CN103512828A - Method for accurately measuring content of primary amine group - Google Patents

Method for accurately measuring content of primary amine group Download PDF

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CN103512828A
CN103512828A CN201310482730.5A CN201310482730A CN103512828A CN 103512828 A CN103512828 A CN 103512828A CN 201310482730 A CN201310482730 A CN 201310482730A CN 103512828 A CN103512828 A CN 103512828A
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primary amine
way cock
amine groups
reactor
gelatin
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李天铎
许静
姜青伟
唐小龙
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Shandong Institute of Light Industry
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Shandong Institute of Light Industry
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Abstract

The invention provides a method for accurately measuring content of primary amine group on the basis of a Van method and particularly relates to a method for accurately measuring the content of the primary amine group in molecules of macromolecular biological compounds such as gelatin, proteins and the like. The method comprises the following steps: a primary amine group quantometer is prepared; the air in a system is emptied; testing conditions of concentration of nitrous acid, a ratio of glacial acetic acid to nitrous acid, concentration of a solution to be measured, a testing temperature, testing time and the like are fixed; the problems of great limitation to the environmental conditions and long macromolecular reaction balancing time of measurement on the content of the primary amine group in biomacromolecules are solved; the method has a calibration error of 0.5 percent on amino acid and has a measurement deviation of 1 percent on the gelatin.

Description

A kind of primary amine groups content Accurate Determining method
Technical field
The present invention relates to a kind of method of primary amine groups content Accurate Determining, relate in particular to the method for primary amine groups content Accurate Determining in a kind of gelatin, protein and other.
Background technology
Formol titration, ninhydrin colorimetry, TNBS colourimetry, Van Slyke method and vapor-phase chromatography are to measure the common method of primary amine groups content.Wherein, H+ that formol titration dissociates after being combined with free primary amine groups by titration formaldehyde calculates primary amine groups content, yet titration jump does not occur in whole titration process, shows that this method can not survey accurate primary amine groups content; TNBS colourimetry is to utilize trinitro-benzene-sulfonic acid and primary amine groups generation alkylated reaction to measure primary amine groups content, yet hydrogen on carbonyl α carbon, active methylene group etc. also can participate in alkylated reaction, and this method subsidiary reaction is many, and experimental error is large; In ninhydrin colorimetry, there is similar problem.Van Sykel method produces N2 volume by measuring primary amine groups and nitrite reaction calculates primary amine groups content.Gas chromatography has utilized Van Slyke method and has passed through gas chromatographic detection N2 content, yet gas chromatography can not gas collection, requiring reaction is being to finish in 5-10 minute, although amino content in the little molecule of the method energy Accurate Determining does not meet long demand of macromolecular reaction time.Comparatively speaking, Van Slyke method has that subsidiary reaction is few, reliability is high, meets the distinguishing features such as macromolecular reaction equilibration time is long, is the best practice of measuring primary amine groups conversion ratio in polypeptide reaction system.
Summary of the invention
The object of the invention is on primary amine groups quantitative instrument device development basis (ZL2012202419656), a kind of method of primary amine groups content Accurate Determining is provided, relate in particular to the method for primary amine groups content Accurate Determining in the large molecular biosciences compound molecule such as a kind of gelatin, protein, for biological macromolecular compound structure determination provides experimental basis.
The technical scheme that the present invention takes is, be ready to primary amine groups quantitative instrument, air in emptying system, prepare certain density liquid to be measured, the fixing proportioning of the concentration of sodium nitrite and glacial acetic acid and sodium nitrite, selected test duration, probe temperature, measure biomacromolecule compound solutions such as gelatin, protein solutions.
Above-mentioned primary amine groups assay method, comprises that step is as follows:
1) prepare primary amino radical quantitative instrument;
2) air in emptying system;
3) prepare liquid to be measured;
4) measure; By following formula, calculate primary amino radical content:
Wherein, n 0for primary amino radical content (mol) in biomacromolecule sample to be measured, n n2for the amount (mol) of the nitrogen that records,
Figure 2013104827305100002DEST_PATH_IMAGE002
for the mass concentration of biomacromolecule sample to be measured, m is biomacromolecule sample gross mass to be measured (g), ρfor the mass density (g/mL) of nitrogen, the temperature T during according to amount gas and atmospheric pressure P table look-up and obtain, vfor measuring the volume (mL) of nitrogen, mfor nitrogen molecule amount (g/mol), C 0for primary amino radical content (mol/g) in biomacromolecule sample to be measured.
