JP2857956B2 - Fatty acid determination method and apparatus therefor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for quantifying free fatty acids present in fats and oils for food use by voltammetry.

[0002]

2. Description of the Related Art The quality of fats and oils is evaluated based on the acid value, the quality of fats and oils having a high acid value is evaluated as poor, and the quality of fats and oils having a low acid value is evaluated as good. Here, the acid value is
It refers to the number of mg of potassium hydroxide required to neutralize 1 g of the sample, and has a certain relationship with the amount of free fatty acid, and serves as an index indicating the amount of free fatty acid. That is, fats and oils
If fresh, the free fatty acid is low and the acid value is low, but if the free fatty acid is generated by storage or processing, the acid value is high.

Conventionally, the method of measuring the acid value in fats and oils has been standardized by the standard fat and oil analysis method, the Japanese Agriculture and Forestry Standard, JIS, the Japanese Pharmacopoeia fat and oil test method, the hygiene test method, the food and drink test method, and the like. Employs an alkali titration using phenolphthalein as an indicator.

[0004]

However, in this alkali titration method, titration may be excessively larger than the value corresponding to the amount of free fatty acid actually contained by fats and oils. It was difficult to determine the end point of the titration. Furthermore, the acid value that can be measured with a phenolphthalein indicator is limited to those whose pKa is attributable to a specific range of fatty acids, so that the acid value corresponding to all fatty acids could not be measured.

The present invention has been made in view of the above points, and it is an object of the present invention to provide a method for quantitatively determining the total free fatty acids from lower fatty acids to higher fatty acids by voltammetry and an apparatus for quantitatively determining the fatty acids. The acid value can be accurately measured from the amount of the obtained fatty acid.

[0006]

Means for Solving the Problems By the way, the present inventor has proposed:
A method for the determination of trace acids in reagents using polarographic reduction waves of quinones has been proposed (Analytical Chemistry, Chapter 18
Vol. 3, Issued by The Japan Society for Analytical Chemistry, 1969).
However, it is unclear whether this quantification method can be applied to fatty acids having a high pKa, and since the calibration curves differ depending on the type of acid, when two or more acids are mixed, first, the polarogram is used. From the above, the wave height of the pre-wave corresponding to each acid is obtained, then the concentration of each acid is individually obtained by each calibration curve, and if the concentration of each acid is not added, the concentration of the whole acid can be obtained. Did not.

Accordingly, the present inventors have made intensive studies on the possibility of applying fatty acids and the optimum conditions thereof in voltammetry of a measuring solution in which free fatty acids and quinone coexist by the voltage regulation method. As a result, the magnitude of the current value of the pre-reduction wave of the naphthoquinone derivative is proportional to the concentration of free fatty acids for all fatty acids from lower fatty acids such as formic acid to higher fatty acids such as oleic acid and linoleic acid. I found it. In addition, it was found that the calibration curves of the respective fatty acids were almost the same, and thus the value obtained by superimposing the current values of the respective fatty acids corresponded to the total concentration of the fatty acids. This is probably because the ionization constants of the respective fatty acids are close in the organic solvent.

The present invention has been made on the basis of the above findings and further by determining the optimum conditions. The present invention provides a method for quantifying free fatty acids in a sample by obtaining a current value of a pre-wave immediately before a reduction wave. Features.

That is, in the method for determining fatty acids of the present invention, a coexisting electrolyte containing a sample to be quantified, a naphthoquinone derivative, and an organic solvent is prepared, and the positive half-wave potential of a naphthoquinone derivative reduction wave in the coexisting electrolyte is prepared. It is characterized by measuring the current value of the pre-reduction wave of the naphthoquinone derivative at a potential near the side.

The fatty acid quantifying apparatus of the present invention comprises a working electrode, a counter electrode, and a reference electrode immersed in a coexisting electrolyte containing a sample to be quantified, a naphthoquinone derivative and an organic solvent; And a control unit for measuring a current value of the pre-reduction wave of the naphthoquinone derivative at a potential near the positive side of the half-wave potential.

