Chromatography: Terms and Definitions

• Analytical instrumentation measures chemical composition as a variable
• Analytical instrumentation studies the operation of instruments
• Analytical instrumentation consists of Chromatography and Spectroscopy

Chromatography

• Chromatography is a method for separating mixtures by distributing compounds between two immiscible phases

Common Terms and Definitions

• Analyte/Sample: The substance to be separated during chromatography
• Bonded Phase: A stationary phase covalently bonded to support particles or the inside of the chromatography column
• Chromatogram: The visual output of the chromatograph, showing peaks or patterns corresponding to separated mixture components under optimal separation conditions
• Chromatograph: Equipment used for sophisticated separation, like gas chromatography
• Eluate: The mobile phase exiting the chromatography column, moving in a defined direction
• Eluent: The solvent carrying the analyte in chromatography
• Eluotropic Series: Solvents ranked by their eluting power
• Immobilized Phase: A stationary phase fixed on support particles
• Retention Time: The characteristic time for an analyte to pass through the chromatography system from inlet to detector under set conditions
• Solute: Sample components in partition chromatography
• Solvent: A substance capable of solubilizing other substances, especially the liquid mobile phase in Liquid Chromatography(LC)

Etymology

• Root words of chromatography are "Chroma" (colour) and "Graphein" (write) from Latin

History

• Mikhali Tswett, a Russian scientist, invented it in the 19th century

Characteristics

• Chromatography is simple, efficient, and has broad applications
• Chromatography can be preparative (for purification) or analytical

Purpose

• Preparative chromatography separates mixture components for further use and purification
• Analytical chromatography quantifies specific components, typically using smaller material amounts

Phases

• Chromatography separates compounds by distributing them between two immiscible phases
• One phase is a stationary bed, either large surface or bulk area
• The other phase is mobile, passing through or along the stationary phase
• The mixture is carried by the mobile phase
• The stationary phase can be solid or liquid; if liquid, it's adsorbed onto solid phases
• Mobile phase is either gas or liquid, passing through solid particles or a capillary with an adsorbed liquid layer
• Chromatography is classified based on mobile phase.

Types of Chromatography by Mobile Phase

• Gas Chromatography: Gas mobile phase with a Solid Stationary phase
• Adsorption Colour Chromatography: Liquid mobile phase with a Solid Stationary Phase
• Displacement chromatography: Liquid mobile phase with a Solid Stationary Phase
• Thin Layer Chromatography: Liquid mobile phase with a Solid Stationary Phase
• Gas-Liquid Chromatography (GLC): Gas mobile phase with a Liquid Stationary phase
• Partition Chromatography: Liquid mobile phase with a Liquid Stationary phase
• Paper Chromatography: Liquid mobile phase with a Liquid Stationary phase

Paper Chromatography

• Paper chromatography uses a stationary phase of liquid adsorbed on paper (Cellulose - hygroscopic)
• Mobile phase moves compounds along in Paper Chromatography.

Classification Methods for Chromatography

• Classification methods include Liquid, Gas
• Sub-types of Liquid Chromatography include Paper, Thin Layer, Column, Liq-solid adsorption, Liq-liq partition, Gel permeation, Ion exchange
• Sub-types of Gas Chromatography include GLC (partition), GSC (adsorption)

Liquid-Solid (Displacement) Chromatography

• Solid is the stationary phase, liquid is the mobile phase
• A glass column (few cm in diameter, 10-20 cm long) packed tightly with inert filling
• Components compete for binding sites in the stationary phase
• A molecule with high affinity for the chromatography matrix will compete effectively for binding sites, this molecule is called the displacer
• The high affinity solution displaces all molecules with lesser affinities.
• Solutions with compounds to be separated need to be added from the top as thin band.

Liquid-Solid (Displacement) Process

• A and B are adsorbed on solid surfaces
• Solution S displaces both A and B, moving them down the column briefly when introduced
• B is re-adsorbed more readily on other available sites since is relatively more adsorbed.
• This causes A to move further down the column.
• Continuous adding of solution S keeps displacement happening
• The solution S displaces B which further displaces A
• A and B move down the column at different rates
• A moves faster and is separated first following a given time known as retention time.

Required Conditions for Liquid-Solid Displacement

• Different adsorption strengths of compounds for separation
• Solution S needs to be more strongly adsorbed than the compounds
• Strongest adsorption means longest retention time

Analysis

• Qualitative (identification) and quantitative (how much) analysis requires additional instruments
• Refractive index, which varies with retention time, can identify compounds
• Given the solution, solid, tube dimensions, and package length, one can determine how long compounds take to emerge from the column, unless the compounds have the same retention time
• Solid packing usually consists of clay or clay-type substances, mainly aluminates and silicates
• These have many highly polar hydroxyl terminals
• Non-polar solvents like hexane are employed for the mobile phase
• Moderate compounds are dissolved in the mobile phase
• The main polar compounds are retained stronger than less polar compounds as they pass to the column packing
• This method is useful for preparative analysis and colored compound separations due to overlapping compound concentrations

Ion Exchange Chromatography

• Known as affinity chromatography
• Solid stationary phase and liquid mobile phase
• Insoluble but permeable solid phase
• Must contain labile ions for exchange
• Exchange of ions occurs between liquid and ionic solid phase
• Separates ions and polar molecules based on charge
• Used for charged molecules like proteins, nucleotides, and amino acids
• Often used in protein purification, water analysis, and quality control
• It uses equipment similar or identical to column chromatography

