Chromatography Forum

Dear All

I want to analysed sucrose from fermentation broth sample.
Can I do sucrose anlysis using RP column and using RI Detector ?
Or Is there any method available for sucrose analysis?
If not then Which column can be used for sucrose analysis, mobile phase and detector ? Can anybody give the method ?

Thanks in advance.

FROM Mumbai - India

Sucrose is much too polar to be run on a reversed-phase column. In order to get retention you need to work with hydrophilic interaction columns. The standard approach for sugars is to purchase an amino column designed for sugar analysis. The mobile phase is about 70 to 75 % acetonitrile and water. The detection method can be RI. Some sample prep may be required also.

This is a common analysis. A more robust column and vastly cheaper eluant (water) would be one of the Biorad carbohydrate columns. The Biorad literature will have examples of fermentations and column recomendations to guide your choice. Use RI or PAD detection.

You find similar information in the Waters catalogue pages 162-165.

Dear all thanks for reply,

Can I used HPLC Carbohydrate Analysis column - Aminex HPX 87K
(Bio Red) column for sucrose analysis ?
What is different between USP L19 and L34 Column ? This column can used for sucrose analysis ?

Narendra - Mumbai - India

Any of the ligand exchange carbohydrate analysis columns can be used for sucrose analysis. Your choice of columns depends on the complexity of your sample. The 87K column is a potassium form column which works well for a simple mixture of sugars, especially if potassium is part of the mixture. The heavier the counter metal on the polymer, the greater the degree of separation of sugars. USP L-19 is a calcium loaded column. USP L-34 is a Pb loaded column. The Pb form column will give the greatest separation of sugars, the calcium form less, and the potassium form the least. You need to review what you need to separate and match it to the best column using either selectivity charts, published methods, or consulting with the technical support group at the column manufacturer. Fermentation broths are complex mixtures which will contain both sugars and organic acids. The column of choice for these mixtures is usually the hydrogen form column (87H) which can separate both organic acids using ion-exclusion and sugars using ligand exchange. Besides Bio-Rad, there are a number of companies (Hamilton, Supelco, Metachem, Transgenomic, etc.) that make these types of columns, so if you are working through a distributor, they should carry one of these lines of columns.

Sucrose analysis- is easily done by Electrochemical detection using a CarboPac PA1 column.
http://www1.dionex.com/en-us/columns_ac ... s4771.html

Dear All thank you very much for your valuable suggestion.

I successfully analysed sucrose from broth sample using RI detector. I have to do some trial and error method for fine tunning the method that is obiviousely anyone has to do. For your reference in short I given my method details:

Column : Aminex HPX 87K ( 300 x 7.8mm) HPLC carbohydrate analysis column

Mobile Phase : ACN : Water (75: 25)

Flow rate : 1ml/min

Column oven temp : Ambient (25°C)

Detector : RI

Sample : I used SPE Cartiges for clean up sample (it depend on your sample)

I given all details about my method because I feel eveyone who used or impliment the suggestion or references given by this site. The advantage of this, it will give confident to the author that he/she is in write direction of suggestion.

From Narendra - India - Mumbai