Low zeta potential standard working liquid and preparation method and application thereof

Abstract

The embodiment of the invention discloses a low-zeta-potential standard working liquid as well as a preparation method and application thereof, and belongs to the technical field of metrology and colloidal chemistry. The low zeta potential standard working liquid comprises a suspension liquid a and an ionic liquid b which are independently subpackaged and stored, and the zeta potential value of the suspension liquid a is + 30 mV to + 50 mV; the ionic liquid b contains Na < + > and / or K < + >, and the total ionic strength is 100-200 mmol.L <-1 >; the volume ratio of the suspension liquid a to the ionic liquid b is 1: (4-9). According to the invention, the problem that the low zeta potential standard substance is easy to flocculate and lose stability is solved through a mode of combining double-liquid independent subpackage preservation (zeta preservation) and instantaneous separation intensity regulation (zeta regulation), and long-term stable storage and high reproducibility are realized; the method has a wide application prospect in research and quality control of low surface charge systems related to nano medicines, biological materials, environmental colloids, food emulsions and the like.

Description

Technical Field

[0001] This invention relates to the fields of metrology and colloid chemistry, specifically to a low zeta potential standard working liquid, its preparation method, and its application. Background Technology

[0002] Zeta potential is an important parameter characterizing the surface charge properties of colloidal particles and is widely used in colloid chemistry, materials science, biomedicine, and other fields. Low zeta potential standard materials are reference materials used to calibrate zeta potential measuring instruments and verify the accuracy of measurement methods. Their zeta potential values ​​are usually at a low level (generally within ±10 mV, or even close to electroneutrality).

[0003] Existing low zeta potential standard material particle suspensions are prone to flocculation, sedimentation, or Ostwald maturation during storage and transportation due to insufficient electrical double-layer repulsion, resulting in zeta drift over time and making it difficult to sell as "traceable" standard materials.

[0004] Public literature only reports sporadically that low-zeta reference samples, such as carboxyl-modified polystyrene, rely on the dissociation of surface acidic groups (-COOH) to generate charge. However, in aqueous storage, the carboxyl groups are easily hydrolyzed or undergo ion exchange, leading to a decay in the zeta value. Experiments show that the zeta value of such samples can drift by ±5 mV within 24 hours in a buffer solution at pH=7. These low-zeta reference samples are still single-use reagents and cannot meet the needs of long-term standard supply. Their zeta values ​​typically change significantly within hours to days after preparation (drifting far beyond the allowable error range of instrument calibration), and consistency in reproducibility between batches or different laboratories cannot be guaranteed. Essentially, they lack the core attributes of standard substances—long-term stability and traceability. Therefore, it is necessary to develop a standard substance that can be stably stored for ≥24 months and can easily, quickly, accurately, and reproducibly reproduce the target low-zeta potential value during use.

[0005] A search revealed no reports on the use of "two-liquid + instantaneous centrifugation control" for the preparation of low-ζ standard materials. Summary of the Invention

[0006] To address this issue, this invention provides a standard working liquid with an absolute zeta potential ≤ 5 mV, comprising a suspension a (or buffer stabilizer) and an ionic liquid b (or activator). By combining independent dispensing and storage of the two liquids (zeta preservation) with instantaneous centrifugation and control (zeta adjustment), the problem of easy flocculation and instability of low-zeta potential standard substances is solved, achieving long-term stable storage and high reproducibility. The standard working liquid provided by this invention is crucial for calibrating instruments based on electrophoresis principles (such as electrophoretic light scattering instruments, microelectrophoresis instruments, and electroacoustic spectrometers) and for inter-laboratory comparisons, and has broad application prospects, especially in research and quality control of low surface charge systems such as nanomedicine, biomaterials, environmental colloids, and food emulsions.

