CN110161137A - A method of measurement sodium-iron-chlorophyllin - Google Patents
A method of measurement sodium-iron-chlorophyllin Download PDFInfo
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- CN110161137A CN110161137A CN201910425081.2A CN201910425081A CN110161137A CN 110161137 A CN110161137 A CN 110161137A CN 201910425081 A CN201910425081 A CN 201910425081A CN 110161137 A CN110161137 A CN 110161137A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention belongs to analytical chemistry fields, disclose a kind of method for measuring sodium-iron-chlorophyllin, the following steps are included: the foundation of (1) calibration curve equation: for known to multiple concentration and concentration has differences each other sodium-iron-chlorophyllin solution standard, maximum absorption wavelength is determined first, then the liquid chromatogram corresponding to them is obtained by liquid chromatograph, then the peak position out of sodium-iron-chlorophyllin is confirmed by Mass Spectrometer Method, and obtains peak area-sodium iron chlorophyllin salinity calibration curve equation；(2) to the concentration mensuration of sample to be tested: measuring the liquid chromatogram of sample to be tested, obtain the peak area of sodium-iron-chlorophyllin, corresponding sodium-iron-chlorophyllin concentration value is calculated based on calibration curve equation, realizes the concentration mensuration of sodium-iron-chlorophyllin.The present invention, by liquid chromatogram, mass spectrometry, can effectively solve the technical issues of sodium-iron-chlorophyllin quantitative determines by exploring to measuring method overall flow technological design etc..
Description
Technical field
The invention belongs to analytical chemistry fields, more particularly, to a kind of method for measuring sodium-iron-chlorophyllin.
Background technique
Sodium-iron-chlorophyllin is a kind of derivative mixture of chlorophyll, by saponification, acid from the substances such as natural silkworm excrement
Change, iron generation, salt-forming steps obtain dark green solid or crystal.For treating hypoferric anemia and food additives.Leaf is green at present
Plain sheet sodium salt detection method has the methods of spectrophotometry, fluorimetry, high performance liquid chromatography, nuclear magnetic resonance.It is divided light
In degree method and fluorimetry due in sodium-iron-chlorophyllin mixing chlorophyll and other derivatives in identical SPECTRAL REGION
Absorption peak having the same, interferes the accurate quantitative analysis of sodium-iron-chlorophyllin, and method accuracy and detection limit are lower.Nuclear magnetic resonance
For method due to expensive equipment, popularity rate is not high.The present invention is by providing a kind of high performance liquid chromatography accurate quantitative analysis detection method, more
The deficiency for mending the detection means of sodium-iron-chlorophyllin, can obtain preferable separating effect, improvement method accuracy and precision.
The measuring method of the high performance liquid chromatography of sodium-iron-chlorophyllin is established, provides analysis detection for the measuring method of Related Research Domain
Method.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, the purpose of the present invention is to provide a kind of measurement phyllo-irons
The method of sodium salt, wherein first determining ultraviolet detection maximum by improving to measuring method overall flow technological design etc.
Absorbing wavelength obtains liquid chromatogram again on this basis, and confirms sodium-iron-chlorophyllin color by liquid chromatogram, mass spectrometry
The position of spectral peak constructs peak area with sodium iron chlorophyllin salinity by known concentration, concentration in gradient distribution standard items
Standard variation relationship solve measurement and obtain the dense of sodium-iron-chlorophyllin contained by sample to be tested according to the peak area of sample to be tested
Degree can effectively solve the technical issues of sodium-iron-chlorophyllin quantitative determines.The present invention is also further to used by chromatography detection
Parameter setting is preferably controlled, and separates sodium-iron-chlorophyllin preferably with other phyllins, so as to avoid
The interference of other phyllins further increases the accuracy and precision of the measuring method.
