CN101368903B - Detection method for measuring amino acid micro-element chelate rate - Google Patents
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Abstract
Disclosed is a detection method for measuring the chelation rate of the trace element chelate in amino acid; the main principle comprises the following steps: based on wavelength difference between the ion-state metal ions (such as sulfate, chloride salts and the like) without chelating reaction and the chelated metal irons at the maximum absorption peak in the UV visible light area, the free metal ions and the chelated metal ions can be distinguished; then the concentration of the chelated metal ions is measured so as to caculate the chelation rate. The method is simple to operate and can be used to measure the chelation rates of the trace element chelate in amino acid at different pH values and under different solvent media environments and study the chelation rates of the chelate product under different conditions to provide a basis for the identification of product quality and also provide a reference for the chelate product application under different environments.
Description
Technical field
The detection field that the present invention relates to the chelation percent of the Chelates of Amino Acids And Trace Elements such as cupric glycinate, zinc, iron, is specially a kind of detection method of measuring amino acid micro-element chelate rate.
Background technology
Chelates of Amino Acids And Trace Elements meets mechanism and the feature that body trace element absorbs, the amino acid of needed by human and trace element are organically combined, become a kind of new and effective micro-element nutrition hardening agent, there is good physicochemical property and biologically active.Glycocoll is the amino acid of molecular weight minimum, and it reacts with trace element the chelate generating is also the Chelates of Amino Acids And Trace Elements of molecular weight minimum, can better be absorbed by human body, utilize.
Chelates of Amino Acids And Trace Elements is the compound with ring texture being formed by one or more amino acid groups and metallic ion generation complex reaction.It has the advantages such as good stability, absorptivity is high, biological value is high, toxicity is little, has been widely used at present in herding production.The height of amino acid micro-element chelate rate is an important indicator of reflection Chelates of Amino Acids And Trace Elements quality, at present the assay method of amino acid micro-element chelate rate is mainly contained to gel filtration chromatography and titrimetry.
Gel filtration chromatography can be by molecular size by different shape trace element separately, for the existence of research Chelates of Amino Acids And Trace Elements provides a kind of important detection means (Brown and Zeringue, Laboratory evaluations of solubility and structural integrity of complexd and chelatedtrace miner al supplements,, 1994; Matsui etc., Supplementation of zinc as aminoacid-chelated zinc for piglets, 1996).Cao (1998,2000) utilize gel chromatographic columns to detect free state and complex state zinc in damping fluid, result shows in pH value is 2 and 5 damping fluid, the eluting peak of organic zinc product is identical with the eluting peak of zinc ion in zinc sulfate, and for the neutral aqueous solution of organic zinc product, before the eluting peak of ionic state zinc, also there is a less eluting peak, illustrate that under this condition, fraction zinc is complex state.Lin Ping (2004) has carried out Preliminary Analysis Results discovery with Sephadex G-10 gel chromatography to the composition of synthetic glycin chelated iron: free amino acid boric acid eluent wash-out 7min just goes out peak, and glycine chelate iron sample uses boric acid eluent wash-out 60min still without absorption peak, explanation thus, in glycin chelated iron, there is no free glycocoll, chelated iron may be to exist with the form of negative ion in solution, and has electrostatic interaction between sephadex.Because glycin chelated iron and free inorganic iron all can not directly wash out in gel chromatography, therefore can not separate by gel chromatography method.Guan Haiyue (2007) adopts gel chromatography to measure the chelation percent of ferrous glycine and glycine zine, but process quite bothers.Therefore, gel chromatography chelation percent process trouble, promotes and has certain difficulty in basic unit.
