The pH value of 10% formalin neutral fixative is mainly controlled by phosphate. Disodium hydrogen phosphate is alkaline, while sodium dihydrogen phosphate is acidic. Both of them mainly exist in the form of crystal water. The former often has 12 crystals (called disodium hydrogen phosphate dodecahydrate Na2HPO4·12H2O, molecular weight 358.142) , the latter often contains 2 crystal waters (called sodium dihydrogen phosphate dihydrate NaH2PO4·2H2O, molecular weight 156.01), there are also anhydrous disodium hydrogen phosphate (Na2HPO₄ molecular weight 142.142) and anhydrous sodium dihydrogen phosphate (NaH2PO4 molecular weight) 120.01) . Different composition ratios of the two can produce different pH values for their aqueous solutions (the aqueous solution is called phosphate buffer). In order to control the pH value of 7.2, different units and experts have their own ratios
Examples are as follows:
  Remark:
1): From the molar ratio of disodium hydrogen phosphate/sodium dihydrogen phosphate, it can be inferred that the pH value in Shanghai is the highest, that in Tongji is the second highest, and that in Fujian is the lowest.
2): From the sum of moles of disodium hydrogen phosphate/sodium dihydrogen phosphate, it can be inferred that Shanghai has the strongest buffering power, Fujian has the second, and Tongji has the lowest.
3): From the sum of the moles of disodium hydrogen phosphate/sodium dihydrogen phosphate, it can be seen that the amount of phosphate used in Shanghai is the largest, that of Fujian is second, and that of Tongji is the lowest.
The most correct way to measure the pH value of the fixative is to use a pH meter that can be calibrated at three points and has temperature compensation. The pH meter must be calibrated before measurement. After the calibration is completed, verify whether the correction is in place. Only after it is in place can the pH meter of the fixative be carried out. H value measurement, the value displayed by the pH meter is the correct value at this time.
Calibration should be carried out using three standard buffer solutions according to the instructions of the pH meter. The pH meter used to measure the fixative solution generally uses pH 4.0 (potassium hydrogen phthalate), pH 6.86 (mixed phosphate), pH 9.18 (sodium tetraborate), and three-purpose standard buffer (sold in finished products on the market). Just do the correction. The calibration process must be carried out in accordance with the instructions of the pH meter. After the calibration is completed, verify whether the calibration is in place.
Verification after calibration of the pH meter: After calibration of the pH meter, rinse it with distilled water, and then put it into the standard buffer for calibration at will, such as pH 6.86 (mixed phosphate) standard buffer. At this time, the pH meter The displayed value should be between 6.84 and 6.88. If it is placed in pH 4.0 (potassium hydrogen phthalate) standard buffer, the value displayed by the pH meter should be between 3.98 and 4.02. If it is placed in pH 9. 18 (sodium tetraborate) standard buffer, at this time the value displayed by the pH meter should be between 9.16 and 9.20, otherwise the pH meter needs to be recalibrated.
Correct operation when measuring the stationary solution: rinse the pH meter (glass bulb part) with distilled water and absorb all the water with absorbent paper. Insert the pH meter into the stationary solution to be measured, gently vibrate the pH meter (about 10 seconds), and let the pH meter stand. After the value on the pH meter is stable (not jumping), read the number. This number is the pH value of the fixative.
The pH value control method of our company's 10% formalin neutral fixative is also controlled by phosphate buffer. The amount of phosphate is formulated based on full consideration of the pH value and osmotic pressure of the cell fluid and interstitial fluid. It is different from the usage of the above three companies. The range (6.90~7.50) specified in the company's product quality standards is formulated through stability experiments. During the validity period, the lowest pH value will not be lower than 6.9 near the expiration date, and the highest pH value will not be higher than 7.5 when it just leaves the factory. The company's products are generally controlled between 7.10 and 7.30 when they leave the factory.
Our company’s technical staff noticed that “when formalin and phosphate buffer are mixed together, there is a big difference between measuring the pH immediately and measuring the pH after 12 hours; the pH measured immediately is often higher than The pH value measured after being left for 12 hours was about 0.3; after being left for 12 hours, the pH value dropped very little." The pH value of our company's 10% formalin neutral fixative is the value measured after it is prepared and left for 12 hours.
Key points in controlling the pH value of 10% formalin neutral fixative:
1. The concentration of phosphate in phosphate buffer cannot be too high or too low. It is recommended to use a concentration of 0.02 mol. The osmotic pressure of this concentration is similar to that of cell fluid and tissue fluid.
2. After mixing phosphate buffer solution with pH 7.2~7.4 and formalin and leaving it for 12 hours, the pH value will drop by about 0.3, and some will drop more. It is related to the formic acid in the raw material (formalin solution) It depends on the content.
3. It is recommended to use a pH meter with three-point calibration and temperature compensation. The pH meter must be calibrated before measurement. After the calibration is completed, verify whether the calibration is in place.
4. pH meter calibration: Generally, pH 4.0 (potassium hydrogen phthalate), pH 6.86 (mixed phosphate), and pH 9.18 (sodium tetraborate) three-purpose standard buffer (sold in finished products on the market) are used. Calibration, the calibration process must be carried out according to the instructions of the pH meter. After the calibration is completed, verify whether the calibration is in place.
5. Verify whether the calibration is in place: After calibrating the pH meter, rinse it with distilled water, and then put it into the standard buffer for calibration at will, such as pH 6.86 (mixed phosphate) standard buffer, and the pH meter will display The value should be between 6.84~6.88. If it is placed in pH4.0 (potassium hydrogen phthalate) standard buffer, the value displayed by the pH meter should be between 3.98~4.02. If it is placed in pH9.18 (Sodium tetraborate) standard buffer, the value displayed by the pH meter should be between 9.16 and 9.20 at this time, otherwise the pH meter needs to be recalibrated.
6. Correct operation when measuring tissue fixative: rinse the pH meter (glass bulb part) with distilled water and absorb all the water with absorbent paper. Insert the pH meter into the tissue fixative to be measured, and gently vibrate the pH meter (about 10 seconds). Leave the pH meter standing, and then read the number after the value on the pH meter is stable (not jumping). This number is the pH value of the fixative.