Comprehensive Study Notes: HPLC & LC-MS Techniques

Core Principles and Components of High-Performance Liquid Chromatography (HPLC)

• Overview of HPLC:

  • HPLC stands for High-Performance Liquid Chromatography.
  • In HPLC, a liquid mobile phase carries a dissolved sample through a column packed with a stationary phase.
  • Physical separation occurs because sample compounds interact differently with the stationary phase and mobile phase, causing them to travel through the column at different migration rates.
  • HPLC is particularly suitable for non-volatile, thermally unstable, and higher-molecular-mass compounds, including peptides and biological molecules.
  • The most common HPLC detector used in standard applications is the UV/Vis detector.

• Systematic Flow Path of HPLC:

  • Reservoir $\rightarrow$ Degasser $\rightarrow$ Pump $\rightarrow$ Injector $\rightarrow$ Column $\rightarrow$ Detector $\rightarrow$ Data system.

• Primary System Components and Main Functions:

  • Mobile-phase reservoir: Stores the solvent or solvent mixtures used as the mobile phase.
  • Degasser: Removes dissolved gases from the mobile phase to reduce bubble formation within the fluidics.
  • Pump: Drives the mobile phase through the system at a controlled high pressure and constant flow rate.
  • Injector / autosampler: Introduces a precise, reproducible sample volume into the pressurized mobile phase stream.
  • Column / column oven: The column contains the stationary phase and performs the actual chemical separation; the column oven controls and maintains standard operating temperatures.
  • Detector: Monitors analyte elution and converts the physical/chemical response into an electronic signal.
  • Data processor: Records, integrates, and processes the raw electronic signals into chromatograms and analytical data.

Chromatographic Calculations and Efficiency Parameters

• Essential Calculations:

  • Retention factor ($k$):     $k = \frac{t_R - t_0}{t_0}$
  • Selectivity factor ($\alpha$):     $\alpha = \frac{k_2}{k_1}$
  • The later-eluting peak is designated as $k_2$, ensuring that $\alpha > 1$.
  • Resolution using baseline peak widths ($R_s$):     $R_s = \frac{2(t_{R,2} - t_{R,1})}{W_{b,1} + W_{b,2}}$
  • Resolution using half-height peak widths ($R_s$):     $R_s = \frac{2(t_{R,2} - t_{R,1})}{1.7(W_{1/2,1} + W_{1/2,2})}$
  • Number of theoretical plates ($N$):     $N = 16 \left(\frac{t_R}{W_b}\right)^2$$N = 5.54 \left(\frac{t_R}{W_{1/2}}\right)^2$
  • Height Equivalent to a Theoretical Plate ($HETP$):     $HETP = \frac{L}{N}$

• Term Definitions and Interpretation:

  • $t_R$: Retention time of the specific analyte.
  • $t_0$: Dead time or unretained compound elution time.
  • $k$: Retention factor, measuring how strongly and how long the analyte is retained by the stationary phase. A larger $k$ indicates stronger retention.
  • $\alpha$: Selectivity factor, describing the column's ability to discriminate between two distinct analytes.
  • $R_s$: Resolution, measuring the extent or degree of quantitative separation between adjacent peaks.
  • $N$: Column efficiency; higher values of $N$ correspond to narrower peak widths and superior efficiency.
  • $HETP$: Height equivalent to a theoretical plate; lower $HETP$ values signify better column efficiency.

• Remediation of Unnecessarily High Resolution:

  • If resolution is excessively high and leads to prolonged analysis times, efficient optimization includes:
  • Reducing column length ($L$).
  • Increasing mobile phase flow rate.
  • Adjusting mobile-phase solvent composition to slightly decrease selectivity ($\alpha$).
  • Increasing column temperature to accelerate analyte elution.

Qualitative and Quantitative HPLC Analysis

• Qualitative Analysis:

  • Focuses on determining compound identity ("What compound is present?").
  • Methods of identification include:
  • Retention-time matching with authentic standards.
  • Spectral data comparison (e.g., UV/Vis spectral matching via photodiode arrays).
  • Standard addition techniques.
  • Multi-detector verification.

• Quantitative Analysis:

  • Focuses on determining compound concentration or amount ("How much is present?").
  • Relies on correlating peak area or peak response to analyte concentration using calibration protocols.

• Step-by-Step Standardization Protocol:

  1. Prepare a series of calibration standards with precisely known concentrations.
  2. Inject each standard and measure the resulting peak area.
  3. Construct a calibration plot of peak area versus concentration.
  4. Apply linear regression analysis to derive the standard calibration equation.
  5. Substitute peak area values from unknown samples into the equation to calculate concentration.