Solubleness according to Chelates of Amino Acids And Trace Elements in the organism such as ethanol is minimum, and the free metal ion of trace and amino acid can be dissolved in the characteristic of organic solvent, utilize the two dissolubility difference in organic solvent, adopt the way of organic solvent processing to carry out purifying microelements aminophenol chelated sample, then adopt titrimetry can measure the chelation percent of Chelates of Amino Acids And Trace Elements.Yi Kai etc. (2007) adopt the method to measure the chelation percent of zinc methionine, copper methionine, methionine iron, cupric glycinate, ferrous glycine etc.But for the Chelates of Amino Acids And Trace Elements sample that contains the negative ion that is insoluble in ethanol, titrimetry can not be used for measuring chelation percent, therefore titrimetry also exists certain defect.
At present, the Chelates of Amino Acids And Trace Elements product of selling on market is many, because it is unsound to detect the method for chelation percent, so great majority are all the total contents of measuring metal in Chelates of Amino Acids And Trace Elements product, do not consider that metallic ion is combination or free state, market situation is more chaotic, and many products are sold as Chelates of Amino Acids And Trace Elements, but its essence is only trace element and amino acid whose potpourri.In order to promote the further investigation of Chelates of Amino Acids And Trace Elements product quality inspection, set up the relevant criterion of amino-acid trace element product, be to be currently necessary very much in order to the producing and selling of the feeding Chelates of Amino Acids And Trace Elements product of standard and use.
Summary of the invention
The object of the invention is the deficiency for existing amino acid micro-element chelate rate detection method, a kind of detection method of measuring amino acid micro-element chelate rate is provided, make testing process not need purifying, easy and simple to handle, be easy to basic unit and promote, saved commercial production cost.
The present invention is achieved in that a kind of detection method of measuring amino acid micro-element chelate rate, and determination step is as follows: get corresponding Chelates of Amino Acids And Trace Elements wiring solution-forming to be measured, survey its ultraviolet-visible absorption spectra figure; Get the metallic compound wiring solution-forming of corresponding metallic ion in Chelates of Amino Acids And Trace Elements, survey its ultraviolet-visible absorption spectra figure, on ultraviolet-visible absorption spectra figure, look for target wavelength λ; Get the typical curve of measuring this Chelates of Amino Acids And Trace Elements after above-mentioned Chelates of Amino Acids And Trace Elements solution dilution under target wavelength λ; Take Chelates of Amino Acids And Trace Elements sample preparation to be measured and become solution, under target wavelength λ, measure absorbance, thereby then obtain chelation percent by calculating corresponding concentration.
Described target wavelength λ can be two wavelength X that absorbance equates
1and λ
2.
The preferred determination step of the detection method of Chelates of Amino Acids And Trace Elements (Fe, Mn, Zn) chelation percent is as follows: get corresponding Chelates of Amino Acids And Trace Elements wiring solution-forming to be measured, survey its ultraviolet-visible absorption spectra figure; Then get the metallic compound wiring solution-forming of corresponding metallic ion in Chelates of Amino Acids And Trace Elements, survey its ultraviolet-visible absorption spectra figure, two wavelength X of looking for absorbance to equate on ultraviolet-visible absorption spectra figure
1and λ
2; Get after above-mentioned Chelates of Amino Acids And Trace Elements solution dilution at λ
1and λ
2under two wavelength, measure the typical curve of this Chelates of Amino Acids And Trace Elements, obtain corresponding △ E; Take m gram, Chelates of Amino Acids And Trace Elements sample to be measured and be mixed with 100mL, the XmL getting is wherein made into 20mL solution again, at λ
1and λ
2lower mensuration absorbance, obtains at λ
1and λ
2the absorbance difference △ A at place, then calculates corresponding concentration C, obtains chelation percent according to following computing formula;
Computing formula is as follows:
In formula: m---the quality (g) of the Chelates of Amino Acids And Trace Elements that takes;
C---the concentration (g/mL) that substitution typical curve calculates.
Described metallic compound can be sulfate and/or chlorate etc.
Described X span can be 1-15.
In described Chelates of Amino Acids And Trace Elements, the metallic compound of corresponding metallic ion can be to account for 20～40% of testing sample.
