1. Physical method, (1. Optical rotation method, 2. Refraction method, 3. Specific gravity method,)
2. Physical and chemical methods, (1. Point method, 2 pole method, 3. Photometry, 4. Chromatography)
3. Chemical methods, (1. Feilin's method. 2. Potassium permanganate method. 3. Iodometric method. 4. Potassium ferricyanide method. 5. Bismuth copper colorimetric method. 6. Carbazole colorimetric method )
There are three major species, and when measuring other carbohydrates, it is often hydrolyzed to sugar and then measured.
I. Determination of total sugar
The total sugar in food mainly refers to reducing glucose, fructose, pentose, lactose and can be hydrolyzed under the conditions of the assay.
Reduced monosaccharide sucrose (1 molecule of glucose and 1 molecule of fructose after hydrolysis), maltose (2 molecules of glucose after hydrolysis) and possibly partially hydrolyzed starch (2 molecules of glucose after hydrolysis). The reducing sugar is reductive because the molecule contains a free aldehyde group (-CHO) or a ketone group (=C=O).
The classical chemical methods for determining total sugars are based on their ability to be oxidized by various reagents. Among these methods, the application range of various methods according to the oxidation of the Ferien's solution is wide. Here we mainly introduce the potassium ferricyanide method, beryllium copper colorimetry, and the Filipino volume method. Due to the complex reaction and many influencing factors, the Feilin's volumetric method is not as accurate as the potassium ferricyanide method, but its operation is simple and rapid, and the reagent is stable, so it is widely used. The beryllium copper colorimetric method requires that the concentration of the sugar liquid in the colorimetric range is within a certain range, but the detection liquid is required to be clarified. In addition, in most cases, the measurement requirements do not include starch and dextrin, which requires starch and paste before the measurement. Fine removal, which complicates the operation and limits its wide application.
(a) Potassium ferricyanide method
1. Principle: The original and hydrolyzed invert sugar produced in the sample have reducing properties, and the potassium ferricyanide can be reduced in an alkaline solution, according to potassium ferricyanide.
Concentration and test titration can calculate the sugar content. The response is as follows:
C6H12O6+6K3[Fe(CN)6] + 6KOH →(CHOH)4·(COOH)2 + 6K4[Fe(CN)6]+ 4H2O
At the end of the titration, a slight excess of invert sugar reduces the indicator methylene blue to a colorless leuco.
2, reagent
1) 1% methylene blue indicator
2) Hydrochloric acid (hydrolysis)
3) 10% and 30% NaOH solution
4) 1% potassium ferricyanide (reservation of special bottles, before use)
Calibration step
Said sucrose 1.000g → constant volume 500ml → take this liquid 50ml → in a 100ml volumetric flask → add hcl5ml → shake → 65-70 ° C water for 15 minutes → take out the cooling → neutralize with 30% NaOH → add water to the scale → pour In the burette→ Take 10ml of 1% potassium ferricyanide in an Erlenmeyer flask → add 10% NaOH 2.5ml and add 12.5ml of water and add glass beads to the glass → heat to boiling → keep for one minute → add 1 drop of methyl blue → immediately The amount of sugar is dropped until the blue color is removed.
Formal titration is less than 0.5ml sugar solution, boil for 1 minute, add a drop of indicator, then use sugar droplets to determine the blue color to fade, calculate the concentration of potassium ferricyanide solution.
A=(W·V)/(1000×0.95)
A: the amount of invert sugar equivalent to 10 ml of potassium ferricyanide solution (g)
V: volume of sugar liquid consumed by titration
W: Weigh the amount of pure sucrose
1000: dilution ratio
0.95: Conversion equals
3. Method of operation: Dilute 10g → use 100ml of water as a solution → in a 250ml volumetric flask → add 20% lead acetate 10ml → until the end of precipitation → add 10ml of 10% NA2HPO4 → until no precipitation occurs → add water to the scale → filter - take the filtrate 50ml → in 100ml In the volumetric flask → conversion, neutralization and titration according to the potassium ferricyanide calibration method
Calculate sugar content
Total sugar (% in terms of invert sugar) = (A × 1000) / (W·V) × 100
A: the weight of the invert sugar equivalent to 10 ml of potassium ferricyanide solution,
W: the weight of the sample
V: volume consumed by the titration sample
4. Experiment should pay attention
(a) At the end point, excess invert sugar reduces the indicator methylene blue to a colorless leuco, which is oxidized by oxygen in the air and quickly becomes the color of the indicator.
