Reasons for the Failure of High Performance Liquid Chromatography Column
As an important part of high performance liquid chromatography, the chromatographic column’s performance affects the result directly. It is necessary to understand well and know why there would be failures. Therefore, HAWACH has summarized 7 key failures and will introduce the reasons for the failure of high performance liquid chromatography column in the following content.
1. Poorly packed column
After a short time, the poorly packed chromatographic columns will have the problem of packed bed compression, making the column head collapse and a sudden drop in column efficiency. Usually, the initial evaluation indicators( for instance, plates number and the asymmetry factor) cannot characterize the stability of the chromatographic column bed well.
2. Reduced column efficiency
It may be caused by contamination of the chromatographic column, partial blockage of the filter, and dead volume in the chromatographic column. For example, the mobile phase pH value or improper composition causes the loss of the stationary phase. The rapid change of the mobile phase causes physical damage to the stationary phase, and mechanical vibration causes cracks in the stationary phase, and the column bed shrinks or dries up. It may also be a problem with the instrument connection. Carefully check whether the injector, detector, tubing, guard column, and online filter are connected properly, or the chromatographic column is not well-balanced, and the injection volume is too large.
Start from the following aspects to improve the column efficiency.
(1) Decrease the mobile phase’s flow rate, but it will cause the analysis time longer.
(2) Reduce the amount of stationary phase, 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. Otherwise, the permeability of the column permeability will decrease too.
(4) Facilitate rapid mass transfer with a low-viscosity mobile phase, 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 will also decrease.
(6) Minimize the volume of the stagnant mobile phase, but speed up the flow rate of the mobile phase.
3. Column pressure factor
If the column pressure is too high, it may be clogged by particles. It is caused by column bed expansion, irreversible adsorption, bacterial growth, etc. It may also be a system back pressure problem, such as clogged damper, clogged sampler, clogged tubing, or connection; the in-line filter is not clean, the pressure sensor is inaccurate, etc.
It will cause poor peak shape and reduced column efficiency if there are sudden mechanical impacts, pressure fluctuations, or temperature changes during the use of HPLC columns. To reduce column damage caused by pressure fluctuations, operators can use well-packed chromatographic columns and operate at lower column pressures.
4. Strong retention of sample components
It will affect the service life of the HPLC columns if the column head packing absorbs strongly adsorbed sample components. For complicated samples, it will cause contamination of the stigma easily and there are fewer problems for the relatively pure samples. In chromatographic column applications, usually, the splitting of chromatographic peaks or severe tailings are manifestations of the adsorption of strongly retained contaminants at the head of the column.
5. Slow column balance
The common reason for the slow equilibrium of the column is that the components have strong adsorption to the column in the old or new mobile phase, or small/zero concentration in the new mobile phase. The mobile phase contains an amine modifier; the mobile phase contains an ion pair reagent; a silica gel column; and the mobile phase contains tetrahydrofuran. Consider using the column for a special method. When not in use, the column should be folded down, filled with an appropriate solvent or mobile phase, sealed for storage, and no other analysis is required.
6. Broadening of the peak
The column itself is degraded during use, and the efficiency of the column is gradually reduced. Extra-column peak width effect. A good column used in another liquid chromatography system caused a decrease in the number of plates, indicating that the new system has a large extra-column peak width effect. Chemical effects are mostly caused by the interaction between the mobile phase and the stationary phase. Changing the mobile phase can improve the broad peaks.
7. Poor repeatability, no peaks, low recovery
It may be that the chromatographic column is contaminated, the mobile phase pH value or composition is not suitable to cause the loss of the stationary phase, the sample solvent is different or the sample itself is unstable, the stationary phase is too polar or the mobile phase is too weak or non-specific adsorption occurs. It is also possible that the equilibration time in the gradient experiment is insufficient, the temperature fluctuates, the mobile phase composition changes, the sample solvent is different, the sample stability is not good, the method development is not good, the pH of the buffer is inappropriate or the buffer capacity is insufficient.
