1. Chapter 5c : LIQUID CHROMATOGRAPHY Department of Chemistry, Faculty of Science Universiti Teknologi Malaysia ___________________________ Analytical Chemistry Course SSC 1293

2. Lecture Outline TLC HPLC IC SEC

3. THIN LAYER CHROMATOGRAPHY (TLC) Simple and inexpensive technique.Simple and inexpensive technique. Often used to judge the purity of a synthesized compound or to indicate the extent of progress of a chemical reaction (qualitative analysis)Often used to judge the purity of a synthesized compound or to indicate the extent of progress of a chemical reaction (qualitative analysis) Small quantity of a solution of the mixture to be analyzed is deposited as a small spot on a TLC plate.Small quantity of a solution of the mixture to be analyzed is deposited as a small spot on a TLC plate. TLC plate consists of a thin layer of silica gel (SiO 2 ) or alumina (Al 2 O 3 ) coated on a glass or plastic (stationary phase).TLC plate consists of a thin layer of silica gel (SiO 2 ) or alumina (Al 2 O 3 ) coated on a glass or plastic (stationary phase). The sheet is then placed in a chamber containing a small amount of solvent, which is the mobile phase.The sheet is then placed in a chamber containing a small amount of solvent, which is the mobile phase.

4. The solvent gradually moves up the plate via capillary action, and it carries the deposited substances along with it at different rates. The desired result is that each component of the deposited mixture is moved a different distance up the plate by the solvent. The components then appear as a series of spots at different locations up the plate. The R f Value R f is the retardation/retention factor, or how far up a plate the compound travels. The R f value for a substance is the ratio of the distance that the substance travels to the distance that the solvent travels up the plate.

5. Visualizing the Spots (Qualitative Analysis) If the compounds are colored, they are easy to see with the naked eye. If not, a UV lamp is used or spray with iodine vapour, ninhydrin, flourescamine.

6. Applications To determine the number of components in a mixture.To determine the number of components in a mixture. To determine the identity of two substances.To determine the identity of two substances. To monitor the progress of a reaction.To monitor the progress of a reaction. To determine the effectiveness of a purification.To determine the effectiveness of a purification. To determine the appropriate conditions for a column chromatographic separation.To determine the appropriate conditions for a column chromatographic separation. To monitor column chromatographyTo monitor column chromatography.

7. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) Evolved from preparative column chromatography. Performance (efficiency and resolution) enhanced by use of stationary phase particles with small diameter (2-5  m). IN ORDER TO OBTAIN SATISFACTORY FLOW RATES, THE LIQUID MUST BE PRESSURIZED TO SEVERAL HUNDRED PSI OR MORE.

8. Solvent Common solvents used are water, methanol, acetonitrile, and tetrahydrofuran. Solvents are filtered (0.45  m membrane filter) and degas. Inlet filter at the line tubing.

9. ADVANTAGES OF HPLC Greater sensitivity - various detectors. Ideal for ionic species and large molecules. Analytical or preparative (highly purified compound in small or large quantities). Applications include analysis of sugars, pesticide residues, organic acids, lipids, amino acids, toxins, and vitamins.

10. COMPONENTS OF AN HPLC SYSTEM Consist of several components with defined functions. Components in modular form or as an integrated unit. Modular system allows users to tailor the instruments to their needs and budget. Components interconnected by transfer tubing with small diameter (0.1 mm) e. g. stainless steel or flexible polymer tubing (polyetheretherketone - PEEK)

11. Solvents (Mobile Phase) Pump Injector Thermostated Column Detector Computer

12. Pumping system The function is to deliver mobile phase through the system at flow rates ranging from 0.1 to 10 mL/min. Needs to be controlled, accurate and precise. System and connecting lines are made from stainless steel (can handle pressure of up to 6000 psi). Pumps are sensitive to particles in the mobile phase and air. Filter and degas mobile phase. Types: reciprocating pumps, screw-driven syringe type, pneumatic pump.

13. Injector The function is to introduce sample into the mobile phase. Valve Injector Trouble free, good precision and can change loop for different volumes. Place in load position and load sample into an external fixed volume loop at atmospheric pressure. Rotate to inject position. Loop becomes part of the eluent flow stream and the sample is carried to the column.

14. Auto Samplers Can inject a large number of samples. Small vials with a septum placed in a tray. Needle penetrates septum and withdraws the sample. Valve introduces the sample into the column.

15. COLUMNS Straight, stainless steel column, typically 3-25 cm length and internal diameter 0.5-5 mm. Stationary phase held in column by a porous disc (frit) at each extremity. Dead volumes within column kept as small as possible. Normal columns allow mobile phase through at a few mL/min. Usually constructed from stainless steel tubing (some glass tubing but <600 psi). Packing materials silica, alumina, porous polymer, ion exchange resins.

16. Precolumn –auxillary column that preceed the analytical column. To increase lifetime of column. Guard column - protect analytical column by removing particulate matter and contaminants. Analytical column Size: 10, 15, or 25 cm long with internal diameter of 4.6 or 5 mm. Packing material particle size - 3, 5, or 10  m particles. Shorter columns (3 cm) packed with <3  m particles increasing popular because of faster separation.

17. DETECTORS Choice is determined by solute type and concentration, detector sensitivity, linear range, compatibility with solvent and elution mode. Most common - UV/VIS, fluorescence, refractive index, diode-array (DAD). In UV/VIS absorption detectors: absorption proportional to analyte concentration. Coupled technique: LC-MS, LC-NMR.

