Measuring pH

Learning Outcomes

When you have mastered this topic, you will:

What is pH?

The function pH is defined as the negative logarithm of the hydrogen ion (H₃O⁺) activity of a solution. Thus, pH = -log a^H+. For practical purposes, the hydrogen ion activity, a^H+ is equated with the hydrogen ion concentration [H⁺] (more properly [H₃O⁺]).

At pH = 7.0, [H₃O⁺] = [HO^–], and the aqueous solution is said to be neutral. pH values less than 7.0 are acidic, while those with a pH value greater than 7.0 are alkaline or basic. Note that pH values may be less than 0 and greater than 14. Biological systems operate in a narrow range of pH values. For most enzymes, extremes of pH leads to irreversible denaturation and loss of biological activity. The need therefore to be able to control the pH values of solutions is therefore critical, and the accurate determination of pH is a routine laboratory operation.

Principles of pH measurements

The instrument used to determine pH is called a pH-meter. It is based on the electrochemical principle that if two suitable electrodes are placed in an aqueous solution, a potential difference will be set up between these electrodes which will depend on the activity of the hydrogen ions in that solution.

By convention, such a cell is described in the shorthand notation anode||cathode. Note that the anode (which supplies electrons to the circuit) is on the left, while the cathode (which accepts electrons from the circuit) is on the right

The electromotive force of an electrochemical cell, E, based on two electrodes immersed in an electrolyte, is given by

E = E_c – E_a

Where E_c and E_a are the electrode potentials of the cathode and anode respectively. In turn, these electrode potentials are given by the Nernst equation:

• E_c⁰ and E_a⁰ are the standard electrode potentials for the electrodes,
• R is the gas constant (8.3144 J.K^-1),
• F the Faraday constant (96485 C),
• n the number of electrons transferred when the electrode is undergoing its redox reaction,
• T the absolute temperature,
• and Q a function dependent on the activities of various substances taking part in the redox reactions of the particular electrodes.

For the hydrogen electrode undergoing the reaction 2 H⁺(aq) + 2e ® H₂(g)
Q = a^H+ (the activity of the hydrogen ion H⁺) ~ [H⁺] (in dilute solution), and we can write

where pH = -log a^h+ = -log [H⁺] (in dilute solution), and both E_T and k are temperature dependent constants. We see therefore that the voltage produced by a pair of electrodes, one of which is a hydrogen electrode (acting as anode) and the other a suitable standard electrode (acting as cathode), will depend both on the pH of the solution into which the electrodes are immersed, and the temperature of the measurement.

where

pH_T⁰ is the pH when the measured voltage, E, is zero,
R’ is a constant (1.984×10^-4 V.K^-1),
and S is a correction factor, taking into account deviations from the theoretical response values of the electrodes.

A graphical representation of the above expression is shown in the diagram on the right.

A pH meter is a device that measures the small voltages (E is of the order of millivolts) produced by an electrochemical cell described above. The output that is read can be either the voltage of the cell, or, more commonly in the sort of laboratory application that you will be involved in, pH units.

Two important observations must be made:

• the pH is temperature dependent, and,
• the pH measured by a pH-meter (which is essentially a millivoltmeter), will not necessarily me a direct measure of the hydrogen ion concentration.

The pH-meters that you will use (Beckman-Coulter F^™ Series 300, shown here on the left) are complex electronic devices that are equipped with ATC (Automatic Temperature Control) and can compensate automatically for temperature differences. This is achieved by calculating the slope of the pH vs. E graph based on the temperature input of a temperature probe and automatically adjusting the measured pH values. The meter is, of course, dependent on electrodes, which are not shown in the picture. The theory behind these electrodes will form the subject of the next page.

pH Electrodes

The electrodes that are used for the determination of pH fall into of three main types:

• reference electrodes – the cathode of the electrochemical cell whose electromotive force is being measured;
• glass electrodes – the anode of the above cell, sensitive to changes in hydrogen ion activity, and
• combination electrodes – incorporating both reference and glass electrodes in a single unit. This is the electrode type most commonly used in the laboratory for routine determination of pH.

Reference electrodes

Reference electrodes are normally silver/silver chloride electrodes or saturated calomel electrodes (Calomel is an obsolete name for mercury(I) chloride, Hg₂Cl₂. The name is still used when describing a certain type of reference electrode). They are required to be robust and to have a stable electrode potential.

