JP2000162128A - Discrimination of polymer compound by metal affinity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily evaluate and discriminate the metal affinity of a polymer compound by utilizing the change in coloring of a metal indicator by the addition of a measuring sample. SOLUTION: In measurement of metal affinity, a regulated quantity of a metal indicator is added to a sample solution of a prescribed concentration or distilled water (reference solution), a metal ion solution is added little by little, and the total concentration of metal ions is read when a fixed coloring (rate) is shown. This method is effective as a method for easily measuring the metal affinity. For example, the measuring method of metal affinity of a high polymer compound is applicable to discriminate a synthetic polymer compound such as polyamino acid or polyether such as polyethylene glycol or a biopolymer compound such as protein, acidic polysaccharide, amino polysaccharide or lignin, and also the derived state of derivatives thereof. The analysis such as orientation or alignment of monomer unit is frequently difficult according to a general analyzing method. According to this measuring method, such polymer compounds can be discriminated, and much knowledge concerning the orientation and alignment of monomer unit can be obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring metal affinity, and more particularly to a method for measuring metal affinity of a polymer compound. The present invention relates to a method for identifying a polymer compound as an application.

[0002]

2. Description of the Related Art At present, various kinds of polymer compounds have been synthesized. Some of them adsorb or bind metals (ions). The affinity for a metal to adsorb or bind a metal (ion) is provided by the presence of a metal-binding functional group (hereinafter, referred to as a metal-binding group).

There are several methods for analyzing the presence of a metal binding group. Many kinds of metal binding groups are known, such as an amino group and a carboxyl group. These can be detected by spectroscopic methods, that is, ultraviolet-visible absorption spectrum, infrared absorption spectrum, NMR spectrum and the like.
In addition, the presence of a metal binding group can be known from measurement of ion exchange capacity and the like.

[0004]

Generally, a synthetic polymer compound is prepared by polymerizing a monomer unit. However, there are many cases where the bonding mode of the monomer depends on the reaction conditions. The case of a polymer compound having a metal binding group is not an exception, and there are many examples in which the bonding mode of the monomer depends on the reaction conditions.

In the analysis of the structure and physical properties of such a polymer compound, it is important to know the presence of a metal-binding group, and a spectroscopic method for measuring a spectrum is effective.
However, the affinity of the metal-binding group for the metal largely depends on the state of the metal-binding group. In the case of a polymer compound having a large number of metal-binding groups, the metal-binding groups may be present in close proximity. In this case, chelate formation can occur, so that the metal affinity increases. Therefore,
In analyzing the structure and physical properties of a polymer compound having a metal-binding group, it is very important to know not only the existence of the metal-binding group but also the state of its arrangement and the like.

In discriminating synthetic polymer compounds having the same monomer composition and different molecular chains, it is often difficult to apply a spectroscopic method using spectra. In the case of a synthetic high molecular compound, even if synthesized by a specific method, the synthetic high molecular compound as a substance is itself a mixture of various kinds of high molecular compounds. For this reason, the spectrum of the polymer compound having a metal-binding group becomes complicated, and it is not always easy to directly relate the existing state of the metal-binding group to the spectrum.

[0007] An important subject is a method for identifying a synthetic polymer compound in which the state of the metal-binding group can be directly related to the molecular structure.

[0008]

Means for Solving the Problems The inventors of the present invention have made intensive studies on a method for identifying a metal-binding polymer compound, and have found that (a sample) the bond between a polymer compound or a polymer and a metal ion is monitored with a metal indicator. It was found that the metal affinity can be measured by the method.

The metal affinity reflects the type of metal-binding group such as a carboxyl group, an amino group, a hydroxyl group and the state of its existence. In the molecular structure of the polymer compound or polymer,
The metal affinity changes when the metal-binding group adopts a configuration that facilitates formation of a chelate structure or adopts a characteristic structure such as being sterically hindered. Further, the present invention can provide a method for detecting a metal affinity to identify a polymer compound.

A feature of the present invention is to examine the bond between a (sample) polymer compound and a metal ion based on the bond between a metal indicator and a metal ion. The assay of the present invention seeks to represent the abundance of a polymer structure as the affinity of a metal that binds to all or part of the structure. Therefore, by the combination of metal indicator and metal ion,
In addition to measuring the abundance of metal-binding groups by adding a factor of affinity to metal, it is possible to focus on detecting changes in the presence of specific metal-binding groups in sample polymer compounds .

That is, the present invention relates to the relationship between the addition amount of a metal ion solution and the color development of a metal indicator, the relationship between the addition amount of a metal ion solution and the color development rate of a metal indicator, the total concentration of metal ions and the color development of a metal indicator. Or a relation between the total concentration of metal ions and the color development rate of the metal indicator, and a method of measuring metal affinity characterized by using such an index that the relation changes with the addition of a measurement sample can be provided. In particular, it is preferable to examine the relationship between the amount of the metal ion solution added and the coloring ratio of the metal indicator, or the relationship between the total concentration of the metal ions and the coloring ratio of the metal indicator.

[0012] Therefore, it is possible to easily identify a high molecular compound having the same monomer composition but different monomer bonding modes.

[0013]

The rationale invention DETAILED DESCRIPTION OF THE INVENTION Metal ion binding strength can be evaluated the affinity of metal by measuring the effect of the sample on the color development of the metal indicator. (1) Equilibrium reaction of the measurement system The metal ion (M), the metal indicator (ind), and the metal binding group (pa ij) of the polymer compound or polymer as the sample are expressed by the following formula (1). , Equation 2 holds. (Equilibrium for metal indicator and metal ion)

[0014]

[Outside 1]

In the formula, kind indicates a binding constant between a metal ion and a metal indicator, and [] indicates a concentration. (Equilibrium between sample metal binding group and metal ion)

[0016]

[Outside 2]

Here, in the formula, k pa ij is a binding constant between a metal ion and a metal binding group of a sample, and [] is a concentration.