The sample to be quantified is not particularly limited as long as it contains a free fatty acid, and includes, for example, edible oils such as safflower oil, pharmaceutical materials such as cocoa butter, soybean oil, fish oil, and machine oil. The free fatty acid contained in the sample to be quantified, that is, the free fatty acid to be quantified may be a saturated fatty acid or an unsaturated fatty acid, and has a pKa of about 4 or more (as a value in water). Those having 1 to 20 or more carbon atoms are exemplified. Specifically, there are lauric acid, myristic acid, palmitic acid, stearic acid and the like as saturated fatty acids, and linoleic acid, oleic acid, eicosapentaenoic acid and the like as unsaturated fatty acids. Further, a plurality of these fatty acids may be contained in the sample to be quantified.
The amount of the fatty acid that can be determined by the fatty acid determination method of the present invention can be reduced to 1 ppm or less.

The naphthoquinone derivative is a kind of quinone, and thus undergoes two-electron reduction at one time or stepwise to be converted to the corresponding hydroquinone, and is converted into a one-stage wave (FIG. 9, line a), or a first wave and a first wave. Two-stage wave consisting of two waves (Fig. 1
0, shows the reduction wave of line a). Then, in the reduction wave, when a fatty acid coexists, a front wave having a wave height (current) proportional to only the fatty acid amount appears. In the present invention,
The fatty acid amount is determined by measuring the wave height.

Similar phenomena are observed with quinones other than the naphthoquinone derivative (benzoquinone, methylbenzoquinone, etc.), but these quinones are unstable and difficult to quantify stably and accurately. That is, quinones are unstable to light even in a crystalline state, and are particularly susceptible to photolysis in a molten state. In particular, an aqueous solution of benzoquinone or the like turns red-violet when exposed to sunlight, and a new absorption maximum occurs in the ultraviolet and visible regions. And, with an organic solvent, this decomposition was further facilitated. Therefore, in the present invention,
The present inventors have found that a naphthoquinone derivative is stable against photolysis in an organic solvent, and focused on this point, and decided to use a naphthoquinone derivative. FIG. 7 shows the absorption spectra of A immediately after the preparation of the benzoquinone solution and B at 2 hours after the preparation, and FIG. 8 shows the absorption spectra of A immediately after the preparation of the vitamin K ₃ solution and B at 2 hours after the preparation. .

Examples of the naphthoquinone derivative include vitamins K ₁ , K ₂ and K ₃ . The concentration of the naphthoquinone derivative is not particularly limited as long as the wave height of the reduction wave of the naphthoquinone derivative is higher than the wave height of the preceding wave. For example, 1
It may be ５5 mM.

The organic solvent may be either protic or aprotic.
For example, propanol, methanol, ethanol and the like can be mentioned, and as the aprotic organic solvent, for example, dimethylformamide, acenitrile, propionitrile and the like can be mentioned. Although it is conceivable to use an aqueous solution as a solvent, it cannot be adopted in terms of solubility. That is, intermediate or higher fatty acids are hardly soluble in water, and fats and oils themselves may be used as a sample for measurement, and fats and oils are also insoluble in water. In the case of a protic organic solvent, the half-wave reduction potential shift of the pre-wave with respect to the change in acid strength becomes remarkable, and simultaneous quantification of two or more kinds of each acid becomes possible. When a protic organic solvent is used, the reduction wave of the naphthoquinone derivative tends to be a single-stage wave, and when an aprotic organic solvent is used, it tends to be a two-stage wave.

The co-existing electrolyte usually contains a supporting electrolyte. Examples of the supporting electrolyte include perchlorate, iodide sulfate, and quaternary ammonium. The concentration of the supporting electrolyte is, for example, the concentration of the fatty acid in the electrolyte is 0.35 to 20.
When it is estimated to be about ppm, 0.05 to 0.1 M is preferable.

Further, the coexisting electrolyte may contain various additives as necessary. For example, a maximum inhibitor (eg, gelatin, Triton X-100, polyacrylamide, etc.) and a surfactant can be included.

