CN105548449A - Method for detecting free amino acid in biogas slurry - Google Patents
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Abstract
The invention relates to a method which comprises the following steps: removing high-concentration ammonia nitrogen and volatile biogenic amine in a way of rotary evaporation after biogas slurry is regulated to be alkaline; removing macromolecular disruptors by applying a 3K Millipore ultra-filtration membrane; performing pre-column derivatization on amino acids by using OPA and FMOC derivatization reagents, so that the amino acid has fluorescence emission characteristics to improve the detection sensitivity and the separation and selection characteristics; then performing qualitative and quantitative analysis on 24 kinds of amino acids through a reverse-high-performance liquid chromatography. Compared with a conventional method, the method avoids the selectivity of an SCX-SPE (Strong Cation-Exchange Solid-Phase Extraction Column) for the amino acids caused by different isoelectric points of the amino acids and overcomes the defects of low recovery rate and poor reproducibility during sample determination. The method provided by the invention is simple in step and strong in operability and provides a technical basis for the study on a protein degradation mechanism in the process of anaerobic digestion and a scientific basis for biogas slurry and biogas residue agricultural resource development and utilization.
Description
Technical field
The present invention relates to the synthetical development technology field of natural pond liquid, relate to the technology of carrying out needed for pre-treatment to sample, and the detection method of 24 kinds of free amino acids in the adaptation modern measure instrument disposable analysis natural pond liquid developed.
Background technology
At present, the generation of excess sludge increases sharply along with the high speed development of urban wastewater treatment ability.Cut-off was to 2013, and the year generation of China's excess sludge just reaches 6,250,000 t(in butt).Major part wherein does not obtain reasonable, safe treatment and disposal.Be rich in the nutrients such as nitrogen, phosphorus, potassium in excess sludge, the content of organic matter can reach 50% ?60%.Anaerobic digestion is the technology that a kind of stabilization realizing mud produces the biogas as the energy simultaneously, can as the suitable selection of specific resistance to filtration.In anaerobic sludge digestion process, the most of nutriment except carbon source is all retained in natural pond liquid and natural pond slag, and especially the content in the liquid of natural pond such as nitrogen, phosphorus and potassium is very high.And after anaerobic digestion, organism major part exists with Small molecular that is simple, that be easy to passive plant absorption or ionic condition.This makes natural pond liquid and the exploitation of natural pond slag agricultural reutilization become one of focus of the outer specific resistance to filtration technical research of Present Domestic.
In anaerobic digestion process, in organic nitrogen, a part is converted into ammoniacal nitrogen, and another part then participates in metabolism or is decomposed into the form of ammonia nitrogen as free amino acid etc.Amino acid to plant growth particularly photosynthesis there is unique facilitation, especially glycocoll, it can increase chlorophyll content of plant, improve the activity of enzyme, promote the infiltration of carbon dioxide, make photosynthesis more vigorous, to raising crop quality, increase content all important roles of vitamin C and carbohydrate.Therefore to instruct comprehensive utilization of mud for understanding nitrogen component composition and existing forms in the liquid of natural pond, set up amino acid method of testing fast and effectively and seem particularly important.
Summary of the invention
The object of the present invention is to provide the detection method of 24 kinds of free amino acids in the liquid of a kind of natural pond.
The present invention is regulated by pH and revolves and boils off except natural pond ammonia nitrogen and volatile bio amine, the novel ultra-filtration centrifuge tube of Millipore is used to remove macromolecular substances, the method of then combining column front derivation-oppositely-efficient liquid phase is analyzed natural pond liquid free amino acid, for protein degradation study mechanism in anaerobic digestion process provides basis, and provide scientific basis for natural pond liquid and natural pond slag agricultural reutilization develop.
