CN103926367B - Method for extracting biotin from molasses and thin layer chromatography (TLC) scanning detection method of biotin - Google Patents
Method for extracting biotin from molasses and thin layer chromatography (TLC) scanning detection method of biotin Download PDFInfo
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Abstract
The invention discloses a method for extracting biotin from molasses. The method is characterized by using molasses and diatomite as raw materials, carrying out extraction with methanol, carrying out extraction with N,N-dimethylformamide, carrying out washing and impurity removal with absolute ethyl alcohol, carrying out impurity removal with acetone, carrying out impurity removal with chloroform and carrying out extraction and impurity removal with butyl acetate and cyclohexane, 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, adding a developer to a developing cylinder, developing the sample solution to be detected in the developing cylinder, 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 a kind of from molasses, extract biotin method and thin-layer chromatography scanning detection method thereof.
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 a kind of method extracting biotin from molasses, present invention also offers a kind of thin-layer chromatography scanning detection method.
The present invention is achieved by the following technical solutions:
From molasses, extract a method for biotin, step is as follows:
(1) with molasses and zeyssatite for raw material obtains molasses diatomite in powder: get molasses and zeyssatite, mixing mixing, 40 ~ 60 DEG C of oven dry, obtain molasses diatomite in powder; Wherein, molasses and diatomaceous weight ratio are 3:1 ~ 3;
(2) in molasses diatomite in powder, add methyl alcohol, stir extraction 30 ~ 60min, filter to obtain filter residue; The addition of described methyl alcohol is: every 1 gram of molasses diatomite in powder adds 3 ~ 5ml methyl alcohol;
(3) in above-mentioned filter residue, add DMF, at water-bath 70 ~ 100 DEG C, stir extraction 1 ~ 2 hour, filter to obtain supernatant and filter residue; Filter residue absolute ethanol washing 1 ~ 3 time, obtains ethanol washes; The addition of described DMF is: every 1 gram of filter residue adds 4 ~ 6mlN, dinethylformamide; The addition of described absolute ethyl alcohol is: the amount of each washing absolute ethyl alcohol used is 0.8 ~ 1.2 times of DMF volume;
(4) combining step (3) gained supernatant and ethanol washes, evaporation and concentration, adds the acetone of 3 ~ 5 times amount (volume multiple), leave standstill, cross and filter precipitation, evaporation and concentration again, add the acetone of 3 ~ 5 times amount (volume multiple), leave standstill, cross and filter precipitation, evaporation and concentration again, add the chloroform of 3 ~ 5 times amount (volume multiple), leave standstill, cross and filter precipitation, obtain extract;
(4) in extract, add butyl acetate and cyclohexane, the addition of butyl acetate, cyclohexane is identical with the volume of extract, stratification; Get supernatant, be biotin solution (solution containing biotin).
In above method, evaporation and concentration adopts Rotary Evaporators to carry out, and the temperature of rotary evaporation is 40 ~ 60 DEG C.
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 × 10cm scribbles silica G F
254high Performance Thin plate) on, add developping agent to expansion cylinder, launch in expansion cylinder (10cm × 12cm × 15cm), the saturation time of developping agent is 60 minutes, development distance is 6cm, and duration of run is 15 minutes, is then taken 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 methylene chloride-chloroform-methanol-glacial acetic acid mixed liquor, and wherein, the volume ratio of methylene chloride, chloroform, methyl alcohol, glacial acetic acid is 1 ~ 3:1 ~ 3:1 ~ 2:0.04 ~ 0.06, preferred 3:3:2:0.06;
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 (orange 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=0.69; 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 of the present invention, simple to operate, reliable, the 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 take molasses as the extraction that raw material carries out biotin
15g molasses and 5g zeyssatite are stirred, obtain dry molasses diatomite in powder, add 60ml methyl alcohol 40 DEG C of oven dry, stir 1 time at normal temperatures, mixing time 30 minutes, filters afterwards.In processed molasses diatomite in powder, add 80mlN, dinethylformamide, in water-bath, carry out stirring extract, temperature is at 75 DEG C, time is 1 hour, filters and obtains supernatant, and use absolute ethanol washing filter residue, each 80ml, washs 3 times, merges supernatant and cleansing solution.Steaming instrument evaporation and concentration to 50ml with revolving after mixed liquor filters, adding 150ml acetone, stir and evenly mix rear leaving standstill, be settled out partial impurities, again steam instrument evaporation and concentration to 40ml with revolving after filtration, then add 120ml acetone wherein, stir and evenly mix rear leaving standstill, be settled out partial impurities, concentrating with revolving steaming instrument after filtration, being concentrated to 30ml, adding the chloroform of 90ml, stir and evenly mix rear leaving standstill, after filtering, concentrated by rotary evaporation is to 3ml; Mix with 3ml butyl acetate and 3ml cyclohexane, stratification, get supernatant concentration to 2ml, be biotin solution (as sample liquid).Described rotating evaporation temperature is 55 DEG C.
