We are working with a morphine derivative and are observing some peculiar chromatography. There is excessive peak fronting for approximately a minute (sometimes more or less depending on the % organic) followed by a sharp upward slope, a sharp downward slope, and a full return to the baseline. There is not any tailing on the backside of the peak. Our sample concentration is 80ug/mL with a 10uL injection volume. We have diluted the sample (1:10) and injected only 1uL but this did not change the symmetry of the peak. We have tried several temperatures as well. We have also tried different columns, e.g., CN, C18, C8, Amide, XTerra RP, ether-linked Phenyl, C8 with an ion-pairing reagent but nothing seems to help. Our MP is Buffer/ACN with only 5% - 10% ACN. We have tried phosphate, acetate at pH 3.0, 4.0, and 5.0. Nothing seems to help. If anyone has suggestions to what is going on or has experienced something similar and can share I would greatly appreciate it.

1. What is the sample dissolved in? Your problem could be caused by to much organic solvent in the sample compared to the mobile phase...
2. What is the buffer concentration? You could be overloading the buffer. However, since you injected at different buffer pH values, this may not be the problem...

Other things are possible as well, but let us exclude the simple things first.

Uwe,

After posting the message I realized that I neglected to mention what the sample was dissolved in. The analyte was first dissolve in water. Afterwards, we dissolved the the sample in 100% buffer, i.e., the buffer we were using in our mobile phase. The concentration of all our buffers is 25mM.

Dear mpaciolla;

The problems you describe remind me of a case I recently worked on. The signals I obtained were very fronted (almost like a foot-shape), then a normal upslope edge and then a downslope almost without tailing.

It is obvious from your message that changing pH, column, or mobile phase have not improved your results. Let me ask you this, is it possible some NH2, OH, O, or COOH groups are present in the molecule just at the right positions to form a chelate?. If any of these groups are placed in such a conformation that a 5 or 6 memeber ring could be formed with a metallic element, then chelation is possible.

Fortunately the solution to this problem could be very simple. Just add a few ppm (perhaps 50) of EDTA disodium salt to the mobile phase. Let the column equilibrate for a while and inject your sample. Keep in mind that some columns do not seem to come back to their normal performance after being exposed to EDTA.

I hope this helps.

Sincerely;

Benjamin

One possibility is overloading. Although tailing is a more common peak symmetry when overloading in reversed phase separations, fronting can also manifest under certain circumstances. Have you tried injecting a lower concentration to ascertain the relationship between peak symmetry and concentration?

Benjamin,

The addition of EDTA is clever, and I have found it to be very useful in SPE to overcome chelation. Do you have any suggestions for a volatile chelator that could be LCMS compatable? I am not familiar with any, and I only do LCMS.

Michele;

Perhaps you can try some sort of ketone like acetylacetone (ACAC). This is well known to chelate many metals and improve chromatography, however it is less commonly used because of its high UV absorption.

Good Luck;

Benjamin

Benjamin,

I was very optimistic after reading your response, unfortunately, it did not go very well. We observed a shift in retention; however, the fronting phenomenon remained and did not look significantly better when compared to our buffers that did not contain EDTA. Any other suggestions? I'm puzzled.

Does the peak shape change somewhat, when you change the pH? How about changing it more drastically, say comparing 2 and 7 with phosphate, or going to ammmonium bicarboate at pH 9 to 10 with XTerra?

May be it is the sample. Are you sure that it is pure? Maybe the whole story is caused by a structurely closely related impurity that nearly always elutes with the main compound no matter what!

Uwe,

Our sample is a USP standard. We looked at pH 9 on the XTerra using ammonium acetate. The peak shape was more broad when compared to the same buffer at pH 5 and pH 4. In addition, the fronting problem still remained. Although our reagent is a USP standard and is considered "pure", it is not. Prior to the fronting of the main component we are able to detect two additional peaks that are roughly 6% by area. I placed a call to the USP but I haven't heard back from them.

The 6% impurities in a USP "standard" make me highly suspicious. Maybe your "fronting" is indeed a closely related impurity of your main compound that behaves pretty much like the real thing. If you can, you should try to run LC/MS on the thing and see if the "front" is really another compound. How about some stereoisomer?

Dear mpaciolla;

I am sorry the EDTA salt addition did not work for you. Obviously chelation is not the problem. As I mentioned in my original message, this possibility was a real one only if the right groups are present with the required stereochemistry.

Some of the suggestions above have mentioned the possibility of a real compound, partially separated, producing the fronting you see. It would help a lot seeing one of your chromatograms. If you think this a possibility try using a different column. My suggestion is to try a Fluophase PFP one. This I have found very useful in the resolution of isomers. Another posssible column to try is just a Phenyl one. If you see an extra signal with no fronting anywhere in the chromatogram your problem has been solved.

Good Luck.

Benjamin

Could you give a defintion of fronting and its description on a chromatogram?

THIS LINE OF DISCUSSION HAS BEEN INTERESTING. THE DATA THAT YOU DESCRIBE SOUNDS A LOT LIKE ON-COLUMN DEGRADATION. IN THAT CASE YOU SEE "NORMAL" PEAK SHAPE ON THE LATE SIDE OF THE CHROMATOGRAM BECAUSE ALL OF THE MATERIAL IS THE NONDEGRADED DRUG. IN ADDITION, I HAVE A VAGUE MEMORY OF THIS DRUG CLASS BEING LESS STABLE IN BASE. TO TEST THIS, EITHER COOL THE COLUMN OR LOWER THE pH. LET US ALL KNOW HOW THIS WORKS OUT.