Ion and Ion-Pair Chromatography

• Miscellaneous chromatography includes ion chromatography, size exclusion chromatography (SEC), supercritical fluid chromatography (SFC), thin layer chromatography (TLC), and electrophoresis.

Ion Chromatography (IC)

• Ionic species don't partition well between mobile and stationary phases in standard HPLC systems.
• Two major approaches for reverse-phase HPLC include Ion-Suppression and Ion-Pair Chromatography.

Ion-Suppression Chromatography

• Ionic species are suppressed by controlling the pH of the mobile phase.
• Acids should be protonated.
• Bases should not be protonated.
• Finding an optimum pH for the mobile phase can be difficult to achieve.

Ion-Pair Chromatography

• A counter ion interacts with both the ionic species and the stationary phase.
• Counter ions improve the chromatography of the analyte.
• Quaternary amines act as counter ions for anions.
• Carboxylic or sulfonic acids act as counter ions for cations.
• This is the preferred choice for large ionic analytes.

Ion Exchange Chromatography

• Ion Exchange Chromatography (IC) is a special application of liquid chromatography.
• IC utilizes liquid mobile phases and solid stationary phases.

HPLC vs IC

HPLC

• Normal Phase: non-polar solvent and polar column
• Reverse Phase: polar solvent and non-polar column
• Solutes partition between the mobile and stationary phases
• Solubility effects predominate the separation
• Solutes are neutral species

IC

• IC uses an electrolyte solution as the solvent and an ion exchange medium as the column
• Both mobile and stationary phases are ionic
• Solute ions are exchanged between mobile and stationary phases
• Charge density effects predominate separation
• Solutes are ions

IC Stationary Phases

• Bare Silica, Alumina, or other hydrous oxides can act as the stationary phase
• Silica is used as a cation exchanger, and alumina can exchange either cations or anions, depending on the pH of the mobile phase.
• Bare Silica and Alumina are pH sensitive.
• Coated Silica have ionic species bonded to particles, similar to C8, C18 in standard HPLC columns.
• Drawbacks of Coated Silica include the limited pH range (2 – 7).
• Silanol groups on silica can irreversibly bind some metal ions.
• Polymeric supports, also known as resins can act as stationary phases
• Resins are most commonly copolymers, like styrene-divinylbenzene or divinylbenzene-acrylic/methacrylic acid.
• Resins have a wide pH range.
• Polymer supports can also serve as a partitioning stationary phase.

Stationary Phase Functional Groups

• Strong exchangers remain ionized as pH varies from 2-12.
• Weak exchangers can lose ionization as a function of pH.

Mobile Phase

• Mobile phases are electrolyte solutions containing competing ions.
• Identity and concentration of the competing ion affects elution strength.
• Adding organic modifiers can help dissolve the analyte
• pH affects both the mobile and stationary phases
• The buffer capacity and complexation (for metal ions) also affect elution strength.

Detector Types

• Universal detectors detect changes in conductivity in the mobile phase
• Conductivity will change with concentration of ions
• Mobile phase conductivity will overwhelm solute conductivity
• Ion suppression can solve the issue with overwhelming solute conductivity

Ion Suppression

• An ion suppressor reduces the conductivity of the eluent to increase detector sensitivity.

Electrolytic Suppressor

• Electrolytic suppressors use a membrane to exchange ions and reduce background conductivity.

Selective Detectors

• Selective detectors include UV/Vis, Fluorimetry, Electrochemical detectors, and Atomic Spectroscopy.

Why Use IC?

• IC excels at low-concentration determination of anions.

Size Exclusion Chromatography (SEC)

• SEC utilizes a liquid mobile phase.
• The stationary phase consists of solid, porous beads packed in a tubular column made of polymeric or silica material.
• Separation depends on size, leading to different elution rates.

Mobile/Stationary Phases for SEC

• The Stationary phase depends on the size of molecules analyzed and solvent used.
• Hydrophilic stationary phases are used with aqueous mobile phases (e.g., Polyvinylalcohols).
• Hydrophobic stationary phases are used with organic mobile phases (e.g., Styrene/divinylbenzene).

What is SEC used for

• It can calibrate using molecules of known size.
• Determine size distribution or absolute size
• Care must be taken as retention depends on molecule functionality and shape
• SEC is used for Polymers, proteins, and other large molecules.

