Analytical Chemsitry-Exam 3-Liquid Chromatography/Supercritical Fluid Chromatography

Introduction to Liquid Chromatography

A chromatographic technique in which mobile phase is a liquid

is a much older technique than GC but over shadowed by the rapid development of GC in the 1950s and 1960s

Currently the dominate type of chromatography and is even replacing GC in its more traditional application

Differences of LC to GC

Has no oven (unlike GC)

Run things at room temperature or a cold room if room temp is too hot

Advantages of LC compared to GC

can be applied to the separation of any compound that is soluble in a liquid phase

Useful in separation of biological compounds, synthetic or natural polymers and inorganic compounds

Liquid mobile phase allows it to be used at lower temperatures than required by GC; better suited for separating compounds that may be thermally liable

Retention of solutes in it depend on their interaction with both their mobile phase and stationary phase; GC retention based on volatility and interaction with stationary phase

It is more flexible in optimizing separations-change either stationary or mobile phase

Most detectors are non-destructive; better suited for preparative or process scale separations

Disadvantages of LC

Subject to greater peak or band-broadening

Much larger diffusion coefficents of solutes in gases vs liquids

Low-Performance Liquid Chromatography

Use large, non-rigid support material

Particles >40 um in diameter

Poor system efficiencies and large plate heights

Characteristics of Low-Performance Liquid Chromatography

Broad peaks

Long separation times

Columns can only tolerate low operating procedures

Column Chromatography

An example of the equipment used in low-performance liquid chromatography

Sample is usually applied directly to the top of it

Detection is by fraction collection with later analysis of each fraction

Advantages of Column Chromatography

Simple system requirements

Low cost

Popular in sample purification

Used in the removal of interferences from sample

Used in some analytical applications; not common due to low efficiency, long analysis times, and large LOD

High-Performance Liquid Chromatography

LC methods that use small, uniform, rigid support material

Particles <40 um in diameter

Usually 3-10 um in practice

<2 um is ultra

Good system efficiencies and small plate heights

Characteristics of High-Performance Liquid Chromatography

Narrow peaks

Low LOD

Short separation times

Columns can tolerate high operating pressures and faster flow rates

A typical HPLC systems

Higher operating pressures need for mobile phase delivery requires special pumps and other system components

Sample applied using closed system (i.e injection valve)

Detection uses a flow through detector

Advantages of HPLC

Fast analysis time

Ease of automation

Good LOD

Preferred choice for analytical applications

popular for purification work

Disadvantages of HPLC

Greater expense

Lower sample capacities

Elution

depends on the interactions of solutes with both the mobile and stationary phase; to describe how well solutes are retained on columns with different solvents.

Strong mobile phase

A solvent that quickly elutes from the column

Has a very similar polarity to stationary phase

Weak Mobile Phase

A solvent that slowly elutes solutes from the column (ie high solute retention or large k’)

Occurs if the mobile phase is very different from the stationary phase in its intermolecular interaction with solutes

What dictates whether a mobile phase is weak or strong

Depends on the stationary phase being use.

ex. Hexane is a weak mobile phase on a polar stationary phase

Isocratic elution

Use of a constant mobile phase composition to elute solutes

Simple, inexpensive

Difficult to elute all solutes with good resolution in a reasonable amount of time

Gradient elution

Changing composition of mobile phase with time; solvent programming

Going to a weak mobile phase to a strong one

Solvent change can be stepwise linear or non-linear

How to chose a mobile phase for LC

Type of stationary phase used: determines what will be a strong or weak mobile phase

Solubility of solute: can’t have phase separation

Viscosity of sample

Type of detector used

Types of LC: Absorption Chromatography

Separates solutes based on their absorption to underivatized solid particles

Similar to gas-solid chromatography in that the same material is used as both stationary phase and support material

SP may be either polar or non-polar

Advantages of Absorption Chromatography

Retain and separate some compounds that cannot be separated by other methods

Disadvantages of Absorption Chromatography

Very strong retention of some solutes

May cause catalytic changes in solutes

Solid support may have a range of chemical and physical environments: non-symmetrical peaks and variable retention times

For Polar Supports Absorption Chromatography

The weak mobile phase is a non-polar solvent and the strong mobile phase is a polar solvent

For Non-polar Supports Absorption Chromatography

The weak mobile phase is a polar solvent and the strong mobile phase is a non-polar solvent

Common applications of Adsorption LC

Purification of synthetic organic compounds from reaction mixtures

Separation of geometrical isomers (ortho/meta/para, cis/trans)

