1. Chromatography: HPLC, HPSEC, MS, LC-MS/MS, GC
Isariya Techatanawat, PhD Director of Bioequivalence Study Group, Research and Development Institute,
The Government Pharmaceutical Organization

2. Chromatography
Chromatography is a technique for separating and/or identifying the components in a mixture.
Components in a mixture have different tendencies to adsorb onto a surface or dissolve in a solvent.
All chromatographic methods require one static part (stationary phase) and one moving part (mobile phase).

3. Type of Chromatography
Adsorption Chromatography
Partition Chromatography
Ion Exchange Chromatography
Size Exclusion Chromatography

4. 1. Adsorption Chromatography
Solid stationary phase
Liquid or gaseous mobile phase
Each solute has its own equilibrium between adsorption onto the surface of the solid and solubility in the solvent, the least soluble or best adsorbed ones travel more slowly.
The result is a separation into bands containing different solutes.

5. 1. Adsorption Chromatography

6. 2. Partition Chromatography
Stationary phase is a non-volatile liquid.
Mixture to be separated is carried by gas or liquid as mobile phase.
Solutes distribute themselves between the moving and the stationary phases, with more soluble component in mobile phase reaching the end of chromatography column first.

7. 2. Partition Chromatography

8. 3. Ion-Exchange Chromatography (IEC)
Ion-exchange chromatography based upon electrical charge.
Likes may repel, while opposites are attracted to each other.
Stationary phases are characterized by nature and strength of acidic or basic functions on their surfaces and the types of ions that they attract and retain.

9. 3. Ion-Exchange Chromatography (IEC)
Opposite charge are electrostatically bound to the surface.
When the mobile phase is eluted through resin, electrostatically bound ions are released as other ions are bonded preferentially

10. Ion-Exchange Chromatography (IEC)
Cation exchange is used to retain and separate positively charged ions on a negative surface.
Anion exchange is used to retain and separate negatively charged ions on a positive surface.

11. 3. Ion-Exchange Chromatography (IEC)

12. 3. Ion-Exchange Chromatography (IEC)
Adsorption based on binding of opposite charges.
Different components (virus, proteins, DNA) have different charges, which means strength of binding varies from component to component.

13. 4. Size Exclusion Chromatography
Mixture passes as a gas or a liquid through a porous gel.
Pore size is designed to allow the large solute particles to pass through uninhibited.
Small particles permeate gel and are slowed down. The smaller the particles, the longer it takes for them to get through column.
Separation is according to particle size.

14. 4. Size Exclusion Chromatography

15. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

16. Principles of Liquid Chromatography

17. HPLC HPLC is improved form of column chromatography.
Smaller particle size for column packing material
Provide greater surface area for interactions between stationary phase and the molecules.
Allow better separation of mixture component

18. HPLC
Instead of solvent being allowed to drip through a column under gravity, it is forced through under high pressures.
To separate, identify, quantitate compounds.
Compounds in trace concentrations as low as parts per trillion [ppt] may also easily be identified.

19. HPLC
Normal Phase HPLC
Reversed Phase HPLC

21. Normal Phase HPLC Stationary phase is polar (eg silica particles).
Mobile phase is non-polar solvent (eg. Hexane).

22. Normal Phase HPLC
Stationary phase is polar and retains polar yellow dye most strongly.
Non-polar blue dye is won in the retention competition by the mobile phase and elutes quickly.

23. Reversed Phase HPLC
Stationary phase is non-polar. Silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface (eg. C8 or C18 carbon atoms).
Mobile phase is polar solvent (eg. mixture of water and methanol).

24. Reversed Phase HPLC
The most strongly retained compound is non-polar blue dye, as its attraction to non-polar stationary phase.
Polar yellow dye is won in competition by the polar, aqueous mobile phase, moves the fastest, and elutes earliest.

25. HPLC

26. HPLC Separation

27. What is Chromatogram?

28. HPLC: Qualitative Analysis

29. HPLC: Quantitative Analysis

30. HPLC Chromatogram

31. Isocratic LC System

32. Gradient LC System

33. Gradient LC System

34. Preparative Chromatography

35. High Performance Size Exclusion Chromatography (HPSEC)

36. Size Exclusion Chromatography (SEC)
Smaller molecules penetrate more of the pores on their passage through the bed.
Larger molecules may penetrate pores above a certain size so they spend less time in the bed.
The larger molecules elute first, while the smaller molecules travel slower [because they move into and out of more of the pores] and elute later.

