1. High Performance/Pressure Liquid Chromatography (HPLC)

2. Preview History Affinities for Mobile and Stationary Phases
Retention Time
Shortcomings of liquid chromatography. (slow, band broadening and overlapping, resolution)
How can efficiency be improved?

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. HPLC System

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. 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.

7. In solvent terminology, a strong solvent is non-polar (organic) and a weak solvent is polar (water). Some commonly used strong solvents (from weak to strongest) are methanol, acetonitrile and tetrahydrofuran.

8. Ideal Properties of Mobile phase:
1. Solvents used in HPLC should be ultra-pure and are labeled as “HPLC grade”.
2. Should be inexpensive.
3. Should be non corrosive.
4. Should be inert with respect to samples under analysis.
5. If a UV detector is used, the mobile phase must be transparent at the wavelength of analysis.
6. If a Refractive index detector is used, the difference between the refractive indices of the mobile phase and the sample should be as great as possible.
7. If the eluate is to be recovered for further processsing , then lower boiling point of the solvent is required.

9. Pumps:
A high-pressure pump [solvent delivery system or solvent manager] is used to generate and meter a specified flow rate of mobile phase. 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.
Relatively low 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.
Analytical chromatography refers to the processes of separating a sample into its components and identifying these components, and is usually a small scale process. Preparative chromatography, on the other hand deals with isolating components of a sample and can be done on larger industrial scale operations. The difference between analytical and preparative HPLC is the amount of sample applied to the column. In analytical HPLC the applied sample amount is very small compared to the amount of stationary phase in the column (less than 1:10000). To purify higher amounts of sample in a single run the loadability of a column has to be increased. So, preparative column is used.
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.

10. A HPLC pump should meet the following criterias:
1. Reliable
2. Constructed of materials inert toward solvents to be used
3. Able to deliver high volumes (flow rates) of solvent (upto 10 mL/min)
4. Deliver precise and accurate flow (<0.5% variation)
5. Able to deliver solvent at high pressure (upto 8000 psi)
6. Deliver pulse free solvent flow

11. There are several types of pumps used for HPLC analysis
There are several types of pumps used for HPLC analysis. Among them the following 3 are most commonly used.
1. Reciprocating piston pump: Consists of a small motor driven piston which moves rapidly back and forth in a hydraulic chamber that may vary from μL in volume. On the back stroke, the piston pulls in solvent from the mobile phase reservoir and on the forward stroke, the pump pushes solvent towards the analytical column.
2. Syringe pump and
3. Constant pressure pump

12. Injector:
The mobile phase mixture is introduced into the chromatographic column by means of a suitable injection device.
In case of manual injectors, the solute mixture is introduced into the chromatography by means of a suitable injection device like septum devices in which the sample solution is injected through a self sealing rubber septum or Teflon disk using a micro-liter syringes. Typical sample volumes for manual injector are 5-20 μL.
Automatic injectors (autosamplers) are able to continuously Inject variable volumes of sample ranging from 1–100 mL. Precision and accuracy are much more than manual injectors but more expensive initially. Modern sophisicated autosamplers not only do sample injection but also can do a number of other functions such as :
1. Diluting,
2. Performing derivatization by mixing with different reagents,
3. Adding internal standard and
4. Transferring reagents and sample to an empty vial for pretreatment etc.

13. Based on Elution Technique :
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.

14. Isocratic

15. Low Pressure Gradient

16. High Pressure Gradient

17. 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. Actually particle size varies from 70µ to 230µ.
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.

18. Based on Scale of Operation
1. Preparative HPLC
2. Analytical HPLC
(Discussion was given earlier)

19. Based on mode of seperation:
Normal Phase vs. Reverse Phase
If the stationary phase is more polar than the mobile phase, the separation is deemed normal phase. If the stationary phase is less polar than the mobile phase, the separation is reverse phase. In reverse phase HPLC the retention time of a compound increases with decreasing polarity of the particular species. The key to an effective and efficient separation is to determine the appropriate ratio between polar and non-polar components in the mobile phase. The goal is for all the compounds to elute in as short a time as possible, while still allowing for the resolution of individual peaks. Typical columns for normal phase separation are packed with alumina or silica. Alkyl, aliphatic or phenyl bonded phases are typically used for reverse phase separation.

20. Preparation of Stationary phase for HPLC
Preparation of Stationary phase for HPLC. The stationary phase of normal HPLC usually involves silica (O=Si=O).
SiO2
O=Si=O Si OH
Polar Group

21. 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

22. 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

23. Mobile Phase for Normal Phase
Primary solvents(non-polar)
-Hydrocarbons (Pentane, Hexane, Heptane, Octane)
-Aromatic Hydrocarbons (Benzene, Toluene, Xylene)
Pet. ether
-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.

