternary pump pulsations could be easily dampened The solvent flow from each channel is less expensive, generally has a down. The HP 1090 pumps provided passes through an in-line filter before larger dwell volume, and has less excellent composition accuracy and entering an automated purge valve compositional accuracy, resulting in precision as a result of complete and a pressure sensor (transducer), reduced retention time precision. decoupling of solvent metering and and then a mixer. The mixer assembly Still, another example of the pressure generation. This concept includes two mixer units, and the low-pressure mixing concept was has since been displaced from user can select a mixer of 35 or found in the HP 1090 instruments the market due to its complexity 100 μL volume during installation introduced in 1983. The HP 1090 PV5 and high manufacturing cost. or by re-plumbing the mixer. www.chromatographyonline.com 415 PERSPECTIVES IN MODERN HPLC

(MPB) spiked with a chromophoric FIGURE 4: Gradient profile trace using a UV detector and running a 5-min gradient for compound such as acetone or caffeine. the measurement of the dwell volume of a UHPLC system with a high-speed pump with a 35-μL mixer. An expanded scale insert shows the interception point at 0.28 min Test results from comparing a binary of the extrapolation gradient trace with the baseline. This time point translates to a dwell pump to a quaternary counterpart volume of 0.14 mL or 140 μL (2) for the system and comprises the delay volume within are available elsewhere (11). A step the pump (mostly the mixer) and the volume of the flow path in the autosampler. The gradient or a similar test is often operating conditions are: mobile phase: A1 is water, and B1 is water with 0.6% acetone; included in operational qualification or solvent prog.: 0–100% B in 5 min; flow rate: 0.5 mL/min; detection: 254 nm, 80 Hz. system calibration procedures (12).

DAD1 A, Sig=254,4 Ref=off (JAN2010\GRADIENT000082.D) mAU Other Desirable Characteristics for Modern HPLC Pumps 1000 Other desirable characteristics for modern HPLC pumps include 800 DAD1 A, Sig=254,4 Ref=off (JAN2010\GRADIENT000082. appearance, compactness, mAU 40 0.28 min x 0.5 mL/min = dimensional compatibility to 0.14 mL = Dwell Volume 600 30 other system modules, ease of 20 operation and maintenance, and 10 400 0 biocompatibility for use with mobile -10 phases with high-salt content. -20 200 Having a small system footprint 0.2 0.4 min is becoming important in today’s

0 laboratories, where bench space

0123456788minis at a premium. For modular HPLC systems, a similarity in the dimension of all modules for “stackability” is an Dwell volumes are important mixing efficiency in high-sensitivity UV expected attribute. Compactness in UHPLC because of the use of analysis (9,10). The volume of the mixer is also desirable for built-in pump smaller-diameter columns at lower flow for acceptable mixing is dependent modules inside integrated systems (2). rates. Conventional HPLC systems on the pump performance, especially Ease of operation and maintenance have dwell volumes around 1 mL or on the magnitude and frequency of is another much sought after attribute. even more, which translates to a composition pulsations, produced by Front-panel accessibility of key gradient delay time of 2 min at 0.5 mL/ the pump. For the Agilent 1290 Infinity components (purge valve, pump min or 5 min at 0.2 mL/min. Thus, II High Speed Pump, the larger 100-μL heads, and check valves) is a standard systems based on binary high-pressure mixer is well suited for most applications. feature for most pumps. Many can mixing pumps with reduced dwell If minimized gradient delay is required be controlled by a front panel keypad volumes of 0.2–0.5 mL or less, for high throughput or at low flow rates, or CDS. Many UHPLC pumps are depending on the pump’s dwell volume the 35-μL mixer is useful. For especially equipped with automated prime and and contributions of other system critical applications (such as using purge valves with direct draining to components, such as autosampler, trifluoroacetic acid [TFA] as an eluent waste receptacles, which can be are preferred for the high-throughput additive, and striving for minimized UV- controlled and operated remotely. screening or low-flow applications. baseline noise) an even larger mixer with For ion-exchange or hydrophobic System dwell volume can be 380 μL volume is optionally available (9). interaction chromatography of measured by replacing the column The compositional accuracy of the biopharmaceuticals, titanium or other with a zero-dead-volume union and pump can be verified by running step alloy-based biocompatible systems measuring the gradient profile with gradient profiles (for example, in 10% with higher corrosion resistance to a UV detector, as shown in Figure 4. composition steps over the entire high-salt mobile phases, are often Although having a small mixer is specified composition range and in preferred. Because rinsing columns desirable for reducing dwell volumes, a finer steps in the extremes, near 0 with basic solvents is also common larger mixer may be required for better or 100% B) with the mobile phase B in these applications, construction

416 LCGC Europe August 2019 PERSPECTIVES IN MODERN HPLC

materials compatible with high pH applications (such as a 6. Program the pump to purge the column with strong piston seal) are often required. For ion chromatography (IC) solvents (such as acetonitrile or methanol), and shut systems, the use of metal-free materials is preferred. Many down the pump after the sample sequence is complete. IC systems are poly-ether-ether-ketone (PEEK)-based, with Cautions When Using Buffered Mobile Phases: pressure ratings limited to about 5000 psi or 350 bar. 7. Do not let buffers sit in the HPLC system, due to the danger of precipitation. Flush buffers from column and Best Practices in Pump Operation, system with water or low organic mix, such as 10% Troubleshooting, and Maintenance methanol in water, depending on your application. Best practices in HPLC operation, maintenance, and 8. Using phosphate buffer is particularly problematic because troubleshooting have been described in detail in it may be immiscible or precipitate with acetonitrile. Use books (2,13–14), journal articles (15), and manuals or a lower concentration of phosphate buffer (<20 mM) and publications from manufacturers (9,16). A summary 85% or less acetonitrile–water as MPB, if possible (17). of these practice for HPLC and UHPLC pumps 9. Use the piston seal wash feature to wash the back is included here as a brief reference guide. of the piston according to the recommendations of Mobile-Phase Selection and Preparation: A recent article the pump vendor, especially when running mobile was published on modern trends and best practices in phases with acidic, basic, or nonvolatile additives. mobile phase selection in reversed-phase chromatography (17), and a summary table is included here in Table 2. The Pump Maintenance reader is referred to the original article for more details. In addition to adopting best practice in pump operation, Pump Operation and Troubleshooting Guide: The periodic preventive maintenance to replace consumable user should read the pump manual and follow the parts in a preventive maintenance program is the general manufacturer’s instructions carefully. Here are some general guidelines for HPLC or UHPLC pump operation (2): 1. Place solvent line sinkers (inlet solvent filters provided by the pump vendor) into the mobile phase in solvent reservoirs; be sure to cap the reservoirs to minimize atmospheric contaminations or evaporation. 2. In general, the degasser should be left on, and all solvent lines including the unused ones should be filled with solvents. 3. If the solvent line is “dry”, perform “dry” primes by opening the manual prime and purge valve, and draw out 10 mL from each solvent line using a syringe. For pumps with an automated prime and purge valve, follow vendors’ instructions. Perform wet prime for 2–5 min at high flow rates (such as 4 mL/min) daily to purge solvent lines, and when changing mobile phases. 4. Purge the column with a “strong” solvent for 1–3 min before equilibrating the column at the initial mobile phase condition. Watch for a steady pressure with minimum pulsation (<1%), or check for other criteria for proper pump operation as recommended by the vendor. Check that there is a flat baseline from UV detectors (<0.05 mAU). Excessive pressure pulsation often means a trapped air bubble in the pump head (or a faulty check valve). Follow the vendor’s recommendations to fix this issue, such as by wet priming with acetonitrile or methanol. Replace the check valve if pump pulsation persists. 5. Periodic UV baseline perturbations synchronous to the pump stroke are often observed due to insufficient mixing (10). The use of a larger mixer is often the solution. www.chromatographyonline.com 417 PERSPECTIVES IN MODERN HPLC

rule in most laboratories. Preventive possible reason, it is a good idea replacement when scratches maintenance is typically performed to exchange the solvent bottle or damage are recognized by the manufacturer’s service and inlet tubing altogether, and on the seal-contact areas. specialist or internal metrologists. The even to flush the pump with a • The seal wash system should laboratory user should stock some disinfecting solvent such as 70% be fed with fresh seal wash key spare parts and learn how to do isopropanol. In-line filters on solvent according to the vendor’s some simple maintenance tasks. the high-pressure side can, in recommendations, and it should rare cases, also get clogged be inspected regularly to verify Maintenance Procedures That due to piston seals wear. its proper operation, unless Can Be Performed by the User • A usual symptom of high-pressure the system is equipped with a Although the details of maintenance filter clogging is an unexplained seal-wash monitoring sensor. procedures differ from vendor to operating pressure increase • Check valves can usually be vendor and the user guides should in the system and a significant replaced by the user in most be followed generally, there are some pressure reading when the systems. Some vendors even maintenance procedures common pump outlet is open, even with offer tool-free replaceable valves. for all pumps that can usually be a moderate flow (either in purge A check valve failure is often performed by the user. These operation or when the pump manifested by sporadic pressure include replacement of solvent filters outlet is disconnected from the drops in the system, or by the (sinkers), in-line filters, check valves, system). The exchange procedure inability of the pump to build and in some cases piston seals. for filters is usually very easy. up high pressure at all (given • There are usually several filters • Most vendors offer the users a that the inlet filter and tubing along the solvent flow path. They procedure to exchange piston are not clogged). Follow vendor are consumables and should seals in the pump heads by recommendations to exactly be replaced regularly and if themselves. Though this operation pinpoint the faulty valve, and to getting clogged. The solvent inlet often does not feel complicated, replace it. The most common filters (sinkers), as well as high it is important to keep in mind reasons for check valve failures pressure in-line filters, can get that the tightness of piston seals are their mechanical contamination clogged prematurely when using is decisive for leak-free pump by particles coming from the insufficiently pure solvents, when operation and thus for flow solvents, or in some cases from not keeping solvents dust-free, or and composition accuracy and the system wear, drying out of when letting “nutritious” solvents precision. Therefore, the vendor’s nonvolatile buffers in the pump, like phosphate, acetate, or recommendations must be strictly or precipitation of solids from ammonium buffers with low organic followed. Besides, it is important incompatible eluent buffers. content stay for a longer time, to replace the seals only in a • When servicing the pump especially under light conditions clean environment, and to only or any other module, always (use amber bottles to prevent algae use non-scratching tools, to avoid follow the prescribed tightening growth). The symptom for inlet damaging the surfaces of the pump torques for any mounting filter clogging is unstable pump head that are in contact with the screws, check valves, Swagelock flow or gas bubble formation in piston seal. When replacing the components, or other fittings the inlet tubing. A simple check seals, the user might also clean and connectors. Overtightening procedure for nontoxic solvents the pistons and inspect the seal connectors or valves does not might be to open the line and let seats in the pump heads, as well improve their tightness, and the solvent siphon down the tube. If as the piston surface for scratches leads nearly inevitably to the solvent does not siphon down or damages. It is important that, their permanent damage. or is flowing only slowly, like a though pistons and other pump droplet every couple of seconds, head parts are not qualified as Summary the inlet filter probably needs to be consumables, they are not usually This instalment provides an replaced. In the case that algae or designed for lifetime operation, are updated overview of the bacteria growth is recognized as a prone to wear, and might require design features, performance

