CN103926369B - Extraction method of biotin in molasses and thin layer chromatography (TLC) scanning detection method thereof - Google Patents
Extraction method of biotin in molasses and thin layer chromatography (TLC) scanning detection method thereof Download PDFInfo
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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
The present invention relates to extracting method and the thin-layer chromatography scanning detection method thereof of biotin in a kind of molasses.
Background technology
Biotin is one of vitamin required in vital movement, is the accessory factor of many enzymes in body, participates in the carboxylation of body, decarboxylation and dehydrogenation reaction, participates in the metabolism of carbohydrate, fat, protein three major nutrient.Biotin has been widely used in the fields such as food, chemical industry, medicine, herding, biofermentation.In glutamic acid fermentation is produced, the control of biotin consumption directly affect produce bacterium growth, propagation, metabolism and cell membrane, cell permeability of the membrane and glutamic acid acid production rate height, because most of glutamic acid producer is all biotin deficiency, so the content of biotin plays vital effect to production, the research therefore carried out biotin detects has great importance.
Molasses are a kind of thickness, pitchy, the dynamic material in semi-fluid, sugar industry to be used for again the waste material of boiling sugar products, but the rich content of biotin in molasses, can be used as raw material and the adjuvant of good glutamic acid fermentation, it is the main source of biotin during current glutamic acid is produced.But due to the wherein content of biotin and the source, production batch etc. of molasses closely related, therefore, for steady production, improve the composition change that acid production rate must pay close attention to molasses, Accurate Determining biotin content wherein, to realize the control to biotin consumption in glutamic acid fermentation.
The detection method of the biotin of prior art bibliographical information has microbial method, fluorescence method, spectrophotometric method, euzymelinked immunosorbent assay (ELISA), vapor-phase chromatography, high performance liquid chromatography, thin layer chromatography scanning etc.At present, to the detection of biotin in molasses, microbial method and high performance liquid chromatography are the most conventional, but there is following defect: microbial method due to most of bacterial strain be not narrow spectrum, therefore can bring larger metrical error, and whole operating process is time-consuming, effort.High performance liquid chromatography investment is large, solvent load is large, cost is higher, again due to molasses thickness, color depth, more containing impurity, therefore the extraction purification process difficult of sample preparation and biotin, and high performance liquid chromatography is higher again to the purity requirement of sample,, impurity high to complicated component, pigment content disturbs large molasses sample, should not detect by this method.
Summary of the invention
For above-mentioned prior art, the invention provides the extracting method of biotin in a kind of molasses, present invention also offers a kind of thin-layer chromatography scanning detection method of biotin.
The present invention is achieved by the following technical solutions:
An extracting method for biotin in molasses, step is as follows:
(1) molasses 10g is got, add 15 ~ 25ml (preferred 20ml) DMF (DMF), stir, filter, rinse filter residue with 20 ~ 40ml absolute ethyl alcohol, filter, filter residue is joined in 70 ~ 100ml absolute ethyl alcohol, at 70 ~ 78 DEG C, 40Hz, ultrasonic process 28 ~ 32min (preferred 30min) under 400 ~ 500W condition, then filter while hot, obtain filtrate, filtrate obtains the thick liquid of 5 ~ 8ml sample through rotary evaporation;
(2) get 2ml phenylhydrazine hydrochloride solution, join in the thick liquid of above-mentioned 5 ~ 8ml sample, mixing, heating water bath 28 ~ 32min (preferred 30min) at 90 ~ 98 DEG C, at being then placed on-10 ~-26 DEG C freezing 30 ~ 40 minutes, takes out, removing lower sediment, obtains supernatant;
Described phenylhydrazine hydrochloride solution prepares by the following method: take phenylhydrazine hydrochloride 0.3g, adds 3ml distilled water, heating for dissolving; Take 0.45g sodium acetate, add 3ml distilled water and dissolve; Two kinds of liquid are merged, to obtain final product;
(3) 0.01 ~ 0.02g adsorbent is got, join in step (2) gained supernatant, stirring and adsorbing 0.5 ~ 1h, then 6000 ~ 8000 revs/min centrifugal, obtain supernatant, be biotin solution (solution containing biotin, rotary evaporation is concentrated into 2ml, as sample liquid).
Described adsorbent prepares by the following method: get zeyssatite 1g, atlapulgite 1g and craboraffin 1g, and mixing mixing, to obtain final product.