24. Reversed Phase HPLC Stationary Phase is Non Polar
Mobile Phase is Polar

25. Preparation of stationary phase:
The stationary phase is silica, chemically bonded through a siloxane (Si-O-Si-C) linkage to a less polar functional group.
The stationary phase for reversed phase partition chromatography is prepared by treating the surface silanol groups of silica with an organochlorosilane reagent-

26. Reverse Phase
Normal Phase
Reverse Phase

27. 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

28. 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

29. Column
It is also known as the “Heart of the chromatograph”. Individual components or molecules are separated in the column based on their differential interaction with the stationary phase.

30. 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

31. Standard Analytical Column
~4.5 mm internal diameter and cm in length.
For small amount of sample; narrow column.
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

32. Semi-preparative Column
7-10 mm internal diameter and cm in length.
Medium sized column
If any sample is passed through analytical column and shows positive result, then it is applied in semi-preparative column.

33. Preparative Column 20-40 mm internal diameter and 10-25 cm in length
Largest size
For large amount of sample
For isolation and purification of compounds in industrial scale.
DynammaR (company) produces column with 2.1 mm internal diameter and 25 cm in length for preparative purpose.

34. 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”.

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. Based on Principle of Seperation
The efficiency of separation, performance and time required for analysis depends on the packing material used. The chromatographic modes based on the packing materials are-
1. Adsorption HPLC
2. Partition HPLC
-normal phase partition HPLC
- reversed phase partition HPLC
4. Ion exchange HPLC
5. Size exclusion HPLC

38. 1. Adsortion HPLC:
The stationary phase used is unmodified silica. The solvents used in adsorption HPLC are almost entirely organic and a single binary mixture of miscible solvents of the correct polarity.
Advantages of using silica gel:
1. Efficiency of separation is improved. We can use four types of silica of either 3,5,7 or 10 micron particle size.
2. Due to porosity of silica, a low operating pressure (2000 psi) can successfully be used.
3. As they are fine powdered materials, surface area is large enough. So a large amount of sample can be separated by small amount of silica gel. For ex- 1-5 mg of sample can be separated by using 1 gm of packing material.

39. Mechanism of separation:
Silanol group (Si-OH) can form hydrogen bond with polar groups of the solute. If a compound forms more hydrogen bond than others, it will elute from the column later than those forming fewer hydrogen bonds. Now solvent with increasing polarity (gradiant elution) is used to elute the compounds. At first polar solvent should not be used as polar compounds may form hydrogen bonds with the solvent molecule as well and will be eluted from the column fast. Moreover silica may also be washed away by the polar solvent. The non-polar compound will elute quickly and polar compounds will be retained by the packing material. To separate the polar compounds the polarity of the solvent should be increased slowly.
What about Reverse Phase??

40. Alumina as packing material:
Alumina is used as an adsorbent less frequently than silica. For separation of aromatic compounds, nitrogenous compounds and structural isomers, greater selectivity is obtained with alumina.

41. 2. Partition HPLC:
In partition HPLC an inner support is used to hold the liquid stationary phase. Silica gel is used as the inner support. This silica gel is soaked and coated with a polar liquid stationary phase e.g. ethane-1,2-diol. Here the mobile phase is a less polar solvent or mixture of solvents. Some examples of packing materials are:
Si-(CH3)3-CN
Si-(CH3)3-NH2
Si- (CH3)3-O-CH2-CH(OH)-CH2(OH)
The order of increasing polarity of these packing materials are as follows:
Diol> cyano> amino.
The less polar compounds will elute quickly and then the polar compounds will come out as the polarity of the solvent is increased.

42. Types of partition HPLC:
According to the polarity of the mobile phase partition HPLC can be classified into two types-
-Normal phase partition HPLC (Stationary phase is polar. Mobile phase less polar. So less polar solutes will elute first)
-Reversed phase partition HPLC (Stationary phase is less polar and the mobile phase is polar. So polar solutes will elute first)

43. Mechanism of separation:
The solutes will be partitioned into the liquid stationary phase according to their polarity. In case of reversed phase HPLC, the less polar solutes will be retained by the less polar stationary phase for longer time. In case of normal phase HPLC, however, the scenario is just the opposite. The time of elution is controlled by the polarity of organic modifiers (methanol or acetonitrile) and their proportion in mobile phase. Methanol is used to increase the polarity whereas acetonitrile is used to obtain a mobile phase of decreased polarity.

