High Performance Liquid Chromatography (HPLC) is a powerful analytical technique used to separate, identify, and quantify components within a mixture. In HPLC, a sample—often a blend of organic compounds—is dissolved in a solvent and injected onto a chromatographic column. As the sample travels through the column, its individual components interact differently with the stationary phase, resulting in their separation. These separated compounds are detected by various detectors, and their concentrations are determined by comparing their peak intensities to a calibration curve generated from reference standards.

HPLC Variations

The principle behind HPLC is straightforward: different compounds exit the column at different times due to their unique chemical affinities. However, a wide range of variables can be adjusted to optimize separation. The main HPLC separation mechanisms include:

  • Reverse Phase: Features a non-polar stationary phase and a polar mobile phase, separating compounds based on hydrophobic interactions; this is the most widely used method due to its versatility and high resolution.
  • Normal Phase: Uses a polar stationary phase and a non-polar mobile phase to separate compounds based on polarity.
  • Hydrophilic Interaction Liquid Chromatography (HILIC): Employs a polar stationary phase and a mobile phase with a high organic solvent content, separating compounds by hydrophilic interactions with complementary selectivity compared to reverse phase chromatography.
  • Size Exclusion (Gel Permeation): Utilizes a porous stationary phase to separate molecules by size.
  • Ion Exchange: Involves an ionically charged stationary phase, separating compounds by ionic charge.

Each method allows for fine-tuning of variables such as the mobile phase composition, gradient, stationary phase, detection mode, and injection conditions—enabling precise control over sensitivity and resolution. Proper sample preparation and dissolution are often critical for removing interferences and achieving optimal results.

HPLC Detectors

After separation, compounds are detected using a variety of detectors, including:

  • Diode Array Detector (DAD): Measures wavelength-dependent absorbance (UV-VIS).
  • Evaporative Light Scattering Detector (ELSD): Sensitive to most compounds less volatile than the mobile phase.
  • Conductivity Detector: Commonly used in ion chromatography.
  • Fluorescence Detector: Offers high sensitivity for naturally fluorescent or derivatized compounds.

While these detectors are effective for quantification, they are limited in their ability to identify chemical structures. For definitive identification, mass spectrometry is required.

LC-MS

Liquid Chromatography-Mass Spectrometry (LC-MS) couples HPLC with a mass spectrometer, providing both separation and detailed mass analysis. This allows for precise molecular weight determination, structural analysis, and quantification at extremely low concentrations—even in complex matrices. LC-MS systems often combine multiple detectors in series, enhancing detection capabilities for a broad range of compounds.

LC-MS vs. GC-MS

LC-MS is especially effective for analyzing semi-volatile and non-volatile organic compounds . In contrast, Gas Chromatography-Mass Spectrometry (GC-MS) is typically used for volatile and semi-volatile compounds. Running both techniques in parallel provides comprehensive screening for unknowns across a wide chemical space.

Ultra-High-Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-QTOF-MS)

UHPLC-QTOF-MS represents the next generation of liquid chromatography-mass spectrometry, combining the speed and resolution of Ultra-High-Performance Liquid Chromatography (UHPLC) with the high-accuracy mass analysis of Quadrupole Time-of-Flight (QTOF) mass spectrometry.

Key Advantages

  • Ultra-Fast, High-Resolution Separations: UHPLC uses columns packed with ultra-fine particles and operates at higher pressures than traditional HPLC, delivering sharper peaks, better resolution, higher sensitivity, and much faster analysis times.
  • Exceptional Mass Accuracy and Structural Insight: QTOF mass spectrometry provides high-resolution, accurate mass measurements and advanced MS/MS fragmentation, making it possible to identify and characterize unknown compounds with confidence.
  • Comprehensive Data Collection: UHPLC-QTOF-MS supports both targeted quantitation and untargeted profiling, capturing detailed information on all detectable components in a sample.
    Enhanced Throughput and Efficiency: Faster analysis and reduced solvent consumption make UHPLC-QTOF ideal for and large-scale studies.

Applications

UHPLC-QTOF-MS is widely used in:

  • Unknown identification or deformulation
  • Degradation product analysis
  • Trace impurity/contaminant characterization
  • Polymer additive analysis
  • Extractables and leachables

This technology is especially valuable for complex sample matrices, where both sensitivity and structural information are essential.

HPLC Applications

HPLC and its advanced variants can be applied to a broad range of analytical challenges, including:

  • API purity and stability assessment
  • Residual monomer analysis
  • Drug delivery characterization
  • Excipient characterization
  • Oligomer analysis
  • Lot comparison, resin equivalency, good/bad comparison
  • Cleaning validation
  • Molecular weight analysis

Contact CPG today to discuss your HPLC, LC-MS, or UHPLC-QTOF testing needs and discover how our advanced analytical solutions can support your project.