High Purity Water for Critical Laboratory Applications Brian Rhoades Water Analysis and Purification Specialist

Overview Explain the standards of water purification Review impurities in water Review technologies to remove those impurities Discuss the technologies utilized to produce different qualities of water In today’s presentation we are going to discuss: Why proper maintenance is important and how it affects the purity of the whole system. What components need to be maintained. Understanding what the different components do in the system will help to identify why they need to be changed. Typical consumable replacement schedule Finally, tips on how to change the consumables in your water system.

Water Purification Standards There are several agencies that publish industry standards for the different types of water We will cover the most commonly referenced standards: ASTM – American Society for Testing and Materials – D1193 CLSI – Clinical and Laboratory Standards Institute ISO – International Organization for Standardization – ISO 3696 There are several agencies that publish industry standards for the different types of water. The most well known is set forth by the American Society for Testing and Materials agency. But there are also other similar but lesser known standards set forth by the Clinical Laboratory Standards Institute and the International Organization for Standardization.

Reagent Grade Water Types

Clinical and Laboratory Standards Institute (CLSI) Laboratory Water Standards Clinical Laboratory Reagent Water - CLRW CLSI also sets standards for clinical analyzer feed water, bottled water, special reagent water, and autoclave water Please refer to the entire guidelines as provided by your client Contaminant Unit of Measure Standard Ions Resistivity (megohm) > 10 Organics TOC (ppb) < 500 Bacteria Bacteria (cfu/ml) < 10 Particles Microns 0.2 micron filter The Clinical and Laboratory Standards Institute (CLSI) standard is for water used in a clinical lab setting but NOT for injectables. Basically the standard published is for Type 2 water which is used for a variety of applications like rinsing glassware or feeding / filling ancillary lab equipment like autoclaves. Sometimes water systems are used to feed clinical analyzer systems but this is a bit of a different beast so it is important to reach out to the analyzer manufacturer if this is the desired use. Water for injectibles is a completely different market that requires FDA regulated water also called WFI (water for injectibles) water and is purchased in bottles not produced on the spot.

Grade 1 (ISO) Grade 2 Grade 3 Water Standards – ISO 3696 ISO standards measure in grades ISO only concerns resistivity and conductivity Type I Grade 1 (ISO) Type II Grade 2 Type III Grade 3 Type IV Conductivity - (max. µS/cm) 0.056 0.100 1.000 0.250 5.0 Resistivity - (min. MΩ•cm) 18.0 10.0 1.0 4.0 0.2 There are three grades from the ISO water standard. The ISO standard only takes into consideration resistivity/conductivity. The resistivity values are in italics, representing that they are calculated from the conductivity values. The ISO standard does not address water for organic trace, biological or medical analysis, so we see it does not provide any values or limits. This was a quick overview of the three primary standards you will encounter. The most common standard you will encounter will be the ASTM standards of ultrapure, pure, and RO. The ISO standard and CLSI standard are almost identical.

Appropriate Use of Water Feed for lab equipment Ultrapure water Water for chromatography Water for cell culture If I used ultrapure water for these applications: **ultrapure water is 18.2 water – it has no ions in it. Thus, once it hits the air or the container, it will try to scavenge ions from wherever it can. It’s quite aggressive. It would pit the stainless steel it comes in contact with. – not a good idea Other applications its perfect – no ions or organics

Appropriate Use of Water Feed for lab equipment Reverse Osmosis water Water for chromatography Water for cell culture If I used reverse osmosis water for these applications: **RO water has some ions and organics in it. It would remove enough ions to be great to have exposed to lab equipment For the ion chromatography or cell culture you would experience adverse effects. Ghost peaks in the chromatographs or variations in the cell culture.

