created with mindgenius business 2005® chromatography chromatography

Created with MindGenius Business 2005Created with MindGenius Business 2005®®

Chromatography Chromatography


Chromatography Chromatography

Principles Principles Techniques Techniques


Principles Principles

Introduction Introduction The Chromatogram The Chromatogram Performance parameters Performance parameters Instrumentation Instrumentation


Introduction Introduction Chromatography: The separation of components of a mixture

using differences in concentration equilibrium between two phases; one stationary, and one mobile.

Technique is >100years old, optical detection>60years….developments include miniaturisation, increased sensitivity, computer control and collection

Chromatography instrumentation represents more than half the world-wide sales of analytical equipment and materials.

Preparative Chromatography: Separation of components of a mixture and collection of isolated components

Analytical chromatography: Observation, quantification and potential identification of different components using chromatographic techniques


Introduction (2)Introduction (2)Fundamental Basis

Movement of a compound through the system is governed by the equilibrium process:

KCompound (stationary) Compound (mobile)

(CS) (CM)

Nernst distribution coefficient K = CS / CM

i.e. rate/distance of movement is governed by compounds’ relative affinity for stationary or mobile phases

Formats

A) Plate/gel (e.g. thin-layer chromatography, gel electrophoresisB) Column (e.g. HPLC, GC)


Introduction (3): Plates/GelsIntroduction (3): Plates/GelsConsists of using a solvent (mobile phase) sometimes in an applied field to move compounds over a flat surface coated with a suitable stationary phase1) Sample is loaded in solution2) Chromatograph is run (developed)3) Spots are identified/analysed

1) Loaded plate 2) Developing plate 3) Developed Plate

Results: Intensity of spot (quantitative) & Retention factor (qualitative)

Sample

Coated Plate

Solvent

Developing tank

Line of solvent front

A B A&B A B A&B


Introduction (4): ColumnsIntroduction (4): Columns

How does it work?

(a) Mixed sample is loaded(b) Elution begins(c) B travels faster than A -

separation occurs(d) B is eluted(e) A is eluted

A and B are separated and either:

analysed “on-line” with a detector

Collected for further analysisResults: Area under peaks (quantitative) & Retention time (qualitative)


The Chromatogram The Chromatogram The output of a chromatograph, continuous trace of amount of

analyte (y-axis) against time (x-axis)

But what does this tell us?1) Retention Time: tR Qualitative data – what is present?2) Area under peak: Quantitative data - How much analyte is present?

Time

Dete

ctor

Rep

sonse

t0

tR

wb

w½

Analyte peak

Peak of unretained solute “solvent peak”

Start

tR’


Performance parameters Performance parameters (basic) (basic) 1) Resolution

Peaks should start and finish at the baseline

NOT

Well resolved peaks Unresolved peaks

2) Peak shapePeaks should be: Symmetrical

NOT

Representative of a Gaussian distribution As narrow as possible Reproducible between “runs”


Performance parameters Performance parameters (basic) (basic)

Poor peak shape and resolution means failed chromatography due to:

Unstable conditions Column damage or wear Column overload (too much analyte) Air in the system (gives artifactual peaks) Interfering matrix Incomplete separation of compounds due to sub optimal conditions

Temperature mobile phase stationary phase

More on improving performance later but remember for now:

RESOLUTION AND RETENTION TIME


Performance parameters Performance parameters (Advanced) Theoretical Plate (Advanced) Theoretical Plate

ModelModelMathematical performance assessment

Based on a series of “snapshots” to mimic continuous behaviour

Retention time of analyte: tR = tS + tM

where tS is the time spent in the stationary phase and rate of movement = 0

tM is the time spent in the mobile phase, rate of movement = mobile

phase rateEach molecule has a chance of being mobile or stationary during any “snapshot”

Distance travelled between periods in the stationary phase = one theoretical plate

Assumes no diffusion in the mobile phase

This gives us simple equations describing chromatographic performance


Performance parameters Performance parameters (Advanced) Chromatographic (Advanced) Chromatographic

