UV-1100 spectrophotometric method for the determination of calcium, iron and zinc in milk - Master's thesis - Dissertation

Determination of calcium, iron and zinc in milk by spectrophotometry

Spectrophotometric method for the determination of calcium, iron and zinc in milk. Key words: spectrophotometry; milk; protein; calcium; iron; zinc; analysis instrument ; V-1300 Abstract: milk nutrition is very high, Rich in protein and trace elements such as calcium, iron and zinc, it is one of the main nutritious foods in people's lives. Therefore, it is necessary to analyze the nutrient content in milk. In the laboratory, the simple and scientific method was used to analyze the content of some components in a brand of milk. 1. Experimental purpose The content of protein, calcium, iron and zinc in a certain milk was measured by spectrophotometry. 2. Experimental principle The main protein in milk is casein with a content of about 35g/L. Casein is a mixture of some phosphorus-containing proteins with an isoelectric point of 4.7. When the pH of the milk is adjusted to 4.7 by the principle of the lowest solubility at the isoelectric point, casein precipitates. The precipitate is washed with ethanol to remove the lipid impurities to obtain pure casein. Biuret (NH ₂ CONHCONH ₂ ) reacts with copper sulfate in an alkaline solution to form a purple-red compound called biuret reaction. The protein molecule contains many peptide bonds which can react with Cu ²⁺ in an alkaline solution. Fuchsia compound. Within a certain range, the depth of its color is proportional to the protein concentration. Therefore, the protein concentration can be determined by colorimetry. The biuret method is one of the commonly used methods for determining protein concentration. The operation is simple, rapid, and less affected by the nature of the protein species, but the sensitivity is poor and the specificity is not high. In addition to -CONH-, in addition to this reaction, groups such as -CONH ₂ , -CH ₂ NH ₂ , and -CS-NH ₂ also have this reaction. Calcium is closely related to the health of the body. Calcium is added to the bone to support the body. It is also involved in the metabolism of the human body. It is the main cation of the cell and one of the most active elements of the human body. Calcium deficiency can cause rickets in children, adolescent growth retardation, and pregnant women. Hypertension, osteoporosis in the elderly. Calcium deficiency can also cause a variety of allergic diseases such as neuropathy, diabetes, and traumatic bleeding. Calcium supplementation is getting more and more attention. Milk contains calcium that is easily absorbed by the body. Some milk products also add calcium to become calcium milk. For the content of calcium in liquid milk, direct measurement can be carried out by EDTA method. Considering that the milk contains interfering ions such as Fe ³⁺ and Al ³⁺ , a small amount of triethanolamine can be added to eliminate them. The pH is adjusted to 12-13, and the chrome blue black R is used as an indicator. The indicator and the calcium form a red network. The compound, when titrated to the metering point with EDTA, freed the indicator and the solution appeared blue. When a beam of light passes through a light absorbing material (usually a solution), the solute absorbs the light energy and the intensity of the light is diminished. Absorbance is a physical quantity used to measure the extent to which light is absorbed. The absorbance is indicated by A. A=abc, where a is the absorption coefficient, the unit is L/(g·cm), b is the thickness of the liquid layer (usually the thickness of the cuvette), the unit is cm, c is the concentration of the solution, and the unit of g/L affects the absorbance. Is b and c. a is a constant related to the solute. In addition, temperature affects A by affecting c. Symbol A indicates the degree of absorption of light by a substance. O-phenanthroline is also called phenanthroline. It is a highly sensitive developer for the determination of trace Fe ²⁺ . Under the condition of pH 2～9, Fe ²⁺ and phenanthroline form a stable orange-red complex. Maximum