Introduction to the determination of total sugar i

Introduction to the determination of total sugar in food

there are many methods for the determination of sugar, which can be roughly divided into three categories

1 Physical method, (1. Optical rotation method, 2. Refraction method, 3. specific gravity method,)

2 Physical and chemical method, (1. Point method, 2-pole method, 3. Photometry, 4. Chromatography)

3 There are three kinds of chemical methods (1. Ferlin's method. 2. Potassium permanganate method. 3. Iodometry. 4. Potassium ferricyanide method. 5. Anthracene copper colorimetry. 6. Carbazole colorimetry)

in the determination of other carbohydrates, it is often hydrolyzed to sugar before determination

I Determination of total sugar

total sugar in food mainly refers to reducing glucose, fructose, pentose, lactose and sucrose (1 molecule of glucose and 1 molecule of fructose after hydrolysis) that can be hydrolyzed into reducing monosaccharides (2 molecules of glucose after hydrolysis), maltose (2 molecules of glucose after hydrolysis) and possibly partially hydrolyzed starch (2 molecules of glucose after hydrolysis). Reducing sugars are reductive because their molecules contain free aldehyde groups (-cho) or ketone groups (=c=o)

the classical chemical methods for the determination of total sugars are based on the fact that they can be oxidized by various reagents. Among these methods, various improved methods based on the oxidation of ferring's solution have the widest application range. Here we mainly introduce potassium ferricyanide method, anthracene copper colorimetry, and ferring's volumetric method. Because of the complex reaction and many influencing factors, the ferring capacity 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. Anthracene copper colorimetry requires the concentration of sugar solution to be within a certain range during colorimetry, but requires the detection solution to be clarified. In addition, in most cases, the determination requirements do not include starch and dextrin, so starch and dextrin should be removed before determination, which complicates the operation and limits its wide application

(I) potassium ferricyanide method

1 Principle: the original invert sugar in the sample and the invert sugar produced after hydrolysis have reducing properties. Potassium ferricyanide can be reduced in alkaline solution. The sugar content can be calculated according to the concentration of potassium ferricyanide and the test titration. The reaction 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 slightly excessive invert sugar will reduce 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 (storage characteristic bottle, calibration before use)

calibration steps

weigh 1.0000g sucrose at a constant volume of 500ml, take 50ml of this solution in a 100ml volumetric flask, add 5ml of HCl, shake well with a water margin of ℃ for 15 minutes, take it out for cooling, neutralize it with 30%naoh, add water on the scale, pour it into a burette, take 10ml of 1% potassium ferricyanide, add 10%naoh2.5ml and 12.5ml of water to a conical flask, add glass bead particles, heat it until it boils for one minute, add a drop of methylene blue, and immediately drop enough sugar to make it blue, and record the dosage

formal titration is less than 0.5ml of sugar solution during titration. Boil it for 1 minute, add a drop of indicator, and then titrate it with sugar solution until the blue color fades, and calculate the concentration of potassium ferricyanide solution

a= (W V)/(1000 0.95)

a: the amount of invert sugar equivalent to 10ml potassium ferricyanide solution (g)

v: volume of sugar solution consumed during titration

w: weigh the amount of pure sucrose

1000: dilution ratio

0.95: convert the equivalent number

3. operation method

dilute 10g with 100ml water in a 250ml volumetric flask, add 10ml of 20% lead acetate until the precipitation is completed, add 10ml of 10% Na2HPO4 until the precipitation is not generated, add water to the scale for filtration - Take 50ml of filtrate and convert it in a 100ml volumetric flask according to the potassium ferricyanide calibration method, Neutralization and titration

calculate sugar content

total sugar (calculated as% of invert sugar) = (a 1000)/(W V) 100

A: the weight of invert sugar equivalent to 10ml of potassium ferricyanide solution,

W: the weight of the sample

V: the volume consumed by the sample solution during titration

4. The experiment should note

(a) when the end point is reached, the excess invert sugar will reduce the indicator methylene blue to a colorless leucocyte, and the capacity of leucocyte is affected by oxygen in the air.Oxidation, Soon it 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 carried out quickly, otherwise the end point is not obvious