[0007] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0008] According to a first aspect of the present invention, the present invention provides a low zeta potential standard working liquid, comprising separately packaged and stored suspension a and ionic liquid b, wherein the zeta potential value of suspension a is +30 to +50 mV; and the ionic liquid b contains Na + and / or K + The total ionic strength is 100-200 mmol·L. -1 The volume ratio of the suspension a to the ionic liquid b is 1:4-9.

[0009] Furthermore, the method for preparing the suspension a includes:

[0010] When hydrated particles with a diameter of 20-600 nm and a PDI < 0.08 are added to ultrapure water, a conductivity of < 50 μS·cm is obtained. -1 Diluent;

[0011] The diluted solution was added dropwise to a pH of 8.0-9.5 and a borate concentration of 10 mmol·L⁻¹. -1 In the boric acid / borax buffer solution, the particle solid content is controlled at 0.2-2.0 wt%, and the solution is filtered through a 0.22 μm filter membrane.

[0012] Research has shown that using particles with a hydrated particle size of 20-600 nm and a PDI < 0.08 as raw materials can ensure excellent particle monodispersity, which is beneficial for obtaining accurate and repeatable zeta potential standard working liquids. The suspension 'a' prepared by the above method has a zeta value of +30~+50 mV, ensuring Coulombic repulsion, while also exhibiting good stability. It can be stable for 24 months at 4℃ in the dark, with zeta drift < 2 mV and particle size change < 5%.

[0013] Furthermore, the particles are selected from one or more of neutral polystyrene (PS), weakly positively charged amino polystyrene (NH2-PS), silicon dioxide (SiO2), and γ-alumina.

[0014] Furthermore, the preparation method of suspension a further includes adding 0.01-0.02 wt% of a preservative to the system before filtration. The preservative exerts an antibacterial effect, thereby preventing microbial growth that could compromise stability. As an example, ProClin 950 can be used. TM preservative.

[0015] Further, the ionic liquid b is a 0.9% NaCl solution (physiological saline, pH=7.0±0.2) or a PBS solution (Na... + 137mM, K + 2.7mM, Cl - 140mM, PO4 3- (10mM, pH=7.4±0.1). Studies have found that PBS solution has better pH buffering capacity and is suitable for calibration scenarios that are pH sensitive or require a closer approximation to the physiological environment. When preparing 0.9% NaCl or PBS, it must be filtered through a 0.22μm filter membrane to remove microbial and particulate impurities, ensure purity, and avoid introducing interference or disrupting the stability of the low-ζ standard working liquid.

[0016] According to a second aspect of the present invention, the present invention provides a method for preparing a low ζ-point standard working liquid as described above, wherein the suspension a and the ionic liquid b are mixed.

[0017] Further, the mixed solution is allowed to stand at room temperature for 3-10 minutes to obtain the low zeta potential standard working liquid. This time ensures that ion diffusion and the electric double layer reach a new equilibrium state, resulting in a good ionic strength regulation effect of the mixture: the ionic strength instantaneously increases to 135-175 mmol·L⁻¹. -1 Furthermore, the zeta potential compression effect is as follows: the zeta potential is compressed to -5mV to +5mV; the zeta drift is <1mV within 10 hours, which meets the instrument calibration operation time limit. The study found that when the volume ratio of suspension a to ionic liquid b is 1:9, the zeta potential can be finely adjusted to 0±3mV.

[0018] According to a third aspect of the present invention, the present invention provides the application of the low zeta point standard working liquid described above in the calibration of electrophoresis instruments.

[0019] This invention proposes a solution of "buffered stable suspension + double dilution to standard state before use", which separates the two processes of "stable preservation" and "precise activation":

[0020] (1) Stable storage phase (suspension a-buffer stabilizer): First, a weakly positively charged particle suspension a (ζ=+30~+50mV) is prepared and stored in a boric acid buffer system with high pH (pH=8.0-9.5) and low ionic strength. Under these conditions, there is sufficient electrostatic repulsion (Coulomb repulsion) between particles, which effectively inhibits aggregation and sedimentation. The boric acid buffer system has good buffering capacity and chemical stability under weakly alkaline conditions. The low ionic strength reduces the potential impact of salt on particle stability and is beneficial for subsequent centrifugation control. The suspension a can be stably stored for ≥24 months under light-protected conditions at 4℃, with minimal changes in key parameters (ζ drift <2 mV, particle size change <5%), meeting the requirements for long-term storage and transportation.

