Liquid Chromatography – Sample Solvent and Inj. Volume vs. Peak Shape

Part 1. Introduction

This month we will discuss the impact of sample solvent and injection volume on the peak shape.  To estimate the peak shape, we will use number of theoretical plates as a peak shape characteristics.

Part 2. Injection volume and Solvent/Diluent

Have you had one or both of the following experiences while performing analysis in a reversed-phase mode?

• You injected the same amount (i.e. 50uL) of Standard Solution (prepared in example using methanol) and Sample Solution (prepared in water) and for some reason the main peak of interest for the Standard injection was much broader!!!

• When you carried injection of Standard dissolved in methanol and then repeated with 2x (twice) the injection volume it had an extremely bad effect on the peak’s separation.

We will try to discuss the above issues to help you understand the impact of solvent/diluent and injection volume on the peak shape.

The sample injected via the autosampler reaches the HPLC/UPLC column through the system tubing. If the mixture of sample solvent and mobile phase is insufficient before the sample solvent reaches the column, the peak shape may deteriorate depending on the type of sample solvent used. In case when the sample solvent/diluent will have a higher elution strength than the mobile phase the peak shape will be broadened. This is even more evident when using UHPLC system with much smaller ID tubing, which is more liable to be affected with inadequate mixing of the sample solvent and mobile phase.

Figure 1 below presents the correlation between number of theoretical plates (TPN) for caffeine peak changing with the change of sample solvent/diluent. The peak shape is theoretically represented in Figure 1 by the theoretical plate number (TPN), which here indicates the sharpness of the peak. In this case mixture of methanol/water in ratio 3/7 was used as mobile phase. Comparing TPNs when using pure methanol as the sample solvent/diluent we observe a clear drop of TPNs with increased injection volume, meaning that the peak of interest gets broader. Methanol has much higher elution strength than the mobile phase. On the other hand, when 100% water was used, a drop in the number of theoretical plates was not observed, even though a relatively large volume (1mL = 1000uL) was injected. In fact, there was a slight increase overall.  Water has a lower elution strength than the mobile phase.

Figure 1. Impact of injection volume on NTP while using different solvents/diluents

Considering above Figure 1 please refer to Figure 2 below for overlay of different injection volumes of caffeine standard and its’ impact on the peak shape. Each injected caffeine standard was prepared in methanol. For an injection volume of 100 uL we can observe that the elution starts very early and continues gradually with a result of N < 100, as shown in Figure 1. On contrary the fantastic peak shape was achieved with lowered injection volume of 10uL, which is also confirmed in Figure 1 with N ~ 1000. In this case we must also consider the column loading issue, meaning that too much of the compound of interest loaded onto the column will cause “spreading” / band broadening of the sample across the HPLC/UHPLC system tubing/column with the effect similar to 100uL injection presented in Figure 2.

Figure 2. Caffeine peak shape comparison – different injection volumes

Figure 3-A and Figure 3-B below presents an image of the analytes and the sample solvents moving through the column. This will help you to understand the band broadening effect when incompatible sample solvent/diluent with mobile phase is used. The secret lies with the diffusion, sample solvent can be considered as part of the mobile phase in the column but using a sample solvent with higher elution strength may move the analytes faster, as if it were a mobile phase. In contrary, as for the use of a sample solvent with lower elution strength, the analyte initially concentrates then once more starts to separate in the mobile phase because the sample solvent acts as a mobile phase with lower elution strength. Using a sample solvent with an elution strength lower than that of the mobile phase can sharpen the peak shape because the diffusion of the analyte can be suppressed in the column.

Figure 3-A. Analyte moving through column dissolved in Sample Solvent with HIGHER elution strength

Figure 3-B. Analyte moving through column dissolved in Sample Solvent with LOWER elution strength

Part 3. Countermeasures and Summary

We can clearly see, that if the sample injection volume is relatively large, the type of sample solvent has a large influence on the peak shape and the retention time. This must be investigated and assessed during method development activities.

Additionally, as mentioned above, it is explained that the difference in the elution strength between the sample solvent and the mobile phase affects the peak shape vastly. Peak can be sharpened by using a sample solvent with a lower elution strength than the mobile phase, but if the sample is prepared with a solvent with a higher elution strength (e.g.; methanol 100% in RP-LC), there is concern about the trouble of further pretreatment such as dilution with more compatible sample solvent/diluent.

Liquid Chromatography – Master the Basics

This article is part of our “Liquid Chromatography – Master the Basics” series, your go-to resource for comprehensive and insightful updates on the world of liquid chromatography. Each month in 2024 we will dive into a Liquid Chromatography topic, offering content that is both accessible to beginners and beneficial for experienced scientists.

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