absorption at 510 nm. The lgk _{stability of} the complex was 21.3 (20 ° C), and the molar absorptivity was ₅₁₀ = 1.1 × 10 ⁴ L·mol ^-1 · cm ^-1 . Before color development, the Fe ³⁺ ion is first reduced to Fe ²⁺ with hydroxylamine hydrochloride. The reaction formula is as follows: 2Fe ³⁺ +2NH ₂ OH·HCl = 2Fe ²⁺ +4H ⁺ +2Cl ^- +N ₂ ↑ +2H _{When the 2} O is measured, it is preferable to control the acidity of the solution at a pH of about 5. When the acidity is high, the reaction proceeds slowly; if the acidity is too low, the Fe ²⁺ ion is hydrolyzed, which affects color development. When the iron concentration is within 5.0 mg/mL, the absorbance is linear with the Fe ²⁺ concentration. Bi ³⁺ , Cd ³⁺ , Hg ⁹⁺ , Ag ⁺ , Zn ²⁺ ions precipitate with phenanthroline at pH = 2~9. The Co ²⁺ , Ni ²⁺ , Cu ²⁺ plasma can form a colored complex with o-phenanthroline, so attention should be paid to their interference. Zinc is one of the essential trace elements in the human body. Zinc deficiency can hinder growth and development, and excessive lead can lead to diseases such as stomach cancer. Humans can get a certain amount of zinc from a diet such as milk. In an aqueous solution of pH 4.0-5.5, zinc ions form a red chelate with dithizone (C ₁₃ H ₁₂ N ₄ S), and the molar absorptivity ₅₃₅ = 9.3 × 10 ⁴ L·mol ^-1 · cm ^-1 Colorimetric quantification after carbon tetrachloride extraction. Under the selected pH conditions, a small amount of sodium thiosulfate can be used to mask a small amount of interfering metal ions such as lead, copper, cadmium, cobalt, ruthenium, nickel, gold, palladium, silver, stannous and the like present in the water. 3. Experimental procedure 3.1. Preparation of reagents, preparation of instruments 3.11 Preparation of pH4.7 acetic acid-sodium acetate buffer: First, mix A and B. Liquid A: 0.2 mol/L sodium acetate solution. Solution B: 0.2 mol/L acetic acid solution. Take 6.5461 g of sodium acetate solid dissolved in water and dilute to 100 ml, dilute 5 mL of glacial acetic acid and dilute to 100 ml with water, and mix to obtain 200 mL of acetic acid-sodium acetate buffer solution of pH 4.7. 3.12. Preparation of ethanol-mixture: Mix 25ml of 95% ethanol with 25ml of anhydrous 3.13, biuret reagent: weigh 0.30g of CuS0 ₄ ·5H ₂ O, 0.9g of potassium tartrate and 0.5g of potassium iodide, dissolve and mix separately Add 6 mol·L ^-1 NaOH 10 mL, and finally add water to 100 mL, store in a brown bottle, avoid light, and store for a long time. 3.14, protein standard solution (10mg·mL ^-1 ), weigh 100.0mg of dried bovine serum albumin in a small beaker, dissolve it in a small amount of physiological saline, pour it into a l0mL volumetric flask, rinse the small beaker several times, and pour it together. In the volumetric flask, add saline to the tick mark. Protein sample to be tested: The milk sample is accurately prepared into a certain concentration solution by using physiological saline, and then determined; the prepared casein is accurately prepared into a certain concentration solution by using physiological saline, and then measured. (If it can not be dissolved, add a few drops of 6mol / L NaOH solution, respectively, use l0mL volumetric flask to make up the volume) 3.15, iron standard solution (40.0μg · mL ^-1 ):: accurately weigh 0.0345g of ammonium ferric sulfate [NH ₄ Fe (SO ₄ ) ₂ ·12H ₂ O] 6 mL of 6 mol·L ^-1 HCl and a small amount of water were added to the beaker, dissolved, and made up to 10 mL with pure water. 1.00 mL of this solution contained 40.0 μg of iron. 3.16 Hydroxylamine hydrochloride solution (100 g·L ^-1 ): Weigh 10 g of hydroxylamine hydrochloride (NH ₂ OH·HCl), dissolve in water, and dilute to 100 m (freshly prepared, effective within two weeks) 3.17, o-diazepine Philippine solution (2g·L ^-1 ):: Weigh 0.20g of phenanthroline (C ₁₂ H ₈ N ₂ ·H ₂ O), dissolve it in pure water with 2 drops of concentrated hydrochloric acid, and dilute to 100mL (fresh Preparation, effective within two weeks) 3.18, sodium acetate solution (1.0 mol · L ^-1 ):: Weigh 8.22030 sodium acetate, dissolved in pure water and diluted to 100mL. 3.19, zinc standard solution (1.00μg·mL ^-1 ): accurately weigh 0.0100g of reference zinc in a 100mL beaker, add 6 mol·L ^-1 HCl 0.5mL, immediately cover the surface dish, after the zinc is completely dissolved, Rinse the surface dish and the beaker wall with a small amount of water, transfer the solution into a 100mL volumetric flask, and make up the volume, which is the zinc standard stock solution. Accurately transfer 1mL zinc standard stock solution, dilute to 100.0mL, that is, 1.00μg·mL ^-1 zinc standard solution. 