To achieve the above object, it is proposed, according to the invention, provide a kind of method for measuring sodium-iron-chlorophyllin, feature exists
In, comprising the following steps:
(1) foundation of calibration curve equation:
For known to multiple concentration and concentration has differences each other sodium-iron-chlorophyllin solution standard, first
230-900nm is carried out under ultraviolet specrophotometer as object using wherein any one sodium-iron-chlorophyllin solution standard
Full wavelength scanner, determine maximum absorption wavelength；Then, using the maximum absorption wavelength as ultraviolet detection in liquid chromatograph
Wavelength is detected using the liquid chromatograph using whole sodium-iron-chlorophyllin solution standards as object, is obtained each
Liquid chromatogram of the sodium-iron-chlorophyllin solution standard of a known concentration corresponding to them；Then, true by Mass Spectrometer Method
Recognize the peak position out of wherein any one liquid chromatogram Determination of Chlorophyll ferrisodium salt；Again for any one liquid chromatogram,
Sodium-iron-chlorophyllin goes out peak position and is integrated, and obtains the peak area of sodium-iron-chlorophyllin corresponding with the liquid chromatogram, such as
This is directed to whole liquid chromatograms, peak area can be obtained with the standard variation relationship of sodium iron chlorophyllin salinity, that is, peak face
The calibration curve equation of product-sodium iron chlorophyllin salinity；
(2) to the concentration mensuration of sample to be tested:
Using the sample to be tested of sodium iron chlorophyllin salinity to be measured as object, is detected, obtained using the liquid chromatograph
To the liquid chromatogram of the sample to be tested；Then, to the liquid chromatogram of the sample to be tested, go out peak position in sodium-iron-chlorophyllin
It is integrated, obtains the peak area of sodium-iron-chlorophyllin；Then, the peak area is brought into peak area-that the step (1) obtains
In the calibration curve equation of sodium iron chlorophyllin salinity, corresponding sodium-iron-chlorophyllin concentration value, the phyllo-iron is calculated
The sodium-iron-chlorophyllin concentration value of sodium salt concentration value namely the sample to be tested, the concentration for being achieved in sodium-iron-chlorophyllin are surveyed
It is fixed；
Also, the parameter of liquid chromatograph and liquid chromatograph described in the step (2) described in the step (1) is set
It sets and is consistent completely.
As present invention further optimization, in the step (1), the parameter setting of the liquid chromatograph includes simultaneously
Sample volume, detector, column oven temperature, chromatographic column selection, the setting of mobile phase and flow rate of mobile phase；
Preferably, sample volume is set as 10uL, and detector is set as UV detector, and column oven temperature setting is 30 DEG C,
Chromatographic column is selected as 4 μm of Poroshell 120EC-C18, and flow velocity is set as 1.0mL/min；
The mobile phase includes the mobile phase A and Mobile phase B being used cooperatively, wherein the mobile phase A is by methanol, water
And acetic acid is mixed by the volume ratio proportion of 70:20:10, the Mobile phase B is by methanol, acetone, water and acetic acid by 60:
The volume ratio proportion of 25:5:10 mixes；Corresponding gradient elution program are as follows: when 0min, mobile phase A accounting 100%, flowing
Phase B accounting 0%；When 5min, mobile phase A accounting 0%, Mobile phase B accounting 100%；When 25min, mobile phase A accounting
100%, Mobile phase B accounting 0%；Sampling interval equilibration time is 5min；
Alternatively, the mobile phase A is to be mixed by methanol, water and acetic acid by the volume ratio proportion of 60:30:10, it is described
Mobile phase B is to be mixed by methanol, acetonitrile, water and acetic acid by the volume ratio proportion of 50:25:15:10；Corresponding gradient elution journey
Sequence are as follows: when 0min, mobile phase A accounting 100%, Mobile phase B accounting 0%；When 15min, mobile phase A accounting 0%, mobile phase
B accounting 100%；When 25min, mobile phase A accounting 100%, Mobile phase B accounting 0%；Sampling interval equilibration time is 5min；
Also, reagent used by the mobile phase is chromatographically pure, and the acetic acid is the acetic acid that mass fraction is 0.2%
Solution, water are ultrapure water.
As present invention further optimization, in the step (1), it is described by Mass Spectrometer Method confirmation wherein any one
Liquid chromatogram Determination of Chlorophyll ferrisodium salt goes out peak position, the MS detection parameters is specifically first arranged, it is green that operation instrument obtains leaf
The mass spectrogram of plain sheet sodium salt solution, the position of sodium-iron-chlorophyllin is confirmed by mass spectral database, while analyzing the purity of target peak.
