The shortest possible considering you have seen all your compounds of interest, for the sake of economy. You can start column wash-up and reequilibration almost as soon as your second peak has finished eluting. (if using a gradient)

Ideally, no longer than needed. That is the true and correct answer.

If everything possible has eluted off the column (plus some additional headroom), then the analysis is finished. Some methods just can not be completed in less than an hour. Others take just a minute or two. If the method is developed properly and run well, then there is no specific time value. It depends on the method and analysis goals.

If you have not done so, please evaluate your method and data to demonstrate that the two compounds have acceptable K primes, the resolution and separation between the two meets acceptable guidelines, no unaccounted for peaks or extraneous changes occurred and the method is repeatable. *If it meets all acceptance guidelines, then ask yourself it the method can be optimized to provide the same or better high quality data in less time? If the answer is yes, will doing so make it easier and less time consuming to run the analysis?

The other considered factor is the equilibration time after last peak eluting out if you run a gradient concentration of mobile phase. You would want to make sure the column reaches back to initial concentration for good repetition.

Thank you all for the answers. The method was developed on isocratic elution mode and the system suitability parameters are within acceptable criteria.

I'm just being careful is setting up a correct run time which will ensure full detection of both analytes and their complete clearance from the system before the next injection. So, if i set the run time to be 15 min, is that reasonable?

Meeting "System suitability" objectives does not by itself insure that a quality method was developed or is being used (the same is true of a "Validated Method"). This is because those parameters are limited by the skill and wisdom of the person whom developed the method which is a variable.

There is no possible way to answer your run time question (15 minutes?). You asked when should you end the run? A: "If everything possible has eluted off the column (plus some headroom, perhaps 1.5 x the last peak's Rt), then the analysis is finished". We have no idea what your exact method is. One key danger of an isocratic method is that it does not allow you to increase the strength of the mobile phase over time to insure that everything has in fact, eluted off the column. What often happens is that other unseen components are retained, long term, on the column. This can result in artifact peaks, unidentified peaks and of course column fouling over time. All of these things may compromise the quality of the overall method and limit its use and/or accuracy. One key thing that you can do right now is to make sure you follow up each isocratic analysis with a properly developed gradient column wash step to both detect retained material (indicating your method is flawed) and wash it from the column so it does not contaminate the next analysis. Addressing these basic fundamentals helps early rather than late will reduce future problems.

• "I'm just being careful is setting up a correct run time which will ensure full detection of both analytes and their complete clearance from the system before the next injection."

If you would like to increase your chance of that happening, then, if possible consider a well designed gradient method to increase your chance of that outcome. If that is not possible, then make sure you follow up each isocratic analysis with a properly developed gradient column wash step as suggested.

Many thanks Dr William for the detailed suggestions, which are very helpful indeed. I will act accordingly.

Dear Dr William,

Based on your column washing recommendation above, pls can you suggest me a gradient program plan for the washing?

My mobile phase compose of acetonitrile and 0.001M citric acid, but I'm using methanol and distilled water for the column washing.

Nafiu: The most appropriate washing gradient to use depends on the actual method used and the sample types you are trying to dissolve/remove.

• For a more detailed answer to your question, please refer to this related Researchgate discussion thread; https://www.researchgate.net/post/Is_there_any_proper_way_in_cleaning_High-performance_Liquid_Chromatography_HPLC_column_C18

Ok, thank you Sir.

I completely agree with William. You need to ensure that nothing remains on the column after the last peak has eluted. This can be done by a gradient column wash as suggested by William. Please note that the run time can vary from sample to sample and there in no standard rule to determine that.

Dear Nafiu

15 min runtime is ok as u already devloped a method with RT 10 and 12. and you know the purity of your standards approx 100% so there may not any extra peak ahead if you go more for time to run with same mobile phase.

The only question arise is economically this method not suitable as if your total run time 15 min means there is wastage of solvent as well as column cost.

Sharad: I disagree. ALL methods must be run for a period of time AFTER the last peak has eluted to reduce missing any new compound or impurity which may have been introduced. Time is needed for the signal to return back to the baseline for both proper integration and to show that nothing else has been detected. This is standard good practice in chromatography and should be followed, esp for an isocratic method (By design, Isocratic methods are less sensitive and more prone to "miss" detecting a sample). BTW: The goal of most methods is to run samples, not certified standards. The assumption is that a sample (or samples) may contain other compounds so the method should be designed to cover a range wider than the the expected time range for the last peak. Proper HPLC method design should optimize the separation to reduce errors.

Solvent wastage would only be a concern if the method was run for an extended time (i.e. 2x last peak). Additionally, the cost of solvent is insignificant when compared to collecting accurate data.

Dear William

for which statment you disagree with me in above line?

Both.

ACC TO MY EXP.

18-20 minute Run time (RT) ll be ok if desired conpounds gave RT at 10 nd 12 min simultaneosly.

Dear William what your opinion for below points

1] If the compound is approx 100% pure no other peak (interferance ) should be there gating good baseline .

2] Economically the developed method should suitable

AS Nafiu asking

After the 12 minutes RT, there was no any peak again, its just a stable baseline so 15 min run is ok.

As i do not understood for what u disagree?

I am unable to understand your post as written (language issue).

• What does "should be there gating good baseline ." mean ????
• What does "Economically the developed method should suitable" mean ????

You support the idea that an isocratic run can be ended after 15 minutes if the last peak elutes at 12 minutes. That is what I disagree with as it ignores good HPLC method development design. Please review my earlier comments above for an explanation of why good laboratory practice requires us to extend the run far past the last peak. As you may or may not know, the Isocratic analysis technique is flawed as it limits what can be eluted off the column during the analysis so we always extend the run time out much further to increase our chances of spotting any unknown compounds (In this example, perhaps to 18-20 min, as a general guideline would be to use 1.5 x the last eluted peak's Rt, but we need more information to be sure). *This is true if the compound studied is "pure" (which if you have any practical experience, you should never assume. Few compounds are 100% pure. Different methods often show different purity values.) or impure.

Ok William

I understood the language issue. beacuse what i mention is narrated by you.

so both the things are same.