JPS632465B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a buffer capacity titration method for a sample solution containing ionodiene and an apparatus therefor. More specifically, the present invention involves titrating a sample solution containing ionodiene with a strong acid or strong base, electrically processing the response of a PH electrode in the solution to determine the value of buffer capacity β, and calculating the value of β. This invention relates to a method and apparatus for obtaining a β-PH curve by self-recording the pH of a sample solution.

The term ``ionodiene'' here generally refers to strong electrolytes such as those whose proton dissociation index is in the range of 0 to 14 when water is used as a medium (``ionodiene'' refers to a substance that generates ions). These generally include weak acids, weak bases, ampholytes (including amino acids, peptides, and proteins), and certain insoluble analytes. our natural and living environment;
Most animals and plants contain ionodiene-containing substances.

From a physicochemical point of view, ionodiene undergoes the following proton dissociation in the medium: For example, HAH ⁺ +A ^- (1) Its equilibrium constant is called Ka.

Ka = [H ⁺ ] [A ^- ] / [HA] ([ ] is the concentration or activity) (2) and pKa = -log Ka (3). In ionodiene, the pKa value is 0~
The range is roughly determined by the characteristic groups in the chemical structure.

The Ka and pKa of bases are similarly defined by expressing them as, for example, RNH ⁺ ₃ H ⁺ +RNH ₂ (4). One of the characteristics of ionodiene is that the pH of the liquid is
In the pKa range of ±1, it is resistant to pH changes caused by the addition of strong acids or strong alkalis. For example, if you add 1 ml of 1N sodium hydroxide to pure water 1 with a pH of 7, the pH will immediately rise to 11. Even if the same amount of sodium hydroxide is added to the liquid, the pH will only increase by about 0.02. This effect is called a buffering effect, and it is well known that solutions having this effect are widely used as buffer solutions.

The theory of this action has already been established, and the pKa
Given the value, concentration, etc., it is possible to calculate the buffering capacity. According to this theory, the buffering capacity β is defined as follows.

β=dB/dpH (5) where B is the concentration (gram equivalent/) of strong alkali added. β takes the maximum value when PH=pKa, and in the case of a monobasic acid, the value at that time is given by β=0.576C _A (6). C _A is the analytical concentration of acid. PH is
If it goes beyond pKa, PH decreases rapidly, and PH = pKa
At ±1, it becomes 1/10 of the above value, and at PH=pKa±2, it becomes 1/100. A similar relationship exists for polybasic acids, although the calculations are more complicated.

As is clear from the above, β is proportional to C, and when PH is taken as the horizontal axis, it is a value that occupies a unique position on it depending on its pKa value.
Figure 1a shows examples of β-PH curves for various acids (0.05M) obtained by computer calculation using theoretical formulas.
- Shown in g. In these figures, a rapid increase in β can be seen at PH<2 and PH>12. This is a manifestation of the buffering capacity of the solvent water as an amphoteric electrolyte, which becomes relatively large if the ionodiene concentration is low. For this reason, it is difficult to measure the original β value within the above range. Therefore, the application of the β-PH curve is practically limited to ionodienes with a pKa of 2 to 12.

β is not only proportional to C, but also additive in the mixture. Therefore, the β-PH curve can be considered similar to an absorption spectrum when the Lambert-Beer law holds. That is, the PH axis corresponds to the wavelength axis, and the β axis corresponds to absorbance.

Regardless of these characteristics, the β-
One of the reasons why PH curves have not been used for analyzes such as spectrum measurements is that there was no known method for easily measuring β in unknown samples. Of course, as estimated from equation (5), β is the normal titration curve (Volume-PH curve)
Since it is equal to the reciprocal of the slope of , it is possible to calculate it from the data of the titration curve of an unknown sample, but this requires considerable effort and cannot be used as a routine work. Data processing by computer saves labor but requires considerable preparation and equipment.

The present inventor has conducted intensive research into a method for simultaneously obtaining a β-PH curve for a sample containing ionodiene with the same ease as a normal titration operation, and has completed the present invention. As used herein, "buffer capacity titration" refers to titrating an ionodiene-containing sample with a strong acid or base as described above and simultaneously obtaining a β-PH curve.

Various methods can be considered for recording the β-PH curve simultaneously with the titration. The simplest method in principle is to digitally store the PH and titrant volume data and calculate the β-PH relationship with regeneration, but this method lacks novelty in principle and requires considerable equipment. It requires a lot of preparation, so it is hard to say that it is easy, at least at present.

