Tips for Achieving Good Peak Shapes During HPLC Analysis

Even for the same HPLC column types, separation performance and method robustness depend on column usage. Here, we present some tips for obtaining better results with chemically bonded silica gel columns, which are the most widely used columns today. Generally, sharp peaks are preferred in chromatography because broad peaks can easily overlap with one another. Additionally, since peak area is proportional to concentration, the narrower the peak, the higher the apex. This is important for sensitivity.

1-1. Minimizing Sample Adsorption

When analytes adsorb onto the stationary phase, peak shapes deteriorate and often become asymmetric. When such a situation occurs, measures such as using an end-capped column must be taken. [caption id="attachment_106765" align="aligncenter"] Figure 1-1. Comparison of elution of a metal chelator[/caption] [caption id="attachment_106766" align="aligncenter"] Figure 2-2. Main causes of adsorption[/caption]  

Cause 1: Adsorption Through Electrostatic Interaction

When ionic functional groups, such as silanol groups SiO, remain on the stationary phase, electrostatic interaction can occur. If this occurs in reverse phase mode, it can cause peak shape distortion.

Cause 2: Adsorption Through Metal Coordination

The addition of metals to the stationary silica can lead to adsorption of metal chelators present in the sample. This adsorption can sometimes prevent the elution of target compounds.

1-2. Selection of Smaller Particles

Smaller particles provide higher column efficiency even for the same stationary phase. However, it must not be forgotten that smaller particles create higher column pressure. For this reason, it is important to keep the system pressure resistance under control. [caption id="attachment_106769" align="aligncenter"] Figure 1-3. Relationship between linear velocity and column pressure[/caption]  

1-3. Effect of Flow Rate

Peak widths depend on the linear velocity of the mobile phase and each column has an optimal velocity. To obtain sharp peaks, a flow rate that provides a linear velocity close to the optimum is desired. [caption id="attachment_106770" align="aligncenter"] Figure 1-4. Van Deemter curves of ODS columns[/caption]  

1-4. Minimizing Dead Volume

If there is dead volume between the tubing and the column, peaks deteriorate. This also occurs when the relative volume of tubing to the column is large. Columns with small internal diameter (ID) or short columns are easily affected. Using longer columns is one solution to reduce the effect of dead volume. Figure 1-7 shows a comparison between a 50 mm long column with 2 µm particle size and a 75 mm long column with 3 µm particle size. The reason longer column length allows better peaks even with larger particle columns is the reduced effect of dead volume.

1-5. Pore Size of Silica Particles

For compounds with molecular weight greater than 5000, sometimes using particles with large pores is necessary to obtain good peak shapes. For example, Inertsil WP 300 C18 is a good choice for these compounds.

1-6. Fast Elution

Generally, the longer the time from injection to detector, the more analytes spread and this results in broader peak shapes. Therefore, sensitivity can be improved by adopting analytical conditions that allow for short analysis. Increasing column durability, shortening column length and changing mobile phase composition are among these methods. However, it must not be forgotten that these methods can worsen separation.

1-7. Sample Solvent Composition

If there is an incompatibility between the sample solvent and the mobile phase, peak shapes can deteriorate. Because the sample solvent acts as the mobile phase immediately after injection. This has a significant effect on peak shapes when the sample solvent has higher elution strength. In this case, changing the composition of the sample solvent or the injection volume can improve peak shapes.

Reference https://www.glsciences.com/technique/technique_data/lc/usage_of_hplc_1/column1.html

   

Doğan Özdemir

Sales Engineer GLSciences Product Manager Ant Teknik Cihazlar