Preferably, preparation primary amino radical quantitative instrument described in step 1), concrete operations are as follows: vacuum silicone grease is smeared in each cock, each interface of good seal, wherein hemisphere Drexel bottle leakage fluid dram (26) and reactor leakage fluid dram (17) clip with tongs, to prevent leak of liquid.
Preferably, step 2), concrete operations are as follows: first by washing lotion, from surge flask (23), slowly pour into, fill with hemisphere Drexel bottle (20), and make washing lotion to 1/3 place of surge flask (23) volume.Regulate inclined hole three-way cock (29) to be filled to inclined hole three-way cock (29) with washing lotion and locate, turn inclined hole three-way cock (29), communicates inclined hole three-way cock (29) Bu Yu each side.From leveling bottle (32), inject deionized water, turn inclined hole three-way cock (29) makes eudiometer tube communicate with leakage fluid dram (22), raise the position of leveling bottle (32), when eudiometer tube (31) is full of after deionized water, turn inclined hole three-way cock (29), communicates inclined hole three-way cock (29) Bu Yu each side.Turn three-way cock (10), makes the washing lotion in hemisphere Drexel bottle (20) be filled to three-way cock (10), then regulates three-way cock (10) that hemisphere Drexel bottle (20) is not communicated with reaction unit (I), retracting device (IV).This operation can the amount of making gas, the air in air-washer is discharged completely, in now hemisphere Drexel bottle (20) and eudiometer tube (31), is all full of liquid, and and isolate from outer air.
ON cycle water-bath, set test temperature required, from charge pipe (3), add glacial acetic acid, then add sodium nitrite in aqueous solution, close charge pipe (3), add deionized water fluid-tight, with a small amount of deionized water, gas in the tubule of charge pipe (14) bottom is caught up with into reactor (11), close charge pipe (14).Turn three-way cock (9), communicates reactor (11) and three-way cock (10).Open magnetic agitation (7), in reactor (11), reaction generates NO gas, keeps the interior air of emptying reactor (11) 5 minutes.
Preferably, step 4), concrete operations are as follows: open magnetic agitation (27), turn three-way cock (10), makes reaction unit (I) communicate with air-washer (II).In charge pipe (14), add liquid to be measured, slowly open charge pipe (14) cock, make the slow inflow reactor of liquid to be measured (11), and with a small amount of deionized water rinsing charge pipe (14), with n-octyl alcohol fluid-tight.Closed charge pipe (14) during reinforced end.Reactor (11), from adding liquid to be measured to start timing, reacts and within 30 ~ 300 minutes, stops reaction.Open charge pipe (3) cock, make liquid in charge pipe (3) enter reactor (11), gas in reactor (11) is caught up with completely into hemisphere Drexel bottle (20).Turn three-way cock (10), makes reactor (11) communicate with waste liquid bottle (36), does not communicate with hemisphere Drexel bottle (20).Open leakage fluid dram (17) the emptying reactant liquor of reactor (11).Increase the stirring rate of hemisphere Drexel bottle (20) below magnetic agitation (27), stir after certain hour, close magnetic agitation (27), standing, the inclined hole three-way cock (29) on turn eudiometer tube (31) top, catches up with the interior gas of hemisphere Drexel bottle (20) into eudiometer tube (31), when gas washing liquid flow to inclined hole three-way cock (29) and locates, turn inclined hole three-way cock (29), the each side that inclined hole three-way cock (29) is connected is not connected.Then standing 5 ~ 20 minutes, improve leveling bottle (32), make the interior liquid level concave surface of leveling bottle (32) equal with the interior liquid level concave surface of eudiometer tube (31), read gas volume V in eudiometer tube (31), record now air pressure P and eudiometer tube circulator bath temperature T, for calculating liquid primary amino radical content to be measured simultaneously.
Preferably, step 2), washing lotion be configured to take 10 ~ 40 grams of potassium permanganate, 0.5 ~ 3 gram of NaOH, is dissolved in the two in 200mL deionized water after mixing; Measuring temperature is arranged between 25 ~ 70 ℃; Glacial acetic acid is 10 ~ 30 milliliters; The mass percentage concentration of sodium nitrite is 10 ~ 60%, and consumption is 10 ~ 30 milliliters.