The detecting step of described cupric glycinate chelation percent is as follows: the cupric glycinate that takes 0.5g is made into 100mL solution, survey its ultraviolet-visible absorption spectra figure, then the chlorination copper and copper sulfate of getting 0.05g, 0.10g and 0.15g is also made into the solution of 100mL, surveys its ultraviolet-visible absorption spectra figure; Known according to ultraviolet-visible absorption spectra figure, the mensuration of chlorination copper and copper sulfate chelation percent to cupric glycinate within the scope of 0.5039g/L～1.501g/L can suddenly be listed as and disregard; Then the above-mentioned cupric glycinate solution of getting 2～9mL is made into 20mL, measures its typical curve under the maximum absorption wavelength of cupric glycinate; Take testing sample m gram and be made into 100mL, get after XmL is made into 20mL solution again and survey its absorbance, reference standard opisometer calculates corresponding concentration C, calculates chelation percent according to following computing formula;
Computing formula is as follows:
In formula: m---the quality (g) of the cupric glycinate that takes;
C---the concentration (g/mL) that substitution typical curve calculates.
The detecting step of described glycine zine chelation percent is as follows: take 0.1g glycine zine and be made into the solution of 100mL, survey its ultraviolet-visible absorption spectra figure (UV absorbs spectrogram); Then get the solution that 0.02g zinc chloride is made into 100mL, survey its ultraviolet-visible absorption spectra figure (UV absorbs spectrogram), two wavelength X that find absorbance to equate on its spectrogram
1and λ
2; Then get above-mentioned glycine zine solution 7～10mL and be made into 20mL solution, at λ
1and λ
2under these two wavelength, measure the typical curve of glycine zine and obtain corresponding △ E; Take m gram, glycine zine sample to be measured and be made into 100mL, get XmL and be made into again 20mL solution, at λ
1and λ
2lower mensuration absorbance, obtains absorbance difference △ A, then calculates corresponding concentration C; Obtain as follows chelation percent;
Computing formula is as follows:
In formula: m---the quality (g) of the glycine zine that takes;
C---the concentration (g/mL) that substitution typical curve calculates.
The assay method of described ferrous bisglycinate chelate chelation percent is as follows: take 0.1g ferrous bisglycinate chelate and be made into the solution of 100mL, survey its ultraviolet-visible absorption spectra figure (UV absorbs spectrogram); Then get the solution that 0.04g iron protochloride is made into 100mL, survey its ultraviolet-visible absorption spectra figure (UV absorbs spectrogram), two wavelength X that find absorbance to equate on its spectrogram
1and λ
2; Then get above-mentioned ferrous bisglycinate chelate solution 9～12mL and be made into 20mL solution, at λ
1and λ
2under these two wavelength, measure the typical curve of ferrous bisglycinate chelate and obtain corresponding △ E; Take m gram, ferrous bisglycinate chelate sample to be measured and be made into 100mL, get XmL and be made into again 20mL solution, at λ
1and λ
2lower mensuration absorbance, obtains absorbance difference △ A, then calculates concentration C; Obtain as follows chelation percent,
Computing formula is as follows:
In formula: m---the quality (g) of the ferrous bisglycinate chelate that takes;
C---substitution typical curve calculates concentration (g/mL).
Cardinal principle of the present invention is that ionic state metal ion based on there is not chelatropic reaction is as (sulfate, chlorate etc.) different at the wavelength of the maximum absorption band in ultraviolet-visible district from the metallic ion of chelating state, can differentiate the metallic ion of free state and the metallic ion of chelating state, measure again on this basis the concentration of chelating state metallic ion, and then calculate chelation percent.The inventive method is simple to operate, be applicable to the mensuration of the chelation percent of glycocoll microelement chelate (containing the raw material that can be dissolved in ethanol or be insoluble in ethanol), and need not carry out the purifying of product, can be that commercial production is cost-saving, easy to utilize.
