Water Purification in the Chromatography

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OCTOBER 2017 ® Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

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Ionic Contamination Why and How to Avoid Ionic Contamina- tion in Water Used for LC–MS Analyses Anastasia Khvataeva-Domanov and Stephane Mabic 4

Hormone Analysis Hormone Analysis by LC– MS and Water Impact A Q&A with Joseph Plurad 12

Tips: Water Purification Lab Water Hints and Tips for the Chromatography Lab, Part 1: Water Purification A Q&A with Joseph Plurad 16

Tips: Handling Purified Water Lab Water Hints and Tips, Part 2: Optimal Handling of Purified Water A Q&A with Joseph Plurad 21

Purification System Selection Selecting a Laboratory Water Purification System Jean-Francois Pilette 27 4 | October 20174 |October |LCGC A Water I Anastasia Khvataeva-Domanov and Stéphane Mabic and Why onic onic na l y C s Us ontamination in in ontamination e s ed for L for ed H chromatography–mass spectrometry (LC– Water plays an essential role in liquid the intensity ofincrease in sodiated adducts.. accompanied was byincreased. This an ion the sodium as concentration the water in intensities of the protonated species decreased presented here, observed it that the signal was process. the experiments In the analytical cationicany contaminating components to were and analyses, clean didnotin contribute reagents, used instrument the MS well as as demonstrates signals that solventsanalyte and ion. Moreover, the occurrence of protonated compared as to asodiatedanalysis, peptide charge enables fragmentation more meaningful mobile proton since the partially analysis, protonated molecular ion, peptide in especially suppression. the preferredIn MS, the is ion type because of ion signals reduced analytical (MS) detection lead to adduct formation and spectrometry mass with (UHPLC) analyses ultrahigh-pressure liquid chromatography Ionic the water in in used contaminants o w to to C A – void void M Sponsor’s content Sponsor’s S

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Table I: Direct infusion experiments First set of experiments Second set of experiments Sample Bradykinin (fragment 1-7) Glu1-fibrinopeptide B Monoisotopic Monoisotopic Mass 756.40 Da Mass 1569.65 Da

[M+H] ion (m/z 757) [M+H] ion (m/z 1570) [M-115+Na] ion (m/z 664) [M+2H] ion (m/z 785) [M-202] ion (m/z 554) [M+Na+H] ion (m/z 796) [M+2Na] ion (m/z 807) [M+3Na-H] ion (m/z 818) Instrument Applied Biosystems API 2000 Waters Synapt HDMS Ionization mode ESI+ ESI+ Flow 10 μL/min 10 µL/min Solvents (a) 96:4 fresh ultrapure water–acetonitrile (a) 50:50 fresh ultrapure water–acetonitrile

(b) 96:4 1 ppm Na+ in ultrapure water/ (b) 50:50 1 ppb Na+ in ultrapure water–acetonitrile acetonitrile (c) 50:50 100 ppb Na+ in ultrapure water–acetonitrile

(d) 50:50 1000 ppb Na+ in ultrapure water–acetonitrile Sample concentration 1 μM 500 pM

MS), where it is used extensively in the data analysis more challenging and workflow. Contaminants in the water can complicated. Metal ions may come from affect the quality of data and instrument several possible sources (3) such as performance; therefore, it is recommended solvent reservoirs, gloves, the analyst, and prudent to use only the highest purity and the solvents used in preparing the solvents. Organic contamination of the mobile phase. Therefore, using ultrapure water used in high performance liquid water free of metal ions will contribute to chromatography (HPLC) is an important the success of any LC–MS analysis. issue and has been addressed accordingly (1), but the ionic purity of the water should Experimental also be considered, especially when MS Experiments to Evaluate the Effect of is used as a detection technique (2). Ionic Ionic Contamination in Water on MS contaminants lead to adduct formation Data and reduced analytical signals because of Two compounds, bradykinin fragment ion suppression. 1-7 and Glu1-fibrinopeptide B, were Electrospray ionization (ESI) remains used to investigate the effect of ionic the most popular MS technique. In contamination on LC–MS analyses. positive-ion analyses, it is ideal to have Different samples of peptides were only protonated peaks of the parent ion prepared and infused directly to a mass or its fragments in the mass spectrum. spectrometer. The experimental details The presence of metal adduct peaks, for peptide analyses are described in such as sodium adducts (M+Na), makes Table I.

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(a) (b) 4 105

4 105 3.5 105 [M+H]+ + 5 [M-115] 3.5 10 3 105 3 105 m/z 538 ??? 2.5 105 [M-115]+ 2.5 105 [M+H]+ 2 105 2 105 1.5 105 1.5 105 [M-202]+ [M-115+Na]+

Intensity (cps) Intensity (cps) 5 5 1 10 m/z 703 ??? 1 10 [M-202]+ + 4 [M+Na] 0.5 104 0.5 10 0 0 500 550 600 650 700 750 800 850 900 500 550 600 650 700 750 800 850 900 m/z (amu) m/z (amu)

Figure 1: Mass spectra of bradykinin (fragments 1-7). Direct infusion, ESI+, using (a) 96% fresh ultrapure water, 4% acetonitrile and (b) 96% 1 ppm Na+, 4% acetonitrile.

Experiments to Identify the Results and Discussion Levels of Ions in Ultrapure Water Effect of Ionic Contamination To evaluate the level of sodium in in Water on MS Data ultrapure water, water samples from an The quality of LC–MS data is influenced MilliporeSigma Milli-Q Advantage A10 by many factors, such as instrumentation, water purification system were analyzed experimental parameters, sample using an Agilent 7700s inductively preparation, the quality of reagents used, coupled plasma–mass spectrometry and the quality of solvents (5). Water is (ICP-MS) instrument (4). The calibration used extensively in reversed-phase LC– standards used in experiments were a MS workflows; therefore, its purity plays mixture of Agilent and Spex CertiPrep, a critical role in instrument performance and containers were all perfluoralkoxy and the quality of data generated. (PFA) polymer precleaned with ultrapure The purity of water for LC–MS work water. is mainly assessed through organic contamination, which can be expressed Water Purification Systems by the level of TOC (6), or by certificates of All ultrapure water samples (resistivity analysis reporting results of suitability tests. of 18.2 MΩ·cm and total oxidizable The second approach to evaluate carbon [TOC] below 5 ppb) from water quality for LC–MS analyses is to MilliporeSigma water purification assess its level of ionic purity. Thus, for systems were analyzed immediately bottled water, certificates of analysis after water collection. usually detail maximum concentrations

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(a) 0 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 100 785.820 1.96e5 786.822 % 776.819 787.835796.826 813.384 816.782 818.784 0 760 765 770 775 780 785 790 795 795 800 805 810 815 820 825 830 835 840 845 850 855 860 m/z

1 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 1.86e5 100 785.820 786.832 % 776.829 796.826 799.605 859.809 0 760 765 770 775 780 785 790 795 795 800 805 810 815 820 825 830 835 840 845 850 855 860 m/z

100 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 100 785.830 1.57e5 786.832

% 796.826 807.815 787.835 804.812 813.615 827.642 0 859.798 760 765 770 775 780 785 790 795 795 800 805 810 815 820 825 830 835 840 845 850 855 860 m/z 1000 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 100 785.830 1.37e5 786.832

% 796.826 807.815 787.835 799.605 859.603 0 771.571 818.805 827.631 760 765 770 775 780 785 790 795 795 800 805 810 815 820 825 830 835 840 845 850 855 860 m/z (b) 0 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 785.820 100 786.315 2.20e5 786.822 % 787.339 0 787.835 785 786 787 788 789 790 m/z 1 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 785.820 100 786.326 2.20e5

% 786.832 787.349 787.845 0 785 786 787 m/z 788 789 790 100 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 100 785.830 786.326 2.20e5

% 786.832 787.339 787.835 0 785 786 787 m/z 788 789 790 1000 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 100 785.830 786.326 2.20e5

% 786.832 787.339 787.835 0 785 786 787 m/z 788 789 790 (c) 0 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 100 4.21e4

% 796.826 797.315 786.825 798.314 0 797.825 796 797 798 799 800 801 m/z 1 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 100 4.21e4 796.826

% 797.325 797.835 798.335 799.605 800.616 0 796 797 798 799 800 801 m/z 100 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 100 4.21e4 796.826 797.325

% 797.825 799.605 796.316 787.335 798.813 800.606 0 796 797 798 799 800 801 m/z 1000 ppb of sodium in the water–acetonitrile mixture TOF MS ES+ 100 796.826 797.325 4.21e4

% 797.825 799.605 798.335 798.824 799.303 800.606 0 796 797 798 799 800 801 m/z

Figure 2: (a) Mass spectra of protonated molecular ion [M+2H] of Glu1-fibrinopeptide B sample, (b) and (c) extracted ion chromatograms of [M+Na+H] ion as examples of the effect of ionic concentration on MS detection.