One method of easily obtaining an approximate value of the β-PH relationship, regardless of the above method, is as follows. In other words, the titrant is added to the sample solution at a constant flow rate, the pH change that occurs is detected as a voltage, and after processing with a differentiator circuit, the reciprocal is taken, and an amount proportional to β = dB/dpH is obtained as an output. This is a method to plot against PH. The basic principle is described below.

Add a strong alkali to Vml of a weak acid HA solution with a concentration of C _A.
Suppose that a solution of MOH with a concentration C _B is injected. The mass balance in the titrant solution is Ca = [HA] + [A ^- ] = C _AV /V + v (1) Cb = [M ⁺ ] = C _B v/V + v (2) Ca and Cb are the mass balance in the titrant solution. Analytical concentrations of acid and strong alkali, v is the volume of lye added. The charge balance, including the dissociation of water, is [M ⁺ ] + [H ⁺ ] = [OH ^- ] + [A ^- ] (3) From this, [A ^- ] = C _B v/V + v + [H ⁺ ] - [ OH ^- ](4) The degree of dissociation α of HA is given by the following formula.

α=[A ^- ]/Ca=[A ^- ]/[HA]+[A ^- ] (5) Also, the titrated rate φ of HA is φ=C _B v/C _A V=m _B /m _A ( 6) m _A and m _B are the number of moles of [HA] + [A ^- ] and alkali in the liquid, respectively.

From the above relationship, φ=α+V+v/m _A ([OH ^- ] - [H ⁺ ]) (7) or m _B = m _A α+ (V + v) ([OH ^- ] - [H ⁺ ]) (8) (7 ) is one of the theoretical equations for the titration curve (PH-φ curve).

In equation (8), the acid dissociation formula Ka = [H ⁺ ] [A ^- ] / [HA] = [H ⁺ ] α/1−α
(9) Inserting α obtained from m _B = Kam _A /Ka + [H ⁺ ] + (V + v) ([OH ^- ] - [H ⁺ ]
) (10) Considering that [OH ^- ]=Kw/[H ⁺ ]
Differentiate equation (10) with respect to PH.

dm _B /dpH=dm _B /d[H ⁺ ]×d[H ⁺ ]/dpH=dm _B /d[H
⁺ ] × [H ⁺ ] ×2.303=2.303 {m _A Ka [H ⁺ ] / (Ka + [H ⁺ ]) ² + (V + v) ([H ⁺ ] + [OH ^- ])} (11) Add alkaline solution If it is added at a constant rate r (ml・S ^-1 ), then m _B = C _B rt, dm _B /dpH=dm _B /dt×dt/dpH=C _B r×dt/dpH(
12) Therefore, dt/dpH=2.303/C _B r{m _A Ka[H ⁺ ]/(Ka+[H ⁺ ]) ² + (V+v) ([H ⁺ ]+[OH ^- ])} (13) =2.303 V/C _B r{C _A Ka[H ⁺ ]/(Ka+[H ⁺ ]) ² +V+v/V([H ⁺ ]+[OH ^- ])} (14) On the other hand, the buffer capacity β _i before titration is β _i =2.303 {C _A Ka [H ⁺ ] / (Ka + [H ⁺ ]) ² + ([H ⁺ ] + [OH ^- ])} Since it is given by (15), in combination with equation (14), dt /dpH=V/C _B r{β _i +2.303×v/V ([H ⁺ ] + [OH ⁻ ])} (16) is obtained. That is, dt/dpH consists of a part directly proportional to β _i and a part proportional to the added liquid volume v, but the latter affects only both ends of the β-PH curve. However, it can be reduced to a negligible level by reducing v using a concentrated alkaline solution, and if necessary, it can be easily eliminated by subtracting the results of a blank test using the same volume of solution. can do. That is, according to this principle, by electrically processing the PH meter output, a good approximation value of the β-PH curve specific to the test liquid can be obtained, thereby making it possible to know the ionodiene distribution.