Preferably, step 3), the mass percentage concentration of biomacromolecule sample solution to be measured is 1 ~ 60%.
Preferably, step 4), the magnetic agitation of hemisphere Drexel bottle (20) below should keep 3 ~ 10 minutes; Close after magnetic agitation standing 5 ~ 20 minutes.
Primary amine groups content in the protein recording with this patent method method, amino acid molecular, compares with the percentage composition of contained primary amine groups in its each molecule, asks calculation relative deviation; Primary amine groups content in the gelatin molecule recording with this patent method method, with measured value calculating mean value repeatedly, then compares with each measured value and mean value, calculates test error.
beneficial effect:a kind of method of primary amine groups content Accurate Determining as claimed in claim 1, relate in particular to the method for primary amine groups content Accurate Determining in a kind of gelatin, protein and other, not only can be primary amine groups content Accurate Determining supplying method in the little molecules such as amino acid, synthetic compound, and be primary amine groups content Accurate Determining supplying method in gelatin, protein and other.This method of testing is started with from the new function of arrangement, combination, increase of apparatus, improved the test precision that installs self, specific as follows: reactor has increased after water-bath chuck, magnetic stirring apparatus, the temperature-controlled precision of reactor is accurate to ± 0.1 ℃, compare with the Examination on experimental operation that utilizes room temperature to control temperature of reactor, primary amine groups content measuring result error is reduced to 2-3% from 7-12%.; With the reactive absorption of magnetic agitation strengthening NO, gas washing efficiency greatly improves, the incomplete problem of gas washing of having avoided vibration gas washing to bring.Temperature control, magnetic stirring apparatus are combined with reaction, air-washer, from experiment effect, give the instrument of measuring for primary amine groups and there is temperature control, stir, be easy to catch up with gas, gas washing is convenient completely, be easy to many new functions such as cleaning reaction device.From point of theory, by above-mentioned a series of combinations, arrange.
From the design feature of biomacromolecule and the gathering behavior aqueous solution as theoretical foundation, the impacts of factor on primary amino radical assay such as sodium nitrite concentration, glacial acetic acid and nitrous acid proportioning, liquid concentration to be measured, temperature of reaction, reaction time have been studied, the problems such as macromolecular reaction needs fully to stir, reacting balance time is long have been solved, the test result of having avoided traditional Fan Si Rec device is subject to environmental baseline to restrict large problem, has greatly improved the mensuration precision of primary amine groups in biomacromolecule compound simultaneously.Test result shows, to its calibrated error of amino acid, is 0.5%, to its measurement deviation of gelatin, is 1%.
Accompanying drawing explanation
Fig. 1 primary amine groups quantitative instrument, wherein: I, reaction unit, II, air-washer, III, amount device of air, IV, retracting device 1, connecting pipe ground 2, reactor ground 3, charge pipe 4, recirculated water interface 5, thin neck feed pipe, 6, magneton 7, magnetic agitation 8, connecting pipe 9, three-way cock 10, three-way cock 11, reactor, 12, reactor wall 13, water-bath chuck 14, charge pipe 15, thin neck feed pipe 16, thin neck glass tube, 17, leakage fluid dram 18, thin neck glass tube 19, the thin neck glass tube 20 of S shape, hemisphere Drexel bottle 21, magneton, 22, leakage fluid dram 23, surge flask 24, recirculated water interface 25, thick neck glass tube 26, leakage fluid dram, 27, magnetic agitation 28, air intake opening 29, inclined hole three-way cock 30, water-bath chuck 31, eudiometer tube, 32, leveling bottle 33, silicone tube 34, six lead to adapter 35, silicone tube 36, waste liquid bottle 37, feed tube, 38, gas outlet 39, rubber plug 40, discharge opeing (gas) mouth 41, discharge opeing (gas) mouthful,
Fig. 2 utilizes primary amine groups quantitative instrument to measure primary amine groups content in gelatin molecule;
Fig. 3 utilizes traditional Van Slyke device to measure primary amine groups content in gelatin molecule;
The measurement result contrast of primary amine groups content data and TNBS colourimetry in Fig. 4 primary amino radical quantitative instrument test gelatin molecule;
The relation of primary amino radical content data in Fig. 5 probe temperature, time and gelatin molecule;
The relation of primary amino radical assay data in the mass percentage concentration of Fig. 6 sodium nitrite and gelatin molecule;
The relation of Fig. 7 gelatin mass percentage concentration and primary amino radical assay data.
