Common Problems, Reasons and Solutions for Solid Phase Extraction Cartridges
Solid phase extraction cartridges are a widely used and increasingly popular sample preparation technique. Most are used to process liquid samples, extract, concentrate and purify the semi-volatile and non-volatile compounds; it can also be used for solid samples, but the solid samples must be processed into a liquid first.
In recent years, the application of solid phase extraction cartridges has been rapidly developed and has been widely used in food, environmental, pharmaceutical, and other industries, becoming one of the effective means of sample pretreatment and purification. Hawach supply Reversed Phase C18 SPE Cartridges, Ion-Exchange SCX SPE Cartridges, Ion-Exchange SAX SPE Cartridges, and so on for you to choose from.
But some problems often occur and HAWACH has made a table for your better understanding of the reasons and solutions for such problems.
|When extracting with a reversed-phase SPE column, there is water in the eluted fraction.||1. The SPE column did not dry well before the analyte was eluted.||1. Dry the SPE column with nitrogen or air: Use 20-100 microliters of water containing 60-90% methanol to remove the residual moisture on the SPE column.|
|The final fraction contains interferences.||1. The interfering substance and the analyte are eluted at the same time.|
2. The interference comes from the SPE column.
|1. Before eluting the analyte, use a medium-polarity solvent to wash the interference out of the SPE column. Two or more compatible solvents can be mixed to achieve different polarities.|
Use SPE columns with greater affinity for analytes and low affinity for interferences. Use two SPE columns of different polarity to remove interferences. Such as reverse phase column and then ion exchange column or silica gel column.
2. Wash the SPE column with the elution solvent before the column pretreatment.
|The flow rate of the SPE column is reduced or blocked.||1. There are too many particles in the sample.|
2. The viscosity of the sample solution is too high.
|1. Filter or centrifuge the sample.|
2. Dilute the sample with solvent.
|A reversed-phase column is used to extract non-polar analytes from solid samples.||1. The analyte is not in the liquid solution.||1. Homogenize the sample with methanol, isopropanol or acetonitrile. Then filter or centrifuge, and then dilute the clear liquid with water into an aqueous solution with a water content of 70-90%.|
|A normal phase column is used to extract the analyte from the solid sample.||1. The analyte is not in the liquid solution.||1. Homogenize the slurry with a non-polar solvent (such as n-hexane, petroleum ether, chloroform, etc.).|
|A normal phase column is used to extract the analyte from the fat sample.||1. Fat can be eluted together with the analyte or reduce the adsorption capacity of the SPE column.||1. Use n-hexane to dissolve fat. Freeze to remove condensed fat.|
|The analyte is extracted from the protein-containing solution (blood, serum, plasma) with a reversed-phase column.||1. The analyte is bound to the protein so that the analyte passes through the SPE column without being retained.||1. Break the protein bond by changing the pH value of the sample or diluting the sample with water.|
2. Add acid to remove protein (such as HCIO4, TFA, TCA).
3. Add organic solvent to remove protein (such as acetonitrile, acetone or methanol). Centrifuge, and then dilute the supernatant with water or buffer solution to an organic solvent content of less than 10%.
|The analyte is extracted from the solution containing the surfactant.||1. Surfactant interacts with the surface of the SPE column.||1. If the analyte is in a non-ionic state, an ion exchange column can be used to remove the surfactant ions.|
2. Use a glycol-based column to remove non-ionized surfactants.
|The recovery rate of protein extraction with conventional columns is low.||1. The protein volume is too large to enter the micropores of the extraction column.|
2. The protein is irreversibly adsorbed on the reversed-phase SPE column. The protein is denatured in the micropores of the SPE column support.
|1. Use a large aperture reverse phase column or ion exchange column.|
|The recovery rate of the analyte is low, and it is adsorbed on the extraction column (for example, the analyte passes through the SPE column together with the base fluid)||1. The SPE column is not well pretreated.|
2. The polarity of the SPE column is inappropriate.
3. The affinity of the analyte to the sample solution is far greater than the affinity to the SPE column.
4. As a large-volume water sample.
5. When passing through the SPE column, the reversed-phase column support loses the methanol left during the column pretreatment.
|1. Reversed phase column: Treat the column with methanol, isopropanol, or acetonitrile, and then treat the column with the solvent that dilutes the sample. Take care not to allow the SPE column to dry out.|
2. Choose an SPE column that has obvious selectivity for the analyte.
3. Changing the polarity or the pH of the sample solution reduces the affinity of the analyte in the sample solution.
4. Add 1-2% methanol or isopropanol or acetonitrile to the sample solution.
|Low analyte recovery|
The analyte is not eluted out of the SPE column
|1. The polarity of the SPE column is inappropriate.|
2. The elution solvent is not strong enough to elute the analyte from the SPE column.
3. The elution solvent volume is too small
4. The analyte is irreversibly adsorbed on the SPE support. The carrier-analyte force is too strong.
|1. Choose other SPE columns with low polarity or weak selectivity.|
2. Change the pH value of the elution solvent to increase its affinity for the analyte.
3. Increase the solvent volume.
4. Reverse phase: Choose a carrier with weak hydrophobicity. If the original C18 is used, change it to C8, C2 or CN.
Cation exchange: Substituting carboxylic acid for benzenesulfonic acid.
Anion exchange: Use primary and secondary amines instead of tertiary amines.
|Poor extraction reproducibility||1. The SPE column has dried up before adding the sample.|
2. Excessive capacity of SPE column.
3. The sample flow rate through the column is too fast.
4. The flow rate of the eluent is too fast.
5. The solubility of the analyte in the sample is too large, and the analyte passes through the column at the same time as the sample when the sample passes through the column without being retained.
6. The SPE column is treated with a polar solvent and the elution solvent is an incompatible non-polar solvent.
7. The solvent used to wash impurities is too strong, and part of the analytes and impurities are washed from the SPE column at the same time. How much analyte is lost in this step depends on the flow rate of the wash solvent, the characteristics of the SPE, and the volume of the wash solvent.
8. The volume of the eluent is too small.
|1. Perform SPE column pretreatment again.|
2. Reduce the amount of sample or choose a large-capacity column.
3. Reduce the flow rate. In particular, the flow rate during ion exchange should be less than 5 mL/min.
4. Allow the eluent to permeate through the column before applying external force. Two 500 μl elutions may be more effective than one 1000 μl elution.
5. Change the solubility of the analyte by changing the polarity or pH of the sample.
6. Dry the SPE column before using non-polar solvents.
7. Reduce the strength of the washing solvent.
8. Increase elution