44. 3. Ion exchange HPLC:
Mixtures of aqueous buffers and organic solvents are used to carry out ion exchange HPLC. The ionic strength and pH of the mobile phase control the retention of ions on the column. When the ionic strength of the solvent system is increased, greater competition between the solutes and the solvent ions occurs and this leads to reduced retention of the solute ions.
Packing materials for ion exchange HPLC are based on the cross-linked polystyrene-divinylbenzene resins or an ion exchange residue chemically bonded to silica. The widely used cation exchanger contains sulphonate group and the widely used anion exchanger contains tetra alkyl ammonium group.

45. 4. Size exclusion HPLC:
Microspheres (5-15 microns in diameter) of cross linked polystyrene-divinylbenzene resin or silica is used as the packing material. Here compounds are separated according to their molecular weight. Solutes with larger size can travel through the pores quickly and appear in the chromatogram first. Solutes with smaller size travel slowly as they follow tortuous path and are eluted later.

46. 5. Ion suppression HPLC:
In this type of chromatography, ionizble substances may be obtained by altering the pH of the mobile phase.
Substances that are ionized at the pH of the mobile phase are polar and are poorly retained on a reversed-phase column.
Adjustment of the pH to a value where a greater proportion of the substance is in the non-ionized form reduces its polarity and increases its retention in reverse phase column.
Thus, ion suppression chromatography is the chromatography in which the ionization of sample is restricted by altering the pH of the mobile phase and thus providing a better separation.
Here, appropriate pH range of the mobile phase should be 2-7.5

47. Chromatogram: 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.

48. 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.
Reference 3

49. Common HPLC Detectors Refractive Index (RI) - universal
Evaporative Light Scattering Detector (ELSD) – universal
UV/VIS light – selective
Photo-diode Array Detector- Selective
Fluorescence – selective
Electrochemical (ECD) selective
Mass Spec (MS) - universal
Reference 3

50. Refractive Index detector
Analytes change the refractive index of the light in a proportional amount to the concentration.
Heat can change the R.I of the mobile phase so thermo control important
R.I is ideal for analyzing complex sugars and carbohydrates which have no chromophores, fluorescence or electrochemical activities.
Analytes usually have a different RI compared to mobile phase. It is an universal type of detector

51. Evaporative Light Scattering Detector (ELSD)
Light scatters in response to the dimension of the analyte particles.
This universal detector is more sensitive than R.I and shows a response to compound lacking UV absorption or fluorescence.
Downfall is the sample is destroyed.

52. 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

53. Photo-Diode Array (PDA) Detector
Scan a range of UV-Visible light to detect molecules with chromophores.
Usually having a range of 190 nm to 800 nm
Capable of measuring multiple wavelength at a time
Contains a holographic grating which separate the compound light into individual wavelengths and the photo-sensitive diode can sense which wavelength/s were absorbed.

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

55. Mass Detectors:
HPLC-MS is the most powerful type of detector. Widely used in pharmaceutical laboratories and research and development. The principal benefit of HPLC-MS is that it is capable of analyzing and providing molecular identity of a wide range of components. Volatile mobile phases must be used here.

56. Electrochemical Detectors
Selective detection commonly used with reverse phase and isocratic elution with buffers and salts as the mobile phase.
Major advantages of this type of detectors are their high sensitivity, selectivity, simplicity, convenience and widespread applicability. These devices are based upon amperometry, polarography, coulometry, and conductometry. Analytes are usually readily oxidizable or reducible compounds, especially biological samples.
The mobile phase must carry charged electrolytes eliminating normal phase as an option.

57. Ideal properties of an detector:
High sensitivity
2. Low noise level
3. Applicable to most of the solutes
4. Non-destructive
5. Faster response
6. Should not be affected by changes in temperature or mobile phase composition.
7. Wavelength Reproducibility
8. Simultaneous multi-wavelength measurement
9. Wavelength programming
10. Wavelength Scan mode
[Note: no is only applicable for UV-Vis /Photodiode Array detector]

60. Advantages of HPLC over other chromatographic techniques:
1. Faster and efficient separation of mixtures (high resolution power).
2. Suitable for analyzing very complex mixtures.
3. Accurate quantitative measurements (Qualitative analysis is also possible).
4. Repetitive and reproducible analysis using the same column.
5. HPLC is more versatile than GC in some respects. Only volatile and thermally stable solutes can be analyzed using GC but with HPLC there is no such restriction. The choice of mobile and stationary phases is also much wider in HPLC than GC.
6. It is possible to analyze multiple components of a mixture in a single analysis.