What could be the reason for this HPLC analysis?

[Peter Apps]

They would have a problem with filling the reservoir with clean WATER every day? It's just water! It's not like you're dumping out the acetonitrile! In any case, exchanging the water is more precautionary than anything. However, if the reservoir was 'topped off' with either water or methanol (as your prof posited), the only solution is to discard the contents of the reservoir and re-fill with a known liquid (either 1% MeOH or 100% water).

The water level in there was low. I refilled it today.

Hi Sandra

When you refilled, you first threw away what was in the reservoir, you did not just top it up, right ?

Peter

[bisnettrj2]

Yes, it is 4.6 mm.

Today, first, I ran 100%MeOH before doing any analysis (different conc: of the same std compound). I used 5 mins for post time. It worked out fine. Next time, I will do the same, i.e. run 100% MeOH using the same method before I do any analysis.

Thanks.

Hopefully you DID empty and refill the A reservoir, as Peter Apps asked above.

To summarize, your retention time issues have been fixed by running a system blank (an injection of Methanol) before you begin your analysis AND by adding extra equilibration time (5 minutes instead of 3) at the end of your gradient run. Correct?

if that's the case, then I do have one tip - you can get better peak shapes if you make your injection solvent weaker than your mobile phase (in terms of eluent strangth). You will need to increase your injection volume, but you should be able to inject more volume if the sample is dissolved in the weaker constituent of your mobile phase.

[Sandra7]

They would have a problem with filling the reservoir with clean WATER every day? It's just water! It's not like you're dumping out the acetonitrile! In any case, exchanging the water is more precautionary than anything. However, if the reservoir was 'topped off' with either water or methanol (as your prof posited), the only solution is to discard the contents of the reservoir and re-fill with a known liquid (either 1% MeOH or 100% water).

The water level in there was low. I refilled it today.

Hi Sandra

When you refilled, you first threw away what was in the reservoir, you did not just top it up, right ?

Peter

There was only a little but left in there and so I left it in there.

[Sandra7]

To summarize, your retention time issues have been fixed by running a system blank (an injection of Methanol) before you begin your analysis AND by adding extra equilibration time (5 minutes instead of 3) at the end of your gradient run. Correct?

Today, this same approach didn't work. I ran 5 different conc: of the same std compound (in ug/ml of 175, 150, 100, 50, 25) to do quantification of one of the 2 compounds in my unknown. In fact, the first one was run two times. Eluted earlier and ret time were differently each time. The ret time were 2.667 min and 3.665 min for the 175 ug/ml one, 4.097 min (for 150 ug/ml ), 2.671 min (for 100 ug/ml ), 3.187 min (for 50 ug/ml) and 4.159 min 9for 25 ug/ml). The 4th one (50 ug/ml)that gives ret time of 3.187 min
was initially used in unknown identification stage. Ret time at the time was 5.419 min which matched with standard mixture of 5 compounds prepared in MeOH.

Since I just needed the peak area, I can still the data but what is happening is quite disturbing. Is the reason all because post time of 5 mins is not enough? BTW, other people who used the instrument left the instrument with eluent composition that is unsuitable to my analysis for a certain time, it'd take a long time (like more than 3 hours) to get the column completely washed off from that mobile phase composition, right?