Embodiment
Protein, amino acid have clear and definite molecular structure, and primary amine groups content in the protein recording with this patent method, amino acid is compared with the percentage composition of contained primary amine groups in its each molecule, asks calculation relative deviation; Gelatin molecule does not have clear and definite molecular structure, with test value calculating mean value repeatedly, then with each test value and mean value, compares, and calculates test error.
embodiment 1:
The method of the invention is measured primary amine groups content results and the contrast of traditional Van Slyke device measurement result in gelatin molecule.
Concrete operations are as follows: prepare primary amino radical quantitative instrument, vacuum silicone grease is smeared in each cock, each interface of good seal, and wherein hemisphere Drexel bottle leakage fluid dram (26) and reactor leakage fluid dram (17) clip with tongs, to prevent leak of liquid.
First by washing lotion, from surge flask (23), slowly pour into, fill with hemisphere Drexel bottle (20), and make washing lotion to 1/3 place of surge flask (23) volume.Regulate inclined hole three-way cock (29) to be filled to inclined hole three-way cock (29) with washing lotion and locate, turn inclined hole three-way cock (29), communicates inclined hole three-way cock (29) Bu Yu each side.From leveling bottle (32), inject deionized water, turn inclined hole three-way cock (29) makes eudiometer tube communicate with leakage fluid dram (22), raise the position of leveling bottle (32), when eudiometer tube (31) is full of after deionized water, turn inclined hole three-way cock (29), communicates inclined hole three-way cock (29) Bu Yu each side.Turn three-way cock (10), makes the washing lotion in hemisphere Drexel bottle (20) be filled to three-way cock (10), then regulates three-way cock (10) that hemisphere Drexel bottle (20) is not communicated with reaction unit (I), retracting device (IV).This operation can the amount of making gas, the air in air-washer is discharged completely, in now hemisphere Drexel bottle (20) and eudiometer tube (31), is all full of liquid, and and isolate from outer air.
ON cycle water-bath, setting probe temperature is 30 ℃, from charge pipe (3), add 20 milliliters, glacial acetic acid, then adding mass percentage concentration is 20 milliliters of the sodium nitrite in aqueous solution of 40wt%, close charge pipe (3), add deionized water fluid-tight, with a small amount of deionized water, gas in the tubule of charge pipe (14) bottom is caught up with into reactor (11), close charge pipe (14).Turn three-way cock (9), communicates reactor (11) and three-way cock (10).Open magnetic agitation (7), in reactor (11), reaction generates NO gas, keeps the interior air of emptying reactor (11) 5 minutes.
Open magnetic agitation (27), turn three-way cock (10), makes reaction unit (I) communicate with air-washer (II).In charge pipe (14), adding mass concentration is the aqueous gelatin solution of 5wt%, slowly opens charge pipe (14) cock, makes the slow inflow reactor of aqueous gelatin solution (11) of 5wt%, and with a small amount of deionized water rinsing charge pipe (14), with n-octyl alcohol fluid-tight.Closed charge pipe (14) during reinforced end.Reactor (11), from adding liquid to be measured to start timing, reacts and within 60 minutes, stops reaction.Open charge pipe (3) cock, make liquid in charge pipe (3) enter reactor (11), gas in reactor (11) is caught up with completely into hemisphere Drexel bottle (20).Turn three-way cock (10), makes reactor (11) communicate with waste liquid bottle (36), does not communicate with hemisphere Drexel bottle (20).Open leakage fluid dram (17) the emptying reactant liquor of reactor (11).Increase the stirring rate of hemisphere Drexel bottle (20) below magnetic agitation (27), stir after certain hour, close magnetic agitation (27), standing, the inclined hole three-way cock (29) on turn eudiometer tube (31) top, catches up with the interior gas of hemisphere Drexel bottle (20) into eudiometer tube (31), when gas washing liquid flow to inclined hole three-way cock (29) and locates, turn inclined hole three-way cock (29), the each side that inclined hole three-way cock (29) is connected is not connected.Then standing 5 ~ 20 minutes, improve leveling bottle (32), make the interior liquid level concave surface of leveling bottle (32) equal with the interior liquid level concave surface of eudiometer tube (31), read gas volume V in eudiometer tube (31), record now air pressure P and eudiometer tube circulator bath temperature T simultaneously, for calculating the primary amino radical content of the aqueous gelatin solution of 5wt%, measurement result is shown in Fig. 2.