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200,000

150,000

100,000

50,000

0 785 (M+2H) 796 (M+Na+H) 807 (M+2Na) 818 (M+3Na-H)

0 ppb 1 ppb 100 ppb 1000 ppb

Figure 3: Signal intensities of 500 pmol Glu1-fibrinopeptide B in 50:50 (v/v) acetonitrile–water. The mixture was spiked with different amounts of sodium ions and injected directly into the mass spectrometer.

for certain ions because when stored Table II: Freshly produced ultrapure water sources in a glass bottle, the water will leach Experiment Water purification system used out contaminants from the container Bradykinin direct infu- Elix + Milli-Q Advantage A10 (Q- sion and ICP-MS Gard and Quantum TEX cartridge, even though the water may have been Millipak final filter) of very high purity immediately after Glu1-fibrinopeptide B Milli-Q Integral (Quantum TEX production. For example, standard glass direct infusion cartridge) bottles leach out alkali, contaminating conductivity parameter, or alternatively ultrapure water and leading to a higher its inverse, resistivity, can be used to count of adducts. Since the quality of characterize the ionic purity of water. bottles can vary strongly from one to Thus, at 25 °C, conductivity of 0.055 µS/ another, the nature of ions selected cm, or resistivity of 18.2 MΩ·cm, implies to report in the certificate of analysis that the water is ultrapure, whereas at 1 depends on the quality of the bottle ppb of Na+ in ultrapure water, resistivity used to store the water. Table III decreases to 17.6 MΩ·cm, and at 5 ppm, compares the specifications for some dramatically drops down to 0.093 MΩ·cm; metal ions in ultrahigh-pressure liquid these values can be calculated based on chromatography (UHPLC)–MS-grade, the concentration of sodium, its charge, and LC–MS-grade bottled waters from three mobility (7). Therefore, when fresh ultrapure vendors, and in fresh ultrapure water. water of 18.2 MΩ·cm resistivity measured Because ionic impurities present in via an in-line monitor was used to dissolve water increase its conductivity, the a peptide sample (bradykinin fragment 1-7),

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Table III: Specifications for some metal ions in UHPLC–MS-grade or LC–MS-grade water and fresh ultrapure water produced by a laboratory water purification system specifically for LC–MS practice* Maximum Concentration (ppb) UHPLC–MS Grade LC–MS Grade Water LC–MS Grade Water Fresh Ultrapure Water for Metal Ion Water Brand X Brand Y Brand Z LC–MS Analyses Aluminum 20 500 10 1 Calcium 50 100 100 1 Iron 30 100 5 1 Potassium 50 100 10 1 Magnesium 20 100 20 1 Sodium 50 100 200 1 *Specifications of different sources of laboratory water dedicated for MS analyses were collected from official websites of manu- facturers but their names are not provided to avoid direct comparison.

and infused directly to a mass spectrometer, and presented as a function of Na+ the resulting spectrum was clean (Figure 1a). concentration in water (Figure 3). The parent and fragment peaks represented Specifically, the presence of 1 ppb of only protonated species. However, when the Na+ decreased the [M+2H] signal intensity water used was contaminated with sodium by 5%. But the decrease in signal was ions, the spectrum was more complex 20% with 100 ppb Na+, and 30% when it with the presence of sodium adduct peaks was 1000 ppb. Altogether the presence (Figure 1b). of sodium results in more complex The presence of metal ions can suppress spectra, leading to difficulties in data the signal of the protonated ion peak of characterization, analyte quantification, interest. The effect of the sodium ion on the and subsequently more time spent on signal intensity of Glu1-fibrinopeptide B was data analysis. Therefore, using water in LC– analyzed by varying the ion concentration in MS analyses that is free of ions is of high the water–acetonitrile mixture that was used importance. In addition, there are a few to dissolve the peptide. It was observed key considerations to keep in mind when that signal intensity of the [M+2H] molecular choosing the source of ultrapure water, ion decreased with the increase of sodium and also when handling it to minimize ion concentration in the water. In parallel, contamination. it was observed that signal intensities of the sodium adducts increased with the Five Tips to Avoid Ionic increase in Na+ concentration (Figures 2a, Contamination in LC–MS Practice 2b, and 2c). 1. Choose the Best Further signal intensities of Glu1- Source of Ultrapure Water fibrinopeptide B such as the [M+2H], Common choices are freshly produced [M+Na+H], [M+2Na], and [M+3Na-H] ions ultrapure water from a laboratory water were recorded for each analyzed sample purification system, and bottled water,

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Table IV: Concentration of sodium ions in fresh ultrapure water Quality That Leaches the and in the same ultrapure water that was stored in a glass bottle for one day Minimum Amount of Contamination Sample Na+ (ppb) It is also recommendable to have Freshly produced ultrapure water 0.020 dedicated glassware for LC–MS practice, Ultrapure water in a glass bottle after one day 0.212 and glassware should be cleaned thoroughly before use. such as UHPLC–MS grade or LC–MS grade. Fresh ultrapure water produced 3. Keep Your Work Area Clean using an optimal combination of Moreover, since laboratory air is purification technologies is usually of very characterized by a lot of contamination and high purity, with ionic levels below 1 ppb as ultrapure water absorbs contamination at 18.2 MΩ·cm of resistivity, which can be from the air including volatile molecules (8), monitored online at the moment of water the working area should be kept clean (9). collection. In the case of bottled water, Appropriate covers and caps for mobile- specifications of ionic purity for laboratory phase reservoirs and glassware can help water are usually provided and can be to avoid contact between ultrapure water easily consulted to assess the potential risk and the laboratory air, as well. It is also a of ionic contamination on LC–MS analyses. good idea to wear gloves, and choose Because ultrapure water is a very ones that are powder-free and have the aggressive solvent, it tends to absorb fewest metal or ion extractables (for ions and organic compounds from the example, polyethylene) because simply container. Thus, maximum concentrations touching glassware with a bare hand can of ions in final ultrapure product water transfer enough salt to cause a significant depend significantly on the level of appearance of metal adduct ions. extractables released by the containers used to collect freshly produced ultrapure 4. Select Reagents and Organic water, or to store it, in the case of bottled Solvents of the Highest Purity water. Table IV shows the concentration Here, to evaluate the purity level necessary of Na+ in freshly produced ultrapure water for sensitive MS applications, a certificate and ultrapure water after storage in a of analysis is a good source of information. glass bottle for one day. Also, it is recommended to obtain details As shown in Table IV, the Na+ from the supplier beforehand concerning concentration in freshly produced the level of leachables and extractables ultrapure water was 0.020 ppb. from solvent and reagent packaging. After storage in a glass bottle, the concentration increased to 0.212 ppb. 5. Routine Maintenance When using a laboratory water purification 2. Choose Glassware of the Highest system, make sure that it undergoes

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Table V: Water purification technologies used in the production of ultrapure water from tap water Pretreatment (filter, activated carbon) • Filtration • Removal of oxidizing agents Reverse osmosis • Removal of the bulk of contaminants (95–99%): organics, ions, microorganisms, particulates Electrodeionization • Further removal of ions, and charged organic species UV (254 nm) • Prevents bacterial contamination Virgin ion-exchange resins • Removal of trace amounts of ions Synthetic activated carbon • Removal of trace levels of organics UV (185, 254 nm) • Photo-oxidation to further remove organics Point-of-use end filters • Prevents particle and bacterial contamination • Allows removal of trace levels of organics

routine maintenance to ensure the highest mobile phases. It is also important to water quality. apply best laboratory practices. Ion Removal in Laboratory Water Purification Systems Acknowledgment The laboratory water purification The authors would like to acknowledge systems used in this study combine Jens Baumgaertner of Merck KGaA in the technologies shown in Table V to Darmstadt, Germany, and Maricar Tarun- efficiently remove different types of Dube of MilliporeSigma Corporation in contaminants from tap water feed. The Danvers, Massachusetts, for their help removal of ions is carried out by using with this article. reverse osmosis, electrodeionization, and ion exchange resins. References (1) M. Tarun, C. Monferran, C. Devaux, and S. Mabic, LCGC The Peak, To ensure the efficiency of the 7–14 (2009). purification process and make sure (2) B.O. Keller, J. Sui, A.B. Young, and R.M. Whittal, Anal. Chim. Acta 6(27), 71–81 (2008). that the ultrapure water contains the (3) M. Oehme, U. Berger, S. Brombacher, F. Kuhn, and S. Kolliker, lowest possible ionic content, resistivity TrAC, Trend Anal. Chem. 21, 322–331 (2002). (4) A. Khvataeva-Domanov, G. Woods, and S. Mabic, “Choosing Opti- monitoring can be used. Ultrapure mal High Purity Water Source in Accordance to ICP-MS Application Needs and Laboratory Environment,” presented at the Winter Con- water of 18.2 MΩ·cm resistivity refers to ference on Plasma Spectrochemistry, Amelia Island, Florida, 2014. ultrapure water free of a significant level (5) M.J. Capdeville and H. Budzinsk, TrAC, Trend Anal. Chem. 30, 586–606 (2011). of ions for LC–MS analyses. (6) C. Regnault, I. Kano, D. Darbouret, and S. Mabic, J. Chromatogr. A. 1030, 289–295 (2004). (7) E. Riché, A. Carrié, N. Andin, and S. Mabic, High-Purity Water and Conclusion pH Am. Lab. (Boston) 16, 22–23 (2006) (8) N. Nioradze, R. Chen, N. Kurapati, A. Khvataeva-Domanov, S. The presence of ions in the water used Mabic, and S. Amemiya, Anal. Chem. 89, 4836–4843 (2015). for LC–MS analyses influences the quality (9) I. Rodushkin, E. Engstrom, and D.C. Baxter, Anal. Bioanal. Chem. 396, 365–377 (2010). of data by forming metal adducts and suppressing analyte signals. To avoid Anastasia Khvataeva-Domanov and Stéphane Mabic ionic contamination, it is recommended to are with Millipore S.A.S. in Saint-Quentin-en-Yvelines, France. Direct use fresh ultrapure water when preparing correspondence to: [email protected] ◾

