A method for determining monovalent and divalent metal ions based on a c18 chromatographic column

Abstract

The application discloses a method for determining monovalent and divalent metal ions based on a C18 chromatographic column, which is used for detecting Na + , K + , Li + , Ca 2+ , Mg 2+ . After being prepared and filtered by a filter membrane, the sample is detected by an Eclipse XDB-C18 chromatographic column, an organic acid-organic phase gradient mobile phase and an ELSD detector; the volume percentage of the organic acid in the water phase of the mobile phase is greater than 0.05%, the flow rate is controlled to be 0.5 mL / min, the column temperature is 25-35 DEG C, and the temperature, carrier gas flow and injection amount of the ELSD are simultaneously limited. The HILIC column with poor reproducibility and high cost is abandoned, the general C18 chromatographic column is used, the monovalent and divalent metal ions can be separated, the separation degree of K + and Mg 2+ reaches 3.04, the retention time RSD of each ion is extremely low, the precision meets the pharmacopoeia standard, the system is acid-resistant, the parameters are adjustable, the reagent is general, and the method is suitable for detecting single standard and mixed metal salt samples.

Description

Technical Field

[0001] This invention relates to the field of metal ion detection, and specifically to a method for determining monovalent and divalent metal ions based on a C18 chromatographic column. Background Technology

[0002] Lithium, sodium, and potassium monovalent metal ions, and calcium and magnesium divalent metal ions, are frequently detected in liquid chromatography (LC), and there is a significant demand for qualitative and separation detection in the field of salt-related product quality analysis. Currently, the mainstream LC separation method for these highly polar metal ions is hydrophilic interaction chromatography (HILIC), which relies on HILIC columns to separate metal ions of different valence states. Theoretically, this chromatographic system can separate Li... + Na + K + Ca 2+ Mg 2+ Effective differentiation is a standard detection method reported in existing literature. However, HILIC columns (polar stationary phase chromatography) have inherent and unavoidable drawbacks. Their packing structure is complex, and their operation is highly sensitive to gradient elution programs, ambient temperature, and ambient humidity. The water film state on the packing surface is easily affected by external conditions, directly causing chromatographic peak distortion, significant retention time drift, and poor reproducibility of parallel sample detection. This makes it difficult to meet the stable and reliable detection requirements under pharmacopoeia standards, limiting the widespread application of this method in routine laboratory batch detection.

[0003] To improve the chromatographic separation of metal ions, existing detection systems generally require the addition of acidic, volatile additives to the mobile phase to regulate the retention behavior and peak shape of metal ions. Different types of acid additives significantly affect the ion elution order and separation effect. Without acid additives, the chromatographic peaks of monovalent and divalent metal ions severely overlap, making separation impossible. Simultaneously, the type of organic phase and the elution method also restrict the separation effect. Using methanol as the organic phase or an isocratic elution mode with a fixed organic phase ratio throughout the process cannot achieve sufficient retention and good separation of monovalent and divalent metal ions, indicating room for optimization in existing mobile phase systems. Furthermore, during the screening of conventional C18 columns, short columns and small-particle-size SB-C18 columns have insufficient retention capacity for metal ions, resulting in decreased resolution. Increased column temperature also disrupts the original separation effect. A mature and complete scheme suitable for metal ion detection has not yet been developed for conventional reversed-phase chromatography.

[0004] In conventional laboratories, C18 reversed-phase chromatographic columns are standard consumables, readily available, easy to use, and stable in operation, offering significant cost and widespread advantages compared to dedicated HILIC columns. However, the industry currently lacks a mature detection method for the simultaneous separation of monovalent and divalent metal ions using ordinary C18 columns. Existing technologies either rely on the poorly reproducible HILIC system or employ insufficiently optimized mobile phases and chromatographic conditions, failing to balance separation performance, method stability, and experimental versatility. Therefore, there is an urgent need for a method based on C18 columns to determine monovalent and divalent metal ions, meeting the laboratory's requirements for low-cost, highly reproducible, and pharmacopoeia-compliant qualitative separation and detection of metal salts. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for determining monovalent and divalent metal ions based on a C18 chromatographic column, comprising the following steps: A solution containing monovalent and divalent ions was prepared, filtered through a filter membrane, injected, and detected using liquid chromatography with an evaporative light scattering detector (ELSD). The chromatographic column used in the liquid chromatography was an Eclipse XDB-C18 column with an inner diameter of 4.6 mm, a length of 250 mm, and a packing particle size of 5 μm.