Types of LC: Partition Chromatography

Separates solutes based on their partioning between a liquid mobile phase and a liquid stationary phase coated on a solid support

Partition Chromatography support material

Usually silica, originally, involved coating this support with some liquid sp that was not readily soluble in the mobile phase

Types of Partition Chromatography: Normal Phase

SP is polar

column strongly retains polar compounds

weak mobile phase is a non-polar liquid-organic solvent

strong mobile phase is a polar liquid-water or methanol

SP must have low miscibility with the mobile phase so the stationary phase is not dissolved on the column

Examples of NPLC stationary phases

Dimethyl sulfoxide

Ethylene glycol

Water

Ethylene diamine

SP slowly bleed from the column, changing properties and solute R.T

Common applications of NPLC

Purification of synthetic organic and inorganic compounds from reaction mixtures

General purpose of separation of polar/non-polar compounds when the sample is in a non-polar solvent

Types of Partition Chromatography: Reverse Phase

Stationary phase is non-polar

Retains non-polar compounds most strongly

Weak MP is a polar liquid: water

Strong MP is a more non-polar liquid: methanol or acetonitrile

SP must have a low miscibility with the MP so the stationary phase is not dissolved in the column

Example of SP Reverse Phase Chromatography

Heptane

squalene

hydrocarbon polymers

dimethylpolysiluxane

Common applications of RPLC

Most popular type of LC

Separation of a wide variety of non-polar and polar solutes

Ideal for the separation of solutes in aqueous-based samples, such as biological compounds

Purification of biological and organic compounds present in aqueous solutions

Pharmaceutical analysis (drug quantification and quality control)

Protein and peptide mapping

Analysis of soil and water samples

clinical analysis of blood and urine samples

Ion-Exchange Chromatography

Separates solutes by their absorption on a support containing fixed charges on it surface. A high concentration of a competing ion is often added to the MP to elute the analytes from the column

Stationary Phases IEC: Cation Exchangers

Have fixed negatively charged groups, used to separate positively charged ions

Two general types of SP can be used in IEC: Anion-exchangers

Have fixed positively charged groups, used to separate negatively charged ions

The charged groups that make up SP can be placed on several different types of support materials

Cross-linked polystyrene resins: for use with the separation of inorganic ions and small organic ions

Carbohydrate-based resins: for low-performance separations of biological molecules (dextran, agarose, cellulose)

Silica-based supports: for high-performance separations of biological molecules

Strong MP in IEC

Contains a high concentration of a competing ion for displacement of the sample ion from the stationary phase or a solvent that has a pH which decreases ionization of the analyte of stationary phase

Factors that affect MP strength

MP pH: especially for weak acid or base analytes and weak acid or base sp

MP conc. of competing ion

Type of competing ion

Common application of IEC

Removal or replacement of ionic compounds in samples (sample pretreatment)

Separation of inorganic ions and organic ions

Analysis/purification of charged biological compounds

Types of LC: Affinity Chromatography

Separates based on the immobilized biological molecules (and related compounds) as the stationary phase

Based on the selective, reversible interactions that characterize most biological systems

Binding of an enzyme with its substrate or a hormone with its receptor

Immobilize one of a pair of interacting molecules on a solid support

Immobilized molecule on column is referred to as the affinity ligand

Due to the very selective nature of most biological interactions, the solute of interest is often retained with little interference from other components of the sample

Two main types of Affinity Ligands used in Affinity Chormatography: High-specificity ligands

Compounds which bind to only one or a few very closely related molecules

Two main types of Affinity Ligands used in AC: General or group specific ligands

Molecules which bind to a family or class of related molecules

The affinity ligand does not necessarily have to be of biological origin

Weak Mobile Phase: Affinity chromatography

Usually a solvent that mimics the pH, ionic strength, and polarity of the solute and ligand in their naturally binding environment

Strong Mobile Phase: Affinity Chromatography

A solvent that produces low retention for the solute-ligand interaction by decreasing its bind constant or displaces solute by the addition of an agent which competes for solute sites on the column

Biospecific Elution: Affinity Chromatography

Solutes are eluted by a MP that contains a compound which competes with sample solutes for the ligand’s active sites

Very gentile

Useful in purification of active biological molecules

Produces slow elution with broad solute peaks

Non-Specific Elution: Affinity Chromatography

Change conditions in the column to disrupt the interactions between the sample solutes and immobilized ligand