In order to realize technical purpose of the present invention, adopt following technical scheme: the detection method of 24 kinds of free amino acids in the liquid of a kind of natural pond, comprise after regulating natural pond liquid pH to 10.2 and revolve the ammonia nitrogen and volatile bio amine that boil off high concentration in the liquid of natural pond, 0.5ML3KMillipore ultra-filtration centrifuge tube is used to remove large molecule chaff interference, using o-phthalaldehyde(OPA) (OPA) and chloro-carbonic acid 9-fluorenyl methyl ester (FMOC) two kinds of derivatization reagents to carry out pre-column derivatization to amino acid makes amino acid have fluorescence emission characteristic to improve detection sensitivity and to be separated selectivity characteristic, then oppositely-relative 24 seed amino acids of high-efficient liquid are used to carry out qualitative and quantitative analysis, concrete steps are as follows:
(1), the selection of instrument and reagent
Instrument is Agilent-1260 liquid chromatograph, is furnished with online degasser, quaternary gradient pump, standard automatic sampler, fluorescence detector (FLD) and Agilent chem workstation, the hybrid standard of 17 seed amino acids: aspartic acid, serine, tryptophane, glutamic acid, glycocoll, histidine, arginine, threonine, alanine, proline, halfcystine, tyrosine, valine, methionine, isoleucine, leucine and phenylalanine, supplement amino acid (asparagine, glutamine, citrulline, norvaline, lysine, hydroxyproline, methyl amimoacetic acid) and derivatization reagent o-phthalaldehyde(OPA) (OPA, HPLC), 3-mercaptopropionic acid (3-MPA, and chloro-carbonic acid 9-fluorenyl methyl ester (FMOC HPLC), HPLC) Sigma (StLouis is, MO, USA) product.Methyl alcohol, acetonitrile are HPLC level reagent; Experimental water is Milli-Q water (Millipore, USA); It is pure that other reagent are top grade, internal standard compound α-aminobutyric acid;
(2), the preparation of sample
(2.1) pre-treatment of sample
The filter membrane that natural pond liquid crosses 0.22 μm is removed particle, the filtrate pH value to 10.2 after filtering is regulated with 1M NaOH, get above-mentioned natural pond liquid 5ml to revolve to steam in eggplant type bottle in 50ml vacuumize rotary evaporated to dryness at 40 DEG C, to remove ammonia nitrogen in high density and volatile bio amine, with 5ml0.1M hydrochloric acid again stripping, vortex 30s mixes, and obtains dissolution fluid again; Described in getting, dissolution fluid 200 μ L is in 0.5ML3KMliipore ultra-filtration centrifuge tube again, and 13000g is centrifugal to be passed through to all liq, vortex mixing be placed in 4 DEG C stand-by;
(2.2) derivatization treatment of sample
In step (2.1) the gained sample of 0.5 microlitre, mix the OPA of 0.5 microlitre and the FMOC of 0.5 microlitre, then add the mixed solution dilution of the phosphoric acid of 400 microlitres by every 100ml mobile phase A of 32 microlitres, gained solution is used for RT-HPLC and analyzes; Incorporation time is 2min, automatically mixes, strictly control each reaction time by Agilent HPLC auto injection program.
(3), RT-HPLC chromatographic condition
Chromatographic column: AgilentZorboxEclipseAAA post (3.5 μm, 4.6mm × 150mm);
Mobile phase A is 0.04mol/LNaH
2pO
4(10mol/LNaOH is adjusted to pH=7.8); Mobile phase B is ultrapure water: acetonitrile: methyl alcohol is 10: 45: 45 (V/V/V);
Fluoroscopic examination wavelength: Ex=340nm, Em=460nm, PMT=10; 15.25min changes wavelength Ex=266nm, Em=305nm, PMT=9;
Flow velocity: 2ml/min.
Column temperature: 40 DEG C;
Mobile phase gradient is in table 1.
Table 1
| Time/min | A% | B% |
| 0 | 100 | 0 |
| 1.9 | 100 | 0 |
| 18.1 | 43 | 57 |
| 18.6 | 0 | 100 |
| 22.3 | 0 | 100 |
| 23.2 | 100 | 0 |
| 26 | 100 | 0 |
(4) the sample examination with computer after processing step (2) by the described RT-HPLC chromatographic condition of step (3), obtains amino acids distribution spectrogram.Adopt retention time method qualitative to amino acid in sample, namely under identical chromatographiccondition, in sample, amino acid whose chromatographic peak is identical or close with the chromatographic peak retention time of amino acid standard, then think and contain this amino acid in sample.Before each derivative reaction, internal standard compound is joined in sample, adopt internal mark working curve method to carry out quantitative test to the concentration of sample Free Amino Acids.