Another example of a method for easily obtaining an ionodiene distribution map based on a β-PH curve will be described. In other words, in the case of titration with a strong base, a strong base solution (solution volume △v) is added to raise the pH of the sample solution by a minute amount △PH, for example 0.1, and the required liquid volume is determined by an electrical signal. , calculate △v/△PH from that value, and record it for the set PH. The theory in this case is exactly the same up to equation (11) above, but m _B = C _B
Since v, dm _B /dpH=dm _B /dv×dv/dpH=C _B ×dv/dpH(17
) Therefore, dv/dpH=2.303/C _B {C _A Ka[H ⁺ ]/(Ka+[H ⁺ ]) ² +V+v/V([H ⁺ ]+[OH ^- ])}=V/c _B {β _i +2.303×v/V([H ⁺ ]+[OH ^- ])} (18) Therefore, △v and △PH are finite values, but if they are sufficiently small, the obtained curve will be , gives a good approximation of β _i under the conditions described above for dt/dpH. However, making these values too small will lead to measurement errors, so it is best to empirically select the smallest possible value. These operations and calculations can be performed continuously by electronic quantitative equipment,
A β-PH curve is obtained.

That is, the present invention titrates a sample solution containing ionodiene with a strong acid or strong base, electrically processes the response of the PH electrode in the solution to determine the value of buffer capacity β, and calculates the value of buffer capacity β by calculating the value of β of the sample solution. The present invention provides a buffer capacity titration method characterized by obtaining a β-PH curve by self-registering the pH.

Since the method of the present invention is a completely new method for measuring ionodiene distribution in unknown samples, there are many areas where its application remains to be developed and applied in the future. Judging from the results obtained so far, this appears to be quite reliable. Explanation is added below.

As mentioned above, ionodienes are widely distributed in our environment. Among these, when looking at foods, the majority of foods are mixtures of ionodienes consisting of organic acids, organic bases, amino acids, proteins, etc., and the distribution of these is closely related to their taste, nutritional value, commercial value, etc. Involved.

The method of the present invention is characterized in that the ionodiene distribution can be grasped as one overall image, and provides a new method for characterization. Another feature is that sample pretreatment, which is required in many conventional chemical analyses, is often not required, thereby avoiding possible loss or oversight of components. Of course, with the method of the present invention, it is also possible to perform qualitative analysis from the peak position and quantitative analysis from the peak height, but the feature of the method of the present invention is that in the case of mixtures such as foods, it is possible to conduct qualitative analysis from the peak position, and quantitative analysis from the peak height. It's about seeing the big picture.

Therefore, according to a preferred embodiment of the present invention, the buffer capacity titration is carried out after the pH of the sample liquid is adjusted to the limit of the pH range of the desired β-PH curve by adding a strong acid or a strong base to the sample liquid in advance. It will be done. By performing such an operation, a β-PH curve can be obtained arbitrarily within the maximum range of PH2 to PH12.

In a specific example of the present invention, a strong acid or a strong base is added to a sample solution while controlling the injection rate under constant conditions, and the ratio of the titrant volume corresponding to a minute change in pH is extracted and recorded as an electrical signal. be exposed. More specifically, a strong acid or a strong base is poured into a sample solution at a constant flow rate while stirring, and the response electrical signal of the PH electrode in the solution is processed in a differential circuit and then in a reciprocal circuit. The signal to be measured is the vertical axis signal, and the measured PH
The β-PH curve is obtained by recording the horizontal axis.

The present invention also provides an apparatus for carrying out the above-described method of the present invention. The device of the present invention includes a PH measuring means, a means for electrically processing the response of the PH electrode to determine the value of the buffering capacity β, and a means for determining the value of the buffering capacity β.
and a means for self-recording.

Preferably, the means for determining the value of the buffer capacity β includes a differentiating circuit and a reciprocal circuit.

An example of an apparatus for automatically recording β-PH curves based on the above theory will be described in more detail.

As is clear from Figure 2, the principle of this device is to amplify the voltage output of a PH meter equipped with a glass electrode and a reference electrode, use a differentiator circuit to obtain an output proportional to the differential dE/dt of its time change, and then After amplification, an output proportional to its reciprocal, dt/dE, is obtained by a multiplication/division module. Since dE is an amount proportional to dpH, dt/dE is proportional to dt/dpH. This output is input to the Y axis of the XY recorder, and the output of the PH meter is input directly to the X axis. If recording is started after starting the viewer, a β-PH curve with PH on the horizontal axis and a quantity proportional to β on the vertical axis will be obtained.

This device can be made extremely small and lightweight by using a commercially available titrator and is inexpensive. The difficulty of operation is almost the same as recording a normal titration curve. The drawback of this type of device is that it cannot be applied when the reaction itself requires a long time due to the operation of obtaining a time differential, or when the response of the glass electrode is extremely slow. It is not suitable for suspensions of liquids or titrations in certain non-aqueous solvents. Furthermore, if the alkali solution is not mixed quickly, the pH will not change smoothly and undesirable noise will occur.

