What do you know about the overload for HPLC columns?
Column overload is a problem we all try to avoid when doing analytical method development because column overload will cause distortion of peak shapes and low theoretical column numbers.
Especially when we are doing method development, it is often encountered that a group of analytes contains both a high concentration of the main component (the active ingredient of the drug) and a very low concentration of impurities (process impurities, degradation impurities). In order to take into account the sensitivity of the low-concentration impurities, the injection volume needs to be increased or the injection concentration needs to increase to ensure the accuracy of quantification. Therefore, sometimes it will cause the overloading of the column.
Mass Overload
Mass overload, also known as concentration overload, is a phenomenon that occurs when the stationary phase near the peak band of a spectrum is saturated due to a high injection concentration in a small volume (volume not overloaded), i.e., the injection of a component exceeds the loading capacity of the column and overload occurs. This is generally manifested as a trailing peak shape and low column efficiency.
In the case of a small volume injection, if the mass is not overloaded, as the sample volume increases (a<b<c), the spectral peak area increases accordingly (Aa<Ab<Ac), but the peak width does not increase accordingly. The spectral peaks also appear normal.
As the amount of sample continues to increase, the sample entering the column reaches saturation and the spectrum is distorted.
From Figure 2, it can be seen that the peak width of the spectral peak increases as the sample feed mass continues to increase, and eventually becomes a right triangle shape, while the theoretical tower number of the spectral peak also continues to decrease. Usually, for a column with an inner diameter of 4.6 mm, the sample loading capacity is <50 μg. If the method transfer to other columns is done, the injection volume needs to be adjusted to prevent overloading.
The sample loading is mainly related to the nature of the stationary phase of the column and the analyte to be analyzed. Firstly, different stationary phase types and brands can cause large differences in sample loading. Secondly, the nature of the analytes to be analyzed also directly affects the column sample loading. For example, basic compounds in an ionic state can be easily overloaded, not only to the order of <50 μg, but sometimes even 1 μg can cause overloading. Therefore, in the conventional hydrophobic reversed phase, the molecular state of basic compounds has a higher sample loading than the ionic state of basic compounds. The reason for this is that the mutual repulsion between ionic states makes it easier to saturate the stationary phase surface with the component to be analyzed and causes overload. Also, the positively charged basic compounds in the ionic state are more likely to have secondary interactions with the unbonded active silica hydroxyl groups in the stationary phase. The simplest way to address the mass overload problem is to reduce the injection volume.
In addition, overloading in mixed sample feeds can lead to changes in peak retention times.
For example, in Figure 3, A' and B' are the retention separations for the AB component alone, and A, and B, are the retention separations for the mixed sample. The overloaded B remains stable regardless of whether the sample is fed alone or mixed, while A has an earlier retention time when mixed due to the presence of excess B.
Although the overload of each component is relatively independent, the separation problems are caused during the mixed sample injection. This is due to the influence of the environment on the components. Among other things, the overloading of a component is likely to cause a change in retention time during the mixed injection relative to that during the separate injection.
Volume Overload
Too large an injection volume usually results in a wide flat-headed peak.
As can be seen from Figure 4, the peak width gradually becomes wider and the peak separation decreases as the injection volume increases.
In addition, the volume overload also affects the peak retention time, with the volume overload having a greater effect on the components with short retention time and a smaller effect on the peaks with later outflow.
The dashed line in Figure 5 shows the normal volume injection, and the solid line chromatogram shows the chromatogram after volume overload.
So, how to choose the proper injection volume?
The maximum volume of sample injection allowed at the optimal flow rate is as below.
Where: L is the column length (mm), dc is the inner diameter (mm), dp is the particle size (μm)
The commonly used parameters of each specification column are as follows.
| Column Inner-diameter/mm | Column Length/mm | Particle Size/um | Maximum injection volume /uL |
| 4.6 | 250 | 5 | 100 |
| 4.6 | 250 | 3 | 80 |
| 4.6 | 150 | 5 | 80 |
| 4.6 | 150 | 3 | 60 |
| 4.6 | 50 | 3 | 35 |
| 2.1 | 100 | 1.8 | 8 |
Although the data in the above table is calculated strictly according to the formula, this data is not absolute.
Chromatography is an empirical science that need more practice.