418 LCGC Europe August 2019 PERSPECTIVES IN MODERN HPLC

characteristics, and best practices of operation of chromatography and the optimization aspects and troubleshooting of modern HPLC pumps. of separation performance in both liquid and supercritical fluid chromatography. He is the author Acknowledgements of more than 60 research papers. He received the The authors thank the following colleagues for their 2019 LCGC Emerging Leader in Chromatography review of the manuscript: Cornelia Vad of Agilent Award, and is a member of the editorial advisory Technologies, Davy Guillarme of the University boards of Journal of Chromatography, LCGC North of Geneva, Frank Steiner of Thermo Scientific, America, and Journal of Pharmaceutical Analysis. and Giacomo Chiti of Manetti and Roberts. Michael W. Dong is a principal of MWD Consulting, which provides training and consulting services References in HPLC and UHPLC, method improvements, 1) L.R. Snyder and J.J. Kirkland, Introduction to Modern pharmaceutical analysis, and drug quality. He was Liquid Chromatography (John Wiley & Sons, Hoboken, formerly a Senior Scientist at Genentech, Research New Jersey, USA, 3rd Ed., 2010), Chapter 3. 2) M.W. Dong, HPLC and UHPLC for Practicing Scientists (John Wiley & Sons, Fellow at Purdue Pharma, and Senior Staff Scientist Hoboken, New Jersey, USA, 2nd Ed., 2019), Chapters 4, 7, 8, and 11. at Applied Biosystems/PerkinElmer. He holds a 3) S. Finali, P. Haddad, C. Poole, P. Schoenmakers and PhD in analytical chemistry from City University of D. Lloyd, Eds., Milestones in the Development of Liquid New York. He has more than 100 publications and a Chromatography, in Liquid Chromatography (Elsevier, best-selling book in chromatography. He is an editorial Amsterdam, The Netherlands, 2013), Chapter 1. 4) C.H. Arnaud, Chem. & Eng. News 94(24), 29–35 (2016). advisory board member of LCGC North America and 5) D. Guillarme and M.W. Dong, Eds., Trends the Chinese American Chromatography Association. in Anal. Chem. 63, 1–188 (2014). Direct correspondence to: [email protected] 6) J. De Vos, K. Broeckhoven, and S. Eeltink, Anal. Chem. 88, 262 (2016). 7) J.W. Dolan, LCGC Europe 29(5), 258–261 (2016). 8) M.W. Dong, LCGC Europe 30(6), 306–313 (2017). 9) Agilent 1290 Infinity Binary Pump: User Manual, G4220-90006

c® atote cele m bra ro t h es Rev. E, Agilent Technologies, Waldbronn, Germany, 2015. C Online Gas Analyzer Experts 10) M.W. Dong, LCGC North Am. 35(11), 818–823 (2017). 40 YEARS OF EXPERTISE i n n o n o - a lin nt 11) A.G. Huesgen, Agilent 1290 Infinity Quaternary Pump, Technical e instrume Overview, 5991-1193EN, Agilent Technologies, 2012. ONLINE AUTOMATIC GAS CHROMATOGRAPHY 12) S. Ahuja and M.W. Dong, Eds., Handbook of Pharmaceutical Analysis ЇЉЈЃЎЉЌЃЈЁЉ 92&ЎЊЊЎІАІ by HPLC (Elsevier, Amsterdam, The Netherlands, 2005), Chapter 11. ЃЈІЎЂЈЇЃЈ 13) J. Dolan and L.R. Snyder, Troubleshooting LC Systems

(Humana Press, Totowa, New Jersey, USA, 1989). VOCs 6 + 3 - 14) P.C. Sadek, Troubleshooting HPLC Systems: A Bench Manual PCB (John Wiley & Sons, New York, New York, USA, 2000). BTEX 15) J. Dolan, “HPLC Troubleshooting”, Columns in LCGC 1983–2016. ' 292& 3$+ 16) U. Neue, HPLC Troubleshooting Guide, Waters Applications Notes, 3 720000181EN, Waters Corp., Milford, Massachusetts, USA, 2002. 17) M.W. Dong and B.E. Boyes, LCGC Europe 31(10), 572–583 (2018).

5REXVW3URFHVVPDVV Konstantin Shoykhet is a Senior R&D Scientist VSHFWURPHWHUXVLQJVSHFL¿F TXDGUXSROHFRXSOHZLWK at Agilent Technologies, in Waldbronn, Germany, DXWRPDWLF*&),' focusing on liquid chromatography and solvent 9HU\ ORZ PDLQWHQDQFH delivery. He holds a Ph.D. in chemistry from the RQOLQH*&06 University of the Saarland (Germany) in the field of 6SHFL¿FLQWHJUDWHGDOJRUWLWKPIRUDXWRPDWLF liquid chromatography and capillary electrophoresis. VHOHFWLRQRIWKHULJKWFRQFHQWUDWLRQZLWKRXW Ken Broeckhoven is an Associate Professor at DQ\GDWDWUHDWPHQW the Vrije Universiteit Brussel in the department of chemical engineering and bioengineering sciences. His research focuses on fundamental aspects www.chromatotec.com - [email protected]

www.chromatographyonline.com 419 POLYMER ANALYSIS FOCUS

Sizing Up Size-Exclusion Chromatography

André Striegel spoke to LCGC Europe about innovations in size-exclusion chromatography (SEC) in polymer analysis, including the benefits of hyphenating SEC with on-line multi-angle static light scattering (MALS) and differential refractometry (DRI) detection, the non-trivial nature of a “trivial” solution, the complementary value of “interaction” polymer liquid chromatography (LC) techniques, and the role of quintuple detection in practice.

Interview by Alasdair Matheson, Editor-in-Chief, LCGC Europe

André Striegel is a Q. What are the benefits of using SEC– Q. What are the limitations Research Chemist MALS–DRI in polymer analysis and of SEC–MALS–DRI? in the Chemical where is this technique being used? A: First, it is important to remember that, Sciences Division A: Size-exclusion chromatography because separation in SEC is based on of the Material (SEC) with on-line multi-angle static the size of the analytes in solution (their Measurement light scattering (MALS) and differential so-called hydrodynamic volume) (1), Laboratory. He received his Ph.D. in refractometry (DRI) detection is a very analytes with different architectures and analytical chemistry in 1996 and his powerful technique. The most common chemistries can coelute if they occupy B.S. in chemistry in 1991, both from the application is for obtaining the “absolute” the same hydrodynamic volume as University of New Orleans. From 1996 molar mass (M) averages and molar each other. I will talk about how to deal to 1998 he performed postdoctoral mass distributions (MMDs) of polymers, with this in more detail when I discuss research for the U.S. Department of which is more accurate than reporting interaction polymer chromatography. Agriculture, at the National Center for values that are relative to a calibration Second, and again because SEC is Agricultural Utilization Research. For standard with little architectural and/ a solution-phase technique, the solvent the next six years he worked for Solutia or chemical relation to your analyte. (or carrier liquid, in the case of colloidal (now Eastman Chemical), achieving SEC–MALS–DRI can also be suspensions) must be compatible the rank of Research Specialist. used to glean information about with the SEC column packing material From 2004 to 2011 he was assistant macromolecular architecture, and should not cause appreciable professor of both analytical and including polymer topology to (because there will always be some) materials chemistry in the Department assess whether a polymer is chemical or, more appropriately, of Chemistry and Biochemistry at linear or branched; dilute solution enthalpic interactions between the Florida State University (FSU). In conformation to confirm whether a analyte and the column packing. September 2011 he joined the National linear polymer adopts a “rod-like” The solvent should also allow for Institute of Standards & Technology or “random-coil-like” conformation analyte solutions to have good optical (NIST), where he is a Research at a given set of solvent and contrast with the neat solvent, so that Chemist in the Chemical Sciences temperature conditions; and dilute the detectors can generate peaks with Division of the Material Measurement solution thermodynamics to identify adequate signal-to-noise ratios (S/N). Laboratory. His research interests are in if a dilute polymer solution is at Another limitation of the technique the area of polymer characterization, in good, poor, or theta conditions. is that most non-destructive particular applying separation science SEC–MALS–DRI can also be used chromatographic detectors, including to determining structure-property to measure the size distribution of refractometers, light scattering relations of complex macromolecules, macromolecules and the statistical photometers, and viscometers, are and in the fundamental aspects of averages of this distribution, and differential in nature, that is, they separation and detection methods. a host of other polymeric and measure the response difference dilute solution properties. between the analyte solution

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and the solvent. Non-differential refractive index n0 of the solvent (for to that of the light sources in the MALS detectors are usually both the DRI it’s a first-power dependence and DRI detectors (which, it should be destructive and display a non-linear, on n0, whereas for MALS it’s a squared mentioned, if not identical should match empirically-governed response. dependence). Consequently, if one each other pretty closely to avoid For example, a differential can find an isorefractive solvent pair, introducing an additional source of refractometer measures the difference that is, a pair of solvents with the same error). There are tables of isorefractive between the refractive index of the refractive index as each other, then solvents in the literature, but it must solution being analyzed and that the problem of preferential solvation is be kept in mind that isorefractivity of the solvent used to prepare the obviated. In this case, for our generic does not imply miscibility. So, we first solution. This tends to preclude the polymer dissolved in a 50:50 mix of had to find solvent pairs with fairly 20 use of mixed solvents for accurate generic solvents A and B, it won’t similar nD values, for which we used determination of molar mass averages matter if, within the hydrodynamic the published tables. Second, we and distributions, inter alia, not only in volume of the polymer in solution, minimized the difference between the SEC but also in any macromolecular the solvent ratio is or isn’t 50:50: In refractive indices of the two solvents liquid chromatography (LC) or either case, the refractive index of the by slowly changing the temperature, fractionation method. Let me elaborate solvent mix within the hydrodynamic in 1 ºC increments, until the refractive on this. When using mixed solvents, volume of the polymer will be the index difference was less than 0.001; we encounter what is known as the same as that outside this volume. This this took advantage of the fact that our preferential solvation problem: the means that the solvent baseline now differential refractometer also has the polymer prefers to be solvated more by represents an accurate contribution of ability to measure absolute refractive one of the solvents in the mix compared the solvent mix to both the MALS and indices. Third, we had to make sure the to the other solvent. For example, if DRI detector peaks. I refer to this as two solvents were miscible with one the polymer is dissolved in a 50:50 the “trivial” solution to the problem. another in all proportions, for which mix of solvents A and B, within the we initially relied on both published hydrodynamic volume of the polymer The most common solvent miscibility charts and our own the solvent ratio may be 80:20, or 35:65, experience when making choices, or, basically, something not 50:50. application of SEC– followed by miscibility experiments in Given the aforementioned differential MALS–DRI is to the laboratory at various solvent ratios. nature of most macromolecular LC obtain the “absolute” Fourth, we had to make sure that the detectors, in the case of preferential molar mass (M) individual solvents, as well as mixes solvation the solvent baseline will averages and molar thereof, would actually dissolve our no longer accurately represent the mass distributions intended analytes. For the latter, we solvent mixture’s contribution to the chose polystyrene (PS) and poly(methyl (MMDs) of polymers. detectors’ analyte peaks. This will result methacrylate) (PMMA) because there in the erroneous calculation of many are well-characterized narrow-MMD parameters, most notably in erroneous As to the challenges behind this standards for both of these spanning molar mass averages and distributions. seemingly trivial approach and how a wide molar mass range. we went about overcoming them, Our particular experiments covered Q. You attempted to combat the first one must find a pair of solvents a fourfold range in M, and we could preferential solvation problem isorefractive at a particular temperature do a “quick-and-dirty” evaluation mentioned previously using an and wavelength (remember that, in of solubility using an ultrahigh M isorefractive solvent pair as a most tabulations, refractive indices sample of each. If a standard with 20 6 -1 mixed solvent (2). Can you explain are usually given as nD values, M > 10 g mol dissolved, it would be the concept behind this approach, meaning that they have been natural to assume that samples with the challenges you encountered, determined at 20 ºC at a wavelength of M lower than this would also dissolve. and how you overcame them? 589.29 nm corresponding the average This assumption was turned on its A: Well, with an SEC–MALS–DRI wavelength of the sodium D-line head by one of our supposedly atactic approach one has two detectors, the doublet). Unfortunately, in the case of PMMA samples, which led to our response of which depends on the wavelength, we are pretty much limited discovery that it contained a significant www.chromatographyonline.com 421 POLYMER ANALYSIS FOCUS

