CN101122589A - Urea and its impurity high performance liquid chromatography analysis method - Google Patents

Urea and its impurity high performance liquid chromatography analysis method Download PDF

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CN101122589A
CN101122589A CNA2007100706344A CN200710070634A CN101122589A CN 101122589 A CN101122589 A CN 101122589A CN A2007100706344 A CNA2007100706344 A CN A2007100706344A CN 200710070634 A CN200710070634 A CN 200710070634A CN 101122589 A CN101122589 A CN 101122589A
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urea
test
impurity
melamine
water
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CN101122589B (en
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刘忠发
徐海伟
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Abstract

Warm methanol with water (3+97) is used for sample dissolution. Methanol with water (3+97) is used as mobile phase. A stainless steel column with Kromasil-5Mu m-C18 as filler and an ultraviolet detector are used. A reversed phase liquid chromatography separation is implemented on urea, methylene diurea and biuret in the sample. Melamine is used as an internal standard for internal standard quantification. The precision and the recovery of the method exceed GB2440-2001 test method requirements. The method has the advantages of simple operation method and high automation degree. And the method is a significant innovation for the products detection.

Description

The HPLC analytical method of a kind of urea and impurity thereof
(1) technical field
The present invention relates to high efficiency liquid phase chromatographic analysis method, specifically, relate to the content of analyzing amide-type product and impurity thereof with high efficiency liquid phase chromatographic analysis method.
(2) background technology
The urea formal name used at school is a carbonyl diamide, and it is applied to agricultural production widely as base fertilizer, simultaneously again as the raw material of melamine and fine chemistry industry; In urea production process, because strengthening the material viscosity, the needs of granulation usually add industrial formol, reaction generates MDU, generates biuret simultaneously when high temperature; Urea contains MDU and exceeds standard, and very harmful production to fine chemistry industry causes quality to reduce to Production of Cyanuramide; The urea biuret-containing exceeds standard and will crop seed and seedling be produced murder by poisoning.
The analytical approach of regulation MDU is to adopt naphthalene disulfonic acid disodium salt spectrphotometric method for measuring (GB/T 2441.9-2001) in the urea national standard (GB2440-2001); The analytical approach of biuret is to adopt copper complex salt light spectrphotometric method for measuring (GB/T 2441.2-2001).Time-consuming, the effort of spectrophotometric analysis MDU and biuret requires height to the reviewer, and operating cost height and efficient are low.For utilizing the MDU in the high-efficient liquid phase chromatogram technique analysis urea product and the analytical approach of biuret, also there are not relevant criterion issue and article to report.
(3) summary of the invention
The purpose of this invention is to provide a kind of high performance liquid chromatography that can fast, accurately measure urea and impurity content thereof.
Technical scheme of the present invention is as follows: the HPLC analytical method of a kind of urea and impurity thereof, it is characterized in that adopting the reversed-phase high-performance liquid chromatography internal standard method, and the high performance liquid chromatography operating conditions is:
The stainless steel column of chromatographic column: 250mm * 4.6mm (id), Kromasil-5 μ m-C 18Filler;
Moving phase is formed volume ratio: methyl alcohol: water=10: 90～3: 97, and recommending volume ratio is 3: 97;
Flow velocity: 0.30ml/min～0.40ml/min, the recommendation flow velocity is 0.35ml/min;
Detect wavelength: 205nm～215nm, the recommendation wavelength is 210nm;
Internal standard compound: following polymkeric substance of the ternary of melamine or acid amides or condensation product, the recommendation internal standard compound is a melamine.
The column temperature and the sampling volume of high performance liquid chromatography operation adopt conventional parameter, and the recommendation column temperature is a room temperature, and sampling volume is 6 μ l.
Recommend described moving phase to adopt desalted water, and through boiling, to remove O 2And CO 2Deng dissolved gas; Described moving phase before use with 0.45 μ m membrane filtration removing insoluble salt or organic matter (suspension), and use ultrasonic degas.