However, these can be overcome to a considerable extent by technical improvements, for example by devising a mixing device that minimizes noise sources and by reducing the addition rate, or by using computer-controlled methods. It is something.

The output of the function generator was input to this device, and it was confirmed that it operated normally. We also recorded the β-PH curves of liquids with various concentrations and volumes, and confirmed that the relationships in equations (14) and (16) were satisfied.

Furthermore, instead of using the analog method described above, if signal processing and recording are performed using a digital computer, it becomes possible to perform curve smoothing, automatic background correction, etc. A schematic diagram of an example is shown in FIG. The titration apparatus is the same as shown in FIG.

At point a in the diagram, a fixed volume of titrant in an adjustable volume is added from the biuret (for example 10μ). Adjustable waiting time t (e.g. 1
seconds), the PH meter output is stored as a digital signal (memory). When the same thing is done again and the second memory is entered, the injection amount/(memory) - (memory) is calculated using the digitalized and memorized injection amount (one time). It is calculated and the result is stored in memory (memory A).
When the cycle is repeated, the third PH memory becomes memory and the second PH memory that was in memory is
The PH memory becomes a memory, and the above calculation and memory (memory B) are performed again. Similar operations are repeated thereafter.

Memories A, B......the following are converted into analog one after another and enter the Y axis of the XY recorder. The X-axis is connected to the PH meter output.

According to this method, dv/dpH is recorded as opposed to dt/dpH using an analog method, so by extending t, it is possible to stabilize the amount proportional to pH even in a system with a slow response. You should be able to record it. Also, memory A, B...... performs advanced arithmetic processing to perform smoothing, etc.
It is also possible to expand the observation range by subtracting separately stored blank values (increases at both ends of the curve) from memories A, B, etc. Also, in order not to make t unnecessarily long, it is convenient to adjust t by feedback on the dv/dpH value. All of these are technically not difficult.

However, this equipment, at least at present, is unavoidably large-scale and expensive.

These devices are for measuring the PH distribution of buffering capacity, but other uses are also possible. In other words, the basis of buffering capacity is the Henderson equation PH = pKa + log [salt] / [acid], and PH is converted into voltage at a glass electrode, but for example, redox titration using an inert substance such as platinum as an electrode In this case, E = Eo + RT / nFln [oxidized form] / [reduced form] = Eo + 0.059 / nlog [oxidized form] / [reduced form] The basis of this is n = number of electrons, and these two correspond completely. . That is, in redox titration as well as in PH titration, it is possible to define the redox buffering capacity by the presence of a reversible redox system. It should be noted that this opens up the possibility of using this device for research on systems including complex redox systems, such as biological materials.

The present invention will be explained below using specific examples, but these are not intended to limit the present invention in any way.

The apparatus used in the following examples is that shown schematically in FIG.

PH meter: manufactured by Toa Denpa, HM-5A type Recorder: manufactured by Riken Denshi, type D8-CP Titration was performed by adding 1.00 N of caustic soda at a constant rate of about 10 μ/sec. The mixture was stirred with a magnetic stirrer (approximately 1300 rpm), and the liquid was kept in a water bath at 25°C.

First, as an example, a comparison between actual measurements and calculations for known solutions is shown in FIG. The solution is 0.053M acetic acid (pKa
= 4.76) and 0.053M Tris(tris(hydroxymethyl)aminomethane) hydrochloride (pKa = 8.06)
To enable observation at a low pH, a small amount of hydrochloric acid was added in advance to bring the pH to 2 or less before titration. The measured values and calculated values are in good agreement.

Next, the following specific example shows that the β-PH curve obtained by the method of the present invention can be used for quality determination of various foods.

Aging Process of Pickled Chinese Cabbage As an example, we will examine the relationship between changes in the composition of salt-cured vegetables due to fermentation and aging by observing changes in the β-PH curve obtained by the method of the present invention, and also consider the relationship between changes in the taste of pickled cabbage in conventional foods of this kind. We will discuss the differences and advantages and disadvantages between PH measurement and acidity titration, which have been used for many years.