Accompanying drawing explanation
Fig. 1 is that the UV of cupric glycinate and cupric chloride absorbs spectrogram (wherein, a cupric chloride is 0.503g/L, b cupric chloride 1.002g/L, c cupric chloride 1.501g/L, d cupric glycinate 5.004g/L);
Fig. 2 is that the UV of glycocoll copper and copper sulfate absorbs spectrogram (wherein, a copper sulphate 0.502g/L, b copper sulphate 1.001g/L, c copper sulphate 1.500g/L, d cupric glycinate 5.004g/L);
Fig. 3 is that the UV of glycine zine, zinc chloride and zinc sulfate absorbs spectrogram (wherein, a zinc sulfate, b zinc chloride, c glycine zine);
Fig. 4 is that the UV of ferrous bisglycinate chelate, iron protochloride and ferrous sulphate absorbs spectrogram (wherein, a ferrous sulphate, b iron protochloride, c ferrous bisglycinate chelate).
Embodiment
Below in conjunction with the drawings and specific embodiments, a kind of detection method of measuring amino acid micro-element chelate rate of the present invention is described in detail.
To utilize the absorption of the metallic compound of corresponding metallic ion under the maximum absorption band of cupric glycinate to be suddenly listed as and to disregard for the mensuration of cupric glycinate chelation percent, while measuring the chelation percent of cupric glycinate sample to be measured, only need to survey its absorbance after wiring solution-forming, can calculate chelation percent after obtaining concentration according to typical curve.
For glycocoll trace element (Fe, Mn, Zn) assay method of chelation percent is that the ultimate principle that the absorption dual wavelength elimination method such as utilization is eliminated Interference absorb is measured: because absorbance has additive property, two wavelength places that absorptivity is identical on the absorption spectrum of interfering component measure the absorbance of blending ingredients, can eliminate Interference absorb: if there were significant differences for the absorptivity of tested component, can make error at measurment reduce, in the case, can directly measure the absorbance difference of potpourri at this two wavelength place, this difference is directly proportional to testing concentration, and it is irrelevant with interferent concentration.
Be expressed as follows by mathematical expression:
△A
a+b＝A
1 a+b-A
2 a+b＝A
1 a-A
2 a+A
1 b-A
2 b
＝c
a(E
1 a-E
2 a)×1+c
b(E
1 b-E
2 b)×1
Because E
1 b=E
2 b
So △ A
a+b=c
a(E
1 a-E
2 a) × 1=△ E
a× c
a× 1
Establishing b is herein iron protochloride or zinc chloride etc., in selected wavelength X
1, λ
2the absorbance at place is equal, so
△A
b＝0
△ A
a+bbe directly proportional to determinand a concentration, irrelevant with the concentration of chaff interference b (iron protochloride or zinc chloride etc.).
Therefore, the assay method of the chelation percent of glycine zine and ferrous bisglycinate chelate is the solution that the sample of getting about 0.1g is made into 100mL, survey its ultraviolet-visible absorption spectra figure (UV absorbs spectrogram), then get the solution that the zinc chloride or the iron protochloride etc. that account for sample 20%～40% are made into 100mL, survey its ultraviolet-visible absorption spectra figure (UV absorbs spectrogram), two wavelength X that find absorbance to equate on its spectrogram
1and λ
2, as shown in Figure 3 and Figure 4, then under these two wavelength, measure the E of glycine zine and ferrous bisglycinate chelate, can obtain △ E.