According to the present invention, a coexisting electrolytic solution as described above is prepared, and the current value of the pre-reduction wave of the naphthoquinone derivative at a potential near the positive half-wave potential of the naphthoquinone derivative reduction wave in this coexisting electrolytic solution is measured. I do. The measurement of the current value of the pre-reduction wave of the naphthoquinone derivative is performed by creating a voltammogram, reading the current value at a predetermined potential from the voltammogram, applying a voltage to the predetermined potential, and corresponding to the applied voltage. The current value may be measured. The potential near the positive side of the half-wave potential of the naphthoquinone derivative reduction wave is the potential between the half-wave potential of the naphthoquinone derivative reduction wave and the naphthoquinone derivative reduction pre-wave closest to the reduction wave. Further, the current value of the reduction prewave at this potential is, in other words, a current value corresponding to the wave height of the naphthoquinone reduction prewave closest to the naphthoquinone reduction wave.

For example, in the case of a protic organic solvent,
The voltammogram of the toquinone derivative is shown in FIG.
FIG. 10 shows the case of a neutral organic solvent. Fat in the sample to be determined
Reduction waves of naphthoquinone derivatives in the absence of any acid
Is line a in each case. However, the sample to be quantified
When fatty acids are present in the water, the wave height (current) i_HbThe pre-wave b of
Appears (note that in FIG. 10, the first wave of the line a has a prefix wave b
Is appearing. ). If the sample to be quantified contains more fatty acids
If higher wave height i_HcBecomes the pre-wave c. Accordingly
The half-wave potential E of the naphthoquinone derivative reduction wave_1/2More positive
When the current at the potential Ei near the side is measured,
Is the wave height i of c_HbOr i_HcCan be requested. Also,
When two kinds of fatty acids are contained, for example, in FIG.
A prefix wave d and a prefix wave e appear as indicated by broken lines. this
Time, wave height i_HcIs wave height i_HdAnd wave height i _HeWith
The peak height i at the potential Ei_HcBy seeking
The wave height i of all reduction pre-waves d and e_Hd, I_HeAsk for
be able to. And these wave heights are only fatty acid amount
Depending on the amount of naphthoquinone derivative and the type of fatty acid
Because there is no, it is possible to calculate the fatty acid concentration from this current value.
Wear.

More specifically, voltammetry is performed on various solutions in which fatty acids have been accurately adjusted to various concentrations in advance, and the current value at each concentration is measured to prepare a calibration curve of current value-concentration. . Next, the actual voltammetry of the electrolyte solution prepared by dissolving the quantitative sample, measuring the wave height i _H. Then, if read from a calibration curve prepared with a fatty acid concentration corresponding to the height i _H,
The amount of fatty acids in the sample is determined. From this fatty acid concentration, the acid value can be converted from a predetermined conversion formula.

The measurement conditions for measuring the reduction wave of the naphthoquinone derivative by voltammetry are appropriately selected. For example, when the measurement temperature is 20 to 30 ° C., the scanning speed is 2 to 5 mV / s, and the action when ethanol is used as a solvent, Electrode potential -0.3 to -0.4
V _VS .SCE.

The above fatty acid determination method comprises a working electrode, a counter electrode, and a reference electrode immersed in a coexisting electrolyte containing a sample to be quantified, a naphthoquinone derivative, a supporting electrolyte, and an organic solvent; Fatty acid quantification device having a control unit for measuring the current value of the pre-reduction wave of the naphthoquinone derivative at a potential near the negative side of the half-wave potential of the reduction wave, and a display unit for displaying the current value measured by the control unit Can be implemented.

As the working electrode, a solid electrode (eg, a glassy carbon electrode, a gold electrode, a platinum electrode, a mercury-plated microelectrode, etc.), a dropping mercury electrode, or the like can be used, but it is easy to handle and avoids the use of mercury. Therefore, it is preferable to use a solid electrode. As the counter electrode, a stainless steel electrode, a platinum electrode, a mercury sulfate electrode saturated calomel electrode, or the like can be used. As the reference electrode, a saturated calomel electrode (SCE), a silver / silver chloride electrode, or the like can be used.

The working electrode, counter electrode and reference electrode as described above are immersed in the above-mentioned coexisting electrolyte.
As a form of the immersion, even if a working electrode or the like is provided in the electrolytic cell, and the electrolytic cell is filled with a coexisting electrolytic solution, a working electrode, a counter electrode, and a sensor including only a reference electrode are configured, It may be soaked in a coexisting electrolyte filled in a separate container. The electrolysis cell may be any one used for normal voltammetry. For example,
Examples include a bulk cell, an H-type cell, and a beaker type. In order to keep the naphthoquinone derivative stable for a long period of time, a light-shielding cell is preferable.

