Distinguishing Key Time Concepts in HPLC Analysis

In everyday chromatography analysis, the standard methods you refer to and the chromatography software you use often involve various time concepts such as retention time, adjusted retention time, dead time, relative retention time, and delay time. While these concepts all seem to be related to time, they are quite different, and some aren't related to time at all.

Today, let's review some of the common time concepts in chromatography analysis.

1. Retention Time :

This refers to the time elapsed from the injection of the sample to the appearance of the peak of a specific component in the chromatogram. It is commonly measured in minutes (min). In the example below, the retention time of the component is 10.0 minutes.

In chromatography analysis, retention time is primarily used for qualitative analysis to identify target components. Retention time can vary within a certain range due to factors such as the concentration and type of buffer salts in the mobile phase, the pH of the mobile phase, the ratio of components in the mobile phase, column temperature, and matrix effects. Small variations in these factors, especially for projects sensitive to detection conditions or when analyzing complex samples, can affect the separation of adjacent components.

In preparative chromatography, the concept of retention time is less utilized because samples are typically loaded in excess, leading to peak shapes such as tailing or fronting peaks. These peaks often exhibit chromatographic behaviors that deviate from linear ranges. Additionally, the extrusion effect of adjacent components further diminishes the guiding significance of retention time in preparative chromatography.

2. Dead Time  :

This refers to the time taken for the mobile phase (or solvent) to pass through the chromatographic column, specifically for components that are not retained by the stationary phase of the column. It is represented by t0. In the example below, the dead time is 1.4 minutes.

1. Adjusted Retention Time 

This term refers to the additional time a component spends in the column due to its dissolution (or adsorption) in the stationary phase compared to components that do not dissolve (or adsorb). It is calculated as the retention time minus the dead time. In the example below, the adjusted retention time of the component is 8.6 minutes.

1. Relative Retention Time (RRT) is not expressed in units of time. In chromatographic analysis, the retention time of sample components can vary due to various factors, but components typically show similar trends of variation within a defined range. To avoid misidentification of peaks during routine testing, the peak least likely to be misidentified (often a component with a known structure and purity, or one with a higher concentration) is chosen as a reference. The relative retention time is then calculated as the ratio of the retention time of other peaks to the retention time of the reference peak. This approach mitigates the impact of variations in retention times on the results.

1. Delay Time , also known as delay volume or gradient delay volume, refers to the time it takes for the solvent mixture (typically within the mixer of a liquid chromatograph or within a proportioning valve) to reach the inlet of the chromatographic column. It is primarily determined by the dead volumes of the mixer and connecting tubing between the mixer and the column.

The delay volume has a significant impact on the accuracy and reproducibility of liquid chromatography analysis, especially under gradient elution conditions. The instrument configuration directly influences the size of the delay volume during gradient elution, thereby affecting the method's resolution, retention times, and other parameters of separation.

As the delay volume increases, the time for the mobile phase to reach the inlet of the chromatographic column after changing the gradient ratio also increases. This effect is more pronounced at lower flow rates, resulting in delayed peak elution times for components. Additionally, it can lead to broader chromatographic peaks, thereby reducing sensitivity and column efficiency.

Due to varying delay volumes among different instruments, these differences can affect the reproducibility of gradient methods. They may impact resolution and retention times, which can further influence method transfer or validation processes.