That the method that the present invention adopts high performance liquid chromatograph to analyze amide-type product and MDU and Biuret Content impurity has is highly sensitive, detection limit is low, precision good, speed is fast, the efficient advantages of higher, requiring operating personnel to learn molten (notes) sample just can accurately analyze, be applicable to factory's On-line Control analysis and batch samples analysis, further perfect along with this method, to cause national urea analytical standard modification, that will have far reaching significance to inspection body's umpire analysis; Also will cause simultaneously modification to GB/T9567-1997 " industrial melamine " standard.
(4) description of drawings
Fig. 1 is the canonical plotting of MDU.
Fig. 2 is the canonical plotting of urea.
Fig. 3 is the canonical plotting of biuret.
Fig. 4 is the test result spectrogram of first group of sample.
Fig. 5 is the test result spectrogram of second group of sample.
Fig. 6 is the test result spectrogram of the 3rd group of sample.
(5) embodiment
Below in conjunction with the drawings and specific embodiments the present invention is further described.
Embodiment one
Experimental apparatus:
LC-10ATVP high performance liquid chromatograph (day island proper Tianjin);
SPD-10ATVP UV, visible light detecting device (day island proper Tianjin);
The Sepu 3000P of Hangzhou Pu Hui Science and Technology Ltd. chromatographic work station;
Ultrasonic degas device (the ultrasonic company limited in Shanghai);
Filtrator: filter membrane aperture 0.45 μ m.
Chromatographic condition:
The stainless steel column of chromatographic column: 250mm * 4.6mm (id), interior dress Kromasil-5 μ m-C 18Filler;
Moving phase is formed volume ratio: methyl alcohol: water=3: 97, and water foot desalted water wherein, and through boiling, methyl alcohol is pure for analyzing, moving phase is also used ultrasonic degas 30min with 0.45 μ m membrane filtration before use;
Flow velocity: 0.35Ml/min;
Column temperature: room temperature;
Detect wavelength: 210nm;
Sampling volume: 6 μ l;
Internal standard compound: melamine.
Medicine:
Biuret sample: content 〉=99.5%, national chemical fertilizer inspection center (Shanghai) standard sample;
Melamine: content 〉=99.8%, Shandong Hualu Hengsheng Chemical Industry Co. Ltd. produces; Urea sample: content 〉=99.0%, Tianjin analytical reagent;
MDU: content 0.09%, Shandong Hualu Hengsheng Chemical Industry Co. Ltd. adds the urea of aldehyde explained hereafter.
The selection of chromatographic condition:
1. the selection of moving phase: through screening, be under the condition of 3: 97 (volume ratio) in the methanol-water ratio, each composition can reach baseline separation.
2. detection wavelength: the wavelength of regulating the UV, visible light detecting device is at 210nm.
Determination step:
1. the preparation of internal standard compound deposit mark liquid:
0.1000g/L melamine solution preparation:
Take by weighing 0.1000g technical grade melamine and be dissolved in about 300ml methanol-water (3+97) solution, move in the 1000ml volumetric flask and be diluted to scale.
2. the preparation of determinand stock solution:
(1) preparation of .1100.00g/L urea liquid:
Take by weighing 100.00g and analyze pure urea (Tianjin) and be dissolved in about 300ml methanol-water (3+97) solution, move in the 1000ml volumetric flask and be diluted to scale.
(2) the .2.0000g/L preparation of two or two urea solutions of contracting:
Take by weighing 2.0000g biuret (national chemical fertilizer inspection center sample) and be dissolved in about 300ml methanol-water (3+97) solution, move in the 1000ml volumetric flask and be diluted to scale.
(3) preparation of .0.1900g/L MDU solution:
Take by weighing the urea (Shandong Hualu Hengsheng Chemical Industry Co. Ltd.'s production sample) that 100.00g contains MDU and be dissolved in about 300ml methanol-water (3+97) solution, move in the 1000ml volumetric flask and be diluted to scale.
3. the preparation of standard series
(1). the preparation of urea standard series:
Get respectively 0.25ml, 0.5ml, 0.75ml, 1.00ml, urea storing solution (100.00g/L) in the 100ml volumetric flask, respectively add 20ml melamine storing solution (0.1000g/L) and use methanol-water (3+97) solution dilution to scale, being equivalent to urea content is 0.99 * 25%, 0.99 * 50%, 0.99 * 75%, 0.99 * 100%.
