I'm having a problem separating a single compound using 100% water as a mobile phase in HPLC (ODS). My compound is more polar and it is eluted within 3 minutes when I used 100% water as a mobile phase in aqueous C18 types of reversed column in HPLC. My active compound and another compound are co-eluted at near the same time (2.54 minutes and 2.75 minutes). I don't separate one compound using HPLC because of co-eluting of the two compounds. So, how can I separate my active compound or how can I extend the retention time between these compounds? My sample is only water soluble and it does not bind in HP20 resin or an SPE cartridge. Can someone share their experience regarding this?
There is a rule indicating the advantage of using a mobile phase weaker than the solvent used to pre-dissolve the extract
And do not be afraid to use solvents that could not dissolve the material as a mobile phase
Reduce the dissolving strength of the materials as they travel through the separation column to obtain a clear separation
you can tried many eluents (e.g., ACN, MeOH, EtOH, phsphate buffer pH 7, water)
It is important to known what funtional groups are in your substance. Without any others details i suggest two ODS colums in series. good luck
HILIC column are best for the separation of very polar compounds as suggested by M. Farooq but not all labs have HILIC columns. You can try Cyano or phenyl end capped C18 columns (widely used for separation of polar compounds) for the separation keeping 98-100 % aquous buffer initially for 1-1.5 min then play with the gradient accordingly. Hope this will help you.
Have you tried the simplest solution - changing the pH value of the mobile phase? There are other stationary phases better suited for separation of polar compounds such as CN, NH2 or even HILIC (as others suggested) but these phases are not nearly as robust and reproducible as a good c18 column.
On the other hand polar compounds are VERY susceptible to change of the pH value. Of course if this doesn't help you can always try using ion-par reagents. For positively charged compounds use one of the alkyl sulfonates, for negatively charged try tetrabutylammonium hydroxide. Either way you are better off than using a polar stationary phase.
Polar compounds often have an ionizable group in a molecule whose bearing charge makes the molecule even more polar and difficult to retain and separate. One of the limitations of reverse-phase C 18 columns is lack of retention of highly polar compounds on conventional stationary phases. To overcome these limitation, new generation HPLC columns consisting mixed-mode stationary phases that provide multiple types of interactions with analytes suitable for separations of polar and non-polar compounds at both analytical and preparative scales in isocratic and gradient modes. These stationary phases allow for a great degree of flexibility in the separation of a broad range of analytes on one stationary phase platform using simple mobile phases that are compatible with multiple detection modes. Ionizable compounds interact with the stationary phase by reverse-phase, ion-exchange or ion-exclusion mechanisms. The amount of the acid in the mobile phase influences the retention attributed to the ion-exchange interaction to the same degree as the organic modifier affects the retention in reverse-phase separation. Thus, the amounts of organic and acidic modifiers are both important for control of retention of ionizable analytes. In addition to hydrophobic interactions, neutral compounds participate in different polar interactions with highly polar column functional groups. The behavior of polar groups can be modified by varying the mobile phase. Basic functional groups on such column form salts with different acid residues (sulfate, perchlorate, trifluoroacetate, etc.), and each salt participates differently in polar interaction with neutral analytes. Analytes themselves can be ionized in many ways depending on the pH of the mobile phase, and retention time of your compounds can also be substantially altered by changing the pH of the mobile phase. All these properties offer multiple ways to adjust selectivity of the column. If two or more peaks are not resolved with traditional reverse phase technology, the mixed-mode column can be a powerful tool to find optimum separation conditions.
You can try mixture of water/aqueous buffer and organics solvent(soluble in water-Methanol/acetonitrile) as a mobile phase in a ratio of 90:10 or 95:5 for elution...And as suggested above also try for pH change of mobile phase..You can try gradient elution between 90:10 (aqueous:organic) to 100% aqueous...
Good luck..
Dr Mohammad Nazrul Islam Bhuiyan
I have exactly the same problem, please i would like to know how did you do to resolve this problem. Thanks
There is a rule indicating the advantage of using a mobile phase weaker than the solvent used to pre-dissolve the extract
And do not be afraid to use solvents that could not dissolve the material as a mobile phase
Reduce the dissolving strength of the materials as they travel through the separation column to obtain a clear separation
you can tried many eluents (e.g., ACN, MeOH, EtOH, phsphate buffer pH 7, water)
HILIC is a good technique if you have the time, money, and or the experience. Derivatization will allow you to use your reverse phase columns.
Please see the following link, it will definitely help you out:
https://www.waters.com/webassets/cms/library/docs/wa20756.pdf
There is not enough information to fix the issue here. It is like you ask me how to buy a thing in the store. There are many polar compounds in the world and if you want to try the methods offer by Waters or others such as HILIC, embedded polar reversed-phase column or even ion-exchange mode, it will be just a trial-and-error or try it and see what will work. It will be helpful if you can tell us the name of the compounds or the structures so we can narrow down the solution. What kind of detector you will use is also important to formulate the mobile phase.
If you don't have resources to try another column, I suggest you try change pH of mobile phase or use ion pairing. The C-18 column (RP-HPLC) is made preferentially to separation of sligthly polar compounds.
Hope this help
The best solution for your problem is using a HILIC column. There is a process known as de-wetting for RP columns when you use only water as a mobile phase. If you use water in RP separation you will have this problem and therefore no retention of your compounds. HILIC columns is used to separate very polar compounds like yours.
Hope this help
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