Embodiment 1
The assay method of cupric glycinate chelation percent: the cupric glycinate that takes 0.5g is made into 100mL solution, measures its ultraviolet-visible absorption spectra figure.Then the chlorination copper and copper sulfate of getting 0.05g, 0.10g and 0.15g is also made into the solution of 100mL, surveys its ultraviolet-visible absorption spectra figure, as depicted in figs. 1 and 2.Known according to Fig. 1 and Fig. 2, the mensuration of chlorination copper and copper sulfate chelation percent to cupric glycinate within the scope of 0.503g/L～1.501g/L can suddenly be listed as and disregard, survey so the chelation percent of cupric glycinate and only need under the maximum absorption wavelength of cupric glycinate, measure its typical curve, as long as testing sample is surveyed its absorbance after taking a certain amount of dissolving, reference standard opisometer just can be measured chelation percent after calculating concentration.The above-mentioned cupric glycinate solution of getting 2～9mL is made into 20mL, under the maximum absorption wavelength of cupric glycinate, measuring its typical curve is: A=-0.00148+0.20242C (R=0.99977), take testing sample 0.5g and be made into 100mL, get after 5mL is wherein made into 20mL solution again and survey its absorbance, reference standard opisometer calculates after concentration, the following formula of substitution:
Computing formula is as follows:
In formula: m---the quality (g) of the cupric glycinate that takes;
C---the concentration (g/mL) that substitution typical curve calculates.
The assay method of propylhomoserin zinc chelation percent: the solution that takes 0.1g glycine zine and be made into 100mL, survey its ultraviolet-visible absorption spectra figure (UV absorbs spectrogram), then get the solution that 0.02g zinc chloride is made into 100mL, survey its ultraviolet-visible absorption spectra figure (UV absorbs spectrogram), two wavelength X that find absorbance to equate on its spectrogram
1=190.35nm and λ
2=193.70nm, as shown in Figure 3; Then under these two wavelength, get above-mentioned glycine zine solution 7～10mL and be made into 20mL solution, measure the typical curve of glycine zine and obtain corresponding E, can obtain △ E.Take glycine zine sample 0.1g to be measured and be made into 100mL, get 8mL and be made into again 20mL solution, at λ
1=190.35nm and λ
2under=193.70nm, measure absorbance, obtain △ A, then calculate concentration C, substitution chelation percent computing formula just can be obtained chelation percent.
The assay method of chelation percent is as described below:
The typical curve of glycine zine under 193.70nm: A=0.3715+1.37425C (R
2=0.99818)
The typical curve of glycine zine under 190.35nm: A=0.64429+0.21976C (R
2=0.99697)
△E＝1.37425-0.21976＝1.15449
△A＝△E×C＝1.15449C(1)
Therefore can obtain:
In formula: m---the quality (g) of the glycine zine that takes;
C---the concentration (g/mL) being calculated by formula (1).
Embodiment 3
The chelation percent assay method of ferrous bisglycinate chelate: the solution that takes 0.1g ferrous bisglycinate chelate and be made into 100mL, survey its ultraviolet-visible absorption spectra figure (UV absorbs spectrogram), then get the solution that 0.04g iron protochloride is made into 100mL, survey its ultraviolet-visible absorption spectra figure (UV absorbs spectrogram), two wavelength X that find absorbance to equate on its spectrogram
1=190.40nm and λ
2=195.10nm, as shown in Figure 4; Then under these two wavelength, get above-mentioned ferrous bisglycinate chelate solution 9～12mL and be made into 20mL solution, measure the typical curve of ferrous bisglycinate chelate and obtain corresponding E, can obtain △ E.Take ferrous bisglycinate chelate sample 0.13g to be measured and be made into 100mL, get 10mL and be made into again 20mL solution, at λ
1=190.40nm and λ
2under=195.10nm, measure absorbance, obtain △ A, then calculate concentration C, substitution chelation percent computing formula just can be obtained chelation percent.
The assay method of chelation percent is as described below:
The typical curve of ferrous bisglycinate chelate under 195.1nm: A=0.94883+1.40944C (R
2=0.99922)
The typical curve of glycine zine under 190.35nm: A=0.831+0.7239C (R
2=0.99848)
△E＝1.40944-0.7239＝0.68554
△A＝△E×C＝0.68554C(2)
Therefore can obtain:
In formula: m---the quality (g) of the ferrous bisglycinate chelate that takes;
C---the concentration (g/mL) being calculated by formula (2).