Limitations & Capabilities

Ion Impurities in Water Dissolved Ionized Solids Major Calcium - Ca++ Cations Magnesium - Mg++ Sodium - Na+ ------------------------------------------------------------------- Chloride - Cl- Major Sulfate - SO4= Anions Bicarbonate - HCO3- Silica/Silicate - HSiO3 - Highest purity Resistivity: Deionization with Semiconductor grade High grade mixed bed resin formulated to have a low TOC Service DI: regenerated resins – not high purity Distillation: Ionized gases could reabsorb RO: % removal technology Dissolved ionized solids and gases make up the bulk of the impurities found in water. The most commonly known of the dissolved solids are calcium and magnesium ions that create “water hardness”, and bicarbonate ions that make up “water alkalinity”. Calcium, magnesium and sodium all originate from the rock and soil areas that water passes through. Silica is another dissolved solid with a weak ionic charge that is commonly found in water. We typically see ionized solids in tap water range from 30-500 ppm and is fairly consistent over time. Water quality can vary from city to city and well to well. Areas with typically high ionized solids are the Midwest states, southern CA, TX. For example Marietta OH has about 260 ppm of total ionized solids. Locations that have lower ionized solids are the state of Washington, Northern CA, NY, NC, and GA. To remove ions from the water – Deionization is best, followed by distillation and reverse osmosis.

Monitoring Resistivity - Ions The easiest ways to monitor the purity of water is by measuring the resistivity or conductivity. Since this is what most water systems display for purity, it is important to understand what this measurement means. Resistivity and conductivity measure ionized particles in water. High ions in water, make it the easier it is to conduct an electrical current. For example Tap water has high number of ions. It has high conductivity, or low resistance. Ultrapure water has very low number of ions in the water. Therefore it is has low conductivity or high resistance. . The theoretical highest purity of water 18.2 resistivity at 25 degrees Celsius. This does NOT measure organic compounds or particles in the water. Ultrapure water is not a natural so it is known to be more aggressive to reach an equilibrium by readily absorb contaminants it can back into it. Conductivity Resistivity Conductivity Resistivity

Organics Total organic carbon monitoring organic compounds. Low TOC = higher purity NOT the same as bacteria! Sources include: Plant and animal decay Agricultural and manufacturing byproducts Phthalates or plasticizers increase flexibility and transparency of plastic Best removed from water with UV and carbon. Total organic carbon is a measurement of organics including volatile organic carbon (VOC) or semi- volatile organic carbon( S-VOC) , whose sources include: Plant and animal decay Agricultural and manufacturing byproducts Phthalates or Plasticizers which are added to plastics to increase their flexibility and transparency. These include Endocrine disruptors which can interfere with human development and hormones. Tap water TOCs can range from 1-5 ppm. While trace organic analysis often involves levels of less than 5 ppb. There are different ways to measure TOC content in water. One way is to measure TOC indirectly by breaking down the organic compound with a UV light. The results is CO2 and water. By measuring the change in resistance caused by the CO2 is directly related to the level of TOC in the water. It is important to monitor the TOC level for any changes in the sample.

Bacteria Found: Everywhere – air and water Potentially interferes with: Lab equipment – clogs filters (HPLC) Sensitively biological methods such as cell culture, tissue culture, and bacterial studies Source for pyrogens/endotoxins Source for nuclease (RNase/DNase) Removed with final filter, RO, UV Likes to grow in water systems! Bacteria Measured off-line by micro culture methods A natural contaminant in feed water Measured in CFU/mL Pyrogens – Also know as endotoxins, are by-products of bacterial growth and degradation. Pyrogen contamination can also retard cell or tissue growth or interfere with other research applications. Pyrogens (specifically bacterial endotoxin) are. Bacteria – It can be easy to contaminate water, so there are multiple technologies employed to control bacteria. Bacteria can create havoc with molecular biology applications.

Nuclease and Pyrogens (Endotoxins) RNase/DNase Measured off-line by fluorometric assay Enzyme that cleaves bonds within nucleic acids Measured in ng/mL (ppb) or pg/mL (ppt) Endotoxins (pyrogens): Pieces of Gram negative bacteria Interfere with growth of mammalian cell or tissue cultures Nucleases: Enzymes RNase and DNase Degrade and breaks down RNA and DNA Sensitively biological methods such as cell culture, tissue culture, and bacterial studies Pyrogens Measured off-line by LAL spectrophotometric assay Bacterial cell walls Can retard cell or tissue growth Measured in units/mL (EU/mL) Pyrogens – Also know as endotoxins, are by-products of bacterial growth and degradation. Pyrogen contamination can also retard cell or tissue growth or interfere with other research applications. Pyrogens (specifically bacterial endotoxin) are. Bacteria – It can be easy to contaminate water, so there are multiple technologies employed to control bacteria. Bacteria can create havoc with molecular biology applications.

