Chromatography: Separation and Analysis

• Chromatography is an advanced method for separating, purifying, identifying, and quantifying mixture components
• Chromatography separates a mixture's components based on their distribution between stationary and mobile phases

Types of Chromatography

• Planar chromatography uses paper or thin layers
• Column chromatography uses columns
• High performance liquid chromatography (HPLC), gas chromatography (GC), and supercritical fluid chromatography (SFC) are based on the mobile phase's physical state
• The Russian botanist Mikhail S. Tswett first described chromatography in the early 1900s
• Tswett separated plant pigments using a calcium carbonate column with petroleum ether as the mobile phase
• Various industries can use chromatography to separate and purify products from complex mixtures
• Chromatography can separate similar components precisely, such as proteins differing by a single amino acid
• Chromatography can purify any soluble or volatile substance with the right material, fluid, and conditions
• Chromatography separates delicate products under typically mild conditions

Preparative and Analytical Chromatography

• Analytical chromatography measures the relative proportions of analytes in a mixture on a small scale
• Preparative chromatography isolates separated components on both small and large scales
• Chromatography is used for purification
• Liquid chromatography is used in biotechnology
• Liquid chromatography separates the target molecule from host-related contaminants
• Liquid chromatography analyzes the final product for purity according to governmental regulatory groups like the FDA

Basic Terminologies in Chromatography

• Chromatography separates components between a stationary and a mobile phase
• A chromatogram is a detector response graph showing analyte concentration vs. effluent volume or time
• In planar chromatography, "chromatogram" refers to the paper or layer with the separated zones
• The stationary phase is a solid, gel, or liquid within the chromatographic system
• A bonded phase is covalently bonded to support particles or column tubing
• An immobilized phase is immobilized on support particles or column tubing, often through in situ polymerization
• The mobile phase is a fluid percolating through the stationary bed
• Liquid chromatography uses liquid mobile phases, gas chromatography uses gas mobile phases, and supercritical-fluid chromatography uses supercritical fluid mobile phases
• Efluent (or eluate) is the mobile phase exiting the column
• The sample is the mixture to be separated
• The baseline is any chromatogram part with only mobile phase emerging from the column
• The peak maximum is the highest point of the peak

Key Time/Position Definitions

• The injection point marks sample placement time on the column
• The dead point is where an unretained solute peaks
• Dead time (to) is elapsed time between the injection and dead points
• A dead time marker is an unretained substance like uracil, used to find the dead time
• Retention time (tr) is the time between injection and peak maximum; each solute has a unique retention time
• The dead volume (Vo) measures the mobile phase volume passing through the column between the injection and dead points
• Vo = Q * to, where Q is the flow rate in ml/min
• The retention volume is Vr, the mobile phase volume passing between the injection point and the peak maximum, where Vr = Q * tr
• Corrected retention time (t'r) indicates time elapsed from the dead point to the peak maximum
• Corrected retention volume (V'r) is mobile phase volume between the dead point and peak, which equals the retention volume minus the dead volume: V'r = Vr - Vo = Q(tr - to)
• The retention factor (k) is the ratio of time an analyte spends in the stationary vs. mobile phase
• k' = (tR - t0)/t0 calculates the capacity factor, where tR is the retention time of the peak and t0 is the column's dead time
• Peak height (h) is the vertical distance from peak maximum to baseline
• Peak width at base (WB) is distance between the intersections of tangents at peak sides with the baseline
• Resolution (Rs) quantitatively measures separation of two elution peaks as Rs=2[(tB-t₄)] / (WA+WB)
• B denotes the later-eluting species, with t and W indicating the retention time and peak width, respectively
• Resolution above one typically indicates effective separation
• The selectivity factor (α) measures relative retention of solutes, defined using α = kB / kA

Chromatography Theories

• Plate theory describes equilibrium between stationary and mobile phases, assuming infinitely fast equilibration
• Rate theory accounts for the time needed for solute to equilibrate between phases

