Often the question arises asking how can I extract both acids and bases with the same Supported Liquid Extraction (SLE) procedure. Is this possible? And how?
The whole idea of pre-treating samples for SLE is that you want to have the analyte of interest in a neutral form. When an analyte is in its neutral form it becomes more hydrophobic and will better diffuse into the non-polar extraction solvent used in the SLE extraction step. It is helpful to remember that SLE works on the same principle as liquid – liquid extraction (LLE) most of us did in organic chemistry.
"ISOLUTE SLE+ is an excellent option - it allows us to perform liquid-liquid extractions that yield cleaner samples and require less labor" says Mitsuhiko Kawabata at Shin Nippon Biomedical Laboratories in Wakayama, who uses ISOLUTE® SLE+ to prepare biological samples for pharmacokinetic testing.
The traditional method is to pre-treat your analyte to allow it to exist in its neutral charge state. Acidic compounds are treated with an acid to bring the pH ≥ 2 units below the analyte’s pKa. While with a basic analyte, you would pH ≥ 2 units above the analyte’s pKa where it will be neutral and no longer possess an ionic charge.
Compounds that are non-ionizable usually do not require pre-treatment, although they often benefit by dilution with water to reduce the viscosity. Other pre-treatments may be required if protein binding is an issue.
Below are two individual strategies that can be used to extract both acids and bases in the same procedure.
Load aqueous sample (~pH 5) (50% of total well/column capacity). Elute with first aliquot of elution solvent. Add 10 µL conc. Ammonium hydroxide and wait 5-minutes, then elute with second aliquot of elution solvent.
Increase sample pH using ammonium hydroxide (1-2%), load sample, then elute using solvent containing up to 1% (v/v) trifluoroacetic acid (TFA). Evaporate to dryness (>40°C) to remove any traces of TFA and avoid any ion pair effect in subsequent chromatography.
Do you want to know more about sample preparation techniques? We have prepared for you plenty of interesting material.