Cut 500g of Chinese cabbage into pieces about 7mm wide horizontally.
Mix with 20 g of common salt and store at room temperature under pressure in a commercial pickle pot. This amount of salt corresponds to what is called overnight pickling. According to the literature, the changes that occur when vegetables are salted are complex, but they can be summarized as follows. In other words, aerobic bacterial groups (Pseudomonas,
Flavobacterium, Achromobacter, Escherichia coli,
Bacillus) proliferate first, but within a few days lactic acid producing bacteria (Leuc. mesenteroides, Sc. Faccalis,
When aerobic bacteria such as Pediococcus begin to grow, the growth of aerobic bacteria is suppressed by a decrease in pH, and completely stops when lactic acid increases considerably. Then lactic acid bacteria Flora
Also changes, and in the later stages L.plantarum or L.brevis becomes dominant. (“Food Microbiology”. Yoshii, Kaneko, Yamaguchi, Gihodo. 1980). Of course, not all of the organic acids produced by lactic acid have been detected, but there are cases where a large number of fatty acids have been detected [Tanami et al. Journal of Japan Food Industry Association. 25 , 9 (1978)]. 〓Water is quickly leached from the pressed vegetables, and the supernatant juice and the liquid inside the vegetables are evenly distributed. Collect 20 ml of the supernatant juice on the 3rd, 5th, 6th, and 8th day after the start, and perform the buffer capacity titration and PH of the present invention.
Measurement and acidity titration were performed.

The β-PH curve shown in FIG. 5b has the following characteristics.

(1) Buffer capacity band centered around PH 3.8 (2) Buffer capacity band centered around PH 9.5 (3) Weak buffer band around PH 7 The heights of all three increase significantly with the number of days. ing. Standard sample solution (acetic acid + Tris)
If we calculate the value of β by titrating the same volume of
It becomes 0.032. Assuming that each is due to a single ionodiene, both of these
This corresponds to 0.056 mol/L. If (1) is considered to be the buffering capacity of lactic acid and the lactic acid concentration is calculated from the molecular weight of lactic acid (90.1), it corresponds to approximately 0.5%. However(2)
Regarding the peak, no description of the corresponding component can be found in the conventional literature. Considering that the peak value is at pH ~ 9.5, this substance has (a) amino acid - NH ₂ group,
(b) Heteroaromatic -OH, (c) Phenols (d) Purines (e) Saturated heterocyclic N (f) Aliphatic amines, but considering the raw materials and other factors, ( a)
Or (f). However, if (a), PH2-3 due to -COOH group of α-amino acid
At present, it is reasonable to consider (f) because the buffering capacity of
What is noteworthy is that such information cannot be obtained at all using conventional test methods (PH measurement, acidity titration).
This fact became clear for the first time in the method of the present invention.

Furthermore, there has been no previous description of substances corresponding to the weak buffering capacity band around PH7 described in (2). By inference, it could be a second dissociation of a dibasic acid, or an unsaturated amine. In any case, chemical methods are required to confirm the substance, but one major feature of the method of the present invention is that the pK distribution of various ionodienes can be seen at a glance.

Regarding the relationship with taste, in this experiment, the pickles had a moderate sourness and crispness after 3 to 5 days, but after that, the sourness took over and the taste became unpleasant. Therefore, in the β-PH curve
It is optimal when the peak of β in (1) and (2) reaches 0.01 to 0.02. Furthermore, if abnormal fermentation were to occur, it would probably be easily detected by the β-PH curve.

If you simply measure the PH change using the conventional method, you will notice that there is a sudden change in pH within 3 to 5 days, as is clear from the pre-titration PH value shown by the black circle on the curve in Figure 5b. It shows a decline, but the change after that is slight. This appears as if the production of organic acids through fermentation had subsequently stopped, and although there are some papers that give this interpretation, this is actually incorrect. That is, as seen in the β-PH curve, the actual situation is that the total amount of ionodienes such as organic acids continues to increase even after 8 days. The reason why the PH is almost constant is probably because the ratio of lactic acid to basic substances (which seems to give a peak at PH9.5 in the β-PH curve) is maintained in balance. Probably.

Ordinary acidity measurement by alkaline titration is a method in which, as mentioned above, an indicator is used to add alkaline solution to reach a certain pH, and the volume is determined. This method is widely used as a so-called ``quantitative method for organic acids'' (see, for example, Ohara, ed., ``Food and Nutritional Chemistry Experiment Book'', Kenpakusha, 1972).
This method, however, does not give the amount of organic acids, for example in the sense of the amount of lactic acid in the pickle juice. It is only in special cases that the titration value is equal to the amount of organic acid. That is, (1) all of the organic acids are present in the free state;

(2) Proton dissociation of all organic acids is complete at the color change point of the indicator.