Here, the subscript i of pa ij indicates that it is the i-th type of metal-binding group among the m types of metal-binding groups present in the polymer compound, and the subscript j indicates that the state of existence is m. It means the j-th kind of existing state among the n kinds.
More specifically, for example, in a polymer compound, when an amino group is present at a terminal of a molecule and a carboxyl group is present at a side chain and a terminal of the molecule, the type (m) of the metal-binding group is 2, for example, the carboxyl group can be the first type and the amino group can be the second type. When only the side chain and the molecular terminal of the carboxyl group can be distinguished from each other, the type (n) of the existing state becomes 2 and 1
The presence state of the type is assigned to a side chain, and the existence state of the second type is assigned to a molecular end or the like. When the presence of a carboxyl group is distinguished in detail, the type of existence state (n) is the number of distinct states. As for the amino group, the type (n) of the existing state is 1 when it is characterized by being present only at the molecular end, but when there are several existing states that can be distinguished by phenomena such as molecular asymmetry. This is the total number of states. This notation specifies the type and the state of the metal-binding group present in the polymer compound. Equation 2 summarizes the equilibrium for the metal binding group and the metal ion.

The total concentration (Tm) of metal ions is represented by the sum of free metal ions, metal ions bonded to a metal indicator, and metal ions bonded to a metal-binding group. Here, the metal ion bonded to the metal binding group needs to be considered for each type and existence state of the metal binding group. Therefore, the total concentration of metal ions is expressed by Equation 3.

[0020]

(Equation 3)

The symbol Σ is a symbol of the sum, which is the same as that used in ordinary mathematical expressions.

Among the metal indicators (total concentration Tind), the concentrations of the free state and the state bound to the metal ion are defined as [ind] and [Mind], respectively, and the ratio of the combined state / free state ([Mind] / [Mind]) Ind]) is represented by f, and the relationship between f and the coloring ratio (R) is represented by the following three relational expressions: f = [Mind] / [in
d], from R = [Mind] / Tind, Tind = [ind] + [Mind]
R = f / (1 + f). Here, the coloring ratio can theoretically be freely selected within a range of greater than 0 and smaller than 1, and it is advantageous to set the coloring ratio as high as possible because the amount of change in the Tm value increases. However, under a large coloring ratio, the influence of the measurement error becomes large. Therefore, in practice, it is preferable that the coloring ratio be around 0.5 (the value of f is around 1). (2) Theoretical consideration based on equilibrium reaction Relationship between the ratio of the bound state / free state of the metal indicator ([M ind]
= F [ind]) into Expression 1, the relational expression of k ind = f / [M] is obtained, and [M] = f / kind. [M] means the concentration of metal ions in a free state, and is substituted into Equation 2 and rearranged to obtain Equation 4.

K pa ij = kind · [M pa ij] / (f · [pa ij]) (Equation 4) The concentration of the sample polymer compound (the total concentration of the metal ion binding group) is represented by [s]. Formula 5 is satisfied when the abundance ratio of the i-th group and the j-th kind of the metal-binding group is defined as r ij.

[Pa ij] + [M pa ij] = [s] · r ij (Equation 5) [Pa ij] is eliminated from Equation 4 using the relation of Equation 5, and [M pa
ij], Equation 6 is obtained. [M pa ij] = k pa ij · f · [s] · r ij / (k pa ij · f + kind) (Equation 6) Accordingly, Equation 3 is given by the following Equation 7.

[0025]

(Equation 4)

Tm in this case is shown as Tm sample. When no sample was added (control system, blank), Tm = f /
Since k ind + R · Tind, this is expressed as Tm blank.

Therefore, if the concentration of the metal indicator and the coloring ratio are determined, the total concentration of the added metal ion is determined by the state of the metal-bonding group and the abundance ratio, the binding between the metal ion and the metal-binding group based on the metal indicator. It deviates (deviation) from the blank value depending on the constant and the concentration of the sample polymer compound (Equation 8).

[0028]

(Equation 5)

Under the measurement conditions, when most of the metal binding groups are not bonded to a metal ion, the following equation 9 can be approximated. This is the case where all k pa ij · f are negligible compared to k ind in Equation 8, and the present inventor considers k pa ij · f
The standard is that / kind is 0 to 0.02.

[0030]

(Equation 6)

Since equation 9 depends on the ratio of the bound state / free state of the metal indicator and the value of ind, the relative value with respect to the free metal ion concentration ([M] = f / kind) is obtained by the following equation. It becomes 10.

[0032]

(Equation 7)

In Equation 10, since the variable other than the sample-specific property is the sample concentration, it is converted into a unit sample concentration and this is called a metal ion binding force (Equation 11).

[0034]

(Equation 8)

The value of the metal ion binding force is a property inherent to the sample. The relationship between the metal ion binding force and the experimental data processing is expressed as Equation 12.

[0036]

(Equation 9)

Metal indicator concentration of control system (Tind blank)
Is determined from the measured values of the absorbance and the amount of the metal indicator added. In addition, a common value is designated for the color development rate R in a range from greater than 0 to less than 1 in the measurement system to which the sample is added and the control system.

Since the theoretical background described here is based on the formula 2, the molar concentration (M) of the metal binding group is used as the unit of the concentration of the sample polymer compound of the formula 12 or the formula 13 described below. That is reasonable. However, when processing actual measurement data, Equation 12 or Equation 13 is used. Therefore, in the case of a high molecular compound, a concentration such as a molar concentration (M) of a monomer unit can be used. In these cases, the unit of metal ion binding force is M- ¹ . Practically
It is possible to use a unit indicating the sample concentration such as g / l. In this case, the unit of the metal ion binding force is 1 / g (liter / gram). In the case of a high molecular compound, it is possible to use the molar concentration (M), but it is necessary to consider the molecular weight dependency in displaying the metal ion binding force. In addition, in the selection of the metal ion indicator described later, k ind / k pa max
Therefore, the molar concentration (M) of the metal-binding group is most preferable as the unit of the sample concentration.