percentage of isotactic triads. That chromatography (GPEC)—which by size utilizing, for example, is a story for another day, however! some people have recently taken to SEC in the second dimension. We also had to choose a column calling solvent gradient interaction Employing both physical (MALS) packing material that did not chromatography (SGIC)—and and chemical (IR, NMR) detectors, interact chemically with the polymer temperature gradient interaction this 2D-LC experiment will map the solutions, which ensures elution by a chromatography (TGIC), among others. physicochemical phase-space of the predominantly entropically-controlled, material, showing the co-dependence size-exclusion-based mechanism. of polymer chemistry on molar mass, Lastly, we needed solvents with a SEC–MALS–DRI can both as continuous functions of refractive index different enough from also be used to glean each other. This is a very powerful that of the polymer so that the dilute information about approach to understanding and solutions would show good optical macromolecular solving macromolecular problems, contrast with respect to the solvents architecture, including though still a challenging one from to generate large S/N MALS and DRI a method-development standpoint peaks. As you can see, the challenge polymer topology. really was anything but “trivial”! Q. You also developed a method The main advantage of these to characterize copolymers and Q. Are you planning to explore the techniques is their ability to separate blends by “quintuple detector” use of other isorefractive solvent polymers based on chemical SEC (4). What detectors did you pairs, or develop this research differences. As such, they are most incorporate and why did you further in any other way? advantageous for separating the embark on this research? A: We will probably leave more applied different chemistries present in a A: This method included on-line exploration of isorefractive pairs to macromolecular sample (for example, MALS, quasi-elastic light scattering those more heavily involved than we separating the methyl methacrylate (QELS, also sometimes referred to are in interactive polymer LC. At the from the styrenic component in a as dynamic LS), viscometry (VISC), moment, my focus in this area in on PS-PMMA copolymer or blend). One DRI, and UV detection. Each detector trying to solve the “non-trivial” problem, can find wonderful applications of brings its own individual advantages that is, on how to obtain accurate M these techniques in the literature into the mix—and its own challenges. averages and MMDs by SEC when (3). One major challenge to their Equally, or more importantly, is using a mix of non-isorefractive widespread use, however, has how the detectors can combine solvents. We are just starting to scratch been the difficulty in laying out a with each other synergistically to the surface of this problem. All I can first-principles-based, non-empirical better inform our understanding of tell you right now is that calling it approach to method development. complex polymers and of blends. “non-trivial” is definitely appropriate! This is another area in which we are What this particular SEC–MALS– working here at the National Institute QELS–VISC–DRI–UV set-up allowed Q. What are “interaction” polymer of Standards and Technology (NIST). us to do, when studying a random LC techniques and how do they Interaction LC methods can truly copolymer of acrylamide and N,N- complement a more popular complement size-based separations dimethylacrylamide as well as blends technique such as SEC? such as SEC, HDC, and flow of the individual homopolymers, was A: Interaction polymer LC refers to field-flow fractionation (flow FFF) to see how both the monomeric techniques that separate predominantly for the so-called comprehensive ratio and the polymer architecture on the basis of an enthalpic difference characterization of complex samples, changed continuously across the (chemical interaction) between the which include most polymers either MMD, providing a window into the solution and the stationary phase (as employed by, or produced by, industry. underlying chemical causes behind opposed to SEC and hydrodynamic One can separate the components the architectural changes. One chromatography (HDC), where the of a sample based on its chemical reason for embarking upon this separation is based on entropic constituents by interaction LC in the research was to more fully apply differences). This includes methods first dimension of a two-dimensional our available suite of detectors such as gradient polymer elution separation (2D-LC), then separate to obtain as comprehensive as

422 LCGC Europe August 2019 POLYMER ANALYSIS FOCUS

possible (within the framework of understand both processing and introduction of on-line MALS and the instrumentation in our laboratory end-use properties of the materials viscometry in the 1980s and their at the time) a macromolecular-level examined. For example, chemical popularization in the 1990s, the picture of a polymer, so that others heterogeneity is directly related ability to determine absolute molar could later take our approach to properties such as conductivity, masses was realized (in the case and apply it to their analytes. adsorptivity, and interfacial strength, of viscometry, by applying Benoit’s while molar mass and architecture universal calibration principle [8]). Q. What were the main analytical can affect elongation, tensile strength, Since then, most practitioners have challenges you overcame encapsulation ability, and diffusion, fully embraced or, at least, understand developing quintuple detection? among many other properties. the need for multiple detection, A: Part of the challenge involved trying So, as I mentioned previously, one especially for the combination of to find the proper solvent conditions to of our prime motivators for embarking physical and chemical detectors minimize the polyelectrolytic character upon this research was to provide within a single analysis. Nowadays, I of the solutions, which can influence others in the field with a method, think the most exciting developments both the separation as well as the data or an example of a method, that have been in incorporating seemingly quantitation. We then had to make would give them a deeper, more unusual analytical techniques as sure that, for each chromatographic comprehensive understanding of SEC detection methods (for example, slice eluting from our SEC columns, complex polymers. We have recently small-angle X-ray scattering, dynamic all five different detectors “spoke to seen the same quintuple-detector surface tension, depolarized each other” and did so for the same approach we employed with SEC light scattering), thus expanding slice. Lastly, after quantitating the used with FFF—also to study complex our characterization toolbox, and chemical changes across the MMD polymers (5)—so our work does in the integral role SEC plays as well as the architectural changes, already appear to be helping polymer in most 2D-LC macromolecular we had to try and discern the causal analysts gain a better understanding separations. While the abundance relationship between the former and of different types of macromolecules. of unreliable SEC molar mass data the latter. The project thus involved, in the literature is alarming, I am at a fairly deep level, both analytical Q. Do you have any general heartened by the growing realization chemistry and polymer science, which comments on the evolution of SEC? among polymer scientists that has been a hallmark of most of my Are there any recent developments this needs to change and that the career. Fortunately, I had in my group that you find particularly innovative twin pillars of analytical chemistry, at the time (this was a project that or new application areas where it precision and accuracy, form the began during my time in academia, is being more commonly used? foundation upon which our materials and finished at NIST) a brilliant A: At 55 (6), SEC is decidedly a knowledge can build and flourish. graduate student, Steven Rowland, “middle-aged’ technique. Back in now at the National Renewable Energy 1972, after SEC had been around References Laboratory In Colorado, who was not for only eight years, Gus Ouano 1) A.M. Striegel, W.W. Yau, J.J. Kirkland, and D.D. Bly, Modern Size-Exclusion Liquid only up to the challenge but also took wrote that “With the introduction of Chromatography, 2nd ed., (Wiley, 2009). this project way beyond what I had gel-permeation chromatography 2) A.M. Striegel and P. Sinha, Anal. Chim. originally foreseen. In the end, I’m [size-exclusion chromatography] Acta 1053, 186–195 (2019). certain I learned more from Steven (GPC) by Moore, molecular weight 3) H. Pasch and B. Trathnigg, Multidimensional on this project than he did from me! distribution data for polymers took a HPLC of Polymers (Springer-Verlag, 2013). sudden turn from near nonexistence 4) S.M. Rowland and A.M. Striegel, Anal. Chem. 84, 4812–4820 (2012). Q. Can you illustrate the to ready availability” (7)—such was 5) U.L. Muza and H. Pasch, Anal. Chem. (in benefits of quintuple detection the immediate impact of the technique. press). DOI: 10.1021/acs.analchem.9b01384 and how it benefits polymer In those days, and for a couple 6) J.C. Moore, J. Polym. Sci. A 2, 835–843 (1964). analysts in practice? decades thereafter, most molar mass 7) A.C. Ouano, J. Polym. Sci. A-1 10 A: The type of chemical and information was calibrant-relative , 2169–2180 (1972). 8) Z. Grubisic, P. Rempp, and H. Benoit, J. physical information obtained by and, thus, had to be taken with Polym. Sci. C Polym. Lett. 5, 753–759 (1967). this approach can be used to better more than a hint of caution. With the www.chromatographyonline.com 423 COMPANY PROFILE Advertisement Feature

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commercial-scale chromatographic separations. Chiral Company Description Technologies Europe also provides column selection services, Chiral Technologies Europe (CTE) is a subsidiary of method development, and custom separation services Daicel Corporation, a world leader in the supply of chiral using HPLC, SFC, and simulated moving bed systems. chromatography columns. The company offers the largest portfolio of chiral phases for the separation of pharmaceutical, Facilities agrochemical, and fragrance molecules. Its range of A team of expert chromatographers develop methods phases includes immobilized and coated polysaccharides, and new applications and provide technical support protein-based, crown ethers, and ion-exchange columns. The from the ofﬁ ce, research lab, and separation service company is the technical support and supply centre for Europe centre in Illkirch, France. There are similar facilities in and provides a free method development service, in addition West Chester, Pennsylvania, USA; Hyderabad, India; to custom separation services. Daicel Corporation products are and Shanghai, China, in addition to facilities in Japan. the most widely used and the most referenced chiral phases worldwide for the separation and analysis of chiral compounds.

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Chief Chromatographic Techniques Supported • High performance liquid chromatography (HPLC) • Gel permeation chromatography (GPC) • Gas chromatography (GC) Facilities • Capillary electrophoresis (CE) DataApex ofﬁ ces are located in Prague, the Czech Republic. • Mass spectrometry (MS)

Markets Served DataApex’s chromatography software can be used in any market where chromatography instruments are deployed. DataApex also focus on OEM cooperation with middle-sized chromatography instruments’ manufacturers.