Plate Theory

• The plate model assumes the column contains many layers called theoretical plates
• Sample equilibrations occur separately in these plates
• The analyte moves through the column via transfers of mobile phase
• This theory measures column efficiency via plate number (N) or Height Equivalent to a Theoretical Plate

Rate Theory

• The rate theory accounts for factors affecting plate height and band broadening and describes the equilibrium processes at work in a column
• HETP = A + B /u + Cu expresses the Van Deemter equation for plate height
• 'u' represents average mobile phase velocity, and A, B, and C are factors contributing to band broadening

Band Broadening Factors

• Eddy diffusion causes band broadening because molecules take varied, random paths through the stationary phase
• Longitudinal diffusion broadens the band as analyte molecules diffuse from the band's center to its edges
• A higher mobile phase velocity reduces longitudinal diffusion effects
• Resistance to mass transfer broadens bands when high mobile phase velocity and strong analyte affinity for the stationary phase prevent equilibration
• The higher the velocity of mobile phase, the worse the broadening becomes

Van Deemter Plots

• Van Deemter plots help determine optimum mobile phase flow rate
• Plate height is assessed against average linear velocity of mobile phase

Classification of Chromatographic Methods Based on Bed Shape

• Column chromatography places the stationary phase in a tube, and the mobile phase moves through under gravity or pressure
• The stationary phase is solid or liquid film on packing or column walls
• The solid stationary phase can either fill the whole tube (Packed Column) or concentrated on the inside tube wall (Open-Tubular Column)
• Planar chromatography coats a flat glass, metal, or plastic plate with the stationary phase; the mobile phase moves via capillary action in a developing chamber
• The bed in planar chromatography might be a paper or a solid particle layer on a support

Classification by Mobile Phase Physical State

• Gas chromatography uses a gas as the mobile phase and is always conducted in a column
• Liquid chromatography uses a liquid, carried out in a column or on a plane
• Supercritical-Fluid Chromatography (SFC) uses a fluid above/near its critical temperature and pressure as the mobile phase

Classification According to Development Procedure

• Frontal chromatography feeds the sample continuously into the chromatographic bed without additional mobile phase
• Displacement chromatography uses a mobile phase containing a strongly retained compound (the Displacer) and feeds the sample as a finite slug
• Elution chromatography (isocratic and gradient) continuously passes mobile phase, feeding the sample as a slug

Classification by Separation Mechanism

• Adsorption chromatography separates based on the adsorption affinities of sample components on an active solid surface
• Separation occurs due to differing attractions to the stationary vs. mobile phase ("solid / liquid or solid / gas") Adsorption is where a gas or liquid solute accumulates on a surface to form an atomic film
• Partition chromatography separates based on the solubility differences in the stationary and/or mobile phases
• It uses distribution between two liquid phases in liquid chromatography to separate

Ion Exchange Chromatography

• Retains analytes based on their charge to separate
• Usually performed in columns but can also be useful in planar mode
• Uses a charged stationary phase to separate charged compounds like anions, cations, amino acids, peptides, and proteins
• Uses an ion exchange resin as the stationary phase, that carries charged functional groups
• Used to purify proteins using FPLC

Size Exclusion Chromatography

• Has no attractive interaction between the stationary phase and solute
• Uses liquid or gas which phases through a porous gel to separate by size
• Allows smaller molecules to pass and excludes larger molecules
• The technique is gel-filtration chromatography (GFC) when using aqueous solutions vs gel permeation chromatography (GPC), used when organic solvents act as mobile phases

Affinity Chromatography

• Employs a specific interaction between one solute molecule and a second molecule immobilized on the stationary phase
• Used in biochemistry to purify proteins that are bound to tags
• Can use immobilized antibodies and fusion proteins
• Eluted by changing ionic strength or pH

Chiral Chromatography

• Separates stereoisomers when conventional chromatography can not
• Makes either the mobile or stationary phase chiral
• Uses chiral chromatography HPLC columns to achieve separation