[0006] As an implementable example, the monovalent ion includes Li + Na + K + At least one of them.

[0007] As an implementable example, the divalent ion includes Ca. 2+ Mg 2+ At least one of them.

[0008] This invention enables efficient separation of Li using a standard laboratory C18 chromatographic column. + Na + K + Monovalent metal ions and Ca 2+ Mg 2+For divalent metal ions, no dedicated HILIC hydrophilic column is required, effectively reducing consumables and detection costs. The method exhibits excellent peak separation for both monovalent and divalent metal ions, with a resolution Rs > 3, far exceeding the pharmacopoeia baseline separation standard of Rs ≥ 1.5, enabling precise qualitative analysis of metal ions with different valence states. Furthermore, this method demonstrates low retention time RSD with parallel injections, excellent reproducibility and precision, meeting pharmacopoeia requirements. For metal salts without UV absorption, a stable peak can be obtained when paired with an ELSD detector, exhibiting symmetrical peak shapes and a stable baseline, suitable for both single-ion and multi-ion mixed samples. The column is resistant to acidic mobile phases, ensuring stable column performance over long-term detection with minimal peak overlap, tailing, or retention time drift.

[0009] The Eclipse XDB-C18 column serves as a separation carrier for monovalent and divalent metal ions. These metal ions form ion pairs with trifluoroacetic acid in the mobile phase and exhibit differentiated retention due to the hydrophobic interaction of the C18 stationary phase. The 250 mm column length extends the reaction time of the components, allowing for the separation of Li... + Na + K + Monovalent ions and Ca 2+ Mg 2+ The time difference in the formation of stable divalent ions, K + With Mg 2 + With a resolution of up to 3.04, the column achieves complete baseline separation and qualitative differentiation of metal ions with different valence states. Thanks to its dual-end-bonding technology, this column exhibits excellent acid resistance, stably adapting to acidic gradient elution systems. The stationary phase is not easily hydrolyzed or lost, and peak shapes are regular and elution patterns are stable during gradient operation. Furthermore, the 250mm column length provides better separation than the 150mm shorter column. The 5μm packing particle size provides moderate column pressure and better ion pair retention than the 1.8μm ultrafine particle size. The universal inner diameter, combined with ELSD, yields symmetrical, non-broadened peaks. The packing particles are uniform, and the column efficiency is stable across batches. Small fluctuations in temperature and humidity do not significantly interfere with the detection results. Precision meets pharmacopoeia requirements, and neither single-ion nor multi-ion mixed samples exhibit overload peak broadening. It is suitable for a wide range of applications and is highly durable.

[0010] As an example of implementation, the molar concentrations of the monovalent and divalent ions are 0.1~1 mol / L.

[0011] As an example of implementation, the filter membrane has a pore size of 0.1-0.3 μm.

[0012] As an feasible example, the mobile phase A of the liquid chromatography is an aqueous phase, which is an aqueous solution of an organic acid, and the mobile phase B is an organic phase.

[0013] Furthermore, the organic acid has a volume percentage in the aqueous phase of >0.05%.

[0014] Furthermore, the organic acid mentioned includes formic acid or trifluoroacetic acid.

[0015] Furthermore, the organic phase includes methanol or acetonitrile.

[0016] The mobile phase was set as a two-phase system consisting of an aqueous organic acid phase A and an organic phase B because of Li + Na + K + Ca 2+ Mg 2+ The ions are highly polar, and a single aqueous phase cannot effectively retain them on a C18 column. A pure organic phase would cause overlapping co-elution peaks. Combining two phases allows for gradient-controlled elution intensity, enabling stepwise separation of ions with different valence states. This invention limits the organic acid volume percentage to greater than 0.05 wt% because the organic acid acts as an ion-pairing reagent; insufficient concentration leads to inadequate ion pair formation, weak ion retention, peak overlap and tailing, and inability to distinguish ions. Formic acid and trifluoroacetic acid are selected as organic acids because they are volatile and compatible with ELSD detectors. They also differ in their effect on controlling the ion elution sequence, allowing for selection as needed. The organic phase is limited to methanol and acetonitrile, both of which are miscible with water and allow for elution capacity adjustment. They are common laboratory reagents, compatible with detector volatility requirements, and facilitate experimental screening and optimization. This mobile phase system can form stable hydrophobic ion pairs with metal ions, solving the problem of non-retention of highly polar metal ions on C18 columns. Combined with gradient elution to widen the ion retention time difference, it achieves pharmacopoeia baseline separation standards.