(2). the preparation of biuret standard series:
The biuret storing solution (2.0000g/L) of getting 2.5ml, 5.0ml, 7.5ml, 10.0ml respectively is in the 100ml volumetric flask, respectively add 20ml melamine storing solution (0.1000g/L) and use methanol-water (3+97) solution dilution to scale, being equivalent to Biuret Content is 0.5%, 1.0%, 1.5%, 2.0%.
(3). the preparation of MDU standard series:
Take by weighing industrial urea 1.00g, the 2.00g, the 3.00g that contain MDU 0.09% respectively, other adds respectively, and pure urea reagent 2.00g, 1.00g are analyzed in the Tianjin that does not contain MDU, 0.00g is dissolved in the 100ml volumetric flask; Add 20ml melamine storing solution (0.1000g/L) more respectively and use methanol-water (3+97) solution dilution to scale, being equivalent to MDU content is 0.09%, 0.18%, 0.27%.
4. linear relationship
(1). the MDU linear relationship:
Application contains the MDU sample of variable concentrations, and detecting the curvilinear equation that obtains MDU is C/C i=-0.107544+4.34738 * A/A i, the related coefficient of linearity curve is 0.99996,
(2). the urea linear relationship
The urea sample of preparation variable concentrations, detecting the curvilinear equation that obtains urea is C/C i=-16.0704+190.34 * A/A i, the related coefficient of linearity curve is 0.99940.
(3). the biuret linear relationship
Selection contains the biuret sample of variable concentrations, and detecting the curvilinear equation that obtains urea is C/C i=-0.00752121+7.53823 * A/A i, the related coefficient of linearity curve is 0.99977.
5. measure
Each 6 μ l sample liquid that from the standard series test solution for preparing, pipette, inject liquid chromatography, carry out input and data processing by the Sepu3000P workstation, carry out three groups of tests, obtain the data shown in following Fig. 4, Fig. 5, curve shown in Figure 6 and table 1, table 2, the table 3, will be used for sample test in the curve store method file.
Table 1 (corresponding to Fig. 4)
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Content [%] The peak type Convert solid
1 Urea 4.693 51810 734149 61.672 9920.4997 BV 99.2
2 Biuret 5.968 8646 139944 11.75599 78.10644 VV 0.78
3 * melamine 6.93 12123 269478 22.6374 20 VB
4 Unknown 11.237 2184 46838 3.93462 2.78097 BB 0.03
Amount to 74763 1190409 100 10021.387
Table 2 (corresponding to Fig. 5)
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Content [mg/mL] The peak type
1 Urea 4.708 37020 510167 58.04149 101.17872 BV
2 MDU 5.51 2330 35900 4.08434 0.14839 VV
3 Biuret 5.945 6736 101779 11.5793 0.83395 VB
4 * melamine 6.872 8379 183718 20.90154 0.2 BB
5 8.826 410 7927 0.90183 0 BP
6 Unknown 11.07 1790 38694 4.40223 0.0337 PV
7 12.267 21 785 0.08927 0 VB
Amount to 56686 878970 100 102.39476
Table 3 (corresponding to Fig. 6)
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Concentration [mg/L] The peak type Normal concentration
1 Urea 4.654 45078 640127 70.67108 9901.365 BV 9900
2 Biuret 5.877 1797 27918 3.08225 16.99449 VV 18
3 * melamine 6.726 10059 235406 25.98923 20 VB 20
4 Unknown 10.912 121 2332 0.25744 0.15849 BB 0
6. method precision test (seeing Table 4)
Table 4
Project First group of test Second group of test The 3rd group of test Standard deviation
Urea -0.40 0.20 -0.70 0.29
Biuret -0.07 0.33 -0.27 0.15
MDU 0.000 0.001 0.004 0.005
7. recovery test
(1). the biuret recovery (seeing Table 5)
Table 5
The replica test result Standard specimen is 0.80%
The result Deviation Recovery %
First group of test 0.796 -0.07 99.5
Second group of test 0.804 0.33 100.5
The 3rd group of test 0.792 -0.27 99.0
Mean value 0.797 0.22 99.7
(2). the MDU recovery (seeing Table 6) table 6:
The replica test result
Standard value Detected value/error Recovery %
First group of test 0.270 0.270/0.00 100.0
Second group of test 0.180 0.181/0.001 100.6
The 3rd group of test 0.090 0.094/0.004 104.4
(3). the urea recovery (seeing Table 7)
Table 7
The replica test result Standard specimen is 99.0%
The result Deviation Recovery %
First group of test 99.0 -0.40 100.0
Second group of test 99.2 0.20 100.2
The 3rd group of test 100.1 -0.70 101.0
Mean value 99.4 0.43 100.4
Embodiment two
Chromatographic condition:
Moving phase is formed volume ratio: methyl alcohol: water=10: 90;
Flow velocity: 0.30Ml/min;
Column temperature: room temperature;
Detect wavelength: 215nm;
Sampling volume: 5 μ l;
Internal standard compound: melamine.
