DK162064B - PROCEDURE FOR CREATING A BUFFER CAPACITY CURVE OF AN IONOGENIC SAMPLING SOLUTION AND APPARATUS FOR USE - Google Patents

Description

in

DK 162064B

The invention relates to a method for producing a buffer capacity curve of an ionic sample solution * and an apparatus for use in the method.

By the term "ionogenic" is meant a weak electrolyte whose proton dissociation exponent (pKa) ranges from 0 to 14 when water is used as the medium. "Ionogen" originally means an ion-producing substance. Examples of ionic compounds include weak acids, weak bases, ampholytes such as amino acids, peptides and proteins, as well as certain insoluble dissociating compounds. Almost all material in our environment, such as the biological fluids and foods, includes mixtures of ionic compounds.

From a physicochemical point of view, an ionic compound 15 dissociates as follows: HA I H + + A "(1)

The equilibrium constant is referred to as Ka, 20

Ka - [H +] [A "3 / [HA] (2) where [] means concentration or activity. The proton dissociation exponent is defined as 25 pKa = - log Ka. (3) Similarly, Ka and pKa for a base can be defined The values for ionogenic compounds are in the range of 0 to 14, and the values of a specific class of a dissociating group, such as carboxyl, are known to cover more or less. less restricted area, depending on the nature of the compound containing the dissociating group.

One of the features of an ionic compound is that of a solution of it

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2 ionic compounds show a tendency to counteract the pH change of the solution by the addition of a strong acid or a base, which effect is considerable when the pH of the solution is in the range pKa ± 1, where pKa refers to it. ionogenic compound dtsociation exponent. For example, when 1 ml of 1N NaOH is added to 1 liter of pure water (pH = 7), the pH increases immediately to 11, while when the same is added to a 0.1M solution (pH = 7) imidazole (pKa = 6.95), of which approx. half has been neutralized with HCl, the increase in pH is only approx. 0.02. As is well known, this effect is called the buffering effect, and a solution exhibiting this effect is called a buffer solution whose use is very widespread.

The theory behind this effect has been established and the buffer capacity can be calculated on the basis of pKa values, concentration and other parameters. This theory defines the buffer capacity β as follows: β = dB / d pH (5) where B is the concentration of added strong base (grams eq / liter).

20 When the pH is equal to pKa, β is maximal. For a monovalent acid, the maximum value is as follows: β = 0.576 C. (6)

25 A

where is the analytical concentration of the acid. Consequently, when the pH values of the solution deviate from the pKa value of the ionic compound, β decreases, e.g. to 1/10 of the maximum value in the case that pH = pKa ± 1, and to 1/100 where pH = pKa + 2, A similar relationship can be used for a polyhydric acid, although the calculation is more complicated.

As shown above, β value is proportional to CA, and when plotting the / J value against the pH, a curve with a peak is produced whose position on the pH axis corresponds to the pKa value of the ionic compound. Figures 1 (a) - (g) show examples of / J-pH curves for different acids (0.05 M), calculated according to the theoretical equations.

In the figures, the / 1 value increases sharply at pH below 2 and above 12.

This shows the buffer capacity of the solution water that is one

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3 ampholyte. For this reason, it is usually more difficult to determine intrinsic / i values in the above range, and consequently the use of δ-pH curves for an ionic solution of mean concentration is usually limited to the pH range from ca. 2 to 5 approx. 12th

The buffer capacity is not only proportional to C, but also additive in a mixture. Therefore, a / f pH curve shows certain similarities with an absorption spectrum where Lambert-Beer's law applies. That is, the pH axis corresponds to the wavelength axis and the / 3 axis corresponds to the absorbance axis.

Despite this property, a / J pH curve is rarely used for analytical purposes. One of the reasons for this has been the lack of a suitable and simple method for measuring β in a sample of unknown composition. Of course, as indicated in Equation (5), it is possible to derive β from the traditional pH-volume titration curve for unknown samples by point to point calculation, because β is equal to the reciprocal value of the slope of the titration curve. But the amount of work required to do so makes the method impractical for routine use.

Modern computers will facilitate this work, but require substantial preparation and extensive equipment.

There are many possible methods for indicating a pH-8 curve at the same time as the titration. The simplest method, in principle, is a method which involves memorizing digital data of the solution's pH value and the titrant volume network, as well as calculating an λ-pH relation in restoring the results. However, this method is not new in principle and requires extensive equipment as well as preparation and therefore cannot be said to be simple at present.

The object of the present invention is to provide a method for generating a buffer capacity curve which is as simple as a conventional automatic titration method. By the term "buffer capacity titration" as used herein is meant a method of simultaneously indicating an γ-pH curve by titrating an ionic-containing sample solution with a strong acid or base.