The present invention compares with detection method with existing pre-treating method, and tool has the following advantages:
1, natural pond liquid is in yellow, there are many tiny solid suspensions, complicated component, interfering component is more, add that ammonia and the volatile amine of very high concentrations can form fluorescence accessory substance with some amino acid to be measured, amino acid whose analysis is disturbed to measure, for the removal of these chaff interferences, have employed of novelty of the present invention regulates low pressure rotary evaporation at latter 40 DEG C of natural pond liquid pH to 10.2 to remove ammonia nitrogen and volatile bio amine, the mode of the novel ultra-filtration centrifuge tube of 0.5ML3KMliipore is used to remove large molecule chaff interference, avoid the shortcoming that conventional (by force) cationic solid phases extraction pillar (SCX-SPE) adopted removes chaff interference and amino acid whose non-selective adsorption in chaff interference process.
2, method involved in the present invention and conventional (by force) cationic solid phases extract pillar (SCX-SPE) and remove compared with the mode of chaff interference, avoid the SCX-SPE that causes because amino acid whose isoelectric point is different to amino acid whose selectivity, and the shortcoming of the recovery low and poor reproducibility during working sample.Step is simple, workable.
3, the present invention adopts two kinds of derivatization reagent OPA-FMOC coupling and combines the total analysis that the novel ultra-filtration centrifuge tube of Millipore achieves 24 seed amino acids in the liquid of natural pond.
Accompanying drawing explanation
Fig. 1 is the spectrogram that amino acid standard model of the present invention obtains.
Fig. 2,3 is the spectrogram that in embodiment 1,5% reactor and 20% reactor generation natural pond liquid sample obtain.
DFAA liquid chromatography comparison diagram in the natural pond liquid that Fig. 4 two kinds of Different front processing methods obtain, DFAA chromatogram in the natural pond liquid that in the natural pond liquid obtained by SCX pre-treatment under (a) pH2.2 condition, the novel ultra-filtration centrifuge tube pre-treatment of DFAA chromatogram (b) Millipore obtains.
Number in the figure: 1-asparatate, 2-glutamic acid, 3-asparagine, 4-serine, 5-glutamine, 6-histidine, 7-glycocoll, 8-threonine, 9-citrulline, 10-arginine, 11-alanine, 12-tyrosine, 13-α-aminobutyric acid, 14-cystine, 15-valine, 16-methionine, 17-norvaline, 18-tryptophane, 19-phenylalanine, 20-isoleucine, 21-leucine, 22-lysine, 23-hydroxyproline, 24-methyl amimoacetic acid, 25-proline.
Embodiment
Further illustrate the present invention below by embodiment, but protection scope of the present invention is not limited to described content.
Embodiment 1
Take solid content be 5% and 20% 6L anaerobic sludge digestion lab scale test unit discharging natural pond liquid and charging excess sludge as experiment material.Particle removed by the filter membrane that above-mentioned all natural pond liquid is crossed 0.22 μm respectively, pH to 10.2 is regulated with 1M NaOH, get each 5ml of above-mentioned natural pond liquid to revolve to steam in eggplant type bottle in 50ml and under 40 degrees Celsius, vacuumize rotary evaporated to dryness to remove ammonia nitrogen in high density and volatile bio amine, with 5ml0.1M hydrochloric acid again stripping, vortex 30s mixes.Get above-mentioned dissolution fluid again 200 μ L in the novel ultra-filtration centrifuge tube of 0.5ML3KMliipore, 13000g is centrifugal to be passed through to all liq, vortex mixing be placed in 4 DEG C stand-by.