11 | October 2017 | LCGC Sponsor’s content 12 | October 201712 |October |LCGC I L A Q&A with Joseph Plurad H m C ormone ormone – p M act S and Water and S A you may have inaccurate results. Water quality is you’re to analyze in trying the water you’re using, the separation orare detection of concern. inused an analysis that have on impact adirect contaminants that remain in the purified water is throughout used the entire workflow.Any chromatography and spectrometry,water mass chemicalused in alaboratory. In liquid Plurad: Water is probably the most chromatography spectrometry? and mass liquid in important why water is quality LCGC: Iknow we’ve heard it before, but contaminants. these fortechniques used interference-free of analysis hormone-free lab water for the LC–MS purification processes that provides ultrapure, incorporates system acombinationMilli-Q of compounds our water in supplies. The these increased the interest identifying in water have significantly drinking through of unintendedrisks consumption of hormones municipal water supplies. The potential health resulted hormones in way their into finding throughout care health in the world has The of increased use hormone-based therapies Also, if there are any traces of the molecules na l y s i s by Sponsor’s content Sponsor’s

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important to avoid interference with the today? And how did you pick the water analytes you’re measuring or identifying samples you chose to analyze? as well as for optimizing instrument Plurad: In the last 20 years, a lot of operation. attention has been given to “persistent organic pollutants” in drinking water LCGC: Which specific contaminants sources. Much of the original focus can affect the LC–MS process? in this area was on organic molecules Plurad: The most obvious are organics, and species that came from so-called and in LC–MS, that’s typically what you’re chemical sources such as pesticides and looking for. Reduction to trace levels is solvents. key, particularly if the organics are similar With advances in health care, however, to what you’re analyzing. pharmaceutical sources of these Water that’s heavy with organics can persistent organic pollutants have also cause issues with column efficiency become more significant. As many of our by coating the separation media, resulting listeners may remember, we were once in poor peak resolution and shifting told to dispose of our expired or unused peaks. Ions can be a problem. Certain pharmaceuticals by flushing them down metals can create adducts resulting in the toilet, which in retrospect was not noisy mass spectra. the best idea because this water, now Particle-free water is important to carrying these drugs, eventually finds ensure proper flow through the system. its way back into the municipal drinking With shrinking columns and tubing, as water supply. well as improvements and changes to With the escalation of use of hormone- separation media and higher pressures, based therapies such as topical steroids, the impact of particles clogging an LC– birth control, and hormone-replacement MS becomes even more magnified. therapies, there may be long-term effects Bacterial contamination is a two-headed if these therapeutics exist in our drinking monster. Bacteria behave as particles, so water, such as effects on human fertility you run the risk of blocking and clogging and actual embryo development, as well tubing or columns. But as bacteria die off, as endocrine and other general health they leech out and reintroduce various issues. organic and ionic contaminants into the Consequently, there’s extremely high previously clean water. interest in identifying what’s in the water and at what levels to determine imminent LCGC: Regarding the work or long-term health risks. Because our MilliporeSigma recently completed, lab water systems rely on potable tap why is there so much interest in water as a feed source and are used analyzing for hormones in water throughout the world, we felt it was

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important to understand what influence higher levels of these persistent hormones could have on our ability to contaminants you can expect to see provide ultrapure water. We also wanted some residual contaminants post to demonstrate that our purification purification. techniques can provide high-purity water Clearly, a combination of techniques for the detection of hormones in drinking is required to ensure full removal of water via LC–MS. these molecules and to have water free Because this is a global issue, we of hormone residues for your analytical selected drinking water samples from work. We can consider purification that various geographies including China, includes activating carbon, reverse France, and Spain. We’re not stating that osmosis, UV photo oxidation of organics, these samples reflect the overall water and ion exchange. And if that’s still qualities in these countries or the safety not enough, we can consider other of the drinking water sources. These are purification media at the point of use single points of analysis chosen to show such as additional activated carbon that that the problem exists to some degree targets specific contaminants. everywhere. LCGC: What were the results of your LCGC: What are the challenges in analyses? analyzing hormones at trace levels? Plurad: We found hormones in all of the Plurad: We found that these hormones water sources we tested. It stands to are everywhere and that simple or reason that in highly industrialized and single-stage purification techniques developed countries you would expect may not be effective in removing them. to see various hormones at various levels. Consider deionization, for example. As Our R&D team found androsterone and a purification technique, it only works estradiol in city water sources in France on contaminants that have an electrical and Spain, and corticosterone was charge. Most organics are neutral or detected in China. very weakly charged. So deionization I’d like to reiterate it’s understood that is not very effective at removing these these municipal drinking water samples contaminants. are safe and suitable and approved for Or, consider reverse osmosis. human consumption. Agencies worldwide Although this is a workhorse in water recognize the existence of this issue and purification, a reverse-osmosis system are taking a hard look at the long-term operating well removes only 95% to effects of having these molecules in the 99% of the contaminants in the water drinking water. feeding that membrane. This means The combination of purification that in water systems that have relatively processes embedded in a Milli-Q water

14 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

purification system allows us to provide to develop purification packs that are hormone-free lab water for the LC–MS adapted to remove very specific classes techniques used to analyze for these of molecules at the actual point of use. contaminants. Being able to control the purification process 100% from tap water feeding LCGC: The sensitivity of analyses the water purification unit gives users the is constantly improving. How is best chance at managing the impact of MilliporeSigma responding to this persistent contaminants. laboratory market demand? Plurad: We now have scientists who are able to analyze and quantify trace levels of contaminants that are far below the detection limits provided by traditional quality measures for ultrapure water; in fact, by orders of magnitude at this point. Joseph Plurad, MilliporeSigma continues to develop North America Field Marketing Manager, more efficient means to remove general MilliporeSigma classes of contaminants and continues

15 | October 2017 | LCGC Sponsor’s content 16 | October 201716 |October |LCGC Purification Lab, Part 1: Water for the A Q&A with Joseph Plurad Lab Water C hromatogra H the chromatography workflow. is for used many of applications in different parts specifically, theit’sof eluent. aspart used Water and often,reactant in the chromatography lab sample. times Other it could areagent be or a to prepare samples, and sometimes, it is the in achromatography laboratory. Water is used laboratory, in virtually every everywhere not just that waterand understand is virtually used Plurad: chromatography laboratory? LCGC: Why is water important in a technologies that will meet any lab’s requirements. MilliporeSigma, aboutvarious purification FieldPlurad, America Marketing North Manager, laboratory. LCGC recently spoke with Joseph aresystems avital tool in the life science quality of purified water.Thus, waterpurification The chromatography lab requires the highest choosing awater purification technology unique lab requirements and applications when recommendsMilliporeSigma your evaluating int We have to take abig back step s and T i Sponsor’s content Sponsor’s ps p hy

shutterstock.com/Olga Nikonova Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

And think about other places where your work. And a lot of companies, both water is used in terms of cleaning the in the chromatography side as well as equipment or cleaning the glassware on the water side, have put a lot of work and the lab ware, such as bottles that go into understanding what actually is in on top of an HPLC or beakers used to the water and what it will actually do in prepare the sample. chromatography. Water quality, in sample In the biological world, water is used in prep, separation or detection, can result the autoclaves. And often will condense in a lot of different problems. Certainly, or collect on what you are trying to contaminants in the water can react with decontaminate. It is used in humidity the sample, which could change the nature chambers as well. These are all touch or degrade it, or potentially enhance points that could impact any experiment, what you’re trying to look for. These but there are some specific contaminants same contaminants could interact with and specific issues that impact the solutions that you’re using. It could chromatography. So the impact of water impact the quality of the eluent, especially becomes much more important if it’s not as the solutions sit out because water properly managed. and organics are good nutrient sources Water is a universal solvent, which for biologics. Particles, and bacteria that means that it can, and will, pick up behave as particles, can block columns, contaminants from the environment, and foul the resin and the media. The water will dissolve gasses from the air impact of that is poor separations, but and change the quality. When you think if you are successful in blocking up a about all of these touch points and all of column, that could result not only in these possibilities where something can column damage but instrument damage as enter the water that is being used in your well because pressure is being placed on experiment, the gravity of its impact on the column and these pumps are designed chromatography specifically, becomes to run at very high pressures. much more important. Post separation, if these contaminants are allowed to flow through, you will LCGC: What is the impact of poor continue to have issues with detection quality water in chromatography? techniques and each detection technique Plurad: I don’t think it’s a coincidence will have a specific impact. that in the ultrapure water world, chromatography is the number one LCGC: What are the key contaminants application demanding highly purified in water that can affect an experiment? water. So any changes or deviations And what would happen? from water quality that is ideal for Plurad: In the chromatography world, chromatography will negatively impact there are generally three classes of