The saturated calomel electrode:

The saturated calomel electrode exploits the redox reaction:

Hg₂Cl₂(s) + 2e ® 2 Hg (l) + 2 Cl^–

It is a half-cell (i.e., a single electrode system consisting of substances that can undergo redox reactions), formally described as:

Hg|Hg₂Cl₂, KCl(satd)||

and having a standard electrode potential of 0.246V at 25°C.

Its basic construction is shown on the diagram on the left. Contact with the solution to be measured is achieved through the porous plug. Commercial electrodes have a more complex design than this!

The silver/silver chloride electrode:

The Ag/AgCl electrode (shown in simplified form on the left) relies on the reaction

AgCl(s) + e ® Ag(s) + Cl^–

The filling solution is saturated KCl chloride solution, sometimes saturated with AgCl as well, as AgCl is slightly soluble in concentrated KCl solutions. It is a half-cell, formally described as

AgCl|Ag, KCl (satd)||

with a standard electrode potential at 25°C of 0.199 V.

It is widely used due to the relative non-toxicity of its components, its ease of manufacture, the stability of its potential, and the high temperature range (up to 130 °C) at which it can be used.
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The glass electrode

When two solutions containing the same ion in different concentrations, H⁺, for example, are separated by a thin membrane, an electric potential difference exists between the two solutions. The glass electrode is based on this.

A simplified of the glass electrode is shown on the left. The membrane is a very thin glass bulb containing a buffer or dilute acid. The glass bulb, typically with a thickness 0f 0.2 mm) is made of special glass whose surface is readily hydrated, thus causing H⁺ ions to be adsorbed on both the inner and outer surface, in layers about 10^-6 mm thick. A membrane potential is therefore set up.

Contact is made by means of a silver wire coated with silver chloride. This in itself is a half cell, and we can write the cell as

Ag(s)|AgCl(s), HCl (0.1M)|glass|test solution||

Great care must be exercised when using glass electrodes. The delicate glass bulb is easily damaged by mechanical shock or exposure to concentrated alkalis.

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Combination electrodes

In a structural biology laboratory, one routinely uses combination electrodes, that is, electrodes that combine both the glass electrode and a reference electrode. The compactness make such electrodes very suitable for determining the pH of small volumes of test solutions.

Such combination electrodes may also incorporate a temperature compensation electrode, which caters for the differences in electromotive force of the system due to temperature differences.

Practical issues

The pH-meter

Calibration

Before taking a pH measurement, the pH meter/electrode assembly should be calibrated using standard buffers. Make sure you are in the pH Mode, indicated by the pH icon to the right of the large numeric display. To enter the mode, press the pH/mV or the pH/mV/Conc key until the pH icon appears.

The pH mode consists of two sub-modes:

• pH Standard Buffers (default mode).
• pH Custom Buffers.

Five standard buffers (pH 1.68, 4.00, 7.00, 10.01, and 12.45) are stored in the system. The system will automatically recognize the above buffers during calibration. For precise results at temperatures other than room temperature (25°C) or varying temperatures, the use of a Temperature Probe (P/N 598115) or a 3-in-1 electrode with integrated temperature probe is strongly recommended. This allows for automatic temperature compensation (ATC) with both standard and replacement buffers.

The calibration buffers or standards can be displayed continuously for reference. To turn the feature on, press the SETUP key, select the View Stds item and press the ENTER key.

Press the CAL key to enter the calibration mode. Auto-Read will automatically be turned on. In the lower left part of the display, the standard buffers of an existing calibration are shown. If there is no calibration, only Stds is displayed. If there is an existing calibration, the elapsed time since the calibration has been performed is displayed.

In order to re-calibrate, the existing calibration must be removed first. An indicator for an existing calibration is the [CALIBRATED] box. Press the CLEAR key to remove existing calibration data. The message Clr will appear in the large numeric display, warning the user that data will be cleared. To confirm the removal of the calibration, press CLEAR again. Otherwise, press the CAL or EXIT key to keep the existing calibration. The number of available calibration points depends on the model.

Immerse the electrode into the standard buffer and press the READ key. During the actual measurement of the standard, pH results are displayed continuously and the Auto-Eye flashes. Wait until the Auto-Eye stops flashing, indicating that the meter has locked onto a stable reading. After the meter has locked on, the pH value is displayed in the large numeric display. The buffer value, at 25°C, also appears in the lower left part of the display. Eo is displayed with the first standard and the % Slope is displayed with the remaining standards.

This procedure is repeated with a second standard buffer (and a third one, depending on the model). The display that is obtained (using the pH = 7.00 standard) is shown on the right.

