CN101122589B

CN101122589B - High performance liquid chromatography analysis method of urea and its impurity carbamylurea, methylene biuret

Info

Publication number: CN101122589B
Application number: CN2007100706344A
Authority: CN
Inventors: 刘忠发; 徐海伟
Original assignee: Individual
Current assignee: Individual
Priority date: 2007-09-05
Filing date: 2007-09-05
Publication date: 2011-01-12
Anticipated expiration: 2027-09-05
Also published as: CN101122589A

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 biuret thereof, MDU impurity

(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 biuret thereof, MDU impurity 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 biuret thereof, MDU impurity content.

Technical scheme of the present invention is as follows: the HPLC analytical method of a kind of urea and biuret thereof, MDU impurity, 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.

Adopt moving phase dissolving sample to be tested to make the determinand stock solution.

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 wherein water is desalted water, 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 .100.00g/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) preparation of .2.0000g/L biuret solution:

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)

Table 2 (corresponding to Fig. 5)

Table 3 (corresponding to Fig. 6)

The peak preface	The component name	During reservation	Peak height	Peak area	Peak area %	Concentration	The peak type	Standard
											Between [min]	[uV]	[uV*s]		[mg/L]		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

(2). the MDU recovery (seeing Table 6) table 6:

(3). the urea recovery (seeing Table 7)

Table 7

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

Table 9

Table 10

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

(2). the MDU recovery (seeing Table 13)

Table 13

(3). the urea recovery (seeing Table 14)

Table 14

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: 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=-0.107541+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

Table 16

Table 17

Method precision test (seeing Table 18)

Table 18

Recovery test

(1). the biuret recovery (seeing Table 19)

Table 19

(2). the MDU recovery (seeing Table 20)

Table 20

(3). the urea recovery (seeing Table 21)

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: 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=-0.107540+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

Table 23

Table 24

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

(2). the MDU recovery (seeing Table 27)

Table 27

(3). the urea recovery (seeing Table 28)

Table 28

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: 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=-0.107552+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

Table 30

Table 31

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

(2). the MDU recovery (seeing Table 34)

Table 34

(3). the urea recovery (seeing Table 35)

Table 35

Internal standard compound also can be selected the following polymkeric substance of ternary or the condensation product 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

1. the HPLC analytical method of urea and biuret thereof, MDU impurity, it is characterized in that: adopt the reversed-phase high-performance liquid chromatography internal standard method, the high performance liquid chromatography operating conditions is:

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: melamine;

2. the HPLC analytical method of urea as claimed in claim 1 and biuret thereof, MDU impurity 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 biuret thereof, MDU impurity, 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 biuret thereof, MDU impurity 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 biuret thereof, MDU impurity 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 biuret thereof, MDU impurity is characterized in that:

Moving phase is formed volume ratio: methyl alcohol: water=3: 97;

Flow velocity: 0.35ml/min;

Detect wavelength: 210nm.

7. the HPLC analytical method of urea as claimed in claim 3 and biuret thereof, MDU impurity is characterized in that:

Moving phase is formed volume ratio: methyl alcohol: water=3: 97;

Flow velocity: 0.35ml/min;

Detect wavelength: 210nm.

8. the HPLC analytical method of urea as claimed in claim 4 and biuret thereof, MDU impurity is characterized in that:

Moving phase is formed volume ratio: methyl alcohol: water=3: 97;

Flow velocity: 0.35ml/min;

Detect wavelength: 210nm.

9. the HPLC analytical method of urea as claimed in claim 5 and biuret thereof, MDU impurity is characterized in that:

Moving phase is formed volume ratio: methyl alcohol: water=3: 97;

Flow velocity: 0.35ml/min;

Detect wavelength: 210nm.