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

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. 

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).

(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. 

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. 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.