1. Chromatography

3. Chromatography
Principles
Techniques

4. Principles Introduction The Chromatogram Performance parameters
Instrumentation

5. 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

6. Introduction (2) Fundamental Basis
Movement of a compound through the system is governed by the equilibrium process: K
Compound (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 electrophoresis
B) Column (e.g. HPLC, GC)

7. Introduction (3): Plates/Gels
Consists of using a solvent (mobile phase) sometimes in an applied field to move compounds over a flat surface coated with a suitable stationary phase
1) Sample is loaded in solution
2) 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

8. Introduction (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 analysis
Results: Area under peaks (quantitative) & Retention time (qualitative)

9. 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? tR
tR’ t
Analyte
peak
Peak of unretained
Detector Repsonse
solute “solvent peak” w
Start
Time w b

10. Performance parameters (basic)
1) Resolution
Peaks should start and finish at the baseline
NOT
Well resolved peaks Unresolved peaks
2) Peak shape
Peaks should be:
Symmetrical
Representative of a Gaussian distribution
As narrow as possible
Reproducible between “runs”

11. Performance parameters (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

12. Performance parameters (Advanced) Theoretical Plate Model
Mathematical 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 rate
Each 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

13. Performance parameters (Advanced) Chromatographic Efficiency
Column Efficiency (measured by plate number: N)
N = (tR/s)2
tR is total retention time, and s is the standard deviation of a gaussian peak
BUT 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

14. Performance parameters (Advanced) Chromatographic Efficiency
Other variants of chromatographic efficiency
Effective N (Neff): If tR is low, then t0 affects apparent efficiency
Neff = 5.54 x [(tR - t0) / peak width at half height]2
Plate Height (H): Used to compare columns of different lengths:
H= L / N
L = column length, N = number of plates
H is a measure of plate size, the smaller (lower H) the better (HPLC ~ 10mm)
Effective plate height: takes account of columns with different dead spaces
Heff = L / Neff
Reduced plate height: allows comparison of columns with different particle sizes
h = H / dp
where dP is the particle diameter (same units as L); (Good HPLC column: h = 3)

15. Performance parameters (Advanced) Capacity Factor (k)
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 k
Assume VR : VM = mT : mM
Then: VR / VM = mT / mM = (mM + mS) / mM
= 1 + k
So: VR = VM (1 + k)
And: tR = tM (1 + k)
Since tM = t k = (tR - t0) / t0
K should be between 1 and 5

16. Performance parameters (Advanced) Selectivity (α) and Resolution (Rs)
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 N
where 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

17. Performance parameters (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.

18. Performance parameters (Summary)
Chromatographic systems must be optimised to give:
Good Peak Shape
Good separation/resolution (N, k, α, Rs)
Flat and horizontal baseline
No “artifactual” peaks
Shortest possible analysis times
Resolution and Retention time are key

19. Instrumentation (1) Origins
Preparative column chromatography (Glass columns)
Internal diameter: cm
Length: cm
Particle size: mm (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: cm, Particle size: 5 -10µm)
N>10,000/m

20. Instrumentation (2)

21. Instrumentation (3)
1) Gas and Solvent Reservoirs: Contain mobile phase
There 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 ( ml/min)
Accurate and reproducible rates, independent of column back-pressure
Be “pulse-free”
Corrosion resistant

22. 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: cm, 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

23. 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

24. 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

25. Techniques Gas Chromatography Liquid Chromatography
Supercritical Fluid Chromatography
Advanced Techniques

26. Techniques

27. From Harris, Quantitative Chemical Analysis, 6e, Chapter 24
Gas Chromatography
From Harris, Quantitative Chemical Analysis, 6e, Chapter 24

28. Gas Chromatography
Principles
Stationary phase types
Detection Systems

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

30. GC Stationary phase types (1)
From Harris, Quantitative Chemical Analysis, 6e, Chapter 24
Formats include WCOT (wall coated Open tube), SCOT (support coated) and PLOT (porous-layer)

31. GC Stationary phase types (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
From Harris, Quantitative Chemical Analysis, 6e, Chapter 24

32. 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

33. GC Detection Systems (2)
c) Electron Capture detector
Senses reduction in standing current
Normally operated at 300ºC
Advantages: Extremely sensitive, Selective for halogens, nitro groups, peroxides, quinones, non-destructive
Disadvantages: 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 possible
Advantages: Extremely sensitive, and selective by mass
Disadvantages: most easily interfaced with a low flow rate system

34. Liquid Chromatography
Stationary phase types
Adsorption Chromatography
Size Exclusion Chromatography
Capillary Electrophoresis
Ion Exchange Chromatography
Detection Systems