(b) The whole process should be carried out on a low-temperature electric furnace, and the titration should be speed, otherwise the end point is not obvious.
(c) Dehydration of sugar and sulfuric acid to form hydroxymethylfurfural, and the product is condensed with yttrium copper to form a blue compound. The color depth is proportional to the concentration of sugar in the solution, and the sugars such as mono- and disaccharides are directly in the reagent. It works, so no hydrolysis is required.
(2) Colorimetric method of beryllium copper
1. Principle: The sugar is dehydrated by reaction with sulfuric acid to form hydroxymethylfurfural formaldehyde. The product is then condensed with ruthenium copper to form a blue compound. The color depth is proportional to the concentration of sugar in the solution, and can be quantified by colorimetry.
2. Reagent
(1) Zinc sulphate solution: dissolve 500g of chemically pure zinc sulphate in 500ml of water
(2) Potassium ferrocyanide solution: dissolve 10.6 g of chemically pure potassium ferrocyanide in 100 ml of water
(3) 0.2% beryllium copper reagent: dissolve 0.2 g of beryllium copper in 100 ml of 95% sulfuric acid, and store in a brown bottle in a cool dark place.
(4) 0.1% glucose solution: accurate dry glucose 0.1000g, constant volume 100ml
3. Method of operation
(1) Standard curve drawing
(2) 100ml volumetric flask number
Heat in a boiling water bath for 6 minutes, take out the cooling → measure the absorbance with a 1cm cuvette → 610nm → make a quasi-curve with the absorbance as the abscissa and the sugar concentration as the ordinate
(3) Sample measurement Weigh 10g sample → add 100ml hot water to 500ml volumetric flask - add 5ml of zinc sulfate → boiling water bath for 5 minutes → remove and shake 5ml of potassium ferrocyanide, then cool → volume to 500ml → filter → 25ml of suction solution → 250ml volumetric flask → constant volume 250ml → take 1ml of diluent, add 10ml bismuth copper reagent in the colorimetric tube → shake → water bath for 6 minutes → cool → colorimetric
Test attention
1, the sample solution must be clear and transparent, there should be no protein precipitation after heating
2, when the sample color is dark, it can be determined by decolorization with activated carbon.
3, this method is related to the concentration of sulfuric acid used and the heating time
4, the concentration of the sugar solution is between 1-2.5mg/100ml
2. Method for determining reducing sugar
Reducing sugars include glucose, fructose, maltose, aldehyde stems containing bruce in glucose molecules, ketone stems containing brunch in fructose molecules, and semi-condensed stalks containing bruce in lactose and maltose. Sex. All methods of hydrolyzing sugars to invert sugars for the determination of total sugars in the determination of reducing sugars can be used to determine reducing sugars.
(1) Filipino volumetric method
1. The principle, reagents and methods of this method are the same as those for total sugar. Only the sample solution does not have to be over-converted, but the filtrate is directly titrated, and the filtrate is titrated. The reducing sugar content in the filtrate is preferably 0.2-0.5%, and can be adjusted by increasing or decreasing the sample amount or changing the dilution factor. The theoretically equivalent amount of reducing sugar when mixing 10 ml of Fering's A and B liquids is as follows:
Glucose (anhydrous) fructose or transforming diabetes 0.050g
Breast Diabetes 0.0678g
Maltose 0.0807g
2 reagent
(1) Feilin's A solution, said 69.8g cp copper sulfate in 100ml water, filtered for use
(2) Feilin's B solution, said 34.6g.cp concentrated sodium zinc and 100gcp NaOH in 1000ml water, filtered for use
3 methods
The sample was weighed 10-20 g: the preparation and conversion of the same potassium ferricyanide method. Pour the sample into the dropper, draw A, B to prepare for pre-titration
Pre-titration: 5ml of A and B respectively → add 15ml of sample solution from the dropper → heat to boiling → continue to add sample solution → to blue color change potential → add 3 drops of methyl blue → titrate to within 1 minute end
Upon reaching the end point, a slight excess of invert sugar reduces the blue-colored methylene blue chromosome to a colorless leuco, while the red color of cuprous oxide appears, and the product of heating the sugar under alkaline conditions is complicated.
