Rentention time of analyte is affected by many factors, e.g. mobile phase used, column & other parameters on the instrument. So to prevent this from happening to need to play around with the mentioned factors until u get good separation of your analyte.

I think it is possible, because the one HPLC system is not guarantee the separation of the huge of number of substances. If u will modify one parameter of the system (i.e. temperature, % of organic component, etc or even a column) those two different molecules will be eluted in the different times

Could you tell us which molecules are involved ? which method and solvents are you using ? which detector ?

Rentention time of analyte is affected by many factors, e.g. mobile phase used, column & other parameters on the instrument. So to prevent this from happening to need to play around with the mentioned factors until u get good separation of your analyte.

Its...No...or...never.There is no violation of basic principles of HPLC separation because there are a lot of factors of separation of molecules viz. mobile phase, column chemistry, temperature etc. During method development, which factor click for separation...dont know. So there will be no violation of basic principle.

I go with robin joshi what he said is absolutely correct becoz one set of combination optimized in one system with good separation but we could n't achieve it in another make( column) becoz of column chemistry ,way of packing ( tight or loose pkg) etc. So no violation of basic principles of hplc

For this you have to try different approaches, change the column first then after temperature,pH and then after you have to make gradient programme for the separationThe method you wll try with a longer run time. There is a lot of factors effects the separation n HPLC. Try these methods first.

it can occur, very common, can u tell me the structure, if both are neutral or both are acidic or basic, try to find the pKa value of both

the best shot to this problem is to change the column and the detector temperatures, I think this is possible since all seperation is governed by Van Deemetar equation and unless otherwise stated the rest can be manipulated.

Also think of making gradient elution or even derivatization of the samples.

Well, thanks everyone for your guidance. For your information tha two pair of molecules are:

A) 1. Pyridoxin HCl & 2. Phloroglucinol

both are found in pharmaceutical products and i want to seperate out them to find their peaks in the same run!

I have only Isocratic facilities :(

Please note that fundamentals or thumb rule is .....you have to exploit the difference to separate .....that difference is affected to great height through solvent combination play...which is prevalent in HPLC only as in GC mobile is nutral..

so you have fundamentals intact..

How is your sample prep? What type of column are you using ? Have you start with only injecting standard solutions of both analytes?

As a start calculate th.e octanol-water partioning (log-P) of your molecules (I do it with Chemdraw). Estimate the pKa of the main polar functional group. Do the same for two other molecules you succesfully separated with your conditions or find literature examples. Put both pairs into a log-P vs. pKa graph. This will get you started.

Two Different molecules with different physical and chemical properties......Okie for me if they elute at same time in similar analytical conditions. May be the polarity of the two be same.............if not identical................and high concentrations making it difficult to resolve them...............better to use low concentration sample......and try to reduce the flow rate as much as one can............then the two may get separated.

I had same problem observed in Nateglinide. Nateglinide related compound A and and one process impurity is found at same retention time by USP method. It isthen separed by Gradient by Changing composition.

Hi Hasan,

Under ideal conditions the elution of 2 different components at same time is rarerest or impossible. But if it had happened then there must be some instrumental error..... Please be sure that there were no error. You know i had this problem but was resolved after characterization and it was resulted to be due to 2 isomers of same compd. 2 diff compd. elution must be an error....

Maybe you can try a different mobile phase, and you can monitor two wavelengths.Good luck!

nothing strange to me. try to build retention curves for the two solutes and plot capacity factor against volume fraction of the organic modifier and see how the two curves look like: they can be linear or quadratic, parallel or convergent/divergent. These two curves can cross each other exactly at the chromatographic condition you are using.

Elution of two compounds at same retention time is not any instrumental error. But there is need to optimise HPLC method. Need to optimise mobile phase, gradient or isocratic, column flow, temp, column length, wavelength etc. There are lot of times that there can be merging of compounds, but there is need to optimise or standerdise the method.

Do not confuse urself, just optimize the separation condition by varying temperature of the column ( by column oven), change the mobile phase by adding the organic and or inorganic modifier, change the flow rate .....( this is what you can do with available facilities )

HPLC separation is all about how different molecules disrupt the interaction between water molecules and has very little to do with actual analyte structure. If the energy that is released by the binding of water molecules to the analyte is less than the energy released by water molecules binding to each other, the analyte molecule will partition into the stagnant eluent layer on the surface of the column packing material.  Organic solvent modulate this process by changing the total energy released by the water-water and water-organic solvent interaction. That is why methanol (stronger water-methanol binding energy) is a weaker organic eluent than acetonitrile (weaker water-acetonitrile binding energy). So it is all about relative energy release and very little about interaction between the analytes and column surface!