Chromatography Notes

Introduction to Chromatography

• Derived from Greek words: "Chroma" (color) and "graphy" (writing).
• Technique for separating mixtures to analyze, identify, purify, and quantify components.

Chromatographic Separations

• Based on forced transport of liquid (mobile phase) carrying analyte mixture through porous media.
• Differences in interactions of analytes with porous media cause different migration times.
• Mobile Phase: Transports the analyte.
• Stationary Phase: Immobile phase.

Uses for Chromatography

• Analyze: Examine mixture components and their relations.
• Identify: Determine identity of a mixture based on known components.
• Purify: Separate components to isolate one of interest.
• Quantify: Determine amount of a mixture and its components.

Classification of Chromatographic Processes

• Gas Chromatography: Gas-Solid, Gas-Liquid
• Liquid Chromatography: Planar (TLC), Column (CEC, RP, IEX, SEC, NP)

Brief History of Chromatography

• 1901: Michael Tswett invented chromatography for plant pigments.
• 1938: Thin Layer Chromatography by N.A. Izamailov and M.S. Shraiber.
• 1941: Liquid-Liquid partition chromatography by Archer John, Porter Martin and Richard Laurence Millington Synge.
• 1944: Paper Chromatography development in biotechnology.
• 1945: Gas Chromatography developed by Fritz Prior.
• A. J. P. Martin and R. L. M. Synge: Awarded Nobel Prize in 1952 for partition chromatography.
• Prof. C. Horvath: Originated the term High Performance Liquid Chromatography (HPLC).

Components of Performance (C. Horvath)

• Reproducibility
• Automation
• Speed
• Versatility
• Efficiency
• Complete Control Over Operational Variables
• Selectivity
• Sensitivity
• Data Handling

Advantages of HPLC

• High speed, resolution, and sensitivity.
• Reusable column; no component destruction.
• Automatic, computerized instrumentation.
• Complete sample recovery.
• More accessible and sensitive Quantitative analysis.

Instrumentation of HPLC

• Reservoir for solvents (mobile phase).
• High-pressure pump.
• Sample inlet device.
• Column.
• Detector.
• Recorder.

HPLC System Components

• Solvent Reservoirs: Store HPLC solvents; may include degassing and filters.

Mobile Phase

• Usually organic, aqueous, or a mixture.
• Placed in glass bottles.

Characteristics of Mobile Phase

• Pure, low viscosity, chemically inert, compatible with the detector.
• Can solubilize the sample; low cost.
• Miscible with water (e.g., acetonitrile, methanol, isopropanol).

Treatment of Mobile Phase

• Filtration before entering the column.
• Degassing (heating with stirring, vacuum, $N_2$ or He, ultrasound).
• Pre-saturation with the stationary phase (liquid-liquid chromatography).

UV Transparency of HPLC Solvents

• Acetonitrile: 190 nm
• Isopropyl alcohol: 205 nm
• Methanol: 205 nm
• Ethanol: 205 nm
• Uninhibited THF: 256 nm
• Ethyl acetate: 268 nm

Solvent Polarity in HPLC

• Water (10.2) > Dimethyl sulfoxide (7.2) > Ethylene glycol (6.9) > Acetonitrile (5.8) > Methanol (5.1) > Acetone (5.1) > Dioxane (4.8) > Ethanol (4.3) > Tetrahydrofuran (4.0) > I-propanol (3.9)

Elution Techniques

• Isocratic: Mobile phase composition remains constant.
• Gradient: Mobile phase composition changes during separation.
  • Continuous (linear).
  • Discontinuous (stepwise).

Advantages of Gradient Elution

• Shortens analysis time.
• Reduces tailing, gives sharp peaks.
• Increases sensitivity.
• Decreases retention of later-eluting components.

Pump

• Provides constant mobile phase flow; modern pumps mix solvents.
  • Syringe Pumps: Pulse-free, small capacity, no gradient elution.
  • Reciprocating Pumps: Widely used, small volume, high-pressure, gradient elution, needs pulse damper.

Injector

• Introduces analyte mixture into mobile phase before column.
• Modern injectors are autosamplers.
• Sample Inlet Device (Injection Port)
  • Manual injection
  • Automated injection
• Rheodyne injector has fixed volume loop, load and inject modes.

Column

• Separates analytes in the mixture.
• Interface between mobile and stationary phases.
  • Analytical: 1-6 mm i.d.
  • Preparative: Up to 3 cm i.d.
  • Material: Stainless steel.
  • Shape: Straight.
  • Length: Variable.

Detector

• Registers physical/chemical properties of column effluent.
• UV (ultraviolet) detectors are common in pharmaceutical analysis.

Detectors

• Refractive index detectors
• U.V detectors
• Fluorescence detectors
• Electrochemical detectors
• Evaporative light scattering detectors
• IR detectors
• Photo diode array detector

Data Acquisition and Control System

• Computer-based; controls eluent composition, column temperature, injection sequence.
• Acquires detector data; monitors system performance.

