1. High Performance Liquid Chromatography

2. Introduction
HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution.
HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector.
Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase.
The mobile phase must be degassed to eliminate the formation of air bubbles.

3. Separation Principles in HPLC
General Rule of Thumb:
Polarity of analytes ≈ polarity of stationary phase ≠ polarity of mobile phase
To achieve good separation, the analytes should interact with the stationary phase, but not too strongly or the retention time will become very long

4. HPLC system

5. FOUR TYPES OF LIQUID CHROMATOGRAPHY
Partition chromatography
Adsorption, or liquid-solid
chromatography
Ion exchange chromatography
Size exclusion, or gel, chromatography

6. COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM
Solvent
Solvent Delivery System (Pump)
Injector
Sample
Column
Detectors (Diode Array)
Waste Collector
Recorder (Data Collection)

7. Picture of HPLC instrument

8. HPLC Chromatography
Pump System. Mobil phase pressures up to 6000 psi are necessary to achieve reasonable column elution times (~ minutes). Typical flow rates are 0.1 to 10 mL/minute.
Injection System. Used to introduce small samples (0.1 to 500 µL) into the carrier stream under high pressure.
Reservoirs (Solvents). Multiple solvents are necessary for performing gradient elution's (i.e. changing the polarity of the mobil phase during a run).
Chromatographic Column. Typically cm in length containing a packing of 5-10 µm diameter. Many types of columns are available, depending on the type of liquid chromatography desired.
Detector. Many types are available including UV, IR, refractive index, fluorescence, conductivity, mass spectrometry, and electrochemical. Diode array detectors are used when wavelength scans are desired.

9. Pump System Desirable Features:
Must generate pressures up to 6,000 psi
To allow for separation in reasonable time frames
Flow-rates range from 0.1 to 10 mL/minute
Limited pulsing in the system
Many HPLC systems have a dual pump system to minimize pulsing
Flow control and reproducibility < 0.5%
Corrosion resistance

10. Sample Injection System
Used to introduce small samples (0.001 to 0.5 mL) into the carrier stream under high pressure

11. HPLC columns
The column is one of the most important components of the HPLC chromatograph because the separation of the sample components is achieved when those components pass through the column.
The High performance liquid chromatography apparatus is made out of stainless steel tubes with a diameter of 3 to 5mm and a length ranging from 10 to 30cm.
Normally, columns are filled with silica gel because its particle shape, surface properties, and pore structure help to get a good separation. Silica is wetted by nearly every potential mobile phase, is inert to most compounds .
Silica can be used to separate a wide variety of chemical compounds, and its chromatographic behavior is generally predictable and reproducible.

12. Picture of an HPLC column

13. PARAMETERS AFFECTS SYSTEM
Column Parameters
Column Material
Deactivation
Stationary Phase
Coating Material
Instrument Parameters
Temperature
Flow
Signal
Sample Sensitivity
Detector

14. Sample Parameters
Concentration
Matrix
Solvent Effect
Sample Effect

15. Several column types (can be classified as )
Normal phase
Reverse phase
Size exclusion
Ion exchange

16. Normal phase
In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute.
For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample

17. Reverse phase
In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample.
Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase.
Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.

18. Size exclusion
In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size.
Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules.

19. Ion exchange
In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase.
Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.

20. Separations High Performance Liquid Chromatograph
Separation in based upon differential migration between the stationary and mobile phases.
Injector
Detector
Column
Solvents
Mixer
Pumps
High Performance Liquid Chromatograph
Waste
Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica
Mobile Phase - carries the sample through the stationary phase as it moves through the column.

21. Separations High Performance Liquid Chromatograph mAU time
Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph

22. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time

23. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

24. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

25. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

26. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

27. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

28. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

29. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

30. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

31. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

32. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

33. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

34. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

35. Separations mAU time Start Injection Injector Chromatogram Mixer Pumps
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

36. mAU time Start Injection Injector Chromatogram Mixer Pumps Column
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

37. The Chromatogram to - elution time of unretained peak
Injection to tR
mAU
time
to - elution time of unretained peak
tR- retention time - determines sample identity
Area or height is proportional
to the quantity of analyte.

38. HPLC Analysis Parameters
Mobile Phases
Flow Rate
Composition
Injection Volume
Column
Oven Temperature
Wavelength
Time Constant

39. Modes of High Performance Liquid Chromatography
Types of Compounds
Mode
Stationary
Phase
Mobile Phase
Neutrals
Weak Acids
Weak Bases
Reversed
C18, C8, C4
cyano, amino
Water/Organic
Modifiers
Ionics, Bases, Acids
Ion
Pair
C-18, C-8
Ion-Pair Reagent
Compounds not
soluble in water
Normal
Silica, Amino,
Cyano, Diol
Organics
Ionics Inorganic Ions
Exchange
Anion or Cation
Resin
Aqueous/Buffer
Counter Ion
High Molecular Weight
Compounds
Polymers
Size
Exclusion
Polystyrene
Silica
Gel Filtration-
Aqueous
Gel Permeation-
Organic

40. Uses of HPLC
This technique is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties.
It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.
In addition, it is used for analyzing air and water pollutants.
Federal and state regulatory agencies use HPLC to survey food and drug products.

41. HPLC Applications Bioscience Chemical Pharmaceuticals
Environmental
Pharmaceuticals
Consumer Products
Clinical
polystyrenes
dyes
phthalates
tetracyclines
corticosteroids
antidepressants
barbiturates
amino acids
vitamins
homocysteine
Bioscience
proteins
peptides
nucleotides
lipids
antioxidants
sugars
polyaromatic hydrocarbons
Inorganic ions
herbicides