As present invention further optimization, the MS detection parameters are as follows: mass spectrum operation condition ESI ion source, point
Son amount scanning range 650-750.
As present invention further optimization, in the step (2), the sodium iron chlorophyllin salinity to be measured to test sample
Product are solid sample, fluid sample or suspension sample；
When the sample to be tested is solid sample or suspension sample, the sample to be tested also have passed through pretreatment work
Skill；When the sample to be tested is solid sample, the pretreating process is specially that first solid sample is dissolved in the water, then leads to
Cross centrifugation and filtering, finally take filtrate be placed in be stored in 4 DEG C in lighttight reagent bottle under conditions of in 30 days as to
Sample is detected；When the sample to be tested is suspension sample, the pretreating process specifically includes centrifugation and filtering,
Finally take filtrate be placed in be stored in 4 DEG C in lighttight reagent bottle under conditions of in 30 days be used as sample to be tested examined
It surveys；
Preferably, it when target substance content is lower than 5mg/L in the sample to be tested, before centrifugal treating, need to first pass through
Extraction carries out concentration.
As present invention further optimization, the filtering is using micropore filtering film suction filtration or micro porous filtration head, filtering
Film is specifically the filter membrane that aperture is 0.22 μm, and filter membrane material is polyether sulfone (PES), polytetrafluoroethylene (PTFE) (PTFE) or Kynoar
(PVDF)；
The lighttight reagent bottle is preferably brown liquid phase bottle.
As present invention further optimization, in the step (1), the multiple concentration is known and concentration each other
The sodium-iron-chlorophyllin solution standard having differences, specifically configuration concentration be 5mg/L, 10mg/L, 20mg/L, 50mg/L,
100mg/L standard solution.
As present invention further optimization, in the step (1), the peak area with sodium iron chlorophyllin salinity mark
Quasi- variation relation, specifically establishing by ordinate, sodium iron chlorophyllin salinity of peak area is the coordinate system of abscissa, then needle
To whole liquid chromatograms, corresponding point is marked in the coordinate system, then fits canonical plotting again, peak face can be obtained
The calibration curve equation of product-sodium iron chlorophyllin salinity.
It is described to be fitted to linear fit as present invention further optimization.
As present invention further optimization, in the step (2), the volume of the sample to be tested is no less than 0.2mL.
Contemplated above technical scheme through the invention, compared with prior art, first based on chlorophyll known to concentration
Ferrisodium salting liquid standard items determine that ultraviolet detection maximum absorption wavelength, cooperation liquid chromatograph obtain liquid chromatogram, recycle
Chromatography, mass spectrometry determine liquid chromatogram Determination of Chlorophyll ferrisodium salt go out peak position (and the purity at peak), pass through calculating
The peak area of sodium-iron-chlorophyllin solution liquid chromatographic figure Determination of Chlorophyll ferrisodium salt, it is green with leaf to obtain peak area known to each concentration
The standard variation relationship of plain sheet sodium salt concentration；Again based on the liquid chromatogram of sample to be tested, by the peak of corresponding sodium-iron-chlorophyllin
Area substitutes into the concentration that unknown sample Determination of Chlorophyll ferrisodium salt can be obtained in standard variation relationship.
The present invention also by the proportion and gradient elution program of the parameter to liquid chromatograph, especially mobile phase, carries out
It is preferred that control, cooperate chromatographic column, can effectively overcome in sodium-iron-chlorophyllin that several main component structures are similar, molecular weight is close
The defect for causing the single mobile phase of liquid chromatograph that can not be separated.The present invention preferably makes mobile phase A and Mobile phase B cooperation
It is used as mobile phase, mobile phase A can be controlled to be mixed by methanol, water and acetic acid by the volume ratio proportion of 70:20:10,
To be mixed by methanol, acetone, water and acetic acid by the volume ratio proportion of 60:25:5:10, corresponding gradient is washed for Mobile phase B control
De- program are as follows: when 0min, mobile phase A accounting 100%, Mobile phase B accounting 0%, when 5min, mobile phase A accounting 0%, stream
Dynamic phase B accounting 100%, when 25min, mobile phase A accounting 100%, Mobile phase B accounting 0%；Alternatively, mobile phase A control is served as reasons
Methanol, water and acetic acid by 60:30:10 volume ratio proportion mix, Mobile phase B be by methanol, acetonitrile, water and acetic acid by
The volume ratio proportion of 50:25:15:10 mixes, corresponding gradient elution program are as follows: when 0min, mobile phase A accounting 100%,
Mobile phase B accounting 0%, when 15min, mobile phase A accounting 0%, Mobile phase B accounting 100%, when 25min, mobile phase A is accounted for
Than 100%, Mobile phase B accounting 0%, sampling interval equilibration time is 5min, realizes the preferable gradient elution journey of separating effect
Sequence separates sodium-iron-chlorophyllin preferably with other phyllins, so as to avoid other phyllins
Interference, improves the accuracy and precision of measuring method.The present invention makes phyllo-iron by suitable mobile phase and instrument parameter
Sodium salt is separated with other derivatives as far as possible, in different times appearance.In addition, the present invention can be by further analyzing mesh
The purity for marking peak, can analyze sodium-iron-chlorophyllin, sodium-iron-chlorophyllin is allowed to separate with other derivatives, realize accurate
Quantitative analysis.