17 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

contaminants. The first is particles, or would recommend a pre-treatment to bacteria as particles in this case. These remove the larger particles. particles can clog frits, fittings, pre- Once you’ve gone through that columns, and the columns themselves. prefiltration, we recommend reverse This will result in column damage; osmosis, which is really the workhorse damage will occur if you try to over- in water purification. This removes 95% pressurize the system. of all the contaminants in the incoming Another contaminant is organics. Trace tap water and across all classes, whether organics in ultrapure water can foul inorganic, organic, or particulate. the media of the columns. It will coat The next step is EDI, or the columns, reducing efficiency. If you electrodeionization, which is similar to reduce the efficiencies of the columns, our Elix technology. EDI technology uses they won’t be as good at separating your membranes and ion exchange media and targets. This results in shifting baselines, electrical current to remove additional or even tailing and masked peaks. ions from the water purification stream. It The last contaminant is ions. Within the builds on the work that reverse osmosis world of ion chromatography, you don’t did and pulls out additional ions. This is want your target ions in the water that going to provide additional benefit for you’re using. However, in LC and LCMS, sodium as well as difficult-to-remove ions alkali metals, and specifically sodium, can like silica. form adducts that will suppress signals The next step is to pass the water in the mass spec. Sodium is a particular through ion exchange media again to concern in water purification and any continue to remove more ions from that purification has to work extra hard to purified water, and hit the water with UV reduce sodium levels prior to dispensing photo oxidation to remove organics. This ultrapure water. will impart a charge to them that can be removed by downstream ion exchange LCGC: What technologies in water media. The UV photo oxidation will also purification can address these deactivate bacteria, and remove them contaminants? from the equation in the ultrapure water. Plurad: At MilliporeSigma, we want to After UV photo oxidation, we will educate people that the best way to chase it with additional ion exchange manage water is to own 100% of the media to do its final polishing to get process at the point of use. Take tap to that ultrapure water quality. And water, and bundle or sequence the finally, at the point of use, where you’re best series of purification techniques to dispensing the water, we recommend a achieve the water quality that you need final stage purification that is specific to for your application. From tap water we your analytes and your application. At

18 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

MilliporeSigma, we have specific activated recommend flushing a few liters from carbon cartridges that are designed to the system if it hasn’t been used in a few remove volatile organic carbons as well as days. If you are using a water purification a separate activated carbon filter that is system daily, or several times a day, as designed for endocrine disruptors. little as 250–500 mL of water can be Other options include a C-18 reverse- flushed from the system to ensure the phase silica cartridge, a .2 micron filter freshest, most recently purified water. at the dispensing point an ultrafilter at The goal is to be prudent about what the point of use. However, the downside you’re picking in terms of purifying water to ultrafiltration, is that it generally does and immediately be using it as it’s being leach out trace organics, which are the made. In doing so, you will most likely get nature of the membrane. the best results in your chromatography. The best combination of techniques specific to what you’re looking for and the About the Life Science Business of specific chromatography technique that Merck KGaA, Darmstadt, Germany you’re using will determine which are the The life science business of Merck KGaA, best techniques for a specific application. Darmstadt, Germany, which operates as MilliporeSigma in the U.S. and LCGC: What else can be done to Canada, has 19,000 employees and 65 optimize results? manufacturing sites worldwide, with a Plurad: Step one is again to use the best portfolio of more than 300,000 products technologies to purify specifically for the enabling scientific discovery. Udit Batra analyte in your application. One thing that is the global chief executive officer of we can do is as simple as producing the MilliporeSigma. right amount of water at the time you’re Merck KGaA, Darmstadt, Germany about to use it. completed its $17 billion acquisition Another practical technique includes of Sigma-Aldrich in November 2015, trying to avoid plastic containers for creating a leader in the $125 billion sample preparation. Plastics typically global life science industry. use organics as release agents in their Merck KGaA, Darmstadt, Germany is a molding process and these release leading company for innovative and top- agents stick to the sides of the containers. quality high-tech products in healthcare, So very fresh glassware, very clean life science and performance materials. glassware, is preferred. The company has six businesses – Finally, as a point of practice, flush a Biopharmaceuticals, Consumer Health, little bit of water from your system before Allergopharma, Biosimilars, Life Science you actually collect it for use. Depending and Performance Materials – and on how often the system is used, we generated sales of €12.85 billion in

19 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

2015. Around 50,000 employees work internationally except in the United in 66 countries to improve the quality States and Canada, where the company of life for patients, to foster the success operates as EMD Serono, MilliporeSigma of customers and to help meet global and EMD Performance Materials. challenges. Merck KGaA, Darmstadt, Germany is the world’s oldest pharmaceutical and chemical company – since 1668, the company has stood for innovation, business success and responsible entrepreneurship. Holding an approximately 70 percent interest, the founding family remains the majority owner of the company to this day. The Joseph Plurad, North America Field company holds the global rights to Marketing Manager, MilliporeSigma the name and the trademark “Merck”

20 | October 2017 | LCGC Sponsor’s content 21 | October 201721 |October |LCGC A Q&A with Joseph Plurad of Purified Water Part 2: Lab Water Op tima H Some organics foul can chromatography need to minimized. or be identify to purify or mask the organic compounds that you seek Organic contaminants that will either compete are for organic compounds and molecules. sincecause chromatographic most analyses clear that the issues organics pretty be can behave in achromatography system. It should of because how they category in the particles ions are Bacteria and particles. also included contaminants ofprimary focus are organics, Plurad: water and what they can do to an analysis? contaminants that could be in ultra-pure LCGC: Can you quickly review some key MilliporeSigma. AmericaNorth Field Marketing Manager at water, LCGC Joseph with talked Plurad, To more learn about of purified the handling order to ensure consistent and reliable results. and the product water in from systems these and manage alab water purification system consider waysDialogue to will handle specific purification technologies, Dedicated this lab water and contaminants which discussed Following on our previous Dedicated Dialogue int As discussed in our last podcast, the in our podcast, last discussed As l s

H and and l T ing i Sponsor’s content Sponsor’s ps ,

shutterstock.com/Yeryomina Anastassiya Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

media or resins which results in reduced technologies to deliver water that meets column efficiency, unstable baselines, technical requirements, regardless of and poor peak resolution. Similarly, whether using tap water or water from ions can cause competing reactions de-ionized, distilled, or reverse osmosis along with separation and resolutions systems. In addition to the standard issues, especially in ion chromatography. ion exchange and UV technologies, for When chromatography is coupled with example, companies like MilliporeSigma mass spectrometry (MS) some ions have contaminant-specific products to will create what are known as adducts, remove specific classes of contaminants, which suppress the MS signals and like trace organics, volatile organics, make molecule identification difficult. persistent organic contaminants like In addition, particles, including bacteria endocrine disruptors, and/or bacteria and can clog frits, fittings, pre-columns particles. There are also filters that can and the columns themselves. In high remove certain enzymes like nucleases pressure systems, while it’s not likely and proteases. However it’s important to that particulate contamination will cause remember that one size does not fit all. the column themselves to rupture or While a nuclease filter may be perfect burst—remember that these columns are for certain life science applications, it designed to take lots of pressure—there could actually complicate things for basic are real risks of having the fittings and organic separation and identification. the pumps fail as a result. At a more basic Once a system is chosen, operate it level, pre-columns and fritted fittings will regularly and keep it well maintained. need to be replaced more frequently if Purification elements like ion-exchange contaminated by particulates, adding cost media and physical filters do need to and time to your analysis. So clearly, a be replaced regularly as they become well-designed and well-managed water fouled or exhausted. Cartridges and system can help reduce the impact of filters should be rinsed prior to collecting contaminants in everyday laboratory water to ensure that any preservatives work. that may remain from manufacturing are flushed out of the system. And of course, LCGC: What are some good practices proactive cartridge and filter replacement in using a water system to generate ensure that you stay ahead of your your chromatography-grade water? contaminants. Remember that organic Plurad: First and foremost, make sure that contaminants can reduce the ion removal the water system you’re using is optimized efficiency of resin, and once all of the for the analysis and the experiments you ion exchange active sites are consumed plan on running. Today’s high purity water or blocked, the cartridge provides no systems employ multiple purification purification power or benefit at all, which

22 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

Tips and Tricks for Handling High Purity Water in the LC-MS Laboratory Anastasia Khvataeva-Domanov#, Stephan Altmaier§, Martin Lange§, and Stéphane Mabic# # Merck Millipore, Lab Water, Lab Solutions, 1 rue Jacques Monod, Guyancourt, France, 78280; § Merck Millipore, Advanced Analytics, Lab Solutions, Frankfurter Strasse 250, Darmstadt, Germany, 64293

Introduction Ultrapure water is highly prone to contamination, e.g. it easily leaches contaminants out of container surfaces and absorbs contamination from the laboratory environment. As ultrapure water is the most frequently used solvent in any LC-MS laboratory, its purity plays a critical role in analyses. There are a number of high purity water handling pitfalls that result in degradation of its quality. To help analysts critically evaluate the potential risks involved in poor ultrapure water handling, we discuss here (I) the effect of laboratory environment and long high purity water storage, (II) the effect of the container material used to collect ultrapure water, (III) the effect of laboratory ware and equipment cleaning, and (IV) the effect of poor practices of water purification system usage.

