Well there is no restriction for RT length of any analyte in RP-HPLC, however mostly suitable RT is within 15-20 minutes, particularly in industrial analysis, where maximum work is to be done in short time.

In research Labs there are works where RT has been stretched upto 250 minutes, so it all depends on your needs and your method of analysis, however the shortest RT should not be less than 2 minutes (though even less than 1 minute RT is also found)

I hope I have cleared your query!

In my opinion, in order to finish your experiment without any doubt, you might run you HPLC for at least 60 minutes by increasing the percentage of organic solvent. For example, if your mobile phase are Water and Acetonitrile, then you might increase the percentage of Acetonitrile from (10-100 %).

It depends on variable factors, mobile phase ratio as "Ly Na" stated, flow rate, number of peak you seek, instrument, its pressure, columns size(s) ett. In other word the method that you wanna use determines the RT. More than 60 and less than 3-4 min is not preferable.

Thank u all..Usually am running for maximum of 60 minutes...but then I moved to preview run for stabilizing column, getting some peaks in longer RT's..Is this any residues of my prevoius samples? Am getting confused bcz am not following any prevoius method, am trying to standardise new methods...

It seems that the behaviour you described might be sample dependent. Could you try the procedure Ly Na suggested in his comment, increasing the content of polar organic modifier?

How long after switching to equilibration step you see the additional peaks? And if possible, could you please specify the properties of your sample (known mixture, plant extract, cell lysate)?

Hello, All the experts have given there opinions, i just want to add small information on the same.

As rightly said by others there is no maximum limit of RT in HPLC. Depending on application of your method like, for dissolution studies, assay, related substance determination or study of degradation products you may have to decrease or increase the retention time. Depending on nature of your sample you can modify the polarity or your mobile phase.

In case when you have analytes of different polarities you can always choose gradient program and you can set your gradient accordingly so that in short time span you can separate all the components.

Ideally selection of retention time will depend on for which analysis you are using developed HPLC method.

thank u all..actually am doing it in plant extract..i got some more ideas through this discussion forums..Today i incresed the polarity, and got some better results..How we can chose buffers as mobile phases?

Hello,

Selection of Buffer is very crucial in case of HPLC. Whenever you want to choose any buffer for RP-HPLC analysis you must know what is the Pka of your drug or analyte which you want to analyse. if you know Pka accordingly you can select pH of your buffer.

If you want to analyse your samples by LC-MS then you must choose buffer which is LC-MS compatible, like acetate, formate etc.

So if possible try to find out what is Pka of your analyte it will surely help you selecting appropriate buffer.

It should be as low as possible but should not compromise with resolution for single component 5 min, two component 10 min and if three 15 min. But this is only for reference because every time it is not possible to set minimum.

Thank u Omkar Sherikar. If I knw the pKa of analyte, should I adjust the pH of buffer alone or the total volume pH of mobile phase?

Hello Mam,

Ideally its a practice which is most commonly followed, always adjust the pH of buffer i.e. aqueous buffer phase, and then you can mix the organic component if you are running samples in isocratic mode. If you are using gradient mode then you can keep buffer in one port and organic in another port and then you can run your gradient.

what is the pKa of your analyte ? if you want i can suggest some buffers for the same.

Hope i have answered your query.

If you go with isocratic mode just add a gradient up to 100% of organics as final step of the run. First, you can follow if any peaks appear at this stage, thus make a decision wheather the run should be extended. Second, you can make sure that the column was flushed enough and is ready to start a new run.

Am expecting some imidazole derivatives (amino carboxy vinyl imidazole)...I followed some published methods for imidazole compounds..but didnt get any peak within 60 RT..And in column stabilisation running time, am getting some peaks in longer RT's..so I came in to this discussion forum..

And when column get stabilized (this can clear from preview run), starts new run..

just try the elution with the elevated column temperature i.e. >55 degree centi. It might be helps to elute the sample from the column....

You have to add some modifiers like trietheyl amine or like that because your componet are very basic they are attached to the silinol groups in the column so to elute such component you have to use large component of organic phase and little aq. Phase. And few drops of triethylamine. Your probable mobile phase will be like methanol: acetonitrile and buffer and triethylamine (60:40:10:0.1) and flow rate of 1.5ml/min.

Hello,

AS you told there is no elution of peak till 60 min. Major reason may be due to non polarity of analyte which is associated with the structure.

Best way to elute your analyte is by increasing the ratio of strong solvent, i.e. Acetonitrile. try ACN/ water or ACN/Buffer in the ratio of 90/10. v/v

As ACN is strong solvent it will help to elute analyte very fast. Additionally due to higher % of ACN you will get very sharp peak.

Still if you see some tailing in peak , then you can add peak modifiers such as triethylamine (TEA) or ammonia in 0.1% - 0.5% concentration.

I think this combination of mobile phase will work.

it seems that the most of compounds of your plant extracts are nonpolar, then u can use much of the Organic content in the mobile phase if after that u r not getting gud result increase the flow u can add some sort of THF also , if u r facing problems till then use some mobile phase modifiers like sulphonic acid salts they r gud options ..

first let me know weather after 60 min how peaks are what is MP conc . and flow .

hi Rajat...Am usually running samples upto 60 RT..For example, if i use MeOH :ACN as mobile phases, thn runs for 60 mnts, thn stabilise the column with the same mobile phase (some cases) for next sample...During this time we should check preview run for knowing whether the column get stabilised or not.. here, in preview run, am getting some peaks...Or if I change the mobile phase, checking preview run, again getting some sharp or broad peaks..(sure, it from the previous samples??)..And if we tried those mobile phase for the same samples, didnt get such a peak..

As per your details i observed that the compounds are having very high retention so this shows tht the compounds are highly nonpolar in this case first change the methanol with buffer of similar pka as of ur compound which is needed and have higher ratio of ACN with buffer use it in the columns have less carbon load Like BDS .

if after tht u will not get the proper result then i m pretty much sure tht you have to move for Normal phase rather thn Reverse phase . and for ur concern about different MP as u have tried it is clear tht compounds are so much retained in column and it require very high run time which is not gud for analysis.

I hope this will help u some what ..