Major Products/Services Clarity Chromatography SW can enable control of hundreds of different instruments (including Agilent, Hitachi, Shimadzu etc.) from one environment. Clarity is multilingual; users can switch among six languages: English, Chinese, Russian, Spanish, French, and German. Clarity offers easy operation, unmatched free user support including free SW updates, optional extensions that support a variety of applications (PDA, MS, GPC, NGA, DHA, SST, etc.) and competitive DataApex pricing. Clarity OEM versions are available. A free demo Petrzilkova 2583, 15800 Prague, The Czech Republic version is available from DataApex’s web pages. E-mail [email protected] Website www.dataapex.com Number of Employees 39 Year Founded 1991

432 LCGC Europe August 2019 Advertisement Feature COMPANY PROFILE

Dr.Maisch HPLC GmbH

Company Description Dr.Maisch offers an Dr.Maisch HPLC GmbH manufactures the ReproSil OEM HPLC/UHPLC Silica. With knowledge of modifying silica, the company column bonding and can offer a solution for every problem in LC or SFC. The packing service and company portfolio offers products from UHPLC through produces a wide to large prep LC; the biggest prepacked columns range of high quality are currently 200 mm internal diameter (i.d.). columns for other ReproSil columns are designed to meet the demands chromatography of chromatographers working in all industries including companies. pharmaceutical and biotechnology companies, universities, hospitals, research institutions, government agencies, Facilities and the environmental and process control areas. Dr.Maisch is a well known company Chief Chromatographic Techniques Supported with extensive • UHPLC HPLC and UHPLC • HPLC column bonding • Prep LC experience. The • SFC manufacturing facility • Process scale is headquartered • Silica bulk in Germany. • Core–shell All products from Dr.Maisch are available • DAC columns through the worldwide distributor network. • MODcol multipacker • LongLife DAC columns • Chiral

Markets Served With a worldwide network of distributors, Dr.Maisch columns are available to chromatography users in laboratories all over the globe. Industries served include the pharmaceutical, chemical, clinical, biotechnology, Dr.Maisch HPLC GmbH universities, food and beverage, environmental, and Beim Brueckle 14, 72119 Ammerbuch, Germany many more. Dr.Maisch HPLC also serves high quality OEM HPLC solutions for their partners. E-mail [email protected] Major Products/Services Dr.Maisch manufactures quality products from UHPLC Website to prep LC and has several patents on their prep www.dr-maisch.com hardware. Dr.Maisch brands include ReproSil, Ultrasep, Number of Employees GromSil, and the recently acquired ranges from Grace/ 30 Alltech—Vydac, Adsorbosphere, Allphabond, Allsphere, Econosphere, GraceSmart, Platinum, and VisionHT. Year Founded With an own patented DAC System end users can pack 1996 their own high-quality prep columns up to 150 mm i.d. www.chromatographyonline.com 433 COMPANY PROFILE Advertisement Feature

Ellutia

Company Description Ellutia offers a range of gas chromatographs, GC accessories, software, and consumables. Ellutia’s instruments are designed and manufactured in the UK at the company headquarters. The instruments are designed to be compact with great energy efficiency, whilst also delivering industry standard analytical performance. Ellutia works closely with its customers and regularly produces fully customized systems tailored to their individual requirements rather than delivering off-the-shelf products. It is this working relationship with customers and the flexibility to adapt • High Purity Gas Analyzer: A rack-mounted GC for that sets Ellutia apart from other GC manufacturers. detecting impurities in gas to extremely low levels. • 800 Series TEA: Nitro, nitroso, and nitrogen detector Chief Chromatographic Techniques Supported that can be interfaced with almost any GC system. • Gas chromatography • Ellution Chromatography Data Station: Software for • Ultra-fast gas chromatography collecting and processing data from a GC system. • Multidimensional chromatography • 7000 Flowmeter: A compact handheld flowmeter • Hyphenated chromatography techniques for accurately setting flows on a GC system.

Markets Served Facilities Ellutia’s products can be found in a wide range of industries Ellutia’s new purpose-built headquarters are located including: just outside Cambridge in the UK. This facility hosts the • Brewing and malting main research and development laboratories and is • Food testing where all of the instruments are manufactured and tested. • Packaging testing Outside of the UK, Ellutia also has an office in Germany • Permeation testing and a sales and service organization in the USA. • Education • Forensic laboratories • Agro-chemical • Cosmetics • Cannabis analysis • Toy production • High purity gas Ellutia • Natural gas Colston House, 200 Lancaster Way Business • Environmental testing Park, Ely, Cambridgeshire, CB6 3NX, UK • Defense and security E-mail [email protected] Major Products/Services Website • Custom chromatography systems: Ellutia will work with the www.ellutia.com customer to develop bespoke chromatography solutions Number of Employees that cannot be addressed by off-the-shelf instruments. Europe: 21 • 200 Series GC: A compact and versatile Outside of Europe: 3 single channel gas chromatograph. Year Founded • 500 Series GC: Unique gas chromatography allowing 1994 conventional, fast, and ultra-fast chromatography.

434 LCGC Europe August 2019 Advertisement Feature COMPANY PROFILE

GERSTEL

Company Description GERSTEL develops and produces automated sample preparation and sample introduction accessories for GC–MS and LC–MS. GERSTEL technology enhances productivity, reduces solvent use, and significantly improves detection limits. GERSTEL is the leading Agilent Technologies Premier Solutions Partner worldwide, providing modules and • Pyrolysis complete systems with integrated software control. GERSTEL • Cooled injection system (CIS), PTV-type inlet accessories are compatible with all standard systems. • Automated Liner EXchange (ALEX) for QuEChERS • Multidimensional GC, olfactory detection port Chief Chromatographic Techniques Supported (ODP), preparative fraction collection (PFC) • Autosamplers for GC–MS and LC–MS • Extraction, solvent evaporation, weighing • Automated sample preparation for GC–MS and LC–MS • GERSTEL SPE based on standard dimension cartridges • Filtration, centrifugation, and evaporation • Online SPE with miniaturized replaceable cartridges • Liquid addition, derivatization, generating standards • MAESTRO software—stand-alone or integrated operation • Automated solid-phase extraction (SPE) • Application support and special solutions upon request • On-line SPE with disposable cartridges (SPExos) • Dynamic headspace, thermal desorption, and pyrolysis Facilities • Headspace and solid-phase microextraction (SPME) Steady growth over 50 years has resulted in the need for • Twister and Stir Bar Sorptive Extraction (SBSE) ever-larger facilities. The energy-efficient GERSTEL headquarters • PTV and large-volume injection in Mülheim an der Ruhr, Germany, were inaugurated in 2007 • Automated Liner Exchange (ALEX) with room to grow. Offices with application laboratories are • Multicolumn and multidimensional GC systems located in Germany, the US, China, Singapore, and Japan. • Olfactory detection and preparative fraction collection These, along with partner laboratories in other territories, enable GERSTEL to support its unique solutions worldwide. Markets Served • Food, beverage, flavour, and fragrance • Personal care and cosmetics • Forensic • Metabolomics • Polymers and packaging • Automotive material emissions GERSTEL • Environmental and industrial hygiene Eberhard-Gerstel-Platz 1, 45473 Mülheim an der Ruhr, • Pharmaceuticals Germany E-mail Major Products/Services [email protected] • MultiPurpose Sampler (MPS): Autosampler and Website sample preparation robot for GC–MS and LC–MS www.gerstel.com • Filtration, Centrifugation, Addition of standards Number of employees • Thermal desorption of VOCs/SVOCs up to n-C40+ Europe: 150 • Headspace and solid-phase microextraction (SPME) Outside of Europe: 50 • Dynamic HeadSpace (DHS) concentration of Year Founded VOCs and SVOCs from liquids or solids 1967 • Stir Bar Sorptive Extraction (SBSE), GERSTEL Twister®

www.chromatographyonline.com 435 COMPANY PROFILE Advertisement Feature

HILICON AB

Company Description HILICON offers a broad range of innovative hydrophilic interaction liquid chromatography (HILIC) products for the separation of polar and hydrophilic compounds. As illustrated in the company name, it focuses on the HILIC chromatography business. Three different column chemistries in UHPLC and HPLC formats, iHILIC®-Fusion, iHILIC®-Fusion(+), and iHILIC®-Fusion(P), provide customized and complementary selectivity, excellent durability, and ultralow column bleeding. The columns are versatile for the LC–MS analysis of polar compounds in “omics” research, food and beverage analysis, pharmaceutical discovery, environmental studies, and clinical diagnostics. In addition, HILICON also offers iSPE®-HILIC cartridges and 96-well plates for HILIC sample preparation in solid phase extraction. These are very useful for studies Facilities in glycans, glycopeptides, and other polar compounds. HILICON started its business in Umeå Biotech Incubator inside Uminova Science Park in Umeå, Sweden, in Chief Chromatographic Techniques Supported 2014. In 2018, we moved out from the incubator and • HILIC steadily grew our HILIC chromatography business and • UHPLC facility size in Uminova Science Park. We will move • HPLC into a new wholly renovated site at the end of 2019. • LC–MS • HILIC SPE

Markets Served Our high-performance HILIC products are used in various applications related to the analysis of polar compounds. The major markets are: • Omics research, that is, metabolomics, proteomics, lipidomics, glycomics • Pharmaceutical HILICON AB • Clinical diagnostics Tvistevägen 48A, SE-90736 Umeå, Sweden • Life science • Food and beverage E-mail • Environmental [email protected] • Forensic Website Major Products/Services www.hilicon.com • iHILIC®-Fusion, silica-based column, pH 2–8; 1.8, 3.5, and 5 μm Number of Employees • iHILIC®-Fusion(+),silica-based column, Europe: Under 10 pH 2–8; 1.8, 3.5, and 5 μm • iHILIC®-Fusion(P), polymer-based column, pH 1–10; 5 μm Year Founded • iSPE®-HILIC, in single cartridge and 96-well plate 2014

436 LCGC Europe August 2019 Advertisement Feature COMPANY PROFILE

KNAUER Wissenschaftliche Geräte GmbH

Company Description Based in Berlin, Knauer has been serving the sciences since 1962. With a highly qualified staff of 140 employees, of which 40% are women, we develop and manufacture scientific instruments of superior quality like liquid chromatography systems and components, including: • Analytical HPLC/UHPLC • Preparative HPLC • Fast protein liquid chromatography (FPLC) • Multi-column chromatography/SMB • Osmometry Knauer valves, pumps, detectors, and other components are also used for many liquid handling processes aside from HPLC and are popular with industry customers who want custom solutions (OEM). • Method development service We support technological advancement • Maintenance programs today and in the future. • KNAUER academy • Rent-an-expert Chief Chromatographic Techniques Supported • Analytical HPLC and UHPLC Facilities • Preparative HPLC Knauer develops, manufactures, and markets its complete • Fast protein liquid chromatography (FPLC) line of instruments at its headquarters in Berlin, Germany. • Simulated moving bed chromatography (SMBC) The products are distributed to more than 60 countries • Sample preparation through a worldwide network of dealers, who receive regular training in Berlin. Sustainability and corporate Markets Served responsibility are a matter of the heart to Knauer. Knauer HPLC systems and components are used in research and development, quality control, and production laboratories around the world. Customers are typically governmental and industrial organizations as well as universities. The main fields of application include chemical, pharmaceutical, life science, and food research (analysis and purification) as well as the environmental sector. Knauer also manufactures most of its product range for a number of OEM customers. KNAUER Wissenschaftliche Geräte GmbH Hegauer Weg 38, 14163 Berlin, Germany Major Products/Services E-mail • AZURA Analytical HPLC and UHPLC systems [email protected] providing powerful and adaptable solutions Website • AZURA Bio purification systems for www.knauer.net protein cleaning tasks (FPLC) Number of Employees • AZURA Preparative HPLC for small molecule purifications 140 • AZURA SMBC systems for continuous chromatography Year Founded • High-pressure dosing/metering pumps 1962 • Flow-through monitoring/detection www.chromatographyonline.com 437 COMPANY PROFILE Advertisement Feature

LECO Corporation

Company Description For over 80 years, industries around the world have trusted LECO to deliver high-performance analytical instrumentation. Today that commitment continues with high-speed time-of-flight mass spectrometry (TOF-MS) for liquid and gas chromatography, as well as comprehensive two-dimensional gas chromatography (GC×GC), all featuring our easy-to-use ChromaTOF® brand operating software. An independent, family-owned company, LECO is dedicated to serving our customers—from concept to design, development to manufacture, to customer service and support after the sale.

