increased organic concentration in buffer - no change in RT

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Hello all,

I am working in reversed phase HPLC using an electrochemical detector. My column is a C18 5um 4.6x250mm. I am working on characterizing peroxides in HPLC and last week when I ran my sample in 25% ACN buffer and then in 60% ACN buffer: I got the same retention time (2.5 minutes which is too short, I would like to lengthen its retention time). This week I thought I would try a new column: C8 5um 4.6x150mm. But I am curious as to why this has happened? Is trying this new C8 column a good idea?
What is your flow? Maybe your compound elute unretained in botg cases?
Flow rate is 1.00mL/min. Should I decrease flow rate to encourage partitioning into the column?
No, you should lower acn. Flow question wast just for my understanding.i think your compounds elite unretained. Dus you check with certificate that came with your column?
Yes, it is an Agilent column that has been working fine for the rest of our group. Perhaps it is the analyte. It is a c10 hydroperoxide so it doesn't make much sense that it would elute so quickly.
First of all, when going to C8 from C18 your RT is nearly have of the C18. Decrease the ACN or if RT is in the dead volume you can change the separation mode or try to change your buffer pH.
Gerhard Kratz, Kratz_Gerhard@web.de
Want i ment is did you check t0 on the certificate
You can estimate the t0 anyway; work out the cross sectional area of the column in mm^2 using pi-r^2, and multiply by length (in mm) to get the volume in uL. Probably only about 60% of this volume is accessible to solvent. All HPLC systems will also have a dead volume of tubing etc. between the autosampler and the detector, which you'll have to guess. Add this to the column volume and divide by flow.
A 250*4.6mm column has a volume accessible to solvent of about 2.5mL, so if you're using a column like this on a system with not too much tubing and 1mL per min flow-rate, then a retention time of 2.5min means the compound isn't retained at all, and any increase in acetonitrile percentage will make no difference. You need to decrease the percentage until you have retention. If you have no idea what percentage is appropriate, one option is to run a gradient from about 3% up to your current percentage and see what happens (I don't recommend 0% because some columns don't like running in 100% aqueous and will "de-wet", temporarily losing the ability to interact with anything, and requiring a long purge in 100% organic to recover).
please can you share some more info like your components to be separated , your buffer pH , from your column length 25 cm , C18 stationary phase , 2.5 min retention i think it may be unretained peak especially as you changed % of the organic solvent its retention didnt change which mean it may be your t0 , from my experience , with this column dimentions usually t0 is around 2.5 min with flow rate 1 mL/min , please check your capacity factor , usually it can be calculated automatically through your syste software
Can someone please tell me if this impact RF...
I am new to HPLC and had to equilibrate Waters c18 3.5x150 column for the first time
Mobile phase A is 20mM ammonium acetate
Mob B is 100%Acn
I have equilibrated column with mob B at the flow rate 0.1 ml a min for 12 h with required t of the column
Then ranked it up to0.6 slowly changing flow every 20 min or so
Then introduced mob A at 40%
Then changed to 95% as required
Could someone tell me if this ok?my RF is a bit on a low side
Thanks
Hi Anka,

It may be that the reason no one has yet responded to your query is that a new thread could have been started with your new question as the topic...just my opinion.

Possibly, the procedure you followed could have lessened the retention of your analytes. A bit more information would confirm this; the time you spent running at an eluent composition of 95%A:5%B (was the flow rate still 0.6 mL/min?) before making injections.

Also, as a check, were the column dimensions 4.6mm x 150mm?
MattM
Hi Matt thank you for ur reply
Yes I tried as a new post but wasn’t successful.
The 95/5 was running for about 30 min and the column is 3.5 by 150
Very well, Anka,

Interesting. The estimated column volume for 3.5mm x 150mm is about 0.98 mL. If this volume is swept with eluent at a rate of 0.6 mL/min, about 16.4 minutes of time would be required to provide 10 column-volumes of eluent.

Normally this would be sufficient to begin a run with adequate column equilibration. That is to say, 30 minutes of equilibration should have been more than enough time to remove excess acetonitrile from the column.

Other possibilities for the decreased retention may be looked into.
MattM
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