The control unit detects the current at a predetermined potential while properly regulating the potential of the working electrode with respect to the reference electrode, even if the control unit is constituted by a potentiostat with a recorder used in conventional voltammetry. A microcomputer or the like that displays the detected value may be used. The current obtained by this control unit can be displayed in various ways according to the purpose, for example, it is directly displayed on an automatic balance recorder, various displays, or the like, or is displayed after being converted into an acid value. In addition, it can be used as switching of connected equipment without displaying.

The fatty acid quantification apparatus of the present invention can be combined with various analyzers. For example, by combining with a flow injection analyzer, it is possible to rapidly and quantitatively determine the fatty acids of a large amount of a sample to be quantified in real time. And high performance liquid chromatography (HP
Combination with LC) enables qualitative and quantitative determination of various fatty acids.

[0027]

The fatty acid determination method of the present invention is based on the fact that the slope (wave height / concentration) of the calibration curve is constant regardless of the type of fatty acid. Therefore, even if a plurality of kinds of fatty acids are contained in the sample, the total fatty acid amount can be obtained with only one calibration curve. The slope of the calibration curve is constant. The degree of dissociation of fatty acids in organic solvents is almost constant, and thus the effect of electrophoresis is almost constant. For fatty acids, the degree of dissociation is generally small, and most fatty acids are present as neutral molecules and are unlikely to cause migration, and the effect of migration itself is small.
It seems to be because.

[0028]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the fatty acid determination apparatus according to the present invention will be described with reference to FIGS. FIG. 1 is a partial sectional view of the fatty acid quantification device, and FIG. 2 is a basic circuit diagram of the fatty acid quantification device.

In FIG. 1, reference numeral 1 denotes an electrolytic cell, and the electrolytic cell 1 is filled with a coexisting electrolytic solution 2 containing a sample to be measured, a naphthoquinone derivative, a supporting electrolyte, and an organic solvent. A lid 3 is fixed on the upper part of the electrolytic cell 1 and the inside thereof is sealed, and a working electrode 4 made of glassy carbon, a counter electrode 5 made of platinum, and a reference electrode 6 made of SCE are fixed to the lid 3. Each of these tips is immersed in the coexisting electrolyte 2. In addition, lid material 3
In order to remove dissolved oxygen, a gas supply pipe 7 for supplying nitrogen gas or the like to the co-existing electrolyte is fixed, and a gas discharge pipe 8 for discharging gas in the electrolytic cell 1 is fixed.
The tip of the gas intake pipe 7 is immersed in the coexisting electrolyte 2.

As shown in FIG. 2, the working electrode 4, the counter electrode 5, and the reference electrode 6 are connected to a potentiostat 9 as a control unit, and the potentiostat 9 is connected to a recorder 10. I have.

FIG. 3 is an overall configuration diagram of another embodiment of the fatty acid quantification device, FIG. 4 is a partial cross-sectional view of the fatty acid quantification device, and FIG. 5 is a partial end view of the fatty acid quantification device.

The fatty acid quantification devices shown in these figures are miniaturized in the form of a sensor, and reference numeral 11 denotes a sensor unit, which is connected to a control unit 13 to which a display 12 is connected. . This sensor unit 1
4, a cylindrical counter electrode 14 made of stainless steel is provided, and a working electrode 15 made of glassy carbon is provided substantially at the center of the counter electrode 11, as shown in FIGS.
Further, a reference electrode 16 made of silver / silver chloride is provided near the working electrode 15. These are connected to the control unit 13. In such a fatty acid determination device, an arbitrary container 17 is filled with the prepared coexisting electrolyte 18, and the sensor 11 is immersed in the coexisting electrolyte 18 to immerse the working electrode 15 and the like.