37. Size Exclusion Chromatography (SEC)

38. Size Exclusion Chromatography (SEC)
Biomolecules could be separated based on their size, by passing, or filtering, them through a controlled-porosity packing.
Stationary phases are synthesized with a pore-size distribution.
Mobile phases are good solvents for analytes and may prevent any interactions [based on polarity or charge] between analytes and stationary phase surface.

39. Size Exclusion Chromatography (SEC)
Large components such as virus typically elute just after void volume.
Contaminating proteins and nucleic acids elute at volume greater than void volume.

40. SEC: Exclusion Limit
SEC resin are often characterized by their exclusion limit.
Exclusion limit is the molecular weight of the smallest molecule which cannot enter the pores of the matrix.
All molecules bigger than the exclusion limit elute in the void volume.

41. Typical steps in SEC
Equilibration – prepare column for binding target biomolecule/virus
Load – apply biomolecule/virus to column
Elution – collect purified biomolecule/virus from column
Cleansing/Sanitization – ensure no viable microorganisms present
Storage – fill column with solution that minimizes biological growth and maintains integrity of resin

42. Separation by SEC

43. HPSEC

44. HPSEC

45. HPSEC
Chromatogram shows how much material exited the column at any one time, with the higher molecular weight, larger polymer coils eluting first, followed by successively lower molecular weight (and therefore smaller) chains emerging later.
The primary separation is according to elution volume.

46. HPSEC
Chromatogram is compared to calibration that shows the elution behavior of a series of polymers for which the molecular weight is known.
Molecular weight distribution of the sample is calculated.

47. Calibration graph used to determine polymer MW from its retention time

48. Average molecular weights of polymer nearly symmetrical

49. Process flow for the preparation of clinical flu vaccine
Expansion of Vero cells
Infection
Harvest and Clearance
Benzonase
TFF
AIEX
SEC
Purified Vaccine Bulk

50. Mass Spectrometry (MS)

51. Mass Spectrometry
Measures mass-to-charge ratio of ions to identify and quantify molecules.

52. Mass spectrometer
1. Ion source: Sample molecules are ionised. 2. Mass analyzer: Sample molecules are separated according to their mass/charge ratio (m/z). 3. Ion detector: Separated ions are detected and sent to data system. m/z ratios and their relative abundance is presented as m/z spectrum .

53. Ionisation methods Electrospray Ionisation (ESI)
Matrix Assisted Laser Desorption Ionisation (MALDI)
Atmospheric Pressure Chemical Ionisation (APCI)
Chemical Ionisation (CI)
Electron Impact (EI)
Fast Atom Bombardment (FAB)
Field Desorption / Field Ionisation (FD/FI)
Thermospray Ionisation (TSP)

54. Mass Analyser
To separate ions formed in ionisation source according to their mass-to-charge (m/z) ratios.
Mass analysers: quadrupoles, time-of-flight (TOF) analysers, magnetic sectors , and both Fourier transform and quadrupole ion traps.

55. Ion Detector To monitor ion current and amplify it.
Signal is transmitted to data system where it is recorded as mass spectra .
m/z values of ions are plotted against their intensities to show number of components in sample, molecular mass of each component, and relative abundance of various components in sample.

56. Mass Spectrometer

57. Mass Spectrum

58. Mass Spectrum

59. Mass Spectrometry
m/z spectrum shows dominant ions at m/z 556.1, which are consistent with the expected protonated molecular ions, (M+H+).
Measured molecular weight is Da.

60. Mass spectrum of sulfamethazine acquired without collision-induced dissociation exhibits little fragmentation

61. Mass spectrum of sulfamethazine acquired with collision-induced dissociation exhibits more fragmentation and more structural information

62. Mass Spectrum

63. Mass Spectrometry
Samples (M) with molecular weights greater than 1200 Da give rise to multiply charged molecular-related ions such as (M+nH)n+ in positive ionisation mode and (M-nH)n- in negative ionisation mode.

64. Mass Spectrometry
Proteins have many suitable sites for protonation as all of the backbone amide nitrogen atoms could be protonated theoretically, as well as certain amino acid side chains such as lysine and arginine which contain primary amine functionalities.