Instrumentation

• SEC equipment is very similar to HPLC.
• Refractometry is often used for detection.
• Other HPLC detectors are also possible.
• Coupling with a light-scattering detector can provide independent size characterization.

Supercritical Fluid Chromatography (SFC)

• Supercritical fluid is a fluid above both its critical temperature and critical pressure (the critical point).
• Above the critical temperature, a gas can no longer condense as a liquid.
• The critical pressure is the pressure required to condense a gas at the critical temperature
• At the triple point, all phases coexist.
• At the critical point, phases become indistinguishable

Supercritical Fluid Properties

• A supercritical fluid has properties of both a gas and a liquid:
• Liquid-like density and solvation properties: Molecules are close enough together to influence solute molecules
• Gas-like transport properties: Very low viscosity, easily diffuses through materials and High flow rates possible

Carbon Dioxide as an SFC Solvent

• Carbon Dioxide is non-toxic and cheap compared to organic solvents.
• Supercritical conditions are not too difficult to achieve.
• Solvent properties can be modified by additives and pressure
• Pressure modifies density
• Pressure gradient works like gradient elution in HPLC

SFC Instrumentation

• SFC is a hybrid between GC and HPLC.
• A high-pressure pump is needed to attain supercritical conditions.
• Packed columns (HPLC) or capillary columns (GC) can be used.
• FID detection is usually used unless an organic modifier is used.
• HPLC detectors can also be used.

Applications of SFC

• SFC is faster than HPLC and can achieve higher resolution
• SFC can achieve high resolution at lower temperatures than GC.
• SFC is especially important for compounds not stable at high temperature and important for large molecules such polymers and biomolecules.

Thin-Layer Chromatography (TLC)

• TLC uses a liquid mobile phase and a solid stationary phase.
• Usually there is silica gel that is deposited on glass, plastic, or metal
• The mobile phase moves through the stationary phase by capillary action.
• Solutes partition between the mobile and stationary phases.

Stationary Phases

• Silica gel is used as the stationary phase.
• The size and porosity of deposited particles influence separation parameters.
• Polarity of phase depends upon the ratio of silanol groups to siloxane groups.
• Modified silica gel can be also used as stationary phase.
• Silica columns can be modified by bonding organic chains to it, like with HPLC.

Solute Identification

• Solute identification in generally done by retention time.
• Retardation factor (Rf) is: distance traveled by compound divided by distance traveled by solvent

TLC for Analysis

• Quantitative analysis is possible with TLC using a densitometer
• TLC's primary strength is as a rapid screening technique.

Electrophoresis

• Electrophoresis does not utilize a moving mobile phase.
• Solutes are separated by differential migration in an electric field.
• Differences are due to differences in the charge/size ratio between solutes
• Electrophoresis is widely used in biological applications, especially DNA sequencing.

Stationary Phase in Electrophoresis

• Paper and polymer sheets are used as substrates within electrophoresis.
• Substrates are usually coated with starch gel (agarose) or polyacrylamide.

Free-Solution Electrophoresis

• "Free-solution" electrophoresis was the original electrophoresis technique.
• Tiselius placed serum in a U-shaped tube and filled it with buffer to purify proteins.

Capillary Electrophoresis (CE)

• Capillaries have been coupled with electrophoresis to make modern CE.
• Cations migrate to the cathode, and anions migrate to the anode.

Movement in CE

• Electrophoretic mobility is movement based upon charge/mass ratio.
• Electro-osmotic flow facilitates that everything tends to move toward the cathode.

Electro-osmotic Flow

• Electro-osmotic flow happens because of presence of silanol groups in the silica capilary

Types of CE

• Capillary Zone Electrophoresis (CZE) uses only buffer in the capillary.
• Micellar Electrokinetic Capillary Chromatography (MEKC) uses micelles to elute neutral molecules. -Solutes partition between micelles and buffer
• Capillary Gel Electrophoresis (CGE) adds media to the capillary.
• Capillary Electrochromatography (CEC) adds an HPLC stationary phase to column.

CE Detectors

• Spectroscopic detectors, such as UV/Vis and fluorescence detectors can be used.
• Electrochemical detectors such as conductometric and amperometric/voltammetric can be used.
• Mass Spectrometry can also be used as a detector.