Sample Preparation Tip
Level: Intermediate
Troubleshooting SPE
This technical tip explores some issues encountered with Solid Phase Extraction and in particular to Analyte Recovery. Recovery relates to the percentage of analyte captured and retrieved through SPE. A couple of other commonly used terms relating to recovery, which may be of interest are:
Relative Recovery: Indirect determination of analyte capture and retrieval during the extraction process based on the recovery of an internal standard (which is a compound with a structure similar to the target analyte) that is added to the crude sample/matrix prior to the actual extraction procedure.
Absolute Recovery: Quantitative method for the determination of the actual amount of analyte captured and retrieved during the extraction process. Analyte recovery is based on an external standard that is added to the eluant at the end of the extraction procedure (rather than being based on an internal standard that is co-extracted with the analyte).
Symptom
Analyte recovery is less than 100%.
Clue
Analysis of the unretained fraction (void volume) indicates that the analyte is present in the “flow through”.
Problem
Analyte binding is not quantitative during the sample loading step.
| Cause | Solutions/ Suggestions |
|---|---|
| A. Column conditioning is improper, incorrect, or not optimized. | 1. Condition column with methanol or isopropanol. 2. Use sufficient methanol or isopropanol to wet the entire sorbent bed; allow more than two column volumes to percolate slowly under low vacuum. 3. Follow the methanol or isopropanol directly with one column volume of a solution with a composition similar to the actual sample (pH adjusted) but without the analyte(s). 4. Do not use too much of this second “conditioning solvent” in (3) above, or allow it to remain in the column for too long. 5. Do not over-dry the column during or after conditioning (use low vacuum for ~1 minute). |
| B. Sample/matrix is in (or contains) a solvent which is “too strong”. | 1. Dilute the sample in a “weaker” solvent/solution to promote binding (less polar for Normal Phase, more polar for Reversed Phase, less salt, buffered and/or pH adjusted for Ion-Exchange 2. Increase the sample dilution with a “weak” solvent. 3. Load less sample. 4. Increase the sorbent mass. 5. Decrease the flow rate during sample loading. 6. Use a “stronger” sorbent with a higher affinity for the analyte. 7. Add an organic modifier (or adjust the pH) to enhance binding affinity for the analyte(s). 8. Adjust the sample pH so that the analyte is neutral for Reversed Phase or charged for Ion- Exchange. 9. Add salt (5 to 10% NaCl) to increase the solvent polarity and to enhance the retention of highly polar analytes in Reversed Phase. 10. Add an ion-pair reagent to enhance binding of charged analytes in Reversed Phase. |
| C. Column “mass overload” (column is too small and/or the total mass of bound solutes/components is too large). | 1. Decrease the volume of sample loaded. 2. Increase the sorbent mass. 3. Use a sorbent with higher surface area. 4. Use a “stronger” sorbent. 5. Decrease the flow rate during loading (to enhance “diffusion”). 6. Decrease the column inner diameter but use the same sorbent mass (to increase the pressure drop, reduce flow, and enhance “diffusion”). 7. Dilute the sample in a “weaker” solvent to improve capacity. |
| D. Flow rate is too high during the sample loading step. | 1. Decrease the flow rate during loading. 2. Increase the sorbent mass. 3. Decrease the column inner diameter to reduce flow. 4. Use a sorbent with a higher surface area. |
| E. Sorbent is “too weak” (has poor or low affinity for the analyte relative to the sample/matrix and/or the dilution solvent). | 1. Switch to a “stronger” sorbent, to one with a higher ligand density, from a non-endcapped to an endcapped sorbent (or vice versa). 2. See “Solutions/Suggestions” for Cause C (“Column mass overload…”), above. 3. See Solutions/Suggestions for Cause B (“Sample/matrix is in (or contains) a solvent which is “too strong””), above. |
| F. Conditioning solvent is “washed away” with large sample volumes. | 1. Add 2% methanol or isopropanol to the sample to prevent alkyl chain collapse during the sample loading step. |
