High-Performance Liquid Chromatography (HPLC) Lectures PDF

Instrumental Analysis Chapter 24: High-Performance Liquid Chromatography (HPLC)  Mechanism of Separation in HPLC Mobile phase is liquid: 1. Partition chromatography 2. Adsorption chromatography 3. Ion-exchange chromatography 4. Size exclusion chrom...

Instrumental Analysis Chapter 24: High-Performance Liquid Chromatography (HPLC)  Mechanism of Separation in HPLC Mobile phase is liquid: 1. Partition chromatography 2. Adsorption chromatography 3. Ion-exchange chromatography 4. Size exclusion chromatography (gel chromatography) 5. Affinity chromatography. 6. Chiral Chromatography Figure: Applications of the most widely types of HPLC for various analyte species. Original work by Tswett was carried out in glass columns (diameter ~ 1-5 cm , length 50 -500 cm). To assure reasonable flow rates, the diameter of the particles of the stationary phase was usually in the 150-200 µm range. (separation time ~ hours) Smaller P.S Leads to: 1-Higher NTP 2-Lower HETP 3-Higher pressure 4-Shorter optimum run time 4-LOD.  HPLC Advantages of HPLC Speed → fast Resolution → high Accuracy → error < 1% Sensitivity → detection limit (LOD) ~ 10-10g Automation → possible (major advantage in industry) Limitations of HPLC Expensive instrumentation Experience required Instrumentation of HPLC Sample injection HPLC Chromatogram Solvent valve mixing Pump valve Detector Recorder Mobile phase reservoir Waste HPLC Chromatograph The Stationary Phase Highly pure, spherical, microporous particles permeable to solvents: - Silica - Polymeric such as polystyrene ❑ Bare silica can be used as the stationary phase for adsorption chromatography. 1.0 - 5.0 µm  Partition chromatography (LLC) Liquid-liquid Bonded (Covalent) chromatography phase chromatography Silica gel solid support The liquid stationary phase is The liquid stationary phase is held on the solid support by bonded chemically to the solid physical adsorption (H2O support. molecules) Normal Reversed phase phase (Mostly) ❑ Most commonly, liquid-liquid partition chromatography is conducted with a bonded stationary phase covalently attached to silica surface by reactions such as: RP-HPLC the pH of the mobile phase within the range of 3-8. If R is a polar functional group, then the stationary phase is polar. Examples of polar stationary phases include those where R contains a cyano (–C2H4CN), a diol (–C3H6OCH2CHOHCH2OH), or an amino (–C3H6NH2) functional group. Bonded-normal phase chromatography Mobile Phases ❑ The elution order of solutes in HPLC is governed by polarity. 1. In a normal-phase separation ❑ st. p. is polar and the m. p. is non-polar (or of low polarity) ❑ The least polar solute is the first to elute from the column. ❑ Increasing Polarity of m.p resulting in decreasing the elution time. (highly polar→bleeding for the column) 2. In a reverse-phase separation ❑ st. p. is non polar and the m. p. is polar (or of high polarity) ❑ The most polar solute being the first to elute. ❑ Increasing polarity of m. p. resulting in increasing the elution time, (highly non polar→ bleeding for the column) Source: “Solvents and Solvent Effects in Organic Chemistry,” Christian Reichardt https://onlinelibrary.wiley.com/doi/book/10.1002/9783527632220 ©2022 Alison Frontier, University of Rochester. Supported by a grant from the National Science Foundation. NSF Funding {+} Safety Disclaimer {+}  Reversed-phase and normal-phase HPLC Effect of mobile Phase 70 % ACN + 30 % H2O 70 % THF + 30 % H2O 75% of all HPLC work (C8 or C18) Observe Sep. efficiency and retention times!!!!!!!! The Elution process of RP-HPLC Isocratic elution: Fixed solvent composition st.p: RP Example: Mixture of 8 aromatic compounds m.p: (% of acetonitrile (B) in water) Gradual Increase of polarity of m.p →tR 5 injections Adv. and Disadv Rs, Long separation time Gradient Elution: Variable solvent composition High polar m.p in the start, then decrease its polarity continuously Adv. Good Rs.& Shorter separation time  Isocratic versus Gradient Elution An isocratic elution in HPLC is one in which the solvent composition remains constant. A gradient elution in HPLC is one in which the composition of the solvent is changed continuously or in a series of steps. Figure 31-4 illustrates that gradient elution frequently improves separation efficiency. 68 The effect of chain length on the separation. Long chains produce packings that are more retentive.  4. Detectors: (according to material to be used): ◼ UV detectors: most widely used detectors. It can detect few nanograms of a solute having a moderate UV absorption. It has constant λ or variable λ e.g. unsaturated organic compounds. ◼ Fluorescence detector: highly sensitive for the substances that have the ability to re-emit the absorbed light. ◼ Refractive index detector: the index of the substance inside the mobile phase can't be used for gradient analysis , because the change of mobile phase causes change in refractive index. Unlike UV detectors, refractive index detector is very temperature sensitive, and very sensitive to flow rate of column effluent. ◼ Electrochemical detector: conductance and potentiometer. There is no two substances have the same conductance or potential