18. HPLC NORMAL PHASE Stationary Phase: Polar Chemically attached polar nonionic functional group (alcoholic hydroxy, nitrile, or amino). Mobile Phase: Nonpolar Nonpolar solvent (e.g. hexane) with a more polar modifier (e.g. methylene chloride). Solvent strength and selectivity is controlled which influences solute retention (weak solvents increase retention and strong solvents decrease retention). Applications Best for compounds highly soluble in organic solvent (fat soluble vitamins) or that have low solubility in an aqueous mobile phase. Also can be used for compound classes, isomers, and highly hydrophilic species like carbohydrates.

19. HPLC REVERSE PHASE Stationary Phase: Non-Polar Silica Chemically bound to silica via surface silanol. Usually an octadecyl (C18). Bonded phase (non polar octadecylsilyl). Chromatographic behavior is influenced by the type of organic group bound to silica, chain length of the organic group (C4 - C18), support particle size and shape. Polymeric Highly cross-linked polystyrene-divinylbenzene (PS- DVB).

20. Mobile Phase: Polar Usually water mixed with methanol, acetonitrile, or tetrahydrofuran. Solute retained due to hydrophobic interactions with the non polar stationary phase. Solute eluted in order of increasing hydrophobicity (least hydrophobic first, most hydrophobic last). Increasing polarity of the mobile phase increases solute retention. Increasing organic solvent content decreases solute retention. Additives: an amine to deactivate silanols, aqueous buffers to suppress ionization of sample components, ion-pair reagents to neutralize charged solutes and make them more lipophilic

21. Elution Mode Isocratic Elution The composition of the solvent remains constant. Gradient Elution The composition of the solvent is changed continuously or in a series of steps. This technique can overcome the general elution problem.

22. Applications of HPLC Reverse Phase Proteins,- including cereal proteins. Water soluble and fat soluble vitamins. Carbohydrates using ion-pairing reagents. Antioxidants, dyes, pigments, and phenolic flavor compounds.

23. HPLC SEPARATION OF PAHS HPLC separation of standard PAHs using a C 18 column at temperature of 60 o C. Acetonitrile-water (70:30) as mobile phase at a flow rate of 1 mL/min. UV detection at 280 nm.

24. OTHER LC METHODS Ion-exchange chromatography (IC) Size-exclusion (gel-permeation, gel- filtration) chromatography (SEC/GPC)

25. ION EXCHANGE CHROMATOGRAPHY An ion exchange technique: low concentrations of anions or cations are separated via column materials that carry an opposite charge. The ionic groups of exchanger columns are covalently bound to the gel matrix and are compensated by small concentrations of counter ions, which are present in the buffer. When a sample is added to the column, an exchange with the weakly bound counter ions takes place.

26. sample resin ligand Counter ion

27. Functional Group ConditionType  SO 3  H + Sulfonic acidStrong acidCation  COO  H + Carboxylic acid Weak acidCation  CH 2 N + (CH 3 ) 3 Cl  Quartenary amino group Strong base Anion  CH 2 N(CH 3 ) 2 H + OH  3  amino group Weak baseAnion Stationary Phase Ion exchanger functional groups

28. When a cation exchanger is immersed in an aqueous solution containing the M+ cations, an exchange equilibrium is establised: xRSO 3  H + + M x+ (RSO3  ) x M x+ + xH + (solid) (solution) where, M: cation, R: part of the resin molecule containing one sulfonic acid group. For an anion exchange resin: xRN(CH 3 ) 3 + OH  + A x- [RN(CH 3 ) 3 + ] x A x- + xOH  (solid) (solution)(solid)(solution) where A x- is an anion.

29. Mobile Phase Solute retention is controlled by changing ionic strength and pH of the mobile phase. Aqueous solution (sometime mix with the organic solvent) which contain ionic solute. Applications Simple inorganic compounds (cation and anion). Amino acids, carbohydrates, organic acids. Preparative purification of proteins, fractionation of peptides.

30. Instrumentation IEC is practiced in two forms. Suppressed IC A second column (suppressor column) is used to remove the buffer ions so that sample ions can be more easily detected. Nonsuppressed IC Weakly conducting buffers at low concentration are carefully selected, and the entire effluent is passed through the detector; ions are detected above the background signal. Supressed column

31. SIZE EXCLUSION CHROMATOGRAPHY (SEC) Also called gel-filtration or gel-permeation chromatography (GPC). Separation of mixtures based on the molecular size of the components. Uses porous particles to separate molecules of different sizes. Molecules that are smaller than the pore size can enter the particles and therefore have a longer path and longer transit time than larger molecules that cannot enter the particles. Molecules larger than the pore size cannot enter the pores and elute together as the first peak in the chromatogram.

32. Molecules that can enter the pores will have an average residence time in the particles that depends on the molecules size and shape. Different molecules therefore have different total transit times through the column. Molecules that are smaller than the pore size can enter all pores, and have the longest residence time on the column and elute together as the last peak in the chromatogram.

33. molecular weight distributions of polymers. Stationary and Mobile Phase Select stationary phase for a matrix pore size that matches the molecular weight range of the species to be resolved. Hydrophilic packing (surface modified silica or methacrylate resins): Use with water soluble samples and an aqueous mobile phase. Hydrophobic packing (polystyrenedivinylbenzene): Use with non-aqueous mobile phase that is chosen based on sample solubility. Applications To separate biological molecules (amino acid, virus, carbohydrate). To determine molecular weights and