EfficiencyEfficiencyColumn Efficiency (measured by plate number: N) N = (tR/s)2

tR is total retention time, and s is the standard deviation of a gaussian peakBUT s requires accurate determination of points of inflection, so we use

N= 5.54 x (tR / peak width at 50% height)2

Large N indicates good column performance (should be ~10,000 for HPLC)

N is increased by: increased temperature, column length decreased stationary phase particle size, flow rate, mobile phase viscosity


Other variants of chromatographic efficiency

Effective N (Neff): If tR is low, then t0 affects apparent efficiencyNeff = 5.54 x [(tR - t0) / peak width at half height]2

Plate Height (H): Used to compare columns of different lengths: H= L / NL = column length, N = number of platesH is a measure of plate size, the smaller (lower H) the better (HPLC ~ 10mm)

Effective plate height: takes account of columns with different dead spacesHeff = L / Neff

Reduced plate height: allows comparison of columns with different particle sizes

h = H / dpwhere dP is the particle diameter (same units as L); (Good HPLC column: h = 3)

Performance parameters Performance parameters (Advanced) Chromatographic (Advanced) Chromatographic

EfficiencyEfficiency


The ratio of mass in the stationary phase (mS) to that in the mobile phase (mM)where total mass: mT = mM + mS

Capacity Factor: k = mS / mM

= K. (VS / VM)where K is the equilibrium constant, VS is the volume of the stationary phase, and VM is

the volume of the mobile phase (dead volume).

Determining kAssume VR : VM = mT : mM

Then: VR / VM = mT / mM = (mM + mS) / mM = 1 + kSo: VR = VM (1 + k)And: tR = tM (1 + k)

Since tM = t0 k = (tR - t0) / t0

K should be between 1 and 5

Performance parameters Performance parameters (Advanced) Capacity Factor (k)(Advanced) Capacity Factor (k)


Selectivity Factor (α): Comparison of interaction with stationary phase

Ratio of capacity factors α = k (B) / k (A)

Primarily affected by changing the stationary or mobile phases Larger α means better separation (but little gain in resolution beyond α=3)

Resolution (RS): A measure of how well separated two peaks are:

RS = 2(tR peak A - tR peak B) / (wA + wB)

Since measuring w is difficult, can use:

RS = 0.25 x [(α - 1) / α] x [ k(B) / (1 + k(B)) x Nwhere B is the last eluting peak, and N is the plate number for B

High Rs is better: should be at least 1.5 for baseline separation

Performance parameters Performance parameters (Advanced) Selectivity ((Advanced) Selectivity (αα) and ) and

Resolution (RResolution (Rss))


Performance parameters Performance parameters (Optimisation)(Optimisation)

Optimisation depends on type of chromatography, usually involves changes in:

Stationary Phase: Hundreds of kinds on offer, choice based on analytes to be separated, cost Differences based on chemical structure, particle size, column bore and length, compressibility of packing

Mobile Phase: Use changes in polarity, pH, viscosity Detector: Use best sensitivity available for analytes Flow rate: affects retention time and diffusion and thus performance Amount of sample: Too much will overload the column, too little will

be difficult to detect accurately Sample Matrix: Avoid incompatible contaminants, use minimum

injection volume, preferable similar to the mobile phase (HPLC) Temperature: increases improve performance (must avoid

decomposition of sample) by increasing solubility and reducing viscosity. Very important in GC.


Performance parameters Performance parameters (Summary)(Summary)

CChromatographic systems must be optimised to give: Good Peak Shape

Good separation/resolution (N, k, αα, R, Rss))

Flat and horizontal baseline No “artifactual” peaks Shortest possible analysis times

Resolution and Retention time are keyResolution and Retention time are key


Instrumentation (1) Instrumentation (1) Origins Preparative column chromatography (Glass columns) Internal diameter: 1 - 5 cm Length: 50 - 500cm Particle size: 150-200mm (large) Flow rate: tenths of ml/min, under gravity N<100/m

