Hey, I'd like to hydrolyze starch with HCl in hot water and quantify as reducing sugar with Feehling reaction.
What are the conditions to have a complete hydrolysis of starch (acid concentration, temperature, time), and avoid degradation?
Is it possible to evaluate the complete hydrolysis of starch during acid reaction, with any reagent which is not an interference with Feehling reaction?
Regards
Stefano
Dear Stefano, the conditions of the hydrolysis that should meet your requirements are as follows: 3M HCl, 3h, 100C. I would suggest to neutralize hydrolysed samples before you add Fehling reagents and this should prevent potential interferences.
Dear Stefano,
as for the starch acid hydrolysis, it proceeds in much higher rate compared to cellulose.
The reason is the absence of highly ordered elementary fibrilles creating microfibrilles /so called cristalline portion/ of cellulose.
Starch ussually consists of two forms, amylopectin - representing space branched macromoleculles consisting prevailingly from 1,4 alpha D-anhydro-glucopyranose, resp. 1,6 alpha D-anhydro-glucopyranose, and of amylose which macromolecules are "linear" /spiral like polymers/ constructed from 1,4 alpha D-anhydro-glucopyranose building units. The both forms are of almost amorphous structure. Average molecular weight of starch is in general much lower than that of cellulose.
Instead of HCl I would recomend you to use 3% H2SO4 for starch hydrolysis. A sufficient temperature of hydrolysis is the temperature of boiling water bath and time 3 hours. Advantage of H2SO4 is its simple removal without dillution of the hydrolyzate via neutralization. You can remove sulphuric acid with addition of slight excess of BaCO3. The products of reaction is water insoluble BaSO4 that can be filtered off, or centrifuged. You can also neutralize the acid on anion-exchange resin in OH- cycle. In both cases there will be no disturbing interference with Fehling solutions.
Dear Stefano
Starch content can be estimated by complete acid hydrolysis method (Kent Johns and A. J Amore, 1960). Flour sample of 2.5 g was suspended in a mixture of 200 ml of water and 20 ml of HCl acid. (Sp. Gravity 1.125) The mixture was heated in a flask provided with a reflux condenser for 2.5 hours. Contents were cooled, neutralized with NaOH (5 N). Volume was made to 250 ml and the sugar formed was determined as dextrose by Lane and Eynon reducing sugar estimation method. The value obtained should be multiplied by 0.9 to get the starch amount. Before the hydrolysis process starch should be mixed with distilled water and discard the filtrate to remove the soluble sugars and other water soluble matter.
Hey Stefano,
according to Haebel et al. 2008 (Anal Biochem 379, 73-79) starch hydrolysis is complete after 3h at 95 degrees C in 0.7M HCl or 2M trifluoroacetic acid (TFA).
Completeness of the hydrolysis can be easily tested by varying the hydrolysis time and checking whether the amount of glucose released still changes.
The advantage of the TFA method is that it's a volatile acid and can be removed using a speedVac (depending on the amount of starch put to hydrolysis, the hydrolysate needs 2-3 round of drying and dissolving in water; takes some time).
The advantage of the HCl method is that it's faster. You hydrolyse up to 0.1 - 5mg starch in as little as 100uL 0.7M (use tight-closing screw-cap vials and shake sample down from time to time). After that you add a little amount of buffer (I use final concentration of 10mM imidazole pH8), neutralize with a small volume of 5M KOH, and dillute if necessary with water.
HCl and KOH only make KCl and H2O which should not interfere with any follow-up procedure. However some buffers do. So you should check whether the imidazole influences the Fehling reaction. You can check that easily using a blank hydrolysate sample (without starch but containing all ingredients of the hydrolysate). If you include different amounts of glucose (which would be result of acid starch hydrolysate) you can subject that to Fehling and see if you can get a linear correlation that you can calibrate your actual starch hydrolysates on. I would generally recommend some form of calibration with glucose.
Good luck.
Felix.
I want to hydrolyse starch into mono, di, tri, tetra and so one glucose mols how can i do it. Will using TFA/H2SO4/HCl at lower conc and low temp will work. Pl suggest.
Does this process affect the proteins? I want to use it for hydrolysis of starch in grain flour samples and then do an in vitro protein digestibility study. I do not want the proteins to be free but not affected by the starch hydrolysis process. Any suggestion is appreciated.
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