Embodiment 2 take molasses as the extraction that raw material carries out biotin
15g molasses and 10g zeyssatite are stirred, obtain dry molasses diatomite in powder 50 DEG C of oven dry, add 100ml methyl alcohol and stir 2 times at normal temperatures, mixing time 45 minutes, filters afterwards.Processed molasses diatomite in powder is added 125mlN, dinethylformamide, in water-bath, carries out stirring extract, temperature is at 85 DEG C, and the time is 1.5 hours, filters and obtains supernatant, and using absolute ethanol washing filter residue, each 125ml washs 4 times, merges supernatant and cleansing solution.Instrument evaporation and concentration is steamed to 80ml with revolving after mixed liquor filters, add the stirring of 320ml acetone and be settled out partial impurities, again instrument evaporation and concentration is steamed to 50ml with revolving after filtration, add 200ml acetone precipitation more wherein and go out partial impurities, concentrate with revolving steaming instrument after filtration, be concentrated to the chloroform precipitated impurities that 20ml adds 80ml, after filtering, concentrated by rotary evaporation is to 3ml.Mix with 3ml butyl acetate and 3ml cyclohexane, stratification, get supernatant concentration to 2ml, be biotin solution (as sample liquid).Rotating evaporation temperature is 60 DEG C.
Embodiment 3 take honey as the extraction that raw material carries out biotin
15g molasses and 15g zeyssatite are stirred, obtain dry molasses diatomite in powder 60 DEG C of oven dry, add 150ml methyl alcohol and stir 3 times at normal temperatures, mixing time 60 minutes, filters afterwards.Processed molasses diatomite in powder is added 180mlN, dinethylformamide, in water-bath, carries out stirring extract, temperature is at 95 DEG C, and the time is 2 hours, filters and obtains supernatant, and using absolute ethanol washing filter residue, each 180ml washs 5 times, merges supernatant and cleansing solution.Instrument evaporation and concentration is steamed to 60ml with revolving after mixed liquor filters, add the stirring of 300ml acetone and be settled out partial impurities, again instrument evaporation and concentration is steamed to 40ml with revolving after filtration, add 200ml acetone precipitation more wherein and go out partial impurities, concentrate with revolving steaming instrument after filtration, be concentrated to the chloroform precipitated impurities that 20ml adds 100ml, after filtering, concentrated by rotary evaporation is to 3ml; Mix with 3ml butyl acetate and 3ml cyclohexane, stratification, get supernatant concentration to 2ml, be biotin solution (as sample liquid).Rotating evaporation temperature is 65 DEG C.
Embodiment 4 take molasses as the extraction that raw material carries out biotin
15g molasses and 10g zeyssatite are stirred, obtain dry molasses diatomite in powder 55 DEG C of oven dry, add 100ml methyl alcohol and stir 3 times at normal temperatures, mixing time 50 minutes, filters afterwards.Processed molasses diatomite in powder is added 100mlN, dinethylformamide, in water-bath, carries out stirring extract, temperature is at 85 DEG C, and the time is 75min, filters and obtains supernatant, and using absolute ethanol washing filter residue, each 100ml washs 4 times, merges supernatant and cleansing solution.Instrument evaporation and concentration is steamed to 60ml with revolving after mixed liquor filters, add the stirring of 240ml acetone and be settled out partial impurities, again instrument evaporation and concentration is steamed to 40ml with revolving after filtration, add 200ml acetone precipitation more wherein and go out partial impurities, concentrate with revolving steaming instrument after filtration, be concentrated to the chloroform precipitated impurities that 20ml adds 100ml, after filtering, concentrated by rotary evaporation is to 3ml; Mix with 3ml butyl acetate and 3ml cyclohexane, stratification, get supernatant concentration to 2ml, be biotin solution (as sample liquid).Rotating evaporation temperature is 65 DEG C.