The concentration of Plays product of the present invention preferably configures range in 5mg/L to 1000mg/L, and concentration is too low, instrument inspection
The summit very little for surveying number, the too high and easy damage liquid phase column even without, concentration.The inspection of present invention detection sodium-iron-chlorophyllin
Survey range is 0.1-25mg/L, and sodium iron chlorophyllin salt derivative detection range is 5-1000mg/L, the opposite mark of recovery of standard addition
Quasi- deviation (RSD) is 98.23%-101.45%, can obtain the beneficial effect that accuracy in detection and precision improve.
The method of the present invention is suitable for various samples to be tested, such as can be and contain from environmental area sewage, sludge etc.
The content of sodium-iron-chlorophyllin involved in the chemical synthesis process of the determinand of sodium-iron-chlorophyllin, chemistry and biological field
The sample of quantitative determination process is particularly suitable for the measurement of sodium iron chlorophyllin saline solution.
Detailed description of the invention
Fig. 1 be mobile phase be acetone when 0.005% sodium-iron-chlorophyllin separate liquid chromatogram.
Fig. 2 be mobile phase be acetone when sodium-iron-chlorophyllin standard curve (concentration range 1-20mg/L).
Fig. 3 be mobile phase be acetonitrile when 0.01% sodium-iron-chlorophyllin separate liquid chromatogram.
Fig. 4 be mobile phase be acetonitrile when sodium-iron-chlorophyllin standard curve (concentration range 1-20mg/L).
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below
Not constituting a conflict with each other can be combined with each other.
Acquire and save invention provides for the scope of application of high effective liquid chromatography for measuring sodium-iron-chlorophyllin, sample,
Reagent and material, instrument and equipment, analytical procedure, calculated result.
The method that the present invention measures sodium-iron-chlorophyllin includes mainly sample pretreatment, determines ultraviolet detection on the whole
Maximum absorption wavelength, liquid chromatograph detection, liquid chromatography mass combination confirmation peak position, make standard curve, sample measurement and
Calculating process.Specifically, this method including the following steps:
(I) it sample pretreatment: is stored in brown or lighttight reagent bottle after extraction, centrifugation, filtering.
(II) it determines ultraviolet detection maximum absorption wavelength: certain density sodium iron chlorophyllin salting liquid is configured, at ultraviolet point
Full wavelength scanner is carried out under light photometer, determines maximum absorption wavelength.
(III) liquid chromatograph detects: instrument parameter setting includes sample volume, detector, column oven temperature, chromatographic column choosing
It selects, the setting of mobile phase.Set ultraviolet in liquid chromatograph for the detection of maximal ultraviolet obtained in step (II) absorbing wavelength
Detection wavelength.Obtain the liquid chromatogram of sodium iron chlorophyllin salting liquid.
(IV) liquid chromatography mass combination confirmation peak position: by chromatogram further progress mass spectrum obtained in step (III)
Analysis.Mass spectrometry parameters are set, and operation instrument obtains the mass spectrogram of sodium iron chlorophyllin salting liquid, (such as by mass spectrogram interpretation of result
Determination can be analyzed by the nucleocytoplasmic ratio on ion flow pattern) confirm the position of sodium-iron-chlorophyllin, while analyzing the pure of target peak
Degree.