De-alkaline fracture is due to different alkalinity, different heating time, and different production of fragments. This fragment brings errors to the subsequent titration. Moreover, this fragment has no relationship with the amount of sugar. Therefore, Lanecrol-Eynon Method Make a data lookup table
Formal titration: suction A, B liquid each 5ml → in a triangular flask → add 0.5-1.0ml less than the predetermined amount → boil for 1 minute in 2 minutes → add 3 drops of indicator → use the sample droplets to clear the blue color
The total boiling time is 3 minutes, ie the titration is completed in 3 minutes.
Calculation:
Reducing sugar = ( F · V2) / (W · V1) × 100
F: the number of sugar returned, that is, the number of milligrams of invert sugar equivalent to 10 ml of Fischer's test solution.
V1: total volume of sample test solution
V2: sample test droplet quantitation
W: sample weight
In the measurement of lactose products, if the content ratio of sucrose to lactose exceeds 3:1, it should be calculated by adding the correction value in the relevant table (Table 9-8 in the textbook) to the titration.
Our examples are as follows:
If the standard fructose solution is 262.5 mg sugar per 100 ml solution. For the 10ml film test solution, you can find that the fructose droplets should be 20ml from 9-5. If it is not 20ml, you can first calculate the A and B liquid correction equals. Then calculate
For example, the concentration of the standard sugar solution is 199.3 ml of sugar per 100 ml of solution. For 10 ml of A, the liquid B is found from 9-4, and the sugar droplets should be 25.00 ml, which can be corrected if there is any difference. If the requirement is not high, the calibration step can be omitted but 1% of the measurement error is required, and the correction is omitted. In addition, sometimes the sugar content of the sample is not calculated according to the search table, but the A and B liquids are calibrated so that the amount of reducing sugar is determined. This error is 0.5%. Below we talk about the standard quantitative A, B liquid
Accurately and accurately weighed and dried AR sucrose 1.5g → dissolved in water and weighed into a 250ml volumetric flask → constant volume → suck 50ml in 100ml quantitative bottle → add HCL5ml → then 65-70 degrees Celsius water for 15 minutes → cool → use 30 % NaOH neutralization → constant volume
Accurately absorb 5 ml of each of B and B in a triangular flask → add about 50 ml of glass beads to the water → heat to boiling → hold for 1 minute → add 1 drop of indicator → cook again for 1 minute → immediately remove the blue color with sugar droplets. Red appears as the end point
Formal titration, first add about 0.5ml less than the pre-drip timing, boil the sugar solution for 1 minute → add 1 drop of indicator → boil for 1 minute → continue to drop to the end point
Calculation: A=W*V/500×0.95
A: the amount of invert sugar equivalent to 10 ml of Filipino A and B solutions
W: Weigh the quality of sucrose
V: the amount of sucrose titrated
500: dilution ratio
0.95: Conversion equals
Calculated after zui:
Total sugar (reducing sugar) in terms of invert sugar%=(A*1000/W*V)*100
A: Same as above
W: the amount of sample taken
V: When the titration is the sample consumption
1000: is the dilution factor (100/50*500)
1. Predicted purpose: There must be a requirement for reducing sugar concentration in the sample solution (about 0.1%). The consumption volume of the sample solution should be similar to the consumption volume of the calibration glucose standard solution. It is determined whether the sample concentration is appropriate by measurement. If the concentration is too large or too small, it should be adjusted so that the amount of sample solution consumed in the measurement is about 10 ml. Secondly, the approximate consumption of the solution can be known by measurement, so that in the official titration, the pre-addition is less than the actual amount. About 1 ml of the sample solution, leaving only about 1 ml of sample liquid to be added at the time of continuous dripping, in order to ensure that the continuous titration work is completed within 1 minute, and the accuracy of the prediction is submitted.