(I) Laboratory Environment and Water Storage (II) Container Material Avoid Plastic Containers (IV) Water Purification System Usage

Avoid long water storage and water exposure to Utilization of plastic devices such as bottles or funnels during Flush before Water Collection laboratory atmosphere handling and storing of water leads to leaching of ubiquitous additives (anti-static agents, stabilizers and plasticizers) and Water stagnating in the water purification system may degrade with time. Also contaminants present in the lab environment can become absorbed by the final polisher membrane and Different types of contamination are present in laboratory environment (organics, alkali causes ghost peaks and increased background noise (Figure 3). contaminate water during its collection. Thus, it is recommended to properly flush a system metals, bacteria, particles), thus water exposure to the lab atmosphere and usage of various It is recommended to collect ultrapure water in either surface prior to water collection, e.g. with several liters after the weekend or ~ 250-500 mL when decanting steps results in water quality degradation. The practice of long-time storage of treated brown glass bottles or in borosilicate glass. In standard system is used every day. ultrapure water increases the risk of water contact with the lab environment. This results glass bottles silica and alkali dissolve and form adducts (see in contamination leading to high MS background noise (Figure 1), adduct formation and sodium effect in Figure 2). signal suppression (Figure 2). Figure 3. Effect of using polypropylene bottles for ultrapure water storage.

MS profiles of two Milli-Q® water samples stored in surface-treated brown glass and polypropylene bottles, respectively (top), and TICs of the same samples (bottom).

The analyses were performed via direct injection of the solvents into the Bruker Esquire 3000+ ion trap MS operated in ESI+ mode. Technical Effects • Ghost peaks and signal suppression in LC-MS Figure 5A. Effect of ultrapure water collection without water purification system flushing when a system • Decrease in sensitivity and increase of LOD was not used for several days (e.g. after the weekend). • Increased background noise • Complexity of MS spectra MS profile of Milli-Q® water collected from an unflushed water purification system directly after the weekend.

Implications of contaminated Technical Effects high purity water Technical Effects • Complexity of MS spectra • Complexity of MS spectra • Decrease in sensitivity • Difficulties in MS data interpretation, e.g. quantification • Decrease in sensitivity • Noisy MS background • Decrease of chromatography column lifetime • Noisy MS background • Signal suppression • Increased maintenance of LC-MS instrument • Signal suppression • Adduct formation • Waste of sample, reagents and solvents • Increase of LOD • Increase of LOD Figure 1. Effect of using ultrapure water stored and exposed to the lab atmosphere and its comparison to • Risk of repeated experiments freshly produced ultrapure water. Figure 5B. Effect of flushing the water purification system prior to ultrapure water collection.

MS profile (top) of LC-MS grade water from competitor stored for four weeks and opened to atmosphere MS profile of freshly produced Milli-Q® water collected from properly flushed water purification system. several times before analysis and MS profile (bottom) of freshly produced Milli-Q® water analyzed on the day of water collection.

Analyses: Bruker Esquire 3000+ ion trap MS system, ESI+, 0.2 mL/min via syringe pump at 25°C. Technical Effects • Decrease in sensitivity and increase of LOD • Increased background noise • Complexity of MS spectra • Ghost peaks in LC-MS • Adduct formation (III) Glassware Cleaning Avoid Dishwashers

Dishwashers are operated utilizing strong bases and surfactants. The former lead to dissolution of alkali and silica from glassware, while traces of the latter remain on the glass surface (see Technical Effects above).

Figure 4. Effect of dishwashers on glassware cleanness. Figure 5C. Effect of water purification system handling in the laboratory.

Comparison of MS spectra and TICs of Milli- Q® water Figure 2. Effect of Na+ presence on increased adduct formation and signal suppression. Comparison of TICs of Milli-Q® water samples collected at different points in time. *Analyses presented in stored in borosilicate bottles cleaned: 1- in dishwasher Figures 5A, 5B, 5C were performed using Bruker Esquire 3000+ ion trap MS system, ESI+, 0.2 mL/min via 1, 2- in dishwasher 2, 3- with a mixture of LC-MS grade A solution of 500 pmol Glu-Fibrinopeptide in 50/50 ACN/water (v/v) was spiked with different amounts syringe pump at 25°C. water and ACN. The mixture was analyzed via direct of sodium (as NaCl) and the mixture was analyzed via direct injection (10 μL/min) into the Synapt® HDMS injection into the MS, ESI+ mode. system operated in ESI+ mode.

Conclusion Reference: LW_P 91 EMD Millipore is a division of Merck KGaA, Darmstadt, Germany. EMD Properly installed and well-maintained Milli-Q® water purification systems meet stringent LC-MS requirements for solvent purity. The best results are obtained when using freshly collected ultrapure water without long storage Millipore, the M mark and Milli-Q are registered trademarks of Merck KGaA, Darmstadt, Germany. All or exposure to the lab environment. Alternatively, ultrapure water should be collected in clean borosilicate glass not processed in a dishwasher. other trademarks are the property of their owners. www.emdmillipore.com/labwater © 2014 EMD Millipore Corporation, Billerica, MA, USA. All rights reserved.

can happen relatively quickly. UV lamps five parts per billion are fine for most should also be replaced when they’re chromatographic techniques. unable to oxidize organics or kill bacteria, and depending on the manufacturer, LCGC: What are some best practices replacement could be required every two for handling the purified water once it’s years. Finally, make sure you’re regularly been dispensed from the system? monitoring and reading the water quality Plurad: One thing to remember is that prior to collecting the water for use; both water is a universal solvent so it will resistivity, which gives you an idea of the absorb contaminants from the air as ionic removal, and Total Organic Carbon well as the container you put it in. To (TOC), which measures trace organics in minimize impact on experiments, a few water, can put the system and subsequent things need to be considered. First, only experiments at risk. Generally, freshly dispense what is needed for a particular produced water that is 18.2 Mega-ohm/ experiment or “run” to minimize the total centimeter resistivity with TOCs less than amount of exposure the water gets to

