Preparative chromatography is a specialized subset of chromatography, where the goal is to purify a target analyte (or analytes in some cases) from a complex mixture of crude sample. The ultimate goal is to purify as much target analyte as possible, in as short of a period of time as possible, with the highest purity achievable. As you can imagine, it’s not easy to balance these goals as they often run counter to one another (e.g. if you try to make a short, fast run method, you will probably sacrifice purity) and the chromatographer must find the perfect combination of column chemistry, mobile phase composition/gradient profile, and sample load to achieve their purity and yield goals. In this article, we are going to be focusing on just one aspect of this complex equation – the role of mobile phase pH and how it can be utilized to maximize the efficacy of purification methods.
Given the fact that the majority of conventional silica-based media are only stable from a pH of about 1.5 to 7, most developed methods use lower pH buffers to maximize LC media lifetime. Historically, one of the most popular mobile phase modifiers for preparative purifications is trifluoroacetic acid (TFA). TFA is a strong acid that helps to ensure that residual silanols on the silica surface remain protonated and less prone to interact ionically with positively charged molecules. TFA is also a reasonable ion-pairing agent, which further improves the chromatographic behavior of basic analytes that might otherwise display peak tailing of adsorption. In addition, the fact that TFA is highly volatile means that it can be easily removed from the collected fractions. While gradient elution pH using volatile acidic modifiers, such as TFA, was routinely used for preparative separations due to the pH limitations of traditional silica-based columns, new organo-silica hybrid media provides increased stability even under alkaline mobile phase pH ranges. This means that you are no longer constrained to these working under neutral pH conditions. The ability to utilize elevated pH volatile buffers can offer improved performance for compounds that have low solubility and/or low resolution under more typical low pH running conditions.
To illustrate this, we made a simple mixture of two strong bases – propranolol and diphenyldramine (Figure 1). Under low pH conditions, strong bases will be protonated and ionized and behave as relatively more hydrophilic molecules than when they are deprotonated and non-ionized. Ionized molecules will display less retention than their de-ionized counterparts and display relatively shorter retention times. As seen in the chromatograms (Figure 1), under low pH conditions (0.5% TFA in water and acetonitrile), both propranolol and diphenhydramine elute relatively early in the gradient profile (2.8 minutes) and are not well-resolved from one another (in fact they almost totally co-elute).
Figure 1. Separation of diphenhydramine and propranolol using a low pH mobile phase (0.5% TFA in water and acetonitrile) gradient and a Gemini®-NX organo-silica hybrid HPLC column. Gradient conditions were standardized using a 5 minute gradient from 5 % to 95 % ACN (Acetonitrile). UV detection at 254 nm was used for all separations.
Now, if we perform this same separation using alkaline running conditions (0.2% ammonium hydroxide; Figure 2), these analytes will be deprotonated and behave as more hydrophobic species and, therefore, retain longer, using typical reversed-phase columns. Using these new running conditions, you can see that both molecules elute later during the same gradient profile (3.8 minutes) and are well-separated from one another. Of course, the critical consideration here is that the majority of silica-based reversed-phase sorbent will NOT be stable at such a high pH and the column will form a void in a brief period of the time as the base silica itself dissolves under the alkaline conditions. In order to take advantage of alkaline mobile phases and the unique selectivity benefits they offer, you will need to use a media that has been specifically designed to withstand these conditions. These types of silica are referred to as organo-silica hybrid particles and have been modified by the manufacturer to resist alkaline silica dissolution, most often through the integration of carbon into the silica backbone.
Figure 2. Separation of diphenhydramine and propranolol using an alkaline pH mobile phase (0.2% ammonium hydroxide (pH 10.5) in water and acetonitrile) gradient and a Gemini®-NX organo-silica hybrid HPLC column. Gradient conditions were standardized using a 5 minute gradient from 5 % to 95 % ACN (Acetonitrile). UV detection at 254 nm was used for all separations.
Under these alkaline mobile phase conditions, the resolution of diphenhydramine and propranolol is significantly improved at the higher pH and the two compounds are now separated by 30 seconds instead of 6 seconds. As you can imagine, the greatly increased resolution between the two analytes will allow you to inject a significantly larger mass on-column without sacrificing purity. Thus, by utilizing mobile phase pH, in this case by going to high pH, we are able to simply and effectively increase both our yield and purity.
In a second illustration of this principle, we have made a more complex mixture containing diphenhydramine, oxybutynin, and terfenadine (Figure 3). As before, we begin our method development using a standard low pH buffer containing 0.5% TFA. Under these conditions, the compounds are separated from each other by approximately 0.5 minutes at the low pH gradient conditions. The three compounds are well resolved with a 53 mg load with the TFA buffer. When more of the diluted sample is loaded, the 1400 μL injection deforms the peak shape due to the high volume of DMSO. If the sample volume is limited to 700 μL, the peak shape is maintained and there is no sample breakthrough. When the sample load is increased to 106 mg the compounds are still resolved, although they elute slightly earlier in the gradient, but there is no sample breakthrough at the solvent front.
Figure 3. Separation of diphenhydramine, terfenadine, and oxybutynin using a low pH mobile phase (0.5% TFA in water and acetonitrile) gradient and a Gemini®-NX organo-silica hybrid HPLC column.
When the mobile phase is increased to pH 10.5 using ammonium hydroxide (Figure 4), these three compounds become deionized and are more strongly retained on the column. The resolution between the compounds is vastly improved as compared to the low pH conditions and is also well-preserved as the sample volume and/or sample mass is increased. There is no loss of resolution and only a very slight shift in retention time as the sample mass increases indicating that more sample could be loaded on the column. Thus, one can expect to achieve better yield and purity using the alkaline mobile phase conditions in conjunction with the organo-silica hybrid column.
Figure 4. Separation of diphenhydramine, terfenadine, and oxybutynin using an alkaline pH mobile phase (0.2% ammonium hydroxide in water and acetonitrile) gradient and a Gemini®-NX organo-silica hybrid HPLC column.
As you have seen, the use of a high pH mobile phase in preparative separations of basic compounds has several advantageous.:
1) At pH 10.5 the basic compounds are un-ionized and more hydrophobic, allowing increased interaction with the stationary phase.
2) Higher concentrations of acetonitrile are required to elute the compounds.
3) Separations are less affected by injection volume allowing more dilute samples to be purified.
4) Peak distortion due to column overloading is reduced and higher sample loads can be purified in a single run.
In all cases studied, the higher pH buffer allowed higher mass loading reducing the number of purification cycles required. The increased resolution at the higher pH also resulted in higher purity and yield for the desired basic compounds. Operating at the higher pH also provides an alternative for the chemist to use a smaller diameter column with higher sample load. The smaller diameter column reduces the amount of solvent consumed and reduces the volume of the collected fractions.
For additional examples and to learn more about the exciting silica based media technology that adds new opportunities to achieve better purifications within the pH range of 1-12, please request the technical note, TN-1050,