Chromatography General. Chromatographic Process Chromatographic Systems.
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Transcript of Chromatography General. Chromatographic Process Chromatographic Systems.
ChromatographyChromatographyGeneralGeneral
Chromatographic ProcessChromatographic Process
Chromatographic SystemsChromatographic Systems
Chromatographic TechniquesChromatographic Techniques
TLC/PC
HPLC
GC/SFC
PC-Paper Chrom
Chromatography – Separation MechanismChromatography – Separation Mechanism
AdsorptionAdsorption PartitionPartition Ion - Exchange & Ion - InteractionIon - Exchange & Ion - Interaction Size ExclusionSize Exclusion Affinity (antibody-antigen interactions; Affinity (antibody-antigen interactions;
chemical interaction; attraction)chemical interaction; attraction) Complexation - ChelationComplexation - Chelation Ion – exclusion (Separation of weak acids) Ion – exclusion (Separation of weak acids)
Sorption problems
ABsorption ADsorption
Different sorptions explained
Chromatograhy – Mechanism of SeparationChromatograhy – Mechanism of Separation
Adsorption Partition Ion exchange
Chromatography – Mechanism of SeparationChromatography – Mechanism of Separation
Affinity Size Exclusion
Chromatogram – Basic ParameterChromatogram – Basic Parameter
tR = retention timetm = dead time
H
1/2HW1/2
unretained
Chromatographic TheoriesChromatographic Theories
Adjusted retention time: Adjusted retention time: ttR’R’ = t = tRR – t – tMM
Plate theoryPlate theory – distillation – plate – distillation – plate numbernumber
N = 5.54[(tN = 5.54[(tRR – t – tMM)/w)/w1/21/2]]22
Plate height Plate height H = L/NH = L/N
This theory did not include interaction of analytes This theory did not include interaction of analytes with stationery phasewith stationery phase
Chromatography – Peak Broadening
Chromatographic TheoriesChromatographic Theories
Rate Theory Rate Theory – kinetic factors – van – kinetic factors – van DeemterDeemter
H = B/u + Cu (+ A)H = B/u + Cu (+ A)
Where:Where: u – velocity of mobile phaseu – velocity of mobile phaseB – effect of molecular diffusion B – effect of molecular diffusion C – Resistance to mass transferC – Resistance to mass transferA – Spreading related to different A – Spreading related to different
distance traveled by distance traveled by molecules molecules in packed columnsin packed columns
Chromatography – Packing Effect on BroadeningChromatography – Packing Effect on Broadening
Chromatography - EquilibriumChromatography - Equilibrium
AAmobilemobile A Astationarystationary
Van Deemter factors:Van Deemter factors:
Molecular diffusion (B)Molecular diffusion (B) – in mobile – in mobile phasephase
proportional to time analyte spends in proportional to time analyte spends in a columna column
affected by diffusion coefficient of affected by diffusion coefficient of analyte in mobile phaseanalyte in mobile phase
affected by temperature and pressureaffected by temperature and pressure not important in LC – low diffusion not important in LC – low diffusion
coefficientcoefficient inversely affected by mobile phase inversely affected by mobile phase
velocityvelocity
Van Deemter factors:Van Deemter factors:
Resistance to mass transfer (C):Resistance to mass transfer (C): Mass transfer in mobile and stationary Mass transfer in mobile and stationary
phasephase Lack of equilibrium – moving phaseLack of equilibrium – moving phase Affected by thickness of liquid phaseAffected by thickness of liquid phase Affected inversely by the diameter of Affected inversely by the diameter of
particles or inner diameter of capillary particles or inner diameter of capillary columncolumn
Lower at higher temperatures (viscosity)Lower at higher temperatures (viscosity)
Van Deemter factors:Van Deemter factors:
Conclusions:Conclusions: Minimum value for H is achieved Minimum value for H is achieved
when:when: stationery phase thickness is minimalstationery phase thickness is minimal column packed with the smallest particlescolumn packed with the smallest particles capillary columns have the smallest capillary columns have the smallest
internal diameterinternal diameter mobile and stationary phases have low mobile and stationary phases have low
viscosity and high diffusion coefficientviscosity and high diffusion coefficient
Chromatography – van Deemter PlotChromatography – van Deemter Plot
Mobile phase velocity
Multipath effect
Mass transfer
Diffusion (Longitudinal)
Pla
te h
eigh
t (c
m)
H
Cu
A
B/u
Chromatography - ResolutionChromatography - Resolution
Response
tR1 tR2tR
Wb1
R = 2(tR1 – tR2)/Wb1 – Wb2
Wb2
Baseline resolution for Gaussianshape peaks = 1.5
100%
Chromatography - ResolutionChromatography - Resolution
Resolution equation where separation Resolution equation where separation parameters are included:parameters are included:
RRss = ½ = ½ xx ( (-1/-1/+1) +1) xx k’ k’22/1+k’/1+k’22xx (L/h) (L/h)1/21/2
Where:Where: – selectivity factor (separation) – selectivity factor (separation) = t= tR1R1/t/tR1R1
k’ – migration term, capacity factor; k’ – migration term, capacity factor;
k’ = mk’ = mss/m/mmm
L – column lengthL – column lengthh – plate height h – plate height
Chromatography - ResolutionChromatography - Resolution
ChromatographyChromatography
Qualitative AnalysisQualitative Analysis Retention data – RT; RRetention data – RT; Rff; RRT; Kovacs ; RRT; Kovacs
IndexIndex
Quantitative AnalysisQuantitative Analysis Peak area and height usually proportional Peak area and height usually proportional
to the amount of componentto the amount of component CalibrationCalibration Internal Standard method Internal Standard method External Standard methodExternal Standard method Area Normalization method Area Normalization method
Chromatogram – Basic ParameterChromatogram – Basic Parameter
tR = retention timetm = dead time
H
1/2HW1/2
unretained
IS α
- C
hol
esta
ne
Ch
oles
tero
l
1
2
3
4
5
RRT1 = RT1/RTIS
RRT2 = RT2/RTIS
RRT3 = RT3/RTIS
Accurate to e few digits (2) at fourthDecimal Point
ENDEND
Chromatography - MethodologyChromatography - Methodology
Peaks Broadening