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PRE-TREAT THE SAMPLE Sample pre-treatment enhances analyte retention. Use the following chart to help determine what pre-treatment may be required: AQUEOUS MATRIX Type of Analyte Type of Sorbent Pre-treatment Neutral or Neutralized Hydrophobic (e.g., C2, C8, C18, ENV+) Add 0.5 to 1 % organic solvent such as methanol to samples having volumes >100 mL. If analyte is ionisable, adjust sample pH to 2 units above pK a for bases and 2 units below pK a for acids. Cationic Anionic Cation exchanger (CBA, SCX, C2, SCX-2) Anion exchanger (NH2, SAX, PSA, PE-AX) Adjust pH with buffer to ensure charge on analyte (2 pH units below analyte pK a ). IMPORTANT NOTES: 1. If CBA phase is used, pH must not be below 7. For C2 phase, pH must not be below 5. 2. The ionic strength of the sample must not exceed 50mM for singly charged cation, or 100 mM for doubly charged cation. Samples with a high ionic strength (e.g. urine) must be diluted. 3. An appropriate buffer should be selected that will not compete with the analyte of interest. The following series lists ions on the left that will displace ions on their right: Ba + >Ag + >Ca 2+ >Zn 2+ >K + >NH 4 + >H + >Li + Adjust pH with buffer to ensure charge on analyte (2 pH units above analyte pK a ). IMPORTANT NOTES: 1. If NH2 phase is used, sample pH must not be above 7.8. 2. The ionic strength of the sample must not exceed 50mM for singly charged anion, or 100 mM for doubly charged anion. Samples with a high ionic strength (e.g. urine) must be diluted. 3. An appropriate buffer should be selected that will not compete with the analyte of interest. The following series lists ions on the right that will displace ions on their left: OH - > acetate> formate> HPO 4 - >HCO 3 - >Cl - > HSO 3 - > Citrate © 2002 Argonaut Technologies, now a Biotage company All rights reserved. 6
HOW TO CONDITION AND EQUILIBRATE THE COLUMN The appropriate solvents and/or buffers should be used to ensure that the sorbent is active. Use Chart A to help in choosing column conditioning and column equilibration solvents and Chart B for determining volumes and flow rates. CHART A Type of Analyte Type of Sorbent Column conditioning/Column equilibration Neutral Hydrophobic (C2, C8, C18) Condition column with methanol. Displace excess methanol by rinsing with reagent water buffered to pH of sample. Neutral Cation or Anion Hydrophobic (ENV+) Cation exchanger or Anion exchanger IMPORTANT NOTE: If ionisable species are being neutralized for a hydrophobic extraction, reagent water rinse should be followed with a buffer rinse at the pH used for sample pretreatment. Not required unless: ionisable species are being neutralized for a hydrophobic extraction. Then use a buffer rinse at the pH used for sample pretreatment. The buffer concentration should be between 10-50 mM to control ionic strength. Condition column with methanol. Rinse with reagent water followed by a buffer rinse at the pH used for sample pre-treatment. The buffer concentration should be between 10-50 mM to control ionic strength. Type of Analyte Silica Based ENV+ CHART B Bed Mass Volumes, mL Flow rates, mL/min 25 mg 0.1-1 0.25-2 50 mg 0.25-1 0.5-2 100 mg 0.5-2 1-4 130 mg 1-2 2-4 200 mg 1-2 2-4 500 mg 3-5 6-10 1 g 5-10 10-20 25 mg 0.5-4 1-8 200 mg 4-8 8-16 IMPORTANT NOTE: The higher flow rates listed here should be used with the greater conditioning and rinse volumes given. © 2002 Argonaut Technologies, now a Biotage company All rights reserved. 7
Page 2 and 3: STEP BY STEP GUIDE TO SPE METHOD DE
Page 4 and 5: SELECT A MECHANISM (given in italic
Page 8 and 9: HOW SHOULD THE SAMPLE BE LOADED? Th
Page 10 and 11: IS A DRYING STEP REQUIRED? Often a

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