With an emphasis on the impact of low temperature on sugars and carbohydrates.
Freezing is probably the best way to store the samples but you should be wary of the impact of thawing. Freezing the sample means that the sample becomes non-homogenous. Usually the last fluid to freeze has the greatest amount of sugar and salt. This is typically in the center of the container. If the entire sample is not melted, you are likely to test liquid that is lower in concentration. But melting the entire sample has its own problems. Once in solution, sugars, especially reducing sugars, can interact with other components in solution. If you freeze your sample but then let it sit on the bench for 3 hours before refreezing it, you may be generating artifacts. The best way to handle this problem is to take your original sample and divide it into aliquots and freeze all the separate aliquots. That way you can ensure that there are no freeze-thaw artifacts in a sample because you will use each sample only once.
Dear Dr. Joe Olechno,
Thank you very much for your help and your advices.
I agree with the answer provided by Joe, but here we have to also consider the extraction method as well as the solvent selected for extraction, since these both things play an important role for breakdown as well as formation of glycosidic linkages which are said to be also temperature dependent.
Prasad s absolutely right! Unfortunately, carbohydrates and sugars are very water soluble and not soluble in much else. But you can get good extraction of smaller carbohydrates with methanol. Extracting with methanol means that you will denature your enzymes and have less biochemically induced degradation. Since the first step of the Amadori rearrangement is the formation of a Schiff base and since we have reduced water concentration during the extraction, there will be a driving force to form the Schiff base. But methanol does not afford as much opportunity for bond transfers, you may find subsequent steps of the rearrangement to slow down. What sugars are you looking for, in particular?
Dear Dr. Joe Olechno and Dr. Prasad vijay Kadam,
0.1 g of the sample (dry matter of plant tissue) was centrifuged twice in 80% hot ethanol at 10000 rpm for 10 min. Supernatants were mixed and stored in a refrigerator.
How long can this extract be stored in a refrigerator?
How many times can this extract be subjected to different temperatures (room and refrigerator temperatures)?
Good method. It should get most sugars and some of the larger oligosaccharides and polysaccharides. Depending on your plant material, the extract is probably acidic. That means that you will slowly modify the sugars. The acid will breakdown some glycosidic linkages and may also generate ethyl glycosides. If this is a problem (and it might not be), you should be able to plot the loss versus time and get an idea of the magnitude of the problem. 80% EtOH will keep bacteria and fungus from destroying your sample. If there is no hydrolysis and no ethyl glycoside generation, this should be very stable. Simple temperature changes should not be a problem. Some of the other compounds in the extract could chemically react with reducing sugars (e.g., the previously mentioned Schiff base formation). As the concentration of water is reduced, the formation of the Schiff base will increase due to LaChatelier's principle.
Are you using GC, CE, size exclusion, HILIC or HPAE-PAD for analysis?
Dear Dr. Joe Olechno,
The procedure was "Phenol-sulfuric acid total carbohydrate determination" according to "Miniaturization of Three Carbohydrate Analyses Using a Microsample Plate Reader" by Jeffrey D. Fox’ and John F. Robyt (ANALYTICAL BIOCHEMISTRY 195, 93-96 /1991). In our work, samples were analyzed by an Epoch Spectrophotometer.
Dear Dr. Pourkhaloee,
I am not sure but I believe that the formation of glycosides and Schiff bases may not have much impact on the Phenol-Sulfuric acid test. I believe that they both still act as carbohydrates in the test. That means that your results should stay constant so long as you 1. prevent microbes from using the carbohydrates as an energy source and 2. sample from your stored material in a way to ensure that there are no local concentrations (or lack thereof) of carbohydrates. That means either keeping the solution liquid or, if frozen, the entire sample is melted and mixed (possibly heated if precipitate has formed that was not there when initially frozen).
Many of the suggestions I gave earlier assumed that you wanted to identify the individual sugar constituents (e.g., glucose, mannose, xylose, turanose, fructose, cellulose, starch, etc.). Some of these compounds can change chemical and chromatographic properties but they all remain carbohydrates, albeit different carbohydrates.
Good luck on your research.
Dear Dr. Olechno,
I am forever grateful for your understanding and I would be very grateful for your kind help.
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