The above object of the method according to the invention is achieved by one

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4, characterized in that it comprises the following steps: (1) titrating the sample solution with a strong acid or base; 5 (2) detecting a pH change as a voltage change during titration of the solution with a constant titanium addition rate , (3) obtaining through a differentiator and a sub-circuit the ^ reciprocal value for the voltage change rate, (4) obtaining the output proportional to β = dB / dpH, and (5) providing the output as a function of pH to obtain the buffer capacity curve (/ J-pH).

The theoretical basis for this is as follows: When a solution containing a weak monovalent acid HA (including the protonated weak base) with a concentration of CA is titrated with an alkaline solution of the concentration Cg, the mass equilibrium and the principle of electron neutrality require:

Ca = [HA] + [A] = CAV / (V + v) (1) Cb = [M +] - CBv / (Vtv) (2) [M +] + [H +] - [OH "] + [A "j (3) where Ca and are the analytical concentrations of the acid and the added base, respectively, both in the titrated solution. V and v are the 30 volumes of the original solution and the added base, respectively. From these relations it follows that: [A'1 - CBv / (V + v) + [H +] - [OH-] (4) 35

By introducing the degree of dissociation, "- [A"] / ([HA] + [A "]), the following relation is obtained, where nA and ng are the amount of titrated acid

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s respectively the added base in moles: nB = nA + (V + v) ([0H '] - [H +] (5) ® Since the degree of dissociation is related to the acid association constant at:

Ka - [Η +] α / (1-α) (6) becomes Equation 5: n = - ^ Λ- + (V + v) ([OH '] - [H +]) (7) B Ks + [H] 15 Differentiation with respect to pH gives: * 1B- = *> B- X ^

dpH d [H +] dpH

• j · = 2.303 / —A — a · ^ - + (V + v) ((H +) + [OH ']) l (8)

20 l (k + [h ir J

α

Since the base solution is added at a constant rate r, 3 -1.

(cm -s), and 25 nB = CBrt where t indicates the time in seconds, the following relations are found: 30 dn dn dt dt - = - x - = CRr x - (10)

dpH dt dpH dpH

dt 2.303 V C-Ka [H +] V + v - = - '---ΓΤ + - (iH] + [OH "])

35 dpH cor (Ka + [H]) ά V

B (11)

The buffer capacity βί of the solution before the titration is according to the theory:

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6 f C Ka [H +] Ί βι = 2.303 i + (h 1 + io "ri -o2) j by combining Equations 11 and 12 to obtain: 5.

dt v f v + - 1 - = - 4 βί + 2,303 X - C [H] + (OH])} (13)

dpH CBr lV J

Thus, the amount of dt / dpH consists of one portion which is proportional to the buffer capacity of the original solution, βί, and another portion which is proportional to the volume of the added base solution v. The latter only works in both the terminal ranges of the pH scale. , and may be neglected when an appropriate concentrated alkali solution is used. If necessary, the second term can be eliminated to some degree by subtracting the result from a blank test. According to this principle, an electrical treatment of the output of the pH meter gives a good approximation to the actual / J-pH curve for an unknown sample solution, thereby obtaining an inogenous distribution.

Another method for obtaining an inogenic distribution by a β-pH curve will be illustrated. For example, titration with a strong base comprises the method of adding a strong base solution (volume Δν) to a sample solution to increase the pH with an addition of ΔρΗ, e.g. 0, that an electrical signal is obtained,? 5 which is proportional to the required amount of titrant, that Δν / ΔρΗ is calculated and that the value is given as a function of the pH value. The theory in this case is the same as described above with respect to equations (1) to (8). Since ng = Cgv is an dnQ dnQ dv dv

dpH dv dpH dpH

and therefore dv is 2.303 f C.Ka [H +] V + v - = - i -½ -— p + - C (H +] + (OH])

35 dpH CB | _ (Ka + [H] Γ V J

V Γ v _ 1 = - βΐ + 2.303 x - ([H] + [OH]) i (15)

C V J

'B L

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7%

Although Δν and ΔρΗ are certain values, the obtained curve, if Δν and ΔρΗ are sufficiently small, gives a good approximation for β \ with respect to dt / dpH under the conditions described above. However, if they are too small, errors and fluctuations in the measurement will increase. The values 5 are experimentally selected as small as possible.

These procedures and calculations can be performed continuously with electronic equipment to produce the λ-pH curve.

As the method of the invention is a completely new method of obtaining an ionic distribution for unknown samples, it is expected that it can be used for many practical purposes as well as scientific studies.