All the other embodiments are all identical with embodiment one.
(1). the MDU linear relationship:
Application contains the MDU sample of variable concentrations, and detecting the curvilinear equation that obtains MDU is C/C i=-0.107551+4.34741 * A/A i
(2). the urea linear relationship
The urea sample of preparation variable concentrations, detecting the curvilinear equation that obtains urea is C/C i=-16.0711+190.29 * A/A i
(3). the biuret linear relationship
Selection contains the biuret sample of variable concentrations, and detecting the curvilinear equation that obtains urea is C/C i=-0.00752131+7.53833 * A/A i
Each 6 μ l sample liquid that from the standard series test solution for preparing, pipette, inject liquid chromatography, carry out input and data processing, carry out three groups of tests by the Sepu3000P workstation, obtain the data shown in table 8, table 9, the table 10, will be used for sample test in the curve store method file.
Table 8
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Content [%] The peak type Convert solid
1 Urea 4.71 67353 954394 61.672 9920.5012 BV 99.2
2 Biuret 6.012 11239 181927 11.7559 78.2016 VV 0.78
3 * melamine 7.00 15760 350321 22.6374 20 VB
4 Unknown 11.321 2839 60889 3.9346 2.5234 BB 0.03
Amount to 97192 1547532 100 10021.225
Table 9
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Content [mg/mL] The peak type
1 Urea 4.705 48126 663217 58.04144 101.20827 BV
2 MDU 5.55 3029 46670 4.08432 0.1348 VV
3 Biuret 6.021 8757 132313 11.5794 0.83390 VB
4 * melamine 6.99 10893 238833 20.90147 0.2 BB
5 8.822 533 10305 0.90184 0 BP
6 Unknown 11.1 2327 50302 4.40218 0.0335 PV
7 12.367 27 1020 0.08926 0 VB
Amount to 73692 1142661 100 102.41047 ---
Table 10
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Concentration [mg/L] The peak type Normal concentration
1 Urea 4.69 58601 831525 70.67108 9901.427 BV 9900
2 Biuret 5.900 2336 36307 3.08225 17.000 VV 18
3 * melamine 6.75 13077 306028 25.98923 20 VB 20
4 Unknown 10.92 157 3032 0.25744 0.16002 BB 0
Method precision test (seeing Table 11)
Table 11
Project First group of test Second group of test The 3rd group of test Standard deviation
Urea -0.38 0.21 -0.3 0.37
Biuret -0.09 0.30 -0.27 0.29
MDU 0.004 0.002 0.002 0.003
Recovery test
(1). the biuret recovery (seeing Table 12)
Table 12
The replica test result Standard specimen is 0.80%
The result Deviation Recovery %
First group of test 0.798 -0.25 99.8
Second group of test 0.801 0.12 100.1
The 3rd group of test 0.795 -0.62 99.4
Mean value 0.798 -0.25 99.8
(2). the MDU recovery (seeing Table 13)
Table 13
The replica test result
Standard value Detected value/error Recovery %
First group of test 0.270 0.272/0.02 100.7
Second group of test 0.180 0.180/0.000 100.0
The 3rd group of test 0.090 0.092/0.002 102.2
(3). the urea recovery (seeing Table 14)
Table 14
The replica test result Standard specimen is 99.0%
The result Deviation With yield %
First group of test 99.1 -0.10 100.1
Second group of test 99.0 0.20 100.0
The 3rd group of test 99.4 0.2 100.4
Mean value 99.2 0.20 100.2
Embodiment three
Chromatographic condition:
Moving phase is formed volume ratio: methyl alcohol: water=4: 96;
Flow velocity: 0.38Ml/min;
Column temperature: room temperature;
Detect wavelength: 205nm;
Sampling volume: 7 μ l;
Internal standard compound: formamide.
