CN103197012B - Method for determining content of 1,3-dihydroxy acetone by adopting liquid chromatography external standard method - Google Patents
Method for determining content of 1,3-dihydroxy acetone by adopting liquid chromatography external standard method Download PDFInfo
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- CN103197012B CN103197012B CN201310143866.3A CN201310143866A CN103197012B CN 103197012 B CN103197012 B CN 103197012B CN 201310143866 A CN201310143866 A CN 201310143866A CN 103197012 B CN103197012 B CN 103197012B
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Abstract
The invention relates to a method for determining the content of 1,3-dihydroxy acetone (DHA) by adopting a liquid chromatography external standard method. The method is characterized by comprising the steps that standard 1,3-dihydroxy acetone serves as an external standard object and an acetonitrile-water solution serves as a solvent to prepare a standard solution, wherein in the acetonitrile-water solution, the volume ratio of acetonitrile to water is 10:90, and pH of the water is adjusted to 10.5 by using ammonia water. The method has the advantages that 1, universal and convenient chromatographic conditions are selected; 2, a very stable mobile phase with the property being close to that of a reactant and a resultant is selected; and 3, the measurement result is accurate, quick and convenient, and the analysis cost is low.
Description
Technical field
The present invention relates to the method for a kind of liquid chromatography external standard method 1,3-Dihydroxyacetone (DHA) content.Be specifically related to a kind of use liquid chromatography external standard method taking formaldehyde as raw material in ionic liquid as catalyzer, organic base are as promotor, at 140 DEG C, under the homogeneous reaction condition of 30 minutes, prepare 1,3-dihydroxyacetone, and measure the method for synthesizing 1,3-Dihydroxyacetone content in mixed liquor.
Background technology
1,3-dihydroxyacetone (1,3-Dihydroxyacetone) be called for short DHA, being polyhydroxy ketose, is the simplest ketose, and outward appearance is with pleasantly sweet white powder crystallization, soluble in water, ethanol, ether and acetone and other organic solvent, fusing point is 75-80 DEG C, and water-soluble >250g/L (20 DEG C) is to stablize for 6.0 o'clock at pH.
1,3-Dihydroxyacetone is a kind of important industrial chemicals, medicine intermediate and functional additive.Its purposes is mainly the ketose that a kind of natural existence has biodegradability, edible and to human body and environment nonhazardous; Be that one has multi-functional adjuvant, can be used for cosmetics, medicine and food service industry etc.Its energy wide participation simultaneously, such as the reaction such as polymerization, condensation, is a kind of important medicine, agricultural chemicals synthetic intermediate.
At present, 1,3-Dihydroxyacetone production method mainly contains chemical synthesis and microbe fermentation method.
Beijing Xuyang Chemical Technology Institute Co., Ltd. independently researched and developed by formaldehyde be raw material ionic liquid be catalyzer, organic base be promotor, at 140 DEG C, under the homogeneous reaction condition of 30 minutes, prepare 1,3-dihydroxyacetone, it is the novel synthesis route of a kind of environmental protection.
Mol ratio=the 30:1:0.02 of formaldehyde, ionic liquid, organic base.
Taking formaldehyde as raw material, ionic liquid is that catalyzer, organic base are that promotor prepares 1, in the research and development of 3-dihydroxyacetone and production run, in reactant liquor 1, the accurate fast quantitative analysis of 3-dihydroxyacetone is extremely important, and this height and ionic liquid catalyst that is related to formaldehyde conversion ratio is optionally determined.But also there is no at present universally recognized analytical approach comparatively quickly and accurately, seriously restricted the optimization of 1,3-Dihydroxyacetone synthesis technique.
The comparison of DHA Main Analysis method
The analytical approach of DHA mainly contains the methods such as thin-layered chromatography, vapor-phase chromatography, high performance liquid chromatography.
Thin layer chromatography agents useful for same costliness and accurate quantitative analysis difficulty.
Zhang Zhihong, Sun little Juan take gas chromatography to carry out quantitative measurement DHA in " gas chromatographic analysis of dihydroxyacetone " (" analytical test journal ", 1999,18 (3) 56-63).
But because the volatility of DHA is not high, need first be derived turn to derivant volatile and that stability is higher just can indirect determination DHA content, but the method pre-service complexity, and in derivatization process, the impact of moisture is larger, need to add a large amount of derivatization reagents.
The high-efficiency liquid chromatography method for detecting of product and the residual glycerol substrate in Chinese biochemical drug magazine (the 28th the 3rd phase of volume of Chinese Journal of Biochemical Pharmaceutics2007) proposes to set up dihydroxyacetone (DHA) sweat such as the Chen Jing of Life Science and Technology institute of China Medicine University, Chen Jianhua.Method mobile phase is acetonitrile-water (90:10), and chromatographic column is Lichrospher5-NH
2(4.6mm*250mm), at 271nm place detection DHA, detect glycerine with differential refraction detector by UV-detector.
