General rule of thumb—collect all fractions to help troubleshoot and method optimization
Method inconsistencies can come from:
a). Sample loading too fast
Allow sample to soak into the bed before pulling a gentle vacuum. This allows more time for analytes of interest to interact with the sorbent
b). Elution flow rate too fast
Allow time for interaction by letting elution solvent soak into the bed, for analytes to partition into the elution phase and then apply a gentle vacuum. Having a vacuum gauge will help identify what the actual vacuum pressure. Leaks from unused wells or manifold ports or tubing can lower vacuum pressure, and cause variable flow rates. Variable flow rates can be solved by using a sealing mat in the 96-well format and shutting the stopcock valve of unused manifold ports. Generally, flow rate of 1 mL/min is good.
c). Loading solvent is too strong
Solvents that provide great solubility to your analytes are likely to pull your entire sample through the cartridge with it. Use a solvent that is just right to dissolve your sample and save the stronger solvents to use for elution
d). Overloaded cartridge
Most of the overloaded portion of the sample travels down the cartridge and comes through with loading. Some overloaded analytes get washed out with washing and some are recovered in elution, resulting in an inconsistent cleanup
e). Phase collapse from drying (applies to silica-based materials only)
Sorbent chemistries can be deactivated in silica-based cartridges, resulting in a loss of retention throughout a protocol. Avoid leaving the vacuum on until after loading is complete
f). Wash solvent is too strong
A strong wash can pull off weakly bound analytes. If time allows, do a small study to optimize wash strength by applying washes with increasing percentage of the stronger modifier, i.e. 5% methanol in water to 50% methanol in 10% increments. Then analyze the degree of cleanup and analyte recoveries
g). Elution strength altered by residual solvents from previous steps
Dry cartridge on full vacuum after washing to prevent any left over solvents from diluting the strength of elution. This is important especially when the wash solvent is significantly different in polarity or strength than the elution solvent. Drying time depends on the bed mass. While a 60mg bed may take about 5 minutes to dry, a 1g bed will take up to 20 minutes to dry
h). Solvent volumes too small or large
Unnecessary large solvent volumes affect analytes/interferences binding to the sorbent and dry down time is also prolonged. Use only recommended volumes
i). Are solvents old?
Solvent strengths are likely to be altered in older solvent mixtures, especially when volatile components are involved
j). Also check your analytical method
Make sure the injection solvent is appropriate and the instrumentation is not the source of inconsistency
k). Analyte may be volatile
Analytes with similar or lower boiling points may be lost during the dry down step. For example, acetonitrile has a boiling point of 82 C, if the analyte boiling points are lower than that, they will be lost. Depending on the analyte properties, they may also have stability problems when the sample is completely dry. Monitor the drying step closely
l). Use of Internal Standard
An internal standard will help keep track of errors that can occur during the SPE method
0 Comments