23 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

High Purity Water: Hints and Tips Good Practices in Using a Water Purification System and Handling High Purity Water Estelle Riché, Stéphane Mabic, Ichiro Kano, Cecilia Regnault, Béatrice Gérion and Julien Bôle Research and Development, Lab Water, EMD Millipore, St Quentin-Yvelines, France Introduction Storage of purified water Flushing the water system The usage of water purification systems is widespread in laboratories. When it comes to utilizing the purification Ultrapure water should not be stored, as it may absorb impurities from the air or the container used for storage. While a water purification system is left idle, the system and the ultrapure water, however, each laboratory and user develops a number of habits. While some practices However, it is often necessary to store pure water before further purification with a polishing system. The water will quality of the water remaining inside the system are rational and based on sound experience, others simply arise and set up over time. Working with high purity water be in contact with the reservoir for an extended period of time. Therefore, the reservoir material should be chosen will slowly degrade. Therefore, it is best to discard actually requires taking a number of precautions and following some simple rules. Some of these “good laboratory carefully as it may leach organic and/or ionic compounds into the water. the first few liters of water produced when water practices” are described here. High-density polyethylene (HDPE) and polypropylene (PP) were tested. Ultrapure water was stored in carefully rinsed is drawn from an idling system, and collect only bottles for 24 h, water was analyzed then discarded. The procedure was repeated 3 times. Table 1 and Figure 4 show freshly purified water. Overview that reservoirs made of HDPE are a better choice than those made of PP. HPLC: Figure 8 demonstrates that some organic Figure 8: Effect of flushing on HPLC (A) and LC-MS (B) baselines UV Ion-exchange resins contaminants may be present in the water initially Pretreatment pack 254 nm Day 1 Day 2 Day 3 HDPE 1 PP + activated carbon Polishing Unit Ions (ppb) drawn from a water purification system. Day 1 Day 2 Day 3 Day 1 Day 2 Day3 Pretreatment unit (Elix®) Table 2 shows a reduction in bacteria levels Bacteria in water (Milli-Q®) Fluoride ND ND ND ND ND ND Bacteria: downstream of the membrane when more water Tap water Chloride 11,7 0,9 0,5 6,0 3,8 2,2 Membrane Reverse osmosis TOC is drawn. This suggests that the bacteria observed Nitrite 2,2 0,5 < 0,1 0,3 ND ND monitor at the outlet of the filter are of airborne origin. Phosphate ND ND ND ND ND ND Pharmaceutical-Grade Bacteria in ambiant air Resistivity TOC Bromide ND ND ND ND ND ND To have bacteria-free water, it is recommended Absolute, 0,22 m Filter UV cell 185/254nm (ppc) TOC Nitrade 1,4 0,3 < 0,1 1,3 1,0 0,7 to draw some water (>1 L) through the filter to Electrodeionization Volume Week 1 Week 2 Week 3 Week 4 Sulfate 6,2 0,4 0,1 1,1 1,1 1,0 remove downstream bacteria. Even better results Reservoir Calcium 9,0 2,0 1,0 5,0 1,0 1,0 can be obtained by locating the filter outlet in a delivered (L) (CFU/ml) (CFU/ml) (CFU/ml) (CFU/ml) 0,22 µm filter 0,0 73 5 35 86 Magnesium 1,7 0,3 < 0,1 0,4 0,3 0,3 sterile area such as a laminar flow hood. 0,5 22 1 2 25 Monitoring the water quality Potassium 2,0 < 1 < 1 3,0 < 1 < 1 Rinsing the cartridges High purity water 1,0 0 0 2 3 Flushing the system Sodium 8,0 < 1 < 1 4,0 < 1 < 1 3,0 0 0 0 0 Careful storage of purified water HDPE 1 PP Figure 4: Total organic carbon (TOC) extractables from Table 1: Ionic extractables from polyethylene and polypropylene. Selection of sampling containers polyethylene and polypropylene. Flushing the water system Rinsing the cartridges Tap water RO cartridge 2 Monitoring the water quality Ultrapure water is an excellent solvent. It has a high affinity for chemicals in its surroundings and can absorb chemicals RO cartridge 3 Water purification cartridges often are manufactured with preservatives RO cartridge 1 readily from storage containers, as well as chemical vapors from the laboratory air. Figure 9 shows that the polyethylene Monitoring water quality ensures experiment repeatability and quality results. Commonly used monitoring tools are for long-term storage. In particular, this is the case for reverse-osmosis carboys and wash bottles commonly used to store water in laboratories can leach organic molecules into ultrapure conductivity and total organic carbon (TOC) meters. water. Glass containers leach less organics, however they may leach inorganic compounds. Polyvinyl chloride (PVC) (RO) cartridges. • Conductivity measures the flow of electrons through a fluid, which is proportional to the concentration of and fluoropolymers (PFA) are also commonly used sample containers. Table 3 shows that many compounds may leach Figure 1 shows the importance of rinsing a newly- installed RO cartridge. from these polymers into ultrapure water, as seen by ICP-MS. Figure 10 shows that chemicals present in laboratory TOC (ppb) TOC ions, their charge and mobility. Initial organic contamination (TOC levels) is high (~500 ppb), then decreases air may also be absorbed by ultrapure water and affect LC-MS results. • TOC measurement indicates the total amount of organic material present in water. with time as rinsing occurs (~95 ppb after 300 min). Polishing cartridges, containing ion-exchange resins and activated carbon, It is therefore important to use freshly produced ultrapure water and to select a sample container compatible with Many organic molecules do not ionize readily and, as a result, do not influence the conductivity of water. Therefore, also must be rinsed initially. Figures 2 and 3 demonstrate the decrease in the analyses to be done Time (min) purified water may contain some organic compounds and have an excellent resistivity reading. Similarly, a low TOC resistivity level and TOC level with rinsing time. Figure 1: Initial rinsing of reverse-osmosis cartridges: evolution of TOC levels of permeate value only indicates that there are no organic molecules present in water without indicating the levels of metals or with time. salts. Combining these two monitoring tools reflects the level of water purity more precisely. Element (ppt) PFA bottle PVC bottle Large amounts of sucrose were added to pure water. Figure 7 shows that it was first retained by the activated carbon B (11) 16,768 68,347 Na (23) 0,782 110,460 of the polishing unit, then broke through the purification system. Resistivity levels remained unchanged, although Mg-2 (24) 0,295 0,800 TOC levels rose with time. Al-2 (27) 0,832 0,788 Monitoring Sucrose K (39) 15,932 28,058 Figure 9: Reverse-phase HPLC chromatogram of ultrapure water stored in polyethylene (A) or in glass (B). Absorbance measured at 254 nm. Ca (40) 0,565 5,834 Polishing

TOC (ppb) TOC Tap Reverse Storage High purity Unit Fe (56) 9,776 4,428 Water Osmosis reservoir water (no UV) Cu (63) 0,883 1,191

Resistivity cell (MOhm.cm) Zn-1 (64) 12,092 18,489 Quantum IX # 1 Ag (107) 0,258 0,266 Quantum IX # 1 Quantum IX # 2 Quantum IX # 3 Quantum IX # 2 Quantum IX # 3 Cd (111) 2,417 1,381 Pb-2 (208) 0,055 0,049 Resistivity Time (min) Time (min) Table 3: Effect of container material on sample purity. After Figure 5: Stand-alone A10 TOC meter (MOhm.cm) Figure 10: Total ESI+ plot (from m/z 150 to 600) of HPLC- grade water kept at room Figure 2: Initial rinsing of polishing cartridges: changes in Figure 3: Initial rinsing of polishing cartridges: rinsing each container 5 times with Milli-Q® Element water, TOC fresh Milli-Q® Element water was collected in the containers and temperature, opened to atmosphere three times a week for 15 minutes during a resistivity levels of effluent water with time. changes in TOC levels of effluent water with time. (ppb) analyzed by ICP-MS. 2-month period. Figure 6: EMD Millipore Time (min) flow-through coaxial Figure 7: Evolution of resistivity and TOC levels in ultrapure water LW_P51 resistivity cell after addition of sucrose in the storage reservoir EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

the air and to the container. Stored high from the manufacturing package or purity water will degrade with simple storage processes. However, if using glass absorption of CO2 from the atmosphere, containers, surface-treated amber glass and if the container itself isn’t well bottles or borosilicate glass containers managed, organics and particles can are recommended. Regular glass bottles enter the bottle. Letting water stand for can leach silica and alkali metals which will days in between runs should be avoided cause problems. So basically, anything since it introduces the risk of bacteria and that can be done to minimize storage and other contamination. Also, be mindful exposure to contamination risks should of the container used. The last podcast become best practice. discussed avoiding plastic containers. Plastic containers, depending on the LCGC: Is there anything else you can material of construction, could include do to minimize the recontamination of anti-static agents, plasticizers, stabilizers ultra-pure water? or even dust and plastic fines that result Plurad: Finding ways to reduce exposure

24 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

is always the best practice. In addition About the Life Science Business of to picking the right size and material of Merck KGaA, Darmstadt, Germany construction, you should really try to use The life science business of Merck KGaA, the smallest container possible should Darmstadt, Germany, which operates be used to help minimize exposure and as MilliporeSigma in the U.S. and contamination and to keep everything Canada, has 19,000 employees and 65 clean. Another thing to consider is to manufacturing sites worldwide, with a minimize exposure and contamination portfolio of more than 300,000 products from the tubing used to introduce ultra- enabling scientific discovery. Udit Batra pure water into the container, especially is the global chief executive officer of if transferring caps and moving from MilliporeSigma. one container to the next. For example, Merck KGaA, Darmstadt, Germany bench top or atmospheric contaminants completed its $17 billion acquisition can easily be introduced if tubing of Sigma-Aldrich in November 2015, or caps are left on the countertop. creating a leader in the $125 billion If experimental work is sensitive to global life science industry. CO2, bottled vent filters or organic Merck KGaA, Darmstadt, Germany is a traps should be considered. Container leading company for innovative and top- cleaning should also be avoided quality high-tech products in healthcare, if possible, especially in automatic life science and performance materials. glassware washers. Most commercially The company has six businesses – available detergents utilize strong bases Biopharmaceuticals, Consumer Health, that can etch the glass and dissolve Allergopharma, Biosimilars, Life Science alkali and silica which will be absorbed and Performance Materials – and into the water. Traces of surfactants generated sales of €12.85 billion in and detergents, which are organic, 2015. Around 50,000 employees work could also remain on the surface of the in 66 countries to improve the quality glassware, only to be leached off with of life for patients, to foster the success the ultra-pure water dispensed into the of customers and to help meet global bottle. The same risks exist for bottled challenges. water. The certificate of analysis (CoA) Merck KGaA, Darmstadt, Germany lists the condition of the water when it is the world’s oldest pharmaceutical was bottled, not necessarily when used. and chemical company – since 1668, The CoA is no longer effective one the the company has stood for innovation, lid is removed, and the result is the business success and responsible same risk of contamination compared entrepreneurship. Holding an to water from any water purification approximately 70 percent interest, the system. founding family remains the majority

25 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

owner of the company to this day. The company holds the global rights to the name and the trademark “Merck” internationally except in the United States and Canada, where the company operates as EMD Serono, MilliporeSigma and EMD Performance Materials.