The rich will of Southern Yangtze University is bright, the beautiful Chinese biological engineering of Xu Mei magazine (China Biotechnology, 2008,28 (1): 61-64) set up high performance liquid chromatography (HPLC) method of mensuration 1,3-Dihydroxyacetone (DHA).Taking Allt ima C18 (250*4.6mm) as separating column, 5% methanol aqueous solution (0.05%H
3pO
4adjust PH to 3.0), flow velocity is 1ml/min, detects DHA by UV-detector at 200nm place.
But above-mentioned two kinds of HPLC analytical approachs one are to measure to adopt microorganism to send out DHA content in fermentation liquor taking glycerine as raw material, and under normal circumstances, fermentation liquor shows faintly acid.
Beijing Xuyang Chemical Technology Institute Co., Ltd. independently researches and develops taking formaldehyde prepares 1,3-Dihydroxyacetone synthesis technique in ionic liquid as catalyzer, and reactant liquor shows alkalescence (PH=9.5-11.0).And chromatographic column is that mobile phase is analyzed under service condition for a long time at water at high proportion, is easy to cause post to fix mutually hydrophobic caving in, DHA retains the phenomenon (serious drift occurs the retention time of DHA) obviously reducing or do not retain completely.
Summary of the invention
The present inventor is for unique synthesis technique of research and development, through concentrating on studies discovery: adopt suitable mobile phase acetonitrile-water (10: 90) water ammoniacal liquor to regulate pH=10.5; DAD diode array detector, ZORBAX Extend-C18.Can measure accurately, fast, reliably by formaldehyde is that catalyzer is prepared 1,3-Dihydroxyacetone content in ionic liquid.Complete thus the present invention.
Object of the present invention is for providing a kind of liquid chromatography external standard method 1, the method of 3-dihydroxyacetone content, it is characterized in that, employing standard 1,3-Dihydroxyacetone is as external standard, and acetonitrile-aqueous solution is solvent preparing standard solution, wherein, in acetonitrile-aqueous solution, the volume ratio of acetonitrile and water is 10:90, and water regulates pH to 10.5 with ammoniacal liquor.
In the application's a preferred implementation, described method comprises the steps, the method comprises the steps:
(1) adopt standard 1,3-Dihydroxyacetone as external standard, acetonitrile-aqueous solution is solvent preparing standard solution, and wherein, in acetonitrile-aqueous solution, the volume ratio of acetonitrile and water is 10:90, and water regulates pH to 10.5 with ammoniacal liquor;
(2), with the peak area of the 1,3-Dihydroxyacetone in liquid chromatograph difference bioassay standard solution and testing sample, calculate the equation of linear regression of typical curve:
C=AX
In formula, the content (g/L) that C is 1,3-Dihydroxyacetone
X is the slope of typical curve
A is the peak area of 1,3-Dihydroxyacetone;
(3) by each component concentration C in equation of linear regression calculation sample
i(g/L)
C
i=4E-07A
In formula, the peak area that A is 1,3-Dihydroxyacetone.
Concentration to each component in standard solution is not particularly limited, and can adjust with the relative situation that product changes according to reactant in real reaction process, to measure accurately.
Reagent 1,3-Dihydroxyacetone is pure for analyzing, and weighs quality and is accurate to 0.0002g.
Distinguish the peak area of the external standard in bioassay standard solution with liquid chromatograph, and calculated the equation of linear regression of typical curve by content and peak area relation.
According in measuring method of the present invention preferred implementation, the optimal detection condition of liquid chromatography is:
Liquid chromatograph: Agilent1260;
Chromatographic column: ZORBAX Extend-C18 (4.6mm × 250mm);
Detecting device: DAD diode array detector, is used wavelength 200nm;
Post case temperature: 25 DEG C;
Mobile phase: acetonitrile-aqueous solution, wherein the volume ratio of acetonitrile and water is 10:90, water regulates pH=10.5 with ammoniacal liquor; – sample size 0.5 μ L;
Flow velocity: eluent gradient changes to be seen as following table-1
Table-1: eluent gradient change list
It is that available large traffic flow is rinsed chromatographic column residual component mutually, and at next sample introduction forward horizontal stand enough time, has also reduced analysis time because retention time determinand and major impurity in the time of 15 minutes have all flowed out detecting device that eluent gradient changes.
Detection method of the present invention has the following advantages:
1, selected general, chromatographic condition easily;
2, selected the mobile phase close and highly stable with reactant, product character;
3, measurement result is accurate, quick and easy, and analysis cost is low.
