Understanding Laboratory Water Purification
By: Jean-François Pilette, Millipore Corporation
The selection of the proper ultrapure water system depends on several parameters such as the
types of contaminants that need to be removed, the quality of the feed water available in the
laboratory, instant and daily volumes of pure or ultrapure water needed, and the quality of
monitoring of water quality. This article discusses each of these parameters in some detail and
how they affect laboratory water use.
Main Classes of Contaminants
Water contaminants can be classified into five somewhat simple and arbitrary categories:
inorganic salts, dissolved organic substances, particulates and colloids, micro-organisms, and
gases. Some of these contaminants are removed to some extent when natural water is processed
into drinking water; however contaminants will have to be removed to a much lower level, and
some additional contaminants removed when tap water is further purified to become laboratory
grade water.
The common inorganic salts dissolved in natural water are composed of negatively charged
anions and positively charged cations. The presence of unknown or unwanted ions in laboratory
grade water, or an excessive concentration of ions, can cause many issues in laboratory
processes. For example, if the water used to prepared standard solutions or wash glassware
contains lead, and lead ions are being analyzed, results will be variable. Therefore ions have to
be kept at a minimum level in laboratory grade water.
The cations present in natural water are typically: sodium, calcium, iron, manganese, lead and
aluminum. The most common anions are chloride, nitrate, sulfate, phosphate, and carbonate.
Silica, which is negatively charged, may also be classified as an anion. But silica is a special
case: it is weakly charged, and silica can exist in different forms. Monomeric silica has a single
silica atom, dimeric two atoms, and polymeric silica with many silica atoms, as well as silica
sheet and silica crystal forms. Silica is therefore a multitude of different contaminants all with a
general common behavior, but also small differences, making silica difficult to remove from
water. It is important to remove ionic contaminants from the laboratory water supply to avoid
positive bias, interferences, and variations of the ionic strength of solutions during analysis. In
addition, ionic contaminants can also affect chromatographic results. In HPLC or Ion
Chromatography (IC), ionic contaminants can result in additional peaks, increased background
levels that could affect sensitivity, or artificially increased values.
Ionic contamination could also be an issue in HPLC analyses involving mass spectrometry
detector, especially when using electrospray positive (ESI +) mode. Instead of getting the
conventional [M+H]+ response, sodium and potassium ion contamination could result in
[M+Na]+ peaks, called “sodiated” peaks, and [M+K]+ peaks, resulting in incorrect masses
assignments Multiple peaks for a single molecule make data analysis more complicated and
difficult.
Dissolved organic substances based on carbon atoms are another source of contamination.
Organic substances can have a natural origin. For instance, during the fall, when leaves fall on
the ground, rain water starts dissolving them. After a while, the natural components of the leaves
and wood are released and dissolve in water. The long cellulose chains and other, smaller
molecules such as the lignin monomers, gallic acid or catechin (part of the tannins of the wood)
can have a wide diversity of nature and size (molecular weight). In addition to the organic
molecules of natural origin, natural water may also be contaminated by different substances
generated by the human activities such as:

Volatile organic compounds (VOC) that are solvents like perchlorethene used in
activities like clothes dry cleaning

Molecules that are part of automobile fuel such as benzene

Pesticides like herbicides, fungicides or insecticides used by the farming industry or in
gardening activities

Polyaromatic hydrocarbons (PAHs), molecules released e.g. from the tar used to make
roads

Polychlorobiphenyls (PCBs), whose usage is forbidden since 1976, an example of a
persistant industrial contaminant

