Attached file may help you, also develop analytical method from literature review.

Best to use other detection methods, but if you functionalize/derivatize them, then some types of UV methods are possible. Otherwise only 190-210nm could be used (low UV), but are NOT selective for the sample type (a poor quality method). As to the mode of chromatography, there are many which may be used (NP/RP/HILIC). Amino (NH) columns are the most popular choice, but many other types may also work too.

• The sample's own chemical and physical properties dictate which analytical techniques are the most scientifically appropriate to use for analysis. In their natural forms, your samples would best be detected using methods other than UV. For example, RID, ELSD, CAD or MS.

Please try a keyword search on the web to find example methods (e.g. https://www.sciencedirect.com/science/article/abs/pii/0076687988610561 ). I found many types of methods using many modes of chromatography with a keyword search in just a few seconds on the web. Please contact your teacher for help and advice (or someone with training using HPLC) and also give the keyword search a try too. Very easy to find examples this way and learn about available options. Learning 'how' to research a question is one of the most important skills you can learn.

William Letter I don't think you would be limited to 190 to 210 nm for the oligosaccharides. I routinely analyze the chondroitin disaccharides at 236 nm. With the amide and esters in your compound, the UV range is broader than with simple sugars. Scan your solution from 200 to 300 nm and see which wavelengths can be used.

Lance, there is a reason few report using low UV for these types of samples. While there are some methods which use low UV at ~ 230nm, as noted earlier, they are not selective for most of samples that the original poster has described. Good quality HPLC methods should use retentive and selective methods of detection to show the method is selective for the sample under analysis and low wavelength UV alone does not provide that basic level of confidence.

I certainly do agree that in all cases, the sample / standards under investigation should be fully scanned (DAD/PDA) to evaluate their spectral characteristics before deciding on an analysis method. This is fundamental to good chromatography method development principles and one we have taught in our professional classes for almost thirty years.

The problem with derivatizing the compounds in the mixture is finding a good reagent to use, and ensuring you don't lose your analytes doing the cleanup.

As far as using a C18 column to separate the analytes, acidic conditions (pH 3 to 5) would protonate the nitrogen's and provide better separation.

William Letter Refractive index is what I see in most papers for analyzing saccharides. RI is worst since it cannot differentiate any compounds, requires higher concentrations of the material, and isocratic methods. The analyst is depending on the column to separate the compounds and being able to use standards to identify the peaks.

We also do not recommend RID (for some, it is all they have, but very poor overall). After establishing a good chromatography method, most samples are analyzed with MS or ELSD/CAD detectors which are a better option. For the analysis to meet basic scientific goals, the method chosen must be appropriate. You can not force a method to work using the wrong tools. The most appropriate methods are noted in my previous replies on this post topic.