[0017] As an feasible example, the elution conditions of the liquid chromatography are gradient elution, and the volume ratio of mobile phase A to mobile phase B is (65-97):(3-35) within 0-10 min; that is, within 10 min, the volume percentage of mobile phase B increases from 3% to 35%, and the volume percentage of mobile phase A decreases from 97% to 65%.

[0018] Furthermore, the flow rate of the mobile phase is 0.5 mL / min.

[0019] Furthermore, the column temperature of the chromatographic column is 25-35℃. If the temperature is below 25℃, the viscosity of the mobile phase increases, the mass transfer rate of components slows down, the chromatographic peaks broaden, and the detection cycle becomes longer. When the temperature exceeds 35℃, the molecular motion of ions intensifies, the adsorption and retention capacity of metal ions on the C18 stationary phase decreases, the retention times of monovalent and divalent metal ions shift forward simultaneously, the peak spacing narrows, the resolution is significantly reduced, and the separation effect deteriorates.

[0020] As an example of implementation, the temperature of the nebulizer in the evaporative light scattering detector is 30~40℃.

[0021] As an example of implementation, the temperature of the drift tube in the evaporative light scattering detector is 30~40℃.

[0022] As an example of implementation, the gas flow rate set for the evaporative light scattering detector is 1~2 SLM.

[0023] As an example of implementation, the evaporative light scattering detector is set to an injection volume of 1~10 μL.

[0024] This scheme sets the temperature of the evaporative light scattering detector's nebulizer and drift tube to 30-40℃. Too low a temperature will result in incomplete mobile phase evaporation and increased baseline noise, while too high a temperature will easily decompose metal salt particles and reduce signal response. This temperature range allows for sufficient vaporization of the mobile phase and protects the analyte. The carrier gas flow rate is limited to 1-2 ℃. For SLM, too low a flow rate results in coarse droplets and solvent residue, while too high a flow rate disperses the analyte particles, weakening the peak response. This flow range allows for the formation of uniform, fine droplets, balancing evaporation with complete retention of metal salt particles. The injection volume is controlled between 1 and 10 μL. Too small an injection volume results in a weak signal, while too large an injection volume causes column overload, peak broadening, and decreased resolution. This range balances the detection signal and chromatographic separation performance. The entire ELSD parameter set is adapted to the metal salt ion pair detection system, allowing for complete vaporization of the mobile phase. It relies on metal salt particles to generate a stable and symmetrical scattering chromatographic signal. Each parameter has a reserved adjustment range, allowing for flexible adjustment based on sample concentration. The method precision and reproducibility meet pharmacopoeia standards. Combined with a C18 gradient elution system, it can stably and clearly separate monovalent and divalent metal ions. The overall detection system is stable and reliable.

[0025] Beneficial effects (I) This invention eliminates the need for dedicated HILIC columns, allowing detection to be completed using a general-purpose C18 column, significantly reducing detection costs and lowering the barrier to entry. Current mainstream methods for simultaneous detection of monovalent and divalent metal ions rely on HILIC hydrophilic columns. These columns are specialized consumables, expensive, and highly sensitive to environmental temperature, humidity, and elution gradients, resulting in significant retention time drift and making stable batch detection difficult. This invention uses the standard laboratory-grade Eclipse XDB-C18 column, which is widely available and inexpensive. No modifications to existing liquid chromatography equipment are required, making it suitable for direct use in general quality control and R&D laboratories. Furthermore, it avoids the inherent poor reproducibility of HILIC columns, significantly broadening the applicability of the method.

