1. Prof. Zoltán Juvancz D.Sc.
Chromatography
Prof. Zoltán Juvancz D.Sc.

2. The first chromatography by Tswett

3. 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.

4. Gas chromatograms of 104 volatile organic compounds (VOC)

5. If the peaks are narrow, more compounds can be separated

6. 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.

7. 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.

8. Schematic view of aGC

9. Cross section of open tubular (capillary)
General parameters
Column length:
5-100 m
Column diameters: 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.

10. 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.

11. Trace analysis

12. Single cell analysis

13. Determination of trace compound from cpmplex matrix

14. Analyses of chlorinated pesticides with GC x GC system

15. Determination 106 compounds during one run

16. Determination of trace impurity of a compounds
Determination of compounds having different
magnitudes in their volume.

17. Fast analysis

18. On-line GC/MS coupling with selected ion monitoring

19. Chromatography is highly automitized
Auotomatic processes allow whole day work without human inspection.
The automated processes have high reproducibility and accuracy.

20. On line HPLC/MS coupling

21. Determination of origin of extasy tablets

22. Recognition of adulteration of bergamot oil

23. Elution profile of a chromatogram
Intensity
Time

24. Chromatographic peak An ideal chromatographic has Gaus shape
Qualitative measure: retention time (tR)
Quantitative measure: Peak area (A)

25. 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).

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

27. 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.

28. 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

29. 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.

30. 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

31. Increasing of capacity ratio with increasing of volume of stationary phase

32. Changing of capacity ratio with decreasing solvent strength of mobile phase
Decreasing solvent strength results
in longer retention times.

33. 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).

34. Distribution of a compound between the mobile and stationary phases

35. Material distribution between the stationary phase and mobile phase
K= p/q

36. Band broadening in mobile phase

37. Stones in river Light stone water flow Heavy stone
base
water flow
Heavy stone
Simulation of separation

38. Material distribution between the stationary phase and mobile phase

39. Separation processes Number of the equilibrium Intensity of signal
Place of materials from injection point

40. If the peaks are narrow, more compounds can be separated

41. 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.

42. 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.

43. Band broadening caused by uneven flow
The flow velocity is not uniform across the column.

44. 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.

45. The ideal flow of mobile phase is result of compromises
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)

46. Resolution vs. efficiency, capacity ratio, selectivity
P. Sandra
JHRC 12 (1989) 82.

47. Resolution-efficiency- selectivity

48. 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

49. Higher efficiency with smaller diameter columnGC
Chirasil-Dex 10 m

50. Higher column length Longer column give better resolution,
but longer analysis times.

51. Increasing of capacity ratio with increasing of volume of stationary phase

52. Weaker mobile phase increases the capacity ratio

53. Selectivity steeply increases with decreasing temperature

54. Selectivity vs. analysis temperature
= tR2’/ tR1’
ln α = Δ(ΔS0)/R- Δ(ΔH0)/RT
α: selectivity
S: entrophy
H: entalphy
R: gas constant
T: absolute temperature

55. Selectivity increase with derivatisationGC
Oszlop: 10 m x 0.1 mm
CSP: Chirasil-Dex
Vivő gáz: H2
Hőmérséklet: 180C

56. Selectivity increase using selectivity stationary phase

57. 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.

58. „Minor peak first” is important with peaks in different magnitudesGC
Nem csak a szórás, de az abszolult érték is más. Ok a tailing, ami a csatlakozás és adszorpciós helyek.
A: 20 m x 0,2 mm, ChNEB, 160° C. B: 20 m x 0,2 mm, ChDA, 150° C.

59. 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

60. Distorted peak shapes

61. The determination of area of badly separated peaks is errorneous
V.R. Meyer, Chromatographia 40 (1995) 15.

62. Asymmetric peaks give false area count.

63. Difficulties in area determination
Noisy baseline Drifting baseline

64. 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

65. Effect of overloading

66. Overloading destroys the resolution

67. Compensation of overloading

68. 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)

69. 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

70. The role of different interaction types in various chromatographic modesGC
SFC
HPLC
EKC
Dispersion
++++
+++ ++ +
 - 
Dipole-dipole
Hydrogen bridge
Ionic /
Repulsion
A királis felismerő kölcsönhatásoknak legjobban megfelelő módot érdemes kiválasztani.
GC a funkciós csoport nélküli szénhidrogén enatiomerek elválasztására is alkalmas.
The GC is mostly (70-95%) boiling point
selective method.

71. 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).