Joseph Plurad North America Field Marketing Manager MilliporeSigma

26 | October 2017 | LCGC Sponsor’s content 27 | October 201727 |October |LCGC Jean-Francois Pilette Sy Water Purification Se l s ecting a Laboratory tem manganese, lead, and aluminum. The most water are sodium, typically calcium, iron, charged ions). The cations present in natural (positively charged ions) and anions (negatively water.purified becometo laboratory-grade removed be must water whentap is further and additional reducedbe further contaminants The levels of contaminants these however must natural water into is processed drinking water. contaminants are removed to someextent when microorganisms, of types and gases. Certain organic substances, particulates and colloids, fivecategories: inorganicsalts, dissolved Water contaminants divided be into can of Contaminants Classes Primary laboratory water use. parametersthese and describes how affect they discusses monitoring article required. This water youof water need, quality and the type volumes of Type 2(pure) or Type 1(ultrapure) available feed water, and daily the instant you need to removed, of the the quality ofthat you contaminants determine the types purification for system your laboratory requires the most appropriateIdentifying water Inorganic are composedof salts cations Sponsor’s content Sponsor’s

shutterstock.com/Africa Studio Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

common anions in natural water are Ionic contamination can also be an chloride, nitrate, sulfate, phosphate, and issue in HPLC analyses involving mass carbonate. The presence of unknown spectrometry detectors, especially when or unwanted ions, or an excessive using electrospray positive (ESI +) mode. concentration of ions, in laboratory-grade Instead of providing the conventional water can adversely affect laboratory [M+H]+ response, sodium and potassium processes. For example, if lead ions are ion contamination can result in [M+Na]+ being analyzed and the water used to peaks (called sodiated peaks) and [M+K]+ wash glassware or prepare standard peaks. These unintended peaks produce solutions contains lead, the results will be incorrect mass assignments. Achieving inaccurate. Ion concentrations therefore multiple peaks for a single molecule have to be minimized in laboratory-grade makes data analysis more complicated water. and difficult. Silica, a negatively charged mineral, Dissolved organic substances, which may also be classified as an anion. As a contain carbon atoms, are another contaminant, silica is a special case. It is source of water contamination. Organic weakly charged and exists in different substances can have a natural origin. For forms. Monomeric silica comprises a example, when leaves fall on the ground single silica atom, dimeric silica is made in autumn, rain water starts dissolving up of two atoms, and polymeric silica is them. In time, components of the leaves composed of many silica atoms, as well as and wood are released and dissolve in silica sheets and silica crystal forms. Silica the rain water. The long cellulose chains therefore encompasses a multitude of and smaller molecules such as lignin contaminants with common behaviors but monomers, gallic acid, and catechin (a also small differences. These differences component of the tannins in the wood) make silica difficult to remove from water. vary widely in nature and in molecular Removing ionic contaminants from weight. laboratory water avoids positive bias, Natural water may also be contaminated interferences, and variations in the by dissolved organic substances such ionic strength of solutions during as the following generated by human analysis. Ionic contaminants can also activities: affect chromatographic results. In high- • Solvents called volatile organic performance liquid chromatography compounds (VOC), such as (HPLC) and ion chromatography (IC), ionic perchlorethene, used in the dry cleaning contaminants can result in additional of clothing peaks, increased background levels • Molecules such as benzene found in that can affect sensitivity, and artificially gasoline increased values. • Pesticides such as herbicides,

28 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

fungicides, and insecticides used by the additional inorganic and organic farming industry and in gardening contaminants. • Polyaromatic hydrocarbons (PAHs), Colloids (such as colloidal silica) are which are molecules released, for stable suspensions of organic and example, from coal tar used as a inorganic particulates (0.1 to 0.001 µm in pavement sealcoat size). The particulates in colloids usually • Polychlorobiphenyls (PCBs), a persistent have the same electric charge, and industrial contaminant whose use has the repulsion prevents the liquid from been forbidden since 1976 settling (unless a coagulating agent of • Plasticizer substances added to the opposite charge is added). Hard polymers to make them softer, for particulates can damage or impair the example, bisphenol-A (BPA), widely operation of laboratory instruments used with polyvinyl chloride (PVC), a such as pumps and injectors, and soft polymer used to make most of the particulates can plug chromatography plastic pipes used to carry tap or columns and frits. Particulates can also act deionized water as a shelter for microorganisms and may operate as a catalyst to accelerate certain As with ionic contaminants, organic reactions. contaminants must be removed Microorganisms such as bacteria and in order to avoid positive bias and viruses are also common natural water interferences during organics analyses. contaminants. Bacteria vary in size and Organic contaminants can also affect shape. Escherischia coli, a microorganism chromatography by coating stationary commonly found in the human intestine phases. This results in slower mass are 2 μm, while Pseudomonas diminuta, transfer (shifting retention times), reduced is the smallest bacteria. When grown resolution, baseline issues, and shorter in a very poor culture medium with column lifetimes. stirring, Pseudomonas diminuta grows Particulates and colloids contaminants as a sphere 0.27 μm in diameter. can be divided into two classes: hard Because it is the smallest bacterium particulates (such as sand and clay) and known, this microorganism is typically soft particulates (such as vegetal debris). used to challenge membrane filters for Hard and soft particulates can offer bacteriological retention. protection against ultraviolet rays and Bacteria can be a nuisance even when chemical agents, while acting as a surface dead because they release byproducts for bacteria to adhere to. Particulates and that can cause problems with scientific colloids are typically between 0.1 and 100 experiments. Gram negative bacteria µm. Hard and soft particulates can slowly can release pyrogens from their walls, dissolve in natural water, generating and other bacteria can release nucleases

29 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

Figure 1: DNA Separation Column: Injection 1

Retention HPLC Grade Bottled Water – TOC > 50 ppb

Figure 2: DNA Separation Column:– Injection 1950

such as RNases and DNases. Bacteria contaminants that can alter biological and byproducts need to be removed experiments such as cell cultures. Bacteria from laboratory water sources because byproducts such as pyrogens and they are sources of ions and organic nucleases can affect cell operation and

30 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

Milli-Q® Water – TOC = 4 ppb

Figure 3: DNA Separation Column:– Injection 6400

molecular biology mechanisms. Bacteria Other gases can dissolve in rain water also effect chromatography in much the and contribute to its acidification,

same way as organic contaminants. including sulfur dioxide (SO2, from Dissolved gases are also natural combustion plants, industrial processes, water contaminants. The primary transportation, and natural origins), gases dissolved in water are typically nitrates (originating primarily from road the main constituents of the air that is transportation and combustion plants),

in equilibrium with the water source: and ammonia (NH3, typically originating oxygen, nitrogen, and carbon dioxide from agriculture activities). Removing (which is in equilibrium with carbonic dissolved gases from water may be acid). The solubility of these gases is required for certain experiments to a function of temperature. Another avoid the formation of bubbles. Bubble common dissolved gas contaminant is formation can affect the measure of radon, a water-soluble gas produced optical density or result in outgassing, by the decay of radium isotopes. It which can cause pump and detector is naturally found in ground water problems in chromatography. and results from granite formations, phosphate deposits, and uranium Water Monitoring Techniques deposits. In addition to being a Conductivity is the most accepted means contaminant, radon may also cause of determining the purity of the water. human health problems, including cancer. By definition, conductivity is a material’s

31 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

The conductivity of a material is directly

DNA Sep Number of related to the temperature; conductivity Column Water Source TOC (ppb) Injections increases—and resistivity decreases—as 1 Bottle Brand B 777 408 temperature rises. The formula used to 2 Bottle Brand E 100 555 calculate conductivity is directly related 3 Bottle Brand C 87 2103 to the temperature of the material so 4 Bottle Brand A 32 1235 a normalized or compensated value 5 Bottle Brand D 16 2167 is often reported in order to have a 6 Milli-Q® Gradient 4 6394 standard point of reference. Typically this 7 Milli-Q® Gradient 4 10685 temperature is 25°C. Therefore, while 18.2 Bottle = HPLC Grade Bottled Water – Various Brands MOhm x cm water is the accepted value