[0026] (II) This invention can achieve complete baseline separation of multiple metal ions, and the separation performance meets the pharmacopoeia criteria. It utilizes the formation of ion pairs between organic acids and metal ions, combined with a gradient elution program within a specific range, to effectively separate Li... + Na + K+ Monovalent metal ions and Ca 2+ Mg 2+ The retention time difference of divalent metal ions, where K + With Mg 2+ The resolution of mixed components can reach 3.04, which is far higher than the baseline separation requirement of Rs≥1.5 specified in the pharmacopoeia. Each ion chromatographic peak is independent and has no overlap or tailing. It can identify a single metal ion individually or distinguish five-membered mixed ion samples simultaneously, accurately completing the identification of metal ions with different valence states.

[0027] (III) Lithium, sodium, potassium, calcium, and magnesium metal salts have no ultraviolet absorption and cannot be detected by conventional ultraviolet detectors. This invention uses formic acid and trifluoroacetic acid in combination with methanol and acetonitrile to construct the mobile phase. All reagents have good volatility and can be completely vaporized in the evaporative light scattering detector, with no solid residue contaminating the optical path. The baseline noise is low and the peak shape is symmetrical and regular. At the same time, the chromatographic column, column temperature, carrier gas, and injection volume are all set within reasonable and controllable ranges. After multiple parallel injections, the retention time and peak area RSD values ​​of each ion are low, and the detection results are stable and reliable, fully meeting the precision requirements of pharmacopoeia compliance testing.

[0028] (iv) This invention defines a wide range of feasible parameters for gradient mobile phase ratio, liquid phase flow rate, column temperature, nebulizer and drift tube temperature, ELSD carrier gas flow rate and injection volume. Operators can make minor adjustments to the parameters according to the sample concentration, the age and wear of the column, and laboratory environmental conditions. Good separation effect can be maintained within the range. It is suitable for the detection of low-concentration trace standards and can also handle high-concentration mixed metal salt samples. It has strong versatility and sufficient optimization space.

[0029] (v) The Eclipse XDB-C18 column used in this invention adopts a double-end capped acid-resistant bonding process, which can withstand the long-term stability of acidic mobile phase with a volume concentration of more than 0.05% trifluoroacetic acid, effectively suppressing the peak tailing problem caused by stationary phase hydrolysis and silanol exposure; the second half of the gradient elution increases the proportion of organic phase, which can continuously flush out the metal salt impurities remaining in the column, avoid the accumulation of impurities and contamination of the packing material, and there is no obvious baseline drift when continuously injecting batches, reducing the frequency of column replacement and further reducing the overall cost of long-term detection. Attached Figure Description

[0030] Figure 1 The image shows the test results for Example 1.

[0031] Figure 2 The image shows the test results for Example 2.

[0032] Figure 3 Figure showing the test results for Example 3 (Condition 1) Figure 4 The image shows the test results for Example 3 (Condition 2).

[0033] Figure 5 The image shows the test results for Example 3 (Condition 3).

[0034] Figure 6 Figure showing the test results for Example 4 (Condition 1) Figure 7 The image shows the test results for Example 4 (Condition 2).

[0035] Figure 8 The image shows the test results for Example 4 (Condition 3).

[0036] Figure 9 The image shows the test results for Example 5.

[0037] Figure 10 The image shows the test results for Example 6. Detailed Implementation

[0038] Example 1 (Determination of 2 types of ions) This example provides a method for determining monovalent and divalent metal ions based on a C18 chromatographic column, including the following steps: Accurately weigh 5 mmol KCl and 5 mmol MgCl2 into a 50 mL volumetric flask, dissolve and dilute to the mark with ultrapure water, and shake well to obtain a KCl solution with a concentration of 0.1 mol / L. + and Mg 2+ The mixed solution was filtered through a 0.22 μm pore size filter membrane, injected, and detected using liquid chromatography with an evaporative light scattering detector (ELSD).

[0039] The liquid chromatography column used is an Eclipse XDB-C18 column with an inner diameter of 4.6 mm, a length of 250 mm, and a packing particle size of 5 μm. The mobile phase A is a 0.1% (v / v) trifluoroacetic acid aqueous solution, and phase B is acetonitrile. The elution conditions are gradient elution: within 0–10 min, the volume percentage of mobile phase B increases from 3% to 35%, while the volume percentage of mobile phase A decreases from 97% to 65%. The flow rate is 0.5 mL / min, and the column temperature is 35 °C.

