1. HPLC

2. The best application fields of various chromatographic modesGC
Volatile, thermostable compounds LC
Polar, non volatile. thermolabile
EKC
Ionic compounds

3. The role of 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.

4. Advantages of various chromatographic modes
Tulajdonság GC
SFC
HPLC
EKC
Efficiency
++++
+++ ++
Analyses temperature +
Variability of mobile phase /
Speed of analyses
Sensitivity
Established instrumentation

6. Resolution as function of other chromatographic parameters

7. Resolution-efficiency- selectivity
HPLC can produce high selectivity,
but moderate efficieny (< tp).
At least, α = 1.3 is required for baseline separation.

8. Band broadening in HPLC
The HPLC uses packed columns.
The diffusion processes are much slower in HPLC than GC.

9. Van Deemter curve in HPLC
The slow diffusion causes increasing HETP values
as function of linear flow of mobile phase.

10. Schematic view of high performance liquid chromatography (HPLC) instrument
Degassing is important to gain smooth baseline.

11. An pp to date HPLC instrument
Pumps upto 300 bar
The degassing is important

12. Pump
Pump head
Motor & Cam
Check valves
Plunger
Plunger seal

13. Pump of HPLC instrument
Pulsation of system is decreased with two pumps,
working in opposite periods.

14. Gradient system Isocratic system Gradient system
Fixed (un-changeable) mixing ratio during analysis
Gradient system
Changeable mixing ratio during analysis
HPGE (High Pressure Gradient, mixing after pumps)
LPGE (Low Pressure Gradient, mixing before pumps)

15. Mobile phase pump with 4 eluents
Low Pressure Gradient

16. Aim of gradient - problems in isocratic mode -
Methanol / water = 6 / 4
Long analysis time, low signal to noise ratio
Methanol / water = 8 / 2
Poor separations
(Column : ODS type)

17. Aim of gradient - solution -
Gradual change of the mixing ratio during analysis
95%
30%
Methanol concentration
in mobile phase
Short analysis time
&
Excellent separation, good signal to noise ratio

18. Polarity of eluents

19. Rotary valve injection in HPLCben
The loop injector introduces exact volume of sample.

20. On-line SPE-HPLC arrangement
Precolumn is in the loop. Precolumn is good for sample concentration.

21. HPLC analyses of polar pesticides with precolumn concentration

22. Integrated precoumn HPLC
The precolumn protect the main column, against the deposition of matrix components, and dissolution of stationary phase.
Main columns have cm length and 2- 4,6mm I.D.

23. Dead volume
Dead volume may cause problems such as poor peak separations and poor reproducibility.
Male nut
Dead volume
Tube
Excellent connection
Poor connection

24. Sample vs. HPLC mode

25. The diameters and porosity of sample influence of efficieny
The efficiency increase with the decrease of packing diameter. However the mobile phase pressure has limits (~ 250 att), wich allows 3-5 µm size of packing material.
The increased porosity increased the loadability. However the deep holes are badly washed.
Spherical particles are the best.

26. Various HPLC packings
Goodnes: monolith > spherical > irregular

27. New type of packings
The limited depths of holes improves the efficiency.

28. New trend the use of 1.8 µm diameter packings
Very high pressure, short columns and fast analyses

29. Different molecular weight molecules requires different poremsizes
Bigger molecules need bigger pore size..

30. Most frequently used HPLC

31. Normal phase / Reversed phase
Stationary phase
Mobile phase
Normal phase
High polarity
(hydrophilic)
Low polarity
(hydrophobic)
Reversed phase

32. Retention order on reverse vs. normal phase packings

33. Polarity of solvent
The strongest mobile phase is hexane in reversed phase mode.
The strongest mobile phase is acetic acid in normal phase mode.

34. Bonded silica (Reversed phase HPLC packing)
Revers phase s are used in 80 % of HPLC analyses.

35. Stationary phase Reversed phase packings: C18 C8 C4
Cinao
Diol
Normal
Specials: chiral, ion exchange, gel
Increasing polarity→

36. Most frequently used HPLC stationary phase C18
Apolar compounds have big retention
Mobile phases are mixture of water, methanol acetonitrile.

37. Condition process of C18 stationary phase
A methanol wash reqires for the activation of
C18 stationary phase.

38. Column polarity - Retention time
C18 (ODS) OH
weak
strong
CH3

39. Mobile phase polarity - Retention time
Mobile phase: Methanol /Water
Methanol / Water
60 / 40
Methanol / Water
70 / 30
Methanol / Water
80 / 20

40. Influence of strength of mobile phes on C18 stationary phase
A decrease of mobile phase strength results in
increases of resolution values and retention times.

41. HPLC analysis of basic herbicides
Amines need specially deactivated packings

42. Ionic compounds analysed as ion pairs on C18.

43. Cianopropyl Stationary phases

44. Stationary phase vs. sample

45. Normal phase, Adsorption chromatography
The molecules of sample is solved in mobile phase, but they touch only in the surface of stationary phase.

46. Ion excange chromatography
The ions of stationary phase interact with the oppositely
charged molecules of sample.

47. Ion chromatogram of anaions
The stationary phase is anionic ionexchange resin.

48. Analysis of anions in ppb level using supressor

49. Size excusion (gel) chromatography
The voluminous molecules elute fast because they are
excluded from the small diameter pores, therefore they
interact in less extent.

50. Size excusion (gel) chromatography

51. Specially designes stationary phase for carbamate pesticides
Carbamate can not be analysed with GC,
because they are thermolabiles.

52. Molecular imprintesd (MIP) stationary phases
They are very selective,
but low efficiency
packings

53. Various HPLC detectors
Electrochemical S
Mass spectrometric U
Fluorescent S
Ultraviolett S
Refractive U
Light scaterring U
S, selective; U, univeral

54. UV/UV-VIS detector Ein Eout l A A = e·C·l = –log (Eout / Ein) C
C : Concentration
Cell
Ein
Eout A C
D2 / W Lamps l
A = e·C·l = –log (Eout / Ein)
(A : Absorbance)

55. External standard Calibration curve Area Concentration A1 C1 A4 A2 A3
Peak area A2 A3 C3 A1 A4 C4 C1 C2 C3 C4
Concentration

56. Internal standard Calibration curve Concentration Internal Area Target
AIS
Calibration curve C1
CIS
A4 /AIS A2
AIS
A3 /AIS C2
CIS
Area: Target / Internal standard
A2 /AIS A3
AIS C3
CIS
A1/AIS A4
AIS
C1/CIS
C2 /CIS
C3 /CIS
C4 /CIS C4
CIS
Concentration: Target / Internal standard

57. Diodarray (DAD) UV-VIS detector

58. HPLC-UV detection of pesticides

59. Recommended detection wave length for various functional groups

60. Light scattering HPLC detector
Universal, sensitive

61. Refractive index detector (RID-10A)
Photodiode
Reference
W Lamp
Sample

62. Ionization in HPLC/MS

64. LC/MS-MS is appropriate for compound identification
First MS→Ionic adduct with soft ionization
Second MS→fragmentation with EI ionization

65. On line HPLC/MS coupling