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Nanjing Aikon Quanxin Analysis and Testing Center is a comprehensive third-party testing institution that integrates testing and analytical technical services. The center currently possesses a series of large-scale analytical instruments, including a high-resolution 400M nuclear magnetic resonance spectrometer, a full set of Agilent series liquid chromatography-mass spectrometers, gas chromatography-mass spectrometers, high-performance liquid chromatographs, gas chromatographs, and various physicochemical analyzers.



Under certain chromatographic operating conditions, the column efficiency of a chromatographic column can be measured by the number of theoretical plates or the height equivalent to a theoretical plate. Generally, the greater the number of plates or the smaller the plate height, the better the separation efficiency of the column. We will demonstrate through experiments how column efficiency is evaluated.



How to evaluate column efficiency?



1. Experimental Instruments and Reagents



Instruments: High-performance liquid chromatograph (with autosampler or equipped with a microsyringe), analytical balance.



Reagents: Benzene, naphthalene, biphenyl (all analytical grade), methanol (chromatographic grade), purified water.



2. Experimental Steps



Chromatographic conditions: Column: C18, 4.6×150mm, 5μm; Mobile phase: methanol-water (80:20, v/v);



Detection wavelength: 254nm; Flow rate: 1mL·min⁻¹; Column temperature: 30°C; Injection volume: 10μL.



3. Operating Steps



(1) Precisely prepare three reference solutions, each 10mL, containing benzene, naphthalene, and biphenyl at concentrations of approximately 1mg·mL⁻¹ each.



(2) Precisely pipette 2mL of each reference solution into a 10mL volumetric flask, dilute with the mobile phase, bring to volume, and mix well to obtain a mixed reference solution containing benzene, naphthalene, and biphenyl.



(3) Operate under the above chromatographic conditions, inject the sample, and record the chromatogram.

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Calculate the theoretical plate number for each chromatographic peak and the resolution between peaks.



How to improve HPLC column efficiency?



To enhance the efficiency of liquid chromatography, the following approaches can be adopted. Below are several internationally popular methods for measuring and calculating column efficiency values.



1. Methods to Improve Liquid Chromatography Column Efficiency



(1) Reduce the flow rate of the mobile phase, though this will extend the analysis time.



(2) Decrease the amount of stationary phase, but this also reduces the sample loading capacity of the column.



(3) Reduce the particle size of the stationary phase, but not excessively, as it may decrease the permeability of the column.



(4) Use a low-viscosity mobile phase to facilitate rapid mass transfer, though this may not be suitable for multi-component analysis.



(5) Appropriately increase the column temperature to reduce the viscosity of the mobile phase, but this may also lower column efficiency and resolution.



(6) Minimize the volume of stagnant mobile phase, though this may increase the flow rate of the mobile phase.



As shown above, various factors in chromatographic analysis are interconnected and mutually restrictive. Only by tracking and calculating column efficiency values and continuously researching and practicing analytical methods can optimal working conditions be identified.



2. Considerations for Tracking and Calculating Column Efficiency Values



It should also be noted that column efficiency values alone are not sufficient to predict column performance under all conditions. For most chromatographers, column performance refers to the ability of the column to achieve specific separations, and high column efficiency does not guarantee this separation capability.



Regardless of the specific testing method used, several parameters can affect the determination of column efficiency. These include the composition and viscosity of the eluent, its linear velocity, the solute used for plate number measurement, temperature, column length, packing method, particle size, and the chosen measurement and calculation methods. The measurement and calculation methods play a significant role in determining column efficiency values.



3. Methods for Measuring and Calculating Column Efficiency Values



Since chromatographic peaks are assumed to follow a normal distribution of sample concentration in the mobile and stationary phases, the peak shape is often treated as a Gaussian curve to calculate the theoretical plate number. Thus, the formula for calculating column efficiency (in terms of theoretical plates, n) is conventionally defined as:



where tR is the retention time of the chromatographic peak; σ² is the variance of the peak measured in time units; a is a constant related to the peak height (measured from the baseline of the peak width); ωb is the peak width, representing the distance between the two points where tangents drawn at the inflection points on either side of the peak intersect the baseline.



4. Conclusion



If chromatographic peaks were truly Gaussian, all calculation methods would yield the same results. However, even with relatively ideal instruments and solvents that tend to produce symmetric peaks, non-Gaussian peak shapes can occur due to channels or voids in the column. Therefore, different calculation methods may yield significantly different n values. Peaks deviating from the Gaussian model are typically described as "fronting" or "tailing." For such peaks, measurements taken higher up the peak will yield larger theoretical plate numbers (with lower accuracy).



In many cases, chromatographers need column efficiency values that reflect the entire peak shape (including tailing), while also requiring good peak symmetry to ensure quantitative reproducibility. In such scenarios, calculation methods sensitive to chromatographic peak asymmetry are suitable. If the goal is to monitor column efficiency from initial use to the end of its lifespan, any of the above methods can be used, and the simplest method should be selected.