Determination of reduced sugar content (Somogyi-Nelson color method)

one. Objective
in plant nutrition and metabolism research, it is often required to determine the content of reduced sugar, and this experiment requires the determination principle of Somogyi-Nelson color method., principle
reduced sugar is a carbide-based (> C s O) sugar, can restore other substances and its own oxidation.
(1) Reducing sugar under alkaline conditions
heating
, in the presence of potassium saline sodium, can be quantitatively reduced to a price of copper ions, that is, to produce brick-red copper oxide precipitation, which itself is oxidized. The reaction is as follows:
CuSO 4 2 NaOH → Na 2 SO 4 Ten Cu (OH) 2 -generated Cu complex reacts with sugar, Cu produces copper oxide (Cu 2 O).
(2) Copper oxide in acidic conditions, ammonium niobium can be reduced, but also prototype ammonium di sodium arsenate to play a role in the production of a blue compound namely arsenic blue, its color depth in a certain range and reduced sugar content (i.e., reduced Cu 2 O amount) in direct ratio, with standard glucose and arsenic niobium acid, compared with the standard, can be measured in the sample reduced sugar content.III, instruments,
reagents
and materials
1 .instrument
(1) hydrometer
(2) water bath pot
(3) plug-scale test tube: 15ml X 10
( 4 ) straw: 1 ml X 1 , 2m1X4 , 5ml X 3
( 5 ) capacity bottle: 100m1 X 2
( 6 ) funnel
( 7 ) research
( 8 ) Electronic top load
balance
2 . Reagents
(1) Copper Reagents
1) 4 % CuS0 4 - 5H 2 0
2) 24g sodium carbonate without water, soluble with 850m1 water In the large
smooth
, add 120g of potassium citrate with 4 molecules of crystalline water, add sodium bicarbonate 16g after total dissolution (suitable heating), then add 120g of sodium aqueous sulfate, fully dissolved and cooled after adding water. To 900m1, precipitation 1 to 2 days, take the liquid (when strict requirements
filter
) can be spared. Mix A and B together by 1: 9 before use.
(2) arsenic niobate reagents: 25g ammonium diphate (N H 4) 6 Mo 7 0 24 . 4H 2 0 ) dissolved in 450ml distilled water (heated dissolved, but easily decomposed when the temperature is close to 150 degrees C), to be cooled and then mixed with 21 ml thick H 2 SO 4. Another 3g sodium arsenate (Na 2 HAs0 4 . 7H 2 0) Dissolve in 25 ml distilled water, then add to the ammonium vanadium solution and place at room temperature in a brown bottle for long-term standby.
(3) 200ug/ml glucose standard liquid: accurately called analysis of pure glucose 200mg, dissolved to l000ml.
3. Materials
fresh plants
organized
.4th, operation step
1 . production of standard curve
in 7 scale test tubes, respectively, add 200ug / ml standard glucose 0, 0.1, 0 . 2 , 0 . 3, 0 . 4, 0 . 5, 0 . 6 ml, respectively. Distilled water 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4 ml are added in order to a solution containing 0, 10, 30, 40, 50, 60ug glucose per milliliter. Each test tube is added to the copper reagent 2m1, heated in a boiling water bath after mixing 10min, immediately cooled, then added 2m1 arsenic niobate reagent, diluted to 15ml after two minutes of oscillation, with a phosphorescometer at 620 nm wavelength to measure its absorbance. The standard curve is drawn with the sugar content (ug) as the horizontal coordinate and the absorbance as the ordinate.
2. Extraction of reduced sugar from plant samples
Wash the sample, absorb moisture from its surface, finely chopped and mixed, weigh 1g into the research, add quartz sand about 0.5g, Grind into a smooth paste, transfer the sample to a 100m1 triangular bottle, add about 70-80m1, shake and place on a
rout temperature
water bath for half an hour.
the sample is cooled, precipitate
protein
: add 5% zinc sulfate 5 ml, then slowly drip into 0.3 mol/L Ba (OH) 2 5m1 to precipitate the protein. Set aside after oscillation, add a drop of Ba (OH) 2 until there is no white precipitation, transfer the solution to a 100m1 capacity bottle and add water to the scale.
3. Determination of reduced sugar content
filters the above-mentioned fixed-capacity sample fluid, takes 5 ml of filter fluid, and then determines the capacity to 100m1 (depending on the sugar content of the sample). Take 2 ml of diluted solution, the same coloration method as standard glucose solution: copper reagent 2 ml, boiling lomin, arsenic tantalum reagent 2m1, oscillation 2min, fixed capacity to 15 ml, 620nm wavelength color ratio, record light absorption., the results of
six, note
due to different materials, should be appropriately adjusted dilution multiply., think about why
precipitated protein when extracting samples..