All the other embodiments are all identical with embodiment one.
(1). the MDU linear relationship:
Application contains the MDU sample of variable concentrations, and detecting the curvilinear equation that obtains MDU is C/C i=-0.107541+4.34744 * A/A i4.
(2). the urea linear relationship
The urea sample of preparation variable concentrations, detecting the curvilinear equation that obtains urea is C/C i=-16.0711+190.32 * A/A i
(3). the biuret linear relationship
Selection contains the biuret sample of variable concentrations, and detecting the curvilinear equation that obtains urea is C/C i=-0.00752128+7.53817 * A/A i
Each 6 μ l sample liquid that from the standard series test solution for preparing, pipette, inject liquid chromatography, carry out input and data processing, carry out three groups of tests by the Sepu3000P workstation, obtain the data shown in table 15, table 16, the table 17, will be used for sample test in the curve store method file.
Table 15
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Content [%] The peak type Convert solid
1 Urea 4.69 51810 734149 61.672 9920.4997 BV 99.2
2 Biuret 6.00 8646 139944 11.75599 78.10644 VV 0.78
3 * melamine 6.95 12123 269478 22.6374 20 VB
4 Unknown 11.24 2184 46838 3.93462 2.78097 BB 0.03
Amount to 74763 1190409 100 10021.387
Table 16
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Content [mg/mL] The peak type
1 Urea 4.708 37020 510167 58.04149 101.17872 BV
2 MDU 5.51 2330 35900 4.08434 0.14839 VV
3 Biuret 5.945 6736 101779 11.5793 0.83395 VB
4 * melamine 6.872 8379 183718 20.90154 0.2 BB
5 8.826 410 7927 0.90183 0 BP
6 Unknown 11.07 1790 38694 4.40223 0.0337 PV
7 12.267 21 785 0.08927 0 VB
Amount to 56686 878970 100 102.39476
Table 17
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Concentration [mg/L] The peak type Normal concentration
1 Urea 4.654 45078 640127 70.67108 9901.365 BV 9900
2 Biuret 5.877 1797 27918 3.08225 16.99449 VV 18
3 * melamine 6.726 10059 235406 25.98923 20 VB 20
4 Unknown 10.912 121 2332 0.25744 0.15849 BB 0
Method precision test (seeing Table 18)
Table 18
Project First group of test Second group of test The 3rd group of test Standard deviation
Urea -0.40 0.20 -0.70 0.29
Biuret -0.07 0.33 -0.27 0.15
MDU 0.000 0.001 0.004 0.005
Recovery test
(1). the biuret recovery (seeing Table 19)
Table 19
The replica test result Standard specimen is 0.80%
The result Deviation Recovery %
First group of test 0.796 -0.07 99.5
Second group of test 0.804 0.33 100.5
The 3rd group of test 0.792 -0.27 99.0
Mean value 0.797 0.22 99.7
(2). the MDU recovery (seeing Table 20)
Table 20
The replica test result
Standard value Detected value/error Recovery %
First group of test 0.270 0.270/0.00 100.0
Second group of test 0.180 0.181/0.001 100.6
The 3rd group of test 0.090 0.094/0.004 102.4
(3). the urea recovery (seeing Table 21)
The replica test result Standard specimen is 99.0%
The result Deviation Recovery %
First group of test 99.0 -0.40 100.0
Second group of test 99.2 0.20 100.2
The 3rd group of test 100.1 -0.70 101.0
Mean value 99.4 0.43 100.4
Table 21
Embodiment four
Chromatographic condition:
Moving phase is formed volume ratio: methyl alcohol: water=6: 94;
Flow velocity: 0.40Ml/min;
Column temperature: room temperature;
Detect wavelength: 213nm;
Sampling volume: 8 μ l;
Internal standard compound: biuret.
