Reverse phase chromatography is based on polarity of the compounds not based on the absorbance of the compounds.

If the compounds are having same absorbance , it means that both are related substances of each other.In that case , if the compounds are not separated , increase the strength of the buffer and utilize ion pair reagent.

Srinivasu Penupothula wrote: "If the compounds are having same absorbance , it means that both are related substances of each other." ???

• That makes no sense. Having a 'similar' lambda max does not mean the compounds are related at all. Even similar spectra may not indicate related. MANY compounds show abs at 280nm (Many compounds have no absorbance too). It only means that they share one max in their spectra and no other conclusions can be made. A full spectra should be collected and compared for each compound (std)

Sonali Ghosh: The "concentration" of any compound by HPLC can not be determined until AFTER you have resolved each sample, compared it to a full calibration table for your sample. Determination 'in solution', as you propose can not be carried out until after they have been resolved apart, then checked against your calibration table for each sample. The fact that each one may show absorbance at a specific wavelength (same or different) is not an issue at all and why it is so important that you first retain, separate and resolve each compound from all others present using a proper HPLC method first.

• Once you have developed a a proper HPLC method which follows good chromatography fundamentals, then using standards, you can create individual calibration curves for each compound to make any qualitative measurements (concentration values). As it takes many years of professional experience to learn the basics of HPLC, please contact an experienced chromatographer at your school for help.

• "Modern HPLC Method Development Tips (PART I)"; https://hplctips.blogspot.com/2016/07/modern-hplc-method-development-tips.html
• "Modern HPLC Method Development Tips (PART II)"; https://hplctips.blogspot.com/2016/08/modern-hplc-method-development-tips.html

First of all you have to separate these compounds with appropriate conditions (mobile phase, stationary phase etc.)/ For choosing of conditions need to know what rind of compound you need to measuring

When faced with the challenge of separating components with similar absorbance wavelengths in HPLC, you can employ the following strategies:

1. Column selection: Choose a column with a different selectivity than the commonly used C18 column. Alternative stationary phases, such as phenyl, cyano, or embedded polar group (EPG) columns, can provide different separation mechanisms and improve resolution.

2. Gradient elution: Implement a gradient elution method where the composition of the mobile phase changes over time. By starting with a weaker solvent and gradually increasing the concentration of a stronger solvent, you can achieve better separation of closely eluting components.

3. Mobile phase pH adjustment: Adjusting the pH of the mobile phase can influence the ionization of analytes and improve separation. By manipulating the pH using buffer solutions, you can alter the retention and selectivity of the compounds, leading to improved resolution.

4. Mobile phase additives: Incorporate additives, such as salts or organic modifiers, in the mobile phase to modify the separation. These additives can affect the solubility, ionization, or interactions of the analyzers, resulting in improved resolution.

5. Temperature control: Adjusting the temperature of the column can influence the separation. By optimizing the temperature, you can enhance the efficiency and selectivity of the separation, leading to better resolution of components.

6. Derivatization: Consider derivatizing the analytics before analysis. Derivatization involves chemically modifying the compounds to create derivatives with different physicochemical properties. This technique can improve separation and detection, especially for compounds with similar absorption wavelengths.

7. Sample preparation: Focus on optimizing sample preparation techniques to reduce matrix interferences and enhance separation. Techniques such as solid-phase extraction (SPE) or sample cleanup procedures can help remove unwanted substances and improve resolution.

You are better off to change your stationary phase, your column; or switch from an HPLC with a UV based detector.

Having both compounds with a similar lambda max is a good thing. They will both give good response at this detector setting. Now it is time for you to do a solubility study and search literature for pKa values and use this information for selecting the right column. Here is a technical reference to help you select the right column and information on how to optimize the components resolutions https://www.agilent.com/cs/library/eseminars/public/Method%20Development%20by%20the%20Numbers.pdf

mobile phase , column , temperature

The gradient, column and the pH of the faces must be adjust to better separation.

I agree with doctor Ahmed

Try to run the individual standards in two separate runs. If the polarity of compounds is different, then the retention times for those compounds will be different. Using the retention time of your reference compound and UV absorption spectrum, you can identify and quantify the targeted compounds from your sample.

What are the compounds (e.g. natural products, pharmaceutical active ingredients, sugars etc.) that you are trying separate? and what HPLC column are you currently using for HPLC method development?

I am tring to separate API (active pharmaceutical ingredient) from coformers. I used C18 column for Hplc.

Initially, you run the standard sample of your mixture if you receive a different retention time. you can select the mobile phase

Hi Sonali, thank you for sharing the details of your work.

If they are very polar API’s, determine their pKa values, so you can estimate the ideal pH of your mobile phase. Polar API‘s typically separate in c18 columns and buffered mobile phase.

Try isocratic flow first and if your HPLC system is equipped with a binary pump, you can try gradient mode.

What experimental conditions or methods can be employed to induce the formation of multiple polymorphs in a cocrystal that previously exhibited various polymorphs with a specific solvent under ambient conditions, but now only forms a single polymorph with the same solvent and other solvents of varying polarity?