15 Brief Description of the Drawing:

Figure 1 shows calculated E-pH curves for various acids, Figure 2 shows a schematic view of an automatic recording system for a pH-8 curve according to the present invention, Figure 3 is a diagram showing a sketch of an apparatus. to obtain δ-pH curves by computer control, Figure 4 shows observed and calculated / J-pH curves which are in good agreement, Figures 5 (a) and (b) show changes in a titration curve and an -curve for the salted and fermented Chinese cabbage juice respectively at 30 different times, Figures 6 (a) and (b) show titration curves for the salted and fermented Chinese cabbage juice and lactic acid respectively, and Figures 7 to 19 show obtained by the method of the present invention: Figure 7 (a): green tea, (b): heated tea, (c): black tea, fig. 8 (a): vinegar; (b): acetic acid and Trishydrochloride; 9: a commercial

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δ amino acid beverage, fig. 10 (a): chemical spice, (b):. glutamic acid, fig. 11 (a): Japanese case, (b): wine, fig. 12 (a): coffee, (b): instant coffee, fig. 13: grapefruit juice, fig. 14: lemon juice, fig. 15: mandarin juice, fig. 16: applesauce, fig. 17: grape juice, fig. 18: pineapple juice, fig. 19 (a): soy sauce, (b): calculated from the amino acid composition of soy sauce, and Figure 20t shows an λ-pH curve of the extracted solution from fully fermented rice straw compost, and Figures 21 (a), (b) and (c ) shows 10 µ3 pH curves for urine.

In Figures 2 and 3, numbers 1 through 6 refer to a burette, a glass electrode, a standard electrode, a sample solution, a stirrer, and an X-Y recording apparatus, respectively.

15

As mentioned above, ionic compounds are widely used in our living environment. In particular, a large proportion of foodstuffs are mixtures which comprise organic acids, organic bases, amino acids, proteins and the like, the distribution of which is closely related to taste, nutritional value, commercial value and the like.

In the process of the present invention, an ionic distribution of unknown samples is produced in the form of an integrated profile.

In this regard, the present invention provides a novel method for characterizing substances. Another feature of the invention is that in most cases the method requires no pretreatment of the sample, which is often required by a traditional chemical analysis, and therefore possible loss or forgetting of ionic components therein may occur. Rather, while it is possible to perform qualitative or quantitative analyzes from top position or height, the features of the present invention should rather be seen as obtaining an ionic distribution in a case where the method is applied to a complex mixture.

According to a preferred embodiment of the invention, the buffer capacity titration is carried out after the pH of a sample solution is adjusted to the terminal regions of the pH range of possible pH curves by the addition of strong acid or base to the sample solution. Such a pH setting makes it possible to obtain one

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9 desired δ-pH curve in the range of pH 2 to pH 12.

In a specific embodiment of the present invention, a strong acid or base is added to a sample solution at a constant rate of addition, and a signal proportional to the ratio of the titrant volume to the infinitely small change in pH is recorded. More specifically, a strong acid or base is added to a sample solution at a constant rate with stirring, and the output signal of a pH electrode in the solution is processed in a differentiator circuit and then in a reciprocal circuit to produce a signal proportional to buffer capacity β. By registering the signal proportional to the λ value of the ordinate axis and the measured pH value of the abscissa axis, a ³3 pH curve is obtained.

15

The invention also relates to an apparatus for carrying out the method of the present invention. This apparatus comprises means for measuring pH, means for electrically processing the output of a pH electrode to obtain the buffer capacity β, and means for autographically recording the δ value as a function of the pH of the sample solution.

Preferably, means for achieving the buffer capacity comprise a differentiator circuit and a reciprocal circuit.

25

An embodiment of the apparatus for autographically recording a / J pH curve based on the above-mentioned theory will now be discussed in more detail.

30

As shown in FIG. 2, the output of a pH meter comprising glass and standard electrodes is amplified and fed to the differentiator circuit, where the input is converted to an output proportional to dE / dt. Output is further amplified and converted to an output proportional to dt / dE (the reciprocal value of DE / dt) at

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using a parts multiplier module. Since the size dE is proportional to the size dpH, dt / dE is proportional to dt / dpH.

Output (dt / dE) is input to the Y input of an X-Y recording apparatus, connecting the X input to the pH meter output. When the burette is started, the record is started to produce a pH curve where

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The pH is indicated on the abscissa and the size proportional to β is indicated on the ordinate.

This apparatus can be compactly and economically manufactured using a commercially available titrator. Operationally, this apparatus is almost identical to the traditional titrator. Disadvantages of this type of apparatus are that due to the method used to obtain time differential, the apparatus cannot be used in cases where the reaction or response of a glass electrode requires a long period of time and therefore cannot be used for the titration in certain suspensions or in certain non-aqueous solvents, and that undesirable noise reveals when a ten is not mixed rapidly with the sample solution, thereby not changing the pH value evenly.

