Chromatography Essentials

Cheatsheet Content

1. Introduction to Chromatography Definition: A laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase , which carries it through a structure holding another material called the stationary phase . Principle: Components of the mixture separate due to differences in their differential partitioning between the mobile and stationary phases. Key Terms: Mobile Phase: Solvent that carries the sample. Can be liquid or gas. Stationary Phase: Material that selectively retards components. Can be solid or liquid (coated on a solid support). Analyte: The substance being separated. Chromatogram: The visual output of the separation. 2. Basic Separation Mechanisms Adsorption: Solutes adsorb to the surface of the stationary phase. Separation based on differential adsorption/desorption. Partition: Solutes partition (distribute) between two immiscible liquid phases (stationary liquid, mobile liquid/gas). Separation based on solubility differences. Ion Exchange: Separation of charged molecules based on their affinity to an ion-exchange resin. Size Exclusion (Gel Filtration): Separation based on molecular size. Larger molecules elute first. Affinity: Separation based on specific biological interaction (e.g., enzyme-substrate, antigen-antibody). 3. Key Parameters & Equations Retention Factor ($R_f$): Used in planar chromatography (TLC). $$R_f = \frac{\text{distance traveled by solute}}{\text{distance traveled by solvent front}}$$ $0 \le R_f \le 1$. Higher $R_f$ means weaker interaction with stationary phase. Retention Time ($t_R$): Time taken for a specific analyte to pass through the column. $t_R = t_M + t_S$ (mobile phase time + stationary phase time) Dead Time ($t_M$ or $t_0$): Time for an unretained component to pass through the column. Adjusted Retention Time ($t'_R$): $t'_R = t_R - t_M$ Capacity Factor ($k'$): Measures retention relative to the column dead time. $$k' = \frac{t_R - t_M}{t_M} = \frac{t'_R}{t_M}$$ A higher $k'$ indicates stronger retention. Selectivity Factor ($\alpha$): Measures the ability of the system to distinguish between two analytes. $$\alpha = \frac{k'_2}{k'_1} \quad (\text{where } k'_2 > k'_1)$$ For separation, $\alpha > 1$. Plate Number ($N$): Measures column efficiency. Higher $N$ means better separation. $$N = 16 \left( \frac{t_R}{W} \right)^2 \quad \text{or} \quad N = 5.54 \left( \frac{t_R}{W_{1/2}} \right)^2$$ (where $W$ is peak width at base, $W_{1/2}$ is peak width at half height) Height Equivalent to a Theoretical Plate ($H$ or $HETP$): $H = L/N$ (where $L$ is column length). Smaller $H$ means higher efficiency. Resolution ($R_s$): Measures the degree of separation between two peaks. $$R_s = \frac{2(t_{R2} - t_{R1})}{W_1 + W_2}$$ $R_s \ge 1.5$ is often considered baseline separation. 4. Common Chromatography Techniques 4.1. Gas Chromatography (GC) Mobile Phase: Inert gas (e.g., He, N$_2$). Stationary Phase: Liquid coated on a solid support or solid adsorbent. Sample: Volatile compounds. Detectors: Flame Ionization Detector (FID), Thermal Conductivity Detector (TCD), Mass Spectrometry (MS). Applications: Separation of organic compounds, environmental analysis, forensic science. 4.2. Liquid Chromatography (LC) Mobile Phase: Liquid solvent. Stationary Phase: Solid particles or liquid coated on solid particles. 4.2.1. High-Performance Liquid Chromatography (HPLC) Principle: Uses high pressure to force mobile phase through a column packed with small particles. Types: Normal Phase (NP-HPLC): Polar stationary phase, non-polar mobile phase. Separates polar compounds. Reversed Phase (RP-HPLC): Non-polar stationary phase, polar mobile phase. Most common type. Separates non-polar/moderately polar compounds. Ion-Exchange HPLC: Separates charged species. Size Exclusion HPLC: Separates based on size. Detectors: UV-Vis, Refractive Index (RI), Fluorescence, Mass Spectrometry (MS). Applications: Pharmaceutical analysis, food analysis, environmental analysis, biochemistry. 4.3. Thin-Layer Chromatography (TLC) Mobile Phase: Liquid solvent (eluent). Stationary Phase: Thin layer of adsorbent (e.g., silica gel, alumina) coated on a plate. Procedure: Sample spotted on plate, plate placed in solvent, solvent rises by capillary action. Visualization: UV light, chemical stains. Applications: Qualitative analysis, reaction monitoring, purity check, rapid screening. 4.4. Paper Chromatography Mobile Phase: Liquid solvent. Stationary Phase: Water adsorbed on cellulose paper. Principle: Partitioning between water and organic solvent. Applications: Separation of inks, amino acids, sugars. (Less common in modern labs). 5. Factors Affecting Separation Mobile Phase Composition: Affects elution strength, selectivity, and retention times. Stationary Phase Chemistry: Determines interaction type (polar/non-polar, ion-exchange). Temperature: Affects viscosity of mobile phase, solubility, and kinetics of interaction. Column Length/Diameter: Affects plate number and back pressure. Flow Rate: Affects retention time and efficiency (Van Deemter curve). Particle Size (LC): Smaller particles lead to higher efficiency but higher back pressure. 6. Van Deemter Equation (Column Efficiency) $$H = A + \frac{B}{u} + Cu$$ $H$: Plate height (HETP) $u$: Linear velocity of the mobile phase $A$: Eddy diffusion term (multiple paths) $B/u$: Longitudinal diffusion term (diffusion against flow) $Cu$: Mass transfer term (resistance to mass transfer in stationary and mobile phases) Optimal Flow Rate: Minimum of the curve, where $H$ is smallest. 7. Troubleshooting Common Issues Broad Peaks: Overloading, poor column packing, detector issues, extra-column volume. Tailing Peaks: Secondary interactions with stationary phase, active sites, column voids. Fronting Peaks: Column overloading, solvent mismatch. Baseline Noise: Detector instability, mobile phase impurities, temperature fluctuations. Retention Time Drift: Mobile phase composition changes, column degradation, temperature changes.