3 - UPLC_Work_Practical_Considerations

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Running U-HPLC: Practical Considerations needle VDD Sample Syringe Wash cycle after injection BSM to column and detector loop Possible contaminated area Wash block VDD Sample Syringe BSM loop Strong needle wash followed by weak needle wash Wash Solvent description • There are two wash solvents – Strong Needle Wash • Tubing flushing • Elimination of components injected • Never injected – Weak Needle Wash • Strong solvent elimination • Injected with the sample in partial loop pressure assist mode Shula Levin, Waters Israel to column and detector 24
Running U-HPLC: Practical Considerations Wash Solvent Considerations • As a general principle, strong and weak solvents should include the same organic species – This may not always be practicable, especially in the case of “sticky” samples. You may, however use a 100% organic strong wash solvent • Do not use salt buffers in wash solvents • Wash volume ratio (weak to strong) – Should be about 3:1, weak wash to strong – Sufficient to ensure the weak wash flushes the strong from the needle and sample loop • For more details on solvents, see the section titled “Selecting weak wash and strong wash solvents” in the ACQUITY Operators Guide Strong Wash Solvent • Flushes internal and external portion of the needle to prevent carryover • Typically stronger than sample and mobile phase to dissolve sample residue • Function performed in the wash station • Strong solvent should be no stronger than the concentration needed to reduce carryover to an acceptable level • Strong wash solvent does not contact the sample Shula Levin, Waters Israel 25
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