1. High Performance/Pressure Liquid Chromatography (HPLC)

3. Definition:
The term HPLC stands for High Performance/ Pressure Liquid Chromatography.
It is a kind of sophisticated and mechanized column chromatography. It uses high pressure to drive the mobile phase through the (column of) stationary phase.
Chromatography: Method
Chromatograph: Machine
Chromatographer: Person
Chromatogram: Data

4. Liquid Chromatography Planer Chromatography Liquid Chromatography
Chromatographic Tree
Chromatography
Gas Chromatography
Liquid Chromatography
Column
Chromatography
Planer Chromatography
High Performance
Liquid Chromatography

5. General Description of System:
HPLC system principally consists of three parts-
A solvent delivery section
A separation section
A detection unit
Chromatography: Method
Chromatograph: Machine
Chromatographer: Person
Chromatogram: Data
Mixture
Pump
Injector
Guard Column
Column
Detector
Record
2nd solvent reservoir
1st solvent reservoir
Fig: Schematic Diagram of HPLC

6. High Performance Liquid Chromatograph
Shimadzu Prominence

7. Solvent Reservoir:
These are glass or stainless steel containers capable of holding upto a liter of mobile phase that may consist of pure organic solvent or aqueous solution of salts or buffers.
The mobile phase should be of the highest purity available since contaminants would be eventually deposited on the column thereby disrupting the chromatography.

8. Pumps:
Pumps are required to deliver a constant flow of mobile phase at pressures ranging from 1 to 550 bar (14.6 to 8000psi).
Such pumps (capable of pressure up to 8000psi) can provide a wide range of flow rates of mobile phase, typically from 0.01 to 10 ml/min.
Low flow rates (10-100µl/min) are used with microbe columns, intermediate flow rates (0.5-2 ml/min) are used with conventional analytical HPLC columns and fast flow rates are used for preparative or semi-preparative columns and for slurry packing technique.
Mechanical pumps of the reciprocating piston type give a pulsating supply of mobile phase.
But a Dual-piston reciprocating pumps produce an almost pulse –free flow because the two pistons are carefully phased so that as one is filling the other one is pumping.

9. Pumps

10. Desirable Pump performance
High Pressure Resistance
Shimadzu LC-10ATvp- 400Kgf
Precise Flow Rate
Shimadzu LC-10ATvp- Within  0.3 (%RSD<0.1)
Low Pressure Fluctuation
Shimadzu LC-10ATvp- 0.5 Mpa
High-accuracy gradient performance
Pulse-free solvent delivery

11. Desirable Pump performance

12. Injector:
The solute mixture is introduced into the chromatography by means of a suitable injection device.
Septum devices are available in which the sample solution is injected through a self sealing rubber septum or Teflon disk using a micro-liter syringes.

13. Desirable Auto Sampler
Performs various pretreatment functions, including addition of sample or reagent, mixing and diluting
Performs derivatization, addition of internal standard or sample dilution
transfer reagents and sample to an empty vial for pretreatment mixing

14. Desirable Auto Sampler

15. Mode of Separation: Isocratic Elution: Gradient Elution:
Elution through HPLC may be of two types
Isocratic elution
Gradient elution
Isocratic Elution:
If a solvent or mixture of solvent, having fixed composition and fixed polarity is used pumped through out the overall analytical procedure, then it is called isocratic elution.
Gradient Elution:
In this type of elution, polarity of solvent is changed gradually and slowly. For gradient elution, there should be two solvent reservoirs, two pumps and one mixturing device.

16. HPLC configuration by eluting mode
Isocratic
Binary
Quaternary

17. Gradient VS Isocratic

19. Isocratic

20. Low Pressure Gradient

21. High Pressure Gradient

22. Facilities of Gradient system
Some analysis required gradient mode
Save the solvents
Save labour
Method development
Accuracy of Analysis

23. Column:
HPLC Columns are made of high quality stainless steel, polished internally to a mirror finish.
1. Standard Analytical Column
2. Semi-preparative Column
3. Pre preparative Column

24. Standard Analytical Column
~4.5 mm internal diameter and cm in length.
For small amount of sample
This column is packed with HPLC grade stationary phase, e.g. silica gel, octadecyl silicyl C18 column.
This is not for actually isolation. It is used for analysis of compounds in industries to see that whether the compound is pure or not and to see particular arrangement of a compound.
DynammaR (company) produces column with 0.46 mm internal diameter and 25 cm in length for analytical purpose

25. Semi-preparative Column
1-2 mm internal diameter and cm in length.
If any sample is passed through analytical column and shows positive result, then it is applied in semi-preparative column.
DynammaR (company) produces column with 1.5 mm internal diameter and 25 cm in length for semi-preparative purpose.