Embodiment 5 biotin thin-layer chromatography scanning detection method
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 × 10cm), with methylene chloride (v): chloroform (v): methyl alcohol (v): the proportions 8.06ml developping agent of glacial acetic acid (v)=3:3:2:0.06, chromatography in expansion cylinder (10cm × 12cm × 15cm), the saturation time of developping agent is 60 minutes, development distance is 6cm, duration of run is 15 minutes, is then taken 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 obvious orange-red standard items spot and sample point with background contrast.
Utilize Camag3 type thin-layer chromatogram scanner to carry out thin-layer chromatography scanning with 530nm absorbing wavelength, sweep velocity is 20mm/s, and data resolution is 50 μm/step, obtains Rf=0.69.Irradiation light is that tungsten lamp carries out detection computations, with the quality of biotin standard items (ng) for horizontal ordinate (X), integrating peak areas value is ordinate (Y), the regression equation Y=67.1435+2.8351X of standard items and integrating peak areas value relation is directly obtained by thin layer chromatograph management software winCATS1.4.1, r=0.99933, RSD=2.58%.Result shows, sample liquid is linearly good at 0.2 ~ 1.6 μ g/ spot, and in molasses, the total content of biotin is 26.525 μ g/g as calculated.
Embodiment 6 biotin thin-layer chromatography scanning detection method
According to the chromatography method of embodiment 5, difference is the biotin sample liquid adopting embodiment 2 gained, with methylene chloride (v): chloroform (v): methyl alcohol (v): the proportions 7.04ml developping agent of glacial acetic acid (v)=3:2:1:0.04, dyeing liquor wherein 2,4-dimethylaminocinnamaldehyde concentration is 0.2%, sulfuric acid concentration is 2%, and the content calculating biotin in molasses according to equation of linear regression is 26.515 μ g/g.
Embodiment 7 biotin thin-layer chromatography scanning detection method
According to the chromatography method of embodiment 5, difference is the biotin sample liquid adopting embodiment 3 gained, with methylene chloride (v): chloroform (v): methyl alcohol (v): the proportions 7.05ml developping agent of glacial acetic acid (v)=2:3:2:0.05, dyeing liquor wherein 2,4-dimethylaminocinnamaldehyde concentration is 0.15%, sulfuric acid concentration is 1.5%, and the content calculating biotin in molasses according to equation of linear regression is 26.545 μ g/g.
Embodiment 8 biotin thin-layer chromatography scanning detection method
According to the chromatography method of embodiment 5, difference is the biotin sample liquid adopting embodiment 4 gained, with methylene chloride (v): chloroform (v): methyl alcohol (v): the proportions 7.05ml developping agent of glacial acetic acid (v)=2:2:3:0.05, dyeing liquor wherein 2,4-dimethylaminocinnamaldehyde concentration is 0.15%, sulfuric acid concentration is 2%, and the content calculating biotin in molasses according to equation of linear regression is 26.535 μ g/g.
Claims (9)
1. from molasses, extract a method for biotin, it is characterized in that: step is as follows:
(1) with molasses and zeyssatite for raw material obtains molasses diatomite in powder: get molasses and zeyssatite, mixing mixing, dries, obtains molasses diatomite in powder; Wherein, molasses and diatomaceous weight ratio are 3:1 ~ 3;
(2) in molasses diatomite in powder, add methyl alcohol, stir extraction 30 ~ 60min, filter to obtain filter residue; The addition of described methyl alcohol is: every 1 gram of molasses diatomite in powder adds 3 ~ 5ml methyl alcohol;
(3) in above-mentioned filter residue, add DMF, at water-bath 70 ~ 100 DEG C, stir extraction 1 ~ 2 hour, filter to obtain supernatant and filter residue; Filter residue absolute ethanol washing 1 ~ 3 time, obtains ethanol washes; The addition of described DMF is: every 1 gram of filter residue adds 4 ~ 6mlN, dinethylformamide; The addition of described absolute ethyl alcohol is: the amount of each washing absolute ethyl alcohol used is 0.8 ~ 1.2 times of DMF volume;
(4) combining step (3) gained supernatant and ethanol washes, evaporation and concentration, adds the acetone of 3 ~ 5 times amount, leave standstill, cross and filter precipitation, evaporation and concentration again, add the acetone of 3 ~ 5 times amount, leave standstill, cross and filter precipitation, evaporation and concentration again, add the chloroform of 3 ~ 5 times amount, leave standstill, cross and filter precipitation, obtain extract;
(5) in extract, add butyl acetate and cyclohexane, the addition of butyl acetate, cyclohexane is identical with the volume of extract, stratification; Get supernatant, be biotin solution.
