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How much buffer to weigh and what's the best way to prepare my buffered mobile phase?

30 December 2013
By
  • Nancy Schwartz
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Occasionally we get this question and sometimes folks do need help with the calculations. We discussed in a previous post what a buffer is composed of. Click on this link if you need to review it:

When should you use a buffer for HPLC, how does it work and which one to use?

For a non-pH-adjusted mobile phase, you will need to weigh a portion of the salt of an organic acid or organic base and then add or measure an equal molar amount of its corresponding conjugate acid or base. For a mobile phase with buffer prepared at a specified pH, the amount of acid or base needs to be adjusted slightly to achieve that pH. Note: Selecting a pH range that is too far away from the pKa of your buffer’s acid or base will result in ineffective buffering.

Examples:

2 mM Ammonium Acetate buffer, not pH-adjusted

Let’s say that according to the pKa of your analytes, you need to prepare a solution with a pH that is slightly acidic, below 5.0. You are using LCMSMS, so you will need to choose a volatile buffer salt. The appropriate choice would be ammonium acetate.  In hopes of keeping the ionic strength on the low side, you might try a 2 mM solution. Since we are not adjusting the pH, it will be close to 4.76, the pKa of acetic acid/acetate buffer.

Now how to do the calculation? Most chemists will remember this from the courses they’ve taken, but technicians often need some help figuring this out. To calculate molarity, first you need to know the molecular weight of the components. In this example, our buffer components are ammonium acetate (MW= 77.08 g/mol) and acetic acid (MW=60.05 g/mol).  Since acetic acid is a liquid, you will also need to know the density, which is 1.05 g/mL, and the concentration of the stock acid solution. You would likely use glacial acetic acid, which is usually 99.8% (not of much consequence for this acid).

If you are going to prepare 2 L of aqueous solution at 2 mM concentration, to determine the weight of ammonium acetate, please see calculation below:
2 mmol/liter×2 liters ×77.08 g/mol×1mol/1000mmol  =0.308 g
If you needed a pH of 4.8 (equal to the pKa), you would add the equivalent molarity of acetic acid. Here is how you would calculate the volume of acid to add:
2 mmol/liter×2 liters ×60.05 g/mol×1mol/1000mmol  ÷(0.998 ×1.05 g/mL)=0.229 mL
 

 

The following table for “Composition of concentrated reagent grade acids and bases” from Sigma-Aldrich may be useful:

http://www.sigmaaldrich.com/chemistry/stockroom-reagents/learning-center/technical-library/reagent-concentrations.html

(For accurate calculations, it is always best to check the concentration as shown on the bottle of the reagent or provided by its manufacturer. Some may vary slightly.)

 

2 mM Ammonium Acetate buffer, pH-adjusted to 4.0

Let’s say that according to the pKa of your analytes, you need to prepare a solution with a pH of 4.0. The most common way to do this is to weigh out the ammonium acetate as described above (0.308 g), then use its liquid acid or base to adjust the pH as needed. The best way to measure the pH is with a properly calibrated pH meter. To get an idea of how much acid you need to add, it might be useful to compare this solution to the previous one we made. If we are adjusting the pH to below 4.76, we know that we will have to add more than 0.229 mL of glacial acetic acid to get to a pH of 4.0. Keep in mind that with concentrated reagents a little can go a very long way, particularly after you’ve reached the point of equilibration (at 4.76 in this case).

If you choose not to use a pH meter for this adjustment, concentrations can be calculated. Since most of us would avoid this complication, it might be useful to use an online calculator such as the one provided by Sigma-Aldrich:

http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-calculator.html

 

Next question: What comes first?

First of all, if you have a written method, follow it to the tee. The important thing for reproducible results is for a method to be clearly written and followed consistently each time.

If you do not have a method that outlines buffer preparation, I have found the most reliable and convenient sequence to be as follows:

 

  1. Make sure you have clean containers of the correct volume. Avoid the use of UV absorbing solvents such as acetone as well detergents or surfactants. My preferred last step is to rinse with purified water and drip dry.
  2. Weigh your buffer salt in a weigh boat and transfer to a volumetric flask. Rinse into your flask with enough water to ensure a complete transfer of the buffering salts. Add a stir bar if pH adjustment is needed. If pH adjustment is not needed, add the appropriate volume/amount of acid or base.
  3. Dilute to volume with purified water. If adjusting the pH, don’t take it all the way up yet.
  4. Mix well by stirring or shaking. Sonicate if required.
  5. Adjust pH with a calibrated pH meter and the appropriate base or acid for your buffer (For example, an acetate buffer calls for acetic acid). Make sure the material in your pH probe is compatible with your analysis before dipping it into your mobile phase. For example, remember to avoid plastics if your analysis includes or is affected by phthalates that could be present. Always make sure that the probe is rinsed with clean water before using and keep the solution.  stirring throughout the pH measurement to ensure an accurate sampling
  6. After you have reached the desired pH, add water to reach the target volume and mix again. If you have to add more than a small amount of water, double check the pH again afterward.
  7. Always filter your mobile phase when it contains buffer compounds. It is best to do this with the aqueous portion before adding the organic solvent, as the pH can be affected considerably by solvents such as methanol. Restek sells the glassware and supplies to do this, shown here: https://www.restek.com/en/products/accessories-labware/lc-accessories/Mobile-Phase-Management/942/. Keep in mind that failure to filter the mobile phase can result in pressure increases, blockages and shorter column lifetimes.
  8. Measure the desired volume of pH-adjusted aqueous mobile phase and add to organic portion if needed.  Mix well and bring to final volume with the organic solvent. Allow to come to room temperature before use.

 

To run an established method, the key (once again) is consistency.  Since modern LC systems can pump from multiple bottles and do the mixing, it has become popular to prepare the aqueous and organic mobile phases separately and program the HPLC to do the mixing.  Assuming your instruments are calibrated and maintained properly, this may be the best way to ensure repeatable results. However, there may be differences between instruments due to dwell volume, calibration and maintenance of the pump modules and LC components, and the flow rate range. Also, if the method you are using was developed with premixed mobile phases, your results should be compared to the premixed mobile phase to make sure the retention times, responses, results, etc. are equivalent. Keep these in mind and be prepared to make adjustments as needed.

I hope that some of the suggestions here have been helpful in getting you up to speed with buffer preparation. Thanks to my colleagues for their assistance with providing/reviewing this information. Thank you for reading.

 

 

Comments

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Thu, Jan 10, 2019

Dear Sir, Let me first thank you for allowing to submit comments. I am writing this to bring to your attention a gross error in the molarity of the buffer described under "2 mM Ammonium Acetate buffer, not pH-adjusted". By definition buffer molarity is the additive of the molarities of acid and its conjugate base. By calculation number of moles of acetate is 0.308/77.08 = 0.00399 and that of the acetic acid is 0.004. Therefore there are total of approximately 8 milli moles of the buffer components in a 2 L volume and the buffer concentration is 4mM not 2mM. Best Regards, Babu V Bassa, Ph.D.

Wed, Aug 02, 2017

Thank you for your input, Brigitte. I agree that usually the aqueous and organic phases are prepared separately. The focus of this post is really on the additives that may be added to the aqueous phase or perhaps both. We welcome your feedback, though.

Wed, Aug 02, 2017

To run an established method, the key (once again) is consistency. Since modern LC systems can pump from multiple bottles and do the mixing, it has become popular to prepare the aqueous and organic mobile phases separately and program the HPLC to do the mixing.

Mon, May 18, 2015

Thanks for your sharing, it's really useful!