[0021] (2) Precise activation phase (ionic liquid b - activation solution): When using, mix suspension a with ionic liquid b (physiological saline or PBS) at the specified volume ratio to instantly increase the ionic strength to 135-175 mmol·L. -1 And by compressing the double layer, the ζ value is reduced to 0±5mV;

[0022] (3) The dual-liquid packaging solves the long-term stability problem, avoids the instability problem of directly storing the low ζ system for a long time, and ensures the reproducibility of the value. After restoration, the target low ζ value can be obtained repeatedly.

[0023] The embodiments of the present invention have the following advantages:

[0024] (1) This invention separates “preserving ζ” from “adjusting ζ”, thereby increasing the shelf life of low ζ reference materials from several days to ≥24 months;

[0025] (2) The “dual liquid dispensing + instant activation” mechanism of this invention ensures that the target low ζ value can be obtained easily, quickly (<10 minutes), accurately (target 0±5mV), and repeatably (RSD<5%) each time it is used, thus meeting the requirements of high-precision calibration.

[0026] (3) The materials of this invention are universal, covering a variety of particle types and a large particle size range (20-600nm), covering the commonly used areas from nanomedicines to latex systems;

[0027] (4) The mixing operation of the present invention is simple, requiring only a simple liquid mixing operation, without special equipment or complex steps, and does not rely on expensive or easily degraded surfactants, thus reducing raw material costs and failure risks;

[0028] (5) The reproducibility of the restored ζ value, conductivity and particle size of the present invention is better than 95%, which meets the calibration requirements of ISO 13099-2. Attached Figure Description

[0029] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0030] Figure 1 A flowchart illustrating the preparation of a low zeta potential standard working liquid provided by this invention;

[0031] Figure 2 This refers to the long-term stability data of suspension a provided in Example 1 of the present invention;

[0032] Figure 3 The data provided are for the long-term stability of suspension a in Example 2 of this invention. Detailed Implementation

[0033] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] SiO2 sol: solid content 30wt%, hydrated particle size 100nm, PDI < 0.08, sourced from products independently produced by Beijing Coast Hongmeng Standard Material Technology Co., Ltd.

[0035] NH2-PS suspension: solid content 10wt%, hydrated particle size 100nm, PDI < 0.08, sourced from products independently produced by Beijing Coast Hongmeng Standard Material Technology Co., Ltd.

[0036] Electrophoretic mobility μ e Measurements were performed using a multi-frequency electrophoretic light scattering instrument calibrated with the National Institute of Standards and Technology (NIST) standard reference material SRM 1980 (polystyrene latex suspension); the dielectric constant (ε) and viscosity (η) of the dispersion medium (water) used in the measurements were traced back to the recommended values ​​of the Committee on Data for Scientific and Technical Data (CODATA) and the national viscosity standard, respectively; and the measurements were performed according to the Smoluchowski equation (ζ = (μ...). e *η) / ε), combined with the ε and η values ​​obtained from tracing the source, the measured μ e Convert to ζ potential value.