(c) sugar reacts with sulfuric acid to dehydrate to form hydroxymethyl furaldehyde, and the product is then condensed with anthracene copper to form a blue compound. Its color depth is proportional to the concentration of sugar in the solution

. Monosaccharides, disaccharides and other sugars act directly on the reagent, so hydrolysis is not required

(II) colorimetry of anthracene copper

1 Principle: sugar reacts with sulfuric acid to dehydrate to form hydroxymethylfuraldehyde, and the product is then condensed with anthracene copper to form a blue compound. Its color depth is directly proportional to the concentration of sugar in the solution, which can be compared quantitatively

2. Reagent

(1) zinc sulfate solution: dissolve 500g of chemically pure zinc sulfate in 500ml of water

(2) potassium ferrocyanide solution: dissolve 10.6g of chemically pure potassium ferrocyanide in 100ml of water

(3) 0.2% anthracene copper reagent: dissolve 0.2g of anthracene copper in 100ml of 95% sulfuric acid, place it in a brown bottle and store it in the cold dark

(4) 0.1% glucose solution: accurately weigh 0.1000g of dried glucose, constant volume 100ml

3 Operation method

(1) standard curve drawing

(2) 100ml volumetric flask number

boiling water bath heating for 6 minutes, take out the cooling, use a 1cm cuvette 610nm to measure the absorbance, and make a quasi

curve with absorbance as the abscissa and sugar concentration as the ordinate

(3) sample determination

weigh 10g sample into a 500ml volumetric flask with 100ml hot water - add 5ml zinc sulfate to the boiling water bath for 5 minutes, take out and then add 5ml potassium ferrocyanide under shaking, Cool 500ml to constant volume, filter 25ml of filtrate, take 1ml of diluent in 250ml volumetric flask to constant volume, add 10ml of anthracene copper reagent to the colorimetric tube, shake well, heat in water bath for 6 minutes, cool the colorimetric

test note

1, the sample solution must be clear and transparent, there should be no protein precipitation after heating

2, and when the sample is dark, use activated carbon to decolorize and then determine

3. This method is related to the concentration of sulfuric acid used and heating time

4, The concentration of the sugar solution is 5mg/100ml

II Determination method of reducing sugar

reducing sugar includes glucose, fructose and maltose. There are bruised aldehyde stems in glucose molecules, bruised ketone stems in fructose molecules, and bruised semicarboxylic stems in lactose and

maltose, so they all have reducibility. When determining reducing sugars, generally when determining total sugars, all methods of hydrolyzing sugars into converted sugars and then determining

can be used to determine reducing sugars

(I) ferring capacity method

1 The principle, reagents and methods of this method are the same as those of total sugar determination. Only the sample solution does not need to be over converted, but directly take the filtrate for titration. The filtrate is titrated, and the reducing sugar content in the filtrate is 0 5% is better, and it can be adjusted by increasing or decreasing the sample volume or changing the dilution multiple. Theoretically, the equivalent amount of reducing sugar when 10ml of ferring's a and B solutions are mixed is as follows:

glucose (anhydrous) fructose or transformed diabetes 0.0500 g

milk diabetes 0.0678 g

maltose 0.0807 g

2 reagent

(1) ferring's a solution, weigh 69.8g CP copper sulfate in 100ml water, filter it for standby

(2) ferring's B solution, weigh concentrated sodium zinc and 100gcp NaOH in 1000ml water.The, Filtration standby

3 method

weigh sample G: preparation and conversion are the same as potassium ferricyanide method. Pour the sample solution into the burette, suck a and B solution for pre titration

pre titration:

suck 5ml of a and B solution respectively, add 15ml of sample solution from the burette, heat it to boiling, continue to drop the sample solution until the blue color changes, add 3 drops of methylene blue, titrate to the end point within 1 minute

when the end point is reached, slightly excess invert sugar will reduce the blue methylene blue chromosome to a colorless Leucosome, and show the red color of cuprous oxide, The products of heating sugar under dealkaline conditions are complex

dealkalinization interrupt cracking is due to different alkalinity, different heating time, and different production of fragments. This kind of fragment brings errors to the subsequent titration. Moreover, this kind of fragment has no relationship with the amount of sugar. Therefore, lanecrol Eynon method makes a data retrieval table

