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Extraction method of biotin in molasses and thin layer chromatography (TLC) scanning detection method thereof

A technology of thin-layer chromatography and extraction method, which is applied in the field of extraction of biotin in molasses, can solve the problems of time-consuming, high cost, and laborious operation, and achieve the effect of simple operation

Inactive Publication Date: 2015-06-17
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, to the detection of biotin in molasses, microbial method and high-performance liquid chromatography are the most commonly used, but there are following defects: microbial method will bring larger detection error because most strains are not specific, and The whole operation process is time-consuming and laborious
High-performance liquid chromatography requires a large investment, a large amount of solvent, and high cost. Because molasses is thick, dark in color, and contains many impurities, sample processing, that is, the extraction and purification of biotin, is difficult, and high-performance liquid chromatography is difficult for samples. The purity requirements are relatively high, and it is not suitable to use this method for detection of molasses samples with complex components, high pigment content, and large interference from impurities.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 extracts biotin from molasses, and detects

[0023] Proceed as follows:

[0024] (1) Preparation of sample crude solution:

[0025] Take 10g of molasses, add 20ml of N,N-dimethylformamide (DMF), stir and filter evenly, rinse the filter residue with 20ml of absolute ethanol and filter. Add 100ml of absolute ethanol to the filter residue, and ultrasonicate at 78°C, 40Hz, and 400W power for 0.5h. Filter while hot to obtain the filtrate. The filtrate was rotary evaporated to 5ml as the sample crude solution.

[0026] (2) Preparation of phenylhydrazine hydrochloride solution:

[0027] Weigh 0.3 g of phenylhydrazine hydrochloride, add 3 ml of distilled water and heat to dissolve. Weigh 0.45 g of sodium acetate and add 3 ml of distilled water to dissolve it. The two liquids are combined, that is.

[0028] (3) Add 2ml of phenylhydrazine hydrochloride solution to 5ml of crude sample solution, and heat in a water bath at 95°C for 0.5h. A pale yellow turbid li...

Embodiment 2

[0035] Embodiment 2 extracts biotin from molasses, and detects

[0036] Proceed as follows:

[0037] (1) Preparation of sample crude solution:

[0038] Take 10g of molasses, add 20ml of DMF, stir and filter evenly, rinse the filter residue with 30ml of absolute ethanol and filter. The filter residue was added to 70ml of absolute ethanol. Sonicate at 75°C, 40Hz, 450W for 0.5h. Filtrate immediately while hot. The filtrate was rotary evaporated to 5ml as crude sample solution.

[0039] (2) Preparation of phenylhydrazine hydrochloride solution: with embodiment 1.

[0040] (3) Take 2ml of phenylhydrazine hydrochloride solution and add it to 5ml of crude sample solution, and heat in a water bath at 90°C for 0.5h. Then freeze at -20°C for 35 minutes. Take out and remove the lower precipitate to obtain the supernatant.

[0041](4) The preparation of the adsorbent was the same as in Example 1, and 0.015 g of the adsorbent was added into the supernatant of (3) to stir and adsorb...

Embodiment 3

[0044] Embodiment 3 extracts biotin from molasses, and detects

[0045] Proceed as follows:

[0046] (1) Preparation of sample crude solution

[0047] Take 10g of molasses, add 20ml of DMF, stir and filter evenly, rinse the filter residue with 40ml of absolute ethanol and filter. The filter residue was added to 80ml of absolute ethanol. Ultrasound at 70°C, 40Hz, and 500W power for 0.5h. Filter while hot to obtain the filtrate. The filtrate was rotary evaporated to 8ml as crude sample solution.

[0048] (2) Preparation of phenylhydrazine hydrochloride solution: with embodiment 1.

[0049] (3) Add 2ml of phenylhydrazine hydrochloride solution to 8ml of crude sample solution, and heat in a water bath at 95°C for 0.5h. Then freeze at -26°C for 30 minutes. Take out and remove the lower precipitate to obtain the supernatant.

[0050] (4) The preparation of the adsorbent was the same as in Example 1, and 0.02 g of the adsorbent was added into the supernatant of (3) to stir an...

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Abstract

The invention discloses an extraction method of biotin in molasses. The method is characterized by using molasses as a raw material, carrying out extraction with N,N-dimethylformamide, washing with absolute ethyl alcohol, carrying out extraction with a phenylhydrazine hydrochloride solution and adsorbing with an adsorbent, thus finally obtaining a sample solution containing biotin. The invention also provides a thin layer chromatography (TLC) scanning detection method of biotin. The detection method comprises the following steps: (1) dropping the sample solution to be detected on a silica gel plate, developing the sample solution to be detected in a developing cylinder to which a developer is added, and then taking out the thin layer plate and putting the thin layer plate in a fume hood to be aired; (2) spray-dyeing the developed and aired plate with a dyeing solution to obtain spots, in obvious contrast to the background, of standard substances and samples; (3) carrying out TLC scanning by utilizing a thin layer scanner in the absorption wavelength of 530nm, and computing the mass concentration of biotin in the sample solution. The methods have the advantages of simplicity, quickness, accuracy, reliability, stability and the like.

Description

technical field [0001] The invention relates to an extraction method of biotin in molasses and a thin-layer chromatography scanning detection method thereof. Background technique [0002] Biotin is one of the essential vitamins in life activities. It is a cofactor of many enzymes in the body. It participates in the body's carboxylation, decarboxylation and dehydrogenation reactions, and participates in the metabolism of sugar, fat and protein. Biotin has been widely used in food, chemical industry, medicine, animal husbandry, biological fermentation and other fields. In the production of glutamic acid fermentation, the control of the amount of biotin directly affects the growth, proliferation, metabolism, cell wall, cell membrane permeability and the rate of glutamic acid production of the producing bacteria, because most glutamic acid bacteria are Biotin-deficient, so the content of biotin plays a vital role in the production, so it is of great significance to carry out re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/90G01N1/28G01N1/34
Inventor 刘代成
Owner SHANDONG NORMAL UNIV