Equation 12 assumes that the metal indicator concentrations in the sample addition and control systems are completely equal. Therefore, in the actual measurement, the concentration of the metal indicator in the sample addition system needs to be the same as that in the control system. If the absorbances (metal indicator concentrations) are not uniform, an error is required in the numerator of equation 12, so correction is required. Such molecular correction does not cause any problem in the numerical calculation even if the concentrations of the Tm sample and Tm blank are matched with the appropriate metal indicator concentration. Therefore, if the concentration of the metal indicator used for sample measurement is described as Tind sample,
Instead of Tm sample (Tm sample-R · Tind sample)
Instead of Tm blank (Tm blank−R · Tind blank)
Can be used (Equation 13).

[0040]

(Equation 10)

As is clear from the above description, this measuring method is a method for comprehensively examining the metal ion binding force, unlike the method for measuring the metal ion binding capacity. As is apparent from theoretical considerations, even if the metal-binding group is dispersed in many independent molecules, there is no problem in measuring the metal affinity. (3) Scatchard plot
Theoretical consideration of the relationship between t) and the present invention In the case of ordinary measurement, the measured data is expressed by Equation 12 or Equation 1
3. Calculate by 3 to determine the metal ion binding force. In the case where another molecule is reversibly bound to a polymer compound, for example, when a substrate or a coenzyme is bound to an enzyme molecule, a Scatchard (type) plot is often used for the analysis. However, the Scatchard (type) plot is based on the assumption that data relating to simple equilibrium between molecules is processed. Scatchard (type) for high molecular compounds with various types of binding sites
Data processing becomes difficult because polymorphism appears in the plot. In addition, Scatchard (type) plots have not been used for the purpose of analyzing equilibrium, such as the method of the present invention. Here, Scatchard (type)
Consider the possibility of obtaining the metal ion binding force by applying the plot.

Given the equilibrium between the metal indicator of formula 1 and the metal ion, consider the possibility of performing a Scatchard (type) plot. [Mind] = R · Tind when the color development ratio, that is, the ratio R of the amount of the metal indicator bound to the metal ion and the total concentration Tind of the metal indicator are used. [ind] = Tind-[Min
d], [ind] = Tind−R · Tind. Tm 'is the total concentration of metal ions not bound to the sample polymer,
That is, [M] = Tm′−R · Tind when the metal ion concentration is in the free state and in the state bound to the metal indicator.
Substituting these relationships into Equation 1 gives Equation 14. Equation 14 can be summarized as Equation 15 on the assumption that Scatchard (type) plotting is performed.

Kind = R · Tind / [(Tind−R · Tind) (Tm′−R · Tind)] (Equation 14) (1 / 1-R) = kind (Tm ′ / R) −kind · Tind (Equation 15) As a control system, when a normal Scatchard (type) plot is performed on the binding between a metal ion and a metal indicator, Tm ′ = Tm because there is no sample polymer compound.
Therefore, in the case of the control system, the relationship of Expression 16 is established.

(1 / 1-R) = kind · (T m / R) −kind · Tind (control system, formula 16) The equilibrium of formula 2 holds for the metal ion binding group present in the polymer compound. . Substituting the ratio f = [Mind] / [ind] of the bound / free state of the metal indicator into Equation 1 and solving for the free metal ion concentration gives [M] = f / kind,
It was mentioned earlier that Equation 4 and Equation 6 are obtained by substituting into Equation 2.

Total metal ion concentration Tm in the sample addition system
And Tm 'is shown by equation (17). Expression 18 is obtained from Expression 17 and Expression 6.

[0046]

[Equation 11]

By substituting Tm ′ in equation (18) into equation (15), equation (19) is obtained.
become.

[0048]

(Equation 12)

The relationship between R and f is R = f / (1 + f). Metal indicators are much easier to bond with metal ions than metal-binding groups (k pa ij · f / kind is 0 to
Equation (19) can be approximated by Equation (20).

[0050]

(Equation 13)

[0051]

[Equation 14]

Expression 21 is a modified version of Expression 20, and a part of the expression is replaced with Expression 22 to make the expression easier to handle.

[0053]

(Equation 15)

Expression 21 can be expressed by using Δ as (1/1 /
1−R) + Δ / (1−R) = kind · Tm (1 / R) −kind
Becomes Tind. Equation 23 is obtained by rearranging the left side, and is further transformed to equation 24.

(1 / 1-R) (1 + Δ) = kind · Tm (1 / R) −kind · Tind (sample addition system, equation 23) (1 / 1-R) = [kind / (1+ Δ)] · Tm (1 / R)-kind · Tind / (1 + Δ) (Equation 24) In Equation 24, {kind / (1 + Δ)} and kind · Tind
/ (1 + Δ) is a constant. Where kexp =
Rewriting equation 24 as kind / (1 + Δ) gives equation 25.

(1 / 1-R) = kexp · Tm (1 / R) −kexp · Tind (Sample addition system, Equation 25) That is, the relationship between Tm (1 / R) and (1 / 1-R) Becomes a simple linear function.

From the above theoretical considerations, it has been shown that Scatchard (type) plots can be used in the analysis for determining the metal ion binding force. Since the slope of the Scatchard (type) plot of the sample addition system is kexp (kind /
The metal ion binding force is determined as kexp-1) / [s]. In addition, kind is a binding constant between a metal ion and a metal indicator, and is obtained by processing data measured in a control system by a Scatchard (type) plot. 4 and 5 attached as examples are examples of analysis of the metal ion binding force using this method. (4) Data processing method of the present invention The data processing method described herein provides a method for determining the metal ion binding force from the difference {Δ / (1-R)} between the sample addition system and the control system. {Δ / (1-R)} is (kind / R) ×
(Deviation) or the difference between the sample addition system and the control system in the Scatchard (type) plot.

