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HPLC mobile phase preperation? - (Oct/11/2011 )

I've to prepare a mobile phase consisting of methanol:water:acetic acid 250:750:3 v/v/v).
If the acetic acid wasn't present I'd usually make it up the following way; 250:750. 250ml methanol in cylinder and add 750ml of h20 and mix. I've been told this way can be inaccurate but don't know any other way any help would be appreciated. Im confused about the acetic acid if all was added together in would be 1003ml ?? or am i wrong can any one explain please.


You have given ratios so final volume is not important.

Normally I would to this like that: 250 mL of methanol measured with 250mL cylinder, 750 mL of water measured with 1000mL cylinder (or 500mL + 250mL if you want to be bit more precise), 3 mL of acetic acid measured with 1000 uL pipette (3x).

If extra precision is required I would use volumetric flasks - 250mL of methanol in 250mL flask, transfer to 1L flask, fill up with water, add 3 mL of acetic acid (3x 1000 uL pipette).


also, keep in mind that 250ml of methanol added to 750ml of water will not equal 1000ml (try it).


Thanks for the advice. I ended up needing 500ml of mobile phase so i ended up doing the following 250/1003 x 500 = 124.6ml Methanol and the same for the other ratios but changing the value for water and acetic acid. But thanks for the reply all seemed to work ok :)