FIG. 6 is also an overall configuration diagram of another embodiment of the fatty acid quantification apparatus of the present invention. The fatty acid quantification device shown in this figure is an example applied to a flow injection analysis method. In this figure, reference numeral 21 denotes a pump, and a naphthoquinone introduction part 22 for introducing a solution of a naphthoquinone derivative and an organic solvent and an electrolyte introduction part 23 for introducing a solution of a supporting electrolyte and an organic solvent are provided upstream of the pump 21. And are connected. A coil 24 is provided downstream of the pump 21.
Are connected to the coil 24 via the naphthoquinone introduction part 22 directly and the electrolyte introduction part 23 via the sample injection part 25 to be measured. Downstream of the coil 24, a detection unit 26 including a working electrode, a counter electrode, a reference electrode, a control unit, and the like is connected, and a recorder 27 is electrically connected to the detection unit 26. Hereinafter, Examples of the method for determining fatty acids of the present invention will be specifically described.

Preparation of Calibration Curve A calibration curve of linoleic acid was prepared. That is, linoleic acid-containing electrolytes of various concentrations were subjected to voltammetry, respectively.
The wave height i _H of each pre-wave was measured from the obtained voltammogram of each vitamin K ₃ , and each wave height i _H was plotted against each concentration to prepare a calibration curve. Voltammetry is
Carried out in the apparatus shown in FIGS. 1 and 2, the measurement conditions, vitamin _{K 3 3mM, L i ClO 4} 38mM, using ethanol as solvent, measurement temperature 25 ° C., was scan rate 5 mV / S. The results are shown in FIG.

Calibration curves for oleic acid and palmitic acid were prepared in the same manner as for linoleic acid. The results are shown in FIGS. 12 and 13, respectively. As is clear from FIGS. 11 to 13, it can be seen that the calibration curves are the same for all fatty acids.

Example 1 Four types of unknown concentrations of linoleic acid were used as samples to be quantified, and respective coexisting electrolytes were prepared. The coexisting electrolyte composition is
Vitamin K _{3 was} 3 mM and LiClO _{4 was} 38 mM, and ethanol was used as a solvent. Then, voltammetry was performed on the four types of electrolyte solutions using the devices shown in FIGS.
A voltammogram of ₃ was obtained. The measurement temperature was 25 ° C,
The scanning speed was 5 mV / s. The results are shown in Figure 14 (Note that is shown only before置波portion of each vitamin K ₃ reduction wave.).

From FIG. 14, it can be seen that the wave heights i _H of the pre-waves in each concentration solution are 130, 420, 880 and 1410 nA, respectively. Then, from the calibration curve created earlier, the above wave heights i
As a result of reading the corresponding concentrations for _H , the linoleic acid concentrations were 1.25, 4.98, 9.90, and 14.8 (×
10 ^-2 ) mM.

[0038]Example 2 Uses commercially available cocoa butter and soybean oil as samples to be determined
Then, these total fatty acid contents were quantified. The electrolyte
Except that the sample concentration in the sample was 0.15 and 2.8%, respectively.
Performed voltammetry in the same manner as in Example 1 above.
Yes, each vitamin K _ThreeWas obtained. Figure 1 shows the results
5 (Note that each vitamin K_ThreeOnly the pre-wave part of the reduction wave
It is shown. ).

From FIG. 15, i _H is 593 and 1017 nA, respectively. Then, from the calibration curve created earlier, each of the above wave heights i
As a result of reading the corresponding concentration for _H , the total fatty acid concentration in cocoa butter and soybean oil was 1176 and 1176, respectively.
It was 36.4 ppm. This fatty acid concentration was converted to an acid value. Table 2 shows the conversion results.

The fatty acid compositions (% by weight) in cocoa butter and soybean oil are as shown in Table 1.

[0041]

[Table 1]

The same cocoa butter and soybean oil as above were subjected to alkali titration with a phenolphthalein indicator based on the acid value measurement method described in the Japanese Pharmacopoeia. Further, in order to determine an accurate titration end point, titration was performed by potentiometric measurement in place of the phenolphthalein indicator. From these, the respective acid values were determined, and the obtained acid values are shown in Table 2. Table 2 also shows the acid values of glycerin monostearate and safflower oil obtained in the same manner.