It is necessary. For example, in the simple titration of an aqueous lactic acid solution shown in FIG. 6b, these two conditions are satisfied, and either indicator can be used to accurately obtain the end point and quantify lactic acid. However, if we look at the titration curves of pickle juice shown in Figures 5a and 6a, both of the above two conditions are not satisfied (these curves show that the titration curves of pickle juice are (This is the titration curve of the one with HCl added.) In other words, when looking at the β-PH curve, by subtracting the blank difference, the presence of ionodiene, which is thought to be a carboxyl group with a pKa of about 3.5, is recognized, but the pH of the pickle juice is 4.1 after 8 days, and it is clear that 60% of it already exists in a neutralized state. Furthermore, even if a mixed indicator is used, the end point will be unclear due to the presence of a buffer capacity band around pH 7, and if phenolphthalein is used,
Not only is the end point significantly unclear, but
Approximately half of the ionodiene with a pKa of approximately 9.5 that appears in the β-PH curve is also titrated, and as a result, the method loses its meaning as a quantitative determination of organic acids.

On the other hand, if we rely on the β-PH curve, as mentioned above, we find that there are two major ionodienes, their pKas are around 3.5 and 9.5, and the approximate value of their amount (8 days together in the eyes
0.056M), and the presence of a small amount of ionodiene around pKa7, making it possible to obtain information that could not be obtained using conventional methods. Of course, chemical methods are required to identify these ionodienes, but it is possible to know the ionodiene distribution at a glance with the same ease as ordinary titration, especially in complex mixtures such as foods. It would be extremely advantageous.

Below, the observation results obtained by applying the method of the present invention to other foods will be briefly described.

Titration uses N-NaOH, injection rate 2×
It was performed at 9.74 μ/sec and Eref = 1.0V. Eref is the × input of the divider, which adjusts the output according to the expected magnitude of the PH change.

Sencha (Figure 7a) Extracted with 20 times the amount of hot water PH6.0 20ml N-HCl 1.5ml The shape of the amino acid mixture is clearly visible,
It is possible that it was added later.

Hojicha (Fig. 7b) Extract with 10 times the amount of hot water (80℃) PH5.43 20ml N-HCl 1.0ml It has a much lower amino acid content than Sencha.

Black tea (Figure 7c) Lipton tea bag (10g), extracted with 20 times the amount of hot water 20ml N-HCl 1ml It is an amino acid type similar to hojicha, but -
The ratio of the portions corresponding to COOH and -NH ₂ seems to be different, and further investigation is required.

Vinegar (Figure 8a) Summitt brewed vinegar (acidity 4.2%) PH2.75 sample solution vinegar 2ml N-HCl 1ml Water 18ml A curve identical to that of completely pure acetic acid (Figure 8b) was obtained.

Amino acid drink (Figure 9) Argin Z (Ajinomoto Co., Inc., PH3.80, L-arginine,
Sodium L-aspartate, fructose,
Glucose, citric acid, dl-maleic acid, honey, vitamin C, niacin, caramel, flavor) 5 ml N-HCl 1 ml water 15 ml It is an amino acid drink, but the peak around PH 9 due to -NH ₂ is lower than the low PH peak. by the addition of organic acids (citric acid, malic acid, ascorbic acid).

Chemical seasoning (Figure 10a) "Hondashi" (Ajinomoto Co., Inc.) 2% solution (centrifuged) (PH6.02) 20ml N-HCl 2ml Glutamic acid (0.05M) (Figure 10b) Extremely close.

Sake (Figure 11a) One cutlet Ozeki (Ozeki Sake Brewery Co., Ltd.) PH4.30 A Sample 20ml 1 ml of N-HCl pH 1.60 B Concentrate 100ml sample to 20ml N-HCl 3ml pH 1.45 A form of amino acid mixture is emerging.

Wine (Fig. 11b) Suntory "Delica" Red PH3.30 Sample 20ml N-HCl 1ml It is close to that of tartaric acid, but the buffering capacity band at PH-11 is due to phenolic acid or compounds with amino groups. Probably.

Coffee (Figure 12a) Hills Brothers Med.fine grind 10g 250ml.
PH5.13 Above sample 20ml N-HCl 1.5ml Instant coffee (Figure 12b) MJB 2%, PH5.00 Sample 20ml N-HCl 1ml Buffering capacity around PH4, which is thought to be due to organic acid,
An unexplained peak around pH 9 appears in both real coffee and instant coffee.

