SPE - How to use the polymeric SPE phases of the PLEXA Family

Created by Dr. Know over 1 year ago | Last modified by Dr. Know over 1 year ago

The Bond Elut Plexa, Plexa PAX and Plexa PCX polymeric sorbents have unique technical properties that yield extremely clean extracts and retain a wide range of polar to non-polar bases and acidic compounds. Several unique applications have been developed on the Bond Elut Plexa, Plexa PAX and Plexa PCX sorbents.

Bond Elut PLEXA

Bond Elut Plexa is a polymer with a hydroxylated ligand on the surface leading to a hydrophobic core of Polystyrene Divinylbenzene with no amide functionality. The benefits are simplified methods and high sensitivity due to the removal of proteins during extraction.

General Specifications:

Functional Group:       ethylvinylbenzene/divinylbenzene with hydroxylated ligand
Particle Size:              45 µm
Pore Size:                  150 Å
pH range:                   1 – 14
Capacity:                   10 % of Sorbent Mass

General Applications:

Bases/Neutrals

Condition with 500 µL of Methanol
Wash with 500 µL of Water
Apply sample: 100 µL plasma diluted (1:3) with 2% ammonium hydroxide
Wash with 500 µL Methanol/Water (5:95)
Elute with 500 µL Methanol

Acids:

Condition with 500 µL of Methanol
Wash with 500 µL of Water
Apply sample: 100 µL plasma diluted (1:3) with 1 % aqueous formic acid
Wash with 500 µL Methanol/Water (5:95)
Elute with 500 µL Methanol

Method Optimization:

Inadequate or no retention after washing

Reduce volume of washing step
Reduce concentration of organics in the washing step
Rinse with either 2 % ammonium hydroxide for basic analytes or 1 % formic acid for acidic analytes to ensure hydrophobic interaction or increase the sorbent mass to retain more polar compounds.

Inadequate Elution:

Decrease flow to 1 mL/min.
Increase elution volume or use multiple aliquots of eluent (2 x 250 µL)
Add a modifier to the elution solvent
- Formic acid for basic analytes
- Ammonium Hydroxide for acidic analytes.

Technical Tips:

If the pKa of the analyte is < 7, use the acid method.
If the pKa of the analyte is >7, use the basic method.
Highly non-polar basic analytes can show improved recovery with the acid load method.
Sorbent may be dried after the conditioning step with no effect on results.
Avoid the following solvents as they tend to cause swelling of the Plexa polymer
- Toluene
- Dimethylsulfoxide (DMSO)
- Tetrahydrofurane (THF)
Halogenated hydrocarbons and their alcohol derivatives.
Molecules with MW > 10,000 will penetrate the pores and molecules with MW >100,000 with not
Temperature tolerance is limited by the melting point of the polypropylene materials (176 °C/348 F) used in the tubes or plates. The melting point of the sorbent polymer is 240 °C/464 F.

Applications:

Bond Elut PLEXA PCX

Bond Elut Plexa PCX is a mixed-mode ion exchanger with a hydrophobic polymer similar to Bond Elut Plexa and strong cation exchange sites that remove neutral and acidic interferences from the matrix, concentrate basic analytes and improve analytical performance for basic analytes.

General Specifications:

Functional Group: ethylvinylbenzene/divinylbenzene/benzene sulfonic acid
Particle Size: 50 µm
Pore Size: 150 Å
pH range: 1 – 14
Capacity: 1 meq/g

General Application (Plasma):

Dilute 100 µL of sample 1:3 with 2 % aqueous H₃PO₄
Condition with 500 µL of Methanol
Wash with 500 µL of Water
Load acidified sample
Wash with 500 µL of 2 % Formic Acid
Wash with 500 µL of Methanol/Acetonitrile (50:50)
Elute with 500 µL 5 % Ammonium Hydroxide in Methanol/Acetonitrile (50:50)

Application Notes:

Acidification is necessary to disrupt drug protein interaction and to adjust the pH of the ionizable basic compounds to at least 2 pH units below the pKa of the functional group. Therefore, to prepare acidified aqueous dilutents: add 20 µL of concentrated H₃PO₄ to 1 mL of water. Dilute the sample 1:3 with aqueous diluent. It may be necessary to centrifuge or filter the sample prior to SPE. Add 10 to 50 µL of a suitable internal standard.

Method Optimization:

Inadequate or no retention after washing

Reduce volume of washing step
Reduce concentration of organics in the washing step
Increase sorbent mass for increased ion exchange capacity.

Inadequate Elution:

Decrease flow to 1 mL/min.
Check solubility of analyte in the eluent
Increase strength of the elution solvent
Increase elution volume or use multiple aliquots of eluent (2 x 250 µL)

Technical Tips:

Neutralize the charge of the analyte by adjusting the pH to 2 pH units below the pKa of the analyte. Elute with a dilute base in an organic solvent that has an efficient solvent for the basic (neutralized analyte). Up to 5 % ammonium hydroxide (28 – 30 %) in solvents such as methanol and acetonitrile is recommended.

Applications:

Bond Elut PLEXA PAX

Bond Elut PLEXA PAX is a mixed-mode nonpolar polymeric anion exchanger offers high level of analyte selectivity. It is for the exclusion of endogenous interferences and offers superior cleanliness and minimizes ion suppression. There is a simple, single method for ease-of-use reduces method development time

General Specifications:

Functional Group:       ethylvinylbenzene/divinylbenzene with quaternary triethylammonium ligand
Particle Size:              45 µm
Pore Size:                  150 Å
pH range:                   1 – 14
Capacity: 0.6-0.9 meq/g

Technical Tips:

Sorbent conditioning: It is very important that the SPE sorbent bed is properly conditioned and equilibrated prior to sample load. 500 µL of MeOH followed by 500 µL of water should be employed for a 30 mg sorbent bed.

Flow Rate: Most recovery related issues in ion exchange SPE can be traced to the flow rate during sample load. Ion exchange processes can be slower than normal non-polar or polar interactions, and as such, flow rates need to be kept at a low level to reduce sample breakthrough. A flow rate of 1 mL per minute is optimal.

Sample Volume: This is very important when conducting sample clean up on biological fluids or complex matrices. For plasma we recommend that the end user does not load more than 100 µL of plasma per 30 mg bed. If more sample is added then secondary interaction with the matrix can occur and clean up performance may be compromised.

Applications:

Bond Elut PLEXA SPE Protocol

PLEXA Method Development Poster

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