35. Liquid Chromatography
From Harris, Quantitative Chemical Analysis, 6e, Chapter 25

36. Liquid Chromatography Which technique to use?
102
103
104
105
106
Non-ionic, polar
Nonpolar
Ionic
Water-soluble
Water-insoluble
Partition
Adsorption
Ion-exchange
(Gel permeation)
(Gel filtration)
Size Exclusion
(Reversed Phase)
(Normal)
Increasing Polarity
Molecular weight

37. Adsorption/Partition Chromatography
Normal Phase
Reversed Phase

38. Adsorption/Partition Chromatography
Choosing mobile and stationary phases
Stationary phase must have a similar polarity to the analyte
Mobile phase is of substantially different polarity
Polarity Series
In 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

39. Normal Phase Stationary phase: normally a solid
Analyte adsorbs to the stationary phase
Packing is usually Silica or Alumina and is therefore polar
Mobile phase:
normally organic (i.e. not aqueous)
wide choice of mobile phase
Retention:
TR increases with polarity of analyte
Increasing the polarity of the mobile phase reduces elution time
Optimisation normally consists of varying the mobile phase
R=OH, CN, NH2 & more

40. 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 buffers
Retention:
TR decreases with polarity of analyte
Increasing the polarity of the mobile phase increases elution time
Optimisation normally consists of varying the mobile phase
R= C18, C8, Ph & more

41. Size Exclusion 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)
Theory
Total 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)

42. Size Exclusion Chromatography (2)
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

43. Size Exclusion Chromatography (3)
Applications
1) Simple separations: e.g. a large protein from low Mw contaminants such as amino acids and salts
2) Separation of oligomers: e.g. Series of fatty acids of increasing size
3) Separation of homologs: e.g. sugars in fruit juice
4) 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

44. Capillary Electrophoresis
Separation of analyte ions via differential migration in an electric field, coupled with electro-osmotic flow of mobile phase
Advantages
Only needs nL sample
High speed and resolution, virtually no band broadening
Instrumentation
Capillary tube ( mM internal diam., cm 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

45. Capillary Electrophoresis
Mobile Phase
Commonly phosphate or borate buffer ( 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 separation
Based 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

46. 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

47. 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
Applications
There 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

48. Ion Exchange Chromatography
Uses displacement of an ion from the stationary phase by a solute ion
K is dependent on relative affinity of the analyte for the surface over the solvent ions
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

49. Cation Exchange 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 Series
For a strong acid column, (e.g. RSO3H) size of K :
Ag+ > Cs+ > Rb+ > K+ > NH4+ > Na+ > H+ > Li+
and
Ba2+ > Pb2+ > Sr2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+ > Zn2+ > Mg2+

50. Anion Exchange 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- > Cr2O42- > I- > NO3- > Br - > Cl- > HCO2- > CH3CO2- > OH- > F-

51. Ion Exchange Chromatography (optimisation)
Stationary phase: “Strong” or “weak”, different pKa’s and pKb’s of ionic stationary phase groups give different strengths
Mobile 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

52. 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) Fluorescence
Mass LOD = 1 -10pg (10fg)
Monitors emission at specific wavelengths, in response to excitation with a specific wavelength

53. 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

54. 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 exchange
The eluent is often sodium carbonate (Na2CO3), and the suppressor column is a strong acid type cation resin.
2Na+(aq) + CO32-(aq) + 2Resin-H+(s) Resin-Na+(s) + H2CO3(aq)
Charged Neutral
Cation Exchange
The 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

55. Supercritical Fluid 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 phases
Source:
SFC is particularly useful for analytes that are:
Non-volatile
Easily thermally-decomposed and/or
Unsuitable for spectroscopic or electrochemical detection
and thus cannot be analysed by GC or HPLC

56. Supercritical Fluid Chromatography
Mobile:Stationary phase equilibrium
Dependent 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)

57. Supercritical Fluid Chromatography
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
Applications
Wide range including: Drugs, foods, pesticides, polymers, surfactants….

58. Advanced Techniques
Immunoaffinity techniques
Tandem techniques

59. Advanced Techniques (1)
Immunoaffinity 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

60. Advanced Techniques (2)
Tandem techniques
LC-MS & GC-MS: Allows separation of species by chromatography, followed by MS of resultant ions
MS/MS: Three main techniques
Product ion scan: Single ion selected and refragmented
Precursor ion scan: Precursor ions which result in an ion of interest identified
Neutral loss scan: identifies ions that lose a particular fragment
Tandem techniques represent the gold standard for most pharmacological, biomedical and environmental analyses today.