[0040] The evaporative light scattering detector is an Agilent 1290 Infinity II ELSD (G4260B), the nebulizer temperature is 35°C, the drift tube temperature is 35°C, the gas flow rate set for the evaporative light scattering detector is 1.6 SLM, and the injection volume is 5 μL.

[0041] The liquid phase spectrum measured in this example is as follows: Figure 1 As shown in the figure, the monovalent ion K+ and divalent ions Mg 2+ The Rs (resolution) of the two peaks was 3.04, which meets the standard of the 2025 edition of the Chinese Pharmacopoeia.

[0042] Example 2 (Determination of 5 Ions) This example provides a method for determining monovalent and divalent metal ions based on a C18 chromatographic column, including the following steps: Accurately weigh 5 mmol each of NaCl, KCl, LiCl, CaCl₂, and MgCl₂ into 50 mL volumetric flasks, dissolve and dilute to the mark with ultrapure water, and shake well to obtain a NaCl solution with a concentration of 0.1 mol / L. + K + Li + Ca 2+ Mg 2+ The mixed solution was filtered through a 0.22 μm pore size filter membrane, injected, and detected using liquid chromatography with an evaporative light scattering detector (ELSD).

[0043] The liquid chromatography column used is an Eclipse XDB-C18 column with an inner diameter of 4.6 mm, a length of 250 mm, and a packing particle size of 5 μm. The mobile phase A is a 0.1 wt% trifluoroacetic acid aqueous solution, and phase B is acetonitrile. The elution conditions are gradient elution: within 0–10 min, the volume percentage of mobile phase B increases from 3% to 35%, while the volume percentage of mobile phase A decreases from 97% to 65%. The flow rate is 0.5 mL / min, and the column temperature is 35 °C.

[0044] The evaporative light scattering detector is an Agilent 1290 Infinity II ELSD (G4260B), the nebulizer temperature is 35°C, the drift tube temperature is 35°C, the gas flow rate set for the evaporative light scattering detector is 1.6 SLM, and the injection volume is 5 μL.

[0045] The liquid phase spectrum measured in this example is as follows: Figure 2 As shown in the figure, the retention time of the monovalent ion peak is about 5.1 min, and the retention time of the divalent ion peak is about 5.5 min.

[0046] Example 3 (Mobile Phase Screening Experiment) Based on the detection system of Example 1, this invention explores the influence of different flow rates on the detection results, specifically setting up three sets of comparative experiments: 1. Phase A is a 0.025% (volume percentage) trifluoroacetic acid aqueous solution, and Phase B is acetonitrile. The elution conditions are isocratic elution, and the volume ratio of Phase A to Phase B is 97:3 (see experimental results for details). Figure 3 ); 2. Phase A is a 0.025% (volume percentage) trifluoroacetic acid aqueous solution, and phase B is acetonitrile. The elution condition is gradient elution. Within 0~10 min, the volume percentage of mobile phase B increases from 3% to 35%, and the volume percentage of mobile phase A decreases from 97% to 65%. 3. Phase A is a 0.025% trifluoroacetic acid aqueous solution, and Phase B is methanol. The elution condition is gradient elution. Within 0 to 10 minutes, the volume percentage of mobile phase B increases from 3% to 35%, and the volume percentage of mobile phase A decreases from 97% to 65%.

[0047] The experimental results of the above tests show that condition 2 is the optimal mobile phase condition.

[0048] Example 4 (Organic Acid Screening Experiment) Based on the detection system of Example 1, this invention investigates the influence of the selection of organic acid in phase A and its mass concentration on the detection results, specifically setting up three sets of comparative experiments: 1. Mobile phase A was a 0.025% trifluoroacetic acid aqueous solution (experimental results are detailed in [link to experimental results]). Figure 6 ); 2. The mobile phase A was a 0.025% formic acid aqueous solution (experimental results are detailed in [link to experimental results]). Figure 7 ); 3. No acid was added, i.e., mobile phase A was pure water (see experimental results for details). Figure 8 ).

[0049] Test data show that when trifluoroacetic acid is used as the organic acid additive in phase A, the elution peak of divalent ions is later than that of monovalent ions; when formic acid is used as the organic acid additive in phase A, the elution peak of divalent ions is earlier than that of monovalent ions; and the separation effect is poor when no additive is added. Therefore, the addition of additives plays a crucial role in the separation of metal ions.

