High-Performance Liquid Chromatography (HPLC) is a widely used analytical technique that separates, identifies, and quantifies components within a liquid mixture. It is a powerful tool across many scientific disciplines, including pharmaceuticals, environmental monitoring, and food science.
Core Principles and Components
HPLC operates on the principle of separating compounds based on their differing interactions with two phases: a stationary phase and a mobile phase. Like a race, molecules travel at different speeds. Those interacting more strongly with the stationary phase move slower, while those with weaker interactions move faster, leading to their separation and resolution.
An HPLC system consists of several integrated components working together to achieve this separation. The solvent delivery system, or pump, precisely controls the flow of the mobile phase through the instrument at high pressures. Next, the sample injector introduces a precise volume of the prepared sample into the flowing mobile phase. This injection is often automated by an autosampler, enhancing efficiency and reproducibility by handling multiple samples without manual intervention.
The separation occurs within the column, which contains the stationary phase, typically tiny, porous particles designed to interact with sample components. As the mobile phase carries the sample through, differing affinities cause components to separate. Finally, a detector positioned at the end of the column measures the separated components as they exit. The detector converts a physical or chemical property of each component into an electrical signal, allowing for their identification and quantification.
Pre-Run Preparations
Before an HPLC run, careful preparation of the mobile phase and samples is essential for accurate results. The mobile phase, the solvent or mixture that carries the sample, must be prepared with high-purity, HPLC-grade solvents. Common solvents include water, acetonitrile, and methanol, often mixed in specific ratios to optimize separation. Accurately measuring and mixing these solvents according to the method’s specifications is important.
After mixing, the mobile phase requires degassing to remove dissolved gases, such as oxygen and nitrogen. Dissolved gases can form bubbles, leading to issues like detector noise, pump cavitation, and inconsistent flow rates, which compromise data quality. Degassing can be achieved through methods like sonication, helium sparging, or by using in-line vacuum degassers integrated into the HPLC system. This step helps maintain a stable baseline and consistent operation.
Sample preparation typically involves dissolution and filtration. Samples must be dissolved in a suitable solvent, ideally one compatible with the mobile phase, to ensure all components are in solution. Following dissolution, samples are filtered to remove any particulate matter.
This filtration step protects the HPLC column and other instrument components from clogging, which can cause increased backpressure and reduce system lifespan. Filters commonly have a porosity of 0.45 µm. The prepared samples are then transferred into appropriate vials or containers.
Executing a Run
With the mobile phase and samples prepared, the next step is setting up the HPLC instrument and initiating the run. Sample vials are loaded into the autosampler, which automatically injects precise volumes into the system, improving throughput and reproducibility compared to manual injection. Injection volumes typically range from a few microliters to tens of microliters, depending on sensitivity requirements.
Analytical parameters are then configured using the HPLC system’s control software. These parameters include the mobile phase flow rate, which dictates how quickly the mobile phase moves through the column, typically ranging from 0.5 to 2.0 mL/min. The column temperature is also set, often controlled within a column oven to ensure consistent separation and retention times. For UV-Vis detectors, the detection wavelength is selected, typically where target compounds absorb light most strongly, to maximize sensitivity. A common wavelength range for UV detection is between 200 and 400 nm.
Once parameters are set, the run can begin. The system begins pumping the mobile phase at the set flow rate, allowing it to equilibrate through the column. After a stable baseline is established, the autosampler injects the sample. The sample then travels through the column, where its components separate, and are subsequently detected. During the run, the system software continuously monitors parameters like pressure and detector signal, providing real-time feedback on the separation process.
Understanding the Output
After an HPLC run is complete, the primary output is typically a chromatogram, which is a graphical representation of the detector’s signal over time. This graph displays a series of peaks, each corresponding to a different separated component that has eluted from the column. The position of a peak along the time axis is its retention time, the specific time it took for that component to travel through the system. Retention time is a characteristic property for a given compound under specific analytical conditions and helps in identifying the components within the mixture.
The height and area of each peak in the chromatogram provide quantitative information about the amount of each component present in the original sample. A larger peak area or height indicates a higher concentration of that particular compound. Chromatograms allow for both qualitative analysis, by identifying compounds based on their retention times, and quantitative analysis, by determining their concentrations. While the chromatogram provides foundational data, further processing and calibration with known standards are often necessary for precise quantitative results.