All the other embodiments are all identical with embodiment one.
(1). the MDU linear relationship:
Application contains the MDU sample of variable concentrations, and detecting the curvilinear equation that obtains MDU is C/C i=-0.107540+4.34735 * A/A i
(2). the urea linear relationship
The urea sample of preparation variable concentrations, detecting the curvilinear equation that obtains urea is C/C i=-16.0709+190.33 * A/A i
(3). the biuret linear relationship
Selection contains the biuret sample of variable concentrations, and detecting the curvilinear equation that obtains urea is C/C i=-0.00752118+7.53822 * A/A i
Each 6 μ l sample liquid that from the standard series test solution for preparing, pipette, inject liquid chromatography, carry out input and data processing, carry out three groups of tests by the Sepu3000P workstation, obtain the data shown in table 22, table 23, the table 24, will be used for sample test in the curve store method file.
Table 22
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Content [%] The peak type Convert solid
1 Urea 4.702 56991 807564 61.68339 9920.4887 BV 99.2
2 Biuret 6.00 9424 153966 11.76023 78.20644 VV 0.78
3 * melamine 6.92 13335 296156 22.621 20 VB
4 Unknown 11.21 2367 51522 3.93535 2.68097 BB 0.03
Amount to 82117 1309208 100 10021.376
Table 23
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Content [mg/mL] The peak type
1 Urea 4.699 40733 562204 58.09657 101.27872 BV
2 MDU 5.71 2563 39490 4.0807 0.15002 VV
3 Biuret 6.000 7403 111977 11.57126 0.82395 VB
4 * melamine 6.88 9217 201906 20.86417 0.2 BB
5 8.83 451 8720 0.90109 0 BP
6 Unknown 11.09 1967 42563 4.39829 0.0327 PV
7 12.367 21 856 0.08845 0 VB
Amount to 62355 967716 100 102.48539
Table 24
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Concentration [mg/L] The peak type Normal concentration
1 Urea 4.754 49586 704140 70.67018 9901.565 BV 9900
2 Biuret 5.977 1975 30715 3.08267 16.79449 VV 18
3 * melamine 6.826 11115 258950 25.98921 20 VB 20
4 Unknown 10.99 132 2570 0.27793 0.15949 BB 0
Method precision test (seeing Table 25)
Table 25
Project First group of test Second group of test The 3rd group of test Standard deviation
Urea -0.20 0.30 -0.40 0.38
Biuret -0.06 0.30 -0.29 0.30
MDU 0.001 0.000 0.002 0.005
Recovery test
(1). the biuret recovery (seeing Table 26)
Table 26
The replica test result Standard specimen is 0.80%
The result Deviation Recovery %
First group of test 0.800 -0.12 100
Second group of test 0.802 0.38 100.25
The 3rd group of test 0.795 -0.5 99.4
Mean value 0.799 0.12 99.9
(2). the MDU recovery (seeing Table 27)
Table 27
The replica test result
Standard value Detected value/error Recovery %
First group of test 0.270 0.272/0.02 100.7
Second group of test 0.180 0.180/0.000 100.0
The 3rd group of test 0.090 0.093/0.003 103.3
(3). the urea recovery (seeing Table 28)
Table 28
The replica test result Standard specimen is 99.0%
The result Deviation Recovery %
First group of test 99.0 -0.40 100.0
Second group of test 99.1 -0.3 100.1
The 3rd group of test 100.0 0.6 101.0
Mean value 99.4 0.40 100.3
Embodiment five
Chromatographic condition:
Moving phase is formed volume ratio: methyl alcohol: water=8: 92;
Flow velocity: 0.32Ml/min;
Column temperature: room temperature;
Detect wavelength: 207nm;
Sampling volume: 6 μ l;
Internal standard compound: triuret contracts.
All the other embodiments are all identical with embodiment one.
