Chromatography

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Chromatography

• Prof. Zoltán Juvancz D.Sc.

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The first chromatography by Tswett
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Aim of chromatography

• The compounds of interest has to be separated
  from matrix components.
• The signal to noise ratio must be as high as
  possible.
• The time consumption of analysis must be short.

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Gas chromatograms of 104 volatile organic
compounds (VOC)
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If the peaks are narrow, more compounds can be
separated
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Definitions

• Chromatography is a separation method.
• (Chromatography is not an identification method
  like NMR, IR, MS)
• Chromatography consist of two phases mobile and
  stationary phase.
• Mobile phase is forced along the column from
  injection to detector as a flowing media.
• Stationary phase is anchored to the column wall
  or to the particles, which are packed into the
  column.

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Chromatographic process

• The injected sample is dragged by mobile phase
  along the column.
• The components of the sample distribute between
  the stationary phase and mobile phase.
• If X compound has bigger affinity to stationary
  phase than affinity of Y compound to stationary
  phase, the X compound elutes later than Y from
  column.
• The sharp injected peaks become broader and
  broader during their run.

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Schematic view of aGC
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Cross section of open tubular (capillary)
General parameters Column length 5-100
m Column diameters 0.1-0.5 mm Film thickness
0.15 5 µm
Fused silica wall has no metal content. The
metals can cause destruction of sample and
stationary phase with their catalytic effects.
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Advantages of chromatography

• Exact quantitative analysis is done even from
  trace compounds.
• Disturbing effects of matrix compound can be
  eliminate.
• Small material consumption
• The quantization has a broad linearity range.
• Analyses of several compound can be done during
  one run.
• Chromatography is a fast analysis method.
• On-line coupling are routinely solved to compound
  identification methods,
• Well establishes instrumentation with high level
  automation is commercially available.

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Trace analysis
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Single cell analysis
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Determination of trace compound from cpmplex
matrix
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Analyses of chlorinated pesticides with GC x GC
system
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Determination 106 compounds during one run
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Determination of trace impurity of a compounds
Determination of compounds having different
magnitudes in their volume.
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Fast analysis
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On-line GC/MS coupling with selected ion
monitoring
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Chromatography is highly automitized
Auotomatic processes allow whole day work without
human inspection. The automated processes have
high reproducibility and accuracy.
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On line HPLC/MS coupling
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Determination of origin of extasy tablets
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Recognition of adulteration of bergamot oil
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Elution profile of a chromatogram
Intensity
Time
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Chromatographic peak
An ideal chromatographic has Gaus shape
Qualitative measure retention time
(tR) Quantitative measure Peak area (A)
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Chromatographic peak

• A certain compound shows same retention times
  under same conditions (column, flow, temperature
  etc.) independently from its injected amount (in
  the linear range of Langmuir isotherm). The
  identity of the compounds is partly based their
  retention time. (Qualitative parameter)
• Peak area is linear function of the quantity of
  certain compound (Quantitative parameter).

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Chromatographic peak
An ideal chromatographic has Gaus shape
Qualitative measure retention time
(tr) Quantitative measure Peak area (A)
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Chromatographic peak

• The given compound shows same retention times
  under same conditions (column, flow, temperature
  etc.) independently from its injected amount (in
  the linear range of Langmuir isotherm). The
  identity of the compounds is partly based their
  retention time.
• Peak area is linear function of the quantity of
  certain compound.

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Chromatographic expressions

• Retention time tR (qualitative measure)
• Hold up time tm (The time, what the compounds
  spend in mobile phase)
• Peak with in half high wh
• Peak area (A)
• (quantitative measure)
• Theoretical plate high N
• (efficiency, sharpness )
• N 5,54 (tR/wh)2
• Capacity ratio k
• (Strength of stationary phase)
• k tR-tm/tm

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Theoretical plate height
The peaks become broader and broader during the
chromatography The later eluting peak are
broader than early ones. The theoretical plate
height is same for all peaks. The theoretical
plate height characterize the chromatographic
system. The capacity ratio shows the interaction
of compound of interests (distribution between
the stationary and mobile phases.
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Chromatographic expressions

• Selectivity ?
• (Measure of interaction ratio between two
  compound and stationary phase)
• tR2/ tR1
• Resolution Rs
• (Measure the separation of two peaks)
• Rs 1,177 (tR1-tR2)/(Wh1Wh2)

Rs 1.5 baseline resolution
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Increasing of capacity ratio with increasing of
volume of stationary phase
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Changing of capacity ratio with decreasing
solvent strength of mobile phase
Decreasing solvent strength results in longer
retention times.
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Distribution of the compounds between two phases

• Kd Cm/Cst p/q
• E KdV/(1 KdV)
• Where
• Kd distribution constant
• C m concentration of a compound in mobile phase
• Cst concentration of a compound in stationary
  phase
• E extraction ratio
• V phase ratio
• Two compounds can be separated if their
  distribution constants are not equal (Kdx ? Kdy).