Grapefruit (Figure 13) Juice, Florida PH3.07 Sample 5ml N-HCl 1ml Water 15ml Shows a peak that seems to be mainly citric acid, but
The peak of citric acid, which shows buffering capacity even around PH10, is out of position with the calculated peak in Figure 1 f, but this is due to the strong ionic strength of tribasic acid ions, and can be corrected by calculation. It is.

Lemon (Fig. 14) Lemon juice (10 times diluted) 2 ml N-HCl 11 ml Water 17 ml It is also thought to be due to citric acid, but the buffering capacity around PH10 found in grapefruit may be due to the high concentration and dilution. The obi is not visible.

Mandarin oranges (Fig. 15) Mandarin orange juice (2 times diluted) 10ml N-HCl 2ml Water 18ml Titration speed 2 Eref-1.00V It seems that acids other than citric acid are contained.
A buffering capacity band around PH10 can be seen.

Apple juice (Figure 16) Kagome Co., Ltd. 100%, PH3.76 Sample 5ml (A), 20ml (B) N-HCl 1ml Water 10ml It is clear that malic acid is the main component, but the pH is over 10 The same is true for youth drinks, which contain a considerable amount of substances with strong buffering capacity.

Grape diuse (Figure 17) Kagome fruit juice 100%, PH3.73 Sample 10ml 1 ml of N-HCl 10ml water A tartaric acid pattern can be seen.

Pineapple (Fig. 18) Pinenut Pulse Youth 10ml PH3.10 N-HCl 1ml Water 10ml According to the literature, citric acid and malic acid are the main organic acids, but the characteristics of citric acid are more pronounced. This will probably be more.

Soybean oil (Figure 19a) Kitsukoman PH4.72 Sample 2ml N-HCl 2ml Water 17ml Soybean oil is a mixture of many amino acids, and β
- The PH curve should be the sum of the buffering capacities of the individual amino acids. The measured curve shows a unique pattern as shown in the figure. As an attempt, we determined the β-
Results of calculating the calculated value of the PH curve (Figure 19b)
shows good agreement with the measured values.

As mentioned above, the results for foods not only confirmed the results expected from known ingredients, but also found a considerable amount of unidentified ionodiene associated with it. The practical meaning of these will become even greater as more empirical information is accumulated. It is also expected to be used in quality control.

On the other hand, organic components are an extremely important field in soil fertilizer science, and for practical purposes, their extensive use is encouraged and compost is produced in large quantities. However, this type of so-called humic substance has received little research due to the complexity of its structure and composition. Also, in the production of compost, the determination of the degree of rot is extremely ambiguous. Since humic acid is a colored polymeric substance exhibiting acidity mainly due to carboxyl groups, the present method can be applied to its qualitative and quantitative determination. An example is shown in FIG. The figure shows the buffering capacity curve of a solution (PH8.60) obtained by extracting 40 g of fully ripened rice straw compost with 100 ml of water and removing insoluble matter (PH8.60), and as expected, it is known to be a mixture of many ionodienes. Its distribution is
It seems that they can be divided into two groups: pKa of about 4-6 and pKa of about 9-11. At this stage, the meaning and practical implications of this are unclear, but will become clearer by comparing a large number of samples.

Application to human urine The composition of urine is extremely variable. In a sense, urine should reflect all aspects of daily life, but it is difficult to actually capture the whole picture, and clinically, only a small number of results are used to aid in diagnosis. do not have. Its components are complex and include an extremely large number of inorganic and organic substances, most of which are ionodienes. Examples include phenols, organic acids, imidazoles, indoles, amino acids, etc. Inorganic ammonia also belongs to ionodienes. Therefore, the buffer capacity curve of human urine faithfully reflects the amounts and balance of these components, and may be extremely useful in medicine and clinically. less than,
An example of three adult males (40-60 years old) (all healthy) is shown below.