The difference between the sample addition system (Equation 23) and the control system (Equation 16) is represented by {Δ / (1-R)} on the left side. ｛Δ /
(1-R)｝ is the metal ion binding force and [s] / (1-R)
Depends on the product of In the measurement for examining the relationship between the addition concentration of the metal ion and the coloring ratio of the metal indicator, [s] / (1
-R) is an easily determined parameter. [S] /
(1−R) and {Δ / (1−R)} are theoretically directly proportional, but the proportionality constant is the metal ion binding force. Therefore,
A method of plotting the relationship between [s] / (1-R) and {Δ / (1-R)} to determine the slope of a straight line or {Δ / (1-R)}
Is divided by [s] / (1-R) to determine the metal ion binding force. In the case where the metal ion binding force of the measurement sample is compared with each other or the correspondence with the Scatchard (type) plot is considered, [s] / (1
−R) and １ ／ Δ / (1-
R) It is also practical to examine the relationship with｝. In this case, the product of the metal ion binding force and [s] is obtained, and the metal ion binding force can be calculated as necessary. From this, in the embodiment, 1 / (1-R) and ｛Δ /
The relationship with (1-R)｝ was examined to determine the metal ion binding force. Hereinafter, a plot showing the relationship between 1 / (1-R) or [s] / (1-R) and {Δ / (1-R)} is also referred to as an order plot. When actually applying this method, the kind is determined by examining the relationship between the metal ion concentration and the coloring ratio of the metal indicator in a measurement system (control system) not containing a sample. In this case, it is preferable to use a Scatchard (type) plot. Tm sample (1 / Rsamp) for the measurement system containing the polymer compound of the sample was also examined by examining the relationship between the metal ion concentration and the color development of the metal indicator.
le) and 1 / (1-Rsample) are calculated. Further, 1 / (1-Rstandard) corresponding to Tm sample (1 / Rsample) is calculated by Expression 26 as a reference for calculating the shift. 1 / (1-Rstandard) = kind · Tm sample (1 / Rsample) −kind · Tind sample (Equation 26) The Tind value used in Equation 26 is for a measurement system including a sample. Here, the suffix sample attached to Tm, R, and Tind indicates that the data is of a measurement system to which a sample is added. Further, the left side of Equation 26 takes into account the correspondence with the Scatchard (type) plot, and 1 / (1-Rstandard) is one variable indicating the value calculated on the right side. ｛Δ / (1
−R)｝ can be calculated by the equation 27 using the experimental value {Δ / (1−Rsample)}.

｛Δ / (1-Rsample)｝ = 1 / (1-Rsample) −1 / (1-Rstandard) (Equation 27) Equations 26 and 27 consider the relation with the Scatchard (type) plot. It was done. Theoretically, the relative deviation in Equation 10 and Δ in Equation 22 are the same. Therefore, the difference between the sample-added system and the control system is calculated by using (deviation) corresponding to Rsample, {Δ / (1-Rsample)} = (kind / Rsample)
X (deviation) may be calculated. However, considering the measurement error of the control system, the calculation of {Δ / (1-Rsample)} is given by Equation 26,
The use of 7 is slightly more advantageous. ｛Δ / (1-Rsampl
e) Since the proportional constant between｝ and 1 / (1-Rsample) is the product of the metal ion binding force and [s], the metal ion binding force is obtained as (proportional constant) / [s]. The plots of the invention described herein are shown in the examples (FIGS. 1 and 2).

In the present method, since the effect of the sample on the coloring ratio of the metal indicator is examined, it is important to accurately measure the control system. Considering the measurement error, if a (regression) line is obtained in this plot, 1 / (1-Rsample)
Should be emphasized for data whose value is around 2. Also, it should be noted that the (regression) line originally passes through the origin (0, 0).

The theoretical basis of the metal ion binding force has been described above. Any of the above three types of data processing methods, that is, Equation 12 or Equation 13, Scatchard (type) plots, and the data processing method (oda plot) of the present invention can be used. Equation 12 is a simple data processing method.
Alternatively, the processing method of Expression 13 is preferable, and the data processing method (Oda plot) of the present invention is preferable in that the analysis can be performed while grasping the state of the measurement error. Method for Measuring Metal Affinity In the present invention, it suffices if the total concentration of metal ions can be measured under the condition that the metal indicator shows a constant coloration rate. Therefore, the basis of the measurement is that a specified amount of a metal indicator is added to a sample solution or distilled water (control solution) having a predetermined concentration, and a metal ion solution is added little by little. To read the total metal ion concentration (Tm sample or Tm blank). This method is effective as a simple method for measuring metal affinity.

In the case of measuring the coloring ratio of a metal indicator in a normal measurement, the accuracy is improved by compensating for an increase in the liquid amount due to the addition of a metal ion solution. It is preferable that the addition amount of the metal indicator is corrected within a range in which the Lambert-Beer's law can be applied so as to correct the dilution by increasing the liquid amount.
In this case, the lower limit of the absorbance by the metal indicator is 0.
It may be considered that the upper limit is practically up to about 1. However, Lambert-
The deviation from Beer's law increases, and the value of the correction term R · Tind blank for the metal indicator in Equation 12 increases, resulting in a large measurement error. When the concentration of the metal indicator is low, the value of the correction term R · Tind blank is small, which is advantageous, but the change in absorbance becomes difficult to measure, and the measurement error increases.

The corrected absorbance Ac is expressed as follows:
0, the addition amount of the metal ion solution is Vm, and the measured value of the absorbance is A
Assuming m, it can be obtained as Ac = Am · (V0 + Vm) / V0. The coloring ratio R = 0 is the absorbance (A0) before adding the metal ion solution. The color development ratio R = 1 is defined as the color development (absorbance after correction for the increase in liquid volume, As) in which no change in color development is observed even when the metal ion solution is added. The coloring ratio is
It can be calculated as R = (Ac-A0) / (As-A0).