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Distribution of a compound between the mobile and
stationary phases
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Material distribution between the stationary
phase and mobile phase
K p/q
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Band broadening in mobile phase
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Stones in river
Light stone
Heavy stone
Simulation of separation
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Material distribution between the stationary
phase and mobile phase
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Separation processes
Number of the equilibrium
Intensity of signal
Place of materials from injection point
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If the peaks are narrow, more compounds can be
separated
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The slow mass transfer between the phases causes
peak broadening
Resistance of mass transfer processes cause that
the material zone are retained in stationary
phase from the material zone in mobile phase.
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Band broadening in open tube
The band of sample is getting broader caused by
slow diffusion from the middle of mobile phase to
the stationary phase.
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Band broadening caused by uneven flow
The flow velocity is not uniform across the
column.
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Band broadening caused by badly washed holes
The compounds come in and out from the holes
slowly. The diffusion processes are much slower
than the flow of the mobile phase.
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The ideal flow of mobile phase is result of
compromises
U
HETP high equivalent theoretical plate, column
HETP N/L (L length of )

• HETP A B/u
  Cu
• AEddy Diffusion, B Molecular Diffusion, C
  Resistance to mass transfer, U Linear velocity
  of mobile phase (cm/s)

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Resolution vs. efficiency, capacity ratio,
selectivity
P. Sandra JHRC 12 (1989) 82.
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Resolution-efficiency- selectivity
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Ways how to improve the resolution

• Increase the efficiency
• Longer columns
• Smaller diameter of column or smaller diameter of
  particles
• Thinner stationary phase
• Optimated flow of mobile phase
• Increase the capacity ratio (3-10)
• Bigger amount of stationary phase
• Weaker mobile phase (HPLC)
• Lower analysis temperature (GC)
• Increased selectivity
• More selective phases
• Lower analysis temperature
• Derivatization

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Higher efficiency with smaller diameter column

• GC
• Chirasil-Dex 10 m

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Higher column length
Longer column give better resolution, but longer
analysis times.
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Increasing of capacity ratio with increasing of
volume of stationary phase
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Weaker mobile phase increases the capacity ratio
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Selectivity steeply increases with decreasing
temperature
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Selectivity vs. analysis temperature

• tR2/ tR1
• ln a ?(?S0)/R- ?(?H0)/RT
• a selectivity
• S entrophy
• H entalphy
• R gas constant
• T absolute temperature

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Selectivity increase with derivatisation
GC

• Oszlop 10 m x 0.1 mm
• CSP Chirasil-Dex
• Vivo gáz H2
• Homérséklet 180?C

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Selectivity increase using selectivity stationary
phase
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Resolution
The small value of resolution can produce false
retention time. Peaks with different magnitudes
need more than Rs 1.5 value for their baseline
separations.
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Minor peak first is important with peaks in
different magnitudes
GC
A 20 m x 0,2 mm, ChNEB, 160 C. B 20 m x 0,2
mm, ChDA, 150 C.
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The peak area is the base of quantitative analysis

• The area of a peak is the sum of regularly
  measured signals
• Threshold value of
• Steepness
• Area
• Sampling frequency
• Mode of baseline
• corrections

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Distorted peak shapes
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The determination of area of badly separated
peaks is errorneous
V.R. Meyer, Chromatographia 40 (1995) 15.
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Asymmetric peaks give false area count.
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Difficulties in area determination
Noisy baseline Drifting
baseline
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Overloding

• If the concentration of a compound exceed the
  saturation value overloading effects occures.
• Errors are caused the overloading
• Incorrect retention times,
• Incorrect peak areas

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Effect of overloading
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Overloading destroys the resolution
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Compensation of overloading
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Different types of chromatography according to
mobile phase

• Gas chromatography, (GC)
• Liquid chromatography (LC), High performance
  liquid chromatography (HPLC)
• Supercritical fluid chromatography (SFC)
• Electro kinetic chromatography (EKC)

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Chromatogry according to the column

• Packed column
• Open tubular column (capillary)
• Chip
• Thin layer (TLC)
• Chromatography according to interaction types
• Distribution
• Adsorption
• Exclusion
• Ion exchange

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The role of different interaction types in
various chromatographic modes
Types GC SFC HPLC EKC
Dispersion
? - ?
Dipole-dipole
Hydrogen bridge
Ionic / /
Repulsion
The GC is mostly (70-95) boiling point
selective method.
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Advantages of various chromatographic modes
Tulajdonság GC SFC HPLC EKC
Efficiency
Analyses temperature
Variability of mobile phase /
Speed of analyses
Sensitivity
Established instrumentation
GC is very efficient (long columns), but less
selective method (no mobile phase selectivity).