Use at ambient temperature traditional Van Slyke device to measure, (see figure 3), comparative determination result simultaneously.
From Fig. 2 and Fig. 3, we can find out, utilize primary amino radical quantitative instrument, under the proportioning of definite temperature, test duration, material concentration, sodium nitrite concentration, sodium nitrite and glacial acetic acid, the stability of data measured is higher, relative error < 1% of data measured.Tradition Van Slyke device is due to cannot fixing test temperature, and the data stability recording is at ambient temperature poor, the relative error of determination data > 5%, be difficult to meet our requirement to data stability.
embodiment 2:
The measurement result contrast of primary amine groups content results and TNBS colourimetry in the method for the invention mensuration gelatin molecule, setting and measuring temperature is 45 ℃, and minute is 40 minutes, and other are with embodiment 1.
In the gelatin molecule that use primary amine groups quantitative instrument is measured, primary amine groups content results is shown in Fig. 4.Meanwhile, utilize TNBS colorimetric method for determining result to compare, see Fig. 4.
Measure, primary amino radical quantitative instrument is better to the data stability of gelatin primary amino radical assay, and relative error is 0.5%.And the data stability of the gelatin primary amino radical content of TNBS colorimetric method for determining is poor, relative error reaches 7%.
Utilize this result to show, under the condition of definite reaction time, temperature, the data accuracy that primary amine groups quantitative instrument records is higher, illustrating that Van Slyke method has the distinguishing feature that subsidiary reaction is few, reliability is high, meet macromolecular reaction equilibration time length, is the best practice of measuring primary amine groups content in biomacromolecule.And the subsidiary reaction of TNBS colourimetry is many, experimental error is large.
embodiment 3:
Probe temperature, the impact of time on primary amino radical assay result in amino acid in amino acid.
Owing to forming, the amino acid kind of gelatin is more, and we have chosen wherein representative alanine, glycocoll, glutamic acid, arginine, lysine and have measured.Before measuring to having carried out respectively purification process with upper amino acid according to its nature difference.For alanine, glycocoll, lysine, arginine, mainly utilize amino acid deliquescent difference in different solvents, with absolute ethyl alcohol, it has been carried out to repeatedly precipitating respectively, then standby after vacuum drying.For glutamic acid, utilize its dissolubility difference in the water of different pH, by regulator solution pH, it is carried out to repeatedly recrystallization, standby after vacuum drying.
Amino acid after purifying is mixed with respectively to the solution of 0.1 mol/L, then getting 2 milliliters measures with primary amino radical quantitative instrument, minute is respectively 30 minutes, 60 minutes, 90 minutes, measure temperature and be respectively 40 ℃, 45 ℃, 50 ℃, sodium nitrite 40wt%, nitrous acid/glacial acetic acid 1:1(V/V), then according to formula, calculate the content of primary amino radical in amino acid.Other are with embodiment 1.
As shown in Table 1, the mensuration of primary amino radical content tested fixed temperature and the impact of time to a certain extent in amino acid, when surveying fixed temperature≤45 ℃, survey fix time≤60 minutes time, in the amino acid that primary amino radical quantitative instrument records, the relative deviation of primary amino radical content is all less than 1%, hence one can see that, and the experiment conditions such as probe temperature, time are to determine that primary amine groups content turns the key element of but measuring.
The relation of primary amino radical assay data in table 1 probe temperature, time and amino acid
embodiment 4:
Probe temperature, the impact of time on primary amino radical assay in gelatin molecule.Preparation gelatin mass percentage concentration is 5%, sodium nitrite mass percentage concentration is 40%, nitrous acid/glacial acetic acid 1:1(volume ratio) under condition, studied and measured temperature and the impact of minute on primary amino radical assay, measure temperature and be respectively 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 70 ℃, minute is respectively 0.5h, 1h, 1.5h, 2h, 3h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h.Other are with embodiment 1.