HPLC System Overview

• Reservoir -> Pump -> Injector -> Column -> Detector -> Data System

Stationary Phases - Classification

• Column packing materials are the media producing the separation.
  1. Type (monolithic; porous; nonporous)
  2. Geometry (surface area; pore volume; pore diameter; particle size and shape; etc.)
  3. Surface chemistry (type of bonded ligands; bonding density; etc.)
  4. Type of base material (silica; polymeric; zirconia; etc)

Type of Packing Materials

• Porous: Most common, diameters between 3 and $10 \mu m$.
• Nonporous: Increases efficiency, decreases adsorbent surface area.
• Monolithic: Increases column permeability, decreases gap in column dual porosity.

Base Material

• Silica ($SiO_2$): Most common, uniform, but soluble at high pH.
• Zirconia: Stable over a wide pH range (1-14), but has low reactivity.
• Polymers: High pH stability and chemical inertness.

Silica as Packing Material

• Used for normal phase chromatography and with chemical modification for reversed phase, ion exchange, chiral, and size exclusion chromatography.
• High surface area leads to high efficiency.

Factors in Manufacturing Silica-Based Columns

• Purity (metal ion content)
• Sol-gel or xerogel silica type
• Deactivation/surface treatment
• Hybridization with alkyl groups
• Shape (spherical or irregular)
• Particle size
• Particle size distribution

Metal Ion Content

• Causes peak tailing due to interactions with metal/silanol groups.
• Surface metal ions act as chelating agents.
• Remedy: Acid wash the silica to remove metal ion contamination.

Particle Type - Sol-Gel Silica

• Formed via agglomeration of small silica particles to form 1.8-10 $µm$ sols.
• Generally have lower surface area, porosity, and surface reactivity.

Particle Type - Xerogel Silica

• Silica sol subjected to heat treatment, creating rigid porous silica.
• Higher surface area and porosity.
• Dissolve readily in the mobile phase at pH 7 and above.

Particle Shape

• Irregular and spherical.
• Irregular particles have poorer efficiency than spherical particles due to packing homogeneity.

Particles Size Distribution

• Analytical columns have particle sizes from 1.8 to 10 $µm$ in diameter.
• Popular particle sizes are 1.8-5 $µm$.
• Narrower distribution increases column efficiency.

Hybridization

• Silica substrate becomes susceptible to hydrolysis at high pH.
  REMEDY: Hybrid Silica
• Manufactured from organic (alkylsiloxane) and inorganic (silane) monomers.
• Good stability at high pH; reduced silanol surface groups improve peak shape.

Particle Size

• Smaller particles = better efficiency (higher plate number, N).
• Smaller particles lead to higher back pressures. *Approach 1: use smaller particles but keep the flow rate the same as the original application. *Approach 2: use the smaller particles in short columns at high flow rates.
  • This type of column is known as a fast or high throughput column and is available for high throughput applications needing moderate efficiency.

Mechanisms of Separation in Liquid Chromatography:

1. Adsorption:
   • The stationary phase is an adsorbent and the separation is based on adsorption-desorption steps.
   • Example: Normal Phase Liquid Chromatography
   • Polar forces are the dominant type of molecular interactions employed in NPLC.
2. Partitioning:
   • The stationary phase is a liquid or a liquid support by a solid.
   • Example: Reversed Phase Liquid Chromatography
   • Principle is similar to solvent-solvent partitioning.
3. Ion-exchange/Ionic Interaction:
   • The stationary phase is an ion-exchanger.
   • Example: Ion-exchange Liquid Chromatography
   • Separation is achieved because of differences in charge densities.
4. Mechanical Entrapment:
   • The stationary phase is a polymer with different pores (pore sizes).
   • Example: Size Exclusion Chromatography
   • Separation is achieved because of differences in sizes.

Scale of Polarity

• Characterized by Structure and Electron Charge Distribution:
  • Polar molecules include salts, acids, alcohols, ketones, ethers
    • Scale: Polar Molecule (+ end) -> Non-Polar Molecule (- end)
    • Sample Analytes: Aliphatic Hydrocarbons, Halogenated/Fluorinated Hydrocarbons

Polarity Scale of Mobile Phase

• Polar: Water, Alcohol
• Non-Polar: Alcohol, Water, Acetronitrile, THF, Hexane

Polarity Scale of Stationary Phase

• Competition between stationary and mobile phase for different analytes creates separation.
• Changes in rates of speed of analytes based upon different attractions.

Chromatographic Retention Behavior

• Competition for analytes between mobile and stationary phases with different polarities affects movement.
• Analytes attracted to the mobile phase will move faster.
• Analytes attracted to the stationary phase will slow down.

Modes of Liquid Chromatography

• NORMAL PHASE LIQUID CHROMATOGRAPHY (NPLC)
• REVERSED PHASE LIQUID CHROMATOGRAPHY (RPLC)
• ION-EXCHANGE CHROMATOGRAPHY (IEC)
• SIZE EXCLUSION CHROMATOGRAPHY (SEC)
• OTHERS

Size Exclusion Chromatography

*Separates molecules based on size.
*Based on sample size relative to pore size.
*No interactions with packing material surface.

