The Role of the Injection Solvent - - Chromatography Online
The Role of the Injection Solvent


LCGC Europe
Volume 25, Issue 10, pp. 564-569

The solvent chosen for injection can affect the appearance of chromatographic peaks.

A recent observation in the laboratory of one of the authors (B.A.) of this column highlights a problem that has been mentioned in "LC Troubleshooting" discussions before, but its importance justifies additional discussion about the selection of the injection solvent to use with a liquid chromatography (LC) method. In this particular case, it was observed that the peak areas were constant with two different injection solvents, but the peak heights were not. Similar results were observed with methods for two different compounds (A and B). Let's see what the likely cause is for this problem.

Background


Table 1: Chromatographic results for two methods A and B.
We can't share the complete details of the methods because they are proprietary, but the key elements follow. A 10 ppm concentration of sample is prepared in distilled water or in 100% methanol, and 20 μL of this solution is injected. A 250 mm 4.6 mm C18 column packed with 5-μm diameter particles is used with UV absorbance detection at 210 nm. For compound A (method A), a mobile phase of 60:40 methanol–water is used at a flow rate of 1.1 mL/min; for compound B (method B), the mobile phase is 70:30 (methanol–water) with a flow rate of 1.4 mL/min. The data are summarized in Table 1. Both compounds (A and B) are well retained with k-values of approximately 2.6 in each case. You can see that the peak area differences between injection in methanol and water are ≤0.1% for both methods. However, the peak heights are approximately 30% larger for the water injections.

Effect of Injection Solvent


Figure 1: Chromatograms for caffeine (first peak of each chromatogram) and salicylamide (second peak). Injections of 30 μL of ~1 mg/mL of each analyte dissolved in (a) acetonitrile, (b) mobile phase, (c) 81:1 water–acetic acid, and (d) water. Column: 250 mm 4 mm, 10-μm dp; mobile phase: 18:81:1 acetonitrile–water–acetic acid; flow rate: 1 mL/min. Adapted from reference 1.
The chromatograms of Figure 1 illustrate the influence of the injection solvent on the appearance of peaks in the chromatogram for another sample (1). In each case, the reversed-phase column was operated with an 18% acetonitrile–buffer mobile phase, where the buffer was 81:1 water–acetic acid. Injection volumes of 30 μL were made in various injection solvent compositions.

First, consider the cases where the injection solvent is no stronger than the mobile phase [Figures 1(b)–1(d)]. In each case, the retention times of each peak are approximately the same. Although we don't have data for the peak areas in each case, we'll assume they are the same, because the same volume of the sample at the same concentration was used in each case. Note, however, that the peak heights differ. Water is the weak mobile-phase solvent in this example, and the less water that is present in the injection solvent, the shorter the peak heights are. These observations are consistent with the problem presented at the beginning, where peak areas and retention times were constant, but peak heights dropped when less water was used in the injection solvent.

Next, notice what happens when the sample of Figure 1(a) was injected in 30 μL of 100% acetonitrile — the peaks are broader, distorted and at shorter retention times. This example continues the pattern of Figures 1(b)–1(d), where the peaks are broader and shorter as less water is used in the injection solvent.


ADVERTISEMENT

blog comments powered by Disqus
LCGC E-mail Newsletters
Global E-newsletters subscribe here:




 

LCGC COLUMNISTS 2014

Sample Prep Perspectives | Ronald E. Majors:

LCGC Columnist Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments in existing technology lines.

LATEST: The Role of Selectivity in Extractions: A Case Study

History of Chromatography | Industry Veterans:

With each installment of this column, a different industry veteran covers an aspect of the evolution and continued development of the science of chromatography, from its birth to its eventual growth into the high-powered industry we see today.

LATEST: Georges Guiochon: Separation Science Innovator

MS — The Practical Art| Kate Yu:
Kate Yu is the editor of 'MS-The Practical Art' bringing her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers.

LATEST: Mass Spectrometry for Natural Products Research: Challenges, Pitfalls, and Opportunities


LC Troubleshooting | John Dolan:

LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan, Vice President of LC Resources and world renowned expert on HPLC, is able to highlight common problems and provide remedies for them.

LATEST: LC Method Scaling, Part I: Isocratic Separations

More LCGC Chromatography-Related Columnists>>

LCGC North America Editorial Advisory Board>>

LCGC Europe Editorial Advisory Board>>

LCGC Editorial Team Contacts>>


Source: LCGC Europe,
Click here