However, the disadvantages mentioned above can be considerably rectified by various technical improvements, e.g. an improved mixing device in which a noise source at the end source is substantially avoided, or a reduced rate of addition, or by a computer controlled process.

20

Output from a function generator was entered into the apparatus to confirm whether the apparatus was functioning or not. Then, it was confirmed that the relationship between equations (14) and (15) is satisfactory for recording δ-pH curves for solutions of different concentration and volume.

Furthermore, it will be possible to make the curves smooth and automatically compensate for the background by performing the recording and signal processing with a digital computer instead of the analog method.

30

An example of this is illustrated in Figure 3. The titration equipment is the same as shown in FIG. 2nd

At point (a) of FIG. 3, an adjustable amount of titrant, e.g. 10 microliters. After the expiry of an adjustable wait time t (e.g., 1 second), the output of the pH meter is stored as a digital signal (memory I). If the second memory is stored (memory II) after repeating the same process, the value is calculated:

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π _meanade introduced_ (memory II - memory I) using the amount of solution that is digitized and memorized and the result stored (memory A). When the same cycle is repeated, the third pH memory is stored in memory II, while the second pH memory stored in memory II is converted to memory I and this calculation and storage method (memory B) is repeated. The same operations 10 are thus repeated.

The contents of the memories A, B, etc. are converted to analog results one by one and introduced to Y input into the X-Y recording apparatus.

X-i nput is connected to the pH output of the titrator.

15

While the value dt / dpH is recorded in the analog method, the value dv / dpH is recorded according to the present method, therefore the value can be recorded proportionally to β for slow response systems. Further, it may be possible to smooth the curve by further processing the contents of memories A, B, ... and expanding the observable range by subtracting a separately stored blank (rising at both ends of the curve) from memories A, B, ... It is preferable to adjust t by regaining the value of dv / dpH so that t is not unnecessarily extended. There are no special technical difficulties in doing this.

However, it cannot be avoided that such an apparatus is relatively large and expensive.

30

These devices are mainly used to determine the pH distribution of the buffer capacity, but they can also be used for other purposes. The measurement of the buffer capacity is based on Henderson's equation: 35 PH - PKa + log, and on the conversion of the pH to an electrical potential by the glass electrode. For example, a redox titration with an electrode of an inert material such as platinum, the measurement is based on the equation:

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12 E = E + £ n f ° x * derated form] o nF [reduced form] = g + 0> 059. foxidized form] g o n 9 [reduced form] where n denotes the number of electrons and these two relations are in complete agreement with each other. In other words, it is also possible in the redox titration to define the redox buffer capacity on 10 basis of the presence of a reversible redox system, as is the case with pH titration. Therefore, it should be noted that the apparatus can be used to study a system including a complex redox system such as biological materials.

The invention will now be described in more detail with reference to the following non-limiting examples.

The apparatus used in the following examples is schematically illustrated in Figure 2.

20 pH meter: Type HM-5A manufactured by Toa Electric Wave Co., Ltd. recording apparatus: Type D8-CP manufactured by Riken Electronics.

The titration was carried out by adding 1.00 N sodium hydroxide at a constant rate of approx. 10 microliters / second. The titrated solution was stirred with a magnetic stirrer (about 1300 rpm) and kept in a water bath at 25 ° C.

Figure 4 shows an example of a comparison between found and o π calculated results for a solution of known concentration. The solution contained 0.053 M acetic acid (pKa = 4.76) and 0.053 M Trish hydrochloride [tris- (hydroxymethyl) -aminomethane hydrochloride] (pKa = 8.06), and a small amount of hydrochloric acid was previously added to lower the pH of the solution to below pH 2, so that the result in the low pH range is also obtained. As can be seen in Figure 4, the found and calculated curves correspond quite well with each other.

It should now be illustrated by means of examples that the pH curve measured

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13 by the method of the present invention can be used for qualitative assessment of various foods and the like.

Aging process in Chinese cabbage pi cles.

5

As an example, the relationship between the change in the constituents of a salted vegetable due to fermentation and aging and the taste thereof in relation to the change in the pH curve obtained by the process of the present invention and the differences 10 between the process of the present invention were investigated. and traditional pH measurement as well as acidic titrometry or its relative values were also investigated as these traditional titremetries have been used to qualitatively measure such foods.

* 5,500 g of Chinese cabbage were cut into strips 7 mm wide and mixed with 20 g of sodium chloride, and the mixture was then stored in a commercial container under pressure and at room temperature.