Above-mentioned sample is as follows through Agilent HPLC1260 automatic sampler auto injection program: 0.5 μ L mixes the OPA of 0.5 μ L and the FMOC of 0.5 μ L, add the mixed solution dilution of the phosphoric acid of 400 μ L afterwards by every 100ml mobile phase A of 32 μ L, realize the derivatization of sample.
Agilent HPLC1260 instrument collocation AgilentZorboxEclipseAAA post (3.5 μm, 4.6mm × 150mm) chromatographic column is used to analyze under following chromatographic condition.
Mobile phase A is 0.04mol/LNaH2PO4 (10mol/LNaOH is adjusted to pH=7.8); B is ultrapure water: acetonitrile: methyl alcohol is 10: 45: 45 (V/V/V).
Fluoroscopic examination wavelength: Ex=340nm, Em=460nm, PMT=10; 15.25min changes wavelength Ex=266nm, Em=305nm, PMT=9.
Flow velocity: 2ml/min.
Column temperature: 40 degrees Celsius.
Mobile phase gradient is in table 2.
Table 2
| Time/min | A% | B% |
| 0 | 100 | 0 |
| 1.9 | 100 | 0 |
| 18.1 | 43 | 57 |
| 18.6 | 0 | 100 |
| 22.3 | 0 | 100 |
| 23.2 | 100 | 0 |
| 26 | 100 | 0 |
Obtain the experimental result as following table 3, the chromatogram of 5% and 20% solid content is shown in Fig. 2,3 respectively.
Table 3
| Amino acid | Retention time (min) | 20% reactor | 5% reactor | Excess sludge natural pond liquid |
| Asparatate | 2.056 | 194.0 | / | 49.9 |
| Glutamic acid | 4.116 | 305.4 | 20.3 | 84.8 |
| Asparagine | 6.325 | / | / | / |
| Serine | 6.612 | 80.4 | 24.2 | |
| Glutamine | 7.222 | / | / | / |
| Histidine | 7.475 | / | / | / |
| Glycocoll | 7.822 | 158.4 | / | 139.3 |
| Threonine | 8.015 | 130.0 | / | 29.0 |
| Citrulline | 8.246 | / | / | 64.6 |
| Arginine | 8.562 | / | / | 236.0 |
| Alanine | 9.212 | 280.9 | 15.3 | / |
| Tyrosine | 10.329 | 54.0 | / | 43.3 |
| Valine | 12.202 | 183.3 | / | 431.6 |
| Methionine | 12.4 | 47.3 | / | 62.6 |
| Norvaline | 12.744 | / | / | 109.8 |
| Tryptophane | 13.2 | / | / | 34.7 |
| Phenylalanine | 13.613 | 69.9 | / | 54.5 |
| Isoleucine | 13.817 | 118.7 | / | 195.8 |
| Leucine | 14.451 | 164.2 | / | 313.8 |
| Lysine | 14.836 | / | / | / |
| Hydroxyproline | 15.37 | / | / | / 4 --> |
| Methyl amimoacetic acid | 17.458 | 54.1 | / | / |
| Proline | 18.061 | 100.0 | / | 130.1 |
| Cystine | 12.028 | / | / | / |
Embodiment 2
Solid content is taked to be that 10% anaerobic sludge digestion lab scale test unit discharging natural pond liquid is as experiment material.Natural pond liquid sample is with 0.22 μm of membrane filtration.Natural pond liquid 1molL after filtration
-1naOH solution regulates pH to 10.2, revolves after steaming evaporate to dryness and uses 0.1molL
-1hCL dissolves again; The sample that strong cation solid phase extraction column (SCX-SPE) is tested passes through 1molL after filtration
-1hCL regulates pH to 2.2 stand-by.