Chromatography Equipment

• Commonly uses a column, even though planar methods exist
• A column usually consists of a glass or metal tube, which contains the stationary phase
• Includes mobile and stationary phases, for which affinities vary
• Materials vary based on chromatography type

Column Types

• Packed bed columns consist of granular stationary phase filling the column homogeneously
• Open tubular columns have a thin film or layer of stationary phase on the column wall
• Their center has a passageway

Basic Operation of Chromatography

• Processes occur within a column made of glass or metal
• Packed columns contains particles to make up the stationary phase
• Open tubular columns are lined up with a thin film stationary phase
• Uses a solvent that moves through the column
• Mobile phases are liquid or gas
• The stationary phase is a viscous liquid coating solid particles
• Solutes travel into and out of the column in a partitioning process

Process Steps

• Sample injection injects the sample into the mobile phase
• Mobile phase flows through the column via a pump or capillary action
• Separation occurs as components adsorb to the stationary phase
• Elution then occurs
• Elution from the column occurs as different components elute at different times, separating the various components
• Detection occurs with equipment to analyse content

Planar Chromatography

• Izmailov and Schraiber discovered it in 1938
• It is useful for detecting and separating amino acids
• Filter paper strips support a stationary water phase
• A mobile organic phase moves down the suspended paper strip in a cylinder
• Separation is based on liquid-liquid partition; essentially, partition chromatography between two phases

Ascending Paper Chromatography

• The solvent travels up the chromatographic paper
• The solution containing a mix of amino acids gets applied three cm from the end
• The filter paper is then equilibrated inside a cylinder containing solvent
• A common solvent ratio is N-butanol / acetic acid / water with ratio 4:1:5

Other Types of Paper Chromatography

• Descending involves the solvent permitting through the filter
• Radial chromatography places filter paper wicks into sample to rise
• Two-dimensional paper chromatography involves 2 separation steps and a square sheet of filter

Paper Chromatography Analysis: Locating Compounds

• Strip is removed when the solvent has migrated the available space and marked
• Completed chromatograms feature as colorless with no obvious separation without being developed
• The strip is first dried, then sprayed with 0.5% ninhydrin in acetone and then heated at 80-100° C
• For radioactive compounds, detection occurs with a Geiger-counter

Identifying and Quantifying Compounds

• Rf = Distance from origin run by compound / Distance from origin run by solvent
• Compare these and run a calorimetric chemical composition to determine concentration

Chromatography Forces in Operation

• In PC and TLC, two counteracting forces act on sample components
• Propelling forces are mainly Capillary action or the solubility
• Retarding is gravitational or partition

Thin Layer Chromatography

• For separating and analyzing
• Components can be analyzed, and the purity of components can be determined
• Is TLC is sensitive, microgram quantities can be analyzed
• It takes around 5-10 minutes
• The stationary phase in TLC is made as a powder or medium

Method Steps

• TLC consists of spotting, development and a plate with the compound
• As solvent moves, the plate will be in competition
• The silica plate tries the spot and the solvent takes the spot across
• Silica is very polar
• Visualization involves a detector that can detect compounds by shining a UV light, this light interferes

How to Measure RF factor

• With measurement of the compounds
• Rf measures extend of molecules along the TLC plate
• The silica might be like if Unknown molecule (X) = (0.12, 0.25, and 0.87)

Silica Gel Use

• Silica gel is great, but the polarity has to be balanced for best efficacy
• For instance, if a silica gel plate contains ethyl acetate
• The gel contains O-H (OH groups) on external surface with OH formula

Equilibrium and Polarity in TLC

• Polarity and size are key factors
• There is need to alter the equilibrium for good measure
• Solvents need to achieve a separation factor
• Non-polar compounds travel in more polar compound

Mobile Phases and Functionality

• Start with a non-polar solvent, but use something more polar to move components more
• You need the right balance between solvent to get a reaction
• Finding this balance can be the hardest bit
• You need to move components

TLC Efficiency

• Selectivity from Adsorbents must be towards certain substances
• Silica is great adsorbent with bonds