Pharmaceutical Analysis: HPLC & Optical Activity

• Pharmaceutical Instrumental Analysis includes Separation Techniques (HPLC) and Optical Activity (Polarimetry).
• The labs covered are Lab 5 and Lab 6 for Separation Techniques and Optical Activity respectively.
• The date is 12/8/2024.

Chromatography

• It originates from the Greek words "chroma" (color) and "graphein" (to write).
• A Russian botanist first developed it for colorful separation of plant pigments using a column of calcium carbonate.
• It has developed into a lab tool used for separating mixtures of substances into their components and identifying compounds.

Separation Techniques

• All forms of chromatography share the same principle, involving a stationary phase (solid or liquid supported on a solid) and a mobile phase (liquid or gas).
• The mobile phase flows through the stationary phase, carrying the components of the mixture.
• Different components travel at different rates.

High Performance Liquid Chromatography (HPLC)

• It is conducted in a liquid phase.
• A sample is separated into its components by distribution between the stationary and mobile phases, with the mobile phase being a flowing liquid pumped at high pressure.
• The mobile phase can be an organic solvent.
• The stationary phase can be porous silica particles packed in a column.
• Essential components of a complete HPLC system consist of:
  • Solvent delivery system (pump)
  • Fixed volume injector loop or autosampler
  • Packed column
  • Solvent reservoirs
  • Detector
  • Data system
  • Recorder

HPLC Column

• The column is the heart of the HPLC system.
• HPLC columns are particle-packed.
• Column selection depends on knowledge of sample chemistry.
• The solvent delivery system, or pump, delivers the mobile phase.

Sample Introduction

• Samples are introduced by syringe injection or autosampler.
• Reproducible and precise injections are important in sample introduction, especially for quantitative analysis.
• Reproducibility of peak response depends on the precision of sample introduction.
• HPLC detectors include UV-Visible, fluorescence, electrochemical, and mass spectrometry detectors.

Chromatographic Efficiency (N)

• It is expressed as the number of theoretical plates.
• N = 16 (tR)² / Wb
  • N = number of theoretical plates
  • tR = retention time
  • Wb = peak width at base

Resolution (Rs)

• It measures the degree of separation between two peaks, calculated by the equation:
• Rs = (t2 - t1) / (Wb2 + Wb1) / 2
• Asymmetry Factor (AF) = A/B, measured at 10% of peak height, with A and B representing the two half widths on each side of the peak center.

Retention Factor (k)

• k = (tR - tM) / tM

Types of Chromatography

• Normal Phase Chromatography employs a stationary phase with polar functional groups and a non-polar mobile phase.
• Reversed Phase Liquid Chromatography (RPLC) uses a non-polar stationary phase and a polar mobile phase.
• Retention in RPLC occurs through hydrophobic interactions between the solute and stationary phase.
• Organic compounds have hydrophobic regions.
• They interact with the stationary phase, explaining RPLC's wide application.
• There are a wide variety of RP-HPLC columns available, mostly silica based for good mechanical stability.
• A typical stationary phase involves chemically bonding a long-chain hydrocarbon group to porous silica.
• Typical ligands include n-octadecyl (C18), n-octyl (C8), and n-butyl (C4).
• Separation in RPLC is affected by:
  • Stationary phase type
  • Column length
  • Type and % of organic solvent in the mobile phase
  • Mobile phase pH
  • Flow rate
  • Temperature

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.
• The solvents are pumped into the LC system.
• The ratio of solvents is varied in programmed way.
• Gradient elution is an effective and less expensive separation technique.
• Its disadvantages are:
  • Longer run time for column equilibrium
  • Incompatibility with some LC detectors (e.g., refractive index detectors)
  • Baseline problems

Example Problem

• Pyridine and t-butylbenzene peaks:
  • HPLC retention times of 1.3 min and 3.5 min
  • Peak widths were 0.4 min and 0.6 min.
• Requires calculation of:
  • Resolution between the two peaks
  • Retention factor for pyridine (tm = 0.45)
  • Efficiency of butylbenzene

Lab 6: Polarimetry

• Covers Optical Activity

Optical Activity (Polarimetry)

• When plane-polarized light passes through a medium, it is retarded to an extent indicated by the refractive index.
• In an optically inactive medium, both circularly polarized components are retarded to the same extent, and the beam emerges polarized in the same plane as the incident beam.
• In an optically active medium, components are retarded to different extents.
• The refractive indices differ for left (nL) and right (nR) circularly polarized light.
• The rotation of plane-polarized light is inclined at an angle α relative to the incident beam's plane of polarization.

Optical Rotation Measurement

• α = (1800 / λ) * Δn
  • l: light path
  • λ: wavelength
  • α: optical rotation of the medium is positive when the plane of polarization is rotated clockwise (dextrorotation) and negative when rotation is anticlockwise (levorotation).
• Measurements are frequently made with sodium D light at 20°C.
• Temperature should be controlled for precise work.

Polarimetry Instruments

• Visual Polarimeter
• Spectropolarimeter
• Optical rotation can be used to study the impurity of optically active materials.

Explores separation techniques in pharmaceutical instrumental analysis, focusing on HPLC. Covers the principles of chromatography, including stationary and mobile phases. Details the basics of High Performance Liquid Chromatography (HPLC).