As can be seen from Figure 5 the gelatin primary amino radical content, recording with primary amino radical quantitative instrument with the increase of temperature of reaction, the growth in reaction time is increase trend.When measuring temperature between 40-50 ℃, minute > during 4h, the primary amino radical content of the gelatin recording tends towards stability; When measuring temperature > 50 ℃, the primary amino radical content recording is along with the increase of minute increases sharply, measure the rising of temperature.Result of study shows, probe temperature, time are the key elements that affects primary amine groups content in the biomacromolecules such as gelatin.Gelatin is obtained by collagen hydrolysate, is biomacromolecule, is polyampholyte, contains various polarity group and non-polar group in its molecule, in aqueous solution, can assemble, and different temperatures state of aggregation is different, causes the variation of primary amino radical exposed amount.Gelatin is biomacromolecule, and chemical reaction occurs needs sufficiently long equilibration time, and therefore, along with the time increases, the primary amino radical content recording presents the trend that first increases back balance.
embodiment 5:
The impact of the mass percentage concentration of sodium nitrite on primary amino radical assay in gelatin molecule, change sodium nitrite mass concentration is respectively 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% time and measures, and other are with embodiment 2.
As can be seen from Figure 6 when sodium nitrite mass concentration<30%, the primary amino radical content recording increases along with the increase of sodium nitrite concentration; When sodium nitrite mass concentration>30% time, the primary amino radical content recording tends towards stability.
embodiment 6:
The impact of gelatin mass percentage concentration on primary amino radical assay, other are with embodiment 1.
As can be seen from Figure 7, in reaction system, the quality table of gelatin minute concentration is less than 10%, the gelatin primary amino radical content recording is more stable, when in mensuration system, the quality table of gelatin minute concentration is greater than 10%, increase along with gelatin material concentration in mensuration system, the primary amino radical content recording is on a declining curve, and it is the key element that guarantees primary amine groups content Accurate Determining in the biomacromolecules such as gelatin that applicable concentration is selected in this explanation.

Claims (7)

1. a method for primary amine groups content Accurate Determining, the method that relates in particular to primary amine groups content Accurate Determining in a kind of gelatin, protein and other comprises the steps:
1) prepare primary amino radical quantitative instrument;
?2) air in emptying system;
?3) prepare liquid to be measured;
?4) measure; By following formula, calculate primary amino radical content:
Figure 237786DEST_PATH_IMAGE001
Wherein, n 0for primary amino radical content (mol) in biomacromolecule sample to be measured, n n2for the amount (mol) of the nitrogen that records,
Figure 675721DEST_PATH_IMAGE002
for the mass concentration of biomacromolecule sample to be measured, m is biomacromolecule sample gross mass to be measured (g), ρfor the mass density (g/mL) of nitrogen, the temperature T during according to amount gas and atmospheric pressure P table look-up and obtain, vfor measuring the volume (mL) of nitrogen, mfor nitrogen molecule amount (g/mol), C 0for primary amino radical content (mol/g) in biomacromolecule sample to be measured.
2. the method for a kind of primary amine groups content Accurate Determining described in 1 as requested, relate in particular to the method for primary amine groups content Accurate Determining in a kind of gelatin, protein and other, it is characterized in that, preparation primary amino radical quantitative instrument described in step 1), concrete operations are as follows: vacuum silicone grease is smeared in each cock, each interface of good seal, wherein hemisphere Drexel bottle leakage fluid dram (26) and reactor leakage fluid dram (17) clip with tongs, to prevent leak of liquid.
