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Chapter 12: Chromatographic and Electrophoretic Methods

General theory, optimizing separations, GC, HPLC, electrophoresis and other chromatographic methods
by

Neil Fitzgerald

on 29 August 2014

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Transcript of Chapter 12: Chromatographic and Electrophoretic Methods

Chapter 12: Chromatographic and Electrophoretic Methods
General Theory
Chromatographic Separations
Chromatography describes a series of separation techniques involving an equilibrium between a
mobile phase
and
stationary phase
The sample, in the mobile phase, separates into its components as it moves through the stationary phases
Mobile phase can be a gas, liquid, or supercritical fluid
Stationary phase is a solid or viscous liquid
Chromatography techniques are often named after the states of mobile and stationary phase e.g. gas-liquid chromatography has a gas mobile phase a liquid stationary phase
Stationary phase can be coated on a flat surface, known as
planar chromatography
, or packed into a column, known as
column chromatography
Elution Time
Chromatogram
t , Void Time
m
t , Retention Time
t , Adjusted Retention Time
r
r
'
W, Peak Width
W1/2
Peak Width at Half Peak Height
Peak Height
Half Peak Height
Peak Area
Resolution
Retention Factor
Selectivity
Column Efficiency and Optimization
A measure of how well two neighboring peaks are separated from each other
Calculated by:



For two similar sized peaks, a Rs value of 1.5 or greater is considered to be fully resolved
The retention factor or capacity factor, k, is a measure of how strongly retained a component is on the stationary phase and can be found by:



Note that a larger retention factor means that the distribution ratio favors the stationary phase leading to a longer retention time
A small k (<1) means that the component is not being retained by the stationary phase and cannot be separated from other poorly retained species. A large k (>20) is likely to cause excessive peak broadening and take a long time. A retention factor between 1 and 5 is considered ideal
Selectivity is a measure of the relative retention of two components, measured as the selectivity factor:



Selectivity factors are 1.0 or greater. A selectivity factor close to 1.0 indicates that the components have similar retention times.
Column Efficiency
An efficient column produces narrow peaks even at long retention times
Column efficiency is measured as the number of theoretical plate, N, using the analogy to fractional distillation
Number of theoretical plates can be calculated by:


The Height Equivalent to a Theoretical Plate, H or HETP, is given by:



Column efficiency depends on the amount of band broadening which can be considered as a combination of three physical process as defined by the Van Deemter Equation

A is the Eddy Diffusion Term
Molecules follow different paths through the stationary phase causing peak broadening
Independent of flow rate
B is the Longitudinal Diffusion Term
Random motion of molecules leads to molecules spreading (diffusion)
Inversely proportional to flow rate (more time allows for more diffusion)
C is the Mass Transfer Term
Combining the terms leads to the Van Deemter plot
Optimization
A chromatographic separation can be optimized by:
-Optimizing the mobile phase flow rate (as shown in the Van Deemter plot)
-Using small spherical stationary phase packing (e.g. HPLC) or removing column packing (e.g. capillary GC) (reducing A term)
-Optimizing the temperature of the separation
-Changing the stationary and/or mobile phase composition (changing selectivity)
-Changing column dimensions (length, internal diameter, film thickness)
-Changing the pH of the mobile phase for acidic or basic solutes
When a band moves through the column in the mobile phase, some molecules will be retained on the stationary phase (b) then coming to equilibrium (c), resulting in a broader peak (d)
Effect is directly proportional to the flow rate (slower flow allows more time to reach equilibrium)
Gas Chromatography
High Performance Liquid Chromatography
Other Liquid Chromatography Methods
Electrophoretic Methods
Mobile Phase
Sample Introduction
Temperature Control
Columns
Detectors
-Mobile phase is an inert gas e.g. nitrogen or helium
-Often called a carrier gas, the purpose is to move the sample through the column. It does not chemically interact with the sample
-Gas must be appropriate for the detector
-Normally supplied from a pressurized cylinder
-Normal flow rates are 1 to 25 mL/min for a capillary column
Typically samples are introduced as liquids into a heated injector to convert to a gas
Normally 1 or 2 uL of sample injected into a capillary column with a GC Syringe
GC syringes have sharp tips in order to pass through a rubber septum
GC can be run in spilt or splitless modes
In split mode, some of the sample is vented in the injector to prevent column overload
Retention time in gas chromatography is strongly dependent on temperature
Columns are enclosed in an oven
The oven must have good temperature control, the ability to be programmed and cool quickly between runs
A GC experiment run at one temperature is called
isothermal
Often
temperature programming
is used to allow for separation of volatile components at lower temperatures before ramping to higher temperatures to elute less volatile components in a reasonable time
GC columns can contain stationary phase material supported on particles (
packed column
) or coated on the inner surface of an open tubular column (
capillary column)
Modern GC columns are fused silica capillary columns with an outer protective coating
Capillary columns are typically 15 to 100 m long, with an internal diameter of 150 to 300 um, coated with a viscous liquid film of stationary phase material about 0.25 um thick
Stationary phase can be non-polar, slightly polar or polar
A typical non-polar stationary phase is polydimethyl siloxane which is nonpolar, it can be made slightly polar by substituting the methyl groups for phenyl groups
An example of a polar stationary phase is polyethlene glycol