Developments Automation Many different types (see later) Engineered columns – can takes extreme pressure and temperature Reduction in column size (Typical Liquid Chromatography: Internal

diameter: 4 -10mm, Length: 10 - 30cm, Particle size: 5 -10µm) N>10,000/m


Instrumentation (2) Instrumentation (2)


Instrumentation (3) Instrumentation (3) 1) Gas and Solvent Reservoirs: Contain mobile phaseThere may be more than one reservoir Isocratic elution uses a constant mobile phase composition (only one

reservoir necessary) Gradient elution uses more than one, two pumps and a mixing system

deliver a mobile phase that varies with time Mobile phase may need pretreatment: e.g. Filtering: Prior to placing in the solvent reservoirs, or using an in-line filter Sparging (degassing): by sonication or by bubbling an inert gas (e.g.

Helium) through the solvent Guard Column (positioned after injection port): Provides filtration and

preconditioning to protect the column

2) Pumping systems (not Gas chromatography) Generate high pressures (up to 6000psi) Generate variable flow rates (0.1 - 800ml/min) Accurate and reproducible rates, independent of column back-pressure Be “pulse-free” Corrosion resistant


Instrumentation (4) Instrumentation (4) 3) Sample addition facility (injection port) Sampling Loop (see Skoog for diagram) Syringe injection, through a septum (<1500psi) Stop-flow, direct injection onto top of column packing Pump (larger volumes)

4) Column (& oven)- See later for stationary phases by type Usually stainless steel (may be glass) Length: 10 - 30cm, up to several m for GC i.d (internal diameter) mm-cm Particle size of packing 5 or 10μm >10,000 plates/m Most common packing is silica based, may also have alumina, zirconium,

polymeric, ion-exchange resin Short columns are quicker to use but have lower N Small particle size gives higher N but needs higher pressure to maintain flow A column oven or a water jacket may be used to provide a stable

temperature or to allow chromatography at different temperatures


Instrumentation (5) Instrumentation (5) 5) Detectors (More detail later) Generally the most expensive part of the instrument Choice depends on analyte properties, and required sensitivity (and

money available) Performance is measured in terms of Mass Limit of Detection (LOD)

which is the mass that gives a signal 5 times the standard deviation of the noise, using 10ml of a sample of Mr =200

6) Output Chart recorder: contains a drive-mounted pen that moves according to

the current supplied by the detector output. May include a second pen, driven by an integrator which “draws” the cumulative signal over time, giving the area under a peak

Integral processor: uses instrument hardware and drop-down menus to allow modifications of conditions, and or inspection/reprocessing of chromatograms

PC - based: output and controls are driven by specialist software written for use on a PC

Can allow the spectrum to be reprocessed and reprinted


Instrumentation (6) Instrumentation (6) Automatic Integration and Reprocessing – treat with

Caution

Automatic processing parameters must be set so that the instrument can “recognise” peaks correctly

This includes setting how the instrument recognises: Baseline Peak Start and Finish (time) Non retained peak (to ignore) Detectivity (size of baseline fluctuation to ignore)

This can be altered after a run to improve peak recognition – but will NOT improve poor chromatographic performance


Techniques Techniques

Gas Chromatography Gas Chromatography Liquid Chromatography Liquid Chromatography Supercritical Fluid Chromatography Supercritical Fluid Chromatography Advanced Techniques Advanced Techniques


Techniques Techniques


Gas Chromatography Gas Chromatography

From Harris, Quantitative Chemical Analysis, 6e, Chapter 24From Harris, Quantitative Chemical Analysis, 6e, Chapter 24


Gas Chromatography Gas Chromatography

Principles Principles Stationary phase types Stationary phase types Detection Systems Detection Systems


GC Principles GC Principles

Requires volatile analytes Utilises gas/liquid partition Most volatile / lowest boiling point normally elutes first Resolution primarily influenced bya) Temperature (can have gradient)b) Flow rate (affects diffusion and interaction with stationary phase)c) Stationary phase (type and distribution)d) Column dimensions


GC Stationary phase types GC Stationary phase types (1) (1)

Formats include WCOT (wall coated Open tube), SCOT (support coated) and PLOT (porous-layer)



GC Stationary phase types GC Stationary phase types (2) (2)

