17 The “Form” Sample name _________________ Analyte of Interest ________________Sample size ____________________Sorbent Bed Mass _______ Cartridge volume_____Sorbent phases to try 1) _____ 2) _____ 3) _____ 4) _____1. Sample pretreatment ___________________________2. Condition column with ____ mL of _________ at ___ mL/min3. Equilibrate column with ____ mL of _________ at ___ mL/min.4. Load sample at ______ mL per minute.5. Remove interference with ____ mL of _________ at ___ L/min.Drying step _____ minutes6. Elute analyte with ____ mL of _________ at ___ mL/min.Remember to include soak step if possible!
56 Analyte BreakthroughCheck pH and ionic strength at each step of extraction procedureSelect phase offering greater interaction
57 Impact of pH Analyte loaded at pH=2 Analyte loaded at pH=7 The analyte loaded at pH=2 is well retained on the column. The analyte loaded at pH=7 bleeds through the column.At a pH=2, the surface of the silica is neutral, since the silanols are protonated (SiOH). At pH=7, the surface is negatively charged, since the silanols are ionized (SiO-). The very water soluble, negatively charged analyte is much more likely to be retained on a surface that is neutral than one that is negatively charged.
58 Selection of Bonded Phase Analyte poorly retained on C2Analyte well retained on C18(EC)Analyte well retained on C8
59 Poor Elution Select stronger elution solvent pH, ionic strength, % organicsSelect less retentive sorbentInclude a soak step
60 Solvent Strength Elution with 5% methanol in water
61 Less Retentive Sorbent Analyte completely eluted from C8Traces of analyte remain on C18(EC)
62 Soak StepThe analyte is completely eluted from the column which was soaked for one minute with the elution solvent.Some analyte remains on the column that was not soaked in the elution solvent.