3. the method for a kind of primary amine groups content Accurate Determining according to claim 1, relate in particular to the method for primary amine groups content Accurate Determining in a kind of gelatin, protein and other, it is characterized in that, step 2) concrete operations are as follows: first by washing lotion, from surge flask (23), slowly pour into, fill with hemisphere Drexel bottle (20), and make washing lotion to 1/3 place of surge flask (23) volume; Regulate inclined hole three-way cock (29) to be filled to inclined hole three-way cock (29) with washing lotion and locate, turn inclined hole three-way cock (29), communicates inclined hole three-way cock (29) Bu Yu each side; From leveling bottle (32), inject deionized water, turn inclined hole three-way cock (29) makes eudiometer tube communicate with leakage fluid dram (22), raise the position of leveling bottle (32), when eudiometer tube (31) is full of after deionized water, turn inclined hole three-way cock (29), communicates inclined hole three-way cock (29) Bu Yu each side; Turn three-way cock (10), makes the washing lotion in hemisphere Drexel bottle (20) be filled to three-way cock (10), then regulates three-way cock (10) that hemisphere Drexel bottle (20) is not communicated with reaction unit (I), retracting device (IV); This operation can the amount of making gas, the air in air-washer is discharged completely, in now hemisphere Drexel bottle (20) and eudiometer tube (31), is all full of liquid, and and isolate from outer air; ON cycle water-bath, set test temperature required, from charge pipe (3), add glacial acetic acid, then add sodium nitrite in aqueous solution, close charge pipe (3), add deionized water fluid-tight, with a small amount of deionized water, gas in the tubule of charge pipe (14) bottom is caught up with into reactor (11), close charge pipe (14); Turn three-way cock (9), communicates reactor (11) and three-way cock (10); Open magnetic agitation (7), in reactor (11), reaction generates NO gas, keeps the interior air of emptying reactor (11) 5 minutes.
4. the method for a kind of primary amine groups content Accurate Determining according to claim 1, relate in particular to the method for primary amine groups content Accurate Determining in a kind of gelatin, protein and other, it is characterized in that, the concrete operations of step 4) are as follows: open magnetic agitation (27), turn three-way cock (10), makes reaction unit (I) communicate with air-washer (II); In charge pipe (14), add liquid to be measured, slowly open charge pipe (14) cock, make the slow inflow reactor of liquid to be measured (11), and with a small amount of deionized water rinsing charge pipe (14), with n-octyl alcohol fluid-tight; Closed charge pipe (14) during reinforced end; Reactor (11), from adding liquid to be measured to start timing, reacts and within 30 ~ 300 minutes, stops reaction; Open charge pipe (3) cock, make liquid in charge pipe (3) enter reactor (11), gas in reactor (11) is caught up with completely into hemisphere Drexel bottle (20); Turn three-way cock (10), makes reactor (11) communicate with waste liquid bottle (36), does not communicate with hemisphere Drexel bottle (20); Open leakage fluid dram (17) the emptying reactant liquor of reactor (11); Increase the stirring rate of hemisphere Drexel bottle (20) below magnetic agitation (27), stir after certain hour, close magnetic agitation (27), standing, the inclined hole three-way cock (29) on turn eudiometer tube (31) top, catches up with the interior gas of hemisphere Drexel bottle (20) into eudiometer tube (31), when gas washing liquid flow to inclined hole three-way cock (29) and locates, turn inclined hole three-way cock (29), the each side that inclined hole three-way cock (29) is connected is not connected; Then standing 5 ~ 20 minutes, improve leveling bottle (32), make the interior liquid level concave surface of leveling bottle (32) equal with the interior liquid level concave surface of eudiometer tube (31), read gas volume V in eudiometer tube (31), record now air pressure P and eudiometer tube circulator bath temperature T, for calculating liquid primary amino radical content to be measured simultaneously.
5. the method for a kind of primary amine groups content Accurate Determining according to claim 1, relate in particular to the method for primary amine groups content Accurate Determining in a kind of gelatin, protein and other, it is characterized in that, step 2), washing lotion be configured to take 10 ~ 40 grams of potassium permanganate, 0.5 ~ 3 gram of NaOH, is dissolved in 200mL deionized water after the two is mixed; Measuring temperature is arranged between 25 ~ 70 ℃; Glacial acetic acid is 10 ~ 30 milliliters; The mass percentage concentration of sodium nitrite is 10 ~ 60%, and consumption is 10 ~ 30 milliliters.
6. the method for a kind of primary amine groups content Accurate Determining according to claim 1, relate in particular to the method for primary amine groups content Accurate Determining in a kind of gelatin, protein and other, it is characterized in that, step 3), the mass percentage concentration of biomacromolecule sample solution to be measured is 1 ~ 60%.
7. the method for a kind of primary amine groups content Accurate Determining according to claim 1, relate in particular to the method for primary amine groups content Accurate Determining in a kind of gelatin, protein and other, it is characterized in that, step 4), the magnetic agitation of hemisphere Drexel bottle (20) below should keep 3 ~ 10 minutes; Close after magnetic agitation standing 5 ~ 20 minutes.
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Citations (2)

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