[0037] Example 1: Preparation of 100nm SiO2 standard material with standard value ζ = -1.2 mV

[0038] This embodiment provides a low zeta potential standard working liquid, the preparation method of which includes the following steps:

[0039] (1) Raw material pretreatment

[0040] Adding 10 mL of SiO2 sol (30 wt% solid content) to 500 mL of ultrapure water yields a solid content of approximately 0.7 wt% and an electrical conductivity < 20 μS·cm. -1 Diluent;

[0041] (2) Preparation of suspension a

[0042] Prepare a solution with a borate concentration of 10 mmol·L -1 Boric acid / borax buffer stock solution: Weigh 0.618 g of boric acid (H3BO3) and 0.954 g of borax (Na2B4O7·10H2O), dissolve in 1 L of ultrapure water, and use 0.1 mol·L⁻¹ -1 NaOH or 0.1 mol·L -1 HCl was finely adjusted to pH=9.00±0.05 (calibrated at 25℃); 10 mL of the diluent from step (1) was added dropwise to obtain SiO2 solid content of about 0.4 wt%, resulting in suspension a. After testing, the ζ of suspension a was +35.5 mV.

[0043] (3) Preparation of ionic liquid b

[0044] To prepare a 0.9% NaCl solution: Weigh 9.00 g of NaCl (analytical grade) and dissolve it in 1 L of ultrapure water. Use a trace amount of 0.1 mol·L⁻¹ NaCl solution. -1 Adjust the pH to 7.00±0.05 with HCl / NaOH (calibrated at 25℃), filter through a 0.22μm filter membrane to remove bacteria and particles, and obtain ionic solution b;

[0045] (4) Preparation of low zeta potential standard working liquid

[0046] The suspension a from step (2) and the ionic liquid b from step (3) are mixed at a volume ratio of 1:9 and allowed to stand at room temperature for 5 min to obtain a low zeta potential standard working liquid.

[0047] The low zeta potential standard working liquid prepared in this embodiment was tested and found to have: ze = -1.2 mV, particle size 102 nm, and PDI = 0.06.

[0048] Example 2: Preparation of 250nm NH2-PS standard material with standard value ζ = -1.2 mV

[0049] This embodiment provides a low zeta potential standard working liquid, the preparation method of which includes the following steps:

[0050] (1) Raw material pretreatment

[0051] Adding 10 mL of NH₂-PS suspension (10 wt% solids) to 200 mL of ultrapure water yields a solids content of approximately 0.6 wt% and a conductivity < 20 μS·cm. -1 Diluent;

[0052] (2) Preparation of suspension a

[0053] Prepare a solution with a borate concentration of 10 mmol·L -1 Boric acid / borax buffer stock solution: Weigh 0.618 g of boric acid (H3BO3) and 0.954 g of borax (Na2B4O7·10H2O), dissolve in 1 L of ultrapure water, and use 0.1 mol·L⁻¹ -1 NaOH or 0.1 mol·L -1 HCl was finely adjusted to pH=9.00±0.05 (calibrated at 25℃); 10 mL of the diluent from step (1) was added dropwise to obtain NH2-PS solid content of about 0.3wt%, resulting in suspension a. After testing, the ζ of suspension a was +41.3 mV.

[0054] (3) Preparation of ionic liquid b

[0055] To prepare a 1×PBS solution, weigh 0.25 g KH₂PO₄, 2.94 g Na₂HPO₄·12H₂O, 8.00 g NaCl, and 0.2 g KCl, dissolve them in 1 L of ultrapure water, and use a trace amount of 0.1 mol·L⁻¹ water. -1 Adjust the pH to 7.00±0.05 with HCl / NaOH (calibrated at 25℃), filter through a 0.22μm filter membrane to remove bacteria and particles, and obtain ionic solution b;

[0056] (4) Preparation of low zeta potential standard working liquid

[0057] The suspension a from step (2) and the ionic liquid b from step (3) are mixed at a volume ratio of 1:9 and allowed to stand at room temperature for 5 min to obtain a low zeta potential standard working liquid.

[0058] The low zeta potential standard working liquid prepared in this embodiment was tested and found to have the following properties: ze = -1.2 mV, particle size 251 nm, and PDI = 0.07.

[0059] Test Example 1: Long-term stability test

[0060] In Example 1, after the suspension a prepared in step (2) was stored at 4°C for 24 months, the ζ value changed from +35.5mV to +34.3mV, and the particle size was 105nm. In Example 2, after the suspension a prepared in step (2) was stored at 4°C for 24 months, the ζ value changed from +41.3mV to +40.8mV, and the particle size was 248nm.

