High Performance Liquid Chromatography-Analysis of Formose Sugar Md. Zafar Iqbal and Senad Novalin* (senad.novalin@boku.ac.at) University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria Abstract- Formose matrix with partial conversion of formaldehyde was analyzed by using HPLC with RP Monolithic-C18 columns and DAD (360 nm) detection. The separation and identification of the compounds were investigated applying a derivatization method. Sugars and unconverted formaldehyde were determined by endowing them with charges through derivatization using DNPH. The required mole ratio of derivatization reagent and Formose sample was approximately 100:1 in order to complete the derivatization process. Keeping the derivatized samples for 24 to 48 hrs at ambient conditions prior to analysis was necessary to complete the derivatization of C5 and consecutive higher sugars. However, the determination of C4 and higher sugars is not possible due to overlapping of peaks, branched-chain sugar etc. Introduction The Formose reaction, an alkaline catalyzed self-condensation of formaldehyde, leads to a very complex matrix containing mainly D and L straight-chain and branched-chain monosaccharides. Analysis of the compounds in this complex solution proved to be a difficult task for the researchers due to uncontrolled product distribution in the matrix. Determination of the neutral monosaccharides and formaldehyde in the Formose matrix was conducted by 2,4-dinitrophenylhydrazine (DNPH) derivatization [1][2]. A detailed analytical procedure for Formose sugar by derivatization has not been discussed in the literature. In this report, characterization of the derivatization procedure for analysis of Formose sugar has been presented. Significant corrections found in the derivatization technique have been reported. Materials and Methods Experiments for producing Formose sugar with partial conversion of formaldehyde were conducted in a batch reactor. All standards, chemicals and reagents were HPLC grade. Hitachi Lachrom Elite HPLC system equipped with Diode Array Detector (360 nm) was used for analysis of the derivatized samples. Three Chromolith RP-C18 Columns each (100 X 4.6 mm) and Chromolith Guard Cartridge (5 X 4.6 mm) were used. Separation of the derivatized samples was performed at ambient temperature (25ºC) and 1.0 ml/min flow rate. Acetonitrile-water (HQ) gradient elution with 5 to 100% acetonitrile was taken as a mobile phase. Derivatization procedure of the samples The target of derivatization reaction is to form a hydrazone product, which absorbs the UV light. The hydrazone development occurs by reacting hydrazine agents (e.g. 2,4-dinitrophenylhydrazine) with the carbonyl group of sugars. The constraint of this derivation procedure is the formation of side-products or various isomers of the derivatized sugars. However, this is the mostly used derivatization procedure for monosaccharides till now [Figure 1: (A), (B)] [3]. (A) (B) Figure 1: (A) 2,4-dihydrophenylhydrazine, (B) Derivatization of reducing sugar (e.g. Glucose) with 2,4-dihydrophenylhydrazine to 2, 4-dinitrophenylhydrazone derivative [3] The Formose sample (0.5 ml) was treated with 0.5 ml of 2,4-DNPH solution at 65ºC for 60 minutes. The reaction mixture was then cooled immediately in a cool-water bath and diluted with 95% ethanol (1 ml). The resulting suspension was then centrifuged (if lead sulfate precipitates appear) for 10 minutes at 5000 g. The supernatant was filtered by a 0.45 µm nylon filter, which was ready to analyze immediately in the first cases. In the other cases, filtered samples were kept at ambient conditions for different durations prior to analysis. Sugars and formaldehyde standards as well were derivatized by the same technique. DNPH concentration was varied from 0.1 to 1.6% (w/v) in all cases. Results and Discussions Following the derivatization procedure described in materials and methods, a mix of ten individual C6 reducing monosaccharides containing equal concentrations (10 mg.L-1 each), were taken under derivatization. The total amount of sugars present in the sample was 278 µmol. DNPH concentration was varied from 0.5 to 1.1% (w/v). The results are shown in Figure 2. Figure 2: Chromatogram overlay of ten mixed C6 reducing sugars Peak area increased gradually with increasing DNPH concentration in the sample. The DNPH/sample mole ratio was varied from approximately 30:1 to 100:1. Other higher monosaccharides starting from C4 also exhibited the same attribute. However, this behaviour was not shown by the C3 and successive lower sugars including formaldehyde where 30:1 was the sufficient ratio to complete the derivatization. Thus, for the appropriate quantitative analysis, approximately 100-fold excess of reagent is required for C4 and higher sugars and approximately 30-fold is sufficient for C3 and lower sugars including formaldehyde. Formose samples were firstly injected immediately after derivatization. A further procedure comprised of keeping the samples at ambient conditions for several hours prior to analysis. Results are shown in Figure 3. Figure 3: Keeping time vs. peak heights of different sugar standards and formaldehyde. 3MixC6 = Allose, Glucose, Gulose In the case of C3 and other lower sugars including formaldehyde, peak height increased slightly up to 12 hrs keeping the samples at ambient conditions. For continued keeping of these samples, peak height decreased slightly until 24 hrs. Within 72 hrs keeping the samples at ambient conditions, the change of peak height was more or less zero. The C4 sugar also showed similar behaviour. On the other hand, mentionable increase of peak height was observed within 4 hrs of keeping time for C5 sugar and approximately 60 hrs for C6 sugar. A further slight increase was observed in C5 sugar. Based on these results, it is recommended to keep the higher sugar samples for 24 to 48 hrs at the ambient storage prior to analysis. Figure 4 shows the typical Formose sugar chromatograms developed in the present experimental conditions. Figure 4: Chromatograms of the derivatized monosaccharides and unconverted formaldehyde in the Formose matrix Table 1: Reducing sugars and unconverted formaldehyde in Formose sample (sugars produced with 35% formaldehyde conversion) determined by 2,4-DNPH derivatization of the samples in HPLC system with RP Monolith-C18 columns; initial concentration of formaldehyde 46 g.L-1; GA = Glycolaldehyde, GCA = Glyceraldehyde and Eryth = Erythrose Table 1 shows the analyzed reducing sugars and unconverted formaldehyde (g.L-1) found in Formose sugar sample with partial formaldehyde conversion. In case of Erythrose, the peak may contain some branched-chain C4 sugar. Conclusions The concentration of used derivatization reagent and the derivatization duration both have significant influences on quantification of higher monosaccharides. Unfortunately, apart from C4 sugar, the analysis of higher sugars in the Formose reaction are still problematic due to overlapping of peaks, branched-chain sugars etc. This in any case needs further investigation and development of analytical methods. References [1] O. Pestunova, A. Simonov, V. Snytnikov, V. Stoyanovsky, V. Parmon; Advances in Space Research 36 (2005) 214 . [2] A. N. Simonov, O. P. Pestunova, L. G. Matvienko, V. N. Parmon; Kinetics and Catalysis 48 (2007) 245 . [3] F. M. Lamari, R. Kuhn, N. K. Karamanos; Journal of Chromatography B. 793 (2003) 15. Sample HCHO GA GCA Eryth formose sugar 30.33 0.75 0.55 1.20