pH influences the rate of many chemical and enzy-matic reactions. Therefore, buffered solutions arecommonly utilized in the laboratory. These solutionsare usually prepared with high-purity water in orderto minimize the risk of contamination with water im-purities. This paper not only describes the difficultiesand alternatives to measuring the pH of high-puritywater, but also the impact of buffer dilution on pH.

pH of high-purity waterThe pH of water is defined as the negative log-arithm of the hydrogen ion activity, which is

often assimilated to the hydrogen ion concen-tration (Eq. [1], Table 1). While the pH ofpure water is approximated to be 7.0 at 25 C,the theoretical value is 6.998. Indeed, the dis-sociation of water and the ionic product forwater (Eqs. [2] and [3], Table 1)1 indicate that106.998 mol/L of H3O+ are present in pure wa-ter. However, one cannot measure the pH ofhigh-purity water simply by dipping the elec-trodes of a pH meter into a beaker full of wa-ter. This measurement is challenging due tothe quasi-absence of ions that would enableelectron transport between the measuring and

High-Purity Water and pHby Estelle Rich, Aude Carri, Nicolas Andin,

and Stphane Mabic

Reprinted from American Laboratory News June/July 2006

Table 1 A quick reference guide of basic definitions and equations

reference sides of the pH electrode. It oftenresults in erratic and meaningless pH readings.

Adding the slightest amount of acid or base topure water will change its pH significantly (Fig-ure 1). Pure water readily absorbs carbon diox-ide (CO2) when exposed to the atmosphere,which forms carbonic acid (Eq. [4], Table 1).Carbonic acid dissociates into bicarbonate thatis in equilibrium with carbonate. Dissolution ofCO2 in water ultimately leads to a pH of ap-proximately 5.8. It is this pH value that iseventually obtained if one measures the pH ofultrapure water without working under con-trolled conditions.

The issues linked to the pH measurement ofultrapure water are addressed in various refer-ences . The Uni ted Sta te s Pharmacope ia(USP) recommends the addition of potassiumchloride (KCl) to alleviate the problem.2 Amore precise and complex experimental setupand method for the pH measurement of purewater are described in an American Societyfor Testing and Materials (ASTM) StandardTest Method.3 It includes the use of a flow-through sample chamber and the addition ofKCl via a reference electrode with positiveelectrolyte leakage. Other standards, such asthe International Organization for Standard-ization (ISO) 3696 standard, simply do notrequire the pH measurement of pure or ultra-pure water. They rely on other physicochemi-cal parameters to characterize water quality,such as conductivity.

Conductivity ofhigh-purity waterA conductivity measurement may beused as an alternative to the pH mea-surement of high-purity water. Con-ductivity measures the flow of elec-trons through a f luid, which isproportional to the concentration ofions, their charge, and mobility (Eq.[5], Table 1). The relationship be-tween resistivity (the reciprocal ofconductivity) and the pH of water isillustrated in Figure 1. When onlypure water is present, a conductivityvalue of 0.055 S.cm1 at 25 C is mea-sured. This results from the water dis-sociation into hydroxide and hydroxo-nium ions. Therefore, at 25 C, aconductivity of 0.055 S.cm1, or a re-sistivity of 18.18 M.cm, implies that

the water is ultrapure and that the pH is inher-ently 6.998.

Diluting buffer solutionsThe pH of a buffer solution composed of aweak acid and its conjugate base is related tothe di s sociat ion constant (pKa ) of theacid/base pair, as shown in Eq. [6], Table 1. Bydef ini t ion, buf fer s are res i s tant to largechanges in pH. However, in the laboratory set-ting it becomes evident that diluting a bufferwith pure water impacts the pH of the solu-tion. Several parameters might explain thesepH variations.

1. Temperature. Activity coefficients and equilib-rium constants vary with temperature, whichcauses the pKa and pH of a buffer to change.Temperature coefficients vary significantlywith the nature of the buffer (0.028 pH/C forTris and 0.0028 pH/C for phosphate). Forthis reason, temperature is usually reportedalong with pH values. The pH of water itselfvaries with temperature: 7.27 at 10 C, 7.00 at25 C, and 6.63 at 50 C.

