What Is a Buffer Solution (and How to Make One)

A lot of lab procedures depend on stable pH. Including calibrating a pH meter, running an enzyme assay, preparing an HPLC mobile phase and testing food samples. Even a small drift off in pH can ruin a result. Buffer solutions are the exact thing that prevent that drift. This guide will explain what a buffer solution is. It will explain how it works. It will explain which buffer to choose and it will explain how to make one accurately in your own lab.

What Is a Buffer Solution?

A buffer solution is an aqueous solution. It's one that resists changes in pH when small amounts of acid or base are added. Or when it is diluted. Most buffers contain one of these two pairings:

A weak acid and its conjugate base - For example acetic acid and sodium acetate. These are usually known as acidic buffers.

A weak base and its conjugate acid - For example ammonia and ammonium chloride. These are usually known as basic buffers.

The two components work together as a pair. One neutralises added acid. The other neutralises added base. This keeps the pH close to its original value.

How Does a Buffer Work?

Take an acetate buffer. One made from acetic acid (CH₃COOH). And take its conjugate base The acetate ion (CH₃COO⁻).

If you add acid (H⁺) - The acetate ions react with it to form acetic acid. The extra hydrogen ions are mopped up. So in this case the pH barely falls.

If you add base (OH⁻) - The acetic acid reacts with it to form acetate and water. The hydroxide ions are neutralised. So here the pH barely rises.

A buffer doesn’t hold pH perfectly constant. What it does is it reduces the change dramatically. As long as you don’t add more acid or base than it can absorb.

The Henderson–Hasselbalch Equation

The pH of a buffer is set by the strength of the weak acid (its pKa). And the ratio of its two components:

pH = pKa + log ([A⁻] / [HA])

Where:

[A⁻] is the concentration of the conjugate base (e.g. acetate)

[HA] is the concentration of the weak acid (e.g. acetic acid)

Two practical rules then follow on from this equation:

1. When [A⁻] equals [HA], pH equals pKa. This here is where a buffer works best.
2. A buffer is effective within about ±1 pH unit of its pKa. If it’s outside that range, one component is too scarce to do its job.

So this means that the first step in choosing a buffer is to pick a weak acid. One with a pKa close to your target pH.

Buffer Capacity

Buffer capacity is the amount of acid or base a buffer can absorb before its pH changes a lot. It depends on two main things:

Concentration - A 0.1 M buffer can absorb more acid or base than a 0.01 M buffer of the same pH.

Ratio - Capacity is highest when the acid and base components are present in roughly equal amounts. For example when pH is close to pKa.

What do you do if your procedure adds a lot of acid or base? You use a more concentrated buffer, but not a different pH.

Common Laboratory Buffers

Two cautions do apply here. Tris is strongly temperature-sensitive. Its pH drops by roughly 0.03 units for every 1 °C rise. Phosphate can precipitate with calcium and some other metal ions. So it is not suitable for every application at all.

Where Are Buffer Solutions Used?

pH meter calibration - Standard buffers are used to calibrate meters before measurement. (typically at pH 4, 7 and 10)

Biology and life sciences - Enzymes, proteins and cells only function within narrow pH ranges.

Pharmaceutical work - Buffers will stabilise formulations. They are used in dissolution testing and analytical methods.

Chromatography - Buffered mobile phases give reproducible retention times in HPLC.

Food and beverage QC - Buffers support pH testing. Also microbiology media and many analytical methods.

The human body - The bicarbonate buffer system keeps blood pH within a pretty tight range of about 7.35–7.45.

How to Make a Buffer Solution

There are two common approaches that people use.

1. Mix calculated amounts of the weak acid and its conjugate base. Using the Henderson–Hasselbalch equation.
2. Titrate one component to the target pH. For example dissolve Tris base and adjust with hydrochloric acid. Or dissolve a weak acid and adjust with sodium hydroxide.