26. Preparative Column 20-40 mm internal diameter and 10-25 cm in length
Largest amount
For large amount of sample
Compound extract from food extract
DynammaR (company) produces column with 2.1 mm internal diameter and 25 cm in length for preparative purpose.

27. Pre-column/Guard Column:
Pre-columns are usually packed with the same material as, but of larger particle size (30-70µm) than, that in the analytical column and are connected between the injector and the analytical column using low dead volume tubing.
The pre-column is used for two reasons as follows:
To saturate the mobile phase (liquid) by the liquid stationary phase i.e. to prevent loss of stationary phase by mobile phase and to retard dissolution. For this reason a pre-column must be used in partition chromatography.
To protect the main column from containing contaminating substances that may be present in the sample or solvent system. Pre-column traps those substances which would be irreversibly adsorbed on the analytical column. That is why it is so called “GUARD COLUMN”.

28. Commonly used solvents Commonly used adsorbents
Packing Materials and Mobile Phases used in HPLC:
Commonly used solvents
Commonly used adsorbents
Pet. Ether
Fuller’s earth
Acetone
Activated charcoal
Ethyl acetate
Activated alumina
Methanol
Activated silica
Water
CaCO3
Ether
K2CO3
CHCl3
Starch
CCl4
Talc

29. Solvents for HPLC should be highly pure
Solvents for HPLC should be highly pure. Generally the solvents are not pure. So it should be distilled double times for use.
For HPLC adsorbents should be finely powdered and be high quality.
Packing materials should be highly packed and their particle size should be 3-10µ.
In case of normal chromatography particle size µ.
The particle sizes are modified according to the use of particles in adsorption or partition chromatography.
Particle size that is used in guard column is 30-70µ.

30. Detectors 1. UV-Vis /Photodiode Array Detector
2. Refractive Index Detector
3. Fluorescence Detector
4. Conductivity Detector
5. Electro Chemical Detector
6. Mass Detector

31. Desirable HPLC Detectors
Highly sensitive
Low Noise Level
Wavelength Reproducibility
Simultaneous dual-wavelength measurement
Wavelength programming
Wavelength Scan mode

32. Desirable HPLC Detectors

33. Desirable Chromatographic Workstation Software
Windows based
User friendly
Excellent Graphical Interface
GLP Compliance
Automated Sequential Run
System Suitability, QC Check
Auto baseline Check
Auto Validation Run
Auto Shutdown
Customize Report
Automation of Analytical Works
Lot of way to calculations
Easy to Upgrade
Easy to reinstall by the customer

34. Chromatogram

35. Separation Technique
Compounds are separated due to the molecules moves at different rates in the column.

36. Separation Technique
Due to different interaction between stationary phase and different sample, the molecules move at different rate, therefore separation can be done.

37. The chromatographic modes
The chromatographic modes based upon the packing materials are-
Adsorption HPLC
Partition HPLC
Normal phase partition HPLC
Reversed phase partition HPLC
Ion suppression HPLC
Ion pair reversed-phase HPLC
Ion exchange HPLC
Size-exclusion HPLC

38. Normal Phase Partition HPLC
Stationary Phase is Polar
Mobile Phase is Non Polar

39. Base material for Stationary Phase
SiO2
O=Si=O Si OH
Polar Group

40. Silica Gel

41. Silica gel

42. Stationary Phase for Normal PhaseSi
Si-OH
Unmodified Silica (USP-L3)
Amino (USP-L11) Si
Si-CH2-CH2-CH2-NH2
Cyano (USP-L10) Si
Si-CH2-CH2-CH2-CN OH Si
Si-CH2-CH2-CH2-OCHCH2
Diol (USP-L) OH

43. Mobile Phase for Normal Phase
Primary solvents(non-polar)
-Hydrocarbons (Pentane, Hexane, Heptane, Octane)
-Aromatic Hydrocarbons (Benzene, Toluene, Xylene)
-Methylene chloride
-Chloroform
-Carbon tetrachloride
Secondary solvents
-Methyl-t-butyl ether (MTBE), Diethyl ether, THF, Dioxane, Pyridine, Ethyl acetate, Acetonitrile, Acetone, 2-propaol, ethanol, methanol
A primary solvent is used as mobile phase. Addition of secondary solvents is to adjust retention time.