Chief Chromatographic Techniques Supported • GC–TOF-MS (low resolution) • GC–TOF-MS (high resolution) • GC×GC–TOF-MS (low resolution) Facilities • GC×GC–TOF-MS (high resolution) Headquartered in the USA, LECO supports nine sales and • GC×GC–FID services offices throughout Europe (Sweden, UK, Germany, • GC×GC–ECD Spain, Italy, France, Ukraine, Czech Republic, and Russia), with additional representation in Dubai. Our European Markets Served Application and Technology Center in Berlin, Germany, offers • Food safety application support, training, workshops, and hands-on • Environmental monitoring demonstrations to our customers throughout Europe. • Metabolomics • Petroleum • Flavour and fragrance • Forensics • Tobacco • Chemical analysis

Major Products/Services LECO Separation Science instruments are ideal for resolving complex samples or pioneering higher laboratory throughput. The Pegasus® GC-TOFMS platform was introduced in LECO Corporation 1994 as one of the first time-of-flight mass spectrometer’s 3000 Lakeview Avenue, St. Joseph, Michigan 49085, USA available in the marketplace. Today, low resolution TOF-MS E-mail is available with the new Pegasus BT GC-TOFMS, while the [email protected] Pegasus GC-HRT+ offers novel ultrahigh resolution TOF-MS. Website Both systems are available in GC×GC configurations. www.leco.com ChromaTOF software provides the tools you need to manage, Number of Employees process, qualify, quantify, and report data to your liking. Europe: 250 LECO also provides customer training, application support, Outside of Europe: 740 and technical service for all our separation science products. Year Founded 1936 in USA; first European office established in 1967

438 LCGC Europe August 2019 Advertisement Feature COMPANY PROFILE

MACHEREY-NAGEL GmbH & Co. KG

Company Description Since 1911, MACHEREY-NAGEL has represented high quality, innovation, and reliability in analytical chemistry and the life sciences. As one of today’s leading manufacturers, MACHEREY-NAGEL, a pioneer in thin-layer chromatography, MN offers a broad range of products for chromatography, is still manufacturing glass plates as well as aluminium and filtration, rapid tests, water analysis, and bioanalysis. plastic sheets coated with silica gels and other sorbents for TLC MN’ expertise in the manufacture of chromatography media and HPTLC. The latest developments, SIL HD and Nano-SIL HD is based on many years of experience in silica technology. The glass plates, are characterized by outstanding dyeability, good development of the spherical HPLC silica NUCLEOSIL® and the wettability, and abrasion resistance as a result of an optimized introduction of commercially available ready-to-use TLC plates binder system. More information: www.mn-net.com/TLC and sheets are just two milestones emphasizing the pioneering Classical CHROMABOND® silica gel phases with tasks of MN in promoting these separation techniques. various modifications (for example, C18, C8, NH2, OH, CN) and modern polymeric resins are widely used for sample Chief Chromatographic Techniques Supported preparation in environmental analysis, food safety control, • SPE and flash chromatography or clinical applications. The hydrophilic-lipophilic balanced • HPLC polymeric SPE adsorbent CHROMABOND® HLB and the • TLC CHROMABOND® HR-Xpert line of polystyrene-divinyl based • GC reversed-phase and mixed-mode ion-exchange resins offer a • Chromatographic accessories full toolbox of innovative SPE products. (for example, syringe filters, vials, and caps) More information: www.mn-net.com/SPE

Markets Served Facilities MN serves laboratories all over the world with HPLC, Branches in France, Switzerland, and the United States, as well GC, and SPE columns, TLC plates and sheets, syringe as a globally operating network of distributors in more than 150 filters or suitable vials and caps. Customers from many countries, ensure worldwide availability of MN products and different industries (for example, the food and beverage services. industry, pharmaceutical industry and healthcare, chemical industry, institutes for environmental analysis, biotechnology, universities, and research institutes) benefit from optimal and reliable solutions for analytical tasks in method development and routine analysis. MACHEREY-NAGEL GmbH & Co. KG Major Products/Services Neumann-Neander-Str. 6-8, 52355 Düren, Germany NUCLEOSIL®, one of the first spherical silica gels in the E-mail 1970s, is still packed in stainless steel columns by MN. These [email protected] columns with various C18 modifications are reliable workhorses Website and are validated in many drug testing methods. In recent www.mn-net.com years, new highly pure and ultra-spherical HPLC and UHPLC Number of employees silica gels followed. Today, the product range of totally porous Europe: 630 NUCLEODUR® and fused-core NUCLEOSHELL® columns Outside of Europe: 20 comprise more than 25 stationary phases, targeted to daily Year Founded routine analysis as well as for method development in HPLC. 1911 More information: www.mn-net.com/HPLC www.chromatographyonline.com 439 COMPANY PROFILE Advertisement Feature

Markes International

Company Description Markes International, an industry leader in technology for trace organic analysis, manufactures a range of instrumentation and software that enhances the analytical capability and productivity of GC–MS systems.

Chief Chromatographic Techniques Supported • Thermal desorption (TD) as a sample concentration • Centri: A multi-technique sample preconcentration and introduction technique for GC and injection platform, delivering enhanced • Automated sample preparation and concentration analytical sensitivity and higher throughput (headspace, headspace-trap, SPME, SPME-trap, and high capacity sorbent extraction) Facilities • Time-of-flight mass spectrometry Markes International’s factory, technical centre, and (TOF-MS) for GC and GC×GC headquarters are located near Cardiff, UK. The company • GC–MS data reprocessing software also has technical centres in Germany, USA, and China. It also supports a global distributor network. Markes is a Markets Served company of the Schauenburg International Group. • Defence/Homeland security • Environmental • Food and fragrance • Forensic and toxicology • Metabolomics • Petrochemical Markes International Ltd. Markes’s global customer base includes major industry, Gwaun Elai Medi-Science Campus, Llantrisant, RCT, UK government agencies, academia, and the contract service Markes International GmbH laboratory sector. Bieberer Straße 1–7, 63065 Offenbach am Main, Germany Markes International, Inc. Major Products/Services 2355 Gold Meadow Way, Gold River, California, USA Markes is globally recognized for its innovation, high-quality Markes Instruments (Shanghai) Co., Ltd products, unrivalled technical expertise, and high level of No. 1 Building, No. 7 Guiqing Road, Xuhui District, customer service within the field of analytical TD. As a global Shanghai 200233, P.R. China technology leader of thermal desorption for GC, Markes has Telephone introduced many highly successful products to the analytical +44 (0)1443 230 935 (UK) laboratory: +49 (0)69 6681089-10 (Germany) • UNITY-xr: A universal thermal desorption +1 866 483 5684 (toll-free) (USA) unit for sorbent sampling tubes +86 21 5465 1216 (China) • TD100-xr: A fully automated TD system for sorbent tubes E-mail • ULTRA-xr: TD autosampler [email protected] • CIA Advantage-xr systems for automated cryogen-free Website analysis of air and gas sampled using canisters www.markes.com • TT24-7 for continuous on-line monitoring Number of employees • The Micro-Chamber/Thermal Extractor for fast 140 sampling of emissions from products and materials Year Founded • Wide range of accessories and consumables for TD 1997

440 LCGC Europe August 2019 Advertisement Feature COMPANY PROFILE

Molnár-Institute for applied chromatography

Company Description Founded in 1981 and located in Berlin, Germany, Molnár-Institute develops DryLab software for high performance liquid chromatography (HPLC and UHPLC). Chromatographers performing analytical method development use DryLab to reduce retention time, increase method robustness, and conform to quality by design (QbD) standards. Molnár-Institute is registered UHPLC-modeling software vendor to the FDA, CDC, CFIA, and other regulatory agencies. DryLab pioneered the analytical quality by design paradigm changes by the FDA in 2002, leading to a new thinking about HPLC quality control and quality assurance.

Chief Chromatographic Techniques Supported • HPLC retention modeling (reversed phase, normal phase, HIC, HILIC, IEX) • Gas chromatography

Markets Served Molnár-Institute is devoted to improving global healthcare and to ensuring the development of safe and effective products in the pharmaceutical, life science, and food industries worldwide.

Major Products/Services • DryLab—visual software for modelling robust and high-quality HPLC methods • Method development services • Training on the application of DryLab software for efficient HPLC method development.

Molnár-Institute for applied chromatography Schneegloeckchenstrasse 47, 10407 Berlin, Germany E-mail [email protected] Website http://molnar-institute.com/ Number of Employees 10 Year Founded 1981

www.chromatographyonline.com 441 COMPANY PROFILE Advertisement Feature

PharmaFluidics

Company Description Introducing micro-chip manufacturing technology in analytical chromatography, PharmaFluidics uses a lithographic etching process to create a perfectly ordered separation bed on a silicon chip. PharmaFluidics wants to bring liquid chromatography to a next level of efficiency in all aspects, by providing separation devices with an unprecedented reproducibility and robustness, in a format that enables extreme user-friendliness and resolution, and opens the way to further miniaturization and integration.

Chief Chromatographic Techniques Supported • Nano LC–MS

Markets Served The μPAC™ micro-chip-based chromatography columns are commercially available for proteomic, lipidomic, and metabolomic profiling, with specific applications in biomarker, diagnostics, and drug research and development. μPAC™ also enables a very detailed characterization of biopharmaceuticals and biosimilars, detecting amino acid substitutions and post-translational modifications.

Major Products/Services To serve your custom separation needs, μPAC™ columns are available in various lengths (50–200 cm) and are equipped with connectors matching your application. Stationary phase chemistry is currently focused on reversed-phase HPLC separations for applications in metabolomics, proteomics, and lipidomics. For fast and easy cleanup and enrichment of peptide samples prior to injection, we have a trapping column that is designed for optimal compatibility with PharmaFluidics’ μPAC™ reversed-phase analytical C18 nano-LC columns. Our μPAC™ capLC column will soon be launched onto the market. It should be used at a flow rate of 1–15 μL/min. PharmaFluidics Technologiepark-Zwijnaarde 82, B9052 Facilities Ghent (Zwijnaarde), Belgium PharmaFluidics’ headquarters are located at the Tech E-mail Lane Ghent Science Park (Campus A) in Ghent, Belgium. [email protected] International commercialization of the Website PharmaFluidics’ products is organized by either www.pharmafluidics.com a direct sales team in Europe or by indirect sales Year Founded channels in the US and the rest of the world. 2010

442 LCGC Europe August 2019 Advertisement Feature COMPANY PROFILE

Postnova Analytics GmbH

Company Description Postnova Analytics offers the worldwide unique FFF-Platform, which is a range of different field-flow fractionation (FFF) and light scattering systems (MALS) for advanced separation, fractionation, and characterization of nanoparticles, proteins, polymers, and bio-macromolecules. Postnova offers a truly complete FFF-MALS product range, which is widely used for applications in biopharmaceutical, nanotechnology, environmental, food, cosmetics, and polymer science. The Postnova FFF-Platform can also be ideally coupled to dynamic light scattering (Malvern Zetasizer Nano DLS) and inductively coupled plasma-mass spectrometry (Agilent Technologies ICP-MS), which provides high resolution size and molar mass separations as well as characterization and element speciation.