Plasticizer substances added to polymers to make them softer (A typical example is
bisphenol-A (BPA) which has been widely used with polyvinyl chloride (PVC), a
polymer used to make most of the plastic pipes used to carry tap or deionized water.
Like ionic contaminants, it is also important to remove organic contaminants, to avoid positive
bias and/or interferences during organics analyses. Organic contaminants can also affect
chromatography by coating stationary phases, resulting in slower mass transfer (shifting
retention times), reduced resolution, baseline issues, and shorter column lifetimes.
Particulates and colloids contaminants can be divided into two classes: hard particulates (sand,
clay) and soft particulates (vegetal debris) .They can offer protection against ultraviolet rays and
chemical agents, while also acting as a vehicle for bacteria to stick to. Particulate and colloid size
is typically between 0.1 and 100 µm. Hard and soft particulates can slowly dissolve, generating
additional inorganic as well as organic contaminants. Colloids are stable suspensions of organic
or inorganic particulates (0.1 to 0.00in 1 um size, e.g. colloidal silica), usually having the same
electric charge, and their mutual repulsion prevents the settling of the liquid (unless a
coagulating agent of opposite charge is added). Hard particulates can damage or impair lab
instrument (e.g. pumps and injectors) operation, and soft particulates can plug chromatography
columns and frits. Particulates can also act as a shelter for micro-organisms, and may operate as
a catalyst to accelerate some reactions.
Micro-organisms like bacteria and viruses are also common natural water contaminants.
Bacteria come in different sizes and shapes, from 2 um Escherischia Coli, a micro-organism
commonly found in the human intestine, to Pseudomonas Diminuta, the smallest bacteria. When
grown in a very poor culture medium, with stirring, Pseudomonas Diminuta grows as a sphere of
0.27 um in diameter. This micro-organism is typically used to challenge membrane filters for
bacteriological retention, as it is the smallest known. Bacteria can be a nuisance even when
dead, because they will release by-products which can cause issues in several different types of
scientific experiments. Gram negative bacteria can release pyrogens from their wall, and other
bacteria can release nucleases such as RNases and DNases. Bacteria and byproducts need to be
removed from laboratory water sources because they are a source of ions and organic
contaminants which can affect many biological experiments such as cell cultures, and specific
bacteria by-products (e.g. pyrogens, nucleases) can affect cell operation and molecular biology
mechanisms. Bacteria also effects chromatography in much the same way as the organic
contaminants mentioned earlier.
Dissolved gases are also natural water contaminants that are important to consider. The main
gases dissolved in water are typically the main constituents of the air that is in equilibrium with
the water source: oxygen, nitrogen, and carbon dioxide, which is in equilibrium with carbonic
acid. The solubility of these gases is a function of temperature. Another common dissolved gas
contaminant is radon, a water soluble gas produced by the decay of radium isotopes. It is
naturally found in ground water from granite formations, phosphate deposits and uranium
deposits, and in addition to being a contaminant, radon may also cause human health problems,
including cancer. Other gases can dissolve in rain water and contribute to its acidification,
including sulfide dioxide (SO2, from combustion plants, industrial processes, transportation and
natural origins), nitrates (main origin is road transportation and combustion plants) and
ammonia-NH3 (typically originating from agriculture activities). Dissolved gas removal from
water may be required in specific experimentations to avoid the formation of bubbles which
affect the measure of optical density, or result in out-gassing which can cause pump and detector
issues in chromatography.
Water Monitoring Techniques
Ion Detection by Resistivity and Organics Detection by TOC
Need Help with this Section, Slides e.g. 31-43.
Resistivity is an excellent technique to assert the absence of ions in ultrapure water. Resistivity
detects all ions that may be present in water and is quite sensitive: a concentration of 1 ppb (=1
µg/L) of any salt is enough to significantly drop the value of resistivity. It is important to check
that the concentration of organics in water remains low and remains constant over time, since
many laboratory experiments can be affected by ions and it is therefore important to measure
resistivity to check that water contains as few ions as possible.
This can only be achieved if a trusted measure of TOC is regularly performed on the water prior
to use.
Figures 1-3 (Slides 50-53) demonstrate the effects TOC can have on a chromatographic
separation. Using HPLC Grade Bottled Water with a TOC > 50 ppb, resolution can be seen to
degrade significantly (Figures 1 and 2) over the course of ~2000 injections. However using
Milli-Q® Water, with a TOC = 4 ppb, the separation remains virtually unchanged in over 6000
injections. Table 1 (Slide 54) further illustrates this point, where the effects of TOC levels on
column lifetime is shown for bottled water versus Milli-Q® Water.
Water Purification Techniques
The production of pure water from tap water is achieved thanks to a series of purification
technologies, as illustrated in Figure 4 (Slide 68). Tap water entering, e.g. the Milli-Q Integral,
first passes through a Progard pre-treatment cartridge whose goal is to protect the reverse
osmosis cartridge. It includes activated carbon for chlorine removal, polyphosphate for
preventing calcium carbonate precipitation and a filter to retain particulates. The conductivity of
the treated water is then measured before entering the reverse osmosis cartridge. The reverse
osmosis cartridge will remove more than 97% of the ions in tap water, as well as 99% of