3.20, 0.1% bismuth sulphate carbon tetrachloride stock solution: Weigh 0.10g of dithizone (C ₁₈ H ₁₂ N ₄ S), dissolve it in carbon tetrachloride in a dry beaker, dilute to 100 mL, pour In a brown bottle. This solution is stored in the refrigerator and can be stable for several weeks. 3.21, Dithizone carbon tetrachloride solution: Before use, draw the appropriate amount of dithizone carbon tetrachloride stock solution, dilute about 30 times with carbon tetrachloride, to absorbance of 0.4 (wavelength 535 nm, 1cm cuvette ). 3.22% 25% sodium thiosulfate solution: 64.60 g of sodium thiosulfate (Na ₂ S ₂ 0 ₃ ·5H ₂ 0) was weighed and dissolved in 100 mL of pure water. 3.23, 1+1 ammonia aqueous solution: 10ml ammonia water plus 10ml water diluted 3.24, V-1300 visible spectrophotometer 3.2, milk casein extraction 3.21, casein crude 50mL fresh milk heated to 40 °C. Slowly add 50 mL of acetic acid buffer preheated to 40 ° C and pH 4.7 with stirring. Adjust the pH to 4.7 with a precision pH test paper or a pH meter. The suspension was cooled to room temperature. Centrifuge for 15 minutes (3000 r · min ^-1 ). Discard the clear solution to obtain crude casein. 3.2.2 Purification of casein The precipitate was washed three times with water, centrifuged for 10 minutes (3000 r·min ^-1 ), and the supernatant was discarded. 30 mL of ethanol was added to the precipitate, stirred for a while, and the entire suspension was transferred to a Buchner funnel for suction filtration. The precipitate was washed twice with an ethanol-mixture. Finally, the precipitate was washed twice and drained. The precipitate was spread on a watch glass and air dried; the casein was pure. 3.3. Determination of casein content 3.11. Drawing of standard curve and determination of sample Take 8 test tubes and operate according to the following table. The tubes were mixed and placed in a 37 ° C water bath for 20 min. 540nm colorimetric, zero point with a blank tube, read the absorbance value of each tube. 3.32. Calculate the absorbance value on the computer as the ordinate and the protein concentration as the abscissa to draw the standard curve. The protein concentration (g/L) of the sample to be tested is found from the standard curve, and the protein concentration in the milk is determined. The true content of casein in 100 mL of fresh milk was calculated from this concentration. 3.4 Determination of calcium content 3.11 Determination of calcium content of calcium preparations Transfer 25ml of fresh milk and calcium milk accurately, transfer them to a 100mL volumetric flask, dilute to the mark with water, and shake. Accurately remove 20.00mL of the above test solution, add 5mL of triethanolamine solution, 4mL of 5mol·L ^-1 NaOH, add 20mL of water, shake well, add RB blue black R8~10 drops, titrate with 0.01mol·L ^-1 EDTA standard solution Add 2 to 3 drops of chrome blue black near the end point. When the solution turns from red to blue, the end point is calculated. According to the volume of EDTA consumed, the calcium content in fresh milk and calcium milk is calculated (g/100 mL). And compare it with the content indicated on the package. 3.42. Precautions The amount of fresh milk and calcium milk is determined by the amount of calcium, and the volume of 0.01 mol·L ^-1 EDTA is 20 to 30 mL. Milk, because calcium milk is milky white, the end point color change is not obvious, add 2 to 3 drops of indicator when approaching the end point. 