Chromatography Forum

I am producing bioethanol from macroalgae, as part of my research group objectives and thus am interested in fermentable carbohydrates such as glucose as well as seaweed specific such as mannitol in the hydrolysate:

Currently our HPLC column in addition to a Shodex Sugar is the Aminex HPX-87H:
The eluant is 5mM sulphuric acid
The flow rate is 0.60 ml/min
The temperature is 50 degrees celsius
The detection is UV 210nm.

This is the existing sample preparation with additional points added as a result of the advice given on this forum:

1)Firstly we are going to adjust the acidic hydrolysate(as a result of the acidic pretreatment phase) to pH 7, using NaOH
2) Then we are going to run the sample through an SPE cartridge filled with a cation exchanger in the hydrogen form to get rid of the sodium which otherwise will accumulate on the analytical column (advice from the forum)
3) We are then going to centrifuge
4) We are then going to take 1 ml of the adjusted sample and dilute with 9 ml de-ionized water for a dilution factor of 1 in 10.
5) We are then going to filter the sample using a 0.45 µm syringe
6) We are going to transfer 1 ml to an HPLC vial for analysis

Now my question is: is this the appropriate dilution factor for this column and carbohydrate analysis (since we have just appropriated the 1:10 dilution factor from our Shodex column methodology). Can we also dilute with de-ionized water or should we use an internal standard and de-ionized water?

Should we perhaps adjust the dilution factor to 1:5 or perhaps use a dilution factor of 1:20 for carbohydrate and organic acid analysis on the Aminex HPX-87H?

First i would MUCH rather (and do) use RI instead of UV... There are a lot of things that will interfere in the UV at low concentration that have so little effect on RI as to be negligible.

Secondly What concentration range are you in? if you are cleaning out the salts first, the Aminex columns can easily handle concentration to 50 g/L with a 10 uL injection and probably higher. Also UV is not very sensitive for sugars.

Thirdly If you are not interested in in organic acids why use the 87H column at all? You will get much better sugar separation on the Shodex 810 or Aminex P (lead) columns.

- Karen