• Standard Quantification Methodologies:

  • External Standard Method: Calibration standards are prepared, injected, and analyzed in separate runs prior to or alongside the sample.
  • Internal Standard Method: A known, constant quantity of a reference compound is added to all standards and unknown samples; concentration is determined using peak response ratios.
  • Standard Addition Method: Known quantities of the target analyte are spiked directly into aliquots of the unknown sample to mitigate and compensate for matrix effects.

Mobile Phase Chemistry and Elution Dynamics

• Mobile Phase Functions and Properties:

  • The mobile phase consists of solvent or solvent mixtures continuously pumped through the stationary phase.
  • Selection directly governs retention time, selectivity, separation resolution, system backpressure, detector response, and overall runtime.

• Solute and Polarity Calculations:

  • Binary-mixture polarity index ($P_{AB}$):     $P_{AB} = \phi_A P_A + \phi_B P_B$
  • Where $\phi_A$ and $\phi_B$ are the volume fractions of solvents A and B, and $P_A$ and $P_B$ are their respective polarity indices.

• Elution Modes:

  • Isocratic Elution: The mobile-phase solvent composition remains constant throughout the entire chromatographic run.
  • Gradient Elution: The mobile-phase solvent composition changes continuously or in step-wise fashion during the run.

• Solvent Strength Rules:

  • Retention Control:
  • To decrease retention time: Apply a stronger mobile phase.
  • To increase retention time (often improving peak resolution): Apply a weaker mobile phase.
  • Reversed-Phase HPLC (RP-HPLC):
  • Stronger solvent: Higher organic/non-polar solvent content.
  • Weaker solvent: Higher water/polar solvent content.
  • Normal-Phase HPLC (NP-HPLC):
  • Stronger solvent: Higher polar solvent content.
  • Weaker solvent: Higher non-polar solvent content.
  • Interactions:
  • Strong solvents interact aggressively with analytes, eluting them rapidly (yielding shorter retention times and often reduced resolution).
  • Weak solvents allow analytes to interact more extensively with the stationary phase (yielding longer retention times and enhanced resolution).

• Mobile Phase Selection Criteria:

  • Solvents must be selected according to key physical and chemical parameters:
  • UV transparency / UV cut-off wavelength.
  • Solvent miscibility.
  • Viscosity.
  • Purity.
  • Chemical stability and shelf life.
  • Safety, toxicity, and flammability profile.
  • Chemical inertness and hardware compatibility.
  • Cost and environmental disposal requirements.

• Miscibility and Degassing Mandates:

  • All solvent components in a mixture must be completely miscible with one another.
  • When purging or switching mobile phases, the incoming solvent must be fully miscible with the previous solvent present in the fluidics.
  • Degassing solvent lines yields reproducible retention times, stable pump operation, smooth baselines, and enhanced detector sensitivity.

Planar and Preparative Chromatography: TLC and Column Chromatography

• Thin Layer Chromatography (TLC):

  • Stationary phase: A thin layer of silica gel or alumina coated onto a solid backing plate.
  • Mobile phase: A liquid solvent or mixture that ascends the plate via capillary action.
  • Separation mechanism: Analytes strongly adsorbed to the stationary phase migrate slowly, whereas weakly adsorbed analytes travel faster.
  • Primary uses: Assessing the number of sample components and confirming compound identity against reference standards.
  • Key advantages: Inexpensive, straightforward execution, rapid processing, and high sensitivity.
  • Retardation factor ($R_f$):     $R_f = \frac{\text{distance travelled by compound}}{\text{distance travelled by solvent front}}$
  • $R_f$ is dimensionless with typical values ranging between $0$ and $1$
  • Visualization techniques: Colourless compounds are visualized under short/longwave UV illumination or by exposure to iodine vapour.

• Preparative Column Chromatography:

  • Used to purify and isolate chemical compounds on a significantly larger mass scale than TLC.
  • Common stationary phases: Silica gel or alumina packed inside a column matrix.
  • Elution profile on polar silica: Less polar components migrate faster and elute before highly polar components.
  • Step-by-step procedure:
  1. Pack the column evenly with stationary phase.
  2. Condition the column matrix with solvent.
  3. Load/introduce the sample mixture onto the top of the bed.
  4. Elute the analytes sequentially using selected solvents.
  5. Collect isolated fractions sequentially across distinct time intervals.
  • Advantages: Cost-effective, disposable stationary phase options, versatile for analytical and preparative purifications.
  • Disadvantages: Labor-intensive, time-consuming setup.
  • Primary applications: Fine chemical synthesis, pharmaceutical manufacturing, food analysis, dye/pigment isolation, fabric processing, and forensic examinations.

