Buffers are chemical solutions with the unique property of being able to consistently maintain their pH regardless of change to their volume or composition. They are used in many chemical, biological, medical, diagnostic, electronic and food preparation laboratories to maintain the pH levels of solutions that have enzymes, cells or other chemicals. They also carry out chemical reactions and can be used to store sensitive metals, nucleic acids or other laboratory items. To dilute a buffer solution, use a process known as "serial dilution."
- Skill level:
- Moderately Challenging
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Things you need
- Tube racks
- Laboratory glassware
- Pipettes, pipette guns and pipette tips
- Personal protective equipment
- Digital pH meters
- Magnetic stirrers and stirring plate
Read the final experimental protocol and decide on the concentration unit of the final diluted buffer. Concentrations can be in molarity (Moles, M; millimoles, mm; and so on) or percentage (percentage weight by volume, w/v; percentage by volume, v/v; or per cent by volume, w/w).
Make a "stock" solution. Using the protocol-determined final concentration as a starting guide, decide on a highly concentrated version of this. For example, if 10mM of the chemical is required in the protocol, make a 1M stock or concentrated solution. Use water or other nonreactive chemical, such as phosphate-buffered saline; however, verify this with your protocol to ensure that it does not interfere with later procedures or reactions. Carefully weigh out the exact quantity or measure out the exact volume of the required chemical. Do not make any inaccuracies at this step as these errors will be amplified in the dilutions and therefore make them incorrect. Dissolve this thoroughly, check the pH and adjust accordingly before beginning the dilution.
Decide on the "serial dilution" to be performed. Serial dilutions are made as ratios of the original stock solution. For example, if setting up a 1:2 serial dilution, then the stock will be diluted half-in-half ; if setting up a 1:100 serial dilution, then 1 millilitre of the stock solution will be diluted in 49 millilitres of diluent used in the final protocol to make up 50 millilitres; and so on.
Set up the serial dilution. Place several test tubes (e.g., 10 tubes) in a rack and label all of these sequentially (e.g. 1, 2, 3, 4 or 1:10, 1:100, 1:100). The manner you perform a serial dilution will depend on the ratio chosen. For 1:10 dilutions, add 1 part of the stock solution to 9 parts of the diluent. For the next tube, take 1 part of the first dilution, and add that to 9 parts of the diluent. Repeat this until the desired dilution is obtained. Therefore to dilute a 1M solution to 100mM, simply add 1 millilitre of the 1M stock to 9 millilitres of a diluent to get the desired 100mM solution. Check the pH of the solution using a digital pH meter for highest accuracy. If this is wrong, prepare the stock solution from the beginning and check the pH of this before preparing a serial dilution.
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