(V) make standard curve: configuring the sodium iron chlorophyllin salting liquid of graded series, examined according to step (III) parameter
It surveys, obtains chromatogram, using peak area as ordinate, sodium iron chlorophyllin salinity is abscissa, makes canonical plotting, is marked
Directrix curve equation.
(VI) sample measurement and calculating process: placing by step (II) pretreated sample, in brown liquid phase bottle, if
Liquid chromatograph parameter is set, chromatogram is obtained, goes out peak position in sodium-iron-chlorophyllin and is integrated, obtain sodium-iron-chlorophyllin
Peak area substitutes into calibration curve equation and obtains the concentration of unknown sample Determination of Chlorophyll ferrisodium salt.
The following are specific embodiments:
Embodiment 1:
(I) determine ultraviolet detection maximum absorption wavelength: purchase sodium-iron-chlorophyllin reagent configures 0.005% phyllo-iron
Sodium salt solution weighs sodium-iron-chlorophyllin solid 5mg, is dissolved in 100mL ultrapure water, crosses 0.22 μm of Kynoar (PVDF)
Sodium iron chlorophyllin salting liquid is stored in brown reagent bottle (sodium-iron-chlorophyllin is a kind of water-soluble substance) by filter membrane.?
240-900nm is scanned with step-length for 1nm under Shimadzu ultraviolet specrophotometer, determines a length of 392nm of maximum absorption wave.
(II) liquid chromatograph detects: being in sample volume by the 0.005% sodium iron chlorophyllin salting liquid configured in step (I)
10uL, UV detector wavelength 392nm, column oven temperature setting are 30 DEG C, and chromatographic column is 4 μ of Poroshell 120EC-C18
M, flow velocity 1.0mL/min, mobile phase: A: methanol: water: acetic acid=70:20:10, B: methanol: acetone: water: acetic acid=60:25:
5:10, gradient elution program 0min, A%=100%, B%=0%；5min, A%=0%, B%=100%；25min,
A%=100%, B%=0%, sampling interval equilibration time are detected under conditions of being 5min, and it is molten to obtain sodium-iron-chlorophyllin
The chromatogram of liquid (see Fig. 1).
(III) liquid chromatography mass combination confirmation peak position: by chromatogram further progress mass spectrum obtained in step (II)
Analysis waits until mass spectrogram using the parameter that liquid phase detects, the position of sodium-iron-chlorophyllin is confirmed by mass spectral database, while analyzing mesh
Mark the purity at peak.Mass spectrum operation condition ESI ion source, molecular weight scanning range 500-750.
(IV) make standard curve: the sodium iron chlorophyllin salting liquid of configuration concentration 1,2,4,5,10,20mg/L, according to step
(II) parameter is detected, and obtains chromatogram, and using peak area as ordinate, sodium iron chlorophyllin salinity is abscissa, makees bid
Directrix curve figure obtains calibration curve equation (see Fig. 2).
Embodiment 2:
(I) determine ultraviolet detection maximum absorption wavelength: purchase sodium-iron-chlorophyllin reagent configures 0.01% sodium iron chlorophyllin
Salting liquid weighs sodium-iron-chlorophyllin solid 10mg, is dissolved in 100mL ultrapure water, crosses 0.22 μm of Kynoar (PVDF)
Sodium iron chlorophyllin salting liquid is stored in brown reagent bottle by filter membrane.Under Mei Puda ultraviolet specrophotometer 220-900nm with
Step-length is scanned for 1nm, determines a length of 395nm of maximum absorption wave.
(II) liquid chromatograph detects: being in sample volume by the 0.01% sodium iron chlorophyllin salting liquid configured in step (I)
10uL, UV detector wavelength 395nm, column oven temperature setting are 30 DEG C, and chromatographic column is 4 μ of Poroshell 120EC-C18
M, flow velocity 1.0mL/min, mobile phase: A: methanol: water: acetic acid=60:30:10, B: methanol: acetonitrile: water: acetic acid=50:25:
15:10, gradient elution program 0min, A%=100%, B%=0%, 15min；A%=0%, B%=100%；25min,
A%=100%, B%=0%.Sampling interval equilibration time is detected under conditions of being 5min, and it is molten to obtain sodium-iron-chlorophyllin
The chromatogram of liquid (see Fig. 3).
