Determination of reducing sugar in candy (2)

6.
Result calculation

The content of reducing sugar (calculated as glucose) in the sample is calculated according to formula (2):

Where x——the content of reducing sugar (calculated as glucose) in the sample, g/100g

m ₁ ——10mL alkaline copper tartrate solution (half each of A and B) is equivalent to the mass of reducing sugar (calculated as glucose), mg

V——The average volume of sample solution consumed during measurement, mL

m——the mass of the sample, g

250——Total volume of sample solution, mL

When the concentration is too low, the content of reducing sugar in the sample is calculated according to formula (3):

Where x——the content of reducing sugar in the sample (calculated as glucose), g/100g

m——the mass of the sample, g

10——The volume of sample solution added during measurement, mL

250——Total volume of sample solution, mL

m ₂ ——The difference between the volume of glucose standard solution consumed during calibration and the volume of glucose standard solution consumed after adding 10mL sample solution is equivalent to the mass of reducing sugar (that is, the mass of reducing sugar contained in 10mL sample solution), mg

m _{2 is} calculated according to formula (4):

m ₂ =p×(V ₁ -V ₂ ).
.
.

.
.

Where p——the mass concentration of reducing sugar (calculated as glucose) standard solution, mg/mL

V ₁ ——The average consumption volume of reducing sugar standard solution during calibration, mL

V ₂ ——The volume of reducing sugar standard solution consumed after adding 10mL sample solution, mL

When reducing sugar content is greater than or equal to 10g/100g, the calculation result retains three significant digits; when reducing sugar content is less than 10g/100g, the calculation result retains two significant digits
.
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed arithmetic 10% of the average

.

7.
Tips

(1) The direct titration method is the first method in the GB/T5009.
7-2008 standard.
It is currently the most commonly used method for the determination of reducing sugars.
It is characterized by a small amount of reagents, simple and fast operation, and an obvious titration end point.
Determination of reducing sugars in similar foods
.
However, when measuring dark samples (such as soy sauce, dark juice, etc.

), the end point is difficult to judge due to pigment interference, which affects the accuracy

(2) Alkaline copper tartrate solution A and solution B should be prepared and stored separately, and mixed before use
.
Otherwise, the potassium sodium copper tartrate complex will slowly separate out the cuprous oxide precipitate under alkaline conditions for a long time, which will reduce the effective concentration of the reagent

.

Cu ₂ O↓+K ₄ Fe(CN) ₆ +H ₂ O=K ₂ Cu ₂ Fe(CN) ₆ +2KOH

(3) Keep boiling during titration.
First, it can accelerate the reaction speed of reducing sugar and Cu ²⁺ ; second, the boiling of the reaction liquid makes the rising steam prevent air from entering the solution, avoiding methylene blue and cuprous oxide from being oxidized and increasing sugar consumption.

.
Because the color reaction of methine blue is reversible, the colorless reduced methylene blue will be oxidized to blue oxidized methylene blue when it meets oxygen in the air

.

(4) When the titration reaches the end point, the methylene blue is reduced by reducing sugars, the blue disappears, and it is light yellow.
After standing for a period of time, it is exposed to oxygen in the air, the methylene blue is oxidized, and the color of the solution turns blue again.
Titrate again
.

(5) This method requires very strict titration operating conditions.
The method for determining the intensity of the heat source, heating time, titration speed, conical flask wall thickness, approximate volume of pre-filling, and endpoint must be as consistent as possible in the determination
.
If the entire titration work must be controlled within 3 minutes, most of the sample solution required for the titration should be added to the alkaline copper tartrate solution to azeotropically react, leaving only about 1 mL for the titration and determining the end point.

In order to reduce operating errors; the operating conditions for the calibration of alkaline copper tartrate solution, sample solution prediction and measurement should all be consistent; do not shake the conical flask at will during titration, let alone remove the conical flask from the heat source for titration , To prevent air from entering the reaction liquid

(6) The difference in sample liquid consumption in parallel tests should not exceed 0.
1 mL
.

(7) Prepare standard solutions with glucose, fructose, lactose, and maltose standards respectively to titrate the same amount of calibrated alkaline copper tartrate solution.
The volume of the consumed standard solution is different, indicating that even if it is the same reducing sugar, the physical and chemical properties There are still differences in the above, so the results of reducing sugars only reflect the overall situation of the sample, and are not completely equal to the sum of the reducing sugar content
.
If the sample is known to contain only a certain reducing sugar, the reducing sugar should be used as the standard, and the result is the reducing sugar content

.