Types of SEC:

*Gel permeation chromatography (GPC)
    *Separation of synthetic polymers.
*Gel filtration chromatography (GFC)
    *Separation of water-soluble biopolymers.

Applications of SEC:

*Determining the m.w. distribution of polymers
*Preparative fractionation of polymers.
*Purification of biological samples.
*Desalting biological materials.

Modes of Separation in SEC:

*Group Separation
*Remove small molecules from a group of larger molecules.
*Often used in protein purification schemes for desalting and buffer exchange.
*Sephadex G-10, G-25, and G-50 are used for group separations.
*High-resolution fractionation
*Separate multiple components in a sample;

Stationary Phases for Gel Filtration Chromatography:

*SEPHADEX
    *made from dextran that had been polymerized with epichlorohydrin
    *for fractionating mixtures of proteins, peptides, and the smaller nucleic acids and polysaccharides
    *By varying the degrees of cross linking, gels of different porosities and different fractionation ranges are obtained.

Lipophilic Sephadex:

*Sephadex LH-20
    * a beaded, cross-linked dextran which has been hydroxypropylated to yield a chromatographic media with both hydrophilic and lipophilic character
    *swell in water and a number of organic solvents.
*Designed for use in aqueous buffer systems, polar organic solvents, and aqueous solvent mixtures.
*fractionation of lipids, steroids, fatty acids, hormones, vitamins, and other small molecules.

Stationary Phases:

• SEPHACRYL prepared by covalently cross-linking allyl dextran with N,N-methylene bisacrylamide to
  give a highly stable matrix.
  *aqueous buffer systems, pH 2-11, in concentrated urea or guanidine HCI, and in a number of organic solvents.

Superdex:

*are media consisting of a composite base matrix of dextran and agarose

*Its low nonspecific interaction permits high recovery of biological material.

Superose

*are SEC media with high physical and chemical stability based on highly cross-linked porous agarose particles.
*mechanical rigidity of Superose allows even viscous eluents, such as 8 M urea, to be run at relatively high flow rates.

Stationary Phases for Gel Permeation Chromatography:

*Highly crosslinked Styrene-divinyl benzene - Organic solvents can be used; therefore, a wide variety of compounds can be separated.
*Bio-gel-P a polyacrylamide polymer cross-linked with methylene bisacrylamide.
*Enzacryl - polymers of nitrogen-containing compounds crosslinked with acrylamide.

Mobile Phases for SEC:

*Usually aqueous; buffer system
*Addition of Denaturing (Chaotropic) Agents and Detergents -Urea or guanidine hydrochloride is very useful for molecular weight determination.

Instrumentation for SEC

*Injection valve
*Tubing
*Sample
*UV/Vis absorbance
*Fraction collector

Applications:

*Proteins are separated by molecular weight.
*Separation of Carbohydrates

ION EXCHANGE CHROMATOGRAPHY (IEX)

*An ion exchange process in which the desired ions are exchanged in sequence and are eluted from a column.
*Separation is based on ionic interaction.

Two Distinct Mechanisms:

*Ion exchange due to competitive ionic binding (attraction)
*Ion exclusion due to repulsion between similarly charged analyte ions and the ions fixed on the chromatographic support

Experiment setup:

*Application of a protein mixture to polymer beads.

Applications:

*Inorganic ions, biomolecules especially oligonucleotides, carbohydrates, carboxylic acids

Ion Exchange Chromatograph consists of:

*a high pressure pump
*an injector
*a column
*a detector
*a data system.

Stationary phases in IEX:

*Charged group involved in exchange process, and matrix on which charged group is fixed.
*Stationary phases are either anion or cation exchangers

Stationary phases used for IEX include:

*Sulfonic acid
*Carboxylic acid
*Quaternary amine
*Primary, Secondary, Tertiary amine

Examples of Materials for the Matrix:

*Polystyrene-divinylbenzene copolymers have very hydrophobic surface and proteins are irreversibly damaged due to strong binding
*Cellulose has a Hydrophilic surface, enhanced stability

Factors which effect chromatographic resolution :

Porosity of matrix effect chromatographic resolution
Size and Size Distribution Factors affect chromatographic resolution
Pore Size directly affect the binding capacity for a particular analyte (protein)

Mobile Phases in IEX

*Consist of an aqueous solution of a suitable salt or mixtures of salts with a small percentage of an organic solvent.
*The competing ion which has the function of eluting sample components through the column within reasonable time is the essential component of the mobile phase.
*at low concentrations, ordinary temperatures, and constant valence, the extent of exchange increases with increasing atomic number of the exchanging ion

Rate and temperature:

*Faster flow rates cause interaction therefore solute elution is more rapid. Increased temperature generally leads to increased interaction also.
*Additives: Usually added to stabilize protein structures and to enhance solubility.

The following buffers are:

Used for Cation-exchange and anion-exchange chromatography

Detection in IEX:

*Electrical Conductivity Detector
*Amperometric detection
*Photometric Detection
*Mass Spectrometry

Applications of of IEX. Anion/Cation using using anion and Cation exchange