Chemistry of Stationary Phase Chemically bonded (as opposed to coated) are the most stable. Less polar polysiloxanes functionalised with methyl, phenyl, trifluoropropyl are common. Polar phases include polyethylene glycols (less thermally stable)

Mobile phase: Hydrogen (best), helium or nitrogen – very little scope for optimisation by mobile phase change



GC Detection Systems (1) GC Detection Systems (1) More than 10 types available, 4 most common are:

a) Flame ionisation detector (FID) Eluent burnt in a hydrogen fuelled flame Leads to release of electrons, dependant on [C] Requires own thermostat at T>column oven

Advantages: Robust, sensitive, semi-universal (C only), wide linear range

Disadvantages: Non-selective, destructive

b) Thermal ionisation detector (or nitrogen detector) Similar to FID Additional alkali metal salt (often rubidium chloride) component

Advantages: More sensitive than FID, Selective for N or P, wide linear range

Disadvantages: needs frequent renewal and careful calibration, destructive


GC Detection Systems (2) GC Detection Systems (2)

c) Electron Capture detector Senses reduction in standing current Normally operated at 300ºCAdvantages: Extremely sensitive, Selective for halogens, nitro

groups, peroxides, quinones, non-destructiveDisadvantages: Radioactive, limited range, easily contaminated

d) Mass selective detector Use mass spectrometry (EI or CI) Focus on monitoring specific molecular ions quantitatively,

although simple spectra are also possibleAdvantages: Extremely sensitive, and selective by massDisadvantages: most easily interfaced with a low flow rate system


Liquid Chromatography Liquid Chromatography

Stationary phase types Stationary phase types Adsorption Chromatography Adsorption Chromatography Size Exclusion Chromatography Size Exclusion Chromatography Capillary Electrophoresis Capillary Electrophoresis Ion Exchange Chromatography Ion Exchange Chromatography

Detection Systems Detection Systems


Liquid Chromatography Liquid Chromatography



Liquid ChromatographyLiquid ChromatographyWhich technique to use?Which technique to use?

102

103

104

105

106

Non-ionic, polar

Nonpolar Ionic

Water-solubleWater-insoluble

Partition

Adsorption Ion-exchange

(Gel permeation) (Gel filtration)

Size Exclusion

(Reversed Phase)

(Normal)

Increasing Polarity

Mol

ecul

a r w

e ig h

t


Adsorption/Partition Adsorption/Partition Chromatography Chromatography

Normal Phase Normal Phase Reversed Phase Reversed Phase


Adsorption/Partition Adsorption/Partition Chromatography Chromatography

Choosing mobile and stationary phases Stationary phase must have a similar polarity to the analyte Mobile phase is of substantially different polarity

Polarity SeriesIn general, polarity of organic compound in increasing order is:

Alkyl < alkenyl < aromatic < halides < sulfides < ethers < nitro < esters ~ aldehydes ~ ketones < alcohols ~ amines < sulphones < sulphoxides < amides < carboxylic acids < phosphates < water

Bold means that these groups can also be substantially affected by pH changes

NB Avoid conditions that could decompose the analyte


Normal Phase Normal Phase Stationary phase: normally a solid Analyte adsorbs to the stationary phase Packing is usually Silica or Alumina

and is therefore polarMobile phase: normally organic (i.e. not aqueous) wide choice of mobile phaseRetention: TR increases with polarity of analyte Increasing the polarity of the mobile

phase reduces elution time Optimisation normally consists of

varying the mobile phase

Si

H3C

H3C

RSi

Si

O

Si

Si

OSi

H3C

H3C

R

R=OH, CN, NH2 & more


Reversed Phase Reversed Phase Stationary phase: normally a liquid Analyte dissolves in the stationary phase Packing is usually modified Silica or

Alumina Mobile phase: normally aqueous, plus MeOH orMeCN wide choice of mobile phase buffersRetention: TR decreases with polarity of analyte Increasing the polarity of the mobile

phase increases elution time Optimisation normally consists of varying

the mobile phase

Si

H3C

H3C

RSi

Si

O

Si

Si

OSi

H3C

H3C

R

R= C18, C8, Ph & more


Size Exclusion Size Exclusion Chromatography (1) Chromatography (1)

