CN101738392B - Method for fast measuring threonine content - Google Patents
Method for fast measuring threonine content Download PDFInfo
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- CN101738392B CN101738392B CN2009101175816A CN200910117581A CN101738392B CN 101738392 B CN101738392 B CN 101738392B CN 2009101175816 A CN2009101175816 A CN 2009101175816A CN 200910117581 A CN200910117581 A CN 200910117581A CN 101738392 B CN101738392 B CN 101738392B
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Abstract
The invention relates to a method for detecting threonine content in production process sample in the biological fermentation engineering technical field, in particular to a method for fast measuring threonine content. The method comprises the following steps: preparing standard threonine solutions of different concentrations, then respectively taking standard threonine solutions and sample solution of certain amount, adding in buffer solution and developing solution respectively, water-bathing at certain temperature, using a spectrophotometer to perform colorimetric estimation to absorbancy, drawing standard curve according to the result of absorbancy estimation, and calculating the threonine content in the sample. The invention has the advantages of fastness, convenience and smaller error value with the measuring time controlled within one hour and the error within 2%.
Description
Technical field
The present invention relates to detect in the bio-fermentation engineering field method of threonine content in the production run sample, especially relate to a kind of method of fast measuring threonine content.
Background technology
In detecting solution, during threonine content, can adopt high performance liquid chromatography or amino-acid analyzer in the laboratory, but, be difficult to popularize at present because the instrument that uses in this kind method costs an arm and a leg.And laboratory method commonly used is the not high paper chromatography of equipment requirements, and paper chromatography uses equipment less, relatively convenient.But the each Measuring Time of paper chromatography is greatly about about 3-4h, and accuracy rate is very low, and error amount can reach about 10%, and because Measuring Time is longer, is difficult to carry out repetition measurement, therefore is difficult to suitability for industrialized production is directly instructed.
Summary of the invention
Purpose of the present invention mainly is to overcome the deficiencies in the prior art, and a kind of method that detects fast, that accuracy rate is high, easy and simple to handle a kind of fast measuring threonine content is provided.
The present invention realizes in the following way:
A kind of method of fast measuring threonine content, it is characterized in that: this method comprises the steps: the standard threonine solution of preparation variable concentrations earlier, the standard threonine solution of getting the above-mentioned variable concentrations that configures of 1ml then respectively joins in the different test tubes with the sample solution that 1ml need measure, add 2ml damping fluid and 1ml triketohydrindene hydrate colour developing liquid more respectively in above-mentioned each test tube, cooling is cooled to room temperature behind the water-bath certain hour, and then in above-mentioned each test tube, add 10ml distilled water, use the spectrophotometric determination light absorption value then, the light absorption value drawing standard curve that the concentration of standard threonine solution is corresponding with it, reacted per sample light absorption value and extension rate just can calculate threonine concentration in the sample solution;
The standard threonine solution concentration of described preparation at 0.1g/L between the 0.5g/L;
Described sample solution dilutes earlier, the concentration that makes dilution back threonine at 0.20g/L between the 0.35g/L;
Described damping fluid is a 0.2mol/L citric acid-sodium citrate damping fluid, and its pH value is 6.00;
In the described triketohydrindene hydrate colour developing liquid, triketohydrindene hydrate concentration is 10g/L, and anhydrous cupric sulfate concentration is 16g/L;
Described bath temperature is 95-100 ℃, and the water-bath time is 8-10 minute;
Described spectrophotometric wavelength is 500-570nm.
The present invention has following effect:
1) method uniqueness, accuracy height: the method for a kind of fast measuring threonine content of mensuration provided by the invention utilizes triketohydrindene hydrate can generate the bluish violet compound with the amino acid reaction about 100 ℃, utilize spectrophotometer to measure light absorption value then, utilize this principle to measure in the method for threonine content, how to reduce the influence of impurity in the sample, and the error of control survey method is the principal element that can this method of decision be promoted.In suitability for industrialized production, because production technology is more stable, can be relative with the dopant species of ninhydrin reaction less, and impurity content is more stable, therefore can eliminate the influence of impurity by introducing corrected value and control reaction conditions, triketohydrindene hydrate can generate the bluish violet material with the threonine reaction under 100 ℃ of left and right sides temperature, utilize spectrophotometer to detect it and have bigger absorption value under 500-570nm, by adding damping fluid control pH value of solution value, make impurity drop to minimum again to the influence of measuring.Carry out threonine content calculating by comparing at last with typical curve.This method is compared paper chromatography and has been saved chromatography and elution step, therefore on minute, shorten significantly, and relatively the paper chromatography sampling amount increase and remove the separating medium that mainly causes error, make the accuracy of this method obtain bigger lifting.