(III) liquid chromatography mass combination confirmation peak position: by chromatogram further progress mass spectrum obtained in step (II)
Analysis waits until mass spectrogram using the parameter that liquid phase detects, the position of sodium-iron-chlorophyllin is confirmed by mass spectral database, while analyzing mesh
Mark the purity at peak.Mass spectrum operation condition ESI ion source, molecular weight scanning range 650-750.
(IV) make standard curve: the sodium iron chlorophyllin salting liquid of configuration concentration 1,2,4,5,10,20mg/L, according to step
(II) parameter is detected, and obtains chromatogram, and using peak area as ordinate, sodium iron chlorophyllin salinity is abscissa, makees bid
Directrix curve figure obtains calibration curve equation (see Fig. 4).
Embodiment 3:
(I) sample pretreatment: naturally silkworm excrement extracts the sodium-iron-chlorophyllin of synthesis by extracting, being centrifuged and crossing 0.22 μm
After Kynoar (PVDF) filter membrane, 4 DEG C of preservations in brown reagent bottle are placed.
(II) it determines ultraviolet detection maximum absorption wavelength: 1mL sodium iron chlorophyllin salting liquid being taken to dilute 10 quilts.In Hash
300-900nm is scanned with step-length for 1nm under DR3900 type spectrophotometer, determines a length of 395nm of maximum absorption wave.
(III) liquid chromatograph detects: measuring 1mL sodium iron chlorophyllin salting liquid in brown liquid phase bottle (volume 2mL).?
Sample volume is 10uL, and UV detector wavelength 395nm, column oven temperature setting is 30 DEG C, chromatographic column Poroshell
4 μm of 120EC-C18, flow velocity 1.0mL/min, mobile phase: A: methanol: water: acetic acid=70:20:10, B: methanol: acetone: water:
Acetic acid=60:25:5:10, gradient elution program 0min, A%=100%, B%=0%；5min, A%=0%, B%=
100%；25min, A%=100%, B%=0%, sampling interval equilibration time are detected under conditions of being 5min, obtain leaf
The chromatogram of green plain sheet sodium salt solution.
(IV) sample measurement and calculating process: go out peak position in sodium-iron-chlorophyllin and integrated, obtain sodium iron chlorophyllin
The peak area of salt substitutes into the calibration curve equation redeterminated (source and reality due to the present embodiment Determination of Chlorophyll ferrisodium salt
Apply example 1, embodiment 2 is all different, therefore calibration curve equation needs redeterminate), obtain unknown sample Determination of Chlorophyll ferrisodium
The concentration of salt.
Embodiment 4:
(I) a length of 392nm sodium-iron-chlorophyllin reagent of the maximum absorption wave purchased sample pretreatment: is added to remaining dirt
It in mud, is improved, after the mixed liquor high speed centrifugation after the completion of improving, crosses 0.22 μm of Kynoar (PVDF) filter membrane.For
The low-down sample of content can refilter processing by first extracting and enriching convenient for detection.
(II) liquid chromatograph detects: measuring (the volume in brown liquid phase bottle of salting liquid containing sodium iron chlorophyllin after 1mL conditioning
It 2mL), is 10uL in sample volume, UV detector wavelength 392nm, column oven temperature setting is 30 DEG C, and chromatographic column is
4 μm of Poroshell 120EC-C18, mobile phase: A: methanol: water: acetic acid=60:30:10, B: methanol: acetonitrile: water: acetic acid=
50:25:15:10, gradient elution program 0min, A%=100%, B%=0%, 15min；A%=0%, B%=100%；
25min, A%=100%, B%=0%.Sampling interval equilibration time is detected under conditions of being 5min, obtains phyllo-iron
The chromatogram of sodium salt solution.
(III) sample measurement and calculating process: go out peak position in sodium-iron-chlorophyllin and integrated, obtain sodium iron chlorophyllin
The peak area of salt substitutes into the calibration curve equation that embodiment 1 obtains and obtains the concentration of unknown sample Determination of Chlorophyll ferrisodium salt.