A thin-layer chromatography scanning detection method for biotin, step is as follows:
(1) get detected sample liquid (i.e. said method prepare biotin solution) 4 μ l, put at silica gel plate that (preferred 10cm × 20cm scribbles silica G F
254high Performance Thin plate) on, in expansion cylinder, add developping agent, launch in expansion cylinder (10cm × 12cm × 15cm), Zhan Cheng 15cm, then takes out to be placed in vent cabinet by thin layer plate and dries; Meanwhile, adopt the biotin standard solution of one point external standard method point 4 μ l variable concentrations, launch simultaneously;
Described developping agent is chloroform-methanol-toluene-acetic acid mixture, and wherein, the volume ratio of chloroform, methyl alcohol, toluene, acetic acid is 4 ~ 4.5:0.5 ~ 0.6:2 ~ 2.2:0.1 ~ 0.15.
Described biotin standard solution comprises concentration and is respectively 0.05 μ g/ μ l, 0.2 μ g/ μ l, 0.25 μ g/ μ l, 0.3 μ g/ μ l, the series of standards product solution of 0.4 μ g/ μ l;
The compound method of described biotin standard solution is: precision takes biotin standard items (available from Sigma) 1.29mg, adds DMF 1.29ml, is mixed with the standard solution that concentration is 1mg/ml; Accurate absorption 50,200,250,300, the standard solution of 400 μ l1mg/ml, is mixed with concentration 0.05 μ g/ μ l, 0.2 μ g/ μ l, 0.25 μ g/ μ l, 0.3 μ g/ μ l respectively, the series of standards product solution of 0.4 μ g/ μ l;
(2) by 2,4-dimethylaminocinnamaldehyde, sulfuric acid and absolute ethyl alcohol mix, be made into dyeing liquor, wherein 2,4-dimethylaminocinnamaldehyde final concentration is 0.1 ~ 0.2% (quality volume fraction, unit g/ml), sulfuric acid final concentration is 1 ~ 2% (quality volume fraction, unit g/ml); The panel of having dried after having opened up, uses dyeing liquor spray dyeing, obtains the spot of the obvious standard items with background contrast and the spot (Chinese red) of sample;
(3) utilize Camag3 type thin-layer chromatogram scanner to carry out thin-layer chromatography scanning with 530nm absorbing wavelength, sweep velocity is 20mm/s, and resolution is 50 μm/step, obtains Rf=0.2; Irradiation light is tungsten lamp, the regression equation that detection computations obtains the relation of biotin standard solution quality and integrating peak areas value (is calculated by thin layer chromatograph management software winCATS1.4.1, for conventional means), then the integrating peak areas value of liquid calculates the mass concentration of biotin in sample liquid per sample.
The extracting method of biotin in molasses of the present invention, simple to operate, reliable, the thin-layer chromatography scanning detection method of biotin of the present invention, has the advantages such as simple, quick, accurate, reliable, stable.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1 extracts biotin from molasses, and detects
Step is as follows:
(1) the thick liquid of sample is prepared:
Get molasses 10g, add 20ml DMF (DMF) and to stir filtration, rinse filter residue with 20ml absolute ethyl alcohol and filter.Filter residue is added 100ml absolute ethyl alcohol, at 78 DEG C, 40Hz, the ultrasonic 0.5h of power 400W.Filter to get filtrate while hot.Filtrate is the thick liquid of sample through rotary evaporation to 5ml.
(2) preparation of phenylhydrazine hydrochloride liquid:
Take phenylhydrazine hydrochloride 0.3g, add 3ml distilled water heating for dissolving.Take sodium acetate 0.45g and add the dissolving of 3ml distilled water.Two kinds of liquid are merged, to obtain final product.
(3) getting 2ml phenylhydrazine hydrochloride solution joins in the thick liquid of 5ml sample, 95 DEG C of heating water bath 0.5h.Yellowish color cloud liquid, put to-10 DEG C freezing 40 minutes.Take out removing lower sediment and obtain supernatant.
(4) adsorbent is prepared:
Get zeyssatite 1g, atlapulgite 1g and craboraffin 1g to mix, obtain adsorbent.Therefrom get 0.01g and add stirring and adsorbing 0.5h in the supernatant of step (3).Through 8000 revs/min of centrifugal supernatants.
(5) supernatant rotary evaporation is concentrated into 2ml as sample liquid.
(6) detect:
Precision takes biotin standard items (Sigma company) 1.29mg, adds DMF 1.29ml, is mixed with the standard solution that concentration is 1mg/ml.Accurate absorption 50,200, the standard solution of 250,300,400 μ l1mg/ml, be mixed with concentration 0.05 μ g/ μ l respectively, 0.2 μ g/ μ l, 0.25 μ g/ μ l, 0.3 μ g/ μ l, the series of standards product solution of 0.4 μ g/ μ l, the sample liquid 4 μ l of Example 1 gained and titer 4 μ l point are scribbling silica G F respectively
254on High Performance Thin plate (10cm × 20cm), with chloroform: methyl alcohol: toluene: acetic acid=4:0.5:2:0.1 (volume ratio) prepares 20ml developping agent, chromatography in expansion cylinder (10cm × 12cm × 15cm), exhibition journey 15cm, then takes out to be placed in vent cabinet by thin layer plate and dries.2,4-dimethylaminocinnamaldehyde, sulfuric acid and absolute ethyl alcohol are mixed, is made into dyeing liquor, wherein 2,4-dimethylaminocinnamaldehyde concentration are 0.1%, and sulfuric acid concentration is 1%, thin layer plate dyeing liquor spray dyeing after drying, obtains standard items spot and the sample point of the obvious salmon pink with background contrast.