Chief Chromatographic Techniques Supported • Field-flow fractionation (FFF) • Asymmetric FFF • Thermal FFF • Centrifugal FFF • Multi-angle light scattering (MALS)

Facilities EMEAI Postnova Analytics GmbH Max-Planck-Strasse 14 86899 Landsberg, Germany Tel: +49 8191 985 688 0 Fax: +49 8191 985 688 99 [email protected]

UNITED KINGDOM Postnova Analytics UK Ltd. Unit 64, Malvern Hills Science Park, Malvern, Worcestershire WR14 3SZ, UK Tel: +44 1684 585167 Postnova Analytics [email protected] Max-Planck-Strasse 14, 86899 Landsberg am Lech, Germany NORTH AMERICA Postnova Analytics Inc. E-mail 230 South, 500 East, Suite 110 [email protected] Salt Lake City, UT 84102, USA Tel: +1 801 5212 004 Website Fax: +49 8191 98568899 www.postnova.com [email protected] www.chromatographyonline.com 443 COMPANY PROFILE Advertisement Feature

PSS GmbH

Company Description Perfect Separation Solutions for characterizing polymers, biopolymers, polysaccharides, and proteins. PSS is fully dedicated to the advancement of macromolecular liquid chromatography by developing true solutions and providing competent and personal support. Based on excellent products and cutting-edge technology, PSS designs easy-to-use and powerful systems for QC and R&D. From a single molar mass reference material, contract analysis, and Instrumentation and Software consulting, to turn-key systems for GPC/SEC multi-detection • SECcurity2 GPC/SEC, IPC, and 2D systems and components with light scattering, viscometry, mass spectrometry, or • Light scattering detectors fully compliant GPC/SEC for the pharmaceutical industry: • Viscometers PSS offers products and services required for successful • WinGPC UniChrom Macromolecular Chromatography Data System macromolecular analysis with expert support. • PoroCheck software for inverse GPC Analytical Service Lab Chief Chromatographic Techniques Supported • Contract analysis • Size-exclusion chromatography (SEC) • Molar mass determination • Gel permeation chromatography (GPC) • Branching/structure information • Gel filtration chromatography (GFC) • Method development and transfer • High temperature GPC • Complete product deformulation • GPC/SEC viscometry/light scattering/triple • Consulting • Interaction polymer chromatography (IPC) GPC/SEC Training and Support • Two-dimensional chromatography (2D) • Full services from installation to validation, operation, and repair • GPC/SEC, IPC, 2D, and software training schools Markets Served • GPC/SEC, IPC, and 2D in-house training Fully featured PSS applications provide solutions for • User meetings the analysis of natural and synthetic macromolecules divided into the following sections: Facilities • Biopharma PSS headquarters are located in Mainz, Germany. PSS also has • Chemical manufacturing offices in the USA and The Netherlands, and works with a network • Food analysis of distributor partners in most other countries around the world. • Medical analysis • Pharmaceutical analysis

Major Products/Services GPC/SEC Standards and Kits PSS GmbH • Certified reference materials In der Dalheimer Wiese 5, D-55120 Mainz, Germany • MALDI kits E-mail • Viscosity and light scattering validation kits [email protected] • ReadyCal kits Websites • Tailor-made polymers www.pss-polymer.com High Resolution Columns www.pss-shop.com • GPC/SEC columns for all organic eluents and aqueous eluents Year Founded • For high- and low-molecular-weight synthetic and biopolymers 1985 • For micro GPC/SEC up to preparative scale

444 LCGC Europe August 2019 Advertisement Feature COMPANY PROFILE

Restek Corporation

Company Description For over 30 years, Restek has been a leader in developing technologies and manufacturing products for gas and liquid chromatography (GC and LC), including columns, reference standards, sample preparation materials, accessories, and more. We have decades of hands-on, practical experience in chemistry, chromatography, and engineering, and our reputation for going the extra mile with Plus 1 customer service and top-performing products is well known • Innovative accessories, instrument throughout the chromatography community. Restek is proud replacement parts, and consumables to assist analysts around the world with monitoring the • Air monitoring canisters and sampling supplies quality and safety of air, water, soil, food, pharmaceuticals, • Sample prep products and petroleum. We proactively offer integrated solutions— • Reference standards: stock and products, applications, and assistance—perfectly matched custom-prepared formulations to your needs, regardless of your industry. www.restek.com • Thousands of innovative products, hundreds of chromatograms Chief Chromatographic Techniques Supported • UHPLC • GC Facilities • HPLC • GC–MS Restek opened for business in 1985 in a small business • LC–MS • GC×GC incubator in central Pennsylvania. Today, more than 500 employee-owners work, play, and celebrate Markets Served milestones in a state-of-the-art 140,000-square-foot • Air monitoring • Forensic facility in Pennsylvania, in our research facility in • Chemical • Industrial hygiene California, and in our subsidiary locations in China, • Clinical • Petrochemical England, France, Germany, Italy, and Japan. • Environmental • Pharmaceutical • Food safety

Major Products/Services Plus 1 Service in everything we do. Living this corporate core value every day ensures we will surpass your expectations Restek Corporation every time you contact us! Our customer service team will 110 Benner Circle, Bellefonte, Pennsylvania 16823, USA suggest time- and money-saving options and is dedicated Telephone to getting your products to you fast. Our technical service (814) 353 1300 chemists can help you from set-up to method development. Fax Visit our website where you can blog with our chemists (814) 353 1309 and review an extensive library of technical publications, E-mail chromatograms, product documentation, step-by-step guides, [email protected] interactive calculators, videos, and educational material. Website Restek’s commitment to continuous innovation in www.restek.com chromatography sets us apart from our competitors. We Number of employees introduce and stock hundreds of new products every year, 500+ designed by chromatographers for chromatographers. Year founded • Exceptional columns for UHPLC, HPLC, LC–MS, GC, 1985 GC–MS, and GC×GC www.chromatographyonline.com 445 COMPANY PROFILE Advertisement Feature

SepSolve Analytical Ltd

Company Description SepSolve Analytical is dedicated to helping analysts select the best equipment for GC and GC×GC analysis of complex mixtures. With many years of experience in the field and access to a range of leading equipment suppliers, we’re very well placed to advise on the most difficult challenges in analytical science—in everything from environmental monitoring to petrochemical analysis and food aroma profiling.

Chief Chromatographic Techniques Supported • GC and GC×GC (and associated software) • TOF-MS • FID • Sample preparation and concentration: Thermal desorption, SPME and SPME–trap, High-capacity sorptive extraction, Headspace and headspace–trap, Large-volume injection

Markets Served • Food and drink • Petrochemical • Fragrance • Environmental • Tobacco and e-cigarettes • Cannabis

Major Products/Services The wide range of products and techniques offered by SepSolve includes the company’s own INSIGHT® flow modulator, ChromSpace® software for GC×GC, and BenchTOF range of time-of-flight mass spectrometers with groundbreaking simultaneous hard- and soft-ionisation SepSolve Analytical Ltd technology—Tandem Ionisation®. 22 Commerce Road, Lynch Wood, Peterborough, SepSolve also offers sample preparation equipment, PE2 6LR, UK robotic autosamplers and thermal desorbers from leading E-mail global suppliers including CTC Analytics, GL Sciences, and [email protected] Markes International. Website www.sepsolve.com Facilities Number of employees SepSolve has offices and demonstration laboratories in 10 Peterborough, UK, and Waterloo, Canada, and works closely Year Founded with partners to support customers worldwide, with facilities 2016 in countries including the US, Germany, and China.

446 LCGC Europe August 2019 Advertisement Feature COMPANY PROFILE

Shimadzu Europa GmbH

Company Description are used in routine and high-end applications, Shimadzu is one of the worldwide leading manufacturers process, and quality control, as well as R&D. of analytical instrumentation, and for over 50 years, the European headquarters have been located in Duisburg, Major Products/Services Germany. The company’s equipment and systems are Nexera LC-40 Series—Experience New Benchmarks used as essential tools for research, development, Shimadzu’s new Nexera series of UHPLC systems offers and quality control of consumer goods in all areas of groundbreaking technology in terms of intelligence, pharmaceutical and environmental industries, food efficiency, and design. Advanced AI capabilities and safety testing, consumer protection, and healthcare, to laboratory management using the Internet of Things contribute to society through science and technology. (IoT) have been integrated to monitor performance and Chromatography, mass spectrometry, spectroscopy, life resource allocation. This makes the Nexera systems sciences, and material testing make up a homogeneous a cutting-edge and user-friendly solution for versatile yet versatile offering. Along with many “industry first” industries, setting new benchmarks in UHPLC. technologies and products Shimadzu has created and invented since 1875, there has also been the exceptional achievement of the 2002 Nobel Prize The new Nexera series of UHPLC systems offers groundbreaking technology in terms of intelligence, efficiency, for Chemistry to Shimadzu engineer Koichi Tanaka and design. for his outstanding contributions in the field of mass spectrometry. Shimadzu is focused on top quality when developing products, including ease of operation and optimum service. The company manufactures according to internationally renowned quality standards, including Pharmacopeia, ISO, FDA, GLP, and GMP.

Chief Chromatographic Techniques Supported • HPLC • SFC • SFE–SFC–MS • GC • LC–MS • GC–MS • Multidimensional chromatography • Comprehensive chromatography • Columns and consumables

Markets Served Shimadzu’s analyzers and equipment are applied in the food industry, clinical and pharmaceutical field, automotive industry, chemical, petrochemical, life LCMS-9030 Q-TOF—Greater Accuracy sciences and biotech, cosmetics, semiconductor, with Higher Sensitivity and nutrition industries, as well as in the flavours The LCMS-9030 quadrupole time-of-flight liquid and fragrances business. Research institutes, chromatograph mass spectrometer is a research-grade privately-run laboratories, administrations, and mass spectrometer designed to deliver high-resolution, universities complete the list of clients. The systems accurate-mass detection with incredibly fast data

www.chromatographyonline.com 447 COMPANY PROFILE Advertisement Feature

acquisition rates, allowing scientists to identify and quantify In the European headquarters in Germany, the Laboratory more compounds with greater confidence. It provides World provides testing and training facilities for customers a new solution for analyzing even the most complex from all over Europe. samples, and integrates the world’s fastest and most sensitive quadrupole technology with TOF architecture.

The new LCMS-9030 Q-TOF system provides greater accuracy with higher sensitivity.