Sample A (Figure 21a) Urine (PH6.70) 10ml N-HCl 1ml Sample B (Figure 21b) Urine (PH5.37) 10ml N-HCl 0.5ml Sample C (Figure 21c) Urine (PH6 .0) 10ml N-HCl 1.5ml When comparing the three cases A, B, and C, a large difference can be seen. A and B are qualitatively and quantitatively similar, but the relative values of each peak (pKa approximately 4.9, 6.3-6.7, 9.3) are slightly different. The first two are probably organic acids,
The last may be due to ammonia or organic amines. In sample C, ionodienes were found to be extremely large and concentrated, with ionodienes of 9.3 being particularly high. For A, B, and C, the presence of ionodiene with a pKa of around 2 to 3 was also observed, and the reason for the disturbance in the high peak in C was that a small amount of solid matter was generated by the addition of alkali. It is. Estimating from the size of β, the molar concentration of each peak substance in sample C is approximately 0.06M for pKa6.2,
The one with pKa9.3 is about 0.12M.

The clinical significance of these findings will depend on more experience, but the fact that such a difference was observed in three people who were all healthy and had similar living conditions suggests that their clinical application is promising. This suggests that

Of course, the method of the present invention is also expected to be applied to other aspects, such as protein and peptide research.

In this field, the PH titration curve has been an important tool for a long time (for example, Katsuya Hayashi, "Electrical Properties of Proteins," University of Tokyo Press, 1971), but the β-PH curve is an effective tool in this field. Probably.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing examples of β-PH curves of various acids determined by calculation, and FIG. 2 is a schematic diagram showing an example of the β-PH curve automatic recording device of the present invention. , FIG. 3 is a schematic diagram showing a method for automatically recording β-PH curves by computer control, and FIG. 4 is a diagram showing a β-PH curve obtained by the method of the present invention and a β-PH curve obtained by calculation. Figures 5a and 5b are diagrams illustrating the temporal changes in the titration curve and β-PH curve of salted Chinese cabbage juice, and Figures 6a and 6b are diagrams illustrating that the figures match well. It is a drawing which shows the titration curve of the said salt pickling juice and lactic acid. Figures 7 to 19 show the β-PH of various foods obtained by the method of the present invention.
It is a drawing showing a curve. Figure 7 a: Sencha, b: Hojicha, c: Black tea, Figure 8 a: Vinegar, b: Acetic acid +
Tris-hydrochloric acid, Figure 9: Amino acid beverage, Figure 10 a: Chemical seasoning, b: Glutamic acid, Figure 11 a: Sake, b: Wine, Figure 12 a: Coffee, b: Instant coffee, Figure 13: Grapefruit Youth, Figure 14: Lemon Youth, Figure 15: Orange Youth, Figure 16: Apple Youth, Figure 17: Grape Youth, Figure 18
Figures: Pineapple oil use, Figure 19a: Mustard oil, Figure 19b: Calculated values from the amino acid composition of soybean oil. FIG. 20 shows the β-PH curve of a fully ripened rice straw compost extract, and FIGS. 21 a, b, and c show the β-PH curves of urine. Explanation of drawing numbers, 1... Viewlet, 2...
Glass electrode, 3...Reference electrode, 4...Sample, 5...
...Stirrer, 6...XY recorder.

Claims (1)

[Claims] 1. While titrating a sample solution containing ionodiene with a strong acid or strong base, the response of a PH electrode in the solution is electrically processed to determine the value of buffer capacity β, and the value of β is determined by A buffer capacity titration method characterized by obtaining a β-PH curve by self-registering the pH of a liquid. 2. Claims characterized in that a strong acid or a strong base is added to the sample solution in advance to adjust the pH of the sample solution to the limit of the PH range of the desired β-PH curve, and then buffer capacity titration is performed. The method described in paragraph 1. 3. A patent claim characterized in that a strong acid or a strong base is added to a sample solution while controlling the injection rate under certain conditions, and the ratio of the volume of the titrant solution corresponding to a minute change in pH is extracted and recorded as an electrical signal. The method described in Scope 1. 4. Pour a strong acid or strong base into the sample solution at a constant flow rate while stirring, and process the response electrical signal of the PH electrode in the solution in a differential circuit and then in a reciprocal circuit, and plot the signal proportional to the buffering capacity β on the vertical axis. signal and measured
4. A method according to any one of claims 1 to 3, characterized in that PH is recorded as a horizontal axis. 5. Ionodiene, which is equipped with a PH measuring means, a means for electrically processing the response of the PH electrode to determine the value of buffer capacity β, and a means for self-recording the value of β against the pH of the sample solution. A device for measuring the buffering capacity of a sample solution containing 6. The device according to claim 5, wherein the means for determining the value of the buffer capacity β includes a differentiation circuit and a reciprocal circuit.