For non-polar columns, the elution order depends on volatility only (compounds with lower boiling points will elute first), for polar and slightly polar columns the polarity of the compound with also have an effect (polar compounds will be retained more than non-polar compounds)
All stationary phase have an upper temperature limit, above this limit the stationary phase will start to be lost, known as
column bleed
Thermal Conductivity Detector (TCD)
Flame Ionization Detector (FID)
Electron Capture Detector (ECD)
Mass Spectrometer (MS)
Makes use of changes in thermal conductivity of the mobile phase
Mobile phase eluting from column passes over a wire filament
Resistance of wire depends on temperature which depends on thermal conductivity of mobile phase
Solutes eluting from column decease the thermal conductivity of the mobile phase
Advantages: cheap, simple, universal (responds to nearly everything), good linear dynamic range
Disadvantages: poor detection limits, can't identify solutes directly (comparison of retention time to known only)
Solutes are burned in a hydrogen/air flame
Ions formed in the flame are detected by a collector (cathode) causing a current flow
Advantages: cheap, simple, good detection limits, good linear range, nearly universal (responds to most organics)
Disadvantages: requires hydrogen and air, cannot identify directly

Radioactive source e.g nickel 63 emits beta particles (electrons) that are detected at the anode causing a current flow (standing current)
Any solutes capable of absorbing electrons cause a decrease in current
Advantages: Simple, cheap, very good detection limits for specific compounds, selective detector (responds to organohalogen and nitro compounds, very useful for environmental analysis)
Disadvantages: Only responds to specific compounds, poor linear range (easily overloads), radioactive (regulations), cannot identify directly
Mobile phase passed directly into a mass spectrometer
Solutes converted to fragment ions by bombarding with electrons
Ion fragments separated by mass to produce a mass spectrum
Mass spectrum is characteristic of the compound
Advantages: Can identify compounds by comparing mass spectrum to a library, good detection limits, good linear range, selective ion monitoring (chromatogram based on one mass fragment) can be used to simplify chromatogram
Disadvantages: Expensive
Mass spectrum of Toluene
Quantitative Analysis
The amount of a solute is proportional to the area of its chomatographic peak
Peak areas are found by integrating the peak above the baseline using a computer or electronic integrator
Integrators are also used to divide overlapping peaks into separate areas
Analyte concentrations can be found by external calibration using a constant injection volume
In gas chromatography, internal standards calibration is often preferred as constant injection volumes can be difficult
Mobile Phase
Stationary Phase
Sample Injection
Pump
Detectors
HPLC experiments are run in normal or reverse phase.
In
normal phase
, the mobile phase is non-polar and the stationary phase polar
In
reverse phase
(the most common) the mobile phase is polar and the stationary phase non-polar
Retention times can be varied by changing the polarity of the mobile phase
For a mixture of two miscible liquids, A and B, the polarity index can be calculated as:


If an HPLC experiment is run with a constant mobile phase it is known as an
isocratic elution
If the mobile phase composition is varied during a run it is known as a
gradient elution
Gradient elution is used to achieve good separation in a reasonable time
Often a buffer is added to the mobile phase to maintain compounds in a form required for optimal separation
An HPLC instrument can have between 1 and 4 solvent reservoirs
Mobile phase solvents must be degassed, mixed together and pumped through the analytical column
Degassing can be achieved by bubbling helium through the solvents or applying a vacuum
Solvents must be pumped at low flow rate (mL/min) and relatively high pressures (up to about 400 bar) with minimal pressure fluctuation
One common pump (a reciprocating pump) incorporates two pistons moving in opposite directions to eliminate pulsations
Pumps must be primed (filled with liquid) before use
HPLC stationary phase is usually contained inside a steel tube known as the analytical column
Stationary phase material is in the form of small spheres. Smaller spheres reduce the A term in the Van Deemter equation but require higher mobile phase pressure and are more difficult to contain in the column
These spheres are made of silica which can be functionalized by chemical reaction:




In normal phase HPLC, the stationary phase is a polar material, for reverse phase it has low polarity
Typical reverse phase materials are long chain hydrocarbons such as -C18H37 (known as C-18 or ODS) or C8H17 (C-8)
Separation order depends on polarity as well as solute size and shape. In general for components A, B, and C where A is the most polar and C the least:


Known volumes of samples injected with a sample loop
Sample loop normally filled using a syringe (like a GC syringe but larger volume and a flat tip)
Sample injected into the mobile phase using a rotatory injection port
Sample added with port in the load position, port rotated to inject.
Spectroscopic
The most popular HPLC detector is the diode array UV/Vis detector
Diode array can collect the entire UV/Vis spectrometer continuously
Single wavelength is monitored to produce chromatogram
Spectrum can be obtain for each chromatographic peak to aid in identification
Fluorescence detectors are also common and provide low detection limits for fluorescent analytes
Electrochemical
Simple, small and cheap
In an amperometric flow cell, the column effluent flows over a working electrode held at a constant voltage relative to a downstream reference electrode
The oxidation or reduction of an analyte causes a current flow that serves as the analytical signal

LC-MS
Similar to GCMS, LCMS (or HPLC-MS) offers the advantage of allowing identification by comparison of the mass spectrum to a library
Interface more difficult than GCMS as column effluent must be converted to a gas for the mass spectrometer
More expensive than other common detectors
Liquid-Solid Adsorption Chromatography
The stationary phase is the column packing
Common stationary phases are porous polar materials such as silica or alumina
Typical mobile phases are non polar solvents such as hexane and isoctane
Commonly used for purification in organic synthesis
Ion Exchange Chromatography
Also known as Ion Chromatography (IC)
Used for separating ionic species
Stationary phase is ion exchange material, a cross-linked polymer with ionic functional groups
Counter ions to functional groups can be displaced by other ions
To separate cations, a cation exchange material with sulfonic acid groups is normally used
To separate anions, a anion exchange material with a quaternary amine may be used
Retention depends on ionic composition and pH of the mobile phase
Mobile phase usually an aqueous buffer
Instrumentation is similar to HPLC with conductivity detector
UV/Vis can be used if analyte absorbs in UV or visible region or if mobile phase absorbs in with case a signal is seen as a decrease in absorbance
Size Exclusion Chromatography
Also known as Gel Permeation Chromatography
Separation based on molecular size
Used for larger molecules such as proteins or polymers
Stationary phase contains porous polymer beads
Small molecules are retained in the pores more than larger molecules
Variety of mobile phases
HPLC type instrumentation
UV/Vis is the most common detector
Supercritical Fluid Chromatography
Uses a superitical fluid (e.g. supercritical CO2) as the mobile phase
Combines the good resolution of GC with the flexibility of HPLC
Supercritical fluid is a good solvent with viscosity similar to gas but requires elevated temperatures and pressures to form
General Theory
Electrophoresis is a separation technique based on the analyte's ability to move through a conductive material under the influence of an electric field
Separation depends on charge and size
In
slab electrophoresis
, and analytes move though a conducting buffer retained of a porous agorase gel
Usually for qualitative analysis particularly in biochemistry and molecular biology
For quantitative analysis, buffer is contained in a capillary column, known as
capillary electrophoresis
In capillary electrophesis sample components move due to
electrophoretic mobility
and
electroosmotic flow
Electrophoretic mobility describes the movement of a solute under the influence of an electric field. Cations will move to the anode, anions to the cathode
Electroosmotic flow describe the bulk movement of a buffer in a capillary column due to an electric field.
Buffer solution moves toward the cathode due to negatively charged capillary walls at low pH. Cations are attracted to the surface leaving move anions than cations in the buffer. The anions are attracted to the cathode dragging the solvent with it
Net result is that cations move fast than anions
Column
Detectors
Sample Injection
Electric Field
Capillary Electrophoresis Methods
Fused silica open tubular columns (similar to GC columns) are normally used
Typically narrower than GC columns
The narrow column reduces heating when the high voltage is applied
Similar to HPLC (e.g. UV/Vis, fluorescence and electrochemical)
Performed on-column before solute elutes
To increase pathlength, a z-cell or internal reflectance can be used

There are two commonly used methods for injecting a sample:
Hydrodynamic injection
uses pressure to inject sample into one end of the column. Either by applying pressure to the sample vial or a vacuum to the opposite end of the capillary
Electrokinetic injection
works by placing the end of the capillary and the anode into the sample vial and briefly applying a potential
A voltages of up to 40kV can be applied to produce electroosmotic flow
Heating is reduced by the use of a narrow capillary column
Capillary Zone Electrophoresis (CZE)
Micellar Electrokinetic Capillary Chromatography (MEKC)
Capillary Gel Electrophoresis (CGE)
Capillary Electrochromatography (CEC)
The simplest method. Capillary column is filled with buffer. Solutes migrate to cathode when voltage applied. Cations elute first. Uncharged species elute as a single band
Allows separation of neutral species. Surfactant added to form micelles (negatively charged spheres). Neutral species equilibrate between the buffer and the micelles. Separation depends on extent of partitioning between buffer and micelles
Capillary filled with a gel. Separation depends on mobility and size. Good for large molecules (e.g. DNA fragments, proteins etc.)
Capillary packed with bonded stationary phase material. Allows for separation of neutral species. Separation similar to HPLC without need for pumps and with better efficiency and shorter analysis times
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