(1). the MDU linear relationship:
Application contains the MDU sample of variable concentrations, and detecting the curvilinear equation that obtains MDU is C/C i=-0.107552+4.34741 * A/A i
(2). the urea linear relationship
The urea sample of preparation variable concentrations, detecting the curvilinear equation that obtains urea is C/C i=-16.0709+190.51 * A/A i
(3). the biuret linear relationship
Selection contains the biuret sample of variable concentrations, and detecting the curvilinear equation that obtains urea is C/C i=-0.00752132+7.53831 * A/A i
Each 6 μ l sample liquid that from the standard series test solution for preparing, pipette, inject liquid chromatography, carry out input and data processing, carry out three groups of tests by the Sepu3000P workstation, obtain the data shown in table 29, table 30, the table 31, will be used for sample test in the curve store method file.
Table 29
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Content [%] The peak type Convert solid
1 Urea 4.693 59582 844272 61.6348 9920.5009 BV 99.2
2 Biuret 5.968 9943 159536 11.64668 78.00644 VV 0.78
3 * melamine 6.93 14063 312594 22.820578 20 VB
4 Unknown 11.237 2533 53395 3.89802 2.68097 BB 0.03
--- Amount to ------- 86121 1369797 100 10021.188 ---
Table 30
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Content [mg/mL] The peak type
1 Urea 4.708 42573 586692 57.99907 101.07872 BV
2 MDU 5.51 2703 41644 4.11683 0.15839 VV
3 Biuret 5.945 7746 117046 11.5709 0.82395 VB
4 * melamine 6.872 9636 211276 20.88627 0.2 BB
5 8.826 467 9116 0.90118 0 BP
6 Unknown 11.07 2058 44885 4.43723 0.0338 PV
7 ---- 12.267 21 895 0.08847 0 VB
Amount to ------ 65264 1011554 100 102.29486 ---
Table 31
The peak preface The component name Retention time [min] Peak height [uV] Peak area [uV*s] Peak area % Concentration [mg/L] The peak type Normal concentration
1 Urea 4.754 51840 736146 70.83114 9901.379 BV 9900
2 Biuret 5.887 2049 32106 3.0892 16.99889 VV 18
3 * melamine 6.736 11668 268363 25.8213 20 VB 20
4 Unknown 10.922 138 2682 0.25805 0.16249 BB 0
Method precision test (seeing Table 32)
Table 32
Project First group of test Second group of test The 3rd group of test Standard deviation
Urea -0.20 0.20 -0.50 0.41
Biuret -0.06 0.30 -0.25 0.28
MDU 0.001 0.002 0.002 0.005
Recovery test
(1). the biuret recovery (seeing Table 33)
Table 33
The replica test result Standard specimen is 0.80%
The result Deviation Recovery %
First group of test 0.800 0.12 100
Second group of test 0.801 0.25 100.1
The 3rd group of test 0.795 -0.5 99.4
Mean value 0.799 0.12 99.9
(2). the MDU recovery (seeing Table 34)
Table 34
The replica test result
Standard value Detected value/error Recovery %
First group of test 0.270 0.271/0.01 100.4
Second group of test 0.180 0.180/0.000 100.0
The 3rd group of test 0.090 0.092/0.002 102.2
(3). the urea recovery (seeing Table 35)
Table 35
The replica test result Standard specimen is 99.0%
The result Deviation Recovery %
First group of test 99.2 -0.20 100.2
Second group of test 99.1 -0.30 100.1
The 3rd group of test 100.0 0.6 101.0
Mean value 99.4 0.40 100.4
Internal standard compound also can be selected the following polymkeric substance of ternary of acid amides for use.
Research is thought by experiment: the precision of this method and the recovery surpass the requirement of the GB2440-2001 method of inspection, and the easy automaticity height of method of operating is a far reaching innovation of such examination and test of products.

Claims (9)

1. the HPLC analytical method of urea and impurity thereof, it is characterized in that: adopt the reversed-phase high-performance liquid chromatography internal standard method, the high performance liquid chromatography operating conditions is:
The stainless steel column of chromatographic column: 250mm * 4.6mm (id), interior dress Kromasil-5 μ m-C 18Filler;
Moving phase is formed volume ratio: methyl alcohol: water=10: 90～3: 97;
Flow velocity: 0.30ml/min～0.40ml/min;
Detect wavelength: 205nm～215nm;
Internal standard compound: following polymkeric substance of the ternary of melamine or acid amides or condensation product.