In above-described embodiment, the ratio of mobile phase each component each means their volume ratio.Liquid chromatograph brand and model
It can be vertical etc. using Agilent, Shimadzu, good fortune.In addition, horizontal, ordinate also can be interchanged when making standard curve.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.
Claims (10)
1. a kind of method for measuring sodium-iron-chlorophyllin, which comprises the following steps:
(1) foundation of calibration curve equation:
For known to multiple concentration and concentration has differences each other sodium-iron-chlorophyllin solution standard, first with it
In any one sodium-iron-chlorophyllin solution standard as object, the complete of 230-900nm is carried out under ultraviolet specrophotometer
Length scanning determines maximum absorption wavelength；Then, using the maximum absorption wavelength as ultraviolet detection wave in liquid chromatograph
It is long, it is detected, is obtained each using whole sodium-iron-chlorophyllin solution standards as object using the liquid chromatograph
Liquid chromatogram of the sodium-iron-chlorophyllin solution standard of known concentration corresponding to them；Then, confirmed by Mass Spectrometer Method
The wherein peak position out of any one liquid chromatogram Determination of Chlorophyll ferrisodium salt；Again for any one liquid chromatogram, in leaf
Green plain sheet sodium salt goes out peak position and is integrated, and obtains the peak area of sodium-iron-chlorophyllin corresponding with the liquid chromatogram, so
For whole liquid chromatograms, peak area can be obtained with the standard variation relationship of sodium iron chlorophyllin salinity, that is, peak area-
The calibration curve equation of sodium iron chlorophyllin salinity；
(2) to the concentration mensuration of sample to be tested:
Using the sample to be tested of sodium iron chlorophyllin salinity to be measured as object, is detected, be somebody's turn to do using the liquid chromatograph
The liquid chromatogram of sample to be tested；Then, to the liquid chromatogram of the sample to be tested, go out peak position in sodium-iron-chlorophyllin and carry out
Integral, obtains the peak area of sodium-iron-chlorophyllin；Then, to bring the peak area into peak area-leaf that the step (1) obtains green
In the calibration curve equation of plain sheet sodium salt concentration, corresponding sodium-iron-chlorophyllin concentration value, the sodium-iron-chlorophyllin is calculated
The sodium-iron-chlorophyllin concentration value of concentration value namely the sample to be tested, is achieved in the concentration mensuration of sodium-iron-chlorophyllin；
Also, the parameter setting of liquid chromatograph and liquid chromatograph described in the step (2) described in the step (1) is complete
It is consistent entirely.
2. the method for measurement sodium-iron-chlorophyllin as described in claim 1, which is characterized in that in the step (1), the liquid phase
Chromatographic parameter setting includes sample volume, detector, column oven temperature, chromatographic column selection, mobile phase and mobile phase stream simultaneously
The setting of speed；
Preferably, sample volume is set as 10uL, and detector is set as UV detector, and column oven temperature setting is 30 DEG C, chromatography
Column is selected as 4 μm of 120 EC-C18 of Poroshell, and flow velocity is set as 1.0mL/min；
The mobile phase includes the mobile phase A and Mobile phase B being used cooperatively, wherein the mobile phase A is by methanol, water and second
Acid is mixed by the volume ratio proportion of 70:20:10, and the Mobile phase B is by methanol, acetone, water and acetic acid by 60:25:5:
10 volume ratio proportion mixes；Corresponding gradient elution program are as follows: when 0min, mobile phase A accounting 100%, Mobile phase B are accounted for
Than 0%；When 5min, mobile phase A accounting 0%, Mobile phase B accounting 100%；When 25min, mobile phase A accounting 100%, stream
Dynamic phase B accounting 0%；Sampling interval equilibration time is 5min；
Alternatively, the mobile phase A is to be mixed by methanol, water and acetic acid by the volume ratio proportion of 60:30:10, the flowing
Phase B is to be mixed by methanol, acetonitrile, water and acetic acid by the volume ratio proportion of 50:25:15:10；Corresponding gradient elution program
Are as follows: when 0min, mobile phase A accounting 100%, Mobile phase B accounting 0%；When 15min, mobile phase A accounting 0%, Mobile phase B
Accounting 100%；When 25min, mobile phase A accounting 100%, Mobile phase B accounting 0%；Sampling interval equilibration time is 5min；
Also, reagent used by the mobile phase is chromatographically pure, and the acetic acid is that the acetic acid that mass fraction is 0.2% is molten
Liquid, water are ultrapure water.
