In High-Performance Liquid Chromatography (HPLC), the elution process is critical to the separation of analytes and the quality of chromatographic results. Elution describes how compounds move through the column and separate based on their interactions with the mobile and stationary phases. This process determines the resolution, peak shape, and retention times in the chromatogram, which are essential for identifying and quantifying components in complex mixtures. Understanding the elution process helps in interpreting chromatographic data and improving separation efficiency.

HPLC utilizes highly pure HPLC-grade solvents to ensure minimal interference from impurities. High-grade solvents are essential because even trace amounts of contaminants can interfere with the elution process, distort chromatogram peaks, and reduce sensitivity during sample detection. Moreover, solvents may contain dissolved gases such as nitrogen and oxygen, which can also interfere with sample detection. Degassing is employed to eliminate these gases, using either a vacuum pump or purging with Helium gas, to maintain the integrity of the separation and detection processes.

The degassed solvents are then pulled from the mobile phase reservoir by a high-pressure pump, with the reciprocating pump being the most commonly used type. The pump consists of a piston and two check valves, which draw the solvent using the back-and-forth movement of the piston. However, a disadvantage of the reciprocating pump is that it produces a pulsed solvent flow. Therefore, modern HPLC systems have a pulse damper connected to the pump to address this issue. Other pumps, such as screw-driven displacement pumps, are available but may have limitations such as less solvent capacity and low-pressure bearing capacity, which make them less convenient for solvent changes.

There are two types of elution processes: isocratic and gradient elution. Isocratic elution uses a single solvent or mobile phase throughout the separation, while gradient elution involves a change in the composition during the separation process. This method improves separation efficiency, reduces analysis time, and enhances resolution, leading to sharper peaks in the chromatogram. Gradient elution provides better resolution and results in more distinct and separated peaks, making it easier to interpret the chromatogram for complex mixtures compared to isocratic elution.

Before the mobile phase enters the column, the sample is introduced using a loop injector with a two-position valve. The valve helps to direct the mobile phase to the column. Due to the high pressure in HPLC, sample injection cannot be performed as in gas chromatography (GC). Sampling loops, with fixed volumes ranging from 0.5 microliters to 2 milliliters, can be interchangeable.