3.5 Determination of iron content 3.51. Drawing of standard curve Take 6 mL colorimetric tube, prepare iron standard series according to the following table and record the measured data (Note: After adding hydrochloric acid, dry it and place it for 2 min, then proceed One step operation), after adding the reagent, shake it, place it in a 37 ° C water bath for 15 min, take the blank solution as a reference, and measure the absorbance value of each solution at 510 nm. Take 25 mL of colorimetric tube and measure the absorbance at 510 nm according to the above table and the above method. 3.52. Calculate the absorbance value as the ordinate on the computer and the standard curve on the abscissa with the concentration of iron mass concentration (μg·mL). The iron content in fresh milk (mg/100 mL) was calculated using a standard curve. 3.53. Determination of the sample 50 ml of fresh milk was added dropwise to the hot pot to avoid boiling evaporation. After removing the water, it is heated to 450~550 °C for 1 hour. After cooling, add 1 mL of concentrated nitric acid to evaporate near-dry, and then burn again at 450~550 °C (can not be spattered). Dissolve the white residue with a small amount of dilute nitric acid, dilute to the mark in a 10mL volumetric flask, adjust the pH to 7~8 with dilute sodium hydroxide solution. 3.6 Determination of zinc content 3.61 Standard curve drawing and sample determination 70ml fresh Add one drop of milk to the hot pot to avoid boiling evaporation. After removing the water, it is heated to 450~550 °C for 1 hour. After cooling, add 1 mL of concentrated nitric acid to evaporate near-dry, and then burn again at 450~550 °C (can not be spattered). Dissolve the white residue with a small amount of dilute nitric acid, dilute to the mark in a 10 mL volumetric flask, and adjust the pH to 7~8 with dilute sodium hydroxide solution. Dilute to 25 ml with distilled water. Accurately absorb 1 ml of water and dilute to 10.0 mL with pure water. Another 8 separatory funnels were added, and zinc standard solutions 0, 0.50, 1.00, 1.50, 2.00, 3.00, 4.00, and 5.00 mL were sequentially added, and pure water was added to 10 mL each. Add 1 drop of methyl red indicator to the water sample and standard series separatory funnel, adjust the solution with 1+1 ammonia water to be yellow, and then add acetic acid solution to red (pH about 4.4). Add 5 mL of carbon tetrachloride, shake to extract methyl red, and discard the organic phase. Add 5.0 mL of buffer solution to each separatory funnel, add 1.0 mL of sodium thiosulfate solution, mix well, add 10.0 mL of dithizone carbon tetrachloride solution, shake vigorously for 4 min, and let stand for stratification. The water in the neck of the separatory funnel was wiped off with absorbent cotton or a finely packed filter paper, and 2 to 3 mL of the organic phase initially discharged was discarded, and the subsequently discharged organic phase was collected in a dry 10 mL colorimetric tube. The absorbance of the sample and the standard series solution was measured at a wavelength of 535 nm using a 1 cm cuvette with carbon tetrachloride as a reference. Draw a calibration curve and find the zinc content in the sample tube on the curve. Note: 1 This method of zinc testing should pay special attention to prevent external pollution, and also avoid operating in direct sunlight. 2 The addition of sodium thiosulfate not only masks the interference of metal ions, but also acts as a reducing agent to protect the dithizone from oxidation. Since sodium thiosulfate can also complex with zinc ions, the amount of sodium thiosulfate added to the standard series should be the same as that of the sample tube. 3 The shaking time must be sufficient, because sodium thiosulfate is a strong complexing agent, and only zinc can be extracted from the complex Zn(S ₂ O ₃ ) ^2- to be extracted by dithizone carbon tetrachloride solution. . The release of zinc is slower, so the oscillation time must be 4 minutes, otherwise the extraction is not complete. In order to make the sample and the standard extraction rate consistent, the shaking strength and the number of times should be consistent. 4. Data processing 4.1 Extraction of casein in milk, casein content: 6.1354g/100ml yield = 4.2 Determination of casein content 4.11, drawing of standard curve and determination of sample