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    Go to Blog What are the 5 steps of SPE?

    What are the 5 steps of SPE?

    Amber Cain

    Understanding the properties of different SPE sorbents can be confusing, refer to selecting sorbent chemistries for a refresher. But what happens at each step of these different SPE sorbents specifically? We have created a simplified table for a reference of what happens in each step of your SPE.

    Steps ABN CX WCX AX WAX
    Pretreatment Dilute viscosity and promote retention for ionizable molecules by adjusting pH to neutralize functional groups. Maximize analyte ionization of basic functional groups by decreasing pH two units lower than the pKa. Disrupt or eliminate protein binding by diluting with 2% formic acid.   Because strong bases cannot be completely neutralized, we focus on controlling the charge of the sorbent. Maximize sorbent ionization by pretreating with aqueous + strong basic buffer, decreasing 2 units below pKa. The sorbent will be 100% ionized.  Maximize analyte ionization of acidic functional groups by increasing pH two units higher than the pKa. Disrupt or eliminate protein binding by diluting with 2% formic acid.    Because strong acids cannot be completely neutralized, we focus on controlling the charge of the sorbent. Maximize sorbent ionization by pretreating with aqueous + strong basic buffer, increasing 2 units above pKa. The sorbent will be 100% ionized.
    Condition Prepare SPE for an even flow through Prepare SPE for an even flow through Prepare SPE for an even flow through Prepare SPE for an even flow through Prepare SPE for an even flow through
    Equilibration Prepare SPE for an aqueous solution and promote retention by maintaining pre-treatment conditions. Prepare SPE for an aqueous solution and promote retention by maintaining pre-treatment conditions. Prepare SPE for an aqueous solution and promote retention by maintaining pre-treatment conditions. Prepare SPE for an aqueous solution and promote retention by maintaining pre-treatment conditions. Prepare SPE for an aqueous solution and promote retention by maintaining pre-treatment conditions.
    Load

    Load neutral analyte 

    ·hydrophobic (non-polar) interactions take place in sorbent

    ·Large proteins cannot enter the pores of the sorbent and pass straight through.

    Load a positively charged analyte to a negatively charged sorbent creating an ionic interaction 

    ·Hydrophobic (non-polar) interactions take place in sorbent    -Large proteins cannot enter the pores of the sorbent and pass straight through.

    Load a positively charged analyte to a negatively charged sorbent creating an ionic interaction retaining analytes. 

    ·Hydrophobic (non-polar) interactions take place in sorbent 

    ·Large proteins cannot enter the pores of the sorbent and pass straight through.

    Load a negatively charged analyte onto a positively charged sorbent creating an ionic interaction. 

    ·Hydrophobic (non-polar) interactions take place in sorbent    

    ·Large proteins cannot enter the pores of the sorbent and pass straight through.

    Load a negatively charged analyte to a positively charged sorbent creating an ionic interaction retaining analytes.

    ·Hydrophobic (non-polar) interactions take place in sorbent   

    ·Large proteins cannot enter the pores of the sorbent and pass straight through.
    Wash 1 Mostly aqueous (5% methanol): removes polar (water soluble) interferences such as salts, small proteins, and some phospholipids. Aqueous with pH 6 to maintain charge: removes polar (water soluble) interferences such as salts, small proteins, and some phospholipids

    Aqueous with pH 10 >: the high pH prevents loss of sorbent charge.

    ·Aqueous wash removes polar (water soluble) interferences such as salts, small proteins, and some phospholipids.

    Mostly aqueous (5% methanol) with a neutral pH will recharge your analyte if the pKa is equal to or less than 5, creating an ionic interaction. 

    ·This will remove polar (water soluble) interferences such as salts, small proteins, and some phospholipids.

    Aqueous with acidic pH: the low pH prevents loss of sorbent charge.

    ·Aqueous wash removes polar (water soluble) interferences such as salts, small proteins, and some phospholipids.
    Wash 2 N/A

    Organic: Analyte is retained by cation exchange interactions. 

    ·Organic wash breaks hydrophobic (non-polar) interactions and removes remaining phospholipids, and neutral and acidic interferences.

    Organic: Analyte is retained by cation exchange interactions.   

    ·Organic wash breaks hydrophobic (non-polar) interactions and removes remaining phospholipids along with neutral, acidic, and weakly basic interferences.

    Organic: Analyte is retained by anion exchange interactions. 

    ·Organic wash breaks hydrophobic (non-polar) interactions and removes remaining phospholipids along with neutral and basic interferences

    Organic: Analyte is retained by anion exchange interactions.   

    ·Organic wash breaks hydrophobic (non-polar) interactions and removes remaining phospholipids along with neutral and basic interferences.
    Elution Organic. Elutes the analytes by breaking hydrophobic interactions.

    Organic with basic pH (95/5): Combination of organic (to break hydrophobic interactions) and basic pH (to neutralize the analyte, breaking the ionic interaction) releases the analyte from the sorbent.   

    ·Refer to the pH rule used in the pretreatment; pH needs to be more basic than the pKa of the sample by 2 units.

    		 Organic with acidic pH. Combination of organic (to break hydrophobic interactions) and acidic pH (to neutralize the sorbent, breaking the ionic interaction) releases the analyte from the sorbent.     

    Organic with acidic pH. Combination of organic (to break hydrophobic interactions) and acidic pH (to neutralize the analyte, breaking the ionic interaction) releases the analyte from the sorbent.

    ·Refer to the pH rule used in the pretreatment; pH needs to be more basic than the pKa of the sample by 2 units.

    		 Organic with highly basic pH.            Combination of organic (to break hydrophobic interactions) and basic pH (to neutralize the sorbent, breaking the ionic interaction) releases the analyte from the sorbent.  

     

    Understanding the goals of your pretreatment, your wash 1 vs your wash 2 will help you determine which solvents are best for your application.

    To read more about practical considerations for SPE method development , please download our Quickstart Guide to SPE linked below.

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