One thing you could try to improve the removal of residual ammonium bicarbonate is to redissolve the lyophilized sample in water and lyophilize it again.

However, some ammonium will remain as the counterion if your glycoprotein has any anionic groups.

If you still get a pH that is too basic, dissolve the dry glycoprotein in a buffer having the desired pH.

Adam B Shapiro thank you for your answer. I have tried repeated lyophilization in the past, but it didn't help, so I guess its indeed ionic interactions between ammonium and some anionic groups. Unfortunately, I don't think I can add any buffers to my glycopeptides, as I intend to use them for a test that relies on free pH changes. Would it be possible to remove the associating ammonium through something like dialysis? Or would the ionic interactions between ammonium and glycopeptides interfere with dialysis?

Hello Agate,

Ammonium bicarbonate is said tobe "volatile with decomposition at +60°C" - which is also the melting point, essentially unless you hear the ammonium bicarbonate up, you will not evaporate it or sublime it away from the sample. Ammonium hydroxide is volatile and can be evaporated or sublimed in a lyophilizer. This may not help you unless you can change your buffer before freeze drying.

If using an evaporation system, be warned, ammonium hydroxide will foam when you first pull a vacuum on it, so reduce the pressure slowly. Same process if you have a basic freeze dryer and rely on vacuum to freeze the samples. [Think about opening a soda after shaking it ... you open it slowly, little by little!]

Point of note with freeze drying and buffers. When you freeze, it is common to see pH shift on freezing. You can see what happens, use some buffer in a small sample tube or vial, add some universal indicator, put in the freezer and watch the colour change when they freeze. As you freeze the sample, the buffer salts are forced out of solution and the pH of the solution can change.

Kind regards,

Rob

Rob Darrington thank you for the answer! I had read that ammonium bicarbonate decomposes at +60C, and because of it I tried centrifugal evaporator. But unfortunately the ones we have around don't allow for precise temperature control. When I did a run with heating on, the samples felt very hot to touch and the resulting pellet was transparent and hard as a piece of plastic. The pH, however, did drop to 6.6, but I'm not sure if overheating didn't damage the glycopeptides. How would one exchange the buffer from ammonium bicarbonate to ammonium hydroxide? Is there some cheap method that doesn't involve dialysis or affinity binding? I have to use the ammonium bicarbonate at the beginning to keep the right pH for my protease.

You might be able to exchange the ammonium counterions for sodium by mixing the glycopeptides with NaCl, the dialyzing extensively against water to remove excess salts.

Dialysis can be a good option if your glycopeptides has over than 1KDa molecular weight. I use dialysis to remove the excess amount of ionizing agents from ionic polymers and it works perfectly.

Use SnakeSkin dialysis tube available in various molecular weight cutoff or pore sizes, choose as per your needs.

-> use water as solvent for the dialysis

-> change the water in every 2 to 3 hrs to maintain the low concentration at outside the membrane.

-> continue it for 24-36 hrs.

Later on to recover your compound use freeze dryer

How about running the peptides through HPLC with ACN and Water with 0.1% TFA? Any salts will elute first, then you can freeze dry the peptide containing fractions, afterwards your peptides should only have TFA adducts.

Swati Tanwar add to your dry peptide (after purification) glacial acetic acid, agitate until it dissolves, freeze in liquid nitrogen and freeze-dry. It may not remove all the TFA, but most of it.