(1): SCX solid phase extraction column is tested;
In experiment, SPE solid phase extraction column used activates and balance through 6ml methyl alcohol and 6ml water, and coutroi velocity is less than 3mlmin
-1.Getting 5ml natural pond liquid sample regulates pH to 10.2 to cross SCX-SPE solid phase extraction column, and coutroi velocity is less than 1mlmin
-1, this step does not collect filtered fluid, then rinses solid phase extraction column with 6ml2% formic acid, finally first uses 5ml methanol-eluted fractions (this step does not also collect eluent), rear use 5% ammoniated methanol wash-out, collect eluent.Get eluent 5ml to revolve in steaming bottle in 50ml eggplant type, load onto explosion-proof bottle, negative pressure rotary evaporated to dryness under 40 DEG C of water-baths.Then 5mL0.1molL is used
-1hCL dissolves again.
(2): the pre-treatment of 3KMillipore ultra filtration membrane;
The above-mentioned natural pond liquid 5ml being adjusted to 10.2 through pH revolves in 50ml eggplant type and steams in bottle, and load onto explosion-proof bottle, under 40 DEG C of water-baths, negative pressure rotary evaporated to dryness, then uses 5mL0.1molL
-1hCL dissolves again.Vortex 30S to mix, after get aforesaid liquid 200 μ L, adopt centrifugal mode to cross 3KMillipore ultra-filtration centrifuge tube, collect filtrate.
After above-mentioned process, sample is as follows through Agilent HPLC1260 automatic sampler auto injection program: 0.5 μ L mixes the OPA of 0.5 μ L and the FMOC of 0.5 μ L, add the mixed solution dilution of the phosphoric acid of 400 μ L afterwards by every 100ml mobile phase A of 32 μ L, realize the derivatization of sample.
Agilent HPLC1260 instrument collocation AgilentZorboxEclipseAAA post (3.5 μm, 4.6mm × 150mm) chromatographic column is used to analyze under following chromatographic condition.
Mobile phase A is 0.04mol/LNaH2PO4 (10mol/LNaOH is adjusted to pH=7.8); B is ultrapure water: acetonitrile: methyl alcohol is 10: 45: 45 (V/V/V).
Fluoroscopic examination wavelength: Ex=340nm, Em=460nm, PMT=10; 15.25min changes wavelength Ex=266nm, Em=305nm, PMT=9.
Flow velocity: 2ml/min.
Column temperature: 40 degrees Celsius.
Mobile phase gradient is in table 4.
Table 4
| Time/min | A% | B% |
| 0 | 100 | 0 |
| 1.9 | 100 | 0 |
| 18.1 | 43 | 57 |
| 18.6 | 0 | 100 |
| 22.3 | 0 | 100 |
| 23.2 | 100 | 0 |
| 26 | 100 | 0 |
Obtain the experimental result of following Fig. 4.After SCX pre-treatment, partial amino-acid runs off cannot quantitative (Fig. 4 (a); Through removing the pre-treatment of ammonia nitrogen and ultrafiltration, amino acid loss few (Fig. 4 (b)), chromatogram successful is better than Fig. 4 (a).
Claims (1)
1. the detection method of a natural pond liquid Free Amino Acids, the ammonia nitrogen and volatile bio amine that boil off high concentration in the liquid of natural pond is revolved after it is characterized in that comprising adjustment natural pond liquid pH to 10.2,0.5ML3KMillipore ultra-filtration centrifuge tube is used to remove large molecule chaff interference, using OPA and FMOC two kinds of derivatization reagents to carry out pre-column derivatization to amino acid makes amino acid have fluorescence emission characteristic to improve detection sensitivity and to be separated selectivity characteristic, then uses oppositely-relative 24 seed amino acids of high-efficient liquid to carry out qualitative and quantitative analysis; Concrete steps are as follows:
(1), the selection of instrument and reagent