This amount of sodium chloride is equal to the amount of sodium chloride used for salting Chinese cabbage overnight. According to the literature, the change in salted vegetables during preservation is very complicated, but it can be summarized as follows: Aerobic bacteria found in the constituents and the like, such as Pseudomonas, Flavobacterium, Achromobacter, Escherichia coli and Bacillus multiply, but after several days, and 25 if the lactic acid producing bacteria such as Leuc.mesenteroides,

Sc.faecal ice, Pediococcus begins to multiply, inhibits the proliferation of the aerobic bacteria by lowering the pH, and they cease to multiply as the amount of lactic acid increases substantially; then the flora of lactic acid bacteria changes and eventually 30 mainly L. plantarum or L.brevis multiply (see "Shokuhim Biseibutsugaku" (Foods Microbiology), written by Yoshii,

Kaneko and Yamaguchi, published by Gihodo, 1980). Of course, during the preservation, not only lactic acid but also other organic acids can be produced, as different

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kinds of fatty acids in salted vegetables [Takanami et al., Bull.Foods Industry, Japan, 25, 9 (1978)]. When the vegetable was pressed with added salt, water quickly was squeezed from the vegetable and the amount of liquid extruded was approx. equal to the amount of water left in the vegetable. 20 ml of the supernatant was extracted after 3, 5, 6 and 8

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14 days after the vegetable was salted and the extracted samples were subjected to the buffer capacity titration of the present invention, pH measurement and acid titration.

5 In the pH curve shown in Figure 5 (b), the following characteristics can be observed: (1) a buffer capacity range around pH approx. 3.8; (2) a buffer capacity range about pH ca. 9.5; (3) a weak buffer capacity range around pH 7.

The height of all these three areas increases significantly with time.

When calculating the value of β by performing the titration of the standard sample solution (acetic acid + tris) with the same volume after 8 days, the / J values in the regions (1) and (2) are approx. 0032. Assuming that the resulting / J values are respectively based on a single ionogenic compound, the amount of each ionogenic compound corresponds to 0.056 moles per ml. liter. If the value obtained for the region (1) is attributed to the buffer capacity due to lactic acid, its concentration is equal to about 0.5% (with the mole weight of lactic acid being 90.1). However, in the literature there is no compound corresponding to the peak (2).

Since the peak is located at a pH of approx. 9.5, it can be assumed that the material corresponds to (a) an amino acid (NHg group j, (b) a heteroaromatic compound (OH group), (c) phenol is, (d) purines, (e) N in a saturated heterocyclic compound or (f) fatamine. Given the known composition of the vegetable and its transformation, (a) is most likely the component of the supernatant responsible for the peak at a pH of 9.5. strong buffer effect at pH about 3 is due to the carboxylic acid groups of the amino acids, since the buffer effect around pH 3 is not substantially higher than at pH 9.5, it is assumed that the content of the carboxylic acid group belonging to organic acids in the supernatant succession is less than the This type of information has never been obtained by the traditional pH measurement or the acidimetric method and has required more elaborate and time consuming methods including multiple chromatographic

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Separation. Even with the latter, it is not possible to obtain the integrated profile of the ionic distribution obtained by the buffer capacity titration. It should be noted that such information has never been obtained by any conventional test method (e.g. pH measurement, acid titration) and is detected for the first time by the method of the present invention.

Furthermore, the component which can produce the weak buffer capacity range (2) around pH 7 has not been discussed in the known literature. It is believed that such compounds as hi stidine and other imidazole compounds as well as phosphate esters may be responsible for this buffer capacity range.

In any case, one of the important features of the present invention is that the distribution of various types of ionic compounds according to their respective pKa values can be understood immediately, although chemical methods are required to identify the individual materials giving rise to each peak. .

20 As for the relationship between taste and the # -pH curve, pickled salt for from 3 to 5 days has a favorable sour taste and is crispy, but then the acidity gradually increases and the taste also deteriorates. This means that the taste is most favorable at the time when the values of β in the regions (1) and (2) are in the range of 0.01 to 0.02. The method 23 also seems to be able to easily detect possible abnormal fermentation by an λ-pH curve.

Where the pH change is only measured according to a traditional method, a sudden decrease in pH is observed after 3-5 days, but then the pH change 3® is very small. This is evident from the initial pH value shown by fully drawn circles in Figure 5 (b). This may be conceived as if the production of organic acid by fermentation has ceased at this time, and some of the known articles mention such a conclusion, but such an explanation is not true. Indeed, the total amount of ionogen, 33 such as organic acids, continues to increase even after 8 days, as evidenced by the β-ρΗ curve. The fact that the pH remains almost constant is due to the proton equilibria between the acidic and basic materials produced simultaneously.