Scan with 530nm absorbing wavelength with CAMAG3 thin-layer chromatogram scanner.Sweep velocity is 20mm/s, and resolution is 50 μm/step, by thin-layer chromatogram scanner software wincats1.4.1 process regression equation Y=72.1356+2.0456X, r=0.99953, RSD=1.99%.Calculate biotin content average out to 25.5 μ g/g in molasses after testing.
Embodiment 2 extracts biotin from molasses, and detects
Step is as follows:
(1) the thick liquid of sample is prepared:
Get molasses 10g, add 20ml DMF and to stir filtration, rinse filter residue with 30ml absolute ethyl alcohol and filter.Filter residue is added in 70ml absolute ethyl alcohol.At 75 DEG C, the ultrasonic 0.5h of 40Hz, 450W.Filter to get filtrate immediately while hot.Filtrate rotary evaporation is the thick liquid of sample to 5ml.
(2) preparation of phenylhydrazine hydrochloride solution: with embodiment 1.
(3) getting 2ml phenylhydrazine hydrochloride solution joins in the thick liquid of 5ml sample, 90 DEG C of heating water bath 0.5h.Then put into-20 DEG C freezing 35 minutes.Take out removing lower sediment and obtain supernatant.
(4) prepare adsorbent with embodiment 1, get 0.015g adsorbent and to add in the supernatant of (3) stirring and adsorbing 45 minutes.Through 7000 revs/min of centrifugal supernatants.
(5) using supernatant rotary evaporation to 2ml as sample liquid.
(6) detection method is with embodiment 1, unlike, developping agent used is chloroform: methyl alcohol: toluene: acetic acid=4.5:0.6:2.2:0.15 (volume ratio).In molasses, biotin content is 26.00 μ g/g after testing.
Embodiment 3 extracts biotin from molasses, and detects
Step is as follows:
(1) the thick liquid of sample is prepared
Get molasses 10g, add 20ml DMF and to stir filtration, rinse filter residue with 40ml absolute ethyl alcohol and filter.Filter residue is joined in 80ml absolute ethyl alcohol.70 DEG C, 40Hz, power is the ultrasonic 0.5h of 500W.Filter to get filtrate while hot.Filtrate rotary evaporation is the thick liquid of sample to 8ml.
(2) preparation of phenylhydrazine hydrochloride solution: with embodiment 1.
(3) getting 2ml phenylhydrazine hydrochloride solution joins in the thick liquid of 8ml sample, 95 DEG C of heating water bath 0.5h.Then be placed on-26 DEG C freezing 30 minutes.Take out removing lower sediment and obtain supernatant.
(4) preparation of adsorbent is with embodiment 1, gets 0.02g adsorbent and adds stirring and adsorbing 1h in the supernatant of (3).Through 6000 revs/min of centrifugal supernatants.
(5) using supernatant rotary evaporation to 2ml as sample liquid.
(6) detect with embodiment 1, unlike, developping agent used is chloroform: methyl alcohol: toluene: acetic acid=4.2:0.55:2.1:0.12 (volume ratio).In molasses, biotin content is 25.00 μ g/g after testing.
Claims (4)
1. the extracting method of biotin in molasses, is characterized in that: step is as follows:
(1) molasses 10g is got, add 15 ~ 25ml DMF, stir, filter, rinse filter residue with 20 ~ 40ml absolute ethyl alcohol, filter, filter residue is joined in 70 ~ 100ml absolute ethyl alcohol, at 70 ~ 78 DEG C, 40Hz, ultrasonic process 28 ~ 32min under 400 ~ 500W condition, then filter while hot, obtain filtrate, filtrate obtains the thick liquid of sample through rotary evaporation;
(2) get 2ml phenylhydrazine hydrochloride solution, join in the thick liquid of above-mentioned sample, mixing, heating water bath 28 ~ 32min at 90 ~ 98 DEG C, at being then placed on-10 ~-26 DEG C freezing 30 ~ 40 minutes, take out, removing lower sediment, obtains supernatant;
Described phenylhydrazine hydrochloride solution prepares by the following method: take phenylhydrazine hydrochloride 0.3g, adds 3ml distilled water, heating for dissolving; Take 0.45g sodium acetate, add 3ml distilled water and dissolve; Two kinds of liquid are merged, to obtain final product;
(3) get 0.01 ~ 0.02g adsorbent, join in step (2) gained supernatant, stirring and adsorbing 0.5 ~ 1h, then 6000 ~ 8000 revs/min centrifugal, obtain supernatant, be biotin solution;
Described adsorbent prepares by the following method: get zeyssatite 1g, atlapulgite 1g and craboraffin 1g, and mixing mixing, to obtain final product.