[0043]

[Table 2]

In the case of cacao butter, the results obtained by the three methods were almost in agreement, but in the case of soybean oil, glycerin monostearate and safflower oil, no agreement was observed. In the alkali titration, no discoloration of the phenolphthalein indicator was observed at the equivalent point apparent from the potentiometric titration, and the endpoint was reached only after adding a considerable amount of alkali, which caused a large positive error in the results of the alkali titration. It has become. As is clear from the results of cocoa butter and glyceryl monostearate, the results of the fatty acid quantification method of the present invention and the results of potentiometric titration showed a very good agreement, confirming that the accuracy of the fatty acid quantification method of the present invention was high. In the case of soybean oil and safflower oil, the potentiometric titration curve did not show a clear jump, and it was impossible to determine the titration end point from the inflection point of the curve. This is considered to be because these samples contain several kinds of fatty acids having different dissociation constants, and thus have a broad potentiometric titration curve in which several jumps overlap. Even in such a case, the fatty acid determination method of the present invention could reliably determine the acid value. Moreover, since the value corresponds to the value when the last part of the broad jump of the potentiometric titration curve is considered as the end point, it is considered to indicate an acid value corresponding to the sum of several fatty acids.

From the above results, it was confirmed that the fatty acid quantification method of the present invention can reliably determine the end point and has high accuracy.

[0046]

According to the present invention, fatty acids can be quantified with extremely high accuracy even in the case of a dark-colored sample and a mixture of a plurality of fatty acids.

[Brief description of the drawings]

FIG. 1 is a partial cross-sectional view of one embodiment of a fatty acid determination device according to the present invention.

FIG. 2 is a circuit configuration diagram of an embodiment of the fatty acid determination device according to the present invention.

FIG. 3 is an overall configuration diagram of another embodiment of the fatty acid determination device according to the present invention.

FIG. 4 is a partial cross-sectional view of another embodiment of the fatty acid determination device according to the present invention.

FIG. 5 is a partial end view of another embodiment of the fatty acid determination device according to the present invention.

FIG. 6 is an overall configuration diagram of still another embodiment of the fatty acid determination device according to the present invention.

FIG. 7 shows an absorption spectrum of benzoquinone.

FIG. 8 is a diagram showing an absorption spectrum of vitamin K ₃ .

FIG. 9 is a diagram showing a typical voltammogram of a naphthoquinone derivative when a protic organic solvent is used.

FIG. 10 shows the results when an aprotic organic solvent was used.
The figure showing the typical voltammogram of a naphthoquinone derivative.

FIG. 11 shows a calibration curve of linoleic acid.

FIG. 12 is a diagram showing a calibration curve of oleic acid.

FIG. 13 shows a calibration curve of palmitic acid.

FIG. 14 is a diagram showing a pre-wave portion of a vitamin K ₃ reduction wave in an electrolytic solution containing four types of samples containing linoleic acid having unknown concentrations.

FIG. 15 is a diagram showing a pre-wave portion of a vitamin K ₃ reduction wave when cocoa butter and soybean oil are used as a sample.

[Explanation of symbols]

 2: Coexisting electrolyte 4, 15 ... Working electrode 5, 14 ... Counter electrode 6, 16 ... Reference electrode 9, 13 ... Potentitostat (control unit) 10, 12 ... Recorder 26 ... Detection unit

──────────────────────────────────────────────────の Continuing from the front page (72) Inventor Kiyoko Takamura 3-31-16 Azamino Midori-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Fumiyo Kusunoki 3-2-1, Minami-Osawa, Hachioji-shi, Tokyo (56) Reference Document JP-A-5-93713 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) G01N 27/48

Claims (2)

(57) [Claims] 1. A coexisting electrolyte containing a sample to be quantified, a naphthoquinone derivative, and an organic solvent is prepared, and the naphthoquinone derivative of the naphthoquinone derivative at a potential near the positive half-wave potential of the naphthoquinone derivative reduction wave in the coexisting electrolyte is prepared. Fatty acid determination method characterized by measuring current value of reduction prewave 2. A working electrode, a counter electrode, and a reference electrode immersed in a coexisting electrolyte containing a sample to be quantified, a naphthoquinone derivative, and an organic solvent; A controller for measuring a current value of a pre-reduction wave of a naphthoquinone derivative at an electric potential;