How do weakly acidic analytes and EVOLUTE®AX work?

We know that compounds can be acidic, basic and neutral. Looking at pK_as and functional groups can help us understand the best approach for SPE selection. Let’s take a look at the compound, methylmalonic acid.

Figure 1. Structure of methylmalonic acid.

When we look at the structure we can see there is no conjugation. Without this conjugation, we will have very little potential for hydrophobic interactions. Now let’s look at the pK_as of the functional groups.

Figure 2. Methylmalonic acid pK_as per Chemicalize.com.

In figure 2 we can see our most acidic functional group has a pK_a of 2.48 and we don’t have any basic functional groups. Remembering some fundamentals of general chemistry, we know strong acids and strong bases are ionized over pretty much the entire usable pH range. Well, the opposite is true for weak acids and weak bases. Ionization for weak acids and bases can be controlled (effectively turned on or off) by manipulation of pH. So what constitutes strong vs weak? Let’s take a look at the sorbent chemistry selection guide in the EVOLUTE Express User Guide.

Figure 3. Looking at the analyte functionality column we can see the pKa ranges for each categorization, we will use this to help us determine the best sorbent chemistry for analysis.

 The acidic pK_a of 2.48 falls within the “weakly acidic” category. For a quick refresher on pH adjustment refer to the pH adjustment sample prep blog. Since we have a weakly acidic functional group, we will exploit that ion exchange potential by using the EVOLUTE®AX sorbent.

Figure 4. EVOLUTE AX Sorbent, the modified polysystrenedivinylbenzene polymer allows the sorbent to be water-wettable.

The AX sorbent has a dual non-polar (hydrophobic) and strong anion exchange functionality. The modified quaternary amine functional group is positively charged and therefore retains negatively charged analytes. We can see in Figure 4 the ionic interactions. Quaternary amines are strongly basic which tells us they are going to be 100% ionized over the entire usable pH range. We can also see that the aromatic polymer backbone of the sorbent allows for hydrophobic interactions.

Figure 5. Hydrophobic and ionic interactions occur simultaneously between the AX sorbent and the methyl malonic acid molecule.

Now let’s see how each step occurs in the AX as seen in Application Note AN847. In this example we are working with human serum and will use the EVOLUTE® Express AX 30 mg 96- well plate part number: 603-0030-PX01.

The typical process for AX sample preparation is based on both reverse phase and ion-exchange interactions. In figure 4 we mentioned the added benefit of the polymeric sorbent. Why is this important? In traditional silica-based SPE, you had to condition and equilibrate your sorbent and frits, which were hydrophobic. With this polymeric SPE sorbent, both the sorbent and frits are water wettable and with an aqueous sample, the sorbent is able to interact with your sample without conditioning and equilibration. This also has the benefit of removing the variability of wells drying out during your analysis. This is especially beneficial if you are working with a 96-well plate and well A1 might be solvated several minutes before H12; the water-wettable capability of the EVOLUTE Express sorbent minimizes the risk of wells drying and well-to-well variability in your application results.