Liquid Chromatography - HPLC PDF

Separation techniques BCMC 2304 Prof. Dr. Faten A. Nour El-Dien Liquid Chromatography LC Prof. Faten A. Nour Eldien Classification of chromatographic methods Column chromatography 12/30/2024  Liquid Chromatography The types...

Separation techniques BCMC 2304 Prof. Dr. Faten A. Nour El-Dien Liquid Chromatography LC Prof. Faten A. Nour Eldien Classification of chromatographic methods Column chromatography 12/30/2024  Liquid Chromatography The types of liquid chromatography are often classified by the separation mechanism or by the type of stationary phase. These include :  (1) adsorption, or liquid-solid, chromatography;  (2) partition, or liquid-liquid, chromatography;  (3) ion-exchange, or ion, chromatography;  (4) size-exclusion chromatography;  (5) affinity chromatography; and  (6) chiral chromatography. Prof. Faten A. Nour El-Dien 12/30/202 4 Prof. Faten A. Nour Eldien  A Well Backed Column Column efficiency increase by:  Decreasing the volume of the sample.  Decreasing the thickness of stationary phase.  Decreasing the particle size of packing material. High pressure enable the use of stationary phase of small particle size to allow proper separation at reasonable flow rate Prof. Faten A. Nour Eldien High P ressure L iquid C hromatography High Performance L iquid C hromatography  In high-performance liquid chromatography (HPLC) we inject the sample, which is in solution form, into a liquid mobile phase. The mobile phase carries the sample through a packed or capillary column that separates the sample’s components based on their ability to partition between the mobile phase and the stationary phase. Prof. Faten A. Nour El-Dien 12/30/202 4  liquid mobile phase + Sample Sample (solution) carries the sample through Inject into capillar Pumping pressures y liquid mobile colum phase n the mobile partition between phase and the stationary phase. Prof. Faten A. Nour El-Dien Detecto 12/30/202 4 r  Mobile Phases Instrumentation Flow Rate Composition Injection System Column Oven Temperature Detetor  HPLC instrumentation The instrument consists of: 1- Mobile-Phase Reservoirs 2-Pumping Systems 3- Sample Injection Systems 4- Columns 5- Detectors A modern HPLC instrument is equipped with one or more glass Sparging is a process in which dissolved gases reservoirs. are swept out of a solvent by bubbles of an inert, insoluble gas. Prof. Faten A. Nour Eldien Prof. Faten A. Nour El-Dien 12/30/202 4  Elution process isocratic elution 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. Prof. Faten A. Nour El-Dien 12/30/202 4  Gradient elution has many advantages over isocratic elution. It shorten the time of separation Prof. Faten A. Nour El-Dien 12/30/202 4 Gradient elution separation of a mixture of flavonoids. Mobile phase A is an aqueous solution of 0.1% formic acid and mobile phase B is 0.1% formic acid in acetonitrile. The initial mobile phase is 98% A and 2% B. The percentage of mobile phase B increases in four steps: from 2% to 5% over 5 min, beginning at 0.5 min; from 5% to 12% over 1 min, beginning at 5.5 min; from 12% to 25% over 15 min, beginning at 6.5 min; and from 25% to 60% over 20 min, beginning at 21.5 min. Sample Injection Systems  The most widely used method of sample introduction in liquid chromatography is based on a sampling loop. Prof. Faten A. Nour El-Dien 12/30/202 4 Columns for HPLC An HPLC typically includes two columns: an analytical column responsible for the separation and a pre- column. The pre- column is placed before the analytical column, protecting it from contamination. 1- Analytical Columns  The most common type of HPLC column is a stainless steel tube with an internal diameter between 2.1 mm and 4.6 mm and a length between 30 mm and 300 mm. Prof. Faten A. Nour El-Dien 12/30/202 4  Backing of stationary phase Normal phase packing Reversed phase packing  Polar stationary phase  Non polar stationary  Low polar mobile phase phase.C18 columns (e.g. octadecyl siloxane, -C18H37)  Polar mobile phase Prof. Faten A. Nour El-Dien 12/30/202 4 Two problems tend to shorten the lifetime of an analytical column.  First, solutes binding irreversibly to the stationary phase degrade the column’s performance by decreasing the available stationary phase.  Second, particulate material injected with the sample may clog the analytical column. To minimize these problems we place a pre-column before the analytical column. Prof. Faten A. Nour El-Dien 12/30/202 4  Precolumns Scavenger column Guard column Guard columns usually contain the same particulate packing material and stationary phase as the analytical column, but are significantly shorter and less expensive Prof. Faten A. Nour El-Dien 12/30/202 4 Precolumns Two types of precolumns are used.  A precolumn between the mobile phase reservoir and the injector is used for mobile- phase conditioning and is termed a scavenger column. A scavenger column is used to condition the mobile phase. (Saturates mobile phase with liquid of stationary phase ) Prof. Faten A. Nour El-Dien 12/30/202 4  Precolumns A second type of precolumn is a guard column, positioned between the injector and the analytical column. The purpose of the guard column is to prevent impurities. A guard column removes particulates and other solvent impurities. The guard column increase the lifetime of the analytical column. Prof. Faten A. Nour El-Dien 12/30/202 4  Detectors Solute property : (property of the analyte is detected)  UV-visible absorbance  Fluorescence  Electrochemical  Mass spectral (MS/MS) Bulk property: (property of the solvent is detected)  Refractive index  Conductivity  Density Prof. Faten A. Nour Eldien  Classification of Liquid chromatographic methods We can divide chromatography into categories based upon the interaction of the solute with the stationary phase.  