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

As many of you know, buffers are usually used in mobile phases to hold the pH constant. They are composed of a weak acid or weak base, combined with its conjugate base or acid in solution that is at least partially aqueous (at least 50%).  This pair of compounds exists in equilibrium, so that when an acid or base is added, the equilibrium shifts to compensate, thus maintaining the same pH.

Buffering is commonly needed when analyzing ionizable analytes with reversed phase LC. For compounds like these, the pH of the mobile phase determines whether they exist in the ionized or non-ionized form.  This does make a difference because the ionized species always elute from the column earlier for reversed phase LC.  If the pH varies, the results would be inconsistent. Buffers are also sometimes necessary for applications because impurities or interfering compounds are ionizable.  In this case, the buffer allows for consistent separation of the peaks of interest from the interferences.

The biggest question is usually which buffer to use. There are a couple of rules of thumb to follow:

#1

For best results, use a buffer that has a pH at least 2 units away from the pKa for the analyte of interest. If the pH of the mobile phase is too close to the analyte’s pKa, you might observe split peaks or shoulders. If your analyte is acidic, then selecting a pH below its pKa results in the non-ionized form, while a pH above the pKa will result in the ionized species.  Conversely, if your analyte is basic, then selecting a pH above its pKa results in the non-ionized form, while a pH below the pKa will result in the ionized species.  If you have several analytes of interest, it usually works best to set your pH so that all of your analytes exist in the same form, either ionized or non-ionized.  For our reversed phase columns, best results are achieved when acids are neutral and bases are charged.

 

Here are pka’s and pH ranges for some HPLC buffers that are commonly used with Restek columns:

 

Buffer

pKa

  Operating pH range
Phosphate,   pk1

2.1

  1.1-3.1
                   pK2

7.2

  6.2-8.2
                   pK3

12.3

  11.3-13.3 (not advisable)
Citrate,        pK1

3.1

  2.1-4.1
                   pK2

4.7

  3.7-5.7
                   pK3

5.4

  4.4-6.4
Formate

3.8

  2.8-4.8
Acetate

4.8

  3.8-5.8
Tris (hydroxymethyl)- aminomethane

8.3

  7.3-9.3  (use at 8.0 or below)

 

#2

With increasing use of LC/MSD and LC/MS/MS, volatility of buffers is critical. Introducing mineral salts into a mass spec system is usually detrimental and not advisable. Examples of acceptable volatile buffers are ammonium acetate, ammonium formate and ammonium citrate. Although modifiers like formic acid and acetic acid are not technically buffers, they are sometimes used to help control pH as well as ionization for LCMS.

 

#3

The next question is usually what concentration to use. Generally, the lowest concentration that works reproducibly for your application is best.  Higher concentrations are more prone to precipitating out of solution when the organic content increases, or perhaps even a drop in temperature occurs. For reversed phase, generally, concentrations of 2-50 mM are adequate. Concentrations are determined experimentally, keeping in mind that for reversed phase LC, increasing ionic strength of the buffer usually results in decreased retention for ionic species, while decreased strength results in increased retention.

Stay tuned for the next blog post, where we will explore preparation of these mixtures, potential pitfalls and share some helpful tips and good lab practices. Thank you for reading.

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

  1. Reem says:

    Would you please provide a reference for information under the #1 description? Thanks

  2. Hello Reem, the information tabulated under #1 in the blog post can be considered common knowledge, since it can be obtained from multiple sources. The pH operating ranges are derived from the pka for each of the buffer compounds. The pKa’s can be found in reference books, such as the CRC Handbook of Chemistry and Physics.
    https://www.crcpress.com/CRC-Handbook-of-Chemistry-and-Physics-97th-Edition/Haynes/p/book/9781498754286

  3. Natalia says:

    Hi, in general what ratio of aqueous buffer to solvent would you recommend for RP when analysing hydrophobic components that are dissolved in oil? Thanks

  4. Nilesh LD says:

    Hello, I am working with plant extract for the screening of antimicrobials. Can anyone tell me what is importance of UV spectrophotometric analysis of extract and what is its relation with HPLC

  5. Dnyandev says:

    If three pka values are,2.1,7.2,12.3 Then which Ph consider for mobile phase.

  6. Dnyandev, most often we would use the pKa value of 2.1 or 7.2 because it would control the pH with a range that is acceptable for the columns that Restek sells. (We do not sell any columns that tolerate pH above 8.0). For a polyprotic acid like this, the pka of the first proton that is ionized is the one at 2.1 and the second one that is ionized is at 7.2, with ionization increasing as the pH increases. pKa values of 2.1 and 7.2 allow us to control the pH at 1.1-3.1 and 6.2-8.2, respectively. I hope that helps to answer your question.

  7. Natalia, I apologize for the long delay in responding to your question. To analyze a sample as you describe on HPLC, you may need to do a liquid-liquid extraction to eliminate some of the oil for the analysis, depending on the solubility of the oil and the concentration. If it is a very small amount of oil or a more soluble oil, you could dissolve it in mobile phase prior to LC injection. You will need to determine the ratio of organic:aqueous based on the needs for solubility as well as the ability to retain the compounds on the column. Personally, I would start with the compounds and no oil to first determine what mobile phase works to help your retain those the column and how much organic you can have present and still get that retention. I hope that helps.

  8. Nilesh, I am sorry for the long delay in response to your question. I do not have experience specifically with the analysis you describe. Generally, spectrophotometric methods are only used with LC as part of the detection system, as in UV detection and photodiode array detectors. Some applications may use a spectrophotometer for a quick screening method, but I do not have direct knowledge of any that are used specifically for antimicrobials in plant material.

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