Official titration:

suck 5ml of a and B solution respectively in a triangular flask, adding 0 less than the predetermined amount 0ml of sample solution is required to boil for 1 minute within 2 minutes, add 3 drops of indicator, titrate with sample solution, and the blue color disappears

the total boiling time is 3 minutes, that is, the titration is completed in 3 minutes

calculation:

reducing sugar = (f V2)/(W V1) 100

f: the number of times of sugar conversion, that is, the number of milligrams of converted sugar equivalent to 10ml of Feilin's test solution,

v1: the total volume of sample test solution

v2: the titration amount of sample test solution

w: the weight of sample

when measuring lactose products, if the content ratio of sucrose to lactose exceeds 3:1, Then it should be calculated after adding the correction value in the relevant table (table in the textbook) to the titration.

our example is as follows:

if the standard fructose solution degree is 262.5mg sugar per 100ml solution. For 10ml Feilin test solution, it can be found that the titration of fructose solution should be 20ml. If it is not 20ml, calculate the correction of liquid a and B first. Then calculate

for example, the concentration of standard sugar solution is 199.3ml of sugar per 100ml of solution. For 10ml of solution a and solution B, it is found that the titration amount of sugar solution should be 25.00ml. If there is any discrepancy, it can be corrected. If the requirement is not high, the correction step can be omitted, but if 1% of the measurement error is required, the correction is omitted. In addition, sometimes the sugar content of the sample is not calculated according to the key table, but the solution a and B are calibrated to determine the equivalent reducing sugar content. This error is 0.5%. Next, let's talk about the standard quantity of a and B liquid

accurately weigh, dry and cool, get 1.5g of A.R sucrose, dissolve it with water, weigh 50ml in a 250ml volumetric flask, add 5ml of HCl to a 100ml quantitative flask, and then add a degree Celsius Water Margin for 15 minutes, cool it, neutralize it with 30%naoh, and then absorb a and B liquid accurately, 5ml each in a triangular flask, add about 50ml of water, heat three glass beads, keep it boiling for 1 minute, add 1 drop of indicator, and then boil for 1 minute, and titrate it with sugar immediately until the blue color fades, When red appears, it is the end point

formal titration. First add about 0.5ml less sugar than the pre dripping timing, boil for 1 minute, add 1 drop of indicator, and then boil for 1 minute, and continue to drip to the end point

calculation: a = w*v/500 0.95

a: equivalent to 10ml of fillings a The amount of invert sugar in solution B

w: weigh the quality of sucrose

v: titrate the amount of sucrose

500: dilution ratio

0.95: convert the equivalent number

finally calculate:

total sugar (reducing sugar) is calculated as invert sugar% = (a*1000/w*v) *100

a: the same as above

w: the amount of sample preparation

v: sample consumption during titration

1000: is the dilution multiple (100/50*500)

1 The zigzag experiment is often used to evaluate the zigzag strength and plastic deformation of materials in production. The purpose is to have certain requirements for the concentration of reducing sugar in the sample solution (about 0.1%), and the consumption volume of the sample solution should be similar to that of the calibration glucose standard solution during the determination. Through the determination, we can know whether the sample concentration is appropriate, and if the concentration is too large or too small, we should adjust it, Make the consumption of sample solution about 10 ml during determination; Second, the approximate consumption of this solution can be known through measurement, so that during the formal titration, about 1ml of sample solution less than the actual consumption can be added in advance, leaving only about 1ml of sample solution to be added at the time of continuous dripping, so as to ensure that the continuous titration work can be completed within 1 minute and the accuracy of prediction can be submitted

2. This reality