10. What affects separation?

Separation of the compounds on the stationary phase (column) is influenced by several factors. We will discuss some below.

 

1. Particle Size Effect

The size of the column packing particles has an effect on the separation. The smaller the particles, the larger the surface area of the packing material is – this results in more interactions between the sample compounds and the stationary phase typically resulting in a better resolution. As you can see below, smaller particles (1.9um in diameter) result in thinner and better-separated peaks compared to larger particles (5um in diameter). It is intuitive to use smaller particle packing, however, this comes at the expense of significantly higher pressures due to the column being more tightly packed. This also results in longer retention times, hence small particle columns tend to operate at higher pressures and lower flow rates. To compensate for this fact and to shorten the analysis time, these columns tend to be shorter (for instance 100mm instead of 150mm).  

 

2. Column Length Effect

Typically, longer columns yield better resolution as the compounds spend more time interacting with the packing material in the column. This common knowledge is shown below where it can be observed that longer column results in a better peak resolution.

 

3. Solvent Effect

Mobile phase composition has an effect on sample partitioning between the mobile and stationary phase. Solvent impacts many things in the analysis, including retention time, pressure (i.e. water is more viscous than acetonitrile resulting in much higher pressure). Solvent with its polarity can block active sites on the stationary phase lowering the interactions between the stationary phase and analyzed compounds – this results in a lower resolution; or can interact directly with the sample compounds forming bonds with these compounds more strongly than the stationary phase does.

 

4. pH Effect

Mobile phase pH has an effect on elution of some compounds – especially acids and basis. Below is an example where analyzed acid elutes very quickly at pH 7.0 in the form of acetate; while at pH 3.0 it exists in the acidic form and elutes late in the analysis.

 

5. Temperature Effect

Column temperature often has an effect on the retention of compounds in the chromatographic column. As an example below, peaks 11,12, 13 are clearly separated at 40°C as opposed to 20°C, while at 60°C  paeks 11 and 12 do not resolve at all. 

As can be seen above, the chromatographic method design takes into account all these factors, and many things are considered, such as column packing material chemistry, particle size, column length and internal diameter, mobile phase composition and pH, analysis temperature, gradient vs. isocratic flow, flow rate, detector settings (i.e. wavelength).