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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
Time
Inte
nsity
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Chromatographic peak
Qualitative measure: retention time (tR)Quantitative measure: Peak area (A)
An ideal chromatographic has Gaus shape
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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
Qualitative measure: retention time (tr)Quantitative measure: Peak area (A)
An ideal chromatographic has Gaus shape
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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 expressionsRetention 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 chromatographyThe 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)/(Wh1+Wh2)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 stoneLight stone
Heavy stoneHeavy stone
base
water flowwater flow
Simulation of separation
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Material distribution between the stationary phase and mobile phase
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Separation processes
Place of materials from injection point
Inte
nsity
of s
igna
l
Num
ber
of th
e eq
uilib
rium
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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
HETP =A +B/u + CuA=Eddy Diffusion, B =Molecular Diffusion, C =Resistance to mass transfer, U =Linear velocity of mobile phase (cm/s)
HETP: high equivalent theoretical plate, column HETP: N/L (L: length of )
U
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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 resolutionIncrease the efficiency•Longer columns•Smaller diameter of column or smaller diameter of particles•Thinner stationary phase•Optimated flow of mobile phaseIncrease 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 α = Δ(ΔS0)/R- Δ(ΔH0)/RT
α: selectivityS: entrophyH: entalphyR: gas constantT: absolute temperature
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Selectivity increase with derivatisation
Oszlop: 10 m x 0.1 mm
CSP: Chirasil-Dex
Vivő gáz: H2
Hőmérséklet: 180C
GC
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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
A: 20 m x 0,2 mm, ChNEB, 160° C. B: 20 m x 0,2 mm, ChDA, 150° C.
GC
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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 SteepnessArea
Sampling frequencyMode 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).