1. High Performance Liquid Chromatography
( HPLC )

2. History
The origins of Liquid Chromatography began in the early 1900’s with the work of the Russian botanist, Mikhail S. Tswett.
His famous studies focused on separating compounds (leaf pigments), which were extracted from plants using a solvent.

3. This was followed by pure solvent.
Two specific materials that he found useful were powdered chalk (calcium carbonate) and alumina.
He poured his sample (solvent extract of homogenized plant leaves) into the column.
This was followed by pure solvent.
As the “sample” passed down through the column by gravity, different colored “bands” could be seen separated, because some moved faster than others.
Fig.1

4. Definition Tswett named his process: Chromatography
from the Greek words “chroma”, meaning “color”, and “graphy”, meaning “writing” (literally “color writing”) to describe his “colorful” experiment

5. This was followed by pure solvent.
Two specific materials that he found useful were powdered chalk (calcium carbonate) and alumina.
He poured his sample (solvent extract of homogenized plant leaves) into the column.
This was followed by pure solvent.
As the “sample” passed down through the column by gravity, different colored “bands” could be seen separated, because some moved faster than others.
Fig.1

6. Stationary phase Mobile phase
The immobile phase involved in the chromatographic process. The stationary phase in liquid chromatography can be a solid, a bonded or coated phase on a solid support, or a wall coated phase. The stationary phase used often characterizes the LC mode.
Mobile phase
The solvent that moves the solute through the column.

7. Chromatography Theory

8. Distribution of analytes between phases
A mobile A stationary
K=[A]s / [A]m
K: partition coefficient

9. tR: retention time
k'A = t R - tM / tM
K': capacity factor
α: selectivity factor
α = k 'B / k 'A

10. The Theoretical Plate Model of Chromatography
HETP: Height Equivalent to a Theoretical Plate
HETP = L / N

12. The Rate Theory of Chromatography
HETP = A + B / u + C u
Van Deemter equation

13. A - Eddy diffusion The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths.
Fig.2

14. B - Longitudinal diffusion The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening.
Fig.3

15. C - Resistance to mass transfer The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase.
Fig.4

17. Resolution

18. The selectivity factor, α, can also be manipulated to improve separations. When α is close to unity, optimising k' and increasing N is not sufficient to give good separation in a reasonable time. In these cases, k' is optimised first, and then α is increased by one of the following procedures:
Changing mobile phase composition
Changing column temperature
Changing composition of stationary phase
Using special chemical effects (such as incorporating a species which complexes with one of the solutes into the stationary phase

19. Types of Chromatography
Method
Stationary Phase
Mobil Phase
Liqiud Chromatography
Liqiud Liquid Chromatography (LLC)
Liqiud
Liquid
Liqiud Soild Chromatography (LSC)
Solid
Gas chromatography
Gas Liquid Chromatography (GLC)
Gas
Gas Solid Chromatography (GSC)
Soild

20. Types of Liquid Chromatography (LC)
Liquid chromatography can be performed in three primary approaches:
1. Thin Layer Chromatography (TLC)
2. Paper Chromatography
3. Column Chromatography

21. 1. Thin Layer Chromatography (TLC)
The sample is “spotted” onto, and then flowed through a thin layer of chromatographic particles fixed onto the surface of glass plates

22. Fig.5

23. 2. Paper Chromatography
The sample is “spotted” onto paper to which solvent is added to create flow.

24. Fig.6

25. 3. Column Chromatography
The sample passes through a column or a device containing appropriate particles.

26. Fig.7

27. What are the parts of an HPLC instruments?

28. Fig.8

29. HPLC System Diagram
Fig.9

30. 1. Solvent :
The HPLC uses a liquid to push the sample. We call this liquid the “Mobile Phase”—or sometimes, we just call it the “solvent.” It sits in a bottle right next to the HPLC, so we have plenty of it to use.

31. Isocratic and Gradient Operation
There are 2 basic types of chromatographic mobile phase operating modes which can be used in HPLC.

32. Fig.10

33. Fig.11

34. 2. Pump :
We need a pump to pump the solvent through the HPLC. We use very high pressures in HPLC. We pump some mobile phase solvents at 1900 psi.

35. 3. Sample injection loop
Fig.11

36. 4. Columns:
The goal of the column is to contain the chromatographic packing material (stationary phase), which will be used to effect the separation.

37. Fig.12

38. Separation Performance
Fig.13

39. 5. Detectors:
The instrument component in the HPLC system that senses the separated compounds eluting from the column, and provides the electronic signal to the Computer Data Station or Recorder to create the chromatogram.

40. 1. UV (Ultraviolet) Lamp FlowCell
Grating/Lens - Wave length
FlowCell
PhotoDiode - Differential Light Output

41. 2. RI (Refractive Index ) Universal analyte detector
Solvent must remain the same throughout the separation
VERY temperature sensitive
Somewhat difficult to stabilize baseline

42. 3. FD (Fluorescence )
Excitation wavelength generates fluorescence emission at higher wavelength
Analytes must have fluoropore group
Can react analyte with fluoropore reagent
Very sensitive and selective
More difficult methods transfer
Results vary dependent upon separation conditions

43. 4. MS ( Mass Spec ) Mass to charge ratio (m/z)
Allows specific compound ID
Several types of ionization techniques
electrospray, atmospheric pressure chemical ionization, electron impact

44. Separation Mechanism Adsorption chromatography
Partition Chromatography
Size Exclusion Chromatography ( Jel permeation chromatography)
Ion exchange chromatography

45. For separation of compounds with low polarity
1. Adsorption Chromatography
Stationary phase:
Silica gel
Alumina
Calcium carbonate
For separation of compounds with low polarity

46. 2. Partition Chromatography
Stationary phase: bonded phase
ODS or C18 C8
-NH2
-CN

47. Partition Chromatography Mechanisms
Normal Phase
For separation of compounds with different functional groups
2. Revered Phase
For separation of homologue compounds

48. Fig.14

49. Revered Phase
Fig.15

50. Normal Phase
Fig.16

51. 3. Size Exclusion Chromatography
An HPLC technique where the different sizes of the compounds are used to create the separation. There is no chemical attraction involved.
Molecules, such as proteins, DNA and peptides, can also be separated by size.

52. 4. Ion exchange chromatography
The stationary phase surface displays ionic functional groups that interact with analyte ions of opposite charge.
Cation exchange chromatography: retains positively charged cations because the stationary phase displays a negatively charged functional group such as a phosphoric acid.
Anion exchange chromatography: retains negatively charged anions using positively charged functional group such as a quaternary ammonium cation

53. Fig.17

54. Analysis using HPLC Qualitative analysis:
peak identification, sample identification.
2. Quantitative analysis:
peak integration, area normalization, external standards, internal standards.

55. Qualitative analysis:
Fig.18

56. Quantitative analysis:
Fig.19

57. External standards
Fig.20