Separates molecules with a high molecular weight. on the basis of size

Packing consists of small (~10mm) porous particles made of silica or a polymer

Separation is dependent on selective penetration of analytes into pores (requires at least 10% difference in molecular weight)

TheoryTotal column volume Vt = Vg + Vi + V0 , where

Vg is the volume occupied by the packing

Vi is the volume of solvent in the pores, and

V0 is the free solvent volume (similar to injection volume)



Analytes may:1) be too large to enter the pores at all, and elute at V0

2) enter the pores completely, and elute at V0 + Vi

3) partially (extent determined by K) interact with the pores and elute at V0 + Kvi

Exclusion limit: the molecular weight beyond which no interaction with the pores is possible. All analytes beyond exclusion limit elute together at V0*

Permeation limit: the molecular weight below which complete penetration of the pores occurs. All analytes below permeation limit elute together at (V0 + Vi)*

Selective permeation region: size between permeation limit and exclusion limit, where 0<K<1 Elution volume dependent on K*

*Assumption: no other interactions taking place



Applications1) Simple separations: e.g. a large protein from low Mw contaminants such as amino acids and salts2) Separation of oligomers: e.g. Series of fatty acids of increasing size3) Separation of homologs: e.g. sugars in fruit juice4) Determination of molecular weight: e.g. of a polymer with behaviour calibrated for the conditions used

Advantages Short analysis time Well defined separation times Narrow bands and good sensitivity Few problems with column contamination or sample loss

Disadvantages Limited number of peaks Requires ~10% difference in molecular weight


Capillary Electrophoresis Capillary Electrophoresis

Advantages Only needs nL sample High speed and resolution,

virtually no band broadening

Instrumentation Capillary tube (10 - 100mM

internal diam., 40-100cm long) Two buffer reservoirs, with

platinum electrodes DC potential (20-30 kV)

applied along capillary Sample introduced one end,

detector at other Direct of potential depends on

charge (+/-) of analyte

Separation of analyte ions via differential migration in an electric field, coupled with electro-osmotic flow of mobile phase


Capillary Electrophoresis Capillary Electrophoresis Mobile Phase Commonly phosphate or borate buffer (20-100 mM) pH and Ionic strength must be controlled Can add detergents to transport neutral molecules in a micelle (MEKC)

Stationary Phase No stationary phase for true CE Newer developments introducing a stationary phase combine CE and HPLC to give electrochromatography

Principles of separationBased on interaction of analyte with electric field

Migration velocity v = (µe + µeo) E where µe and µeo are the electrophoretic mobilities of the analyte and buffer, and E is the applied field strength


Capillary Electrophoresis Capillary Electrophoresis Retention and Resolution: dependent on

Charge / size ratio is primary separation factor Charge gives v and thus RT Size gives v and thus RT

Interaction with buffer ions / molecules pH and ionic strength of buffer affects ionisation of analyte – and thus RT Applied field Column Length Diffusion

Optimisation: may involve buffer pH - alter charge status of analyte buffer ionic strength - change capacity to produce electro-osmotic flow organic modifiers: influence ionic strength, and can “capture” analytes selectively to affect charge/size ratio


Capillary Electrophoresis Capillary Electrophoresis Detection: normally similar to LC detectors but Peak area is dependent on rate of movement through the detector Peak area not independent of retention time Mostly “on-column”, i.e. capillary forms flow cell Short path length gives reduced sensitivity Indirect methods may be needed to increase sensitivity

ApplicationsThere are a variety of named techniques, each suitable for different analytical problems. You may see mentioned: Capillary zone electrophoresis Capillary gel electrophoresis Capillary isotachophoresis Capillary isoelectric focusing Capillary electrochromatography Micellar electrokinetic capillary chromatography


Ion Exchange Ion Exchange Chromatography Chromatography

Principles Small K means low affinity – so

low RT K is often quoted vs. a common

reference ion (e.g. H+) K affected by:

