LC System Optimisation for UHPLC Performance on any HPLC

The Importance of the HPLC Method

Enhancing performance for all column separations can be achieved through the following method-related optimization tips. Specifically, these suggestions can significantly boost your method's performance when using core-shell columns.

When optimizing HPLC methods, our main efforts should focus on ensuring the minimization of sample dispersion in order to mitigate peak broadening and improve the sensitivity and resolution of your peaks. This entails properly concentrating the sample as a tight band at the head of the column when injecting and then working to minimize sample dispersion through the column.

Adjusting injection volumes and selecting a proper sample diluent can greatly help reducing injection-related sample dispersion. Additionally, integrating a simple injector programming into the method can reduce dispersion due to undesired extra-column volume. These suggestions will greatly improve peak shape and efficiency, especially for rapid core-shell LC separations.

Problem: All Peaks are Broad

1. Check Sample Volume

Errors in sample loading may negate the performance gain the core-shell particle can achieve. If you inject the sample in the same organic composition as the mobile phase under isocratic conditions, the initial bandwidth on column will be directly proportional to the injection volume. As such, a large volume sample is injected as a wide band on the column and gives low peak efficiency.

Solution: To reduce the efficiency loss of large volume injections, it is possible to pre-concentrate the sample on the head of the column by using a weaker diluent than the mobile phase. You can also reduce your sample injection volume. When high efficiency columns are used often, lower sample amounts are needed to get good signal response.

2. Check Sample Diluent

If the organic strength of the diluent is greater than the HPLC mobile phase, the sample will load as a diffuse band at the head of the HPLC column. This may occur in either isocratic or gradient methods and can negate much of the performance advantages that the column delivers.

• Gradient methods where the sample is injected in a stronger injection solvent than the mobile phase, the compound exhibits surfing effects which will cause peaks to broaden and split.
• Isocratic methods where the diluent is stronger than the mobile phase will result in excessive sample dispersion.

Solution: Inject a weak solvent. It is best to use a diluent either equal to or weaker than the organic strength of the mobile phase. This focuses the sample on the head of the column, resulting in sharper peaks (i.e., higher efficiency).

Goal: Further shorten the runtime and peak capacities in gradient methods

Check Injector Program

Extra-column volume before the column will cause dispersion of the sample on the way to the column; the injector loop is a major source of extra-column volume.

Solution: It is possible to bypass the loop with an injector program during the analysis method (after the sample has left the injector loop). Table 1 and Figure 1 show an example where the injection loop is bypassed after injection to reduce system dwell volume.

Steps | Commands | Comments

1 | DRAW | Draw volume of sample (injection volume) from vial.
2 | INJECT | Introduce sample into flow path.
3 | WAIT | Flush sample loop after injection (wait time = 6× (injection volume + 5 µL) / flow rate).
4 | VALVE bypass | Direct flow from pump to column, bypassing injection valve to exclude delay volume ~200 – 500 µL from auto-injector path
5 | WAIT 1.5 min | The period of VALVE bypass time (Wait time = Run time – 1 min).
6 | VALVE reinject | Switch valve from bypass to injection position.

Phenomenex: LC System Optimisation for UHPLC Performance on any HPLC

* Mainpass peak is an injector artifact due to switching the injector valve back to include the injector loop in the flow (to flush injector in preparation for the next injection).