2. the HPLC analytical method of urea as claimed in claim 1 and impurity thereof is characterized in that: the column temperature of high performance liquid chromatography operation is a normal temperature, and sampling volume is 6 μ l.
3. the HPLC analytical method of urea as claimed in claim 1 or 2 and impurity thereof, it is characterized in that: the water that described moving phase adopted is desalted water, and through boiling.
4. the HPLC analytical method of urea as claimed in claim 1 or 2 and impurity thereof is characterized in that: described moving phase is before use with 0.45 μ m membrane filtration and use ultrasonic degas.
5. the HPLC analytical method of urea as claimed in claim 3 and impurity thereof is characterized in that: described moving phase is before use with 0.45 μ m membrane filtration and use ultrasonic degas.
6. the HPLC analytical method of urea as claimed in claim 1 or 2 and impurity thereof is characterized in that:
Moving phase is formed volume ratio: methyl alcohol: water=3: 97;
Flow velocity: 0.35ml/min;
Detect wavelength: 210nm;
Internal standard compound: melamine.
7. the HPLC analytical method of urea as claimed in claim 3 and impurity thereof is characterized in that:
Moving phase is formed volume ratio: methyl alcohol: water=3: 97;
Flow velocity: 0.35ml/min;
Detect wavelength: 210nm;
Internal standard compound: melamine.
8. the HPLC analytical method of urea as claimed in claim 4 and impurity thereof is characterized in that:
Moving phase is formed volume ratio: methyl alcohol: water=3: 97;
Flow velocity: 0.35ml/min;
Detect wavelength: 210nm;
Internal standard compound: melamine.
9. the HPLC analytical method of urea as claimed in claim 5 and impurity thereof is characterized in that:
Moving phase is formed volume ratio: methyl alcohol: water=3: 97;
Flow velocity: 0.35ml/min;
Detect wavelength: 210nm;
Internal standard compound: melamine.
CN2007100706344A 2007-09-05 2007-09-05 High performance liquid chromatography analysis method of urea and its impurity carbamylurea, methylene biuret Expired - Fee Related CN101122589B (en)

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CN101413929B (en) * 2008-11-21 2011-09-14 山东省化工研究院 Liquid phase chromatography analysis method of biuret in compound mixed fertiliser
CN101446574B (en) * 2008-10-10 2012-01-04 上海市徐汇区中心医院 LC-MS quantitative detection method for melamine and tricyanic acid in human urine
CN103954722A (en) * 2014-05-21 2014-07-30 江南大学 Detection method aiming at trace urea in white spirit
CN104655738A (en) * 2013-11-21 2015-05-27 宁夏启元药业有限公司 Measurement method of content of urea in mother liquid of tetracycline/urea double salt
CN109633008A (en) * 2018-12-28 2019-04-16 沈阳中科新型肥料有限公司 A kind of method of a methylenediourea and dimethylene triurea in detection urea-formaldehyde fertilizer
CN110954640A (en) * 2020-01-02 2020-04-03 四川金象赛瑞化工股份有限公司 Method for detecting trace urea in melamine

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CN101446574B (en) * 2008-10-10 2012-01-04 上海市徐汇区中心医院 LC-MS quantitative detection method for melamine and tricyanic acid in human urine
CN101413929B (en) * 2008-11-21 2011-09-14 山东省化工研究院 Liquid phase chromatography analysis method of biuret in compound mixed fertiliser
CN104655738A (en) * 2013-11-21 2015-05-27 宁夏启元药业有限公司 Measurement method of content of urea in mother liquid of tetracycline/urea double salt
CN103954722A (en) * 2014-05-21 2014-07-30 江南大学 Detection method aiming at trace urea in white spirit
CN109633008A (en) * 2018-12-28 2019-04-16 沈阳中科新型肥料有限公司 A kind of method of a methylenediourea and dimethylene triurea in detection urea-formaldehyde fertilizer
CN110954640A (en) * 2020-01-02 2020-04-03 四川金象赛瑞化工股份有限公司 Method for detecting trace urea in melamine
CN110954640B (en) * 2020-01-02 2022-08-23 四川金象赛瑞化工股份有限公司 Method for detecting trace urea in melamine

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