(8) In this method, the solution in the burette is changed from the reducing sugar standard solution to the sample solution, which is troublesome.
For the back titration method when measuring the sample solution with dilute concentration, the solution in the burette does not need to be replaced.
Standard reducing sugar solution is sufficient
.
Therefore, the back titration method can be used to determine the reducing sugar content of the sample solution.

The specific operation is: directly add a certain volume of the sample solution when predicting and determining, and then titrate with the standard reducing sugar solution; at the same time, calculate the consumption when calibrating the alkaline copper tartrate solution The difference between the volume of the reducing sugar standard solution and the volume of the reducing sugar standard solution consumed after adding a certain volume of sample solution is equivalent to the mass of reducing sugar (that is, the mass of reducing sugar contained in a certain volume of sample solution), and then proceed according to formula (3) For calculation, replace 10 (mL) in formula (3) with the volume of the sample solution added

(9) Treatment methods of different samples:

①General food: Weigh 2.
5～5g of crushed solid sample or 5～25g of mixed liquid sample, accurate to 0.
001g, put it in a 250mL volumetric flask, add 50mL water, and slowly add 5mL zinc acetate solution And 5mL potassium ferrocyanide solution, add water to the mark, mix well, let stand for 30min, filter with dry filter paper, discard the initial filtrate, and take the subsequent filtrate for later use
.

②Alcoholic beverages: weigh about 100g of the mixed sample, accurate to 0.
01g, place it in an evaporating dish, neutralize it with sodium hydroxide (40g/L) solution to neutrality, and evaporate to the original volume on a water bath After 1/4 of the volume, transfer it into a 250mL volumetric flask, and follow the procedure in ① from "slowly adding 5mL zinc acetate solution.
.
.
"
.

③Foods containing a lot of starch: Weigh 10-20g crushed or mixed sample, accurate to 0.
001g, and place it in a 250ml volumetric flask, add 200ml of water, heat it in a water bath at 45℃ for 1h, and vibrate from time to time Shake
.
After taking it out and cooling, add water to the mark, mix well, let it stand, and settle
.
Pipette 200 mL of the supernatant liquid into another 250 mL volumetric flask, and follow the procedure in ① from "slowly adding 5 mL of zinc acetate solution.
.
.
"
.

④Carbonated beverages: Weigh about 100g of the mixed sample, accurate to 0.
01g, place the sample in an evaporating dish, stir in a water bath to remove carbon dioxide, transfer it to a 250mL volumetric flask, and wash the evaporating dish with water , The lotion is combined into the volumetric flask, and then add water to the mark, after mixing, set aside
.

(10) Clarification of the sample extract: The pretreated sample also contains pigments, a small amount of protein, soluble pectin, organic acids, amino acid tannins, etc.
, which will cause the extract to become turbid and affect the determination, so a certain amount of clarifying agent must be added Mix the precipitate and filter to obtain a clear extract after standing
.

Commonly used clarifying agents are neutral lead acetate (saturated concentration is 30%, the use concentration is self-determined
.
Used to remove protein , soluble pectin, organic acids, tannins, etc.
, but also to condense other colloids), zinc acetate and ferrocyanide potassium solution (for removal of clarified protein ), 0.
3mol / L copper sulfate , and 1mol / L aqueous sodium hydroxide solution (for clarifying remove protein) and the like
.

When using a clarifying agent, the choice should be made according to the type of sample liquid, the type and content of interference components, and the analytical method used should also be considered
.
For example, copper sulfate-sodium hydroxide solution cannot be used for direct titration to avoid introducing Cu ²⁺ into the sample solution ; for potassium permanganate titration, zinc acetate-potassium ferrocyanide solution cannot be used to avoid Fe ²⁺ is introduced into the sample solution
.
In addition, the amount of clarifying agent should be appropriate
.
Too little dosage will not achieve the purpose of clarification; too much dosage will cause errors in the test results
.
For example, when using lead salt clarifiers, excessive lead will distort the analysis results, and even during hydrolysis conversion heating, lead will react with sugars to produce lead sugars, causing errors
.
To minimize the error, excess lead needs to be removed by adding a de-lead agent to improve the accuracy of the analysis results
.
Commonly used lead removal agents are potassium oxalate , sodium oxalate, sodium sulfate , disodium hydrogen phosphate, etc.
The amount should not be too much, and should be used as little as possible on the premise of ensuring the complete precipitation of lead
.