Brief description of the drawings
Fig. 1 is DHA standard working curve.。
Embodiment
Key instrument and reagent are in Table-2
Table-2: key instrument and reagent
According to detection method of the present invention, 5 kinds of standard solution according to the formulated variable concentrations in following table-3:
Table-3 mark liquid concentration
The calculating of typical curve
According to the concentration in upper table-3, measure respectively the peak area of above-mentioned standard solution with liquid chromatograph, table-4:
Table-4 peak area tables
Calculate equation of linear regression C
i=4E-07A
Linearly dependent coefficient R
2=0.9991
DHA assay in sample
Taking formaldehyde as raw material in ionic liquid as catalyzer, organic base are as promotor, at 140 DEG C, under the homogeneous reaction condition of 30 minutes, prepare 1,3-Dihydroxyacetone.
After reactant liquor is crossed the organic film of 0.45um, direct injected.Obtain the peak area of object, draw analysis result by above typical curve.
Determining of precision
Get testing sample, measure 10 times with said method, determination test result is as shown in table-5.
Table-5:1, the precision test data of 3-dihydroxyacetone
Can be found out by table-5, the reproduction repeatability of the method is fine, can meet the analysis requirement in experiment.
The inspection of accuracy
In order to check the accuracy of this analytical approach, to adding respectively a certain amount of sterling in actual sample and carrying out liquid-phase chromatographic analysis with said method, average for parallel 6 times, calculate respectively the recovery of standard items, result is as shown in table-6.
Table-6:1, the content of 3-dihydroxyacetone and the recovery
Can be found out by upper table-6, sample recovery rate measured value, between 99%-101%, can meet the requirement of routine analysis precision.Show to use this test condition to do the analytical approach of DHA content.
The present invention uses versatility ZORBAX Extend-C18 chromatographic column, develop with 1,3-dihydroxyacetone is external standard, at acetonitrile: water=10:90(volume ratio, wherein water regulates pH=10.5 with ammoniacal liquor) under mobile phase condition, measure the analytical approach of 1,3-Dihydroxyacetone, this analytical approach mobile phase sample stability is good, and measurement result can meet scientific research and industrial needs from now on accurately and reliably.
In liquid chromatography external standard method reactant liquor of the present invention, the analytical approach of 1,3-Dihydroxyacetone content is quick and easy, accurate and analysis cost is low, can meet the needs of research and production.
Claims (2)
1. a liquid chromatography external standard method 1, the method of 3-dihydroxyacetone content, the method prepares 1 for measuring taking formaldehyde as raw material in ionic liquid as catalyzer, organic base under the condition of promotor, homogeneous reaction, in the synthetic mixed liquor of 3-dihydroxyacetone 1, the content of 3-dihydroxyacetone, it is characterized in that, employing standard 1,3-dihydroxyacetone is as external standard, acetonitrile-aqueous solution is solvent preparing standard solution, wherein, and in acetonitrile-aqueous solution, the volume ratio of acetonitrile and water is 10:90, and water regulates pH to 10.5 with ammoniacal liquor;
Wherein, the testing conditions of described liquid chromatography is:
Liquid chromatograph: Agilent1260;
Chromatographic column: ZORBAX Extend-C18,4.6mm × 250mm;
Detecting device: DAD diode array detector, is used wavelength 200nm;
Post case temperature: 25 DEG C;
Mobile phase: acetonitrile-aqueous solution, wherein the volume ratio of acetonitrile and water is 10:90, water regulates pH=10.5 with ammoniacal liquor;
Sample size 0.5 μ L;
Flow velocity: eluent gradient changes to be seen as following table
2. method according to claim 1, the method comprises the steps:
(1) adopt standard 1,3-Dihydroxyacetone as external standard, acetonitrile-aqueous solution is solvent preparing standard solution, and wherein, in acetonitrile-aqueous solution, the volume ratio of acetonitrile and water is 10:90, and water regulates pH to 10.5 with ammoniacal liquor;
(2), with the peak area of the 1,3-Dihydroxyacetone in liquid chromatograph difference bioassay standard solution and testing sample, calculate the equation of linear regression of typical curve:
C＝AX
In formula, the content that C is 1,3-Dihydroxyacetone
X is the slope of typical curve
A is the peak area of 1,3-Dihydroxyacetone;
(3) by each component concentration C in equation of linear regression calculation sample
i
C
i＝4E-07A
In formula, the peak area that A is 1,3-Dihydroxyacetone.
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| CN112946096A (en) * | 2021-01-15 | 2021-06-11 | 上海晓创检测技术有限公司 | Method for determining DHA in cosmetics and application thereof |
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