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Buffers are an important part of most laboratory experiments, as they help keep mixtures between a 5.0 and 8.2 pH level. A Sorensen’s phosphate buffer is a non-toxic option for your projects and doesn’t contain any harmful substances, unlike a cacodylate buffer. [1] X Research source This type of buffer isn’t difficult to make, though you do need a background in chemistry and some basic laboratory supplies to get the job done. [2] X Research source You can make a simple buffer by mixing exact amounts of dibasic sodium phosphate and potassium phosphate together, or by using a combination of dibasic sodium phosphate and monobasic sodium phosphate.
Steps
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Pour 2 L (8.5 c) of distilled water into 2 large, reusable containers. Double-check that you’re using exactly 1 L (4.2 c) for your buffer. Always use distilled water instead of tap so the pH can be as accurate as possible.
- You can find distilled water at most stores selling laboratory supplies.
- Each container should have 1 L (4.2 c) of distilled water in it.
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Stir 35.76 g of Naâ‚‚HPOâ‚„ into 1 container of water. Mix in the powder until it’s completely dissolved in the water. Label this as dibasic sodium phosphate, so you don’t get your buffer ingredients mixed up.Advertisement
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Add 9.08 g of monobasic potassium phosphate into the other container. Stir in the ingredient until it’s completely mixed with the distilled water. Be sure to label this container as well, so you can tell your ingredients apart.
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Add 71.5 mL (0.302 c) of Naâ‚‚HPOâ‚„ into an empty container. Set out a large, reusable glass container on your workspace, then pour in a large amount of dibasic sodium phosphate. Measure exactly 71.5 mL (0.302 c) into the container so the mixture can be exact. [3] X Research source
- The container needs to be able to hold at least 1 L (4.2 c) of fluid.
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Mix in 28.5 mL (0.120 c) of KHâ‚‚POâ‚„ with the Naâ‚‚HPOâ‚„. Stir a small amount of potassium phosphate into the container. Check that you’re adding exactly 28.5 mL (0.120 c) to the mixture, so the buffer’s pH can be as accurate as possible. [4] X Research source
- The ratio needs to be exact, or else your buffer may not have the right pH.
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Check the pH to see if it’s 7.2. Take a pH tester and stick it into the mixture. Look at the testing electrode to see if the pH is 7.2, or if it’s higher or lower. If the pH isn’t exact, you may need to create a new mixture using more exact ratios of dibasic sodium phosphate and potassium phosphate. [5] X Research source
- You can get a pH testing electrode at most places that sell laboratory supplies.
Alternative pH Ratios
5.0 pH: 1 mL (0.0042 c) of NAâ‚‚HPOâ‚„ and 99 mL (0.42 c) of KHâ‚‚POâ‚„
6.0 pH: 12 mL (0.051 c) of Naâ‚‚HPOâ‚„ and 88 mL (0.37 c) of KHâ‚‚POâ‚„
8.0 pH: 94.5 mL (0.399 c) of Naâ‚‚HPOâ‚„ and 5.5 mL (0.023 c) of KHâ‚‚POâ‚„
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Store your mixture for 2-3 weeks at 4 °C (39 °F). Seal the top of your phosphate buffer container and label it with the current date. Keep it in a cool area for safekeeping, and use it whenever you need to neutralize a mixture. After a few weeks, toss out the buffer and make a new one, since phosphate buffers can be a breeding ground for small organisms over time. [6] X Research source
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Stir 35.61 g of Naâ‚‚HPOâ‚„ in 1 L (4.2 c) of distilled water. Pour a large amount of distilled water into an empty, reusable container and stir in the measured powder. Wait for the ingredient to dissolve completely, then label the container. [7] X Research source
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Mix 27.6 g of NaHâ‚‚POâ‚„ into 1 L (4.2 c) of distilled water. Measure the powder into the distilled water and stir both ingredients together. Label the container so you can tell all of your buffer ingredients apart. [8] X Research source
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Make a basic solution with a large amount of Naâ‚‚HPOâ‚„. Think about what you’d like the pH of your phosphate buffer to be. You can make your mixture as acidic as 5.8 or as basic as 8.0, depending on the ratio of your buffer. Pour in a 47.35 mL (0.2001 c) of Naâ‚‚HPOâ‚„ in a separate container eto make your buffer more basic, and a smaller amount to make it acidic. [9] X Research source
- For instance, a buffer with 47.35 mL (0.2001 c) of Naâ‚‚HPOâ‚„ would yield a buffer with a pH of 8.0, while 4 mL (0.017 c) would give you a pH of 5.8.
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Add in a small amount of NaHâ‚‚POâ‚„. Pour in 2.65 mL (0.0112 c) of NaHâ‚‚POâ‚„ into the container with the liquid Naâ‚‚HPOâ‚„. Mix both ingredients together thoroughly to finish making the buffer. [10] X Research source
- For example, 46 mL (0.19 c) of NaHâ‚‚POâ‚„ and 4 mL (0.017 c) of Naâ‚‚HPOâ‚„ give you a buffer with a 5.8 pH, while 2.65 mL (0.0112 c) of NaHâ‚‚POâ‚„ and 47.35 mL (0.2001 c) of Naâ‚‚HPOâ‚„ give you a pH of 8.0.
- For something more neutral, like 7.2, mix 36 mL (0.15 c) of Naâ‚‚HPOâ‚„ with 14 mL (0.059 c) of NaHâ‚‚POâ‚„ together.
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Dilute the buffer with 50 mL (0.21 c) of double distilled water. Pour a large amount of purified water into your mixture to help dilute the buffer and make it more effective. As a rule of thumb, the buffer and diluted water need to be at a 1:1 ratio. [11] X Research source
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Label and store your diluted buffer at 4 °C (39 °F). Seal the top of your reusable container and label it as a phosphate buffer. Keep the mixture in a really cool area for 2-3 weeks, using the buffer as needed. Make a new batch after a few weeks, as phosphate buffers tend to foster unwanted growth. [12] X Research source
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Tips
- You can purchase a premade Sorensen’s Phosphate Buffer from companies that sell laboratory supplies.Thanks
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Warnings
- It’s not a great idea to make your own buffers and mixtures unless you have a good background in chemistry.Thanks
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Things You’ll Need
Making a Buffer with Potassium Phosphate
- Dibasic sodium phosphate (Naâ‚‚HPOâ‚„)
- Potassium phosphate (KHâ‚‚POâ‚„)
- 3 containers
- pH tester
Working with Only Sodium Phosphate
- Dibasic sodium phosphate (Naâ‚‚HPOâ‚„)
- Monobasic sodium phosphate (NaHâ‚‚POâ‚„)
- Double distilled water
- 3 containers
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References
- ↑ https://www.ou.edu/research/electron/bmz5364/buffers.html
- ↑ https://www.weberscientific.com/phosphate-buffers-here-s-what-you-need-to-know
- ↑ http://cshprotocols.cshlp.org/content/2010/10/pdb.rec12327.full
- ↑ http://cshprotocols.cshlp.org/content/2010/10/pdb.rec12327.full
- ↑ http://www.globalw.com/support/ph-measurement.html
- ↑ https://www.ou.edu/research/electron/bmz5364/buffers.html
- ↑ https://www.ou.edu/research/electron/bmz5364/buffers.html
- ↑ https://www.ou.edu/research/electron/bmz5364/buffers.html
- ↑ https://www.ou.edu/research/electron/bmz5364/buffers.html
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