Solid phase extraction (SPE): also known as liquid-solid extraction. It refers to the process in which the analyte in the liquid sample is retained on the adsorbent by adsorption and then eluted by a certain solvent.
SPE method is a separation technology which combines selectively retention and selectively elution, mainly used in separation, purification and concentration of samples. Compared with conventional liquid-liquid method, SPE improves the recovery rate of analytes and effectively separates analytes and interfering components to simplify the pretreatment process, indicating its advantages of time-saving, convenient and low-cost.
According to some frequently asked problems during the use of SPE column, they can be summarized in several aspects including low recovery rate, poor reproducibility and purification effect. So how to address these problems?
Low Recovery Rate
Possible reasons and solution
1. Insufficient retention of object substances on packing materials
Judgement method: Blank spiked solvent, recycle effluent and flushing liquid, if the content of object substances in effluent or flushing liquid exceeds 5%, that means insufficient retention.
|Inappropriate activation condition of column or with activation
|Activate SPE column according to the type of stationary phase accurately
|Column is dried up in the operating process
|Reactivate or equilibrate the column to ensure the column is not dried before finishing flushing(except SPE columns based on polymers)
|Inappropriate selection of SPE column
|Choose the column with strong retention to object substances
|Elution strength of sample solvent or flushing liquid is too strong
|Reduce the elution strength of sample solvent and flushing liquid
|Overloading and permeation caused by large solution volume of high concentration
|Reduce the sample loading or concentration, increase the packing materials
2. Incomplete elution of object substance
Judgement method: blank spiked solvent, recycle effluent and flushing liquid, if object compounds don’t appear in effluent and flushing liquid of sample or they exist in eluent of a small amount volume, that means insufficient retention.
|Stationary phase has too strong selectivity to object compounds
|Choose column which has weak retention to object compounds; Adjust the pH of eluent; Eliminate the selectivity of stationary phase to object components
|Too weak elution ability
|Choose stronger elution solvent
|Incomplete elution led by eluent solvent
|Increase the elution solvent volume
|Incomplete elution led by too fast flow rate
|Let the eluent permeates column, vacuumize the column or add pressure, then set the flow rate at 1-2ml/min
3. Other reasons
Judgement method: blank spiked solvent, recycle effluent, eluent, flushing liquid, if object compounds don’t appear in above liquids or they exist in eluent of a large amount volume with insufficient recovery rate, that means insufficient retention.
|Extration efficiency of SPE column is insufficient
|Choose the appropriate extraction solvent
|Unstable object substances dissemble or decompose
|Change extraction way or transferring way; Decrease heating temperature
|Too many steps of pretreatment or loss of object substance
|Simplify pretreatment process
|Object substances combine with impurities
|Remove impurities in an appropriate method
|Interfering impurities impact too much
|Change pretreatment method or compare with spiked substances
|Too many impurities or exceed the highest retention
|Change pretreatment method;
Reduce sample loading or increase the packing materials of column
|Complicate operation steps and operative error
|Optimize pretreatment method or make SOP method
|Complex sample matrices, poor effect of cleaning impurities
|Optimize or change the whole pretreatment method;
Add internal standard or contrast with spiked data of matrices
|Reactivate or equilibrate the column to ensure the column is not dried before finishing flushing (except SPE columns based on polymers)
|Column blockage or too fast flow rate
|The former changes the pretreatment method, the latter uses flow control valves to change flow rate.
Poor Purification Effect
- Change the pretreatment method
- Change the strength of flushing and eluent
- Choose a suitable SPE column
- Operate SPE experiment correctly
Inappropriate Flow Fate
|Reason of Slow Flow Rate
|Too small particle size
|Choose SPE column with large particle size
|After pretreatment of sample, suspension insoluble substances still exist, which leads to blockage of frit
|Freeze and centrifuge before sample loading to make sure the supernatant is clear.
|Sample viscosity is too high
|Dilute the sample
|The SPE resistance is excessive, liquids can not drop naturally
|Use manual SPE device; Control valve by regulating flow; Adjust flow rate by giving positive or reducing negative pressure
|Reason of Fast Flow Rate
|Too large particle size
|Choose SPE column with small particle size
|Flow rate is too fast when gravity works
|Use manual SPE device to adjust controlling valve; Control valve by regulating flow
|Flow rate is too fast when reducing negative pressure
|Regulating flow control valves and valves; Control vacuum gauge pressure to be constant within a certain range
Welchrom SPE Column
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