The OK icon that appears at the bottom left-hand corner indicates that the electrodes are functional, that is, the slope is within 90-110%. From the display shown here, the slope is 98.7%, well within acceptable limits.

pH measurement

To take pH measurements, a calibration should have been performed earlier. An indicator for an existing calibration is the [CALIBRATED] box. If there is no calibration, you can still obtain results but the calculation is based on a theoretical slope. In this case, the results may be unreliable, especially if they differ significantly from the neutral pH 7.00.

Immerse the probe into the sample and press the READ key. During the measurement, calculated pH values are displayed continuously and the Auto-Eye flashes, if Auto-Read has been turned on. Wait until the result has stabilized or the Auto-Eye stops flashing. This may take 30-60 seconds or longer, depending on the sample.

Notice that the STO icon is shown next to the memory status bar, when Auto-Read is turned on, indicating that the result can be saved by pressing the STORE key (for models with RS-232 interface only).

Continue with the next sample.

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The electrodes

Routine use of the electrode

• Remove the electrode from its storage solution.
• Rinse it thoroughly with distilled or de-ionised water.
• Dry the bulb by gently dabbing with a tissue.
• Immerse it in the test solution.

Rinsing and drying should be carried out for each solution being measured. When finished, the rinsed, dried electrode should be returned to its storage solution.

Using the electrode for the first time

Remove the special Performance PAC^TM protective vial. This vial keeps the glass pH-sensing bulb and reference junction in a fresh, ready to use condition. Note that any white residue or crystals are KCl crystals. Simply rinse them off with deionized water.

Hold the cap and unscrew the via] until you can pull it straight off. Remove the O-ring and then the cap. Save these items for later use for long-term storage.

Glass bodied electrodes have a sleeve that covers the fill hole. Pull this sleeve down and then remove the tape covering the hole.

Epoxy bodied electrodes have a stopper that covers the fill hole. Remove the tape covering the hole and stopper.

Fill the electrode with filling solution to the bottom of the fill hole. The filling solution for your electrode can be found in the list below. It is important to keep the electrode filled to insure proper response as the solution flows through the reference junction.

Rinse the electrode with deionized water. Rinse the glass pH-sensing bulb and junction area clean with deionized water to remove any salt deposits. Connect the electrode to the keeper cable and then to the pH meter.

Measurement of samples

Insert the electrode into the sample or standard to he measured. The solution must cover the glass pH-sensing bulb and the reference junction (which is the white porous material just above the bulb). Make sure that the fill hole is open, allowing the flow of filling solution through the junction.

Take a reading or calibration point by following the directions in the pH meter manual. After the measurement is stable, remove the electrode from the sample or standard and rinse it thoroughly with deionized water to minimize contamination. You can then proceed to your next sample or standard.

Storage of the electrode

Beckman Futura^TM refillable combination electrodes should he stored in the appropriate filling or storage solution when not in use.

• For short-term storage, up to 3 days: store the electrode upright, with the glass pH-sensing bulb and reference junction in Beckman storage and soaking solution, part number 566576. Make sure that the filling solution is replenished and the electrode fill hole covered to prevent the electrode from drying out.
• For long-term storage, 3 days or longer: return the electrode to the Performance PAC^TM vial for storage. First, place the tip of the electrode in the vial and add filling solution to within 1¼ cm from the top. Next, remove the electrode, rinse it with deionized water and dry the body with a towel or tissue. Then, slide the cap and O-ring onto the electrode body. Place the electrode lip into the vial and screw the cap on securely. (Be careful not to splash filling solution onto the electrode body, as this may prevent proper sealing of the cap and O-ring.) Finally, seal the electrode fill hole with tape. As long as there is no evaporation from the vial or fill hole, the electrode can be stored indefinitely in the Performance PAC^TM vial.

Note: never store an electrode in deionized water, tap water, pH buffers, or other “obvious” solutions. These solutions all have properties that tend to clog reference junctions and contaminate the filling solutions.

How to achieve the best results

• Standardize your meter and electrode by following the procedures in the meter instruction manual. For the most accurate results, choose pH buffers that are close to the value of the sample and perform a two-point calibration. The buffer values should be above and below the sample value.
• Use fresh pH buffers and replace them at regular intervals, or if suspect that they may have become contaminated.
• Have the pH buffers and samples at the same temperature so the electrode does not have to adjust to a large temperature difference.
• Keep the electrode filled with filling solution so that the reference junction maintains a steady flow. Keep it filled to the bottom of the fill hole and make sure to keep the fill hole open when measuring.
• The biggest choice you have is whether or not to stir your sample. The important thing is to be consistent. It is recommended that solutions not be stirred while taking pH measurements.