bacteria, particulates and organics larger than 200 Dalton. A scanning electron microscope
picture of a reverse osmosis membrane cross-section is shown in Figure 5 (Slide 59). The
membrane is separated into two areas: a very thin, one micron thick, active layer on the top,
which is responsible for the removal of contaminants, and underneath, a thicker (100 µm) porous
support whose function is to provide mechanical support to the thinner active layer. The reverse
osmosis membrane is able to reject contaminants by two combined mechanisms related to size
and electric charge. In this way the reverse osmosis membrane effectively rejects ions, and
organics, even if they are not electrically charged, as well as particulates, colloids and bacteria.
The rejection performance of the RO membranes will depend on the nature of their material;
most are made of cellulose acetate or polyamide. They will also depend on the manufacturer
process and quality. For instance, in the Millipore system RiOs 5, the reverse osmosis cartridge
is fed by a flow rate of 47 L/ hour to produce 5 L/hour permeate water. Consequently the
cartridge rejects 42 litres per hour. However, out of the 42 L/hour rejected by the cartridge, the
system sends only 15 L/hour to the drain and recycles 27 L/hour upstream of the reverse osmosis
cartridge. As a result, the system only needs to take an additional 20 L/hour of tap water to
provide the 47 L/hour required to feed the reverse osmosis cartridge. At the outlet of the reverse
osmosis cartridge, a conductivity meter measures the quality of the water produced and the
reverse osmosis cartridge rejection efficiency.
The permeate water then enters the Elix module whose purpose is to remove most remaining
ions in order to produce type two water with a resistivity greater than 5 MegOhm/cm, and TOC <
30 ppb. The quality of the water produced by the Elix is measured by a resistivity meter placed
at the outlet of the EDI module. Elix Technology is based upon electro-de-ionization technology
developed and patented by Millipore that uses permanent regeneration by a weak electric current.
Elix Modules can last several years with no need for water softening upstream. The resulting
pure water is stored in a reservoir and can either be used directly, or further processed to produce
ultrapure type one water.
The production of ultrapure type one water from type two pure water is achieved with a system
as outlined in Figure 6 (Slide 87). The type two pure water first goes though a 185 nm UV lamp
module for oxidation of organics into electrically charged organic ions and bacteria destruction.
The water then goes through a Quantum cartridge which contains activated carbon and as ionexchange resin for removal of the remaining traces of inorganic and organic ions in order to
produce ultrapure type 1 water. The synthetic activated carbon, produced from the pyrolysis of
styrene beads, has a small (<150 Å) pore size, a surface area of 1100m2/g, and is relatively
homogeneous. Consequently adsorption kinetics are rapid for low molecular weight organics,
with very low release of mineral ions. The activated carbon cartridges are used in these
polishing cartridges mainly for the removal of trace organics. Small organic molecules diffuse
inside the pores and link to binding sites by weak Van der Waals forces. The quality of the
ultrapure water produced is monitored by an in-line resistivity meter (ionic concentration
detection) and an on-line TOC monitor (organic contaminants concentration measure).
Typically, the sum of all ions is below 1 ppb and the sum of all organics is below 5 ppb.
The produced ultrapure water can then be conveniently dispensed though a Q-POD with a PODPak polisher (Figure 7 (Slide 85)) at the outlet in order to further adapt the water quality to the
user needs. The POD-Pak polisher can be adapted to each particular application, and is the most
critical contaminant removal before water leaves the system
Selecting, Operating and Maintaining a Water System
There are several parameters to consider when choosing a laboratory water system. Certainly
quantity is a primary consideration, but perhaps more importantly, the quality of water desired
must be considered; is type two water or Type one ultrapure water required? If type one
ultrapure water is required, the type, sensitivity and calibration of the resistivity and TOC
monitors used are key to ensure water quality. Daily usage should also be considered, as well as
ergonomy, bench space, volumetric dispensing, the flow rate (so that the water level can be
accurately adjusted). It should also be intuitive to use. Quality Assurance (ISO9001, cGMP) is
also important: the availability of certificates for calibration, conformity, consumables quality,
following the established norms of ASTM (resistivity), or USP (resistivity and TOC suitability
tests). CE and cUL certification, and a Consumables Validation Guide should also be included.
Qualification services, installation, calibration, maintenance, repair, service, and technical
service, should also be provided. Operating costs and the environment should also be considered;
over the systems’ whole life, not only the original capital expenditure.
During routine operation, storage of ultrapure water should be avoided, as it can be quickly and
easily contaminated. For best results, avoid storage in carboys & produce water on demand, to
prevent container extractables and bacteria buildup. Unnecessary connections like tubing at the
outlet after the consumable filter should be also avoided, and the manufacturer recommended
maintenance schedules should be followed for consumable replacements on time to reproducibly
obtain high water quality.
Since purified water is the most common reagent found in most laboratory facilities, a properly
configured and maintained lab 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 or cell culture analyses, to complex analytical techniques like HPLC or LCMS, to ensure the success of projects and maintain productivity.