lecture 2- chromatographic theory

2 types of chromatography

planar and column

planar chromatography

• solid stationary phase attached to a flat, 2 dimensional backing,

• Sample applied to the stationary phase and transported through mobile phase

• Sample separated due to affinity with mobile and stationary phase

column chromatography

- solid stationary phase packed into column

- sample forced through column using mobile phase or carrier gas

-sample separates based on its affinity to the stationary phase

Concentration distribution

- conc of analyte in mobile phase/analyte in stationary phase

- An ideal isotherm implies a linear relationship between the analyte's concentration in the two phases

- resulting in symmetrical, bell-shaped: gaussian peak shape

- non-linear response= results not in gaussian peak shape - tailing or fronting

causes of non-linear isotherms

• Overloading: Too much analyte saturates the stationary phase → reduced interaction → peak fronting (analyte less retained, earlier elution).

• Column Voids: Detached or damaged stationary phase → inconsistent analyte interactions → peak broadening and tailing.

• Strong Analyte-Phase Interactions: Excessive adsorption to active sites → peak tailing (analyte more retained, delayed elution).

causes of fronting and how to solve

Fronting: diffused front and sharp tail

- sample overload → use split injection or reduce sample concentration

- low temp increases analyte interaction with the stationary phase → increase temp

- sample solvent not compatible with mobile phase causes poor mixing → dissolve sample in mobile phase or choose a more compatible solvent

langmuir isotherm

As mobile phase concentration increases, analyte interaction with the stationary phase decreases

due to overloading of the stationary phase

causes convex curve in absorption isotherm

indicates peak tailing

anti-langmuir isotherm

• Initial Phase: Slow increase in stationary phase concentration at low analyte concentration (many free sites, low adsorption rate).

• Rapid Increase: As analyte concentration rises, stationary phase overloads but still adsorbs more analyte rapidly due to high analyte availability.

• Overloading: Adsorption becomes inefficient; sites fill quickly, causing a steep rise in stationary phase concentration.

• Causes peak fronting and a concave isotherm curve.

causes of tailing and how to solve

- saturation of stationary phase causes uneven binding → decrease sample concentration

- wrong pH of mobile phase, affects charge of analyte, affecting interaction with the stationary phase → change pH by adding ionisable acids or bases

- column voids cause uneven flow → fill void or replace column

- blocked frit causes uneven flow of mobile phase and analyte → replace frit

- mobile phase contamination affects analyte behaviour → replace mobile phase

causes of split peaks and how to solve

• Improper Sample Injection: Sample is injected too quickly or with too much volume → Use split injection, reduce sample volume, and optimise injection speed.

• Contamination on Guard Column: Impurities affect sample flow → Replace guard column (cheaper than replacing entire column).

• Column Void: Empty space or air bubble in column → Fill the void or replace the column.

• Contamination on Column Inlet: Impurities disrupt sample entry → Replace the column or clean the inlet.

• Wrong mobile phase or Sample Solvent Incompatible with Mobile Phase: Poor mixing → Use mobile phase as the sample solvent for better mixing.

• Unresolved Analytes: Poor separation due to similar characteristics → Optimise mobile phase, use a more selective column, reduce sample concentration, and increase column length.

causes of ghost peaks and how to solve

• Contamination or Impurities: Unexpected peaks appear from previous samples or system contamination.

• Impurities in Injector: Leftover sample causes ghost peaks → Flush injector thoroughly between analyses.

• Late Eluting Peaks: Analytes stuck on the column elute later than expected → Run a strong solvent through the column to wash them out.

column separations

- dead time (time it takes for solvent to pass through stationary phase)

- retention time: How long an analyte takes to pass through the column.

- retention factor (k'): Measures how long an analyte is retained relative to dead time; assesses separation quality.

retention factor interpretation

- if 0 = analyte has no affinity to stationary phase and travels at same rate as mobile phase - dead time

- if less than 2 = all samples elute quickly, lots of peaks at start but nothing at other points, peaks may crowd together

- if 2-5 = ideal gaussian peaks

- if above 10 = analytes elute slowly, peaks become broadened due to long time in column

phase ratio

Controls how long analytes interact with the stationary phase.

