Open Column Chromatography, GC, TLC, and HPLC Murphy, B. (2017). Introduction to Chromatography: Lecture 1. Lecture presented at PHAR 423 Lecture in UIC College of Pharmacy, Chicago. USES OF CHROMATOGRAPHY AND SPECTROSCOPY Chromatography = process of separating compounds to isolate a specific molecule Spectroscopy = identifies a specific molecule Starting with a mixture of unknowns, you can use chromatography and a reference standard to try and identify substances Starting with a mixture of compounds, can use chromatography to separate and isolate a molecule and then spectroscopy to identify this molecule If retrieving a molecule from a biological system, use spectroscopy to localize the molecule and deduce a mechanism of action CHROMATOGRAPHY Utilizes a stationary phase and a mobile phase to separate compounds in a mixture Most common mechanisms of separation: o Partition method utilizes like-dissolves-like theory o Adsorption interaction at a surface (stationary phase) with certain compounds, while others get pulled out with the stationary phase Interactions are relatively weak, including dipole-dipole, dipole-induced dipole, and induced dipole- induced dipole o Exclusion porous stationary phase separates molecules by size Liquid chromatography (LC) vs. Gas chromatography (GC) o LC = solution is passed through a system with an adsorbent stationary phase and a liquid mobile phase. Solutes in the solution will separate based on if they are adsorbent to the stationary phase or not. Mobile phase passes through the column using pressure or just gravity Utilizes differing POLARITIES to separate these solutes Utilizes SOLUBILITY of the solute to keep in mobile phase o GC = gas passes through a system with an adsorbent stationary phase and a gaseous mobile phase. Volatiles in the gas will separate based on if they are adsorbent to the stationary phase or not. Mobile phase passes through column using pressure Utilizes differing POLARITIES to separate these volatiles Utilizes VOLATILITY of the gas to keep in mobile phase
Column can be glass or metal o Metal used for gas and high performance liquid chromatography o Glass used for column chromatography Stationary phase o Composed of organic molecules fused to particles (usually silica) o In normal phase chromatography the stationary phase is POLAR and HYDROPHILIC Groups = -OH, -NH2, CN, etc o In reverse phase chromatography the stationary phase is NON-POLAR and HYDROPHOBIC Groups = C8, C18, phenyl, phenyl-hexyl, etc Mobile phase o Liquid that is composed of different chemicals to alter polarity o In normal phase chromatography the mobile phase is relatively (in comparison to stationary phase) NON-POLAR and HYDROPHOBIC Groups = isopropanol > ethyl acetate > chloroform > dichloromethane > hexanes (starting from most hydrophobic to least) o In reverse phase chromatography the mobile phase is relatively (in comparison to stationary phase) POLAR and HYDROPHILIC Groups = water > acetonitrile > methanol (starting from most hydrophilic to least) o Important note** - the strength of the solvent (mobile phase) is based on how closely it resembles the stationary phase. The more closely it resembles the stationary phase, the stronger it is (b/c can compete with stationary phase for solutes) o Flows: Isocratic flow same composition throughout the entire duration of the separation Step gradient flow isocratic steps (gets progressively more polar or nonpolar with each addition of solvent) Gradient flow almost the same as step-gradient flow except this continues progressively over time rather than in steps (needs a machine to be accomplished, so HPLC or GC) Band widening occurs as one analyte separates from another throughout the column o If different bands overlap (broadening is too slow) won t be able to separate the analytes o To improve band separation, may need a stronger solvent (mobile phase)
o **Important if you want to use a stronger solvent to increase band separation, do not go TOO much stronger. If needed something to be more polar, for example, for a normal phase chromatography, still wouldn t want to use an actual polar solvent. Just need something slightly more polar (but still relatively non-polar in comparison to the stationary phase) than the current solvent THIN LAYER CHROMATOGRAPHY Stationary phase (usually silica gel) coats a plate, and is placed in a container with the mobile phase (liquid) The mobile phase travels up the plate and solutes bind to the stationary phase depending on relative polarity The plate can be visualized with various methods in order to see where different analytes have bound to the stationary phase COLUMN CHROMATOGRAPHY Different column types: o Open/gravity column = pack column with solid stationary phase and let gravity pull down the mobile phase o Flash column = uses pressure from the top to push the mobile phase down o Vacuum liquid column = uses vacuum at the bottom of the column to pull the mobile phase down
Solid phase extraction column = comes pre-packed with stationary phase o Usually used to quickly run a solution through and get rid of unwanted material before running through an HPLC column o Is disposable Small particle size gives better resolution, but would need more pressure to pull mobile phase down HPLC High Performance Liquid Chromatography uses moderate to high pressure to push solvent through the column Ultra Performance Liquid Chromatograph uses extremely high pressures to push solvent through the column with very small particles in the stationary phase As column length increases, resolution increases o There is more time for the solvent to be in contact with the stationary phase As column particle size decreases, resolution increases o There are more chances for the solvent to interact with the stationary phase (more particles) COMPONENTS OF HPLC INSTRUMENTATION Injection port to inject the analyte Mobile phase Controller to set parameters of the experiment Pumps hooked up to the solvent to push it through Column UV detector to identify the samples Degasser use to get rid of bubbles in the solvent Autosampler allows you to run multiple samples in the same sequence SCALES FOR HPLC Preparative o For larger sample sizes > 5 mg o Use flow rate > 10mL/min Semi-preparative o For sample sizes > 0.5-5 mg o Use flow rate > 1-3 ml/min Analytical o For smaller sample sizes > 0.01-0.5 mg o Use flow rate > 0.2-1 ml/min
THE CHROMATOGRAM Gives you a readout for what s in your sample o X-axis is time o Y-axis is absorbance Can compare your readout with reference compounds to try and identify the analyte o MUST be using the same system in order to compare (same stationary phase, same mobile phase) Can use to isolate pure compounds GEL PERMEATION/ SIZE EXCLUSION CHROMATOGRAPHY Particles in the stationary phase has holes in them so that smaller solutes in the mobile phase will get trapped there and larger molecules will bypass the stationary phase Used for protein separations o If you know the MW of the protein you want to isolate, can determine which size pores you will need in your stationary phase AFFINITY CHROMATOGRAPHY Stationary phase is made custom to bind a particular analyte