Facilities Shimadzu operates production facilities and distribution centres in 74 countries. In the European headquarters Shimadzu Europa GmbH in Germany, the Laboratory World provides testing and Albert-Hahn-Str. 6-10, 47269 Duisburg, Germany training facilities for customers from all over Europe. With E-mail over 1500 m2 floor space, Shimadzu’s entire product range is [email protected] available—from chromatographs, spectrophotometers, TOC Website analyzers, mass spectrometers, and balances to material www.shimadzu.eu testing machines. In Europe, Shimadzu runs subsidiaries Number of Employees and branches in Austria, Albania, Belgium, Bosnia, Europe: 700 Bosnia-Herzegovina, Bulgaria, Croatia, Czech Republic, Outside of Europe: 11,000 France, Germany, Hungary, Italy, Luxembourg, Macedonia, Year Founded Montenegro, Moscow, The Netherlands, Russia, Romania, 1875 Serbia, Slovakia, Switzerland, and the United Kingdom.

448 LCGC Europe August 2019 Advertisement Feature COMPANY PROFILE

Tosoh Bioscience GmbH

Company Description Tosoh Bioscience is a global leader in the fi eld of liquid chromatography with a focus on bioseparations. This year, we are celebrating 30 years of providing solutions for the life science, pharmaceutical, and chemical industries. The portfolio encompasses (U)HPLC columns and downstream processing solutions for the analysis and purifi cation of biomolecules. The bioseparation product line is supplemented by EcoSEC GPC/ SEC instruments with matching columns for the characterization of natural and synthetic polymers. Tosoh has a proud history of innovation in size-exclusion chromatography (SEC). The TSKgel SW series has become the industry’s standard for QC of antibodies by SEC. column based on FcγIIIa receptor for fast evaluation Chief Chromatographic Techniques Supported of biologic activity and glycoforms of antibodies. • HPLC and UHPLC columns • Media for purifi cation by preparative chromatography Facilities • GPC systems and columns Headquartered in Griesheim, Germany, Tosoh • Size-exclusion chromatography (SEC) Bioscience’s European operations offer extensive technical support such as application development Markets Served and workshops. Tosoh Bioscience is part of the Tosoh The key markets served are life sciences and the Group, a Japanese chemical and specialty products biopharmaceutical industry. Our products are used in group that comprises over 100 companies worldwide R&D, manufacturing, and quality control. Typical and a workforce of more than 12,000 people. applications are the purifi cation of therapeutic proteins at lab, pilot, and production scale, as well as their characterization by (U)HPLC. The GPC instruments and columns for polymer analysis are applied in chemical and petrochemical industry.

Major Products/Services We offer a comprehensive line of (U)HPLC columns and Tosoh Bioscience GmbH downstream processing solutions, such as screening Im Leuschnerpark 4, 64347 Griesheim, Germany tools, prepacked columns, and purifi cation media. These are complemented by EcoSEC GPC/SEC instruments E-mail with matching columns for polymer characterization. [email protected] TOYOPEARL® and TSKgel® are renowned for their quality and reliability. The stationary phases cover all Website common LC modes, including ion exchange, hydrophobic www.tosohbioscience.de interaction (HIC), reversed phase, hydrophilic interaction (HILIC), size exclusion (SEC), mixed mode, and affi nity. Year Founded The latest developments comprise SEC columns 1989 for highly effi cient UHPLC analysis and an affi nity

www.chromatographyonline.com 449 COMPANY PROFILE Advertisement Feature

UCT, Inc.

Company Description UCT is a vertically integrated manufacturer of high-quality sample prep and U/HPLC column products. We combine this with world-class technical support. Product lines include solid-phase extraction (SPE) cartridges and well plates, QuEChERS tubes, Selectra® U/HPLC columns, extraction manifolds, Selectrasil® reagents and enzymes, and Clean-Up® Metal Scavenging phases.

Chief Chromatographic Techniques Supported • Solid-phase extraction • QuEChERS • Filtration • Enzyme hydrolysis • HPLC/LC–MS/GC–MS

Markets Served Founded in 1986, UCT has grown to be a respected leader in the drug testing, pharmaceutical, clinical, environmental, agricultural, specialty chemical, and cannabis testing markets. Our wide range of highly reproducible solid-phase extraction columns allow the chromatographer a consistent extraction technique, and our expertise in silane manufacturing allows greater control of the chemical processes involved in producing our high-quality bonded phases.

Major Products/Services Product lines include solid-phase extraction (SPE) cartridges and well plates, QuEChERS tubes, Selectra® U/HPLC UCT, Inc. columns, extraction manifolds, Selectrasil® reagents and 2731 Bartram Rd., Bristol, Pennsylvania 19007, USA enzymes, and Clean-Up® Metal Scavenging phases. UCT also offers world-class technical support for all product lines. E-mail [email protected] Facilities UCT was founded in 1986 in Horsham, Pennsylvania, Website United States. Headquarters was relocated to Bristol, www.unitedchem.com Pennsylvania following the acquisition of the specialty chemical branch of Huls America (formerly Petrarch®) to Number of Employees allow for vertical integration. In 1999, UCT expanded to a Europe: Approximately 10 second manufacturing facility in Lewistown, Pennsylvania. Outside of Europe: Approximately 120 Lastly, in 2015, UCT Ireland was established in Wexford, Ireland, as a secondary manufacturing facility to better Year Founded service our European customers and partners. 1986

450 LCGC Europe August 2019 Advertisement Feature COMPANY PROFILE

VICI AG International

Company Description Founded in 1968 by Stan Stearns, VICI and its worldwide associated companies, VICI Valco Instruments, VICI AG, VICI Metronics, VICI Precision Sampling, and VICI DBS, have been leading the development of and providing solutions for chromatography and the separation sciences across industries. A company that started with improving instruments, grew to develop valves, fittings, and more efficient components with VICI now producing more than ten thousand different parts. What began as Valco Instruments became VICI (Valco Instruments Company Incorporated), a legacy dedicated to analytical science 50 years later. the Jour line of HPLC fittings and laboratory safety products. VICI DBS specializes in the production of laboratory hydrogen, Chief Chromatographic Techniques Supported nitrogen, and zero air gas generators. Newest products include • High pressure liquid chromatography the C82 line of UHPLC valves, fittings that permit a direct • Liquid chromatography connection of 360-μm tubing, the universal valve actuator, and • Gas chromatography the C52 platform integrated HPLC injectors and selectors. • Flow injection analysis • Supercritical fluid chromatography Facilities • Gas permeation chromatography With over 150 state-of-the-art CNC machines, VICI’s instruments • Process gas chromatography and components are manufactured to exacting standards at facilities in Schenkon, Switzerland, and Houston, USA. Markets Served Other manufacturing facilities include VICI Metronics • Pharmaceutical in Washington, USA, VICI Precision Sampling in • Chemistry Louisiana, USA, and VICI DBS located in Italy. VICI • Environmental protection Canada operates from its offices in Brockville, Ontario. • Food and beverage ® • Petrochemical • Medical • Cosmetic • Aerospace

Major Products/Services VICI produces a complete line of valves for analytical chemistry. VICI AG International The company specializes in zero dead volume fittings and filters Parkstrasse 2, 6214 Schenkon, Switzerland for the separation sciences. The original manufacturers of the E-mail pulse discharge detector (PDD), the miniPDD consumes less [email protected] than one-fifth the amount of helium required by the PDDs used Website on complete GC systems. The VICI Metronics division produces www.vici.com PEEK, FEP, PFA, and ETFE tubing, ambient temperature gas Number of Employees purifiers, ValcoBond GC capillary columns, and Dynacal gas Europe: 145 calibration standards. VICI Precision Sampling manufactures Outside of Europe: 350 analytical quality syringes, sampling probes, as well as standard Year Founded and custom formed stainless and special alloy tubing. VICI 1981 AG International produces some Valco products as well as www.chromatographyonline.com 451 COMPANY PROFILE Advertisement Feature

Wyatt Technology

Company Description Wyatt Technology, a 35-year-old family-owned and operated enterprise, is the world’s largest company dedicated to SEC-MALS instruments for absolute macromolecular characterization. Our products provide techniques for characterizing macromolecules and nanoparticles in solution, in order to determine molar mass, size, charge, and interactions. Wyatt’s extensive applications laboratories and its full-time staff (including more than 30 PhD scientists) means our customers receive deep technical expertise and support. To ensure the personal touch, “Light Scattering University” (LSU) is included with most instrument purchases. We bring you to Santa Barbara, the same wavelength of light as the light scattering instrument. California, USA, to demystify light scattering and explain • microOptilab: The world’s first refractive index how to get the most from your Wyatt Technology equipment. (RI) detector for UHPLC. Operates at the same Because we don’t just build instruments, we build relationships. wavelength of light as the microDAWN. • WyattQELS: A dynamic light-scattering module Chief Chromatographic Techniques Supported integrated with the DAWN, miniDAWN, or microDAWN • SEC and GPC to determine particle sizes as small as 1 nm. • UHPLC and APC • ViscoStar viscometer: A state-of-the-art intrinsic viscosity • Field-flow fractionation instrument that can be operated above or below ambient temperature with its precise Peltier thermostatic controls. Markets Served Wyatt’s products are the most widely used multi-angle Facility light scattering (MALS) instruments in the world. They are WTC maintains more than 60,000 square feet of employed in thousands of laboratories, including companies R&D, manufacturing, and application laboratories involved in biotechnology, pharmaceutical development, in Santa Barbara, California, USA. and chemical/petrochemical research, in addition to government national laboratories and academic facilities.

Major Products/Services WTC’s family of instruments include: • DAWN: An 18-angle SEC-MALS instrument used at ambient, elevated, and below ambient temperatures for polymers, particles, and biopolymers. • miniDAWN: A SEC-MALS instrument ideal for proteins and Wyatt Technology peptides and molar mass materials below 1 million Da. 6330 Hollister Ave., Santa Barbara, California 93117, USA • microDAWN: The world’s first MALS instrument Telephone for UHPLC. Compatible with all UHPLC systems (805) 681 9009 for molar mass and size determinations. E-mail • Eclipse FFF system: For separation of [email protected] macromolecules and nanoparticles in solution. Website • Optilab: The most advanced RI detector in the world with www.wyatt.com 256 times the detection power and 50 times the dynamic Year Founded range of any other RI detector. The Optilab can be operated 1982 below or above ambient temperature and determine dn/dc at

452 LCGC Europe August 2019 Advertisement Feature COMPANY PROFILE

YMC Europe GmbH

Company Description YMC offers a wide range of innovative chromatography products, which includes UHPLC/HPLC columns (YMC-Triart), BioLC columns (YMC-SEC MAB, BioPro IEX/HIC), chiral columns (immobilized/coated CHIRAL ART), bulk media for preparative chromatography, glass columns for MPLC, and pilot columns. In addition, YMC provides an on-demand service for application support and method development. This product range, developed and engineered in YMC facilities, is available worldwide and is supported by dedicated YMC product specialists. YMC’s extensive distribution network guarantees availability of all YMC products in countries all over the world.