65. Applications for Mass Spectrometry
Field of Study
Applications
Proteomics
Determine protein structure, function, folding and interactions
Identify a protein from the mass of its peptide fragments
Detect specific post-translational modifications throughout complex biological mixtures
Quantitate (relative or absolute) proteins in a given sample
Monitor enzyme reactions, chemical modifications and protein digestion
Drug 
Discovery
Determine structures of drugs and metabolites
Screen for metabolites in biological systems
Clinical 
Testing
Perform forensic analyses such as confirmation of drug abuse
Detect disease biomarkers (newborns screened for metabolic diseases)
Genomics
Sequence oligonucleotides
Environment
Test water quality or food contamination
Geology
Measure petroleum composition
Perform carbon dating

66. Tandem (MS/MS) mass spectrometers
More than one analyser.
quadrupole-quadrupole
magnetic sector-quadrupole
quadrupole-time-of-flight geometries.

67. LC-MS LC-MS/MS

68. LC-MS vs LC-MS/MS

69. LC-MS/MS

70. Full scan mass spectrum of ginsenoside Rb1 showing primarily sodium adduct ions

71. Full scan product ion (MS/MS) spectrum from the sodium adduct at m/z 1131.7

72. Subsequent full scan product ion spectrum (MS3) from the ion at m/z 789.7

73. LC-MS/MS

74. Identification of proteins

75. Identification of proteins

76. Biochemical Applications
Rapid protein identification using capillary LC/MS/MS and database searching

77. Molecular Weight Determination

78. Determining molecular weight of green fluorescent protein
27,000 Dalton with 238 amino acids.

79. Rapid protein identification using capillary LC/MS/MS and database searching

80. Protein identification
Full scan MS/MS spectra from doubly charged parent ion m/z and matching theoretical sequence identified by database searching

81. Identification of structurally similar aflatoxins

82. Analysis of peptides using CE/MS/MS

83. Gas Chromatography (GC)

84. Gas Chromatography
Mobile phase is gas. Stationary phase can either be solid or non-volatile liquid.
GC involves a sample being vapourised and injected onto the chromatographic column.
Sample is transported through the column by the flow of inert gaseous mobile phase.

85. Gas Chromatography

86. GC: Instrumental components
Carrier gas
Carrier gas must be chemically inert.
Commonly used gases: nitrogen, helium, argon, carbon dioxide.
Sample injection port
Temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample.

87. GC: Instrumental components
Columns
Packed columns
Capillary columns
Column temperature
Oven temperature is kept constant for a straightforward separation

88. GC: Instrumental components
Detectors
Concentration dependant detectors:
Signal is related to concentration of solute
Not destroy the sample
Dilution of with make-up gas will lower response
Mass flow dependant detectors
Destroy sample
Signal is related to rate at which solute molecules enter the detector.
Response is unaffected by make-up gas

89. Detector
Type
Support gases
Selectivity
Detectability
Dynamic range
Flame ionization (FID)
Mass flow
Hydrogen and air
Most organic cpds.
100 pg
107
Thermal conductivity (TCD)
Concentration
Reference
Universal
1 ng
Electron capture (ECD)
Make-up
Halides, nitrates, nitriles, peroxides, anhydrides, organometallics
50 fg
105
Nitrogen-phosphorus
Nitrogen, phosphorus
10 pg
106
Flame photometric (FPD)
Hydrogen and air possibly oxygen
Sulphur, phosphorus, tin, boron, arsenic, germanium, selenium, chromium
103
Photo-ionization (PID)
Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics
2 pg
Hall electrolytic conductivity
Hydrogen, oxygen
Halide, nitrogen, nitrosamine, sulphur

90. Gas Chromatography

91. References http://www.waters.com/ http://www.chemguide.co.uk
Chromatography. The Royal Society of Chemistry.
An Introduction to Mass Spectrometry. The University of Leeds.
Mass Spectrometry in Biotechnology
Gary Siuzdak , Academic Press 1996 SiuzdakBiotechnology”
An Introduction to Gel Permeation Chromatography and Size Exclusion Chromatography. Agilent Technologies.
Size-exclusion Chromatography of Polymers. Encyclopedia of Analytical Chemistryilent
Chromatography for influenza vaccine manufacture: Principles, Equipment and Design. NC State University
Basics of LC/MS. Agilent Technologies.
Gas chromatography. Sheffield Hallam University.