Every chromatographer aspires to achieve the narrowest peak width at the peak's apex. But why is this quest for a minimal peak width so crucial? The answer lies in achieving precise peak integration and, subsequently, accurate quantitation. Additionally, it plays a pivotal role in lowering the detection and quantitation limits, vital aspects of chromatographic analysis.
However, the pursuit of an ultra-narrow peak width is not without its challenges. As retention time increases, the peak width at the base also expands. Similarly, a drop in plate count results in a broader peak width at the base. So, how can one navigate this dilemma and obtain those razor-thin peaks we desire?
In this article, we will delve into an essential concept known as "peak compression." This concept, harnessed through gradient elution, offers a solution to the peak width predicament, resulting in sharper chromatograms.
Understanding Gradient Elution:
Gradient elution involves altering the mobile phase's strength over time. In this example, we employ a gradient program where percent A represents a weaker diluent (e.g., aqueous) and percent B signifies a stronger diluent (e.g., organic solvents like methanol or acetonitrile).
Table 1: Gradient run conditions
During the initial phase (0 to 10 minutes), it's isocratic, but after 10 minutes, a gradient is applied. At 10 minutes, it's 90% A, and at 14 minutes, it's 10% A. The proportion of weaker diluent (e.g. Aqueous) decreases over time, while the proportion of the stronger diluent (e.g. acetonitrile or methanol) increases.
In gradient elution, the mobile phase's strength increases as the proportion of the organic solvent grows. At 14 minutes, the mobile phase is significantly stronger than it was at 0 or 10 minutes. As a result, the sample band moves more rapidly in the stronger mobile phase compared to the weaker mobile phase. This results in the rear end of the sample band moving in a stronger mobile phase than the front, causing peak compression.
The Peak Compression Process:
Imagine a column with a sample band represented in blue.
Fig 1: Column with the sample band
The gradient program begins just before the 10-minute mark. By gradually increasing the proportion of the stronger diluent (B) and reducing the weaker diluent (A), the column experiences a change in mobile phase composition. This change causes the rear side of the sample band to move more quickly in the stronger mobile phase. As a result, the band becomes narrower, leading to peak compression.
Fig 2: Column with and without peak compression
Peak compression is achieved by exploiting the difference in solute migration velocities in weak and strong mobile phases. The weaker mobile phase, found at the front of the peak, results in slower solute migration, while the stronger mobile phase at the rear allows for faster migration. This difference in migration velocities narrows the peak, achieving peak compression.
Practical Applications:
Peak compression proves invaluable in situations where isocratic runs are not feasible, often due to complex sample matrices or the presence of interfering substances. This concept can enhance peak height and minimize peak width for analytes with low response, such as in the development of methods for analyzing mutagenic impurities or highly diluted sample solutions.
Example: 4-hydroxyquinoline
In the present example, the peak compression began when the sample band migrated to the exit of the column to avoid further on-column diffusion after peak compression.
(a) Under non-compression:
Elution conditions: 97% H2O and 3% ACN isocratic elution;
Fig 3: Under non-compression (Isocratic elution)
(b) Peak compression (Gradient elution):
Elution conditions: The first was isocratic elution of 97% H2O and 3% ACN, the second was an abrupt gradient from 3% ACN to 20% ACN, and the third was 80% H2O and 20% ACN.
Fig 4: Peak compression (Gradient elution)
Have you ever applied peak compression in your chromatographic work? Share your experiences, thoughts, and insights in the comments below. We'd love to hear about your journey in mastering the art of peak compression.
Thank you for joining us in exploring this essential concept in chromatography.