Maintenance of the electrode

Most problems encountered with an electrode are due to deposits on the glass pH-sensing bulb or by a blockage of the junction.:

Observed symptoms

• Slow response (takes longer than 45 second), to recognize a buffer) can be caused by either a contaminated bulb or a clogged junction. Clean the bulb or junction.
• Drifting, or unstable readings are normally caused by a clogged junction. Clean the junction.
• Low span (standardize at pH 4 then read pH 10 buffer, if it does not read at least 9.7 span is low) is caused by a contaminated bulb. Clean the bulb.
• Zero span or no span, that is, the meter always reads pH 7 no matter what the buffer or sample may be. This can be the result of a cracked bulb, a completely blocked junction, or a bad cable. Replace the defective cable or electrode.

To clean the bulb, use one of these procedures

1. Soak the electrode in 1 M HCI for up to one hour. rinse thoroughly in deionized water.
2. Cycle the electrode several times between 1 M HCI and 1 M NaOH solutions, soaking in each solution for 1 minute. Rinse and soak in pH 4 buffer for 10 minutes.
3. Prepare a solution of 50% acetone and 50% isopropyl alcohol and rinse the electrode with it. Then rinse with deionized water and soak in pH 4 buffer for one hour. This works well with grease or oil solutions or other solutions that act as surfaciants.
4. Prepare a 10% bleach solution(10 parts deionized water to 1 part bleach) and soak the electrode for 15 minutes. Rinse thoroughly in deionized water and soak in pH 4 buffer for 15 minutes. Rinse again in deionized water and use. This works well to remove protein build-up.

To clean the junction, use one of these procedures

1. Soak the electrode in 3-4M NH₄OH (ammonium hydroxide) for 20 to 30 minutes. Rinse thoroughly with deionized water, and then soak for 15 minutes in pH 4 buffer.
2. Soak the electrode in a heated solution of KCl. Heat the solution to a maximum of 50°C and allow the electrode to soak until the solution is at room temperature. This also works well to eliminate salt build-up inside.

For salt build-up inside the electrode

Empty the electrode of as much fillin solution as you can. It may be necessary to hold it carefully and shake it over a sink. Fill the electrode with warm (not greater than 50°C) deionized water and try to get the salt to return to solution. Do not leave the water in the electrode for more than 15 minutes. Empty the electrode of this solution and refill it with the correct filling solution.

Disposal of electrodes

Group 3 electrodes, with a calomel reference junction contain mercury, a hazardous material. Dispose of these electrodes according to applicable laws and regulations.

If you experience problems with your Beckman electrode that these procedures cannot resolve, please call your local Beckman dealer. For additional information on special applications or difficult samples or other Beckman electrodes, please ask for a copy of the Beckman Electrochemistry Handbook, or visit our website at http://www.Beckman.com.

Electrode filling solutions

Group one electrodes are Ag/AgCl electrodes that utilize a 4M KCl reference solution with AgCl, Part number 566467:

• 511060 standard size 12 x 130 mm epoxy body
• 511061 standard size 12 x 130 mm glass body
• 511062 thin long 7 x 245 mm epoxy body
• 511063 thin long 5 x 225 mm glass body
• 511064 standard size 12 x 130 mm epoxy body rugged bulb
• 511065 standard size 12 x 130 mm glass body rugged bulb
• 511066 standard size 12 x 160 mm epoxy body flat bulb**
• 511067 thin long 9.5 x 250 mm epoxy body
• 511068 thin long 1 0 x 200 mm glass body.

Group two electrodes are STAR^TM electrodes that utilize a 1 M KCl reference solution with AgCl, part number 598943:

• 511070 standard size 12 x 130 mm epoxy body
• 511071 standard size 12 x 130 mm glass body.

Group three electrodes are calornel electrodes that utilize a 4M KCl reference solution, PIN 566468:

• 511080 standard size 12 x 130 mm epoxy body
• 511081 standard size 12 x 130 mm glass body
• 511082 thin long 5 x 178 mm glass body
• 511083 thin long 5 x 225 mm glass body
• 511084 thin long 7 x 245 mm epoxy body
• 511085 thin long 10 x 225 mm epoxy body.

All of these electrodes require a separate Futura^TM keeper cable, except the flat bulb electrode (marked **) which comes with an attached cable.

References

1. pH Theory and Practice, Radiometer Analytical SAS.
2. Problems with electrodes (Web-based resource).