Figure 3: DNA Separation Column:– Injection 6400 for Type 1 (ultrapure) water, it is only valid if it is measured at or compensated to 25°C. Since many laboratory experiments ability to conduct electricity. However, can be affected by ions, it is important to since Type 1 (ultrapure) water itself does measure resistivity to assess the presence not conduct electricity, conductivity, is of ions. by proxy a measurement of all of the ions Because there can be multiple that remain after the purification process. sources of organic contamination and As the conductivity value becomes very because it can be difficult to completely small and approaches the requirements removeremove trace organics due to of Type 2 (approaching pure) water, the theirthe complex nature, it is important to purified water is said to be characterized ensure that the concentration of organics by resistivity (the inverse of conductivity). in water remains low and constant over Resistivity is a technique that attempts time. This can only be achieved if a to assert the absence of ions in Type 1 trusted measure of total organic carbon (ultrapure) water. Resistivity detects ions (TOC) is regularly performed on the water present in water with a high degree of prior to use. sensitivity: A concentration as low as 1 ppb Figure 1 through Figure 3 (1ug/L) of a salt causescause a significant demonstrate the effects TOC can have drop in the value of resistivity. Assuming on a chromatographic separation. Using that water fully and equally dissociates HPLC-grade bottled water with a TOC intoto H+ and OH– in equilibrium (resulting greater than 50 ppb, resolution can be in a pH of 7), the theoretical conductivity of seen to degrade significantly (Figure a solution of H+ and OH– is 0.055 uS/cm at 1 and Figure 2) over the course of 25°C, or 18.2 MOhm x cm at 25°C. For this approximately 2000 injections. However reason, Type 1 (ultrapure) is often referred using Milli-Q® water, which has a TOC of to as 18.2 MOhm x cm water. 4 ppb, the separation remains virtually

32 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

UV Tank Progard® Reverse EDI Lamp for for RO Osmosis (Elix®) 254 Type 2 protection cartridge Module nm Water

Figure 4: Water Purification Techniques: Pure Water

unchanged after more than 6000 filter to retain particulates. injections. Table 1, which compares the The conductivity of the treated water effects of TOC levels on column lifetime is then measured before it enters the for bottled water and Milli-Q® water, reverse osmosis cartridge. The reverse further illustrates this point. osmosis cartridge removes more than 97% of the ions in tap water, as well Water Purification Techniques as 99% of bacteria, particulates, and The production of Type 2 (pure) water organics larger than 200 Dalton. A from tap water is achieved using a series scanning electron microscope picture of a of purification technologies, as illustrated reverse osmosis membrane cross-section in Figure 4. Tap water entering a water is shown in Figure 5. The membrane purification system such as the Milli-Q® is separated into two layers: a one- Integral system first passes through a micron-thick active layer that removes Progard pretreatment cartridge, whose contaminants on top, and a thicker (100 goal is to protect the reverse osmosis µm) porous support underneath that cartridge. The pretreatment cartridge provides mechanical support to the contains activated carbon to remove thinner active layer. The reverse osmosis chlorine, polyphosphate to prevent membrane is able to reject contaminants calcium carbonate precipitation, and a using two combined mechanisms related

33 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

Organics Particulates, Rejection is based on Colloids & Size (~ MW) Ions Bacteria Electric Charge

Active layer (1 m)

Porous Support

Mechanical Backing SEM Picture of a RO membrane cut through

Figure 5: RO Membrane: Structure & Properties

to the size and electric charge of the 15 L/hour to the drain and recycles 27 L/ contaminants. The reverse osmosis hour upstream of the reverse osmosis membrane therefore effectively rejects cartridge. As a result, the system only ions, organics (even if they are not needs an additional 20 L/hour of tap electrically charged), particulates, colloids water to provide the 47 L/hour required and bacteria. to feed the reverse osmosis cartridge. The rejection performance of the At the outlet of the reverse osmosis reverse osmosis membranes depends cartridge, a conductivity meter measures on the nature of their material; most are the quality of the water produced and made of cellulose acetate or polyamide. the reverse osmosis cartridge rejection Rejection performance also depends on efficiency. the manufacturer’s process and quality. The permeate water then enters the For instance, in the RiOs™ 5 water Elix® module, which removes most of purification system, the reverse osmosis the remaining ions in order to produce cartridge is fed by a flow rate of 47 L/hour Type 2 (pure) water with a resistivity to produce 5 L/hour permeate water. greater than 5 MOhm/cm, and a TOC Consequently, the cartridge rejects 42 L/ greater than 30 ppb. A resistivity hour. However, of the 42 L/hour rejected meter at the outlet of the EDI module by the cartridge, the system sends only measures the quality of the water

34 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

UV 185 nm AC / IonEx Polisher

MILLIPORE

Ultra Pure Water

Pure Recirculation Water

Figure 6: Purification Techniques to Ultrapure Water

produced by the Elix®. Elix®’s capabilities The water then moves through a are based on electrodeionization Quantum™ cartridge, which contains technology developed and patented activated carbon and an ion-exchange by MilliporeSigma that uses permanent resin to remove the remaining traces of regeneration by a weak electric current. inorganic and organic ions to produce Elix® modules can last for several years Type 1 (ultrapure) water. The synthetic and the water does not need to be activated carbon, produced from the softened upstream. The resulting Type 2 pyrolysis of styrene beads, has a small (pure) water is stored in a reservoir and (<150 Å) pore size, has a surface area of can be either used directly or further 1100m2/g, and is relatively homogeneous. processed to produce Type 1 (ultrapure) Consequently, adsorption kinetics are water. rapid for low-molecular-weight organics, The production of Type 1 (ultrapure) and very few mineral ions are released. water from Type 2 (pure) water is achieved Activated carbon cartridges are used using the system outlined in Figure 6. in these polishing cartridges mainly to The Type 2 (pure) water first flows through remove trace organics. Small organic a 185 nm UV lamp module to oxidize molecules diffuse inside the pores and organics into electrically charged organic link to binding sites by weak van der ions and to destroy bacteria. Waals forces. The quality of the Type 1

35 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

Figure 7: Polisher Packs

(ultrapure) water produced is monitored and calibration of the resistivity and by an inline resistivity meter and an online TOC monitors are key to ensuring TOC monitor. Typically, the sum of all ions water quality. Daily usage should also is lower than 1 ppb and the sum of all be considered, as well as ergonomy, organics is lower than 5 ppb. bench space, volumetric dispensing, The produced Type 1 (ultrapure) water and flow rate (so that the water level can can then be dispensed though a Q-POD® be accurately adjusted). A laboratory with a POD-Pak polisher (Figure 7 at water system should also have intuitive the outlet in order to further adjust the interface. water quality to meet the user’s needs. Quality assurance (ISO9001 certification The POD-Pak polisher can be adapted as and cGMP compliance) is also important. needed for each application. Certificates for calibration, conformity, and consumables quality must be Selecting, Operating, and Maintaining available, and established norms of a Water System ASTM (resistivity) and USP (resistivity and Several parameters are important to TOC suitability tests) must be followed. consider when choosing a laboratory CE certification, cUL certification, and a water system. Quantity is a practical consumables validation guide should also consideration, but perhaps more be included. important is the quality of the water Qualification, installation, calibration, needed—Type 2 (pure) water or Type maintenance, repair, and technical 1 (ultrapure) water. If Type 1 (ultrapure) services should be provided. Operating water is required, the type, sensitivity, costs over the system’s lifespan—not only

36 | October 2017 | LCGC Sponsor’s content Ionic Hormone Tips: Water Tips: Handling Purification Contamination Analysis Purification Purified Water System Selection

the original capital expenditure—should Materials and Life Science—that be considered. Finally, impact on the together are asking very big questions environment should be considered. and tackling some of the world’s most During routine operation, storage pressing scientific challenges. of Type 1 (ultrapure) water should be As Merck KGaA, Darmstadt, Germany’s avoided, as it can be quickly and easily Life Science business, MilliporeSigma is contaminated. For best results, avoid proud to be part this legacy of discovery storing water in carboys. Produce and innovation. With the company’s water on demand to prevent container other businesses in the United States, extractables and bacteria buildup. we’re celebrating our collective 125-year Unnecessary connections—such as U.S. history and the boundless curiosity tubing at the outlet after the consumable behind our past—and future—success. filter—should be avoided. Manufacturer- We invite you to join us in this recommended maintenance schedules celebration and learn how you can be should be followed for consumable part of our journey of discovery. Because replacements to reproducibly obtain high Smarter, Together describes how we water quality. collaborate not only internally, but Since purified water is the most also with our customers, vendors and common reagent found in most partners across the global life science laboratory facilities, a properly industry. configured and maintained laboratory water system is critical to the success of almost every laboratory experiment. The appropriate grade of water is essential for everything from washing glassware to buffer preparation and cell culture analyses to complex analytical techniques such as HPLC or LC-MS. Appropriate purified water ensures the success of projects and maintain Jean-Francois Pilette productivity. Group Product Manager (Retired) MilliporeSigma In 1890, Merck KGaA, Darmstadt, Germany established its U.S. presence in New York City. Today, the company employs approximately 5,000 people in the United States across three businesses—Healthcare, Performance

37 | October 2017 | LCGC Sponsor’s content