Done by changing pH or Ionic Strength

Harder than biospecific elution

Gives narrow peaks and faster run times

Commonly used in analytical applications of AC

Common applications of Affinity Chromatography

Purification of enzymes, proteins and peptides

Isolation of cells and viruses

Purification of nucleic acids

Specific analysis of components in clinical and biological samples

Study of bimolecular interaction

Size Exclusion Chromatography (SEC)

Separates molecules according to differences in that size

Is based on the use of a support material that has certain range of pore sizes

Since larger molecules sample a smaller volume of the column, they elute before smaller molecules

Separation based on size or molecular weight

Based on different interactions of solutes with the flowing mobile phase and the stagnant mobile phase

No true stationary phase is present in this system

Stagnant mobile phase acts as the “stationary phase”

does not have a strong or weak mobile phase since retention is based only on the size/shape of the analyte and the pore distribution of the support

Size Exclusion Gel filtration Chromatography

If an aqueous mobile phase is used (hydrophilic)

Size Exclusion gel permeation chromatography

If an organic mobile phase is used (usually tetrahydrofuran) (hydrophobic)

Common applications of Size Exclusion Chromatography

Separation of Biological Molecules (e.g. proteins from peptides)

Separation/ analysis of organic polymers

Molecular-weight determination

Common types of LC Detector

·      Refractive Index Detector

·      UV/Vis Absorbance Detector

·      Fluorescence Detector

·      Conductivity Detector

·      Electrochemical Detector

LC Detectors: Refractive Index Detector

Measures the overall ability of the mobile phase and its solutes to refract or bend light:

            One of the few universal detectors available for LC

Refractive Index Detector: Advantages

Non-destructive and universal detector

·      Applicable to the detection of any solute in LC

Applicable to preliminary LC work where the nature and properties of the solute are unknown

·      Provided concentration is high enough for detection

Refractive Index Detector: Disadvantages

High limits of detection (10^-6 to 10^-5 M)

Difficult to use with gradient elution

Refractive Index Detector: Process

Light from source passes through flow-cells containing either sample stream or a reference stream

When refractive index is the same between two cells, no bending of light occurs at the interface between the flow cells

·      Maximum amount of light reaches the detector

As solute elutes, refractive index changes between reference and sample cell

·      Light is bent as it passes through flow cell interface

·      Amount of light reaching detector is decreased

LC Detectors: UV/Vis Absorbance Detector

Measures the ability of solutes to absorb light at a. particular wavelength(s) in the ultraviolent (UV) or visible (Vis) wavelength range

·      Most common type of LC detector

Three common types:

·      Fixed Wavelength Detectors

·      Variable Wavelength detectors

·      Photodiode array detectors (not common)

Fixed Wavelength Detectors: UV/Vis Absorbance Detector

absorbance of only one given wavelength is monitored by the system at all times (usually 254 nm)

·      Simplest and cheapest of the UV/Vis detectors

·      Limited in flexibility

·      Limited in types of compounds that can be monitored

Variable Wavelength Detector: UV/Vis Absorbance Detector

A single wavelength is monitored at any given time, but any wavelength in a wide spectral range can be selected

·      Wavelengths vary from 190-900 nm

·      More expensive, requires more advanced optics

·      More versatile, used for a wider range of compounds

Photodiode Array Detector: UV/Vis Absorbance Detector

Operates by simultaneously monitoring absorbance of solutes at several different wavelengths.

·      Uses a series or an array of several detector cells within the instruments, with each responding to changes in absorbance at different wavelengths.

·      Entire spectrum of a compound can be taken in a minimum amount of time

·      Useful in detecting the presence of poorly resolved peaks or peak contaminants

Application of UV/Vis Absorbance Detector

- can be used to detect any compound that absorbs at the wavelength being monitored

-       Common wavelengths:

o   254 nm for unsaturated organic compounds

o   260 nm for nucleic acids

o   280 or 215 for proteins or peptides

-       Absorbance detectors can be used with gradient elution

o   Wavelength being monitored is above the cutoff range of the solvents being used in the mobile phase

-       Limits of detection for fixed and variable UV/Vis absorbance detectors are ~10^-8M

-       Limits of detection for photodiode array detectors are ~10^-7

LC Detectors: Fluorescence Detector

A selective LC detector that measures the ability of eluting solutes to fluoresce at a given set of excitation and emission wavelengths