2. a thin-layer chromatography scanning detection method for biotin, is characterized in that: step is as follows:
(1) get detected sample liquid 4 μ l, put on silica gel plate, add developping agent to expansion cylinder, launch in expansion cylinder, Zhan Cheng 15cm, then thin layer plate is taken out to be placed in vent cabinet and dry; Meanwhile, adopt the biotin standard solution of external standard method point 4 μ l variable concentrations, launch simultaneously; Described developping agent is chloroform-methanol-toluene-acetic acid mixture, and wherein, the volume ratio of chloroform, methyl alcohol, toluene, acetic acid is 4 ~ 4.5:0.5 ~ 0.6:2 ~ 2.2:0.1 ~ 0.15;
(2) 2,4-dimethylaminocinnamaldehyde, sulfuric acid and absolute ethyl alcohol are mixed, be made into dyeing liquor, wherein 2,4-dimethylaminocinnamaldehyde final concentrations are 0.1 ~ 0.2%, and sulfuric acid final concentration is 1 ~ 2%, all in quality volume fraction g/mL; The panel of having dried after having opened up, uses dyeing liquor spray dyeing, obtains the spot of obvious standard items and the spot of sample with background contrast;
(3) thin-layer chromatogram scanner is utilized to carry out thin-layer chromatography scanning with 530nm absorbing wavelength, irradiation light is tungsten lamp, detection computations obtains the regression equation of the relation of biotin standard solution quality and integrating peak areas value, and then the integrating peak areas value of liquid calculates the mass concentration of biotin in sample liquid per sample;
Described sample liquid prepares by the following method:
(1) molasses 10g is got, add 15 ~ 25ml DMF, stir, filter, rinse filter residue with 20 ~ 40ml absolute ethyl alcohol, filter, filter residue is joined in 70 ~ 100ml absolute ethyl alcohol, at 70 ~ 78 DEG C, 40Hz, ultrasonic process 28 ~ 32min under 400 ~ 500W condition, then filter while hot, obtain filtrate, filtrate obtains the thick liquid of sample through rotary evaporation;
(2) get 2ml phenylhydrazine hydrochloride solution, join in the thick liquid of above-mentioned sample, mixing, heating water bath 28 ~ 32min at 90 ~ 98 DEG C, at being then placed on-10 ~-26 DEG C freezing 30 ~ 40 minutes, take out, removing lower sediment, obtains supernatant;
Described phenylhydrazine hydrochloride solution prepares by the following method: take phenylhydrazine hydrochloride 0.3g, adds 3ml distilled water, heating for dissolving; Take 0.45g sodium acetate, add 3ml distilled water and dissolve; Two kinds of liquid are merged, to obtain final product;
(3) get 0.01 ~ 0.02g adsorbent, join in step (2) gained supernatant, stirring and adsorbing 0.5 ~ 1h, then 6000 ~ 8000 revs/min centrifugal, obtain supernatant, be biotin solution, be sample liquid;
Described adsorbent prepares by the following method: get zeyssatite 1g, atlapulgite 1g and craboraffin 1g, and mixing mixing, to obtain final product.
3. the thin-layer chromatography scanning detection method of biotin according to claim 2, is characterized in that: described silica gel plate is for scribbling silica GF254 High Performance Thin plate.
4. the thin-layer chromatography scanning detection method of biotin according to claim 2, it is characterized in that: described biotin standard solution comprises concentration and is respectively 0.05 μ g/ μ l, 0.2 μ g/ μ l, 0.25 μ g/ μ l, 0.3 μ g/ μ l, the series of standards product solution of 0.4 μ g/ μ l.
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| CN1857434A (en) * | 2006-04-10 | 2006-11-08 | 山东鲁泰环中制药有限公司 | Qulity control method for new compound isatis leaf preparation |
| WO2009153327A1 (en) * | 2008-06-18 | 2009-12-23 | IfP Privates Institut für Produktqualität GmbH | Detection of an analyte in aqueous media |
| WO2010107716A2 (en) * | 2009-03-15 | 2010-09-23 | Ribomed Biotechnologies, Inc. | Abscription based molecular detection |
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