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

2. 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.

3. 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.

4. Reagent Grade Water Types

5. 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.

6. 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.

7. 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

8. 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.

9. Limitations & Capabilities

10. Ion Impurities in Water
Dissolved Ionized Solids Major Calcium - Ca++ Cations Magnesium - Mg++
Sodium - Na Chloride - Cl- Major Sulfate - SO4= Anions Bicarbonate - HCO 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 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.

11. 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

12. 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.

13. 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.

14. 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.

15. 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

16. Limitations & Capabilities

17. 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 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

18. Ion Exchange and Regeneration

19. Electrodeionization
Removes ions from water using electricity and resin.
Continuously regenerates automatically (though has a finite life)
The technology that produces 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

20. 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

21. Distillation

22. 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

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

24. Reverse Osmosis Spiral Wound Membrane
(Permeate)
(Concentrate)

25. 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.

26. 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.

27. 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.

28. 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.

29. 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.

30. Limitations & Capabilities

31. 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

32. Thank you
Questions?