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16 t

The usual acidity measurement for all potassium titration involves adding base solution to obtain the desired pH value in the presence of an indicator and determining the volume of the solution. This method is widely used as a so-called "quantitative method for organic acids" (see, for example, "Experimental Textbook of Foods, Nutrient Chemistry", ed. By Obara, ed. By Kenpakusha (1980)). This method determines the amount of organic acids, but it is not the amount of lactic acid included in the supernatant, such as the pickle liquid. The titer and the amount of organic acid correspond to only 10 in those cases where the following requirements are met: (1) all organic acids present are in the free state.

(2) proton dissociation of all organic acids is complete at the indicator point used.

15

These two requirements are e.g. fulfilled by the titration of an aqueous solution containing lactic acid alone as shown in FIG. 6 (b), where the end point is clearly determined with both the indicators used and thus the titration of lactic acid can be carried out. However, from the pixel liquid titration curve shown in Figures 5 20 (a) and 6 (a), neither of the two requirements is met (these curves are titration curves for a solution in which a small amount of HCl has been previously added, so that titration curve is obtained at the low pH range). From the / 3 pH curve, the presence of the ionogenic compound, which is believed to be carboxylic acid groups with pKa of ca.

3.5, after subtracting the blank, however, it is evident that the pH of one of the pickle liquid after 8 days is 4.1 and that 60% of it is already neutralized. Although a mixed indicator is used, the end point becomes weak due to the presence of a buffer capacity range near pH 7. If phenolphthalein is used, the end point becomes quite weak and furthermore, approx. half of the ionic compound with pKa of approx. 9.5 shown on the / in-pH curve also be titrated. As a result, the quantitative determination of the amount of organic acids will lose its importance in such a case.

In contrast, the β-pH curve gives the following information: That as mentioned above, there are 2 essential ionogenic compounds, that the pKa values thereof are at ca. 3.5 and 9.5, respectively, that the approximate amount thereof can be calculated (after 8 days, the values being approx.

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17 0.056M in both cases), and that a small amount of ionogen is present near pKa 7. The present invention thus makes it possible to obtain various information which has never been obtained by the traditional methods. Although the chemical methods are obviously necessary to identify the individual ionogenic compounds, the present invention will be quite advantageous, particularly in the analysis of complex mixtures such as foodstuffs, the ionogenic distribution being as easily determined as conventional titration.

10

The results obtained with other foods by the process of the present invention are briefly discussed hereinafter.

The titration was performed using 1 N NaOH solution at a rate of addition of 2x9.74 microliters per liter. second and at an E f of 1.0 V.

Eref means X input to the divider and adjusts the output in relation to possible pH change.

20

Medium quality green tea (Fig. 7 fall

Sample extracted with 20 volumes of hot water (pH 6.0): 20 ml 25 N-HCl: 1.5 ml

The pattern of the observed curve clearly seems to correspond to the mixture of amino acids, but some of them may be some added during the preparation of the tea.

30

Toasted (heated1 by 7 (b))

Sample extracted with 10 volumes of hot water (pH 5.43): 20 ml 35 N-HCl: 1.0 ml

It was found that the amino acid content of roasted tea is substantially lower than the content of medium quality green tea.

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18

Sort the fig. 7 (cΠ,

Sample solution obtained from Lipton tea bags (10 g) by extraction with 20 volumes of hot water: 20 ml of 5 1N-HCl: 1 ml

The result shows that black tea has the same amino acid pattern as roasted tea, but the ratio between the peak corresponding to -C00H and the peak corresponding to -NH9 differs, however.

10 &

Vinegar fig. 8 (all "Summit fermented vinegar" (trademark) with an acidity of 4.2% and pH 2.75 (1/20 dilution): 2 ml 15 1N-HCl: 1 ml

Water: 18 ml

The curve obtained was essentially the same as the curve obtained with the completely pure acetic acid (Fig. 8 (b)).

20

Amino grease ffia. 91 "Algin Z" (available from Ajinomoto Co., Inc.; pH 3.80; containing L-alginine, sodium L-aspartate, fructose, glucose, citric acid, 25 dl-maleic acid, honey, vitamin C, niacin, caramel and flavors) (diluted to 1/4 with water): 5 ml of 1N-HCl: 1 ml

Water: 15 ml 30

The peak due to -NH 2 near pH 9 is lower than the peaks at lower pHs, due to the presence of added organic acids (citric, malic, ascorbic).

Chemical spice ffia. 10 cases 35

2% solution of "HONDASHI" (Ajinomoto Co., Inc.; centrifuged, pH

6.02): 20 µl 1N-HCl: 2 ml

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19

The shape of the curve obtained is essentially identical to that of glutamic acid (0.05 M; Fig. 10 (b)).

