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

    Structure of 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 pKas of the functional groups.

    Methylmalonic acid pKas per Chemicalize.com

    Figure 2. Methylmalonic acid pKas per Chemicalize.com.

    In figure 2 we can see our most acidic functional group has a pKa 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.

    Evolute Express Sorbent Selection

    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 pKa 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.

    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.

    AX sorbent and methylmalonic acid

    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.

    Step

    Description

    What’s happening?

    Sample Pre-treatment (1)

    10 µL 1 ng/µL internal standard and 100 µL human serum

    Adding the internal standard first is important as you are going to normalize to this during analysis. Typically avoid doing more than 10% volume internal standard addition.

    Sample Pre-treatment (2)

    290 µL HPLC grade water

    Diluting the sample will help decrease viscosity allowing it to more readily flow through the SPE sorbent.

    Condition (optional)

    N/A

      This is optional when using the EVOLUTE Express line due to the water-wettable functional groups (and frits).

    Positive Pressure

    N/A

    Positive pressure avoids variable well-to-well flow-through.

     Nitrogen is typically preferred to prevent oxidation and purity prevents contamination.

    Equilibration (optional)

    N/A

     This is optional when using the EVOLUTE Express line due to the water-wettable functional groups (and frits).

    Positive Pressure

    N/A

    Positive pressure avoids variable well-to-well flow-through.

     Nitrogen is typically preferred to prevent oxidation and purity prevents contamination.

    Sample Loading

    400 µL pretreated sample

    This is within the recommended load capacity.

    Positive Pressure

    Recommend positive pressure

     

    Positive pressure avoids variable well-to-well flow-through.

     Nitrogen is typically preferred to prevent oxidation and purity prevents contamination.

    Wash 1

    1mL HPLC water

    Remove polar interferences such as salts and small proteins.

    Positive Pressure

    Recommend positive pressure

     

    Positive pressure avoids variable well-to-well flow-through.

     Nitrogen is typically preferred to prevent oxidation and purity prevents contamination.

    Wash 2

    1 mL methanol

    Breaks hydrophobic interactions, releasing hydrophobic compounds such as phospholipids and lysophospholipids.

    Positive Pressure

    Recommend positive pressure

     

    Positive pressure avoids variable well-to-well flow-through.

     Nitrogen is typically preferred to prevent oxidation and purity prevents contamination.

    Elution

    1 mL 2% formic acid in acetonitrile

    Adding the acidic buffer will neutralize the analyte, breaking the ionic interactions, and will release the analytes from the sorbent.

    Positive Pressure

    Recommend positive pressure

     

    Positive pressure avoids variable well-to-well flow-through.

     Nitrogen is typically preferred to prevent oxidation and purity prevents contamination.

    Dry Down

    Stream of air or nitrogen

     Nitrogen is typically preferred to prevent oxidation and purity prevents contamination.

    Reconstitution

    100 µL 0.4% formic acid in water

    Mass spectrometry suitable solvent.

     

    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.

     

    Discover more about EVOLUTE AX

     

     


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