Excelling in Buffer Selection for HPLC Method Development: Avoid Critical Mistakes & Achieve Success (Part 2)

In part one we talked about Buffer selection & it’s correlation in terms of solubility, pH & buffer capacity range along with the Buffer concentration & an important term which could affect the baseline in the method development, this term is called UV Cut-off the Method development ,analyte detection & quantification, In this part our main focus would be about the effect of Buffer selection on the Analyte’s retention ,peak shape ,resolution & selectivity on the HPLC column. We would also address here the impact of a varying injection volume on the Buffer capacity & so on the Analyte’s peak shape.

Buffer selection -Impact of pH on method development –(Analyte Retention)

pH is a very crucial factor in HPLC method development of ionizable compounds, since their retention is very sensitive when it comes to the mobile phase pH. As mentioned in the 1^st part, the aim of the buffer is to resist any change in pH upon the addition of minor amounts of an acid or base. There are many different substances that have been used for buffering in HPLC, we would mention the most important additives & salts used for buffer preparation as follows:

As a general note in method development , it is better to start with pH 2-3 range since at such pH , the majority of organic acids ionization would be suppressed-meaning less polar ie more hydrophobic- which indicates a stronger retention on the reversed phase on HPLC column which is the main aim for our method development , also the ionization of the silanol groups on the column would be suppressed which is also very favorable in method development & this would be explained later on.

To further illustrate ,for a molecule to retain on a reversed phase HPLC column ,it needs to be more hydrophobic ,this is guaranteed in case of a non-ionizable molecule so for an acid molecule in order to remain non-ionizable ,we need to prepare a mobile phase pH that is 2 units below the analytes pKa ,in such case the molecule would be >99% unionized & vice versa for a bases where in such case we need to prepare a mobile phase pH that is higher 2 units than the analyte’s pKa ,so to summarize acids would be more retained at low pH(figure 1a) while for bases they would be more retained at high pH(figure 1b)

Impact of pH on method development –(Analyte separation & selectivity on HPLC column)

The more the mobile phase pH is near to the analyte’s pKa ,the more it would be obvious for a small change in pH to give a significant change in retention which is unfavored for a method development since it indicates the lack of a robust separation. Below in figure 2a ,you can see the impact of any minor change in pH even if it was as small as just 0.1 units such as in this case-knowing that this amount of error is very common in many laboratories in pH adjustment- ,yet you can see how even such a minor change in pH made a huge difference which was impacted on retention & thus resolution as well. At pH 5, retention is less sensitive to pH than it is at pH 3 in case of the acid molecule or pH≥6 for the base.

From the above figure ,It is obvious that not only retention time is affected when the pH is near the pKa ,but also selectivity could change in case of molecules of similar structure were found.

HPLC column stability & type of Silica

As mentioned earlier in the 1^st part for this topic , most of the HPLC columns are stable in the pH range of 2-8. Below pH 2 ,hydrolysis could occur causing bonded phase loss while above pH 8 the silica backbone. Adding to the previous ,the major cause for basic compounds peak tailing has been their interaction with the silanol groups that have been ionized by a cation exchange process yet nowadays the newly used Type B Silica is of high purity with a pKa of >7 which eliminates the factor of silanol sites ionization to a great extent ,that’s the reason behind why the high purity silica gives a way high better peak shape for bases than the older type of silica ,In the below graph you can see how the usage of a high purity silica in the HPLC column has improved the peak tailing for bases to a great extent.

Moreover, an improvement of reproducibility is obviously seen upon the usage of a high purity silica compared to a low purity one because of the reduction of such unpredictable secondary silanol groups. As a practical implication to summarize the above-mentioned data.

So to summarize all of the above ,it is generally recommended based on the characteristics of the column& of the samples in general to start the development of the method with a mobile phase pH within pH 2-3 ,normally at this pH suppression for the ionization of organic acids would occur so does the silanol groups on the column ionization as well yet at such pH Bases would be ionized ,suppressing for the ionization for such compounds of usually having pKa>7 would require operating at higher pH which usually would case deterioration of most of HPLC columns. If operating at high pH is crucial, then be sure to choose a durable HPLC column at such pH.

Generally, adjusting the organic content of the mobile phase(%B-solvent) is usually more fruitful at first to begin with regarding obtaining an acceptable retention for either neutral &non-ionized compounds, then to ensure pH adjustment to obtain suitable retention for the ionic analytes.

Correlation of Sample Injection volume with Buffer

The aim of using a buffer is to keep the analyte at the required pH, considering this aspect of buffering so a very minimal amount of buffer is required. In our analytical work, most of the samples are within a µg/ml to a ng/ml range, so in such case where a sample being injected is less than 100µl ,this would mean that the mass on the column won’t be more than a few hundred nanograms.

Moreover, the buffer should also be able to achieve a constant pH on the HPLC column ,along analysis large volume of buffer passes through HPLC column ,this means the silica on the HPLC column is constantly exposed to the buffer. It should be mentioned that nowadays HPLC columns have less acidic silanol groups because of having much more high purity silica which would require less buffer amount than those older HPLC columns having lower purity silica.

Below you would find various chromatograms illustrate the impact of using an additive like Trifluoroacetic acid-TFA- & how much does it’s concentration affects peak shape. TFA is used to keep the pH low along as also acting as an ion pairing reagent, both effects are not isolated from each other. Below you can observe the difference in peak shape among the usage of 0.1% TFA & when it’s concentration is decreased to 0.01%, also in the chromatograms you can observe a correlation between the purity of the silica & the peak shape.

You can see that upon decreasing the concentration of TFA using various columns of different silica purity, you can see how much the peak shape has shown a degradation in peak shape especially upon the usage of moderate & low purity silica in HPLC columns, so from the previous we can conclude that TFA has great effect on buffering the column to avoid the strong secondary interactions between the analyte & the column ,such interactions usually result in peak tailing unfortunately.

Insufficient buffer usually affect all peak shapes regardless the silica purity, for this it is recommended to use more than the minimum amount of buffer meaning for acid additives it is recommended to be in the concentration of about 0.1% v/v, for buffers the recommended molarity to be used is within the range of 5-10 mM.

Most importantly, the aim of the buffer is to buffer the sample being injection in order to attain the pH of the mobile phase. The less amount of sample being injected, the easier the buffer’s ability to act in contrast to a high sample volume in an injection where the pH in such case would differ greatly in the sample than that of the mobile phase. You can avoid the need for preparation of a high molarity buffer if your sample was priorly pH adjusted before injecting.

References:

5989_5672.qxd (agilent.com)

The LCGC Blog: Buffer Choice for HPLC Separations (chromatographyonline.com)

(711) How to choose the correct HPLC buffer? – YouTube