1- Adsorption Chromatography Solutes separate based on their ability to adsorb to a solid stationary phase. Prof. Faten A. Nour El-Dien 12/30/202 4  2- Partition Chromatography The most widely used type of HPLC is partition chromatography in which the stationary phase is a second liquid that is immiscible with the liquid mobile phase. A thin liquid film coating a solid support serves as the stationary phase. Separation is based on a difference in the equilibrium partitioning of solutes between the liquid stationary phase and the mobile phase. Prof. Faten A. Nour El-Dien 12/30/202 4  Difference between Normal and Reveres phase The order of polarities of common mobile phase solvents are: water. acetonitrile. methanol. ethanol. tetrahydrofuran 27. propanol. cyclohexane. hexane. Prof. Faten A. Nour El-Dien 12/30/202 4 Prof. Faten A. Nour El-Dien 12/30/202 4 Prof. Faten A. Nour El-Dien 12/30/202 4  Ion Exchange Chromatography Stationary phases consisting of a solid support with covalently attached anionic (e.g., –SO3 –) or cationic (e.g., –N(CH3)3+) functional groups are used. Ionic solutes are attracted to the stationary phase by electrostatic forces. Prof. Faten A. Nour El-Dien 12/30/202 4 Structure of a cross linked polystyrene ion-exchange resin Prof. Faten A. Nour Eldien Prof. Faten A. Nour El-Dien 12/30/202 4 Polyvalent ions are much more strongly retained than singly charged species  For monovalent cation the order is : Ag + > Cs+ > Rb+ > K+ > NH4+ > Na+ > H+ > Li+.  For divalent cations the order is : Ba2+ > Pb2+ > Sr2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+ > Zn2+ > Mg2+ > UO22+ Prof. Faten A. Nour El-Dien 12/30/202 4 Prof. Faten A. Nour Eldien Applications Typical applications of ion chromatography. (a) Separation of anions on an anion-exchange column. Eluent: 0.0028 M NaHCO3 /0.0023 M Na2CO3. Sample size: 50 mL. Prof. Faten A. Nour El-Dien 12/30/202 4 (b) Separation of alkaline earth ions on a cation-exchange column. Eluent: 0.025 M phenylenediamine dihydrochloride/ 0.0025 M HCl. Sample size: 100 mL. Prof. Faten A. Nour El-Dien 12/30/202 4  4- Molecular Exclusion Chromatography In size-exclusion chromatography, porous gels are used as stationary phases, in which separation is due to differences in the size of the solutes. Large solutes are unable to penetrate into the porous stationary phase and so quickly pass through the column. Smaller solutes enter into the porous stationary phase, increasing the time spent on the column. Prof. Faten A. Nour El-Dien 12/30/202 4  Size-Exclusion Chromatography Gel filtration Gel permeation  is a type of size  is a type of size- exclusion exclusion chromatography in chromatography in which the packing which the packing material is hydrophilic. material is hydrophobic.  It is used to separate  It is used to separate polar species. nonpolar species. Application SEC  The separation of fatty acids with molecular mass M from 116 to 344  Column: polystyrene based, 7.5 x 600 mm.  Mobile phase: tetrahydrofuran Prof. Faten A. Nour El-Dien 12/30/202 4 Application of SEC  Separation of high-molecular-weight , natural- product molecules from low-molecular-weight species.  Separation of proteins from amino acids and low-molecular-weight peptides. Prof. Faten A. Nour Eldien Advantage of SEC 1 - Short separation time 2 - Good sensitivity 3 - Freedom from sample loss 4 - Absence of column deactivation Prof. Faten A. Nour Eldien 5-Affinity Chromatography Affinity ligand molecule : antibodies, enzyme inhibitors, …….etc The stationary phase: is a solid, such as agarose, or a porous glass bead Selectively bind solute molecules of interest. Prof. Faten A. Nour El-Dien 12/30/202 4  After the undesired molecules are removed, the retained analytes can be eluted by changing the mobile phase conditions.  The stationary phase for affinity chromatography is a solid, such as agarose, or a porous glass bead to which the affinity ligand is immobilized. Prof. Faten A. Nour El-Dien 12/30/202 4 The mobile phase in affinity chromatography has two distinct roles to play.  First, it must support the strong binding of the analyte molecules to the ligand.  Second, once the undesired species are removed, the mobile phase must weaken or eliminate the analyte-ligand interaction so that the analyte can be eluted. Often, changes in pH or ionic strength are used to change the elution conditions during the two stages of the process. Prof. Faten A. Nour El-Dien 12/30/202 4 Example: When a mixture of many proteins is passed through the column only the protein that binds the antibody is retained.  Once other proteins have eluted, the pH or ionic strength can be changed to dislodge the analyte protein from the antibody. Prof. Faten A. Nour El-Dien 12/30/202 4 Applications of liquid chromatography. Methods can be chosen based on: solubility and molecular mass  In many cases, for small molecules, reversed-phase methods are appropriate. Prof. Faten A. Nour El-Dien 12/30/202 4 Prof. Faten A. Nour El-Dien 12/30/202 4  Questions Prof. Faten A. Nour El-Dien Prof. Dr. Faten A. Nour El-Dien