2. The main operational factors affecting the determination results of this experiment are the alkalinity of the reaction liquid, the heat source intensity, the boiling time and the titration speed. The boiling time is generally short and the consumption of sugar is small. Conversely, the consumption of sugar liquid is small, the titration speed is too fast, and the consumption of sugar is large. Conversely, the amount of sugar consumed is small. In addition, the higher the alkalinity of the solution, the faster the reduction of divalent copper, so the volume of the reaction must be strictly controlled to make the alkalinity of the reaction system uniform. The heat source is generally 800W electric furnace, and the reaction liquid boils in 2 seconds.
(2) KMNO4 (potassium permanganate method)
1. The principle is that the reducing sugar reduces the copper salt to cuprous oxide in an alkaline solution. Under acidic conditions, the cuprous oxide can reduce the ferric sulfate to ferrous sulfate, and then titrate the ferrous sulfate with the KMNO4 solution to mark the reduction. The amount of sugar.
2. Method of operation
(1) Sample processing
a. Lactose: including dairy products and cold foods containing protein
Weigh 2-5g (liquid sample 25~50ml) → in 250ml volumetric flask → add water 50ml → add liquid A 10ml+1N NaOH 4ml → constant volume → let stand for 30 seconds → filter → discard the initial liquid → measure reducing sugar and For sucrose.
b. Low-alcoholic beverages: beverages such as wheat extracts and various sparkling wines.
First remove the CO2 → take 100ml → in the evaporating dish → neutralize with 1 N NaOH → boiling water to the original volume of four minutes → transfer to a 250ml volumetric flask → add 50ml of water → shake → (add A liquid 10ml → Add 1 N NaOH 4 ml) → add water to the scale → let stand for 30 seconds → filter.
c. Foods containing a lot of starch: baby food, cake powder, baby music, milk replacer, biscuits, bread, cakes, etc.
Weigh 10-20g → 250ml volumetric flask → add water 200ml → 45 degree water bath for 1 hour → keep shaking → add water to the scale after cooling → let stand → suck out 200ml in another volume bottle (250ml) → add liquid A 10ml+ 1N NaOH 4ml → let stand for 30 seconds → filter.
d. Soda, fruit, domestic seven cola and Coca Cola
Treat CO2 → aspirate liquid 100ml → in a 250ml volumetric flask → add water to the scale → measure reducing sugar and sucrose.
(2) Measuring method
Take 50ml of treated sample → in 400ml beaker → add 25ml of A and B liquid → heat to boil for about 4min → cook for another 2min → heat and filter → wash the beaker and precipitate with 60°C water → until the washing liquid is not alkaline → Filtered paper (or asbestos) and Cu2O → transfer to the original beaker → rinse the filter bottle with 25ml of ferric sulfate solution → wash all the rinse solution into the original beaker → add 25ml of water → dissolve Cu2O → use 0.1N KMnO4 standard To a reddish color, a blank experiment was performed as described above using 50 ml of water.
(3) Calculation
3. Note:
(1) The boiled solution is red and not blue, indicating that the amount of sugar is high, which can reduce the sampling volume.
(2) During the whole process of washing Cu2O, the upper layer of the precipitate should be kept a layer of water to isolate the air and prevent Cu2O from being oxidized by oxygen in the air.
(3) This method is applicable to the determination of reducing sugars in various foods. The colored sample liquid is not limited, and the accuracy is high and the reproducibility is good. Accuracy and reproducibility are superior to direct titration, but the operation is complicated and time-consuming, and a special potassium permanganate sugar search table is needed.
(2) Direct titration (Filin's solution method)
Principle
After the sample is treated to remove impurities such as proteins, hydrochloric acid is added to hydrolyze sucrose to a reducing monosaccharide under heating, and the direct titration method is used to determine the hydrolysis.