Japanese saké ffia. Π fall 5 "One Cup Ozeki" (Ozeki Shuzo K.K.) (pH 4.30) was used as a sample, A Sample 20 ml 1N-HCl: 1 ml pH 1.60 B 100 ml of the sample concentrated to 20 ml.

1N-HCl: 3 ml pH 1.45 The pattern of the amino acid mixture was observed.

15

Wine (Fig. 11 (b)) "DELICA" Red (Suntory Limited; pH 3.30): 20 ml 1N-HCl: 1 ml 20

The shape of the curve obtained is similar to the curve of tartaric acid. The presence of a buffer capacity range at pH 11 appears to be due to the presence of a compound with a phenolic acid or an amino group.

25 Plain coffee fio. 12 la))

The sample, which was extracted by percolation with 250 ml hot water (pH

5.13) from 10 g of Hills Brothers Med. finely ground: 20 ml of 1N-HCl: 1.5 ml of 30

Instant coffee fig. 12 (bl)

The sample prepared using MJB instant coffee (concentration 2%; pH 5.00): 20 ml 35 1N-HCl: 1 ml

Both coffee samples yielded a buffer capacity range near pH 4, which appears to be due to the presence of organic acid (s), and a peak near 20

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pH 9 due to an unknown, possibly phenol in sk and amino-containing compound.

Grapefruit through. 131 5

Florida Grapefruit Juice Juice (pH 3.07) (Diluted to 1/4): 5 ml 1N-HCl: 1 ml

Water: 15 ml 10

A peak presumed to be due to citric acid was observed and furthermore a buffer capacity was observed near pH 10. The position of the former peak was slightly offset from the calculated result shown in FIG. 1 (f). This is due to the strong ionic activity due to the trivalent acid ions and can be corrected by the calculation.

Lemon (fig. 141 20 Lemon juice (diluted to 1/10)): 2 ml of 1N-HCl: 1 ml

Water: 17 ml

The peak observed also appears to be due to citric acid, while there is no buffer capacity range near pH 10, which may be due to dilution.

Mandarin juice (Fig. 151) Mandarin juice (diluted to 1/3): 10 ml of 1N-HCl: 2 ml

Water: 18 ml

Acids other than citric acid appear to be included in the sample and a buffer capacity range near pH 10 was observed.

5

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21 Apples (from 16) Apples (manufactured by Kagome Co., Inc .; 100%, pH 3.76) was used as a sample after 2/3 dilution.

Sample 5 ml (A); 20 ml (B): 1N-HCl: 1 ml water 10 ml

It is clear that the main component is malic acid and that the material (s) with a strong buffer capacity at pH above 10 is present in a considerable amount. The same result was observed for prepared apple drinks.

1® Grape Juice (via 171.

As a sample, 100% grape juice made by Kagome Co., Inc. was used.

(pH 3.73) after 1/2 dilution.

20 Sample 10 ml 1N-HCl: 1 ml

Water: 10 ml The tartaric acid pattern was observed.

25

Pineapple (via. 181

Pineapple juice (pH 3.10): 10 ml 1N-HCl: 1 ml 30

Freshly made juice was used without dilution.

According to the literature, the most important organic acids are citric and malic acid, while the characteristics of the citric acid were stronger in this example than for malic acid.

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'/ 22

Sleeping Sauce ffiq. 19 (al)

As a sample, soy sauce made by Kikkoman Co., Ltd. was used. (pH 4.72). 2 ml of 5N-HCl: 2 ml

Water: 17 ml

The soy sauce includes various amino acids, and the δ-pH curve should therefore be a curve obtained by superimposing the buffer capacity of the individual amino acids. The curve found actually shows a unique pattern. In this regard, the curve found is quite consistent with the theoretical curve obtained by calculating the ratio of β to pH using the known pKa values as well as the content of IS types of amino acids in view of the known composition of the soy sauce [see "Table of the Ingredients of

Japanese Foods "published by Medical, Dental and

Pharmaceutical Books Publishing Co., Ltd., (1976)].

Among the aforementioned results for foods, the results that can be deduced from the known components can be confirmed by the method of the present invention, and in some cases smaller but presumably important ionogenic compounds were also found. The practical significance of these results will increase more and more as the empirical results of PC accumulate through the application of the method.

The method of the present invention can also be expected to be used in the qualitative control of various materials.