Claims (3)
1. measure the detection method of cupric glycinate chelate rate for one kind, it is characterized in that, detecting step is as follows: the cupric glycinate that takes 0.5g is made into 100mL solution, survey its ultraviolet-visible absorption spectra figure, then the chlorination copper and copper sulfate of getting 0.05g, 0.10g and 0.15g is also made into the solution of 100mL, surveys its ultraviolet-visible absorption spectra figure; Known according to ultraviolet-visible absorption spectra figure, the mensuration of chlorination copper and copper sulfate chelation percent to cupric glycinate within the scope of 0.5039g/L～1.501g/L is ignored; Then the above-mentioned cupric glycinate solution of getting 2～9mL is made into 20mL, under the maximum absorption wavelength of cupric glycinate, measuring its typical curve is: A=-0.00148+0.20242C, wherein R=0.99977, take testing sample 0.5g and be made into 100mL, get after 5mL is wherein made into 20mL solution again and survey its absorbance, reference standard opisometer calculates out corresponding concentration C, calculates chelation percent according to following computing formula;
Computing formula is as follows:
In formula: m---the quality g of the cupric glycinate that takes;
C---the concentration g/mL that substitution typical curve calculates.
2. a detection method of measuring glycine zine chelate rate, is characterized in that, detecting step is as follows: take 0.1g glycine zine and be made into the solution of 100mL, survey its ultraviolet-visible absorption spectra figure; Then get the solution that 0.02g zinc chloride is made into 100mL, survey its ultraviolet-visible absorption spectra figure, two wavelength X that find absorbance to equate on its spectrogram
1and λ
2; Then get above-mentioned glycine zine solution 7～10mL and be made into 20mL solution, at λ
1and λ
2under these two wavelength, measure the typical curve of glycine zine and obtain corresponding △ E; Take glycine zine sample mg to be measured and be made into 100mL, get XmL and be made into again 20mL solution, at λ
1and λ
2lower mensuration absorbance, obtains absorbance difference △ A, then calculates corresponding concentration C; Obtain as follows chelation percent;
Computing formula is as follows:
λ
2the typical curve of glycine zine: A=0.3715+1.37425C under=193.70nm, R
2=0.99818
λ
1the typical curve of glycine zine: A=0.64429+0.21976C under=190.35nm, R
2=0.99697
△E=1.37425-0.21976=1.15449
△A=△E×C=1.15449C
In formula: m---the quality g of the glycine zine that takes;
C---the concentration g/mL that substitution typical curve calculates.
3. a detection method of measuring Ferrous glycinate chelation percent, is characterized in that, detecting step is as follows: take 0.1g ferrous bisglycinate chelate and be made into the solution of 100mL, survey its ultraviolet-visible absorption spectra figure; Then get the solution that 0.04g iron protochloride is made into 100mL, survey its ultraviolet-visible absorption spectra figure, two wavelength X that find absorbance to equate on its spectrogram
1and λ
2; Then get above-mentioned ferrous bisglycinate chelate solution 9～12mL and be made into 20mL solution, at λ
1and λ
2under these two wavelength, measure the typical curve of ferrous bisglycinate chelate and obtain corresponding △ E; Take ferrous bisglycinate chelate sample mg to be measured and be made into 100mL, get XmL and be made into again 20mL solution, at λ
1and λ
2lower mensuration absorbance, obtains absorbance difference △ A, then calculates concentration C; Obtain as follows chelation percent,
Computing formula is as follows:
λ
2the typical curve of ferrous bisglycinate chelate: A=0.94883+1.40944C under=195.10nm, R
2=0.99922
λ
1the typical curve of ferrous bisglycinate chelate: A=0.831+0.7239C under=190.40nm, R
2=0.99848
△E=1.40944-0.7239=0.68554
△A=△E×C=0.68554C
In formula: m---the quality g of the ferrous bisglycinate chelate that takes;
C---substitution typical curve calculates concentration g/mL.
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