Reversibility

• It is a reversible process in the sense that ions may move from solids to liquid and vice versa
• The quantities of the elements involved have to be equivalent

Instrument and Materials

• Materials used are Na2Al2Si3O10-2 where 2 out of 10 atoms carry a negative charge
• These materials are the same as sponges but with charges sticking out all over

Usage

• Instead of inorganic exchangers, use organic resins to avoid getting destroyed by strong acids and bases as inorganic exhcnagers have limited capacity

Process

• Dissolved ions dissolve in solid surface with mobile liquid help, it is fast.
• The ions diffuse through solid matrix to exchange sites inside the solid matrix, speed depends on the solid matrix and is slow.
• Exchange of ions at exchange sites is reversible, ions move between phases.
• The diffusion of displaced ions happens through solid matrix to the surface.
• There is desorption of ions too including their diffusion into mobile phase

Selectivity

• Some ions are exchanged more easily than others
• Exchange increases with concentration but it eventually reaches a leveling-off
• Separation is determined by selectivity co-efficient

Selectivity Co-Efficient

• Consider equilibrium between cation exchange resin H+R (where R represents negative exchange sites of resin matrix and H+ the labile cation) and an ionic solute M with valency n
• M ions in solution exchange with H⁺ for solid

Retention

• The higher the sc, the longer it takes an ion too move through the resin bed
• Ions with low sc emerge first from the column and vice versa

Analysis Types

• Qualitative, Quantitative

Other features

• Cation exchange chromatography retains positively charged stationary phase displaying a negatively charged functional group.
• Anion exchange chromatography retains anions
• Positively charged stationary phase

Liquid-Liquid Chromatography (Partition)

• Separates the sample based on the differences in solubility between sample and differences between the components in the mobile and stationary pahses
• Both the stationary and mobile phases are all liquids
• same equipment as displacement (short glass) is used
• Inert packing is coated with a highly boiling film that strongly adsorbs to the particle surface
• A mobile liquid is passed through the column that has to be selected in such a way that:
• Not dissolve/mix well with stationary phase
• Not adsorb into inert phase as stationary phase
• Components are then partitioned between the mobile and stationary phases

Process

• Start with a solution with added compounds A and B
• then mobile phase S id added on top of the column
• Compounds then make their way down the column slowly
• Due to molecular structure variations between compound A and compound B whereby (b is stronger), the compounds dissolves in the stationary phase at different rates

Advantages

• Because all components are mixed with eluting agent and the process is continued to the point of of completion, partition is suitable for qualitative analysis

Separation

• Kd = Cs (conc. in stationary)/Cn (conc. in mobile)
• The higher the Kd, the longer the retention time and vice versa

4. Gel Permeation Chromatography (GPC)

• GPC separates based on sizes, known as size exclusion chromatography
• GPC is primarily used in polymer separation considering molecule weight and Polydispersity index (PDI)
• Other factors include number average molecular weight (Mn), weight average molecular weight (Mw), size average molecular weight (Mz), or viscosity molecular weight (Mv)
• GPC is the best method to remove food extract fats and waxes when determining pesticide residues
• GPC is a slow process, may take hours since it involves low low rates
• GPC is similar to gel filtration except it utlizes a different polymer gel type which constitutes dextran polymers and cross-linked styrene-divinyl benzene

Phases

• The stationary phase contains solid packing and windows leading into its tortuous internal passages for the solutes in it to get out.(porous bed)
• Any molecule too big cannot pass through the molecule, it get completely washed by by mobile phase

Other features

• In GPC, the void volume is where mobile liquid begins to emerge.
• In GPC, smaller particles take long to pass within the separation range
• In GPC, very small materials merge together and are unseparated if they fail to reach the necessary seperation range
• The column washes out by Vo or large columns
• In GPC, X7,the exclusion limit i.e. X7, is not separated but rather mixes together in Vo.
• Below, we have a lower VO, seperation range and the particles are emerging based on size
• For GPC, The total volume which is defined as (Vo+Vi), this is the permeation volume
• We can measure the absorption and transmision of radiation where there is an instrument at the end and there are compounds 1 and 2 at the end

Analysis

• The instrument analysis is shown on peaks which indicate the compounds emerging including absorbed radiation levels
• The peaks show different compounds emerging and absorbing rediation differently
• O represents a large fraction of the compounds that cannot pas through the window and require the volume, V0 to wash

Capacity factor

• Compound 1 have particles greater than 2
• This is a measure of a compound's retention in terms of the column volume
• It the compound, V1=V0, it essentially means that all compunds were all unretained
• if large K' then compound takes long to emerge

Separation factor

• á is the ratio of net retention time for two given capacity factors
• The factor = 1, it essentially means that 0 seperation between the 2

Evaluation

• There are ways to evaluate cchromatographic systems
• There exists Number of the oretical Plates is another way to evaluate chromatographic system
• where theoretical plate measure efficiency.
• Has borrowed number concept of distillation theory

Resolution

• Distrinution between peak centers of peaks divided by respective bases. This = the difference of peaks divided by bases

Characteristics for evaluation

• It R has distinct peaks then the resolution is high, compounds are clear
• It resolution is not visible, then there might be danger in separation