The metal indicator used for the measurement can be freely selected according to the metal ion and its purpose, provided that the color change of the metal indicator changes due to a change in the metal ion concentration.
Equation 12 is logically derived from Equation 9, which is an approximate equation, and the theoretical accuracy of the metal ion binding force (Equation 12) is
Is determined. For this reason, in the measurement of the metal ion binding force, it is necessary that the metal indicator has a sufficiently higher affinity for the metal ion than the sample. Under normal measurement conditions, f = 1 is about 1. Therefore, kind / k pa max is 20 or more for the metal ion binding group (the largest one among all k pa ij is described as k pa max). Is desirable. If k ind / k pa max is large, the approximation accuracy of Equation 9 is improved, but in actual measurement, the deviation is small, and the measurement error is large. From this point, it is considered that the upper limit of k ind / k pa max is about 10 ⁸ even when an extremely high-precision measuring device is used. K ind for normal measurement
It is preferable that / kpa max be 50 or more and 10,000 or less. In this case, it is desirable to obtain a change (deviation) of the total concentration of metal ions (claim 2), preferably a metal ion binding force, as an index of metal affinity. This is because quantitative comparison and examination can be performed in the identification of a polymer compound and the like.

As an index of metal affinity, the deviation of equation 8 is also effective for intercomparison of samples. In this case, it is necessary to keep the measurement conditions constant, but this is advantageous in terms of errors because the number of calculations between the measurement values is small. For k ind / k pa max, the upper limit described above does not change, but the lower limit is gradual. If there is no problem in the detection accuracy of the measuring device, the lower limit should theoretically be larger than 0.

When k ind / k pa max is close to 1, Equation 8
It is advantageous if only the mutual comparison is sufficient because the deviation of

The property relating to the deviation of the formula 8 can be used for the purpose of preferentially detecting the presence (amount) of a specific (state) metal binding group (pa ij). In this case, k ind / k pa i
It is preferable that the deviation is compared with the deviation of the reference substance while setting j to 1 as much as possible. In this case, it is preferable to use the change (deviation) of the total concentration of metal ions due to the addition of the measurement sample (claim 2) as an index of metal affinity.

Accordingly, the combination of a metal ion and a metal indicator effective in the present measurement method can be other than the combination in the case of ordinary chelate titration. k ind / k pa ma
If x is appropriate, as shown in Equation 11, the value specific to the metal indicator, k ind, does not theoretically affect the metal ion binding force. On the other hand, selection of the metal ion is important because it directly affects the metal ion binding force as shown in Equation 11.
Examples 1 and 3 are specific examples, and when indicating the metal ion binding force, it is necessary to indicate the metal ion used for the measurement.
Preferred combinations of metal ions and metal indicators to be used are: 1) Alkaline earth metals (Be, Mg, Ca, Sr, Ba, Ra)
For ions, BT, MX, XO, BPR, MTB,
NN (2-Hydroxy-1- (2-hydroxy-4-sulfo-1-naphthylaz
o) -3-naphthoic acid, also called NANA), TPC, PC
2) Heavy metals, etc. (V, Cr, Mo, W, Mn, Tc, Re,
Fe, Co, Ni, Pd, Pt, Cu, Ag, Au, Zn, Cd, H
BT, MX, PAN, XO, BPR, M for ions of g, Al, Ga, In, Tl, Sn, Pb, Bi)
TB, TPC, PR, PV, etc. 3) Rare earths (Sc, Y, La, Ce, Pr, Nd, Pm, Sm, Eu, G
d, Tb, Dy, Ho, Er, Tm, Yb, Lu, Ac, Th, Pa,
B for ions of U, Np, Pu, Am, Cm)
T, MX, PAN, XO, BPR, MTB, TPC, P
C, PR, PV, etc. 4) Arsenazo III (Arsenazo-III) for alkali metal (Li, Na, K, Rb, Cs) ions
Thorin, Nitrophenylazo-15-crown-5, 4TF (4 '-(2,6
-Dinitro-4-trifluoromethylphenyl) aminobenzo-15-cro
wn-5), 6TF (4 '-(2,4-Dinitro-6-trifluoromethylphe
nyl) aminobenzo-15-crown-5). Here, the metal indicator names indicated by abbreviations are BT for eriochrome black T, MX for murexide, PAN for pyridylazonaphthol, XO for xylenol, BPR for bromopyrogallol red, MTB for methyl tomol blue, and TPC for thymol lid. Rain Complexon, PC
Is phthalein complexone, PR is pyrogallol red, and PV is pyrocatechol violet.

In Examples 1 and 3, the metal indicator
NN was used. NN is commonly used as an indicator dedicated to Ca ions in chelate titration, and is not normally used for direct titration of Sr. The first embodiment is a combination of NN and Ca ions, which is generally considered, while the third embodiment is a combination of NN and Sr ions. In both cases, the metal ion binding force can be measured.

The type of metal ion used in the present measurement method is preferably determined depending on the use of the polymer compound or the purpose of identification. The metal indicator may be any as long as it satisfies the condition that the coloring ratio changes depending on the concentration of the metal ion used. However, as one of the guidelines for the combination of a metal ion and a metal indicator, a combination used for chelate titration or colorimetry is preferable.

In the examples, the purpose of using the polymer compound was to consider the capture of alkaline earth metal ions such as Ca ions. Therefore, in this case, Ca ion, Sr
Selection of ions and the like is preferred. The metal indicator in this case is
NN, TPC, etc., whose coloring ratio changes depending on the concentrations of Ca ions and Sr ions, are preferred.