Particulates Ruins RO membranes Lab equipment – can leave a deposit, plug valves Affect gravimetric methods Prefilters remove particulates Measured by turbidity meter – should be below 1 N.T.U. Rust Sediment Plumbing debris Typical tap water Particulates in water, such as rust, sand, silt, and plumbing debris may be visibly floating around in water or settling at the bottom of a beaker. These particulates can plug valves and filters, and tear holes in RO membranes. Some particulates are submicron, not visible to the naked eye, yet can have the same affect. Colloids are another particulate in water. They have a slight negative charge and do not settle out of water. Colloids can come from the feed water source or can often come from regenerated service DI tanks. Particulates are measured by turbidity or silt density index and can be removed by consumables such as filters and reverse osmosis membranes NaCl clip art from biology.arizona.edu

Limitations & Capabilities

Deionization or Ion Exchange Removes ions from water through the use of synthetic resins. Divided into two classifications – cation and anion. Only technology that produces 18.2 megohm water. Mixing cation and anion resins achieve maximum purity. Limitations Benefits Removes dissolved inoragnics (ions), very effectively. Water resistivity above 18.0MΩ -cm Limited capacity – once all ion binding sites are occupied ions are no longer retained Does not remove organics, particles, pyrogens or bacteria

Ion Exchange and Regeneration

Electrodeionization Removes ions from water using electricity and resin. Continuously regenerates automatically (though has a finite life) The technology that produces 5- 15 megohm water. Limitations Benefits Removes dissolved inoragnics (ions), very effectively. Water resistivity 5-15Megohm -cm Finite capacity Expensive replacement Does not remove organics, particles, pyrogens or bacteria

Distillation Water is heated to the boiling point. Water vapor rises to the condenser where it is cooled and condensed. The purified water is stored for use. Benefits Offers the broadest removal capabilities of any single form of water purification Requires no consumables Pure water Limitations Requires periodic maintenance and cleaning of system to maintain water purity Requires cooling water

Distillation

Reverse Osmosis Most economical method of removing up to 99% of all contaminants. Water is pushed through a semi permeable membrane Most impurities do not pass through the membrane, they collect on the surface and are flushed Benefits Limitations Limited flow rates through the membrane require intermediate storage devices to meet user demand Membrane susceptible to: Scaling – CaCO3 Fouling – Organics & Colloids Piercing – hard particles To varying degrees, removes all types of contaminants. Requires minimal maintenance

Reverse Osmosis - Rejection Characteristics Good for pretreatment of tap water (Concentrate)

Reverse Osmosis Spiral Wound Membrane (Permeate) (Concentrate)

Carbon Adsorption Process: Water is passed through activated carbon which organics and chlorine adhere to Benefits: Carbon can be combined with resin to achieve low TOC’s or is a good first step to clean up feed water high in chlorine Limitations: Activated carbon will not remove ions and particulates So, once water has gone through a cartridge to remove ions, then the UV lamp to oxidize organics, trace organics and chlorine must be removed. This is actually accomplished in the same cartridge containing the DI resin using activated carbon via the process of adsorption. In organic adsorption, the organics and chlorine in water form a low energy chemical bond with the surface of the activated carbon. Adsorption is useful because it is easy to incorporate into an existing cartridge with resin in it or in some cases pure activated carbon cartridges are put in front of a system to clean up high levels of chlorine in the feed water. Commonly you see “activated carbon” cartridges for home water treatment systems. Adsorption is very limited in its’ removal capabilities though so its use is very specific.

Depth and Screen Filtration The depth filtration process is the mechanical straining of solid particulate impurities through multiple layers of filtration material. A depth filter can be made of sand in a bed or fiber wound around a hollow core. Depth filtration occurs through the entire filter, and therefore effects a high particle retention. The pore size of the depth filter is nominal; meaning that its designated pore size (for example 5 microns) represents the average pore size. Depth filters are best used for raw water prefiltration.

Final Filters 0.2 micron POU designed to removes bacteria and particles Important Tips: Rinse filter before sampling Change frequently minimum 1-2X per year Purge – upon installation or if flow rate from dispenser, release air with vent port purge air bubbles. Slow flow rate can also indicate filter needs replacement. Avoid adding tubing to final filter, touching tip of final filter. release air bubble from POU filter when 1st time installed or long time no dispense. And when customer find a smaller dispense flow rate, he can make sure if there is air bubble in the filter then he can move POU filter away to see if the flow rate become bigger if release air from POU filter doesn’t work. Photo shows final filter that is plugged with carbon fines/debris from feed water supply.