ΟΛ

In soil and fertilizer science, on the other hand, organic soil components are quite important. Frequent use of organic components has been recommended and a large number of composts are produced and sold. However, there has been no significant progress in the study of the so-called humus materials because they are extremely complicated in structure composition. It is quite difficult to determine the degree of humidification during the production of the compost. The humic acid comprises a colored polymeric material which exhibits acidity mainly due to carboxylic acid groups, and therefore the process of the present invention can be used to

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23 perform the quantitative and qualitative analyzes thereof. One of the examples is shown in Figure 20. Figure 20 shows a buffer capacity curve for a solution (pH 8.60) prepared by subjecting 40 g of a fully humidified rice straw compost to extraction with 100 ml of water and removal of insoluble materials. The curve shows, as predicted, that the solution includes a mixture of different types of ionic compounds. The resulting curve shows 2 buffer capacity ranges at pKa approx. 4-6 and approx. 9-11. Although the significance of these results and their practical application is not clear at the present time, it will become more clear by comparison with a large number of samples.

Use and human urine ^ The composition of urine is highly variable reflecting all living conditions. A large number of point-to-point tests are regularly used for diagnostic purposes, but it will be almost impossible to summarize the overall picture of its varying compositions due to its complexity. Most of the organic urine constituents are the ionic compounds mentioned herein, including organic acids, amino acids, heterocyclic compounds, peptides and proteins. It should therefore be understood that the buffer capacity curve for human urine reflects the amount of the components as well as the balance between them.

The results obtained with 3 normal 25 adult people (40- to 60-year-olds) are then illustrated.

Person A (Fig. 21 (a))

Urine (pH 6.70) 10 ml 1N-HCl 1 ml Person B (Fig. 21 (b))

Urine (pH 5.37) 10 ml 1N-HCl 0.5 ml

Person C (Fig. 21 (c))

Urine (pH 6.0) 10 ml 1N-HCl 1.5 ml

When comparing with the results of these 3 samples, significant differences can be observed. The curves of persons A and B were similar in quantitative and qualitative terms, while the relative values for each 24 '

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peak (pKa approx. 4.9, 6.3 to 6.7 and 9.3) differed slightly.

It is believed that two of the peaks (pKa about 4.9 and 6.3 to 6.7) can be attributed to organic acids, and that the remainder is due to ammonia or an organic amine. The results for the person C show that a remarkable amount of ionic compounds is present in a fairly concentrated state, and in particular a high peak is observed (pKa 9.3). In all the 3 cases, the presence of an inogenic compound near pKa 2-3 was also observed, and the peak for C was the highest. The fluctuation (noise) in the highest peak for C was observed due to the dissolution of a small amount of solid by the addition of alkali.

The molar concentrations of the materials corresponding to each peak of the person C can be calculated from the magnitude of the value β such that the material having pKa 6.2 is equal to approx. 0.06 M and the material having pKa 9.3 equals approx. 0.12M.

15

The significance of this information will be clarified during the accumulation of empirical results in the future, with the fact that such a difference is observed between the 3 persons who are similar in terms of health and living conditions, reflecting that the method of the present invention will be effective for clinical purposes.

The process of the invention will, of course, be expected to be applicable in other fields, such as the exploration of 25 proteins and peptides.

Although the pH-volume titration curve has long been an important agent in these areas [see e.g. "Electric Properties of

Proteins "written by Katsushige Hayashi, published by the Tokyo University Publishing Department (1971)], determining the pH curve will be a useful agent in these areas.

35

Claims (6)

A method of generating a buffer capacity curve of an inogenous sample solution, characterized in that it comprises the following steps: (1) titrating the sample solution with a strong acid or base, (2) detecting a pH change as a voltage change during titration 10 of the solution with a constant rate of titrant addition, (3) obtaining through a differentiator and a sub-circuit the reciprocal value for the voltage change rate, (4) obtaining the output proportional to β = dB / dpH, and (5) ) that the output is indicated as a function of pH to obtain the buffer capacity curve (island pH). 20 Process according to claim 1, characterized in that the titration is carried out after the pH of the solution is adjusted to the terminal end of the usable pH range of an expected / J pH curve. Method according to claim 1, characterized in that the acid or base is added to the solution at a controlled rate and a signal proportional to the ratio of the infinitely small volume of added titrant to the infinitely small change in pH is recorded. 30 Method according to claim 1, characterized in that the acid or base is added to the solution at a constant rate and with stirring, that the output signal from the pH electrode in the solution is processed electronically through a differential circuit and then through a reciprocal circuit to obtain a signal. that is * proportional to the buffer capacity β and that the signal proportional to the buffer capacity β is recorded on the ordinate and the measured pH of the abscissa. DK 162064Β Apparatus for use in the method according to claim 1, characterized by means for measuring pH, means for electronically processing an output from the pH electrode in the solution to obtain the buffer capacity β, and means for autographing the J8 value against the pH of the sample solution. Apparatus according to claim 5, characterized in that the means for obtaining the buffer capacity β comprises a differentiator circuit and a reciprocal circuit. 10 15 20 25 30 35