3. the method for measurement sodium-iron-chlorophyllin as described in claim 1, which is characterized in that described to pass through in the step (1)
Mass Spectrometer Method confirms the peak position out of wherein any one liquid chromatogram Determination of Chlorophyll ferrisodium salt, and specifically first setting mass spectrum is examined
Parameter is surveyed, operation instrument obtains the mass spectrogram of sodium iron chlorophyllin salting liquid, the position of sodium-iron-chlorophyllin is confirmed by mass spectral database,
The purity of target peak is analyzed simultaneously.
4. the method for measurement sodium-iron-chlorophyllin as claimed in claim 3, which is characterized in that the MS detection parameters are as follows:
Mass spectrum operation condition ESI ion source, molecular weight scanning range 650-750.
5. the method for measurement sodium-iron-chlorophyllin as described in claim 1, which is characterized in that described to be measured in the step (2)
The sample to be tested of sodium iron chlorophyllin salinity is solid sample, fluid sample or suspension sample；
When the sample to be tested is solid sample or suspension sample, the sample to be tested also have passed through pretreating process；When
The sample to be tested be solid sample when, the pretreating process be specially first solid sample is dissolved in the water, then by from
The heart and filtering, finally take filtrate be placed in be stored in 4 DEG C in lighttight reagent bottle under conditions of in 30 days as to test sample
Product are detected；When the sample to be tested is suspension sample, the pretreating process specifically includes centrifugation and filtering, finally
Take filtrate be placed in be stored in 4 DEG C in lighttight reagent bottle under conditions of in 30 days be used as sample to be tested detected；
Preferably, when target substance content is lower than 5mg/L in the sample to be tested, before centrifugal treating, extraction need to be first passed through
Carry out concentration.
6. the method for measurement sodium-iron-chlorophyllin as claimed in claim 5, which is characterized in that the filtering is using micro porous filtration
Film filters or micro porous filtration head, and filter membrane is specifically the filter membrane that aperture is 0.22 μm, and filter membrane material is polyether sulfone (PES), poly- four
Vinyl fluoride (PTFE) or Kynoar (PVDF)；
The lighttight reagent bottle is preferably brown liquid phase bottle.
7. the method for measurement sodium-iron-chlorophyllin as described in claim 1, which is characterized in that the multiple in the step (1)
The sodium-iron-chlorophyllin solution standard that concentration is known and concentration has differences each other, specifically configuration concentration are 5mg/
L, 10mg/L, 20mg/L, 50mg/L, 100mg/L standard solution.
8. the method for measurement sodium-iron-chlorophyllin as described in claim 1, which is characterized in that in the step (1), the peak face
Product is specifically established dense as ordinate, sodium-iron-chlorophyllin using peak area with the standard variation relationship of sodium iron chlorophyllin salinity
Degree is the coordinate system of abscissa, then for whole liquid chromatograms, marks corresponding point in the coordinate system, is then fitted again
Peak area-sodium iron chlorophyllin salinity calibration curve equation can be obtained in canonical plotting out.
9. the method for measurement sodium-iron-chlorophyllin as claimed in claim 8, which is characterized in that described to be fitted to linear fit.
10. as described in claim 1 measurement sodium-iron-chlorophyllin method, which is characterized in that in the step (2), it is described to
The volume of sample is no less than 0.2mL.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115950845A (en) * | 2023-03-09 | 2023-04-11 | 国能龙源环保有限公司 | Method for determining tetravalent vanadium content in pickle liquor of waste denitration catalyst |
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| CN104678000A (en) * | 2013-12-02 | 2015-06-03 | 镇江出入境检验检疫局检验检疫综合技术中心 | Method for detecting sodium copper chlorophyllin in olive oil through LC-MS/MS (liquid chromatography-tandem mass spectrometer) |
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| CN115950845A (en) * | 2023-03-09 | 2023-04-11 | 国能龙源环保有限公司 | Method for determining tetravalent vanadium content in pickle liquor of waste denitration catalyst |
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