2) assay method is quick: minute of method provided by the invention is greatly about about one hour, can rapidly threonine content be fed back, this method is drawn by typical curve and sample measurement carries out simultaneously, in test tube, add standard threonine solution, sample and damping fluid and triketohydrindene hydrate developer respectively and carry out water-bath, reaction cooling back adding distilled water dilutes and shakes up, and gets final product colorimetric.The processing time of sample is also shorter, and it is easier to handle sample and instrument cleaning etc. with respect to amino-acid analyzer; And the chromatography of paper chromatography and elution time are longer.Minute of the two kinds of methods in back is substantially all about three hours.
3) step is easy, and is less demanding to operating personnel, and the lab technician is grasped fast: the step that assay method provided by the invention relates to is simpler, grasp easily, even it is shorter that the operator carries out the time of this work, academic level is not high, can comparatively fast grasp yet.Be mainly concerned with the reagent preparation in this step, transfer pipet sampling and water-bath and spectrophotometer colorimetric etc. is all laboratory technical method commonly used.
4) required cost is lower: assay method equipment needed thereby provided by the invention, instrument etc. are all the laboratory often equipment, and reagent also is laboratory reagent commonly used, also has greater advantage on cost.
5) better by the collimation that experimental results demonstrate this method, the same sample repeatedly difference of measurement result can well instruct production in 2%.
6) control impurity interference effect is better, sum up by analysis a large amount of test findings, and in the process that threonine is produced, can be less in the sample to the dopant species of this method affect, influence also less.
7) the assay method accuracy is higher, by with amino-acid analyzer contrast, this method measurement result and amino-acid analyzer measurement result basically identical, difference is all within operate miss.
Embodiment
Embodiment one: a kind of method of fast detecting threonine content, this method comprises the steps: to prepare respectively as required the standard threonine solution of 0.20g/L, 0.25g/L, 0.30g/L, 0.35g/L, according to threonine concentration in the sample of estimating, sample is diluted, make concentration after the dilution at 0.20g/L between the 0.35g/L.The sample solution of getting respectively after 1ml standard threonine solution and the dilution joins in 20 * 200 the test tube, and then add the 0.2mol/L citric acid-sodium citrate damping fluid of 2ml pH=6.00 and 1ml triketohydrindene hydrate colour developing liquid respectively, put into 96 ℃ of heating of four hole water-baths 10min, after taking-up is cooled to room temperature, adding 10ml distilled water also shakes up, and measures absorbance with wavelength 530nm then under spectrophotometer.According to standard threonine solution concentration with measure absorbance drawing standard curve as a result, threonine content in absorbance and the extension rate calculation sample per sample then.
Embodiment two: a kind of method of fast detecting threonine content, this method comprises the steps: to prepare respectively as required the standard threonine solution of 0.20g/L, 0.25g/L, 0.30g/L, 0.35g/L, according to threonine concentration in the sample of estimating, sample is diluted, make concentration after the dilution at 0.20g/L between the 0.35g/L.The sample solution of getting respectively after 1ml standard threonine solution and the dilution joins in 20 * 200 the test tube, and then add the 0.2mol/L citric acid-sodium citrate damping fluid of 2ml pH=6.00 and 1ml triketohydrindene hydrate colour developing liquid respectively, put into 96 ℃ of heating of four hole water-baths 8min, after taking-up is cooled to room temperature, adding 10ml distilled water also shakes up, and measures absorbance with wavelength 540nm then under spectrophotometer.According to standard threonine solution concentration with measure absorbance drawing standard curve as a result, threonine content in absorbance and the extension rate calculation sample per sample then.
Embodiment three: a kind of method of fast detecting threonine content, this method comprises the steps: to prepare respectively as required the standard threonine solution of 0.10g/L, 0.20g/L, 0.30g/L, 0.40g/L, according to threonine concentration in the sample of estimating, sample is diluted, make concentration after the dilution at 0.10g/L between the 0.40g/L.The sample solution of getting respectively after 1ml standard threonine solution and the dilution joins in 20 * 200 the test tube, and then add the 0.2mol/L citric acid-sodium citrate damping fluid of 2ml pH=6.00 and 1ml triketohydrindene hydrate colour developing liquid respectively, put into 97 ℃ of heating of four hole water-baths 8min, after taking-up is cooled to room temperature, adding 10ml distilled water also shakes up, and measures absorbance with wavelength 530nm then under spectrophotometer.According to standard threonine solution concentration with measure absorbance drawing standard curve as a result, threonine content in absorbance and the extension rate calculation sample per sample then.
Embodiment four: a kind of method of fast detecting threonine content, this method comprises the steps: to prepare respectively as required the standard threonine solution of 0.10g/L, 0.20g/L, 0.30g/L, 0.40g/L, according to threonine concentration in the sample of estimating, sample is diluted, make concentration after the dilution at 0.10g/L between the 0.40g/L.The sample solution of getting respectively after 1ml standard threonine solution and the dilution joins in 20 * 200 the test tube, and then add the 0.2mol/L citric acid-sodium citrate damping fluid of 2ml pH=6.00 and 1ml triketohydrindene hydrate colour developing liquid respectively, put into 98 ℃ of heating of four hole water-baths 10min, after taking-up is cooled to room temperature, adding 10ml distilled water also shakes up, and measures absorbance with wavelength 510nm then under spectrophotometer.According to standard threonine solution concentration with measure absorbance drawing standard curve as a result, threonine content in absorbance and the extension rate calculation sample per sample then.
