clumn equilibration and maintenance and pressure limit

[fredj]

Hi every body
I just start working with Hypersil ODS (C18) Column and I don't have any idea about the column maintenance and the equilibration volume before each run, knowing that the volume is about 7 ml...I checked on the internet and i didn't find any information about the max pressure that the column can support. the other thing is that I am working with a mobile containing 1M of ammonium acetate 13% of ACN and I don't know wish solvent to use in order to wash my column to take out the salt trace from it.
if anyone have any idea please answer to my question
thanks

[tom jupille]

A general rule of thumb is that it takes about 10 times the column volume to get complete wash-out. Full equilibration may take longer (e.g., sometimes hours in the case of ion-pair reagents); the only way to tell for sure is to do a series of test runs with different equilibration times.

Re ammonium acetate: that is highly water soluble, so my suggestion would be water with just enough ACN to avoid dewetting of the stationary phase. Given that you are only using 13%, the easiest thing would be just 13% ACN in water.

Most silica-based packings don't really have a pressure limitation. You will blow the ferrules off the end fittings before you will hurt the packing.

[fredj]

thanks for the precious information...
do u have any idea about how can I avoid taling peak in my chromatograme knowing that the PH of my buffer is about 5.16?

[tom jupille]

Do a search of the Forum on the key word "tailing" and you will find a long list of discussions about tailing problems. Some of those may point you in the right direction.

Without knowing:

• what your analyte is (acid or base? pka?),
  what type of chromatography you're doing (reversed phase? hilic? ion-pair?),
  the organic solvent (methanol? acetonitrile?),
  the buffer (acetate? formate?),
  the buffer concentration,
  the *exact* column you are using,
  the age of the column,
  the temperature,
  what the sample is dissolved in,
  the sample concentration,
  what the injection volume is, and
  whether the tailing problem began suddenly or has been present consistently
  in addition to the pH

there is no way to make a specific suggestion. All of the above factors can contribute to tailing problems.

[fredj]

thanks for reply.
I found some informations that they may help.
thank you so much
best regards

[fredj]

Dear sir
I think that I need more your help
i will provide you all the information about my sample and my method hoping that i may help you to provide a suggestion
my analyte are porphyrins in free acid form
what type of chromatography you're doing: reversed phase
the organic solvent: acetonitrile
the buffer: ammonium acetate
the buffer concentration: 0.1 M ( normallyI have to use 1 M of ammonium acetate but because i don't know about the effect on the column i tried to start with this concentration)
the *exact* column: Hypersil ODS C18
new column.
the temperature: 40 degree Celsius
what the sample is dissolved: in 0.1 M HCL
the sample concentration: I tried different concentration where i 've seen the tailing in all the chromatograme
what the injection volume is: 20uL
the tailing problem has been present consistently
the flow is 0.9ml/min
The pH is 5.16
I hope that those information will help
best

[Durga malleswar]

U can reduse peak tailing by changing the pH and temperature or by changing the buffer concentration and if above all not worked lastly we can reduse peak tailing by adding TEA to the buffer

[fredj]

thank you sir

[fattyacid]

Dear sir
I think that I need more your help
i will provide you all the information about my sample and my method hoping that i may help you to provide a suggestion
my analyte are porphyrins in free acid form
what type of chromatography you're doing: reversed phase
the organic solvent: acetonitrile
the buffer: ammonium acetate
the buffer concentration: 0.1 M ( normallyI have to use 1 M of ammonium acetate but because i don't know about the effect on the column i tried to start with this concentration)
the *exact* column: Hypersil ODS C18
new column.
the temperature: 40 degree Celsius
what the sample is dissolved: in 0.1 M HCL
the sample concentration: I tried different concentration where i 've seen the tailing in all the chromatograme
what the injection volume is: 20uL
the tailing problem has been present consistently
the flow is 0.9ml/min
The pH is 5.16
I hope that those information will help
best

Maybe I´m a bit late but I also doing a work regarding porphyrins. I have no problem with tailing peakshapes BUT when i used HCl to dissolve the standard tube (I know that this one is suggested from the manufacturor) I got tailing and bad peakshapes. I use 6M formic acid instead and thar work awesome.

Best regards,

/Jonas