High Performance Liquid Chromatography (HPLC)

Chromatography

• Chromatography separates mixtures of substances into components and identifies compounds.
• All forms of chromatography have a stationary phase (solid or liquid on a solid) and mobile phase (liquid or gas).
• The mobile phase flows through the stationary phase to carry mixture components.
• Components travel at different rates.

High Performance Liquid Chromatography (HPLC)

• It is a physical separation technique conducted in liquid phase.
• A sample separates into components by distributing between stationary and mobile phases (a flowing liquid pumped at high pressure).
• The flowing liquid may be an organic solvent.
• The stationary phase can be porous silica particles packed in a column.
• Essential components of an HPLC system:
  • Solvent delivery system (pump)
  • Fixed volume injector loop or autosampler
  • Packed column
  • Solvent reservoirs
  • Detector
  • Data system
  • Recorder
• The column is the heart of the HPLC system.
• HPLC columns use particle-packed columns.
• Column selection depends on knowing the chemistry of the sample.
• The solvent delivery system (pump) delivers the mobile phase.
• Samples typically introduce by syringe injection or autosampler.
• Sample introduction is important and must be reproducible and precise.
• Precision is highly important for quantitative analysis, where the reproducibility of the peak response depends on the precision of sample introduction.
• HPLC detectors:
  • UV-Visible
  • Fluorescence
  • Electrochemical
  • Mass spectrometry

Chromatographic efficiency

• Expressed as the number of theoretical plates (N).
• N = 16 (tR)2/Wb, where N is the number of theoretical plates, tR is the retention time, and w is the peak width at base.

Resolution

• Resolution (Rs) is calculated using the equation Rs = t2-t1/Wb2-Wb1/2
• It means the degree of separation between two peaks

Asymmetry factor (AF)

• AF = A/B at 10% of peak height, where A and B are the two half widths at each side of the peak center.

Retention factor

• Defined by k = (tR - tM) / tM
• tR is retention time
• tM is dead time

Types of Chromatography

• Normal phase: uses a polar stationary phase and a non-polar mobile phase.
• Reversed phase liquid chromatography (RPLC): uses non-polar stationary and polar mobile phases.
• Retention in RPLC occurs by hydrophobic interactions between the solute and the stationary phase.
• Most organic compounds have hydrophobic regions that can interact with the stationary phase.
• RP-HPLC columns are available and are silica based for good mechanical stability.
• A typical stationary phase is formed by chemically bonding a long-chain hydrocarbon group to porous silica.
• Typical ligands:
  • n-octadecyl (C18)
  • n-octayl (C8)
  • n-butyl (C4)
• Separation in RPLC is affected by stationary phase type, column length, organic solvent type and %, mobile phase pH, flow rate, and temperature.

Types of elution modes in HPLC

• Isocratic elution: uses a single solvent (or a mixture in one reservoir) as the mobile phase.
• Gradient elution: uses two or more solvents separately as a mobile phase, pumped with a varied ratio.
• Advantages of HPLC: effective and less expensive separation technique.
• Disadvantages of HPLC: gradient elution takes longer for column equilibrium and cannot be used with some LC detectors (e.g., refractive detector); also, baseline problems are common.

Problem

• Pyridine and t-butylbenzene peaks by HPLC have retention times of 1.3 min and 3.5 min, respectively, and peak widths of 0.4 min and 0.6 min, respectively.
• Resolution between the two peaks, retention factor for pyridine (tm 0.45), and efficiency of butylbenzene is what needs to be calculated.

Optical activity (Polarimetry)

• When plane-polarized light passes through a medium, it is retarded based on the refractive index of the medium.
• For optically inactive media, both circularly polarized components are retarded, and the beam emerges polarized in the same plane as the incident beam.
• For optically active media, components are retarded differently due to differences in refractive indices for left (nL) and right (nR) circularly polarized light.
• The rotation of plane-polarized light is inclined at an angle α to the plane of polarization of the incident beam.
• α = (1800 / λ) * Δn, where l is the light path, λ is the wavelength, and Δn is the difference in refractive indices.
• Optical rotation (α) is positive when the polarization plane rotates clockwise (dextrorotation) and negative when it rotates anticlockwise (levorotation).
• Measurement of optical activity frequently occurs with sodium D light at 20°C.
• Temperature control is important for precise work.
• Instrumentation:
  • Visual Polarimeter
  • Spectropolarimeter
• Optical rotation:
  • Used to study the impurity of optically active materials.

HPLC is a separation technique where a sample separates into components by distributing between stationary and mobile phases. The mobile phase is a flowing liquid pumped at high pressure. Common components include a solvent delivery system, injector loop, packed column, solvent reservoirs, detector, data system and recorder.