Sugar analysis: description of the steps and theoretical

The sugar analysis, based on acidic hydrolysis, is a technique used to assess the relative massic proportions in which the various monosaccharides are present in the analysed carbohydrates. It is one of the most common analysis techniques when dealing with carbohydrates. However, it fails to give information about the polysaccharides’ structures and bonds.

45 This analysis was carried out in the laboratories of Builidng 29, at the

Universidade de Aveiro. The analysis was carried out following the protocol in force in said department and here descripted.

Polysaccharides are polymers consisting of more than two monosaccharides linked covalently by glycosidic linkages. (Muhamad, et al., 2017)

The analysis is conceptually subdivided in two phases: in the first the polysaccharides are “broken” into the elementary sugars by which they are

composed; said sugars are then converted in alditol acetates that can be volatized in an appropriate eluent; the second phase is the analysis of the alditol acetates by means of a gas-chromatography.

The first step is the acid hydrolysis reaction, conducted in presence of strong acid and heat; this causes the glycosidic linkage between two adjacent linked

monosaccharides to be cleaved (Cui, 2005), “freeing” the different monosaccharides.

Figura 5-2: Glicosidic linkage hydrolysis of AX

A sample between 1-2 mg is weighted on an electronic scale. The hydrolysis agent in our case was sulphuric acid (72% aqueous solution). 200 µL of sulphuric acid are added to the sample and mixed: then, 2,2 mL of distilled water are added.

The heat is provided by a heating bloc, which heats the tubes at 100°C for 2,5 hours.

This time has been estabilshed considering that, during hydrolysis, the

monosaccharides can undergo degradation by the acid; however, not all glycosidic linkages are broken at the same rate, and so the time must be sufficient for all the linkages to be hydrolyzed. By doing analysis on the same sample subjected to different times of heating, we would that the amounts of monosaccharides measured for different heating times would reac. a maximum; by these

considerations, we infer that the time must be a compromise between the two factors: not too short (so we can “free” al the monosaccharides) but not too long (as to avoid degradation) (Cui, 2005).

After that, the tubes are cooled in an ice bath. The next step is to add 200 µL of internal standard; in this case, a solution of water and 2-deoxyglucose in

concentration 1 mg/mL.

The internal standard is necessary as a reference to integrate the

gas-chromatography results. Knowing the area of the peak relative to the internal

46 standard, the areas of the peaks relative to the monosaccharides, and the

quantity of internal standard, we obtain the quantities of the various monosaccharides.

The next step consists in transferring of 1 mL of the sample in a culture tube, and neutralizing it with 200 µL of ammonia 25% aqueous solution; it’s important to confirm that the pH is above 7. That’s because the next step is the reduction, where the reducing agent is 𝑁𝑎𝐵𝐻₄ (sodium borohydride). But sodium

borohydride in an acid solution would decompose (Banfi, et al., 2001), so we neutralize it. Sodium borohydride reduces the aldehydic group of the

monosaccharides, converting them in alditols (Cui, 2005). 𝑁𝑎𝐵𝐻₄ is put into a solution of 𝑁𝐻₃ 3M, in concentration 150 mg/mL. 100 µL of this solution is added to the sample in the culture tube. The tubes are then put in an ice bath, where 2 x 50 µL of glacial acetic acid are added, to decompose the excess sodium borohydride left from the reduction step.

Figure 5-3

At this point the aldehydes have been converted to alditols, so now we proceed to acetylate them. We transfer 300 µL of sample in SOVIREL tubes with inert teflon caps. The tubes are now put into an ice bath, and 450 µL of

1-methylimidazole are added, along with 1 mL of acetic anhydride. The acetic anhydride is the reagent, whereas 1-methylimidazole is the catalyst. The ice bath is needed because of the exothermic nature of the acetylation reaction

(Wachowiak & Connors, 1979). The prepared sample is then shaked and put onto a heating bloc at 30°C for 30 minutes to allow the acetylation reaction to take place.

After that, 3 mL of distilled water and 2.5 mL of dichloromethane are added to the sample. Dichloromethane is used for the liquid extraction of the

monosaccharides from the aqueous phase; that’s because dichloromethane is a good solvent for organic compunds and is hydrophobic (Rossberg, et al., 2006).

The samples are then centrifugated for 30 seconds at 3000 rpm, to cause the separation of the organic phase from the aqueous one. When this is done, the aqueous phase is removed by means of a pasteur pipette. To remove the un-desired components, another step of addition of distilled water and

dichloromethane, followed by centrifugation and water removal is repeated as before. Finally a last washing similar to the last two, but conducted with only distilled water, is performed. The dichloromethane is evaporated in a speedvac

47 (vacuum centrifuge). Two separate additions of 1mL anhydrous acetone, each one followed by evaporation in the speedvac, are performed.

The reactions phase are now concluded. The dry sample obtained is ready for analysis in a gas-chromatographe. The injection is performed using 2 µL of anhydrous acetone as solvent. The samples obtained are analysed in a Flame Ionisation DetectionGas Chromatograph (GC-FID); this tipe of equipment is able to provide a signal which can be correlated to the concentration of internal standard to infer the concentration of the various compunds in the sample. The compunds can be distinguished by their characteristic elution time. The fraction upon which this analysis was carried out were: the 3 protein fractions, the 4 AX fraction , the two polyphenolic fractions, the starting BSG and the finale residue from the final AX extraction with water. The analysis as descripted provided un-satisfactory results for the AX, especially the first fraction, because of their high purity; for this reason, for the AX fraction the analysis was repeated with a doubled concentration of internal standard with respect to what previously desccripted in this paragraph.