Charge of ion: charge gives K Size of hydrated ion

Uses displacement of an ion from the stationary phase by a solute ionK is dependent on relative affinity of the analyte for the surface over the solvent ions


Cation Exchange Cation Exchange Chromatography Chromatography

Use organic acid functional groups as ionic species “Strong acid” type uses sulphonic acid (RSO3H) (more common) “Weak acid” type use carboxylic acids (RCOOH)

xRSO3H + Mx+ (RSO3-)xMx+ + xH+

Stationary Mobile Stationary Mobile

Ion SeriesFor a strong acid column, (e.g. RSO3H) size of K :

Ag+ > Cs+ > Rb+ > K+ > NH4+ > Na+ > H+ > Li+

andBa2+ > Pb2+ > Sr2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+ > Zn2+ > Mg2+


Anion Exchange Anion Exchange Chromatography Chromatography

Use amine groups as ionic species “Strong base” type uses quaternary amines (e.g. RN(CH3)3+OH-) “Weak base” type uses secondary or tertiary amines

xRN(CH3)3+OH- + Ax- (RN(CH3)3)x+Ax- + xOH-

Stationary Mobile Stationary Mobile

Ion Series

For a strong base column, affinity series for K is usually :

SO42- > Cr2O4

2- > I- > NO3- > Br - > Cl- > HCO2

- > CH3CO2- > OH- > F-


Ion Exchange Ion Exchange ChromatographyChromatography(optimisation) (optimisation)

Stationary phase: “Strong” or “weak”, different pKa’s and pKb’s of ionic stationary phase groups give different strengthsMobile phase pH: can affect both

a) the proportion of protonated stationary phase sites,b) the proportion of ionised analyte,

but b) is more influential so in mobile phase pH gives: in retention time for cation exchange in retention time for anion exchange

Ionic Strength: (concentration of the mobile phase), mostly increases selectivity between ions of different charge Buffer salt: i.e. the relative position of the competing counter ion in the series for K Organic modifier: resin supports have a non-ionic component so reverse-phase interactions can occur. Affected by adding an organic solvent Temperature: affects buffer solubility, viscosity and mass transfer kinetics


LC Detection Systems (1) LC Detection Systems (1) Generally the most expensive part of the instrument Depends on analyte properties, and required sensitivity (and money) Choice may be assisted by obtaining spectra on traditional instruments A) Ultraviolet/Visible absorption (may extend to IR) Most commonly used, Mass LOD = 100pg-1ng (1pg), 0.1-1mg for IR Three major types:

The simplest (and cheapest) uses a mercury source & can only detect 254nm and 280nm

Tungsten and Deuterium sources used, together with interference filters

The best (and most expensive) uses a diode array to monitor the spectrum of the eluent over a wide range of wavelengths

B) FluorescenceMass LOD = 1 -10pg (10fg)Monitors emission at specific wavelengths, in response to excitation with a specific wavelength


LC Detection Systems (2)LC Detection Systems (2)C) Refractive Index Mass LOD = 100ng-1mg (10ng) Monitors changes in the refractive index of the solvent, caused by the

presence of analytes Universal, but non-specific, not very sensitive

D) Mass Spectrometric Mass LOD = 100pg-1ng (1pg) Often found as a tandem or “hyphenated” technique

E) Nuclear Magnetic Resonance Spectroscopic A recent innovation in tandem techniques

F) Electrochemical Mass LOD = 10pg-1ng (100fg) May be potentiometric, conductometric or amperometric Most common is conductivity (esp for ion exchange and capillary

electrophoresis) – see over


LC Detection Systems (3)LC Detection Systems (3)Conductivity: Simple cheap robust but……need to solve the problem of detecting analyte ion in presence of large quantities of other ions (eluent)

Suppressor Column Ion exchange column of the opposite type to the analytical column Converts eluent ions (but not analyte) to a non-charged (non-conductive) form

Anion exchangeThe eluent is often sodium carbonate (Na2CO3), and the suppressor column is a strong acid type cation resin.2Na+