This measurement method can be applied to inorganic polymers and inorganic polymer ions. For example, in oxides and sulfides, or ions whose structure is the main structure, atoms such as metals and oxygen or sulfur atoms are covalently bonded,
There are inorganic polymers (ions) linked by coordination bonds, ionic bonds, and the like. LiCoO ₂ , Li (Ni, Co) being researched and developed as materials for lithium ion batteries
O ₂ , LiMnO _{2, and the} like are also considered to be a kind of inorganic polymer, and bonding with Li ions is related to battery performance. In this case, as the metal ion, Li ion is preferable, and as the metal indicator, Arsenazo-III and Trin (Thori), which develop color in accordance with the concentration of Li ion.
n), nitrophenylazo-15-crown-5 (Nitro
phenylazo-15-crown-5) and the like are preferred.

The metal ion concentration of the metal ion solution to be used can be freely determined to some extent. However, in order to prevent the liquid volume of the measuring system from largely changing and to simplify the measuring operation, 1 m
It is generally appropriate to set the range to about M to 1M.

In the case of a polymer compound used in an aqueous solvent, it is preferable that the polymer compound is dissolved or suspended in water or an aqueous solvent before the measurement. Polymer compounds used in an organic solvent such as an organic electrolyte include, for example, alcohols such as methanol, ethanol, propanol and ethylene glycol, ethers such as diethyl ether and dimethoxyethane, and organic solvents such as pyridine, benzene and ethyl acetate. It is preferable to dissolve or suspend in an organic electrolyte containing these for measurement. The concentration (content) of the polymer compound in the measurement system can be selected freely, but is practically preferably in the range of 0.2 to 500 mg / ml. When measuring a sample containing metal ions, when the sample contains metal ions, the sample is subjected to treatments such as dialysis, electrodialysis, ion exchange, and precipitation to remove the metal ions, and then subjected to measurement.

The sample has a low metal ion content and EDTA
If there is no problem even if a strong chelating agent coexists, such as the case where the bond between the sample or the metal indicator and the metal ion is sufficiently weak compared to the bond between the chelating agent and the metal ion (kind / kchel and kpa max / kchel is 0.0
01 or less, kchel is EDTA, CyDTA, DTPA,
Equilibrium for chelating agent chel such as EGTA and metal ion M

[0076]

[Outside 3]

The equilibrium constant [M chel] / [M] [chel] can be easily preprocessed. In this case, titration is performed with the minimum necessary amount (amount) of the chelating agent until the color of the metal indicator becomes almost 0%. The sample after titration can be treated in the same manner as a sample containing impurities having extremely high affinity for metal ions described below. Contains metal-binding groups and compounds with high affinity for metal ions
For the measurement of a sample, a metal binding group (pa ij) has a strong metal affinity (k
In the case of ind <k pa ij), the metal binding group is detected in accordance with the affinity as a portion where the color development rate of the metal indicator in the initial stage of adding the metal ion is hard to change. When a partial structure having a strong metal affinity such as EDTA or a compound having a strong metal affinity such as EDTA is present as an impurity in the sample, the initial coloration of the metal indicator when adding a metal ion is performed. Give the part where the rate hardly changes. In this case, it is possible to obtain the metal ion binding force for the portion where the color development rate of the metal indicator that appears continuously changes. For such a sample, the operation of adding a metal ion little by little and measuring the change in absorbance can be applied, so the measurement operation itself is the same. When a metal ion is added, a partial structure or an impurity having a strong metal affinity first binds to the metal ion preferentially. As a result, the starting point of the change in the absorbance of the metal indicator (hereinafter, referred to as the starting point) is shifted, and the change in the coloring ratio of the metal indicator near the starting point becomes gentle. The obtained data is a graph of the total concentration of metal ions minus the coloring ratio of the metal indicator. For the portion where the coloring ratio of the metal indicator near the starting point changes slowly, the curve portion following it is extrapolated to the original starting point. And determine
Relative Tm with original starting point as origin (Tm sample = 0)
Process by reading the value of sample. In the operation of extrapolating the curve portion, it is recommended to refer to a graph of the total metal ion concentration versus the color development ratio of the metal indicator in the case of no sample (control system). The amount of the partial structure or compound having strong metal affinity present as an impurity is
It can be determined from the amount of metal ions added up to the original starting point.

In the case of these samples, the choice of the metal indicator is important. Regarding the affinity for the metal ion, it is preferable that the metal indicator is located between the impurity and the metal binding group. When examining the existence state of the metal-binding group, it is possible to measure using only one kind of metal ion. In addition, a plurality of metal ions can be used in the present measurement method, and more knowledge can be obtained on the presence of metal-binding groups such as a carboxyl group, an amino group, and a hydroxyl group. Examples 1 and 2 evaluate the metal affinity (metal ion binding force) for Ca ions, and Example 3
This is an evaluation of metal affinity for ions. The sample used was the sodium salt of polyaspartic acid and was designated CF110, M or E depending on the synthesis method. The metal affinity for Ca ions and Sr ions differs depending on the sample, but there is a difference in the degree of difference. In the embodiment, the Ca ion binding force of E / the Ca ion binding force of CF110 =
0.38, the Sr ion binding force of E / CF110
Is Sr ion binding force = 0.134. As a result, metal affinity can be evaluated from many aspects, and detailed identification of a polymer compound can be performed. In other words, the metal ion selectivity can be quantitatively analyzed.

The identification according to the metal affinity in the method of the present invention can be carried out without any particular limitation as long as it is a polymer or a polymer compound exhibiting metal binding properties. For example, methods for measuring metal affinity include synthetic high molecular compounds such as polypeptides, polyamines, polycarboxylic acids including polyacrylic acids, polyamino acids, and polyethers such as polyethylene glycols and polypropylene glycols. , Proteins, acidic polysaccharides, amino polysaccharides, lignins and other biopolymer compounds, derivatives of biopolymer compounds (modifications,
Modifications) can also be applied to identify the state of derivatization.

