The instrumentation of High-Performance Liquid Chromatography (HPLC) is composed of several components, each designed to perform a specific function to enable the entire process.
The core components of an HPLC instrument include:
1. Solvent Reservoir:
This holds the mobile phase or solvents.
In gradient systems, multiple reservoirs are present to store different solvents which are mixed in specific proportions during the chromatographic run.
2. Pump or Solvent Delivery System:
This moves the mobile phase from the reservoir, through the column, and onto the detector.
The pump ensures a consistent and precise flow rate, usually at high pressures, hence the name "high performance."
3. Sample Injection System:
Introduces the liquid sample into the flowing mobile phase stream.
Common injection systems include manual injection loops and auto-samplers for higher throughput and consistency.
4. Column:
The heart of the HPLC system where separation of compounds occurs.
Consists of a steel or glass tube packed with stationary phase particles. The choice of stationary phase and column dimensions (length, diameter, particle size) depends on the analysis requirements.
Sometimes, a guard column is attached before the main column to protect it from contaminants or sample particulates.
5.Detector:
Registers a signal when compounds pass through after eluting from the column.
Common detectors include:
UV/Vis Absorbance Detector: Measures UV or visible light absorbance of eluting compounds.
Fluorescence Detector: Used for compounds that naturally fluoresce or are derivatized to fluoresce.
Refractive Index Detector: Detects changes in refractive index.
Mass Spectrometric Detector (LC-MS): Offers mass-based detection and identification.
Electrochemical Detectors: Measure current resulting from the oxidation or reduction of eluting compounds.
6. Data System (or Recorder):
Records the detector's output to produce a chromatogram, which is a graphical representation of the detector's response against time or volume.
Modern HPLC systems are interfaced with computers and software for data acquisition, analysis, and storage.
7. Column Oven or Temperature Control System:
Maintains the column at a constant temperature or allows for temperature programming.
Temperature affects the separation, retention times, and peak shapes, making this an essential component for reproducibility.
Additional Components:
Degasser: Removes dissolved gases from solvents, as bubbles could interrupt flow and detection.
Gradient Mixer: In gradient systems, this mixes solvents from multiple reservoirs in specific proportions.