• Higher β → Shorter retention time, more peak broadening

• Small β → Better resolution, higher sensitivity, more efficient

selectivity factor

a measure of how well the chromatography system can separate two analytes

α= retention factor of first analyte (less retained) / retention factor of second analyte (more retained)

• α > 1: Good separation (analytes have different retention).

• α = 1: No separation (same retention time).

• α < 1: Poor separation (co-eluting analytes).

how to change selectivity in RP-HPLC

• Change Temperature:

  • Higher Temp: Shorter retention time (lower interactions).

  • Lower Temp: Longer retention time (stronger interactions).

• Change Mobile Phase pH:

  • Affects the charge of analytes.

  • Alters interaction strength with the stationary phase.

• Change Stationary Phase:

  • Different stationary phases have different polarities.

  • Changes how analytes interact with the phase

• Change Solvent Strength:

  • Stronger solvents: Faster elution, less interaction.

  • Weaker solvents: Stronger retention, more interaction.

• Change Polarity:

  • Alters interactions between analytes and stationary phase.

factors affecting column resolution

1. Injection Volume

   • Large Volumes: Can overload the column, leading to poor resolution.

   • Small Volumes: Improve separation and resolution.

2. Distribution Constants (K)

   • High K: Stronger retention in the stationary phase can result in longer retention times, but if too high, it can lead to band broadening and poor resolution.

   • Low K: Faster elution and less retention in the stationary phase, but too low can result in poor separation and overlapping peaks.

   • Ideal K: Moderate values allow for good separation and sharp peaks, without the drawbacks of overly long or short retention times.

3. Band Broadening

   • Causes wider peaks and reduced resolution.

   • Minimize band broadening for sharper peaks and higher resolution.

4. Number of Theoretical Plates (N)

   • More plates = Better resolution, sharper peaks, and more efficient separation.

5. Height of Theoretical Plates (HETP)

   • Lower HETP = Better resolution (sharper, more distinct peaks).

   • Higher HETP = Poorer resolution (broader peaks).

Column resolution

ability of a chromatography column to separate two analytes based on their retention times.

Higher resolution means sharper, well-separated peaks.

• optimum value is 1.5, anything below gives unresolved peaks

improving column resolution

- increase number of plates improves separation

- particle size in packing: Smaller particles create more surface area, leading to better separation

- reduce solvent viscosity improves flow rate and enhances resolution.

- GC= change temp programming

- HPLC= change solvent programming

- change column: different dimensions, stationary phase, or packing material can improve resolution

resistance to mass transfer (Cu)

resistance that an analyte experiences as it moves between the mobile and stationary phases

If analytes don’t move efficiently between phases, resolution and separation quality is reduced

Reduced by:

• Smaller Particles: Increases surface area, improving interaction between analyte and stationary phase.

• Slower Flow Rates: Provides more time for analytes to diffuse and interact.

• Increased Temperatures: Reduces viscosity, making it easier for analytes to move through the column.

• Smaller Diameter Columns: Reduces the distance analytes need to travel, improving mass transfer efficiency.

eddy diffusion (A)

dispersion of analytes caused by multiple flow paths through a packed column, leading to uneven movement of the analyte.

Caused band broadening and reduces resolution

Minimised by:

• Smaller stationary phase particles for more uniform flow paths.

• Proper packing of the column to avoid voids and uneven flow.

• Uniform column design with consistent particle sizes.

- problem in HPLC

longitudinal molecular diffusion (B/u)

- analyte molecules move from areas of high concentration to low concentration along the length of the column

- causes band broadening

- problem in GC

- minimised by:

• Low Temperatures: Reduce molecular movement, limiting diffusion.

• High Pressure of Carrier Gas (GC): Increases flow rate, reduces diffusion time.

• Fast Flow Rate: Reduces the time for analytes to diffuse.

• Short and Narrow Tubing: Decreases the distance analytes travel, reducing diffusion.

Van Deemter Equation

describes the relationship between column efficiency (measured in terms of the height equivalent to a theoretical plate (HETP)) and several factors that influence chromatographic separation. It is a mathematical model that helps us understand and optimise the factors affecting band broadening in chromatography.

