ISI 27-1e Determination of Reducing Power and DE by Lane and Eynon's method.

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1. Scope	The method is applicable to all starch hydrolysis products.			LT 7/12 1966 Rev.: LT 3. May 1999
2. Principle	The Lane and Eynon constant titre method. Mixed Fehling's solution is titrated with sample using methylene blue as indicator.
3. Reagents	3.1 Fehling stock solution A. Dissolve 69.3 g copper (II) sulphate pentahydrate (CuSO4, 5H2O) in distilled water to 1000.0 ml. 3.2 Fehling stock solution B. Dissolve 346 g potassium sodium tartrate tetrahydrate (KNaC4H4O6, 4H2O) and 100.0 g sodium hydroxide (NaOH) in distilled water to 1000,0 ml. Decant solution from sediment if necessary before use. 3.3 Methylene blue (C16H18CIN3S, 2H2O) indicator, 1% aqueous solution. 3.4 Anhydrous D-glucose pro analyse. Dry two hours at 100 oC and cool in dessicator before use. 3.5 Standard glucose solution. Weigh to the nearest 0.1 mg 1.25 g dried anhydrous D-glucose (3.4) and dissolve with distilled water to 250 ml.
4. Apparatus	4.1 Automatic burette. 4.2 Burette, 50 ml graduated to 0.1 ml 4.3 Bunsen burner 4.4 Desiccator with dry drying agent 4.5 Analytical scale 0.1 mg
5. Procedure	Prepare mixed Fehling's solution and standardise. Mixed Fehling's solution does not keep.
A. Standardisation	Make mixed Fehling's solution by transferring first 12.5 ml of solution A (3.1) and then 12.5 ml of solution B (3.2) to a 250 ml conical flask and mix well.			Use of automatic burette for solution A is strongly recommended.
	Fill 50 ml burette (4.2) with standard glucose solution (3.5). Add from burette about 23.5 ml of the standard glucose solution (3.5) to mixed Fehling's solution. Add a few anti-bumping granules and place on wire gauze with clean asbestos mat. Heat over Bunsen burner. Boil for two minutes and add three drops of methylene blue indicator (3.3).
	Add - without interrupting boiling - 2-3 drops of dextrose solution at about 10 seconds intervals until the blue colour completely disappear.			Detect colour change in the clear supernatant liquid from light blue to colourless
	Artificial indirect lighting is strongly recommended.			Diffuse daylight is not suitable for detecting the end-point.
	Titration should be completed in less than a minute. Repeat titration twice.
	Exactly 24.1 ml of standard glucose solution should be used for the titration - otherwise adjust Fehling's stock solution A appropriately and repeat standardisation.
B. Determination	Weigh accurately into a beaker (g) g sample, where approximate sample weight (g) = 12500 / (expected DE x dry substance). Dilute sample with distilled water to 250 ml Proceed as described under A. Standardisation using sample solution in stead of standard glucose solution for the titration. Note b = ml test solution used for titration.			The test solution should contain approximately 1.25 g dextrose in 250 ml.
6. Calculation	DE (average results of two samples to one decimal) = (250 x a x 10) / (b x g x d) ; where a = factor from table below b = ml test solution for titration g = gram sample dissolved to 250 ml d = % dry solids in sample
7. Notes	Alternative method: Calculation of DE from a sugar distribution obtained by HPLC.
8. Reference	International Standard: ISO 5377 Lane, J.H., Eynon, L. (1923). Determination of reducing sugars by means of Fehling's solution with methylene blue as internal indicator. J. Soc. Chem. Ind. Trans. 32-36.
9. Table	ml of sugar solution required	Dextrose factor ~ mg dextrose corresponding to 25 ml of Fehling's solution	mg dextrose per 100 ml	All figures relate to anhydrous dextrose.
	15	120.2	801
	16	120.2	751
	17	120.2	707
	18	120.2	668
	19	120.3	633
	20	120.3	601.5
	21	120.3	572.9
	22	120.4	547.3
	23	120.4	523.6
	24	120.5	501.9
	25	120.5	482.0
	26	120.6	463.7
	27	120.6	446.8
	28	120.7	431.1
	29	120.7	416.4
	30	120.8	402.7
	31	120.8	389.7
	32	120.8	377.6
	33	120.9	366.3
	34	120.9	355.6
	35	121.0	345.6
	36	121.0	336.3
	37	121.1	327.4
	38	121.2	318.8
	39	121.2	310.7
	40	121.2	303.1
	41	121.3	295.9
	42	121.4	289.0
	43	121.4	282.4
	44	121.5	276.1
	45	121.5	270.1
	46	121.6	264.3
	47	121.6	258.8
	48	121.7	253.5
	49	121.7	248.4
	50	121.8	243.6