HPLC Stationary Phase Modes: Normal Phase vs. Reversed Phase

• Normal Phase (NP) vs. Reversed Phase (RP) Comparison:

  • Normal Phase (NP):
  • Stationary Phase: Highly polar (e.g., bare silica).
  • Mobile Phase: Relatively non-polar (organic solvents).
  • Typical Retention Behavior: Polar analytes interact strongly and are retained longer; non-polar analytes elute rapidly.
  • Usage Frequency: Less common.
  • Reversed Phase (RP):
  • Stationary Phase: Non-polar (most commonly octadecylsilane, C18, chemically bonded to silica particles).
  • Mobile Phase: Polar solvents (e.g., water, methanol, acetonitrile mixtures).
  • Typical Retention Behavior: Non-polar analytes interact strongly and are retained longer; polar analytes elute rapidly.
  • Usage Frequency: Most widely utilized operational mode in HPLC.

• Separation Mechanics:

  • Separation governed by relative polarities according to the principle "like dissolves like."
  • Major operational stationary phase modes in LC include: Normal Phase, Reversed Phase, Size Exclusion, Ion Exchange, and Affinity.

• Physical Column Packing Parameters:

  • Stationary phase selection depends on target analyte chemistry, mobile/stationary phase compatibility, particle size, pore size, and mechanical strength.
  • Particle Geometry: Spherical particles are vastly superior to irregular particles because they produce lower system backpressure, superior mechanical stability, higher column efficiency, and greater analytical reproducibility.

Size Exclusion and Affinity Chromatography

• Size Exclusion Chromatography (SEC):

  • Stationary Phase: Porous polymer or silica particles engineered with precise, controlled pore sizes.
  • Separation Mechanism: Based purely on physical molecular size (hydrodynamic volume). Ideally, no chemical or electrostatic interactions occur between the analyte and stationary phase.
  • Elution Order:
  • Large molecules cannot enter the structural pores $\rightarrow$ experience a shorter total flow path $ ightarrow$ elute first.
  • Small molecules permeate deep into the pore structure $ ightarrow$ experience a longer effective flow path $ ightarrow$ elute later.
  • Sub-types:
  • Gel Filtration Chromatography (GFC): Uses aqueous mobile phases.
  • Gel Permeation Chromatography (GPC): Uses organic mobile phases.
  • Primary Applications: Fractionation of proteins and water-soluble polymers; determination of molecular weight distributions for synthetic, organic-soluble polymers.

• Affinity Chromatography:

  • Separation Mechanism: Relies on specific, highly selective biological or chemical binding interactions between an analyte and a ligand immobilized on the stationary matrix.
  • Process Flow: The target analyte binds specifically to the immobilized ligand while non-target matrix impurities are washed completely out of the bed; elution parameters (e.g., pH, ionic strength) are then altered to decouple and release the purified target analyte.

Ion-Exchange Chromatography

• Ion-Exchange Mechanisms and Resins:

  • Cation Exchange Chromatography:
  • Stationary Phase: Negatively charged resin bed.
  • Target Analytes: Binds positively charged ions (cations).
  • Anion Exchange Chromatography:
  • Stationary Phase: Positively charged resin bed.
  • Target Analytes: Binds negatively charged ions (anions).

• Targeted Analytes and Advantages:

  • Used for charged biomolecules including proteins, polypeptides, nucleic acids, and synthetic polynucleotides.
  • System benefits: Exceptional resolving power, broad applicability, high loading capacity, simple execution, and precise operational control.

• Four-Stage Operational Sequence:

  1. Equilibration: Establish initial column conditions; charged resin sites associate with mobile counter-ions at a defined baseline pH.
  2. Sample introduction & wash: Load sample; target ions bind to resin exchange sites while unbound contaminants are flushed out.
  3. Elution: Release bound analytes sequentially by increasing mobile phase ionic strength or modifying buffer pH.
  4. Regeneration: Flush column with high-salt wash to remove remaining strongly bound species, re-establishing initial charge capacity.

• Detection and Mobile Phase Parameters:

  • Chemical suppression is widely integrated in ion chromatography (particularly anion exchange) to suppress background eluent conductivity and enhance signal response.
  • Critical mobile-phase variables: Competing ion identity and concentration, buffer charge state, ionic strength, pH, and flow rate.
  • Impact of flow rate: Excessive flow rates lower chromatographic resolution because ions have insufficient contact time to interact with matrix fixed charges.
  • Detectors: Conductivity detectors are standard; UV/Vis detectors are employed when analytes possess suitable chromophores.
  • Elution profile is dictated by the analyte's overall net charge, charge density, and surface charge distribution.

Instrumentation: Solvent Delivery and Sample Introduction

• HPLC Pump Mechanics:

  • Considered the primary core engine ("heart") of the HPLC instrument.
  • Core Functions: Delivers mobile phase smoothly through the column, accurately proportions multi-solvent mixtures, and maintains precise, continuous flow against elevated column backpressures.
  • Performance Standard: Must yield continuous, highly reproducible flow rates alongside smooth gradient profiles.
  • Configuration: Reciprocating piston and diaphragm pumps represent the standard modern design.

• Sample Injection Systems:

  • Replaced manual syringes with injection loops attached to multiport rotary valves.
  • Maintains uninterrupted pressurized system flow while simultaneously loading the fixed-volume sample loop prior to inline injection.
  • Stable pumping performance and reproducible sample delivery directly dictate peak symmetry and retention time reproducibility.

HPLC Detection Systems

• Operational Requirements:

  • Function: Continuously monitors the column effluent and converts physical/chemical property changes into proportional electronic outputs.
  • Desirable Characteristics: Minimal baseline noise, exceptional stability, low limit of detection (LOD), wide dynamic linear range, and reproducible output.

• Detector Types and Characteristics:

  • UV/Vis Absorbance Detector: Most common LC detector; sensitive to compounds containing absorbing chromophores.
  • Refractive Index Detector (RI): Universal detector sensitive to bulk property changes; ideal for non-chromophoric analytes such as carbohydrates, but exhibits lower sensitivity and selectivity compared to UV/Vis, and cannot be used with gradient elution.
  • Photodiode Array Detector (PDA): Measures full spectral range simultaneously, generating 3D chromatograms (intensity, wavelength, time); supports peak purity verification and qualitative spectral identification.
  • Conductivity Detector: Primary detector for ionic species and inorganic ions in ion chromatography.
  • Fluorescence Detector: Extremely sensitive and selective detector for naturally fluorescent analytes or derivatives.
  • Evaporative Light Scattering Detector (ELSD): Universal detector for non-volatile analytes lacking chromophores; insensitive to solvent gradient interference.
  • Electrochemical Detector: Specialized for compounds capable of undergoing oxidation or reduction reactions.

System Optimization and Efficiency Factors

• Operational Objectives:

  • Minimize band broadening to produce high efficiency, sharp, and symmetrical chromatographic peaks.

• Column Parameters Influencing Performance:

  • Theoretical plates ($N$), Height Equivalent to a Theoretical Plate ($HETP$), column length ($L$), internal diameter ($i.d.$), particle size ($d_p$), and mobile phase flow rate.
  • Particle Size Effects: Reducing packing particle size increases available total surface area and improves efficiency/resolution, but exponentially elevates system backpressure.
  • Column Length Effects: Longer column beds provide greater theoretical plates ($N$) and higher resolution, but proportional increases occur in run time and backpressure.
  • Mobile Phase pH: Modifying pH alters analyte ionisation state, significantly altering retention factors ($k$) and selectivity ($\alpha$).

• Fundamental Efficiency Equations:

  • Theoretical plate count:     $N = 16 \left(\frac{t_R}{W_b}\right)^2$
  • Plate height:     $HETP = \frac{L}{N}$
  • Performance metrics: Maximum efficiency requires maximizing $N$ while minimizing $HETP$

HPLC Troubleshooting and Peak Asymmetry Analysis

• Peak Asymmetry Factor ($A_s$) Assessment:

  • Measured at $10\%$ of total peak height.
  • Distance from peak center to leading edge = $a$; distance from peak center to trailing edge = $b$
  • Asymmetry Equation:     $A_s = \frac{b}{a}$
  • Evaluation Metrics:
  • $A_s = 1$: Perfectly symmetrical Gaussian peak.
  • $A_s < 1$: Peak fronting.
  • $A_s > 1$: Peak tailing.

• Systematic Failure Diagnostics Table:

  • No Peaks Observed:
  • Root Causes: Sample injection failure, detector off/malfunctioning, zero flow delivery, broken tubing connections.
  • Corrective Actions: Verify sample preparation/autosampler operation, check detector signal/lamp, confirm pump flow, check plumbing integrity.
  • Peak Tailing ($A_s > 1$):
  • Root Causes: Excessive extra-column dead volume, oversized detector flow cell, fouled/damaged/voided column matrix.
  • Corrective Actions: Re-plumb tubing with zero-dead-volume fittings, install smaller detector flow cell, replace damaged column.
  • Peak Fronting ($A_s < 1$):
  • Root Causes: Column mass overloading, compound solubility failure.
  • Corrective Actions: Reduce sample injection mass/volume, alter sample solvent matrix, check column condition.
  • Excessively High System Pressure:
  • Root Causes: Particulate in mobile phase, clogged pump outlet frit, blocked tubing or inline filter, improper fitting installation, contaminated column bed.
  • Corrective Actions: Filter solvents through $0.2\,\mu m$ filters, replace pump outlet frit, flush/purge tubing lines, reinstall fittings, replace column or install guard column.
  • Excessively Low System Pressure:
  • Root Causes: Solvent leaks, unprimed pump head, empty solvent reservoir, incorrect flow setting.
  • Corrective Actions: Tighten/replace leaking fittings, prime pump heads, refill solvent reservoirs, check method flow rate.
  • Noisy or Cyclic Baseline:
  • Root Causes: Outgassing in flow cell, dirty mobile phase, pump pulse damper failure, electrical noise, lamp instability.
  • Corrective Actions: Degas solvents thoroughly, replace mobile phase, service pump check valves/dampers, replace detector lamp, isolate electrical line.
  • Baseline Spikes:
  • Root Causes: Microbubbles passing through flow cell, loose electrical connection, failing detector lamp, external electrical interference.
  • Corrective Actions: Degas mobile phase, clean/tighten electrical leads, replace lamp, connect instrument to dedicated grounded circuit.
  • Baseline Drift:
  • Root Causes: Unstable column/oven temperature, changing mobile phase composition, lamp warming up, column un-equilibrated.
  • Corrective Actions: Thermostat column compartment, seal mobile phase vessels against evaporation, allow proper lamp warmup, extend column equilibration time.

• Root-Cause Isolation Strategy:

  • Follow systematic diagnostic sequence: Observe specific failure symptom $\rightarrow$ Isolate individual module/source $ ightarrow$ Modify only one variable at a time $ ightarrow$ Re-test system to verify corrective fix.
  • Avoid changing multiple parameters simultaneously, as this masks true failure mechanisms.

Liquid Chromatography-Mass Spectrometry (LC-MS) Principles

• Instrument Hybridization:

  • LC-MS combines the physical separation power of liquid chromatography with the mass detection and structural capabilities of mass spectrometry.
  • Operation: LC separates compounds based on retention parameters; MS ionizes gas-phase molecules and separates them according to their mass-to-charge ratio ($m/z$).

• Interfacing Demands:

  • HPLC operates at atmospheric pressures with liquid solvents, whereas MS mass analyzers require ultra-high vacuum conditions ($10^{-5}$ to $10^{-7}\,Torr$).
  • Interface Purpose: Vaporizes mobile phase, removes excess solvent, and converts dissolved liquid analytes into gas-phase ions under atmospheric pressure conditions (API).

• Output Formats and Analytical Capabilities:

  • Total Ion Chromatogram (TIC): Plots combined total ion abundance across all scanned mass channels versus chromatographic retention time.
  • Mass Spectrum: Plots individual ion abundance versus mass-to-charge ratio ($m/z$) at any single time point in the chromatogram.
  • Quantitation: Peak area integration of targeted ion signals yields quantitative mass measurements.
  • Qualification: Structural identity is confirmed using retention time, precise isotopic mass, and ion fragmentation patterns.

• Analytical Strengths and Constraints:

  • Advantages: Superior measurement sensitivity and selectivity, ability to analyze complex matrices without complete chromatographic separation, concurrent qualitative and quantitative output, highly suitable for polar, non-polar, and thermally labile compounds.
  • Limitations: Substantial capital cost, requires specialized operational expertise, moderate sample throughput, fragmentation dependent on source parameters, destructive detection method, matrix cleanup often required.

LC-MS Interfaces and Mass Spectrometry Techniques

• Major LC-MS System Functional Modules:

  • LC Interface / Ion Source $\rightarrow$ Mass Analyzer $\rightarrow$ Ion Detector $\rightarrow$ Data System.

• Atmospheric Pressure Ionization (API) Sources:

  • Electrospray Ionisation (ESI):
  • Ionization Mechanism: Soft ionization; applies high voltage to a capillary spray needle to generate charged aerosol droplets, forming gas-phase ions directly from solution as droplets evaporate.
  • Target Analytes: Highly polar, ionic, thermally soft molecules, and large biomolecules (e.g., proteins, peptides, oligonucleotides).
  • Atmospheric Pressure Chemical Ionisation (APCI):
  • Ionization Mechanism: Soft ionization; sample solution is thermal-vaporized into a gas, followed by corona discharge-induced chemical ionization at atmospheric pressure.
  • Target Analytes: Medium-to-low polarity, smaller molecular weight, thermally stable compounds.

• Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF):

  • Step-by-Step Analytical Workflow:
  1. Co-crystallize target analyte with excess light-absorbing organic matrix on a target plate.
  2. Irradiate spot with a pulsed laser beam, causing rapid localized desorption and ionization.
  3. Accelerate generated ions using an electrostatic field into a field-free flight tube.
  4. Measure time-of-flight (TOF) required for ions to traverse the flight tube to reach the detector, determining mass based on $\text{TOF} \propto \sqrt{m/z}$.
  • Primary Applications: High-throughput microbial identification, clinical proteomics, intact biomolecule characterization, food authenticity screening, and environmental trace monitoring.

LC-MS Spectral Interpretation and Quantification

• Key Terminology:

  • TIC (Total Ion Chromatogram): Summed signal of all detected mass channels over the chromatographic run.
  • XIC / EIC (Extracted Ion Chromatogram): Plot showing signal intensity of one specific chosen $m/z$ value versus retention time. Used to isolate target signals from overlapping matrix background peaks and resolve co-eluting species.
  • Molecular / Precursor Ion: Ion representing the intact molecular species (often protonated $[M+H]^+$ or deprotonated $[M-H]^-$).
  • Fragment Ion: Charged cleavage products generated when precursor ions break apart internally.
  • Adduct Ion: Ion formed by association of intact analyte with matrix species (e.g., $[M+Na]^+$, $[M+NH_4]^+$).
  • Isotopic Pattern: Specific array of relative peak heights reflecting natural isotopic distributions ($^{13}C$, $^{37}Cl$, $^{81}Br$), serving as a unique elemental fingerprint.

• Quantitative & Qualitative Execution:

  • Quantification is performed by integrating peak areas on Extracted Ion Chromatograms (XIC) corresponding to analyte-specific target ions.
  • Identification requires matching both precise chromatographic retention time ($t_R$) and target $m/z$ values against verified reference standards.
  • Calculations for charged species (e.g., fragment loss $[M - H - CO_2]^-$):
  • Subtract exact molecular masses of missing neutral fragments ($H = 1.008$, $CO_2 = 43.99$) from target mass $M$, dividing by absolute charge state value $|z|$:     $\text{Observed } m/z = \frac{M - 1.008 - 43.99}{1}$

High-Yield Comparative Summary and Examination Guide

• Rapid-Recall Diagnostic Comparisons:
  • Strong vs. Weak Mobile Phase:
  • Strong Phase: Decreases retention time ($t_R$), decreases resolution.
  • Weak Phase: Increases retention time ($t_R$), improves resolution.
  • Normal Phase vs. Reversed Phase:
  • Normal Phase: Polar stationary phase + Non-polar mobile phase.
  • Reversed Phase: Non-polar stationary phase + Polar mobile phase.
  • Size Exclusion Elution Order:
  • Large molecules elute first $\rightarrow$ Small molecules elute last.
  • Ion Exchange Resin Polarity:
  • Cation Exchange: Resin bed is negatively charged (binds cations).
  • Anion Exchange: Resin bed is positively charged (binds anions).
  • Efficiency Metrics:
  • Column Efficiency ($N$): Higher value is better.
  • Plate Height ($HETP$): Lower value is better.
  • Peak Asymmetry ($A_s$):
  • $A_s = 1$: Symmetrical.
  • $A_s > 1$: Tailing.
  • $A_s < 1$: Fronting.
  • LC-MS Data Profiles:
  • TIC: Represents total ion current across all scanned masses.
  • XIC: Extracted ion signal for a single specific $m/z$
  • Qualitative vs. Quantitative Analysis:
  • Qualitative: Identification of compound identity.
  • Quantitative: Measurement of compound mass/concentration.