## Insights # How to Make and Dilute Aqueous Solutions

## A step-by-step guide

Michelle Dotzert, PhD Michelle Dotzert, PhD

Michelle Dotzert is the creative services manager for Lab Manager. She holds a PhD in Kinesiology (specializing in exercise biochemistry) from the University of Western Ontario. Her research examined the...

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## Aqueous solutions

This guide will describe the process for preparing aqueous solutions, including how to calculate the appropriate amount of solute for a given volume of solution, how to prepare a complex solution with two or more solutes, and how to dilute a solution—including serial dilutions.

## What is an aqueous solution?

Solutions are homogenous mixtures of two or more substances. They are essential for a variety of biological and chemical experiments. Buffers, cell culture media, and reaction mixtures are just some examples of commonly used solutions. ## What you will need to make an aqueous solution:

• Personal protective equipment (gloves, lab coat, eye protection, etc.)
• Purified water
• Solute(s)
• Analytical balance
• Weigh boats
• Beaker
• Magnetic stir plate and stir bar
• pH meter
• Dilute sodium hydroxide
• Dilute hydrochloric acid ## How to make an aqueous solution

### Step 1: Calculate the mass of the compound needed to achieve a specific molar concentration and volume

Determine the desired concentration and final volume of the solution. If concentration will be reported in molarity or normality, the molecular weight or formula weight of the substance is required (this indicates how many grams of the substance are in one mole). This information can usually be found on the container.

This formula is used to calculate the mass of the compound needed to achieve a specific molar concentration and volume:

Mass (g) = concentration (mol/L) x volume (L) x molecular weight (g/mol) Example:

Prepare 1L of a 5M solution of a substance with a molecular weight of 50 g/mol.

Mass (g) = concentration (mol/L) x volume (L) x molecular weight (g/mol)

Mass (g) = 5 x 1 x 50

Mass (g) = 250

The solution in this example requires 250g of solute.

Do not add 1 L of water to 250g of solute. This will exceed the desired total volume.

Add the solute, then add water to bring the total volume up to 1L (see below).

### Step 2: Weigh and measure

Using a balance and weigh boat, weigh the amount of solute calculated in step 1.

Dispense purified water into a graduated cylinder— obtain approximately three quarters of the final volume of solution. For example, for a solution with a final volume of 1L, obtain approximately 750 ml of water.

Always use purified water when preparing aqueous solutions. Tap water can contaminate the solution and compromise subsequent experiments. ### Step 3: Combine the solute and solvent

Place a magnetic stir bar into a beaker, and place the beaker on a magnetic stir plate.

Begin stirring the water and add the solute to the beaker. ### Step 4: Adjust the pH of the solution

Once the solute is dissolved, measure the pH using a pH meter, and adjust if necessary.

Add dilute sodium hydroxide to increase pH, or dilute hydrochloric acid to decrease pH.

Add NaOH or HCL slowly, as pH can change rapidly. ### Step 5: Bring the solution to final volume

Using a funnel, pour the solution from the beaker into a volumetric flask.

Bring the solution to final volume by topping up with purified water.

To "q.s. the solution" means to bring the solution up to the final volume.

The term "q.s." is the abbreviation of the Latin term quantum satis, meaning as much as is enough.

### Making a complex solution

A complex solution contains more than one solute. To make a complex solution, repeat Step 1 for each solute. Weigh each solute individually, then complete steps 2-5. ## How to dilute aqueous solutions If the amount of solute required for a solution is too small to accurately weigh, you can make a concentrated stock solution and dilute it for use.

This formula is used to calculate the volume of stock solution and buffer (solvent) required to achieve the desired volume and concentration:

Concentration (stock) x Volume (stock) = Concentration (dilute) x Volume (dilute) Example:

Using a 5M stock solution, prepare 100ml of 0.5M solution.

Concentration (stock) x Volume (stock) = Concentration (dilute) x Volume (dilute)

5M x Volume(stock) = 0.5M x 100ml

Volume(stock) = (0.5Mx100ml)/5M

Volume(stock) = 50ml/5

Volume(stock) = 10ml

Add 10ml of 5M stock solution to a container and top up to 100ml with water.

### Dilutions for complex solutions

Complex solutions may also be made from more than one stock solution. To dilute a complex solution with more than one solute, treat each dilution individually. Using the equation above, calculate the volume of each stock solution individually then add them to the container and top up the container to the final volume.

### Serial dilutions

Serial dilutions are stepwise dilutions of substances in a solution. They may be used to reduce the risk of error associated with measuring very small volumes (for example, a serial dilution may be used rather than pipetting 1 µl of the substance in solution). Serial dilutions may also be required to create standard curves, or for other experiments.

### How to perform a serial dilution

Example

Obtain 1mM of a 1M compound in a 1 ml solution.

#### Step 1: Calculate the dilution factor

You can calculate the dilution factor with the following equation:

Dilution factor = (Volume(initial) + Volume(dilute))/Volume(initial)

Begin with an initial volume of 100 µl of concentrated solution (1 M) and 900 µl of diluent.

Dilution factor = (Volume(initial) + Volume(dilute))/Volume(initial)

Dilution factor = (100 + 900)/100

Dilution factor = 10

#### Step 2: Calculate the number of dilution steps

Determine the number of dilutions required to achieve the desired final concentration (x).

(Dilution factor)x = Concentration(initial)/ Concentration(final)

10x = 1/10-6

x = 6

106 is used because Concentration(stock) is in M and Concentration(final) is in mM.

1M = 1000000mM

#### Step 3: Label microtubes

Label 6 microtubes as follows:

C/10                C/104
C/100             C/105
C/103                   C/106

#### Step 4: Pipette the diluent and stock solution

Pipette 900 µl of diluent into each tube.

Pipette 100 µl of 1M stock solution into tube “C/10” and vortex the tube.

Pipette 100 µl from tube “C/10” into tube “C/100” then vortex the tube.

Repeat for the remaining tubes.

Tube “C/106” now has a final concentration equal to the stock concentration (1 M) divided by 106. Tags: