The molecular structure of the simple water molecule, its slight positive charge on the hydrogen side and slight negative charge on the oxygen side, yields a dipole. As such, water molecules are attracted to each other and to other polar molecules via hydrogen bonding. It is because of this phenomenon that water has certain properties such as high melting and boiling points and high surface tension. It is why ice is less dense than liquid water. And it explains why water is often referred to as a “universal solvent.”
Of course, it won’t mix with nonpolar substances, like oil. But when it comes to salts, the positively and negatively charged ions are pulled apart, the dipolar water forming a hydration shell around the solute and dispersing it evenly throughout. These same distinctive molecular characteristics that make water such an amazing solvent, also make it prone to contamination, which is less than ideal for research. This is why you should never use natural water in the lab for experiments. There are too many variables.
The main sources of water contamination include inorganic ions, dissolved organics, particulates, microorganisms, and gases. In the first category, an array of ions, even at minute levels, such as sodium, calcium, magnesium, iron, bicarbonate, chloride, and sulfite, can affect and influence reactions by serving as unintentional catalysts. Dissolved organic material may have biologic or manmade origins. Tannins and lignins, for example, are the byproducts of decayed plants. And phthalate esters from PVC pipes may also seep into the water.
Dissolved organics can disrupt cell culture experiments or, when present in eluents, decrease the sensitivity and resolution in liquid chromatography. Particulate and colloids, such as sand, can interfere with instrument function. Bacteria and their pyrogen byproducts can cause an array of issues and faulty data. And natural gases, such as nitrogen, oxygen, and carbon dioxide can influence pH and impact biochemical reactions. Reagent-grade water, however, has had these key contaminants removed to varying degrees to produce application-specific water types.
“Reagent-grade water has a defined quality that ensures reliable and repeatable results, explains Nadia Brandes, product manager lab water, for Sartorius Lab Instruments. “Reagent-grade water is typically used for lab sensitive, critical analysis. It is used for sample dilution, stock and buffer solutions, which require minimal interference by ions, organics, bacteria etc.”
The four types of laboratory-grade water
“Tap water is typically not suitable for daily laboratory applications or specified processes, as it still contains residues that could have an impact on process results,” says Brandes. “Sensitive analytical quantification needs clear results, and impurities [such as those that cause] ghost peaks in chromatograms, could distort results.” And that’s where reagent-grade water comes into play.
Water purity, as defined by the American Society for Testing and Materials (ASTM) International, ranges from ultrapure type I water to semipure type IV water. Purity levels are determined by specific characteristics, which include resistivity (measured Ω-cm), conductivity (measured in µS/cm), total organic compounds (TOC; for type I water), sodium chloride, and silica, according to Brandes. When bacterial levels need to be controlled as well, these ASTM types can be further broken down and subclassified as grade A, B, or C, depending on the heterotrophic bacteria count (HBC) and endotoxin levels. She notes that ASTM recommends certain production technologies to “purify the water to the required type.”
Resistivity and conductivity are used to get an idea of the water’s ionic content. A high resistivity means low conductivity. Generally, water with more ions is less pure and will conduct more electrical current.
Type I (Ultrapure)—Resistivity of >18 MΩ-cm, a conductivity of a conductivity of<0.056 µS/cm, and <50 ppb of total organic carbons (TOC). Should be used for critical applications such as HPLC (high performance liquid chromatography), gas chromatography, mammalian cell culture and IVF, mass spectrometry, making of reagents for molecular biology applications, such as PCR and DNA sequencing, and preparation of solutions for electrophoresis and blotting.
Type II—Resistivity of >1 MΩ-cm, a conductivity of <1 µS/cm, and <50 ppb of TOCs. Used in general applications, to make buffers, pH solutions, microbiological culture preparation, for use in cell culture incubators, to feed instruments and analyzers, or as feed water to a Type I system, as there is less calcium build-up. Can be used for electrochemistry, sample dilution, and radioimmunoassay.
Type III—Resistivity of >4 MΩ-cm, a conductivity of <0.25 µS/cm, and <200 ppb of TOCs. Between 90–99% of impurities are removed from Type III water by way of osmosis (RO) of tap water. May be used for glassware rinsing, non-critical laboratory applications, heating baths, and filling autoclaves. Can also be used as a feed water source for Type I water systems.
Type IV—Resistivity of 200KΩ and a conductivity of <5µS/cm; produced by RO and used as feed water to a Type I or Type II deionized (DI) system.
Purification technology
There are many ways to purify water. Certain methods will remove most contaminants, while others will focus on a specific kind of impurity. To remove all impurities, a combination of techniques is required. Deionization (DI) is one such process. As the name implies, it is the removal of all ionized particles from water by passing through an artificial mixed bed resin. This involves exchanging cations for hydrogen (H+) and anions for hydroxyl (OH-) ions, which then combine to make water. Brandes points out, “The disadvantage of this single process is that DI systems do not remove uncharged molecules, e.g. organic, bacteria etc.” This is why most water is pre-filtered by being pushed through a reverse osmosis membrane, which will remove the majority of major contaminants before moving on to a DI system.
Distillation is another way to purify water. “The distillation process removes impurities such as ions, bacteria, etc. by phase separation process, which means they are separated by boiling water,” says Brandes. “The water is condensing into steam, whereas the impurities remain as residues in the starting vessel. The advantage of distillation is that this process also removes nonvolatile molecular impurities. The disadvantage of it is the typically higher process costs.”
Brandes says that depending on the volume of purified water required, labs may want to purchase bottled water or purify their own. Although she cautions, “Typically the cost and the risk of contamination is much higher compared to freshly produced water.”
There are lots of choices available depending on your needs, and this is why you should speak with a vendor before making a decision about the type of system to purchase. Says Brandes, “The water systems should be optimized to the amount of daily required water volume and water type. In traditional labs, many people are using one system. Therefore the unit should be easy to operate, have a user-friendly interface, minimized maintenance requirements, and reliable high-quality water production.”