You have to decide what molecular target(s) you want. A hydrolysate can contain 1000s of different molecules. Remember HPLC is a 'comparative technology' which means you need a reference standard for quantitation.

Based upon the previous decision above you have to decide on;

1. Detector

2. Column

3. Mobile Phase(s)

4. Concentration of your molecular targets (HPLC may not be the best instrument)

5. Sample matrix and potential interferences

Flow rate is the least of your problems!

Hi Longfei,

It depends on which products you want to observe, as an example, I use to quantify by HPLC compounds such as 5-HMF, Furfural, Formic and levulinic acid. Hereunder you'll find the parameters used, it could be a basis for your reflections.

Detectors : Refractive Index + Photoarray detector (wavelength of 210 nm for acids and 280 nm for furanics)

Column : Grace prevail organic acids column

Mobile phase : water + phosphoric acid (pH=1.7) followed by acidified water/acetonitrile gradient until 50/50 v/v

Flow rate : 1.0 mL/min

Temperature : 35°C

Concerning the sugars and lignin compounds I have nerver used HPLC to analyze these compounds so I can't help, but I know some columns are dedicated to sugar analysis.

Good luck for your analyses !

Almost everything absorbs at ~200 nm so a hydrolysate will have many peaks. You may have to use a more selective detector like PAD (pulsed amperometric detector) or MS (mass spectrometry).

Or you can try and extensive cleanup like liquid-liquid extraction or SPE (solid phase extraction).

You will need to start by obtaining some formal training in liquid chromatography. There are several classic books on the topic (e.g. Snyder/Dolan) which will provide you with an introduction to the fundamentals. READ THEM first. If you do not have many years to learn this new technique, then please also contact someone at your school for help. Ideally, a mentor or local expert who can guide you through the process. Learning HPLC is not like learning how to use an analytical balance or pH meter. *Note: It takes many years of practical experience and an understanding of the fundamental principles to learn how to develop methods.

Next, do your own research. Search for the key compounds plus the technique to see how others have analyzed similar compounds before. Be thorough. This is one of those times when you need to practice your own researching skills. Use the time to get better at it. Good information gathering skills = a good scientist applying the scientific method to research a question.

BTW: If you already tried to search for existing methods and the ones you found did not provide complete method details in the paper or note, then (1) the articles were not scientific articles; (2) To save space and avoid redundancy, the article's authors may have in fact referenced the detailed method as a citation. All properly written HPLC methods will include the details needed to reproduce them properly in the lab (exact column name, type, dimensions, flow, exact mobile phase composition, temp, inj vol, inj solvent, detection details, K prime, T0 time and so on).

Thank you for your detailed advice. I will scan more papers and find a expert, help me to finish this experiment.

Thanks for all of you.@William Letter, @Bruce Neagle,@ Alban Chappaz .