First, identify what the sugar will be "mixed in" (we refer to this as, "the matrix". ) as this will have a profound effect on which methods and techniques may or may not be applicable. Contact someone at your school for help. Depending on what instrumentation you have access to and of course if you have an experienced chromatographer available, try a keyword search on the web (e.g. Google) and you will find thousands of application notes, papers, articles and white papers with examples. Avoid most detection methods which use UV detection as sugars do not absorb well. Spend some time on your own first researching the question before proceeding. You will learn far more about the techniques used and possible analysis methods in this way.

Another point to regard is certainly the specificity of your method. E.g. if you have mass spec identification, your HPLC separation need not be overly specific. However, If you want to separate D-fructose from for instance other monosacharides and identify the sugar solely based on retention time (compared to authentic D-fructose) a common chromatographic approach is High Permformance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). HPAEC-PAD is usually run on special devices (e.g. Dionex from Thermo) with alkaline eluents. The detection with an electrochemical detector is quite sensitive (less than 0.5nmol of monosaccharide detectable). I have used a Dionex ICS3000 with a CarboPac-PA1 column to separate glucose, arabinose, galactose, rhamnose, mannose, xylose, and ribose. I haven't tried fructose but I am sure this will work, too. I can provide details if you have access to an HPAEC device.

If you do not have access to an HPAEC, it would be very helpful to find out what options of columns and devices you HAVE available. Also, I would need more specific information on the sample type, to give more specific advice.

Mr. Rami,

As the other participants in this discussion have pointed out such analysis can be carried out highly selectively, precisely, accurately and sensitively, only by means of mass spectrometry (MS).

At this point, I should only add to the discussion that the analysis should be carried out within the framework of our own-authored (to me and my co-author's theory according to the authorship, below) innovative stochastic dynamic method and model formulas for quantification ofMS outcomes, which is the only currently available method, worldwide, for an absolute quantification of analytes by mass spectrometry in chemometric terms.

So far, it has been applied to analytes in mixtures at pg.(mL-1) concentration level, yielding to coefficients of linear correlation between theory and experiment with respect to analyte concentration in solution /r/ = 0.99997-1.

Please consider references [1,2].

[1] Stochastic Dynamic Mass Spectrometric Approach to Quantify Reserpine in Solution; Bojidarka Ivanova, Michael Spiteller Analytical Chemistry Letters, 10 (2020) 703-721; Received 13 Oct 2020, Accepted 16 Dec 2020, Published online: 28 Jan 2021 Download citation https://doi.org/10.1080/22297928.2020.1865834

[2] Steroids, 164 (2020) 108750 Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II; Bojidarka Ivanova, Michael Spiteller

Its application to quantify carbophydrates in mixture has resulted to statistical chemometric parameter /r/ = 1 within the framework of six multiple measurements (consider, reference [3].)

[3] Bioorganic Chemistry, 93 (2019) 103308

A mass spectrometric stochastic dynamic diffusion approach to selective quantitative and 3D structural analyses of native cyclodextrins by electrospray ionization and atmospheric pressure chemical ionization methods; Bojidarka Ivanova, Michael Spiteller

An additional comment on: Our approach is the only currently developed quantitative method based on data-processing per span of scan time, instead of analysis of outcomes over the whole time of a measurement. Only this approach provides exact quantification. Why? Please, focus on the attached figure, herein. There are shown experimental data of an analyte, having m/z 271. Over the shown scan time the standard deviation (sd) and the standard error (se) are 0.03016 and 0.00871. Therefore the accuracy of the m/z data are at the second decimal sign (XX.XX). When, however, there are treated the m/z-values per short span of scan time, then the standard deviation and standard error become equal to zero (sd = se = 0 0.)

Therefore, via the latter approach, meaningful are the m/z-data up to the five decimal sign in this case (XX.XXXXX.) It is capable of determining exactly each of analytes in mixture.

Conversely, in the former case a m/z 271.XX can belong to more than one analytes. In particular, talking about carbohydrates, with the latter accuracy you are unable to determine selectively such analytes in mixture even by mass spectrometry. An analysis based on only HPLC or HPLC/UV-detection means highly speculative quantitative data, which are very far from any quantitative criteria or standards of the 'Analytical chemistry' and the Chemical Science, in general.

Hi Chirag , there are many ways to analyse sugars with HPLC or ion chromatography. The choice of LC conditions is based on the actual sample and matrix (other sugars or analyses present in your sample) The choice of detection technique is dependent on the concentration levels you need to quantify. If concentrations are high, refractive index detection is a good choice, if you need to be more sensitive you can use corona CAD detection or ultimately electrochemical detection (pulsed amperometric detection to be more precisely). Here you can find an application note based on HPAEC-PAD (Anion Exchange Chromatography in combination with pulsed ampermetric detection as an example. Although it is focussed on the analysis of trace amounts of Lactose and isomers in dairy products, it shows the capabilities of the technique and also Fructose can be quantified in this manner.

https://antecscientific.com/download/19/food-applications/8049/220_009_06-lactose-free-products

Hope it helps, regards Hendrik

Thanks for the suggestion.

You may visit the following link:

https://www.jpsr.pharmainfo.in/Documents/Volumes/vol9Issue08/jpsr09081703.pdf

http://www.ifrj.upm.edu.my/25%20(02)%202018/(28).pdf

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