(aq) + CO32-

(aq) + 2Resin-H+(s) 2Resin-Na+

(s) + H2CO3(aq)

Charged Neutral

Cation ExchangeThe eluent is often hydrochloric acid (HCl), and the suppressor column is a strong base type anion resin. H+

(aq) + Cl-(aq) + Resin+OH-(s) Resin+Cl-(s) + H2O

Charged Neutral


Supercritical Fluid Supercritical Fluid Chromatography Chromatography

Supercritical fluids Occur above a critical temperature

and a critical pressure, Show both gaseous and liquid

properties for Density Diffusion coefficient and Viscosity

Are very good solvents Are frequently cheap, innocuous, non-

toxic Can easily be removed by evaporation Give GC/LC hybrid behaviour – make

great mobile phasesSFC is particularly useful for analytes that are: Non-volatile Easily thermally-decomposed and/or Unsuitable for spectroscopic or electrochemical detectionand thus cannot be analysed by GC or HPLC

Source:http://www.selectscience.net/products/Source:http://www.selectscience.net/products/analytical+preparative-sfc-instrument/?analytical+preparative-sfc-instrument/?

prodID=12418prodID=12418



Mobile:Stationary phase equilibriumDependent on partition, strongly influenced by solvation power of the supercritical fluid

Instrumentation: modified HPLC equipment: Thermostatted column oven Back-pressure device between column and detector (narrow capillary at end of column) Detection may be conducted in gas phase

Mobile Phase Any supercritical fluid (commonly CO2, N2O, NH3, 1-Butane)CO2 becomes supercritical at T>31ºC, P>7400KPa

Stationary Phase Variety, generally “cannibalised” from HPLC or GC May be packed (like HPLC) Or open-tubular siloxane coated (like GC)



Retention and Resolution Quicker than HPLC N similar to that for GC Suffers from band broadening (diffusion) like GC

Optimisation: Pressure gives solvent power and thus RT . e.g.(Hexadecane at 70atm RT=25min, 90 atm RT=5min). May use gradient or isobaric elution. Temperature (for similar reasons) Organic modifiers (e.g. MeOH)

Detection Often uses HPLC detectors (UV etc) SFC allows easy interface with MS or IR Also allows use of Flame Ionisation Detector (FID) Eluent easily removed for further analysis

ApplicationsWide range including: Drugs, foods, pesticides, polymers, surfactants….


Advanced Techniques Advanced Techniques

Immunoaffinity techniquesImmunoaffinity techniques Tandem techniquesTandem techniques


Advanced Techniques (1) Advanced Techniques (1) Immunoaffinity chromatographyImmunoaffinity chromatography Most often for solid-phase or on-line extraction to preconcentrate analyte

Involves Immobilisation of specific antibodies (soft gel or rigid support) Sample is passed slowly through the column, to allow specific analyte adsorption Sample is desorbed by manipulating mobile phase (pH, ionic strength, organic solvent, competition) Concentrated eluent subjected to further analysis

Advantages: Improves analytical capability via preconcentration Very good specificity and recovery

Disadvantages May not be robust/reusable For analytes, need to produce antibodies


Advanced Techniques (2)Advanced Techniques (2)Tandem techniquesTandem techniques LC-MS & GC-MS: Allows separation of species by LC-MS & GC-MS: Allows separation of species by chromatography, followed by MS of resultant ionschromatography, followed by MS of resultant ions MS/MS: Three main techniquesMS/MS: Three main techniques

Product ion scan: Single ion selected and Product ion scan: Single ion selected and refragmentedrefragmented

Precursor ion scan: Precursor ions which result in an Precursor ion scan: Precursor ions which result in an ion of interest identifiedion of interest identified

Neutral loss scan: identifies ions that lose a Neutral loss scan: identifies ions that lose a particular fragmentparticular fragment

Tandem techniques represent the gold standard for Tandem techniques represent the gold standard for most pharmacological, biomedical and environmental most pharmacological, biomedical and environmental analyses today.analyses today. http://www.speciation.net/md/000/001/878/http://www.speciation.net/md/000/001/878/

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