Determination of total amino acids

The hydrolysis or enzymatic hydrolysis of the protein can be changed from a macromolecule to a small pro component. For example, the hydrolyzed product becomes an amino acid after being subjected to hydrazine, peptide, etc., and the amino acid is a basic substance constituting prozui. The pro-hydrolyzed pro and the pre-hydrolyzed pro are very different in physical properties, chemical structure and degree of absorption and digestion. The difference is closely related to the degree of hydrolysis. The degree of hydrolysis can be known by analyzing the amino acid content. It is possible to evaluate the nutritional value of the food. Amino acid is not a simple substance. It can directly determine 17 kinds of amino acids by using an amino acid analyzer (the instrument is expensive and cannot be widely used). For food, sometimes many kinds of amino acids can exist in one food at the same time, so it needs to be determined. The total amount of amino acids, which cannot be expressed as a percentage of amino acids, can only be expressed as a percentage of nitrogen (amino acid nitrogen) contained in the amino acid. Of course, if the food contains only one amino acid, such as glutamic acid in MSG, The amino acid content can be calculated from the nitrogen content. In the evaluation of the nutritional value of protein, in addition to measuring the content of pro, amino acid nitrogen content, it is necessary to separate and identify various amino acids, especially quantitative and qualitative analysis of eight essential amino acids.

one. Single indicator formaldehyde titration:

1 Principle: Amino acid has two properties of acid and base, because amino acid contains -COOH group, while -COOH group shows acidity, and -NH2, -NH2 shows basicity, due to the interaction of -COOH group and -NH2, amino acid It becomes a neutral inner salt. When adding formaldehyde solution, -NH2 combines with formaldehyde, its basicity disappears, and the existence of internal salt is destroyed. The base can be used to titrate the -COOH group, and the amount of amino acid is determined by indirect method. Three forms exist.

When the -COOH group is completely neutralized with a base, the pH is 8.4 to 9.2.

2 reagents:

(1) 40% neutral formaldehyde solution: using thymol oxime as an indicator, the formaldehyde was neutralized with a 1 N NaOH solution (light blue). (2) 0.1% thymolphthalein ethanol solution.

(3) 0.100 N NaOH standard solution.

3 steps:

Weigh about 20mg of amino acid → in a beaker (if the solid sample is added with 50ml of water) → add two drops of indicator → titrate with 0.1N NaOH solution to light blue → add neutral formaldehyde 20ml → shake → stand for 1 minute → At this time, the blue color should disappear → then titrate the blue color with 0.1N NaOH solution, and record the number of milliliters of alkali consumed by the two titrations.

Calculation: amino acid nitrogen = (N × V × 0.014 × 100) / W × 100

two. Double indicator formaldehyde titration

1 principle:

Same as the monochromatic method, except that two indicators are used in this method. From the analysis results, the dual indicator formaldehyde titration method and the nitrous acid nitrogen volume method (this method is complicated, but accurate, not introduced) is similar. The color titration method is slightly lower, mainly because the single indicator formaldehyde titration method is based on the pH value of the amino acid solution as the end point of neutral red, and the pH value is 7.0. From the theoretical calculation, the two-color titration method is more accurate than the monochrome titration method.

2 Reagents: same as single indicator, one more 0.1% neutral red (50% ethanol solution)

3 steps:

Take the same two samples → respectively in a 100ml triangle bottle → one plus neutral red 2 drops → titrate the end point with 0.1N NaOH (from red to amber), record the amount, another plus thymol phenol ethanol solution 3 drops Neutral formaldehyde 20ml → Shake → Use 0.1N

NaOH drops to light blue.

Calculation:

Amino acid nitrogen = (N × (V2 - V1) × 0.014 × 100) / W × 100

V1 - the volume consumed by the lye when neutral red is used as an indicator

V2——Use of thymol and ethanol solution as indicator

0.014 - milligram equivalent concentration of nitrogen

Precautions:

(1) Monochrome indicator formaldehyde titration method, completely neutralized with alkali - pH of 8.5-9.5 when COOH group

(2) The color of the sample is determined to be dark, and it should be titrated after decolorization with activated carbon.

three. Ninhydrin colorimetric method:

1 Principle: Amino acid can form a blue-violet compound with ninhydrin in a certain pH range. It can be quantitatively determined by colorimetry.

2 reagents:

(1) The preparation method of the phosphate buffer (pH 8.04) is as follows:

It is called 4.5350 g of potassium dihydrogen phosphate. Constant volume 500ml

Said NAH2PO4·12H2O 11.9380g dissolved into a volume of 500ml

A buffer solution of pH 8.04 is prepared by mixing 10 ml of potassium dihydrogen phosphate with 190 ml of disodium hydrogen phosphate.

(2) 2% ninhydrin solution:

Weigh glycerol 1g → dissolve in 35ml hot water → add 40mg stannous chloride (SnCl2 · H2O) and stir (preservative) →

Overnight in cold and dark places → constant volume 50ml

1 amino acid standard solution

Weigh dry amino acid (such as isoleucine) 0.2000g → dissolve and make up 100ml → shake → suck 10ml in another 100ml volumetric flask, make up to 100ml, that is, get 200Ug/ml standard solution

3 operation method:

(1) Draw a standard curve:

Take 7 25ml volumetric flasks, take the standard solution 0 0.5 1.0 1.5 2.0 2.5 3.0ml in 7 25ml volumetric flasks, add water to the volume of 4ml, then add 1ml of ninhydrin and buffer, heat in water bath for 15 minutes, cool After the constant volume of 25 ml, let stand for 15 minutes, measure the extinction value at 570 nm, and draw a standard curve.

(2) Sample measurement:

Take sample 5.00~10.0g (liquid sample 5-10ml)→in the beaker→add 50ml water and activated carbon about 5g→heat filter→washing activated carbon with 30-40ml hot water→sucking clarified liquid 1~4ml→adding three The ketone and buffer were heated in a 1 ml water bath for 15 minutes, cooled to a constant volume, and allowed to stand for 15 minutes to measure the extinction value at 570 nm, and the amino acid content was calculated by the following formula.

Amino acid content (mg/100g)=C/(W×1000)×100

C——the amount of amino acid (Ug) obtained from the standard curve

W——measured that the sample solution is equivalent to the amount of the sample (g)

Precautions:

The ninhydrin is easily oxidized to light red or deep red by sunlight, temperature, humidity, air, etc., and should be purified before use. The method is as follows:

Take 10g ninhydrin in 40ml hot water, add one gram of activated carbon, shake and let stand for 30 minutes, filter, filter the filtrate into the refrigerator, blue crystal appears, filter, wash the crystal with 2ml cold water, dry Dry in a dish and bottled for use.

Thinking questions:

1 Describe the matters that must be paid attention to during the digestion of the sample in the determination of the protein. What changes in the color of the contents during the digestion process? why?

2 Why do you need to add sodium hydroxide before digesting the sample? What happens to the solution at this time? why? If there is no change, what is the problem? What measures must be taken?

3 What role does copper sulfate and potassium sulfate play in the determination?
4 Describe the principle of determination of amino acid nitrogen. ·

5 Why do you want to multiply the protein coefficient in the calculation of the results?

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