2. Buffer capacity. Buffer capacity is the ability of abuffer to resist changes in pH. It depends on thetotal concentration of the acid/base pair andthe concentration ratio. Buffer capacity is highwhen solutions are concentrated, the ratio ofacid to base is close to 1, and the pH is 1 unitfrom the pKa.

3. Ionic strength. Ionic strength describes theamount of electric charges in a solution. It is a

Figure 1 Theoretical resistivity values of water following the addition of hy-drochloric acid (HCl) or sodium hydroxide (NaOH). The resistivity of ultrapurewater is 18.18 M.cm and the pH is 6.998.

function of the concentration and the valenceof the ions present. High ionic concentrationtends to limit the mobility of the hydrogen ion,which decreases its activity and influences thepH measurement.

4. Dilut ion . Related to a decrease in ionicstrength, dilution directly impacts the pH ofa solution. For example, the dilution of a 0.1M buffer system (HA/HA) with an equalvolume of water results in a pH change of0.024 units.4 The pH variation of HA/A2

buffer systems, such as phosphate, is threetimes that value.

Buffers are often prepared by diluting stocksolutions. In this study, two phosphate bufferswere diluted 10 and 100 times with variouswater qualities and pH was measured. Thetwo buffer solutions were a phosphate buffer(1.0 mol/L, pH 7.4) and a phosphate bufferedsaline 10 concentrate (PBS, 0.1 mol/L, pHexpected to be 7.27.6 after a tenfold dilu-tion), both from Sigma-Aldrich (St. Louis,MO). Three types of water were used: 1) purewater delivered by an Elix 10 UV water pu-rification system (Millipore Corp., Billerica,MA), 2) ultrapure water produced by a Milli-Q (Millipore Corp.) Biocel (Velocity11,Menlo Park, CA) purification system, and 3)bottled Gibco distilled water (Invitrogen,Carlsbad, CA). Experiments were performedin triplicate.

Diluting phosphate buffers with water resulted inpH increases (Figure 2). For each buffer, the pHchanges were similar with all three types of water.The tenfold dilution of the 10 PBS solution re-

sulted in a pH close to the expectedvalue of 7.4. The PBS solution had alower buffer capacity than the phos-phate buffer (lower concentration0.1M for PBS versus 1.0 M for phosphateand original pH farther from pKa) aswell as higher ionic strength. This ex-plains the larger change in pH upon ini-tial dilution of PBS compared to phos-phate buffer: 6.77.3 versus 7.37.4.

ConclusionHigh-purity water is often used in thelaboratory to avoid possible artifactscaused by water impurities. Measuringthe pH of this water is challenging andnecessitates specialized equipment.However, using water with high resis-

tivity (18.2 M.cm at 25 C) ensures that thepH is close to 7.0. Diluting buffer stock solutionswith high-purity water leads to pH changes.These variations in pH are unavoidable and canbe explained by changes in ionic strength andbuffer capacity.

References1. Nora, C.; Mabic, S.; Darbouret, D. Ultrapure Water

2002, 5660.2. United States Pharmacopeia National Formulary, USP

23 NF18, 1995, 1637.3. Standard Test Methods for pH Measurement of Water of

Low Conductivity. ASTM D 546493 (reapproved1997).

4. Perrin, D.D.; Dempsey, B. Buffers for pH and Metal IonControl; John Wiley & Sons: New York, 1974.

The authors are with Research and Development, Bioscience Divi-sion, Millipore Corp., Bote Postale 307, F-78094 St. Quentin enYvelines, France; tel.: +33 1 30 12 71 40; fax: +33 1 3 12 71 11;e-mail: stephane_mabic@millipore.com.

Figure 2 Effect of phosphate buffer dilution with high-purity water. Valuesshown are the mean and standard deviation of three samples: a) phosphate buffer(1.0 mol/L, pH 7.4), b) 10 phosphate buffered saline (PBS, 0.1 mol/L).