Chromatography Forum

Hi, there!

I have an Econosil NH2 column which was initially shipped in hexane:ethanol=95:5. Prior to use it in reverse phase mode i washed the column successivelly with n-propanol, acetonitrile and acetonitrile:water=75:5. When i tried to separate some monosaccharides i've got same retention time for each other. It seems that carbohydrates passed through my column without any retention. I think the column must be first equilibrated prior to use it, but i don't know how. Which salt or buffer are suitable for amino bounded silica columns.
Any suggestion will be appreciated.
Thanks,
darth.anghel

What monosaccharides are you running, and how sure are you that they are eluting? Your post said 75:5 acetonitrile: water, which is only about 6% water. Most monosaccharides require between 15% and 25% water.

On the other hand, I can't say I've been that successful with amino silica columns that weren't prestabilized. If you buy another, get one that specifically says its a carbohydrate column, like the Waters Carbohydrate, or look for a polymer based amino column like the Shodex Asahipak.

Hello

Carbohydrates can be unusually difficult molecules to chromatograph, as I have found out in the past few days in our lab.

I've been trying to separate glucosamine on a Kromasil NH2 column. The Kromasil column is a new one and its performance checks out just fine, as one would expect from Kromasil. The problem is not with the column, but with our method...and with carbohydrates in general.

Although NH2 columns are preferred for carbohydrates, they do need to be stabilised carefully before use.

In our case, we were getting a large peak early in the chromatogram, that we mistook for glucosamine. The blank showed the same peak too, and we eventually realised that the peak was not due to glucosamine, but due to the sample solvent - water. Water interacts with an NH2 column, under certain conditions, and presents itself as a regular peak, when one uses refractive index detection.

The other problem was, glucosamine is highly water soluble and it elutes out rapidly, if we use water in the mobile phase. So, we tried 100% acetonitrile, and we also tried phosphate buffers at various pH levels. Retention times have improved somewhat but we're still working on the right mobile phase composition.

I'd suggest you equilibrate the column with the mobile phase for an hour at least. Inject a solvent blank first to rule out ghost peaks. Also, minimise water in the mobile phase, or if possible, do away with it. Most carbohydrates are reasonably soluble in acetonitrile and methanol, so you can expect good retention times and separations, if you're lucky.

If you need to use buffers, you might have to use a trial-and-error approach, as we did. We experimented with KH2PO4 buffers, at pH 2, 4, 6 and 8, with various ionic strengths.

In our case, we think we're finally getting somewhere with pH 4, 50 mM KH2PO4 and acetonitrile (85% buffer: 15% MeCN).

I hope this is useful to you.

It's the simple molecules like carbohydrates that are usually the hardest to separate using HPLC - no UV chromophores, excessively water soluble, amphoteric, can't use gradients because of RI detection, etc..

Good luck.

Thank you for suggestions, Nosser222 and sksrinivas.

Nosser222, it was a mistake in my initial posting; I mean eluent composition was acetonitrile:water=75:25 (v/v).

sksrinivas: indeed, Silica bonded aminopropyl column must be equilibrate for a long time period in order to gain satisfactory retention times for saccharides. In ur case of glucosamine it seems its happend something strange. Usually, glucosamine is commercialized in its hydrochloride form (some times ago, when I need some glucosamine, I performed an acid hydrolysis upon chitosan and I have separated glucosamine in its hydrochloride form). Maybe hydrochloric acid present in ur sample causes troubles in ur initial attempt. More than that, under certain circumstances (usually at pH between 4-6), aldehydes and ketones react with amino group of stationary phase giving so called Schiff bases. In order to avoid that thing I think I must eluate my column with a mixture of acetonitrile and a phosphate buffer at pH=3. Do u think its a good ideea? I don't want at all to spoil my column.

Once again, thank u all.

darth.anghel

I'm not very familiar with carbohydrates and amino phases.

I've just read some literature about HILIC and I think that's what amino phasese are.
Have a look at this Waters presentation:
http://www.waters.com/WATERSDIVISION/Si ... UM=WA31785

Maybe your water content is too high, as in HILIC the water is the strong eluent.

In our lab, we have an application for the separation of glucose and fructose on a NH2-phase and our mobile phase is ACN/Water 80/20.

Yes, at 75:25 you should first try reducing the water content.

It is NOT a good idea to acidify your amino column first. Sugars exist in two anomers, which can be separated from each other due to the slow equilibrium between them under acidic conditions. The equilibrium speeds up significantly under alkaline conditions, and this is the reason why amino columns give you one peak instead of the two anomer peaks.

There are additional issues around the use of amino columns under these circumstances, which is why I recommend to use a column that has been qualified for carbohydrate separations.