The column efficiency of a chromatographic column is an important index to evaluate chromatographic performance. Whether the mixture can be separated in the chromatographic column depends not only on the selection of the appropriate stationary phase but also on the chromatographic operating conditions and the packing status of the chromatographic column. Under certain chromatographic operating conditions, the column efficiency of a chromatographic column can be measured by the number of theoretical plates or the height of theoretical plates. Generally speaking, the more the number of trays, or the smaller the tray height, the better the separation efficiency of the chromatographic column. Today, HAWACH will discuss how to improve HPLC column efficiency.
To improve the efficiency of liquid chromatography can start from the following aspects. 1. Methods to improve the column efficiency of HPLC column (1) Reduce mobile phase flow rate, but the analysis time will be increased. (2) The amount of stationary phase is reduced, but the load of the sample in the chromatographic column is also reduced. (3) Reduce the particle size of the stationary phase, but not too much, the permeability of the chromatographic column will also decrease after too much. (4) The mobile phase with low viscosity is used to facilitate the rapid mass transfer, but it is not conducive to multi-component analysis. (5) Properly increasing the column temperature can reduce the viscosity of the mobile phase, but the column efficiency and resolution also decrease. (6) Minimize stagnant mobile phase volume, but the mobile phase flow rate speed will increase.
Many factors are interrelated and restricted in the chromatographic analysis process. Only through the tracking and calculation of the column efficiency value, and the continuous research and practice of our own analysis methods, can we find the best working conditions.
2. Problems that should be paid attention to in the tracking and calculation of the HPLC column efficiency value We should also remember that column efficiency values are not sufficient to predict column performance under all conditions. For most chromatographers, column performance refers to the ability of a column to be used for a particular separation, and high efficiency alone does not guarantee this separation ability. Regardless of the particular test method used, there are several parameters that affect the determination of column efficiency. These parameters include the composition and viscosity of the eluent and its linear flow rate, the solute used to determine the number of plates, temperature, column length, packing method, particle size, and the measurement and calculation method chosen. The measurement and calculation methods play a great role in the determination of the column efficiency value.
3. Several methods of measuring and calculating HPLC column efficiency Because the chromatographic peak is the sample band distribution obtained by assuming that the sample concentration is normally distributed in the mobile phase and stationary phase, the chromatographic peak shape is often regarded as a normal curve to calculate the number of theoretical plates. Therefore, the formula for calculating the column efficiency (in the unit of theoretical plate number n) is customarily defined as N depending on the type, properties (particle size, particle size distribution, etc.) of the stationary phase, packing conditions, column length, type and flow rate of the mobile phase, and the properties of the substances used to measure the column efficiency.
In the formula, tR is the retention time of the chromatographic peak; σ2 is the deviation of the measured chromatographic peak in units of time; a is a constant related to the peak height (measured from the baseline at which the peak width is measured); ωb is the peak width, which represents the distance between the two points where the tangent line between the apex of the chromatographic peak and the inflection points on both sides of the chromatographic peak and the baseline of the peak bottom intersect.
If a chromatographic peak is truly normal, then each calculation method will yield the same result. However, even with ideal instruments and solutes that tend to produce symmetrical peaks, non-normal peaks can occur due to slots or voids in the column. Therefore, different calculation methods will result in different n values. A peak shape that deviates from a normal model is often referred to as “forward” or “tail”. For these peak types, the higher the peak is measured, the larger (and less accurate) theoretical plate values are calculated.
In many cases, chromatographers need a column efficiency value that reflects the entire peak shape (including tailing), and in order to ensure quantitative repeatability, chromatographic peak symmetry is also required. At this time, the calculation method is most sensitive to the asymmetry of the chromatographic peak. If the purpose is simply to monitor column performance from initial use to end of life, any of the above methods will do, and the easiest method should be chosen.