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| Dinitrophenol Method For Determination of Total Reducing Sugars in Potato Tubers | to Top |
Reagent:
Solution A: Dissolve 7.145 g of 2,4-dinitrophenol in 230 mL of 5 per cent sodium hydroxide. Heat on hot water bath to dissolve. Then add 2.5 g phenol. Heat some more if the solution does not remain clear.
Solution B: Dissolve 100 g of Nak tartrate in 500 mL of distilled water.
To make dinitrophenol solution, mix solution A and B together, then transfer to 1000 mL volumetric flask and bring to mark with distilled water.
Preparation of standard: A stock glucose standard was made by dissolving 1 g of anhydrous glucose in distilled water and making the volume up to 100 mL. A crystal of thymol was added in order to keep this standard solution for long periods in the refrigerator. This gave a solution containing 10 mg per mL and could be diluted to any desired extent.
Procedure:
One gram of homogenized powder sample was washed with 5 mL of distilled water into a 50 mL conical centrifuge tuber, vortexed 45 seconds, and centrifuged at 200 rpm for 10 minutes. The supernatant was used to determine total reducing sugars.
Exactly 1.0 mL of potato supernatant material was pipetted into a 16 cm test tube. Add 2.0 mL of dinitrophenol solution to each tube. Mix thoroughly (vortex 10 seconds) and heat in boiling water for exactly 6 minutes. Cool 3 minutes in cool water (approximately 6C). Keep cold and read at 600 nm before 20 minutes elapse. Sample is read in a cell of one centimeter path length in a Gilford spectrophotometer (Oberlin, Ohio). The meter was first set at zero for the water solution.
The calibration curve was prepared each time by using standard anhydrous glucose solution. In preparing the calibration curve, 1 mL of serial standard glucose solutions with concentrations of 0, 0.2, 0.5 and 1.0 mg/mL were treated respectively exactly as were the potato samples. A linear regression equation was generated using a Hewlett Packard Model 10 Calculator with standards and this equation was used to determine the reducing sugar concentration in samples.
Part II. The Rapid Direct Extraction-Derivation Method For Determining Fructose, Glucose and Sucrose in Potato Tubers
The three major sugars in potato tubers, fructose, glucose and sucrose, were determined by gas liquid chromatography (Long and Chism, 1987). Reducing sugars such as fructose and glucose were analyzed as trimethylsilylated oxime (TMS/OX) derivatives while sucrose was analyzed as its TMS derivative.
Reagent:
1. Silylation-grade pyridine.
2. STOX: oxime-internal standard reagent -- 25 mg/mL hydroxylamine hydrochloride and 6 mg/mL phenyl-beta-D-glucopyranoside in pyridine.
3. Fructose, glucose and sucrose standard.
4. Bis-(trimethysilyl)trifluoroacetamide (BSTFA).
Chromatography condition:
Hewlett-Packard HP 5890A gas chromatograph equipped with a data station, a 6 foot Supelco glass column 2 mm I.D. with 3% SP-2330 on 100/120 mesh Suplecoport and Flame-ionization detector (FID) was used for this analysis. The FID and injector were both maintained at 235C. The oven was programmed from 160C to 225C at 10 degrees/min with a 3 minute delay. Purified helium was the carrier gas at a flow rate of 30 mL/min.
Procedure:
Five mg samples of the ground powder were weighed on weighing paper and transferred to 1.0 mL Teflon-capped reaction vials. 75 uL of pyridine and 75 uL of STOX were transferred into the vials using a positive displacement pipet. The mixture was mixed by vortexing and placed into an 80C block heater for 40 minutes with additional mixng at 10, 20, and 30 minutes. At the end of 40 minutes the samples were cooled in cooled water, 150 uL of BSTFA added, mixed and heated 10 minutes at 80C. One uL samples were injected into the GC column. Each analysis took 15 minutes.
Response factors for standards were determined utilizing a Hewlett-Packard Model HP3392A integrator. Standard fructose, glucose and sucrose were derivatized as described above and analyzed to determine response factors compared to the internal standard phenyl-beta-D-glucopyranoside prior to each assay. Analyses were completed using the data station using a calibration table to determine the fructose, glucose and sucrose content in the sample.
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´ ´ Updated: Wednesday, October 31, 2007.