Instrument is Agilent-1260 liquid chromatograph, is furnished with online degasser, quaternary gradient pump, standard automatic sampler, fluorescence detector (FLD) and Agilent chem workstation, the hybrid standard of 17 seed amino acids: aspartic acid, serine, tryptophane, glutamic acid, glycocoll, histidine, arginine, threonine, alanine, proline, halfcystine, tyrosine, valine, methionine, isoleucine, leucine and phenylalanine, supplement amino acid (asparagine, glutamine, citrulline, norvaline, lysine, hydroxyproline, methyl amimoacetic acid) and derivatization reagent o-phthalaldehyde(OPA) (OPA, HPLC), 3-mercaptopropionic acid (3-MPA, and 9-fluorenylmethyloxycarbonyl (FMOC HPLC), HPLC) Sigma (StLouis is, MO, USA) product, methyl alcohol, acetonitrile are HPLC level reagent, experimental water is Milli-Q water (Millipore, USA), it is pure that other reagent are top grade, internal standard compound α-aminobutyric acid,
(2), the preparation of sample
(2.1) pre-treatment of sample
The filter membrane that natural pond liquid crosses 0.22 μm is removed particle, the clear pH value to 10.2 after filtering is regulated with 1M NaOH, get above-mentioned natural pond liquid 5ml to revolve to steam in eggplant type bottle in 50ml vacuumize rotary evaporated to dryness at 40 DEG C, to remove ammonia nitrogen in high density and volatile bio amine, with 5ml0.1M hydrochloric acid again stripping, vortex 30s mixes, and obtains dissolution fluid again; Described in getting, dissolution fluid 200 μ L is in 0.5ML3KMliipore ultra-filtration centrifuge tube again, and 13000g is centrifugal to be passed through to all liq, vortex mixing be placed in 4 DEG C stand-by;
(2.2) derivatization treatment of sample
In step (2.1) the gained sample of 0.5 microlitre, mix the OPA of 0.5 microlitre and the FMOC of 0.5 microlitre, then add the mixed solution dilution of the phosphoric acid of 400 microlitres by every 100ml mobile phase A of 32 microlitres, gained solution is used for RT-HPLC and analyzes; Incorporation time is 2min; , automatically mix by Agilent HPLC auto injection program, strictly control each reaction time;
(3), RT-HPLC chromatographic condition
Chromatographic column: AgilentZorboxEclipseAAA post (3.5 μm, 4.6mm × 150mm);
Mobile phase A is 0.04mol/LNaH
2pO
4(10mol/LNaOH is adjusted to pH=7.8); Mobile phase B is ultrapure water: acetonitrile: methyl alcohol is 10: 45: 45 (V/V/V);
Fluoroscopic examination wavelength: Ex=340nm, Em=460nm, PMT=10; 15.25min changes wavelength Ex=266nm, Em=305nm, PMT=9;
Flow velocity: 2ml/min;
Column temperature: 40 DEG C;
Mobile phase gradient is in table 1.
Table 1
(4) the sample examination with computer after processing step (2) by the described RT-HPLC chromatographic condition of step (3), obtains amino acids distribution spectrogram; Adopt retention time method qualitative to amino acid in sample, namely under identical chromatographiccondition, in sample, amino acid whose chromatographic peak is identical or close with the chromatographic peak retention time of amino acid standard, then think and contain this amino acid in sample; Before each derivative reaction, internal standard compound is joined in sample, adopt internal mark working curve method to carry out quantitative test to the concentration of sample Free Amino Acids.
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| CN109633018A (en) * | 2018-12-29 | 2019-04-16 | 上海微谱化工技术服务有限公司 | The analysis method of functional component in a kind of cosmetic material |
| CN111189939A (en) * | 2020-01-14 | 2020-05-22 | 上海市农业科学院 | Method for detecting endogenous free amino acids of plants by using ultra-high performance liquid chromatography-tandem mass spectrometry |
| CN111398436A (en) * | 2020-03-02 | 2020-07-10 | 浙江工业大学 | Solid-phase extraction-liquid-phase detection method for simultaneously detecting multiple free amino acids in source water |
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| CN115932139B (en) * | 2022-10-14 | 2024-05-28 | 上海吉奉生物科技有限公司 | Method for detecting purity of fluorenylmethoxycarbonyl-S-dipalmitoyl-L-cysteine |
| CN115754049A (en) * | 2022-11-09 | 2023-03-07 | 农业部沼气科学研究所 | Liquid chromatography-mass spectrometry detection method for amino acids in biogas slurry |
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