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Comments |
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Minimize pressure surges |
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Make rapid injections to reduce pressure changes during sample injection.
Maintain pumps to minimize pressure swings resulting from inconsistent flow rates. |
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Use a guard column and/or an in-line 0.5 µm filter. |
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Place each of these before the column and after the injector. An in-line
filter will catch large particulates and a guard column will prevent strongly adsorbed
materials from reaching your analytical column. |
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Frequently flush columns with a strong solvent. |
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Flushing with 100% acetonitrile is often adequate, but if stronger
solvents are needed, consider methylene chloride (CH2Cl2). Less-polar solvents, like CH2Cl2,
are strong solvents in reversed-phase chromatography. Many strong solvents are immiscible
with aqueous containing mobile phases. Remember to flush the column and HPLC system with
isopropanol prior to and after the use of CH2Cl2.
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Pretreat "dirty" samples to minimize strongly retained
components and particulates |
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Solid phase extraction, filtering sample through 0.45 µm filters, or
high-speed centrifugation are useful pretreatment techniques. |
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Use column temperatures of less than 60°C. |
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Check column manufacturer specifications. |
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Keep mobile phase pH between 3 and 7 for silica based columns. |
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If operating outside the 3 - 7 pH range, choose a column designed for low
pH (StableBond) or high pH (Eclipse XDB). |
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Add 100-200 ppm sodium azide to prevent bacterial growth. |
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Stagnant aqueous reservoirs readily generate bacterial growth which can
cause dramatic baseline disturbances and plug columns. |
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When storing columns, purge out salts and buffers. Leave column in pure
acetonitrile. |
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This prevents precipitation of buffer salts in the column. Acetonitrile
is a good storage solvent because aqueous and alcohol mobile phases can increase the rate
of stationary phase hydrolysis. |