44. What is Interaction HO Si
Si-OH AU OH
Minutes

45. Interaction

46. Reversed Phase Partition HPLC
Stationary Phase is Non Polar
Mobile Phase is Polar

47. Reverse Phase
Normal Phase
Reverse Phase

48. Reverse Phase

49. Column for Reverse PhaseSi
Si-CH2-CH2CH2-----CH3 (C18)
ODS (USP-L1) Si
C8 (USP-L7)
Si-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3
C4 (USP-L26) Si
Si-CH2-CH2-CH2-CH3
CH3 Si
Si-CH3
C1 (USP-L-13)
CH3

50. Mobile Phase for RP Water/buffer + Organic Solvents
-When buffer is used, the concentration and pH are important factors
-Methanol, Acetonitrile or THF are common organic solvents for RP HPLC
Optimization of water/buffer and organic solvents ratio is very important

51. Interaction of RP

52. Interaction of RP

53. Interaction of RP
7 Carbons
8 Carbons
6 Carbons

54. Increase of Solvent Polarity

55. Interaction between Phases

56. Interaction between Phases

57. HPLC Detectors
Most HPLC instruments are equipped with optical detectors.
Light passes through a transparent low volume “flow cell” where the variation in light by UV Absorption, fluorescent emission, or change in refractive index are monitored and integrated to display Retention Time and Peak Area.
Typical flow rates are 1 mL/min and a flow cell volume of 5-50 µL.

58. Common HPLC Detectors Refractive Index (RI) - universal
UV/VIS light – selective
Fluorescence – selective
Electrochemical (ECD) selective
Mass Spec (MS) - universal
Reference 3

59. Refractive Index detector
Analytes change the refractive index of the light in a proportional amount to the concentration.
Heat can change the RI of the mobile phase so thermo control important
RI changes cause a shift in a beam’s focal location which is detected on a photo-sensor.
RI is ideal for analyzing complex sugars and carbohydrates which have no chromophores, fluorescence or electrochemical activities.

60. UV/VIS Detectors
Scan a range of UV light to detect molecules with chromophores.
Usually having a range of 190 nm to 600 nm
Low flow cell volume 1 – 10 µL
Single wavelength spectrophotometers -uses a source lamp to emit a single wavelength (Hg, 254 nm)
Multi wavelength spectrophotometers – a number of monochromatic wavelengths (206 nm, 226 nm, 254 nm, 280 nm etc.)
Variable wavelength spectrophotometers –uses deuterium light source to allow selection in the deuterium continuum ( nm)
Programmable wavelength spectrophotometers

61. Fluorescent Detectors
Greater sensitivity than UV/VIS
Many compounds do not fluoresce and are derivatized with chemicals such as Dansyl chloride. This works well with primary and secondary amines, amino acids and phenolic compounds.

62. Electrochemical Detectors
Selective detection commonly used with reverse phase and isocratic elution with buffers and salts as the mobile phase
The two types of ECD’s are voltammetric and conductometric
The mobile phase must carry charged electrolytes eliminating normal phase as an option.
ECD’s respond to analytes that are oxidizable or reducible at an electrode surface.

63. Detector Summary Detector Type RI UV/VIS Fluorescent ECD MS
LOD (ng/ injection)
100 1
0.01
Selectivity No
Moderate
Very
High
Gradient
Elution
Yes
Reference 3

64. Chromatograms of simvastatin
Reference 6

65. Retention time(tR)
The time required to completely wash out of a particular solute from a column in the chromatographic analysis is known as retention time. It is denoted by tR.
Retention volume (VR)
The volume required of a mobile phase required to elute a compound (solute) from a column during the chromatographic analysis is known as retention volume. It is denoted by VR.

69. Desirable HPLC High Sensitive -Low pressure fluctuation
-High sensitive detector
Reliable: Reproducibility
-Precision of flow rate, injection volume, oven temperature, wavelength or applied potential and data transfer
Flexible: Modular Type