CN105067741A - Method for measuring betaine content - Google Patents

Abstract

The invention relates to a method for measuring the betaine content. According to the method, high performance liquid chromatography (HPLC) is adopted to measure the betaine content. The HPLC has the advantages of simpleness, convenience, high accuracy, safety, and reliability. Moreover, an external standard method is adopted to measure the betaine content quantitatively, the method is accurate and simple, the repeatability is good, and the sensitivity is high. When the betaine content in a range of 0.1 to 2.0 mg/mL, a good linear relationship exists, and the linear correlation coefficient is 0.9993.

Description

A kind of assay method of beet alkali content

Technical field

The invention belongs to chemical analysis technology field, especially relate to a kind of assay method of beet alkali content.

Background technology

Betaine (trimethylammonium guanidine-acetic acid) is a seed amino acid--the trimethyl derivative of glycocoll, chemical formula is C ₅h ₁₁nO ₂, betaine molecule has three effective methyl, and in neutral, fusing point is up to 200 DEG C, very easily water-soluble, is soluble in methyl alcohol.Have bibliographical information, betaine-homocysteine methylferase is to Cobastab ₁₂a large amount of synthesis there is very important effect.Betaine-homocysteine methylferase catalysis betaine shifts a methyl to homocysteine, forms dimethylglycine and methionine respectively, and therefore betaine is at participation Cobastab ₁₂methylating in biosynthetic process has very important effect.In addition, betaine is a kind of good osmotic pressure regulator, can promote that cell surface is to the secretion of metabolic product, thus to Production by Bacteria Cobastab ₁₂there is effect of stimulation.Under the prerequisite keeping enough methyl to supply, the betaine of low concentration is conducive to Cobastab ₁₂synthesis.Find the comparison of production concentration inside and outside born of the same parents, in product a large amount of synthesis phase, low concentration betaine obviously can promote the accumulation of extracellular products.Therefore, for Cobastab ₁₂the mensuration of the beet alkali content in fermentation liquor just seems particularly important.

The structure of betaine is as follows:

The detection method of current betaine has acid base titration, colourimetry, gravimetric method, Quantitative TLC scanning technique, Kjeldahl's method, perchloric acid non-aqueous titration, spectrophotometric method, vapor-phase chromatography, the chromatography of ions etc., and each tool of these methods is good and bad:

" Chinese Pharmacopoeia 2005 version is received betaine thin-layered chromatography assay, but the method disturbing factor is many, and temperature and humidity is comparatively large to chromogenic impact, poor reproducibility, separately have and betaine is made derivant, then carry out the mensuration that high performance liquid chromatography carries out measuring betaine.

Betaine sample and developer generation chromogenic reaction are generated coloring matter by spectrophotometric method, re-use spectrophotometer and carry out quantitative measurement.Sample mixes with the KI of cooling and produces iodide and precipitate, centrifugal rear ethylene dichloride dissolution precipitation, is that 365nm place measures absorbance at wavelength, calculates beet alkali content by working curve, this method disturbing factor is more, particularly larger at mensuration low content sample time error.

In " veterinary medical quality standard 2003 ", " betaine HCL ", NY399-2000, GB/T21515-2008 are beet alkali hydrochlorate and betaine target level of product quality respectively, and content detection adopts perchloric acid non-aqueous titration.This method utilizes beet alkali hydrochlorate for quaternary ammonium salt compound, is solvent with glacial acetic acid, can form the difficult mercuric chloride ionized, and form quaternary ammonium acetate and have obvious alkalescence, take crystal violet as indicator, carry out nonaquepous tration with perchloric acid with mercuric acetate.But this method adopts crystal violet indicator, terminal colour change is not obvious, more difficult judgement terminal; The perchloric acid adopted and glacial acetic acid have very strong corrosivity; Perchloric acid standard solution placed meeting yellowing for a long time, higher to laboratory external environment condition control overflow, comparatively large by environment temperature, humidity effect, often will prepare, demarcate.As long as utilize this method carry out detecting in sample there is basic group compound as the salt of amine, amino acids, nitrogen-containing heterocycle compound, some organic base and salt of weak acid, sodion etc., all can affect the accuracy that perchloric acid non-aqueous titration detects.

Above various methods, not only process is complicated, and can use corrosive reagents, and the mensuration for beet alkali content is not very desirable.Therefore develop a kind of can safety, Accurate Determining betaine the method for content be have problem to be solved always.

