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What is HPLC Equilibration?

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What is HPLC Equilibration?

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The column must be equilibrated, re-equilibrated to the initial high aqueous solvent composition before another analysis can be performed. Normally this re-equilibration is stuck onto the end of the gradient. How much equilibration time is enough? As a rule of thumb we give 20 minutes. In reality it depends on the column length, flow rate and the hydrophobicity of your peptides. Some chromatographers use 10 minutes as their standard equilibration time. Equilibration is all about fitness of purpose. You should determine the the equilibration time experimentally, the criteria will be, does my analyte really stick to the column and chromatograph appropriately and reproducibly with subsequent analyses. If you choose to do this part of the method development you will undoubtedly be rewarded with improved chromatography and better cycle time. Should I Control Column Temperature? Yes. Scientists are control freaks. If you can control a variable, control it! Actually if you are performing auto

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