There are many examples in which the analysis of the orientation and arrangement of the monomer units is difficult even though the composition of the monomer units is known by ordinary analysis methods. The measurement method of the present invention can identify these polymer compounds, and can obtain a lot of knowledge on the orientation and arrangement of monomer units. Also,
When a part of the structure of the polymer compound is modified, it is considered difficult to analyze by a normal method, but the measuring method of the present invention can be easily applied.

In addition, the method of the present invention can be applied to inorganic polymers such as oxides and sulfides, and inorganic polymer ions having oxides and sulfides as main structures. Preferred examples of industrially useful applications include identification of metal oxides containing cobalt, nickel, manganese, etc., which are materials for lithium ion batteries.

[0082]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples. In the examples, polyaspartates having different synthesis methods are used as samples, but the molar concentration of the metal-binding group and the molar concentration of the monomer unit can be treated as being equal. In Tables 1 and 2, the metal ion binding force based on the concentration of the monomer unit is shown, but the value is the same as the value based on the molar concentration of the metal binding group. Example 1 Measurement of Tm sample --- Measurement: 25 mm in diameter (inner diameter 23
mm) using a test tube. 6.50 ml in test tube
Of distilled water and 1.00 ml of a sample aqueous solution (concentration: 2%) were injected, and 1.00 ml of KOH (concentration: 50 w / v) and a metal indicator NN were added. Under these conditions, the absorbance by NN (wavelength 470 nm) was 0.200 ± 0.03,
Since the addition amount of 100-fold diluted NN was 7.0 mg,
The concentration of N was estimated at 18.9 μM. The absorbance was measured by adding 0.010 ml of a 5 mM aqueous calcium chloride solution. 25 mM when the absorbance becomes 0.28
0.20 ml of an aqueous solution of calcium chloride was added. Normally, the color development rate is almost 100% in this state, but it was confirmed that 0.10 ml of a 25 mM calcium chloride aqueous solution was added to make sure that the absorbance did not increase. In this measurement, the absorbance was decreased due to an increase in the amount of liquid during the measurement. Therefore, the absorbance was corrected according to the Lambert-Beer law. In addition, bubbling was performed by slowly blowing nitrogen to suppress fading of NN.

The Ca ion concentration and the corrected absorbance were plotted. The Ca ion addition concentration (Tm sample) at a coloring ratio of 50% was determined.

The results of measuring the metal ion binding force of the polyaspartic acid sample (CF110, E) by the method described here are shown in Table 1. Shown in Measurement of Tm blank --- Tm sa
The operation was performed in the same manner as in the measurement of mple, but distilled water was used instead of the sample aqueous solution. Table 1 shows the results. It was shown to.

[0085]

[Table 1]

The metal ion binding force in Table 1 was calculated by Equation 13 (here, in the case of sample CF110, Tm sample =
44.2 μM, R = 0.5, Tind sample = 19.0 μM
^-1 , Tm blank = 35.3 μM, Tind blank = 19.2 μM
M ^-1 , [s] = 17.18 mM).

The results of processing with Scatchard (type) plots are shown in FIG. 3 (control), FIG. 4 (CF110) and FIG. 5 (E). Here, in FIG. 3, kind = 3
8.1 × 10 ³ M ⁻¹ , kexp = 27.5 × in FIG.
10 ³ M ^-1 , metal ion binding force (kind / kexp -1)
/[s]=22.4M ⁻¹ , kexp = 33.3 in FIG.
× 10 ³ M ⁻¹ , metal ion binding force (kind / kexp−
1) / [s] = 8.4 M ⁻¹

In the data processing method (oda plot) of the present invention,
The result of the analysis is shown in FIG. 1 (CF-110, metal ion binding
Power 20.9M^-1), FIG. 2 (E, metal ion binding force 10.
3M ^-1).

Note that there is a difference in the numerical value of the metal ion binding force obtained by the data processing method due to an error in the measured value itself. Example 2 Measurement of Tm sample--As an example in the case of containing impurities of metal ions, a sample was made of polyaspartic acid containing magnesium. 0.100m for 1.00ml of sample (2%)
1 KOH (concentration 50 w / v), add for 10 minutes,
The precipitate was separated by centrifugation. Precipitation is 1.00m of distilled water
Each wash was performed twice, and the wash was added to the centrifuged sample solution. The sample solution was transferred to a test tube having a diameter of 25 mm (inner diameter of 23 mm), and 4.50 ml of water and 0.90 ml of KOH (concentration: 5) were used.
0 w / v), the metal indicator NN was added. Under these conditions,
The absorbance by NN (wavelength 470 nm) was 0.224. 0.0100 ml of 10 mM EDTA was added. Most of the decrease in absorbance is 0.0300 ml of EDTA
Although it occurred by the time of addition, EDTA was set to 0.
0400 ml was added. Since the magnesium in the solution obtained here is firmly bound to EDTA, it does not affect the following measurements. Therefore, the subsequent measurement operation is the same as the measurement of a sample containing a metal-binding group or compound having a high affinity for a metal ion.

NN was added to the solution to adjust the absorbance (wavelength 470 nm) of NN to 0.205. The absorbance was measured by adding 0.010 ml of a 5 mM aqueous calcium chloride solution. 0.080 ml of calcium chloride aqueous solution
At the time of addition, the absorbance was 0.203. When the absorbance exceeded 0.32, 0.20 ml of a 25 mM calcium chloride aqueous solution was added. A decrease in absorbance due to an increase in the amount of liquid during the measurement was corrected according to the Lambert-Beer law. In addition, bubbling was performed by slowly blowing nitrogen to suppress fading of NN.

The Ca ion addition concentration and the corrected absorbance were plotted, and the Ca ion addition concentration (T
m sample).