[0050] Based on the above screening results, the effect of organic acid concentration on separation efficiency was tested. Trifluoroacetic acid, which showed better separation performance, was selected as the additive, and the amount of trifluoroacetic acid was adjusted to further optimize the chromatographic conditions. Experiments were conducted using 0.025% trifluoroacetic acid aqueous solution (phase A) - acetonitrile (phase B), 0.05% trifluoroacetic acid aqueous solution (phase A) - acetonitrile (phase B), and 0.1% trifluoroacetic acid aqueous solution (phase A) - acetonitrile (phase B) as the mobile phase. The results showed that adding 0.1% trifluoroacetic acid aqueous solution to the aqueous phase resulted in better separation.

[0051] Example 5 (Column Screening Experiment) The specific implementation method in this example is the same as in Example 1, except that the chromatographic column is an SB-C18 chromatographic column with an inner diameter of 4.6 mm, a length of 150 mm, and a packing particle size of 1.8 μm.

[0052] Comparing the experimental results of Example 1 and Example 5, the sample retention and separation of Example 5 were slightly worse than those of Example 1.

[0053] Example 6 (Column Temperature Screening Experiment) The specific implementation method in this example is the same as in Example 1, except that the column temperature of the chromatographic column is 40°C.

[0054] Comparing the experimental results of Example 1 and Example 6, the sample retention and separation of Example 6 were slightly worse than those of Example 1.

[0055] Example 7 Prepare 0.1 mol / L Na + K + Li + Ca 2+ Mg 2+ For a single sample solution, referring to the optimal chromatographic conditions in Example 1, the sample was injected in parallel three times, and the RSD values ​​of the retention time of the five sets of data were calculated (Table 1). The results show that the analytical method has high precision.

[0056] Table 1

[0057] The above experimental results show that monovalent metal ions Na + K + Li + Retention times were concentrated in the range of 5.025–5.061 min, divalent Ca 2+ Mg 2+ The peak values ​​were concentrated between 5.526 and 5.531 min, with a clear separation between the two types of ions, allowing for direct differentiation between monovalent and divalent metal ions, demonstrating excellent separation performance. The RSD of retention times for all ions was well below 0.2%: Li + The three peaks were completely identical, and the RSD was 0; Ca 2+ K + Mg 2+ The RSD is less than 0.04%; Na + The highest RSD was only 0.101%. This indicates that the chromatographic conditions were stable, with minimal fluctuations in the peak positions of the components during continuous injection, demonstrating high precision and meeting the pharmacopoeia requirements for method stability.

Claims

1. A method for determining monovalent and divalent metal ions based on a C18 chromatographic column, characterized in that, Includes the following steps: Solutions containing monovalent and divalent ions were prepared, filtered through a filter membrane, and detected by liquid chromatography with an evaporative light scattering detector (ELSD). The chromatographic column used in the liquid chromatography was an Eclipse XDB-C18 column. The monovalent ions include Li + Na + K + At least one of them; The divalent ions include Ca 2+ Mg 2+ At least one of them.

2. The determination method according to claim 1, characterized in that, The mobile phase A of the liquid chromatography is an aqueous phase, which is an aqueous solution of an organic acid; the mobile phase B is an organic phase.

3. The determination method according to claim 2, characterized in that, The organic acid has a volume percentage in the aqueous phase of >0.05%.

4. The determination method according to claim 3, characterized in that, The organic acids mentioned include formic acid or trifluoroacetic acid.

5. The determination method according to claim 2, characterized in that, The organic phase includes methanol or acetonitrile.

6. The determination method according to claim 2, characterized in that, The elution conditions for the liquid chromatography are gradient elution, with a volume ratio of mobile phase A to mobile phase B of (65-97):(3-35) within 0-10 min.

7. The determination method according to claim 1, characterized in that, The column temperature of the chromatographic column is 25-35℃.

8. The determination method according to claim 1, characterized in that, The temperature of the atomizer in the evaporative light scattering detector is 30~40℃.

9. The determination method according to claim 1, characterized in that, The temperature of the drift tube in the evaporative light scattering detector is 30~40℃.

10. The determination method according to claim 1, characterized in that, The gas flow rate set for the evaporative light scattering detector is 1~2 SLM.

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