2. the method extracting biotin from molasses according to claim 1, is characterized in that: described evaporation and concentration adopts Rotary Evaporators to carry out, and the temperature of rotary evaporation is 40 ~ 60 DEG C.
3. a thin-layer chromatography scanning detection method for biotin, is characterized in that: step is as follows:
(1) get detected sample liquid, put on silica gel plate, add developping agent to expansion cylinder, launch in expansion cylinder, then thin layer plate is taken out to be placed in vent cabinet and dry; Meanwhile, adopt the biotin standard solution of one point external standard method point 4 μ l variable concentrations, launch simultaneously;
Described developping agent is methylene chloride-chloroform-methanol-glacial acetic acid mixed liquor, and wherein, the volume ratio of methylene chloride, chloroform, methyl alcohol, glacial acetic acid is 1 ~ 3:1 ~ 3:1 ~ 2:0.04 ~ 0.06;
(2) 2,4-dimethylaminocinnamaldehyde, sulfuric acid and absolute ethyl alcohol are mixed, be made into dyeing liquor, wherein 2,4-dimethylaminocinnamaldehyde final concentrations are 0.001 ~ 0.002g/ml, and sulfuric acid final concentration is 0.01 ~ 0.02g/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.
4. the thin-layer chromatography scanning detection method of biotin according to claim 3, is characterized in that: described silica gel plate is for scribbling silica GF254 High Performance Thin plate.
5. the thin-layer chromatography scanning detection method of biotin according to claim 3, is characterized in that: the volume ratio of described methylene chloride, chloroform, methyl alcohol, glacial acetic acid is 3:3:2:0.06.
6. the thin-layer chromatography scanning detection method of biotin according to claim 3, 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.
7. the thin-layer chromatography scanning detection method of biotin according to claim 3, is characterized in that: described regression equation Y=67.1435+2.8351X, r=0.99933, RSD=2.58%.
8. the thin-layer chromatography scanning detection method of biotin according to claim 3, is characterized in that: described sample liquid prepares by the following method:
(1) with molasses and zeyssatite for raw material obtains molasses diatomite in powder: get molasses and zeyssatite, mixing mixing, dries, obtains molasses diatomite in powder; Wherein, molasses and diatomaceous weight ratio are 3:1 ~ 3;
(2) in molasses diatomite in powder, add methyl alcohol, stir extraction 30 ~ 60min, filter to obtain filter residue; The addition of described methyl alcohol is: every 1 gram of molasses diatomite in powder adds 3 ~ 5ml methyl alcohol;
(3) in above-mentioned filter residue, add DMF, at water-bath 70 ~ 100 DEG C, stir extraction 1 ~ 2 hour, filter to obtain supernatant and filter residue; Filter residue absolute ethanol washing 1 ~ 3 time, obtains ethanol washes; The addition of described DMF is: every 1 gram of filter residue adds 4 ~ 6mlN, dinethylformamide; The addition of described absolute ethyl alcohol is: the amount of each washing absolute ethyl alcohol used is 0.8 ~ 1.2 times of DMF volume;
(4) combining step (3) gained supernatant and ethanol washes, evaporation and concentration, adds the acetone of 3 ~ 5 times amount, leave standstill, cross and filter precipitation, evaporation and concentration again, add the acetone of 3 ~ 5 times amount, leave standstill, cross and filter precipitation, evaporation and concentration again, add the chloroform of 3 ~ 5 times amount, leave standstill, cross and filter precipitation, obtain extract;
(5) in extract, add butyl acetate and cyclohexane, the addition of butyl acetate, cyclohexane is identical with the volume of extract, stratification; Get supernatant, be sample liquid.
9. the thin-layer chromatography scanning detection method of biotin according to claim 8, is characterized in that: described evaporation and concentration adopts Rotary Evaporators to carry out, and the temperature of rotary evaporation is 40 ~ 60 DEG C.
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