The total amount of reducing sugar in the sample.
2. Reagent
3. Method
(1) Take an excess sample for extraction, put it into a 250ml volumetric flask, add 5ml of zinc acetate and 5ML of zinc ferrocyanide, dilute to volume, filter for 30 minutes, filter, and reserve the filtrate.
(2) Determination
Sample pre-titration:
Take 5ml of each of A and B liquid and add 10ml of water to the flask. The number of glass beads will be boiled in 2 minutes. It will be titrated by hot titration, titrated to blue and faded, and the dosage will be recorded.
Official titration:
Take 5 mL of A and B liquids in a triangular flask → add three beads of glass beads → directly add 0.5-1.0ML sample liquid from the burette directly than the pre-drip timing, boil at 2 minutes, and heat the titration
The color fades, records, and the average value is calculated.
three. Determination of sucrose
1. Principle: After the sample is deproteinized, the sucrose is hydrolyzed by hydrochloric acid into a reducing sugar, and the content of sucrose in the sample is determined by reduction.
the amount.
In fact, the determination of the reducing sugar includes two parts: one is the original reducing sugar in the sample, and the other is the reducing sugar after the acid hydrolysis of the sucrose.
2. method
Suction reducing sugar sample treatment dilution 50mL → in 100ML volumetric flask → add → 15 minutes on 68-70 degree water bath → cooling → add methyl red 2 drops → neutral → constant volume →
Take this solution and measure it by reduction.
2. Calculation
Sucrose%=F(100/V2-100/V1) /(W╳50/250╳1000)╳ 100╳0.95
Where: F: 10 ml of the Ferien's test solution is equivalent to the mass mg of the invert sugar.
V1: The undiluted sample dilution volume consumed during the measurement
V2: The diluted sample volume of the sample that was hydrolyzed during the measurement
w: the weight of the sample when the original reducing sugar is measured (G)
1000: Convert liters to grams
0.95: The molecular sucrose is hydrolyzed to become two molecules of reducing sugar (one molecule of glucose and one molecule of fructose). The molecular weight of sucrose is 342, and later becomes 2×180. Then 342/360=0.95. So the conversion sugar is converted to sucrose. Should be multiplied by 0.95.
4. Paper chromatography.
In food, the composition of sugar is more complicated, and it contains various components such as maltose, glucose, maltotriose and maltotetraose in starch syrup. For each component in these foods, it is impossible to carry out chemical analysis. Determination, and physical analysis method for measurement.
Paper chromatography is applied to the separation and analysis of sugars. It utilizes the difference in physicochemical properties of the components in the mixture to make the components move at different speeds to achieve separation.
Specific shift value Rf==distance of component expansion/distance of solvent expansion
The law of the RF value of sugar is:
Monosaccharide>disaccharide>trisaccharide
Pentose > hexose
Ketoose > aldose
Commonly used developing agents in the laboratory:
n-Butanol: HAc: H2O==4:1:5
Commonly used color developer:
0.1N AgNO3: NH4OH (SN) = 1:1 (high sensitivity, but spots are easy to spread)
AgNO3/acetone: NaOH/ethanol = 1:1 (overcoming the above disadvantages)
(There is a lot on the chromogenic book, you can see it yourself)
The sample can be treated by conventional methods such as sugar extraction and protein removal as seen above.
According to the Rf value, the Rf of various sugars can be compared with the Rf value of the standard sample, and the type of sugar can be determined, and the amount can be determined quantitatively, and directly measured on the filter paper by speckle densitometry. The amount is corrected by the spot area correction. .
In order to make full use of Goji resources in Ningxia and deeply exploit the medicinal health care value, this year, the company started the deep-processing projects of high purity Goji polysaccharide and black Goji Anthocyanin, which have been identified as the Model processing enterprises of Chinese Goji Berry by the local government.
Black Currant Extract,Freeze Dried Mango Powder,Lemon Spray Dried Powder,Mango Fruit Powder
Ningxia Rich Resource International Co., Ltd , https://www.richresources-nx.com