• At low flow rates: Dominated by longitudinal diffusion (B/u), leading to broad bands.

• At high flow rates: Dominated by resistance to mass transfer (C*u), leading to broader bands.

• Optimal flow rate (uopt): Minimum HETP, best column efficiency.

HPLC Types

- high performance partition chromatography

- liquid-liquid

• Stationary Phase: Liquid bonded to silica particles through absorption.

• Limitation: Can be broken under certain conditions.

- liquid-bonded

• Stationary Phase: Liquid bonded covalently to silica particles.

• Advantage: Remains attached within the column, reducing column voids.

core shell packing

Particles have a solid core (usually silica) surrounded by a thin shell of stationary phase.

• Reduced diffusion distance: Faster analyte migration.

• Increased efficiency: Combines small particle advantages with lower flow resistance.

• Higher resolution: Improves column efficiency and resolution.

• Faster separations: Better performance at higher flow rates.

reverse phase chromatography

• Stationary Phase: Non-polar (e.g., C18, C8)

• Mobile Phase: Polar, typically water mixed with organic solvents like acetonitrile or methanol.

• Separation Principle:

  • Non-polar analytes interact strongly with the non-polar stationary phase.

  • Polar analytes elute faster as they interact less with the stationary phase.

• Applications: Used for separating a wide range of compounds, especially hydrophobic compounds such as lipids, peptides, and proteins.

Normal phase

• Stationary Phase: Polar (e.g., silica).

• Mobile Phase: Non-polar, typically organic solvents like hexane,

• Separation Principle:

  • Polar analytes interact strongly with the polar stationary phase.

  • Non-polar analytes elute faster as they interact less with the stationary phase.

• Applications: Used for separating polar compounds, such as small organic molecules, pharmaceuticals, and natural products.

UHPLC or UPLC

• Key Difference: Higher efficiency and speed compared to traditional HPLC due to smaller particle sizes (sub-2 μm) and higher pressure systems.

• Advantages:

  • Faster Separations: Reduced analysis time with higher resolution.

  • Better Sensitivity: Improved detection limits due to sharper peaks.

  • Higher Pressure: Can operate at pressures up to 15,000 psi or more.

  • Enhanced Column Efficiency: Results in improved resolution and peak shapes.

• Applications: Common in pharmaceuticals, biotech, and food analysis for rapid, high-resolution separations.

HPLC instrumentation

• Mobile Phase Storage (Schott Bottles): Holds the solvents used in the mobile phase.

• Degasser: Removes dissolved gases (like air) from the mobile phase. The fixed splitter valve allows helium to flow into the frit, degassing into the mobile phase for smoother flow.

• Solvent Proportioning Valve: Essential in solvent programming; changes the composition of the mobile phase during gradient elution.

• Reciprocating Pumps: Have a small cylindrical chamber, providing high output pressure and ensuring constant flow rates of the mobile phase.

• HPLC Valve & Sample Loop: Used to inject a precise volume of the sample into the column.

• Guard Column: Protects the main column from contaminants and debris, prolonging its lifespan.

• HPLC Columns: Contain the stationary phase (e.g., C18), where the separation of analytes occurs.

• HPLC Detector: Measures and identifies the analytes as they elute from the column.

• UV-Vis Detector: Common detector in HPLC, detects compounds based on their absorbance of UV/Vis light.

UV-vis detector

Measures the absorption of UV or visible light by analytes as they elute from the column.

• Light Source: Commonly a deuterium lamp for UV (190-400 nm) and a tungsten lamp for visible light (400-800 nm).

• Flow Cell: Contains the analyte solution; the light passes through the sample, and the amount absorbed is measured.

• Detector: Measures the difference in light intensity before and after passing through the sample.

Types of UV-vis detector

• Single Wavelength Detector: Measures absorption at a fixed wavelength (e.g., 254 nm).

• Dispersive Monochromator: Selects a specific wavelength using a monochromator.

• Diode Array Detector (DAD): Measures multiple wavelengths simultaneously, useful for spectral analysis.