Citrate Buffer recipe

Table of Contents

 Introduction
 Applications
 pH
 Composition
 Reagents
 Instruments and other requirements
 Procedure
 Storage
 Caution
 Reference

Introduction

The authorship is often contributed to Gomori (1955) but Gomori is in this publication citing an
article that is seven years older (Lillie 1948) as original source. Citrate buffer is a universal
buffer used in various reactions. It is sometimes combined with sodium citrate or completely
replaced by sodium citrate buffer. However, alkalized citrate buffer has a similar composition to
sodium citrate buffer.

Applications

This buffer is commonly used in different fields of biochemistry as a basic pH buffer with
adjustable pH for enzymatic solutions and chromatography. In the era of molecular biology, the
citrate buffer was found to be useful in

 RNA isolation because it prevents the bases from hydrolysis,

 heat-induced antigen retrieval protocols since it breaks the cross-links and in DNA
fluorescent in situ hybridization.

pH

For enzymes: The pH can be adjusted from 3,0 to 6,2 according to the specific needs of your
enzyme by changing the composition or by adding HCl to acidify and NaOH to alkalize the
buffer. However, the usage of strong acids and hydroxides can change the ionic strength, and
therefore, it could affect the experiment.

For antigen retrieval: The pH 6,0 is used, adjusted by NaOH.

Composition

There are several ways of preparation of this buffer.

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Molecular 1X Add for 500ml of Add for 1L of

Reagent
weight molarity 1X 1X

Citric acid 192.1 100 mM 9.6 g 19.2 g

Dihydrate tri-sodium
258.1 100 mM 12.9 g 25.8 g
citrate
*
Tween is polyethylene glycol sorbitan monolaurate. It is used as a detergent in this solution.

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Molecular 1X Add for 500ml of Add for 1L of

Reagent
weight molarity 1X 1X

Citric acid 192.1 100 mM 9.6 g 19.2 g

Dihydrate tri-sodium
258.1 100 mM 12.9 g 25.8 g
citrate
Reagents

Citric acid

Dihydrate tri-sodium citrate

Tween (polyethylene glycol sorbitan monolaurate)

0,1 M NaOH, HCl for adjustment if needed

Distilled water

Instruments and other requirements

Glass Beaker

Weighing balance

Magnetic stirrer and pellet

pH meter

Measuring cylinders
Pipette

Procedure

For antigen retrieval:

Add about ¾ of the final volume of distilled water to the glass beaker.

Prepare the exact amounts of citric acid using balance and put it into the water to dissolve. A
magnetic stirrer can be used to speed up the process.

After the citric acid is dissolved, add distilled water to the final volume.

Adjust the pH to 6 with 0,1 M NaOH.

Add 0,5 ml of Tween and mix really well.

For enzymes, 1 l of the final solution:

First, prepare the stock solutions of citric acid and sodium citrate:

Add about ¾ of the final volume of distilled water to the glass beaker.

Prepare the exact amounts of citric acid/sodium citrate using balance and put it into the water to
dissolve. A magnetic stirrer can be used to speed up the process.

After the citric acid is dissolved, add distilled water to the final volume.

In the next step, mix specific amounts of citric acid solution and sodium citrate solution
according to the table below to obtain the buffer of the required pH.

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Amounts of stock solutions (ml)

Required pH

0.1 M citric acid 0.1 M sodium citrate

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3 930 70

3.2 874 126

3.4 800 200

3.6 740 260

3.8 700 300

4 660 340

4.2 630 370

4.4 560 440

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4.6 510 490

4.8 460 540

5 410 590

5.2 360 640

5.4 320 680

5.6 274 726

5.8 236 764

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6 190 830

6.2 144 856

Storage

It can be stored at room temperature for 3 months. At a lower temperature (4 °C), the time could
be prolonged but the solution should not freeze to avoid precipitation.

Caution

Tween is an irritant so it should not come in contact with skin or mucosae and it should not be
swallowed. It is also critical to mix it well with water when preparing the buffer.

Citric acid is a weak acid but it can cause eye irritation.

Reference

1. Gomori, G. (1955). [16] Preparation of buffers for use in enzyme studies.

2. Citrate Buffer Antigen Retrieval Protocol.
3. Citrate Buffer (0.1 M, pH 6.0) Solution Preparation and Recipe
4. Lillie, R. D. “Histopathologic Technic.” Southern Medical Journal 41, no. 6 (1948):
574.