Summary of the invention

The object of the invention is to the defect overcoming above-mentioned prior art, provide a kind of disturbing factor few, safe and reliable, and method is simple and easy in operation, result is accurate, the assay method of reproducible beet alkali content.

Technical scheme taked for achieving the above object is:

An assay method for beet alkali content, is characterized in that adopting high performance liquid chromatography to detect.

The detailed process that described high performance liquid chromatography carries out detecting is: adopt purified water to be solvent preparation standard product solution and need testing solution, after the sample size sample introduction of 10-20ul, calculates content according to external standard method;

Wherein high-efficient liquid phase chromatogram condition is:

Chromatographic column: C18 chromatographic column,

Detecting device: DAD detecting device,

Mobile phase: 0.05mol/L sodium hexanesulfonate solution: 0.05mol/L potassium dihydrogen phosphate=50.0-60.0:40.0-50.0,

Flow velocity: 0.60-1.00ml/min;

Determined wavelength: 190-196nm.

Described standard solution and need testing solution concentration are 0.1mg/ml-2.0mg/ml.

The present invention adopts high performance liquid chromatography to carry out the content detection of betaine, and its method is simple, easy and simple to handle, safe and reliable.And carry out quantitatively by external standard method to beet alkali content in sample, method is accurate, and simple, reproducible, highly sensitive, when betaine is between 0.1mg/ml-2.0mg/ml, all have good linear relationship, its linearly dependent coefficient is 0.9991.Be better than perchloric acid titration method, thin-layered chromatography and spectra photometric method, the Accurate Determining for betaine provides excellent detection method.

Accompanying drawing explanation

Fig. 1 is the linear relationship chart of peak area and concentration.

Embodiment

In order to the technical scheme making those skilled in the art better understand invention, below in conjunction with specific implementation method, technical scheme of the present invention is described in further detail.

Embodiment 1

The preparation of sample analyzed by betaine:

The preparation of standard solution: take betaine standard items 50mg, accurately weighed, put in 100ml volumetric flask, add mobile phase and dissolve and be settled to scale, shake up.Get the organic filter filtration of solution through 0.45um with 1ml syringe, obtain standard solution.

The preparation of need testing solution: take betaine sample 50mg, accurately weighed, be placed in 100ml volumetric flask, add mobile phase appropriate, be settled to scale after dissolving, shake up.Get the organic filter filtration of solution through 0.45um with 1ml syringe, obtain need testing solution.

Standard solution and each obtained 5 parts of need testing solution.

Chromatographiccondition: Shimadzu LC-20A liquid chromatograph, WatersC18 chromatographic column (250*4.6,5um); Mobile phase: 0.05mol/L sodium hexanesulfonate solution: 0.05mol/L potassium dihydrogen phosphate=(50.0:50.0); Flow velocity: 0.80ml/min; Determined wavelength: 192nm; Sample size 20ul, detecting device: UV-detector; Column oven 30 DEG C.

By above-mentioned chromatographic condition, to all standard items and test sample sample introduction respectively, as shown in Figure 1, sample introduction gained peak area is 641612, RSD to standard solution 5 samples is respectively 0.15%; 5 sample gained peak area mean values of need testing solution are 358933, RSD is 0.09%.

According to formula:

The content of betaine: a=A sample M mark/A marks M sample * 100%.

Adopt external standard method standard items and test sample on average to be substituted into the content of test sample betaine is 50.15%.

The methodological study of detection method described in the embodiment of the present invention 1:

Precision Experiment

Take same betaine sample, carry out 6 revision tests, peak area table 1:

The area of table 1 betaine revision test

Secondary	1	2	3	4	5	6
								357	356	358	358	359	357

Peak area RSD is 0.30%, and result shows, it is high that this method detects beet alkali content precision, favorable reproducibility.

(2) linear relationship is investigated

According to the method for the invention, be the storing solution of 10mg/ml with the volumetric flask compound concentration of 50ml, add the standard solution that mobile phase is diluted to 0.1mg/ml, 0.2mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml respectively, according to this method, data regression is carried out to sample concentration (X) and peak area (Y), equation of linear regression is Y=1.26981 × 106X-6699.7, R=0.9993, result shows that betaine is in 0.1mg/ml-2.0mg/ml concentration range, and peak area and concentration are good linear relationship.