Chief Chromatographic Techniques Supported Facilities • UHPLC/HPLC • YMC Europe GmbH, Dinslaken, Germany • Reversed phase/normal phase/HILIC • YMC Schweiz GmbH, Basel, Switzerland • BioLC • YMC Co., Ltd., Kyoto, Japan • Chiral LC • YMC America Inc., Allentown, USA • SEC • YMC India Ltd, New Delhi, India • IEX • YMC Taiwan Co., Ltd, Taipei City, Taiwan • HIC • YMC Korea Co., Ltd., Korea • Preparative/process LC • YMC Co., Ltd. Shanghai Rep. Office, China • YMC Singapore Tradelinks Pte. Ltd., Singapore Markets Served • Pharma • Food and beverage • Agriculture • Forensics and toxicology • Clinic • Life science • Environment

Major Products/Services • UHPLC/HPLC columns YMC Europe GmbH • Capillary/micro LC columns Schöttmannshof 19, 46539 Dinslaken, Germany • (semi) Preparative columns E-mail • Preparative bulk media [email protected] • Lab-scale glass columns Website • Pilot-scale columns www.ymc.de • Method scouting Number of Employees • Method development 45 • Purification service Year Founded • Seminars and trainings 1993

www.chromatographyonline.com 453 PRODUCTS

Chromatography Accessories Automated SPE System The company is selling bottles, closures, caps, The Gerstel SPE 2 and seals, and syringe fi lters. Crimping and screwing MultiPurpose Sampler (MPS) bottles from 2 mL to 1 L. Closures and seals for enables fully automated SPE all types of bottles and caps. Caps, aluminium based on individual 1-mL, 3-mL, standard caps, fl ip-off caps, fl ip tear-off caps, fl ip and 6-mL cartridges, simplifying tear-up caps, and magnetic caps are available. Electric crimping machines are automation of existing manual also available. Samples and catalogue are available on demand. SPE methods. Evaporative www.sertir.fr concentration, solvent change, Action Europe, Sausheim, France. addition of reagents or standards, and introduction to GC–MS or LC–MS is performed as part of the automated process. Ultra-Pure Gas Delivery www.gerstel.com/en/automated- solid-phase-extraction-spe.htm By starting with ultra-pure gas and delivering it through Gerstel award-winning BIP technology cylinders, gases are GmbH & Co. up to 300 times purer than normal gas cylinders, KG, Mülheim according to the company. Low levels of impurities an de Ruhr, are guaranteed. BIP gases are suitable for gas Germany. chromatography where impurities in the carrier gas can potentially cause baseline noise and damage in the column. www.airproducts.co.uk/BIP Air Products PLC, Hersham, UK.

SEC Mobile Phases HILIC Columns Arg-SEC, the universal mobile Hilicon offers a broad range of HILIC products to separate polar phase for SEC, enhances compounds. Three column chemistries in UHPLC and HPLC, protein separations by reducing iHILIC-Fusion, iHILIC-Fusion(+), and iHILIC-Fusion(P), provide nonspecifi c interaction while customized and complementary selectivity, excellent durability, maintaining protein structure, and very low column bleeding, according to the company. The according to the company. columns are suitable for the analysis of polar compounds in “omics” research, The company reports correct food and beverage analysis, pharma discovery, and clinical diagnostics. determination of protein www.hilicon.com aggregates, which tend to stick Hilicon AB, Umeå, Sweden. to columns, is possible and that column lifetime may also be improved. www.nacalai.com Flow Modulator Nacalai Tesque, Inc., Kyoto, LECO has introduced a new Flux fl ow modulator option for Japan. routine GC×GC analysis. While LECO’s traditional thermal modulation alternative is still available and provides high sensitivity, the Flux fl ow modulator is a cost-effective option that makes GC×GC more accessible and easy to use, according to the company. Another advantage of the Flux is that it does not require cryogens to perform GC×GC, saving the user time and resources in the laboratory. https://info.leco.com/fl ux-lcgc LECO Corporation, Saint Joseph, Michigan, USA.

454 LCGC Europe August 2019 PRODUCTS

HPLC System Multi-Angle Static Light Scattering Shimadzu’s i-Series Plus Introducing the next generation DAWN multi-angle expands the product line and static light scattering (MALS) detector for improves performance and absolute characterization of the molar mass user-friendliness, according to and size of macromolecules and nanoparticles the company. With i-Series Plus, in solution. DAWN offers high sensitivity, a wide pretreatment operations have range of molecular weight, size, and concentration, and a large selection of been automated, such as the conﬁ gurations and optional modules for enhanced capabilities. processes to dilute samples and www.wyatt.com/dawn add reagents. This results in better Wyatt Technology, Goleta, California, USA. reproducibility and measurement accuracy by reducing human error, ensuring reliable data in the analysis of pharmaceuticals and SEC Column foods. The YMC-SEC MAB is a column specially designed www.shimadzu.eu for the HPLC analysis of antibodies, their fragments Shimadzu Europa GmbH, or aggregates, by size-exclusion chromatography. Duisburg, This diol-bonded silica-based phase is the Germany. ideal choice for the high resolution of both the high-molecular-weight and low-molecular-weight species. According to the company, the column ensures excellent lot-to-lot reproducibility. www.ymc.de YMC Europe GmbH, Dinslaken, Germany.

FID Gas Station Range Of Columns The VICI FID gas station combines the reliability of the VICI Analysts that are working with DBS hydrogen and zero-air generators into one compact analytical or preparative HPLC, and convenient package, according to the company. or with substances of interest that Available in high- and ultrahigh purity for all GC detector and carrier gas require normal phase, reversed applications. The generator is available in two styles: ﬂ at for placement under a phase, HILIC, GPC/SEC, or

GC, or the Tower. Available in H2 fl ow ranges up to 1 L/min and 10.5 bar. ion-exclusion separations can www.vicidbs.com be sure that Knauer can offer a VICI AG International, Schenkon, Switzerland. wide range of columns. To extend the lifetime, analytical Knauer columns can be equipped with an integrated precolumn. They are certifi ed in this confi guration to ensure performance and quality. Sample Automation Markes’ new Centri multitechnique platform is an advance in www.knauer.net/columns sample automation and concentration for GC–MS, according to the Knauer Wissenschaftliche Geräte company, and offers four sampling modes: HiSorb high-capacity GmbH, Berlin, Germany. sorptive extraction, headspace, SPME, and thermal desorption. The company reports analyte focusing allows increased sensitivity in all modes, state-of the-art robotics increases sample throughput, and sample re-collection allows repeat analysis without having to repeat lengthy sample extraction procedures. http://chem.markes.com/Centri Markes International Ltd., Llantrisant, UK.

www.chromatographyonline.com 455 EVENTS

15–18 SEPTEMBER 2019 The 12th Balaton Symposium on High-Performance Separation Methods The 30th International Symposium The 12th Balaton Symposium on on Pharmaceutical and Biomedical High-Performance Separation Analysis (PBA 2019) Methods will be held 11–13 September Tel Aviv, Israel 2019 in the Hotel Azúr in Siófok, E: [email protected] Hungary. The Balaton Symposia series W: www.pba2019.org has evolved into one of the major separation science events in Europe. 29 SEPTEMBER–1 OCTOBER 2019 Biannually, the Balaton Symposium SFC 2019 brings together separation scientists Philadelphia, Pennsylvania, USA and friends from all over the world. E: [email protected] The aim of the 12th Balaton Symposium is to provide a forum for scientists, W: www.greenchemistrygroup.org/ experts, users, and decision makers of high-performance separation current-conference/sfc-2019 methods to discuss the latest issues, current requirements, and technological challenges we face in the present and future. Focusing on various analytical, 14–16 OCTOBER 2019 The 11th Conference of The preparative, and industrial separation methods and techniques, the symposium World Mycotoxin Forum and the will place special emphasis on new developments in pharmaceutical, XVth IUPAC International environmental, biomedical, forensic, life, food, refinery sciences, and industrial Symposium on Mycotoxins separation methods. Research trends on various stationary phases and (WMFmeetsIUPAC) column technology, advances in separation techniques, and fundamentals Belfast, Northern Ireland of separation science and applications will also be emphasized. E: WMF@bastiaanse-communication. com The scientific programme includes plenary lectures, keynote W: www.worldmycotoxinforum.org lectures, contributed lectures, and posters. The confirmed invited speakers include: 21–23 OCTOBER 2019 • Boguslaw Buszewski, Nicolaus Copernicus University, Torun, Poland Solutions and Workflows in • Gert Desmet, Vrije Universiteit Brussel, Brussels, Belgium (Environmental) Molecular • Sabine Heinisch, University of Lyon, Lyon, France Screening and Analysis (SWEMSA • Gérard Hopfgartner, University of Geneva, Geneva, Switzerland 2019) • Caroline West, University of Orléans, Orléans, France Erding, Germany E: [email protected] • Jean-Luc Veuthey, University of Geneva, Geneva, Switzerland W: www.swemsa.eu • Koji Otsuka, Kyoto University, Kyoto, Japan • David McCalley, University of the West of England, Bristol, UK 5–8 NOVEMBER 2019 • Wolfgang Lindner, University of Vienna, Vienna, Austria Recent Advances in Food Analysis The attendance of young scientists is encouraged via travel grants. (RAFA 2019) Attractive social events will accompany the scientific programme. Prague, Czech Republic Prominent separation scientists will be honoured at the event with one of three E: [email protected] prestigious awards named in honour of pioneers of the field. The Halász Medal W: www.rafa2019.eu Award, the Csaba Horváth Memorial Award, and the Ervin sz. Kováts Award for Young Scientists will be presented during the opening ceremony of the symposium. 4–9 OCTOBER 2019 Grass Roots 4: Understanding and An exhibition of instrumentation, columns, accessories, chemicals, and literature Application of Biopharmaceutical on various separation techniques is planned in connection with the symposium. Workflows The venue of the symposium, exhibition, and social programme is the Hotel Azúr Longmynd Hotel, Church Stretton, UK located by Lake Balaton, which is the largest lake in Central Europe. Since Hungary E: [email protected] is landlocked, Balaton is sometimes called the “Hungarian Sea”. High tourist W: https://chromsoc.com/event/grass- season extends from June until the end of August. The average water temperature roots-2019 during the summer is 25 °C, which makes bathing and swimming very enjoyable. Please send any upcoming event The distance between the Budapest-Liszt Ferenc International Airport and Siófok information to Lewis Botcherby at is only 130 km on highway M7. Siófok can also be reached conveniently by train. [email protected] E: [email protected] W: www.balaton.mett.hu

456 LCGC Europe August 2019 FREE CHROMACADEMY MEMBERSHIP

Agilent Technologies is offering ﬁve years complimentary access to CHROMacademy for all university students and staff. CHROMacademy is an intuitive, comprehensive e-learning and troubleshooting platform with more than 3,000 pages of content for HPLC, GC, sample preparation, and hyphenated techniques. No other online resource offers separation scientists more live streaming events, a knowledge base, practical solutions, and new technologies in one easy to navigate website. Get your free ﬁve year membership worth US $1,995* by submitting the form at www.chromacademy.com/agilent.

* Five years free access to CHROMacademy only available to customers afﬁliated with an academic or research institution, conditions apply. A valid university e-mail address if required.

(TDU) al desorption Therm … with rugged Automation ated Pyrolysis Autom and Thermal Desorption (PYRO) Integrated with TD-GC/MS TRO PrepAhead MAES Inert, ultra-short sample path Productivity Fractionation, Cryofocusing, SPME Smart Ramp, and Split selectable ) and (SBSE 4-Wire temperature sensing for wister T Highest accuracy and stability for μ-vials ) User-performed Temperature into (ATEX Injection Calibration in sample position al extraction therm QC sample for validation

Thousands of users in leading companies ic Headspace ynam D Headspace world-wide rely on GERSTEL solutions. (DHS), What can we do for you?