Endotoxin-free Type 1 Water Ultrafilters: Remove endotoxins in water <0.001 Eu/ml Polysulfone hollow fibers Large surface area In water line – away from potential environmental contamination Combination of UV light and Ultrafiltration (UF) Excellent way to reduce nuclease and endotoxin: Ultrafilter hollow fiber filters trap impurities System rinse remove them from the system UV light – oxidizes bacteria and organics Point of use Type I ultrapure water purification systems with ultrafiltration (UF) are designed to effectively reduce endotoxin to below detection limits. Ultrafilters used in Thermo Scientific Barnstead water purification systems use polysulfone hollow fibers with a 0.05 micron absolute pore size to provide a powerful and consistent barrier to trap these particles. Systems that also use a UV lamp along with the ultrafilter, will also effectively have bacteria destroyed so they do not act like particulates, being trapped on the surface of ultrafilter. This allows UF to have longer life and to be more effective.

UV Photo-Oxidation and Bacteria Reduction Built into systems with UV photo-oxidation: Type 1 systems – dual wavelength UV: 185/254 nm organic compounds 254 nm controls microorganisms Type 2 systems and storage tanks: Use 254 nm only to discourage growth Effective in the flow path Thermo Scientific UV lamps – up to a 2-year lifetime There is a 20 minute “minimum on time” safety regulation in the software for the UV Bulb. The UV Bulb is only on during the “Non-Stop” or “Production” modes Water systems often incorporate features such as UV photo oxidation. UV lights are only effective where it is in contact with water, so recirculation of the water is important. In Type 1 systems, dual wavelength bulbs are used. The 185 nm wavelength oxidizes organic compounds and 254 nm wavelength kills bacteria. In Type 2 water systems and storage tanks, UV lights are only used to control bacteria. Thermo Scientific Barnstead Type 2 systems recirculate the water from the storage tank back to the Type 2 system for repeated exposure to the UV light. Note, UV lights often need to be built into the unit; it may not be able to added to a system as an accessory. Therefore, it is a good feature to consider when purchasing a new water system.

Limitations & Capabilities

Example Water Treatment Flow from Tap to Type 1 Flow Inside POU System POU Water System being supplied type 2 water from resin bed in basement UV Lamp Deionization Cartridge Conductivity Cell Pump Ultrafilter Final Filter Oxidizes organics / Kills bacteria Moves H2O through System Removes ions and dissolved gases Removes particles, bacteria, pyrogens Measures resistivity and shows on system display Removes particles larger than 0.2 micron END RESULT: 18.2 megohm water with TOC levels below 5ppb, nucleases, pyrogens, bacterial levels extremely low 2nd Floor Lab Bench Resin Bed in Basement producing 8 megohm quality water (type 2) Phew, that is all 7 technologies! Now let’s look at a real life example which hopefully will help put it all together. Let’s assume we have a building with a big mixed resin bed in the basement producing 8 megohm quality feed water. They are pumping this to the multiple floors in the building and this is connected into a “point of use” (POU) water system on the second floor in Dr. Jones’ molecular biology lab. At this point all we have removed are mostly ions and maybe some dissolved gases. By the ASTM standard we have type 2 water going into this POU system that is going to turn the water into type 1 water for us. The water enters the system and first gets zapped by the UV lamp which oxidizes the organics and kills bacteria. Then the water goes through a deionization cartridge which will remove ions to the ASTM Type 1 standard of 18.2 megohm and remove more dissolved gasses if present. Now, the water is pushed into the ultrafilter where those nasty pyrogens and bacteria are. So at this point we are pretty clean (industry lingo is “polished”) so the water resistivity is measured and sent to the display so that the client sees that they have 18.2 water and if they have TOC monitors in their unit they will also see how many TOC’s are in the water, in this case let’s say 2 ppb (that is good). Lastly before the water is put into a vessel by the end-user, the water goes through a 0.2 micron filter for filter particulate capture. The end result is ultrapure, Type 1 water with low TOC’s, low nucleases, pyrogens, bacterial levels so in other words, water suitable for Dr. Jones to run her PCR experiment. Main water line with potable incoming water

Thank you Questions?