[0061] According to the requirements of JJF1343-2022 "Assignment and Homogeneity and Stability Assessment of Standard Reference Materials", to verify the behavior of the linear model, for synchronous studies, at least 3 observation time points are required; for asynchronous studies, at least 4 observation time points are required, and repeated measurements are necessary. The observation interval can be arranged according to the principle of dense first and sparse later. The number of units selected and the number of repeated measurements should be able to provide a small uncertainty for predicting stability changes. In this study, long-term stability assessments were conducted at 0, 1, 3, 6, 12, 18, and 24 months. Two packages were sampled each time, and one subsample was taken from each package. The measurements were performed in parallel three times. The stability test results of suspension a prepared in step (2) of Example 1 are shown in Table 1 below, and the stability trend diagram is shown in Table 1 below. Figure 2 As shown.

[0062] Table 1. Long-term stability monitoring results of suspension a in Example 1

[0063]

[0064] The long-term stability of suspension a prepared in step (2) of Example 2 was tested according to the above method. The test results are shown in Table 2. Figure 3 As shown:

[0065] Table 2. Long-term stability monitoring results of suspension a in Example 2

[0066]

[0067] The results show that the characteristic values ​​of suspension a provided in this embodiment of the invention do not change significantly within 24 months, and no instability was observed, indicating that the substance has good stability.

[0068] After storing the suspension a obtained in step (2) of Example 1 at 4°C for 24 months, a low ζ potential standard working liquid was prepared according to step (4) and repeated 10 times. The results showed that the mean ζ value was -0.8 mV and the RSD was 4.6%.

[0069] After storing the suspension a obtained in step (2) of Example 2 at 4°C for 24 months, a low ζ potential standard working liquid was prepared according to step (4) and repeated 10 times. The results showed that the mean ζ value was -0.5 mV and the RSD was 4.2%.

[0070] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A low zeta potential standard working fluid, characterized in that, It includes separately packaged and stored suspension a and ionic liquid b, wherein the zeta potential of suspension a is +30 to +50 mV; and the ionic liquid b contains Na. + and / or K + The total ionic strength is 100-200 mmol·L. -1 The volume ratio of the suspension a to the ionic liquid b is 1:4-9.

2. The low zeta potential standard working fluid according to claim 1, characterized in that, The preparation method of the suspension a includes: When hydrated particles with a diameter of 20-600 nm and a PDI < 0.08 are added to ultrapure water, a conductivity of < 50 μS·cm is obtained. -1 Diluent; The diluted solution was added dropwise to a pH of 8.0-9.5 and a borate concentration of 10 mmol·L⁻¹. -1 In the boric acid / borax buffer solution, the particle solid content is controlled at 0.2-2.0 wt%, and the solution is filtered through a 0.22 μm filter membrane.

3. The low zeta potential standard working fluid according to claim 2, characterized in that, The particles are selected from one or more of neutral polystyrene, weakly positively charged amino polystyrene, silica, and γ-alumina.

4. The low zeta potential standard working fluid according to claim 2, characterized in that, The preparation method of the suspension a further includes adding 0.01-0.02 wt% of preservative to the system before filtration.

5. The low zeta potential standard working fluid according to claim 1, characterized in that, The ionic liquid b is a 0.9% NaCl solution or a PBS solution.

6. The method for preparing the low zeta potential standard working liquid according to claim 1, characterized in that, The suspension a and the ionic liquid b are mixed.

7. The method for preparing the low zeta potential standard working liquid according to claim 6, characterized in that, The mixed solution was allowed to stand at room temperature for 3-10 minutes to obtain the low zeta potential standard working liquid.

8. The application of the low ζ-potential standard working liquid as described in claim 1 in the calibration of electrophoresis instruments.

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