Applications: Fluorescence Detector

-       Fluorescence can be used to selectively detect any compound that absorbs and emits light at the chosen set of excitation and emission wavelengths

o   Relatively few compounds undergo fluorescence

o   High selectivity, low background signal

-       Limits of detection are ~10^-10 M

-       Typical applications:

o   Drugs

o   Food additives

o   Environmental pollutants

o   Any compound that can be converted to a fluorescent derivative: alcohols, amines, amino acids and proteins

-       Can be used with gradient elution

o   Requires extremely pure mobile phases

o   Trace impurities can affect background signal or quench the fluorescence of solutes

LC Detectors: Conductivity Detector

Used in analytical applications of ion-exchange chromatography for the detection of ionic compounds

-       Detector measures the ability of the mobile phase to conduct a current when placed in a flow-cell between two electrodes

-       Current conducted within the cell will depend on the number and types of ion present in the mobile phase 

-       When ions flow into the sensor cell, the impedance between the electrode’s changes producing an “out of balance” signal

-       Two electrodes are placed in mobile phase each corresponding to one arm of a Wheatstone bridge

Conductivity Application Detector

-       Can be used to detect any compound that is ionic or weakly ionic

o   High selectivity, low background signal

-       Limits of detection are ~10^-6

-       Typical applications:

o   Food components

o   Industrial samples

o   Environmental samples

-       Can be used with gradient elution:

o   Constant ionic strength and pH of mobile phase

o   Background conductance of the mobile phase is sufficiently low

LC Detector: Electrochemical Detector

Used to monitor any compound in the mobile phase that can undergo an oxidation or reduction

·      Generally, includes two or more electrodes which monitor the current that is produced by the oxidation or reduction of eluting compounds at a fixed potential

·      Generally electrical output is an electron flow generated by a reaction that takes place at the surface of the electrodes

Applications of Electrochemical Detector

-       Can be used to detect any solute that can undergo oxidation or reductions

o   Detectors can be made specific for a given compound or class of compounds by properly choosing the conditions at the electrodes

o   High selectivity, low background signal

-       Limits of detection for a electrochemical detector are ~10^-11 M

o   Due to extreme accuracy with which chemical measurements, especially current measurements, can be made

-       Compounds that can be detected by reduction:

o   Aldehydes

o   Ketones

o   Esters

o   Unsaturated compounds

-       Compounds that can be detected by oxidation:

o   Phenols

o   Mercaptans (RSH)

o   Aromatic amines

o   Dihydroxy compounds

Supercritical Fluid Chromatography

A hybrid of gas and liquid chromatography that combines some of the best features of both

Supercritical Fluid

exist at temperatures and pressures above its critical temperature and pressure and have densities, viscosities, and other properties that are intermediates between those of the substance in its gaseous and liquid state.

Critical Temperature

Temperature above which a distinct liquid phase does not exist regardless of pressure

Important Properties of Supercritical Fluids

^-               Remarkable ability to dissolve large, non-volatile molecules

^o     E.g supercritical CO2 can dissolve n-alkanes containing over 30 carbon atoms related to their high densities

^-               Dissolved analytes are easily discovered:

^o     Equilibrate with atmosphere at relatively low temperatures

^o     E.g analyte in supercritical CO2 can be recovered by reducing the pressure and allowing the CO2 to evaporate

^-               No need for organic solvents

^o     Environmentally friendly

^-               Inexpensive, innocuous and non-toxic

^-               Higher diffusion coefficients and lower viscosities relative to liquids

Faster and high resolution separations

Advantages of SFC compared to LC and GC:

-      can separate compounds that are not conveniently handled by GC or LC

o   Non-volatile or thermally liable

-       And

o   Contain no functional group that makes possible detection in LC using spectroscopic or electro chemical techniques

-       Up to 25% of all separation problems fall into this category

-       Examples include polymers, fossil fuels, pesticides, foods, drugs, etc

-       Separations are faster than LC

-       Run at lower temperatures than GC

-       Beneficial in industrial scale purification

Instrumentation: SCF

very similar to ordinary HPLC equipment since the temperature and pressure requirements for supercritical fluids fall within the standard operations

Differences:

-       Thermostated column oven

o   Requires precise temperature control of mobile phase (typically supercritical CO2)

-       Restrictor or back-pressure device

o   Required to maintain desired pressure in column

o   Pressure change to convert from supercritical fluid to a gas for transfer to detector

Effects of Pressure: SFC

Pressure increase results in reduced elution time

Increase in density of mobile phases

Effect retention or capacity factor

Pressure changes-analogous to gradient elution in LC and GC