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Chromatography

Prof. Zoltn Juvancz D.Sc.

The first chromatography by Tswett

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.

Gas chromatograms of 104 volatile organic compounds (VOC)

If the peaks are narrow, more compounds can be separated

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.

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.

Schematic view of aGC

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 m Fused silica wall has no metal content. The metals can cause destruction of sample and stationary phase with their catalytic effects.

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.

Trace analysis

Single cell analysis

Determination of trace compound from cpmplex matrix

Analyses of chlorinated pesticides with GC x GC system

Determination 106 compounds during one run

Determination of trace impurity of a compounds

Determination of compounds having different magnitudes in their volume.

Fast analysis

On-line GC/MS coupling with selected ion monitoring

Chromatography is highly automitized


Auotomatic processes allow whole day work without human inspection. The automated processes have high reproducibility and accuracy.

On line HPLC/MS coupling

Determination of origin of extasy tablets

Recognition of adulteration of bergamot oil

Elution profile of a chromatogram

Intensity

Time

Chromatographic peak
An ideal chromatographic has Gaus shape

Qualitative measure: retention time (tR) Quantitative measure: Peak area (A)

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).

Chromatographic peak
An ideal chromatographic has Gaus shape

Qualitative measure: retention time (tr) Quantitative measure: Peak area (A)

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.

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

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

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

Increasing of capacity ratio with increasing of volume of stationary phase

Changing of capacity ratio with decreasing solvent strength of mobile phase

Decreasing solvent strength results in longer retention times.

Distribution of the compounds between two phases


Kd = Cm/Cst = p/q E = KdV/(1 +KdV) Where Kd distribution constant Cm 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).

Distribution of a compound between the mobile and stationary phases

Material distribution between the stationary phase and mobile phase

K= p/q

Band broadening in mobile phase

Stones in river
Light stone Heavy stone water flow

base

Simulation of separation

Material distribution between the stationary phase and mobile phase

Separation processes
Number of the equilibrium

Place of materials from injection point

Intensity of signal

If the peaks are narrow, more compounds can be separated

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.

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.

Band broadening caused by uneven flow


The flow velocity is not uniform across the column.

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.

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 A=Eddy Diffusion, B =Molecular Diffusion, C =Resistance to mass transfer, U =Linear velocity of mobile phase (cm/s)

Resolution vs. efficiency, capacity ratio, selectivity


P. Sandra JHRC 12 (1989) 82.

Resolution-efficiency- selectivity

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

Higher efficiency with smaller diameter column


GC
Chirasil-Dex 10 m

Higher column length

Longer column give better resolution, but longer analysis times.

Increasing of capacity ratio with increasing of volume of stationary phase

Weaker mobile phase increases the capacity ratio

Selectivity steeply increases with decreasing temperature

Selectivity vs. analysis temperature


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

Selectivity increase with derivatisation


GC
Oszlop: 10 m x 0.1 mm CSP: Chirasil-Dex Viv gz: H2 Hmrsklet: 180C

Selectivity increase using selectivity stationary phase

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.

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.

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

Distorted peak shapes

The determination of area of badly separated peaks is errorneous

V.R. Meyer, Chromatographia 40 (1995) 15.

Asymmetric peaks give false area count.

Difficulties in area determination

Noisy baseline

Drifting baseline

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

Effect of overloading

Overloading destroys the resolution

Compensation of overloading

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)

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

The role of different interaction types in various chromatographic modes


Types Dispersion - Dipole-dipole Hydrogen bridge Ionic
Repulsion

GC ++++ ++ ++ + /
++

SFC +++ ++ ++ ++ /
++

HPLC ++ ++++ ++++ +++ ++


+++

EKC + + ++ +++ ++++


++

The GC is mostly (70-95%) boiling point selective method.

Advantages of various chromatographic modes


Tulajdonsg Efficiency Analyses temperature Variability of mobile phase Speed of analyses Sensitivity Established instrumentation GC SFC HPLC EKC ++++ +++ ++ ++++ + +++ ++++ ++++

/
++++ ++++ +++

+
++ ++ +

++++
+ +++ ++++

+++
+++ + ++

GC is very efficient (long columns), but less selective method (no mobile phase selectivity).