In both of these cases, the final pH must be confirmed with a calibrated pH meter. Calculations give a close starting point. But factors such as ionic strength and temperature mean the measured pH will differ slightly from theory.

Worked Example: 1 L of 0.1 M Acetate Buffer, pH 5.0

Step 1: Find the ratio. pH = pKa + log ([A⁻]/[HA]), so 5.0 = 4.76 + log ([A⁻]/[HA]). log ([A⁻]/[HA]) = 0.24, giving a ratio of about 1.74 : 1 (acetate : acetic acid).

Step 2: Work out each concentration. The total buffer concentration is 0.1 M, so:

• Acetic acid: 0.1 ÷ 2.74 ≈ 0.0365 M
• Sodium acetate: 0.1 − 0.0365 ≈ 0.0635 M

Step 3: Convert to amounts for 1 L.

• Sodium acetate, anhydrous (82.03 g/mol): 0.0635 × 82.03 ≈ 5.21 g. If using sodium acetate trihydrate (136.08 g/mol), use ≈ 8.64 g instead.
• Glacial acetic acid (60.05 g/mol, density ≈ 1.05 g/mL): 0.0365 × 60.05 ≈ 2.19 g, or ≈ 2.09 mL.

Step 4: Prepare the solution.

1. First add about 800 mL of deionised water to a beaker with a magnetic stirrer.
2. Dissolve the sodium acetate completely.
3. Add the glacial acetic acid (in a fume cupboard, wearing appropriate gloves and eye protection).
4. Measure the pH with a pH meter that's just been calibrated. Fine-tune to pH 5.0 with small additions of dilute acetic acid or sodium hydroxide.
5. Transfer to a 1 L volumetric flask and make up to the mark with deionised water.
6. Mix thoroughly. Then label with the buffer name, concentration, pH, date and your initials.

Tips for Accurate Buffers

• Use high-purity water. Deionised or distilled water avoids introducing ions that shift pH.
• Adjust pH at the temperature of use. This matters most for temperature-sensitive buffers. Such as Tris.
• Make up to volume after adjusting pH, not before. This keeps the final concentration accurate.
• Watch for contamination. Phosphate and other near-neutral buffers can support microbial growth. So store them refrigerated and discard any that turn cloudy.
• Keep alkaline buffers sealed. They absorb carbon dioxide from the air. This gradually lowers their pH.
• Use certified buffers for calibration. Homemade buffers are fine for experiments. But meter calibration needs traceable and ready-made standards.

Buffer Solutions from Ocon Chemicals

Ocon Chemicals is an Irish and family-run laboratory supplier founded in 1981. We are ISO 9001 accredited. Also REACH compliant and we supply labs all across Ireland. Contact us today!

Ocon Chemicals stocks ready-made pH buffer solutions for meter calibration and routine use. And ready-to-use biological buffers such as PBS and HEPES. We also have the reagents to prepare your own. Including glacial acetic acid, Tris, hydrochloric acid and high-purity water. Glassware such as volumetric flasks and measuring cylinders are also available to order from us.

Frequently Asked Questions

What is a buffer solution in simple terms?

A buffer solution is a liquid that keeps its pH steady. It contains a weak acid and its partner base (or a weak base and its partner acid). They neutralise small amounts of added acid or alkali before they can change the pH.

What are the two types of buffer solution?

Acidic buffers contain a weak acid and its salt. Like acetic acid and sodium acetate. And they hold pH below 7. Basic buffers contain a weak base and its salt. Such as ammonia and ammonium chloride. They hold the pH above 7.

Why do buffers stop working?

Every buffer has a limited capacity. If you add more acid or base than its components can neutralise, or if you dilute it heavily, the pH will change sharply. Contamination and absorption of carbon dioxide can also shift its pH over time.

How long does a homemade buffer solution last?

It really depends on the buffer and how it is stored. Many simple buffers last several weeks. If they are sealed and refrigerated. But near-neutral buffers can grow microbes faster. You should always check the pH before use. And you should discard any buffer that is cloudy or out of specification.