(3) application of sample recovery experiment

The betaine accurately taking known content is appropriate, precision adds a certain amount of betaine sample 80% respectively again, 100%, 120%, make the betaine concentration in need testing solution in the high, medium and low region of betaine typical curve each 3 parts, according to the operation under item of the present invention, measure, calculate the recovery, result average recovery rate is 99.15% in accordance with the law, RSD is 0.54%, shows that the method average recovery is good.

To sum up show detection method of content good stability of the present invention, precision is high, is applicable to the assay of betaine.

Embodiment 2

Sample and standard items preparation are with embodiment 1.

Chromatographiccondition: Shimadzu LC-20A liquid chromatograph, PhenomenexC18 chromatographic column (250*4.6,5um); Mobile phase: 0.05mol/L sodium hexanesulfonate solution: 0.05mol/L potassium dihydrogen phosphate=(40.0:60.0); Flow velocity: 0.70ml/min; Determined wavelength: 190nm; Sample size 20ul, detecting device: UV-detector; Column oven 30 DEG C.

By above-mentioned chromatographic condition, to all standard items and test sample sample introduction respectively, as shown in Figure 1, sample introduction gained peak area is 356794, RSD to standard solution 5 samples is respectively 0.33%; 5 sample gained peak area mean values of need testing solution are 375545, RSD is 0.35%.

According to formula:

The content of betaine: a=A sample M mark/A marks M sample * 100%.

Adopt external standard method standard items and test sample on average to be substituted into the content of test sample betaine is 51.02%.

Embodiment 3

Sample and standard items preparation are with embodiment 1.

Chromatographiccondition: Shimadzu LC-20A liquid chromatograph, WatersC18 chromatographic column (250*4.6,5um); Mobile phase: 0.05mol/L sodium hexanesulfonate solution: 0.05mol/L potassium dihydrogen phosphate=(60.0:40.0); Flow velocity: 1.00ml/min; Determined wavelength: 196nm; Sample size 20ul, detecting device: UV-detector; Column oven 35 DEG C.

By above-mentioned chromatographic condition, to all standard items and test sample sample introduction respectively, as shown in Figure 1, sample introduction gained peak area is 594123, RSD to standard solution 5 samples is respectively 0.21%; 5 sample gained peak area mean values of need testing solution are 325812, RSD is 0.21%.

According to formula:

The content of betaine: a=A sample M mark/A marks M sample * 100%.

Adopt external standard method standard items and test sample on average to be substituted into the content of test sample betaine is 51.55%.

Embodiment 4

Sample and standard items preparation are with embodiment 1.

Chromatographiccondition: Shimadzu LC-20A liquid chromatograph, PhenomenexC18 chromatographic column (250*4.6,5um); Mobile phase: 0.05mol/L sodium hexanesulfonate solution: 0.05mol/L potassium dihydrogen phosphate=(50.0:50.0); Flow velocity: 1.00ml/min; Determined wavelength: 196nm; Sample size 20ul, detecting device: UV-detector; Column oven 30 DEG C.

By above-mentioned chromatographic condition, to all standard items and test sample sample introduction respectively, as shown in Figure 1, standard solution 5 samples respectively sample introduction gained peak area 601458 are that RSD is 0.25%; 5 sample gained peak area mean values of need testing solution are 384752, RSD is 0.31%.

According to formula:

The content of betaine: a=A sample M mark/A marks M sample * 100%.

Adopt external standard method standard items and test sample on average to be substituted into the content of test sample betaine is 51.25%.

Above-mentioned detailed description of carrying out the detection method of content of this kind of betaine with reference to embodiment, is illustrative instead of determinate, can lists several embodiments according to institute's limited range.Therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention.

Claims (3)

1. an assay method for beet alkali content, is characterized in that adopting high performance liquid chromatography to detect.

2. according to the assay method of beet alkali content according to claim 1, it is characterized in that the detailed process that described high performance liquid chromatography carries out detecting is: adopt purified water to be solvent preparation standard product solution and need testing solution, after the sample size sample introduction of 10-20ul, calculate content according to external standard method;

Wherein high-efficient liquid phase chromatogram condition is:

Chromatographic column: C18 chromatographic column,

Detecting device: DAD detecting device,

Mobile phase: 0.05mol/L sodium hexanesulfonate solution: 0.05mol/L potassium dihydrogen phosphate=50.0-60.0:40.0-50.0,

Flow velocity: 0.60-1.00ml/min;

Determined wavelength: 190-196nm.

3., according to the assay method of beet alkali content according to claim 2, it is characterized in that described standard solution and need testing solution concentration are 0.1mg/ml-2.0mg/ml.