Embodiment five: a kind of method of fast detecting threonine content, this method comprises the steps: to prepare respectively as required the standard threonine solution of 0.20g/L, 0.30g/L, 0.40g/L, 0.50g/L, according to threonine concentration in the sample of estimating, sample is diluted, make concentration after the dilution at 0.20g/L between the 0.50g/L.The sample solution of getting respectively after 1ml standard threonine solution and the dilution joins in 20 * 200 the test tube, and then add the 0.2mol/L citric acid-sodium citrate damping fluid of 2ml pH=6.00 and 1ml triketohydrindene hydrate colour developing liquid respectively, put into 98 ℃ of heating of four hole water-baths 8min, after taking-up is cooled to room temperature, adding 8ml distilled water also shakes up, and measures absorbance with wavelength 520nm then under spectrophotometer.According to standard threonine solution concentration with measure absorbance drawing standard curve as a result, threonine content in absorbance and the extension rate calculation sample per sample then.
Claims (6)
1. the method for a fast measuring threonine content, it is characterized in that: this method comprises the steps: the standard threonine solution of preparation variable concentrations earlier, the standard threonine solution of getting the above-mentioned variable concentrations that configures of 1ml then respectively joins in the different test tubes with the sample solution that 1ml need measure, add 2ml damping fluid and 1ml triketohydrindene hydrate colour developing liquid more respectively in above-mentioned each test tube, cooling is cooled to room temperature behind the water-bath certain hour, and then in above-mentioned each test tube, add 10ml distilled water, use the spectrophotometric determination light absorption value then, the light absorption value drawing standard curve that the concentration of standard threonine solution is corresponding with it, reacted per sample light absorption value and extension rate just can calculate threonine concentration in the sample solution, in the described triketohydrindene hydrate colour developing liquid, triketohydrindene hydrate concentration is 10g/L, and anhydrous cupric sulfate concentration is 16g/L.
2. the method for a kind of fast measuring threonine content as claimed in claim 1 is characterized in that: the standard threonine solution concentration of described preparation at 0.1g/L between the 0.5g/L.
3. the method for a kind of fast measuring threonine content as claimed in claim 1 is characterized in that: described sample solution dilutes earlier, the concentration that makes dilution back threonine at 0.20g/L between the 0.35g/L.
4. the method for a kind of fast measuring threonine content as claimed in claim 1 is characterized in that: described damping fluid is a 0.2mol/L citric acid-sodium citrate damping fluid, and its pH value is 6.00.
5. the method for a kind of fast measuring threonine content as claimed in claim 1 is characterized in that: described bath temperature is 95-100 ℃, and the water-bath time is 8-10 minute.
6. the method for a kind of fast measuring threonine content as claimed in claim 1 is characterized in that: described spectrophotometric wavelength is 500-570nm.
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| CN102928362A (en) * | 2012-07-18 | 2013-02-13 | 新疆天康畜牧生物技术股份有限公司 | Ninhydrin detection method as improvement to free amino quantitative method in solid-phase polypeptide synthesis |
| CN106353308B (en) * | 2016-08-24 | 2021-02-26 | 程玉慧 | Preparation method of p-hydroxyphenylalanine urine detection reagent |
| CN115452751B (en) * | 2022-10-26 | 2023-03-10 | 杭州泽天春来科技有限公司 | Residual chlorine detection method and device |
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| CN1645144A (en) * | 2005-01-25 | 2005-07-27 | 江苏省原子医学研究所 | Preparation and use of fluorescent marking reagent 2-naphthoxy acetyl chloride |
| CN1773264A (en) * | 2004-11-12 | 2006-05-17 | 中国科学院兰州化学物理研究所 | Method for colorimetric detecting and analysing cysteine |
| US7144737B2 (en) * | 2001-09-17 | 2006-12-05 | Albion International, Inc. | Process for determining the percent of chelation in a dry mixture |
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| US7144737B2 (en) * | 2001-09-17 | 2006-12-05 | Albion International, Inc. | Process for determining the percent of chelation in a dry mixture |
| CN1773264A (en) * | 2004-11-12 | 2006-05-17 | 中国科学院兰州化学物理研究所 | Method for colorimetric detecting and analysing cysteine |
| CN1645144A (en) * | 2005-01-25 | 2005-07-27 | 江苏省原子医学研究所 | Preparation and use of fluorescent marking reagent 2-naphthoxy acetyl chloride |
Non-Patent Citations (3)
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| JP特开2004-45174A 2004.02.13 |
| 刘黎.氨基酸混合体系的PLS分光光度法测定.《化学研究与应用》.1999,第11卷(第6期),646-649. * |
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