Table 1 shows the results of measuring the metal ion binding force of the polyaspartic acid sample (M) by the method shown here. Shown in Example 3 Measurement of Tm sample --- Measurement: 25 mm in diameter (inner diameter: 23
mm) using a test tube. 6.50 ml in test tube
Of distilled water and 1.00 ml of a sample aqueous solution (concentration 2%), and 1.00 ml of KOH (concentration 50 w / v) and a metal indicator NN were added (absorbance 0.1 at a wavelength of 470 nm).
9-0.199, concentration 18-18.8 μM). 150m
M strontium chloride aqueous solution in a small amount (0.01
(0 ml) until no increase in absorbance was observed. In this measurement, the absorbance was decreased according to the increase in the amount of liquid during the measurement, so the absorbance was corrected according to the Lambert-Beer law. In addition, bubbling was performed by slowly blowing nitrogen to suppress fading of NN.

The concentration of Sr ions added and the corrected absorbance were plotted. The Sr ion addition concentration (Tm sample) at a coloring ratio of 50% was determined.

Table 2 shows the results of measuring the metal ion binding force of the polyaspartic acid sample (CF110, E) by the method shown here. Shown in Measurement of Tm blank --- Tm sa
The operation was performed in the same manner as in the measurement of mple, but distilled water was used instead of the sample aqueous solution. Table 2 shows the results. Shown in

[0095]

[Table 2]

Example 4 Measurement of Tm sample --- Measurement was performed with a diameter of 25 mm (inner diameter of 23).
mm) using a test tube. 10.00m in test tube
A polyaspartic acid sample (CF110, concentration 50.3% 0.1 ml) was injected into 1 l of distilled water, and 1.00 ml of K
(OH concentration 0.2 M) and a metal indicator TPC were added. Under these conditions, TPC (Thymolphthalein Complexone, 3,3'-Bi
s [N, N-bis (carboxymethyl) aminomethyl] thymolphthalei
The absorbance (wavelength 610 nm) according to n) was 0.205 (concentration 11 μM). A 25 mM aqueous solution of calcium chloride was added and the absorbance was measured. When the amount of addition became 0.2 ml, the absorbance hardly changed. The coloring ratio at this time was set to 100%. In this measurement, the absorbance was reduced according to the Lambert-Beer law, because the absorbance decreased due to the increase in the liquid volume during the measurement.

The Ca ion concentration and the corrected absorbance were plotted, and the Ca ion concentration at a color development rate of 50% was determined. Tm sample was 21.5 μM. Tm blank
Measurement—Operation was performed in the same manner as the measurement of Tm sample, but distilled water was used instead of the sample. The absorbance by TPC was 0.198. Tm blank was 3.9 μM.

As a result of this measurement, it was found that the deviation was 17.6 μM.

[0099]

The metal affinity can be evaluated by utilizing the fact that the color of the metal indicator changes with the addition of the measurement sample. High molecular compounds can be identified using metal affinity as an index. Information on the state of the metal ion-binding group can be obtained.

[Brief description of the drawings]

FIG. 1 is a graph showing an example of an Oda plot for a sample CF-110 according to the method of the present invention.

FIG. 2 is a graph showing an example of an Oda plot for Sample E according to the method of the present invention.

FIG. 3 is a graph showing an example of the relationship between {1 / (1-R)} and Tm / R (where m is Ca) for a control by the Scatchard plot method using NN. .

FIG. 4 shows {1 / (1-R)} and T for sample CF-110 by the Scatchard plot method using NN.
It is a graph which shows an example of the relationship with m / R (however, m is Ca.).

FIG. 5 is a graph showing an example of the relationship between {1 / (1-R)} and Tm / R (where m is Ca) for sample E by the Scatchard plot method using NN. is there.

Claims (5)

[Claims] 1. The relationship between the amount of addition of a metal ion solution and the coloration of a metal indicator, the relationship between the amount of addition of a metal ion solution and the coloration rate of a metal indicator, the relationship between the total concentration of metal ions and the coloration of a metal indicator, or a metal. A method for measuring metal affinity, in which the relationship between the total ion concentration and the coloring ratio of a metal indicator is examined, and the change in the relationship due to the addition of a measurement sample is used as an index. 2. The method according to claim 1, wherein a change (deviation) in the total concentration of metal ions due to the addition of a measurement sample or a metal ion binding force at a specific coloring ratio of the metal indicator is used as an index of metal affinity. 3. The method according to claim 2, wherein the metal ion binding force is determined by the following method. Or How to calculate: where, Tm sample, Tm blank
Is the total metal ion concentration of the sample addition system and control system at the metal indicator color development rate R, [s] is the concentration of the metal binding group, Tind sample and Tind blank are the sample addition system,
Shows the concentration of the control metal indicator. From the relationship between the metal ion addition concentration and the color development rate of the metal indicator, the apparent binding constant k between the metal ion and the metal indicator was determined by Scatchard (type) plot for the sample addition system.
A method of calculating exp, and calculating the metal ion binding force as (kind / kexp-1) / [s]: where, kind is the binding constant between the metal ion and the metal indicator, and [s] is the concentration of the metal binding group. is there. The difference {Δ / (1-R)} between the sample-added system and the control system was examined, and the proportionality constant found in {Δ / (1-R)} and 1 / (1-R), or ｛Δ / ( 1-R)｝ and [s] / (1-R)
To find the metal ion binding force based on the proportionality constant found in the above equation: where {Δ / (1-R)} is (kind / R) ×
(Deviation) or the difference between the sample addition system and the control system in the Scatchard (type) plot, where R is the color development rate of the metal indicator, [s] is the concentration of the metal binding group, and Kind is the metal ion and the metal indicator. And shows the binding constant. 4. The method according to claim 1, wherein NN is used as the metal indicator.
4. The method according to any one of the above items 3. 5. The method according to claim 1, wherein the measurement sample is a polymer compound.
5. A method for identifying a polymer compound using the metal affinity measurement method and index according to any one of items 4 to 4.