Chromatography is an analytical method in which compounds are physically separated and measured. The main purpose of chromatography is to separate and quantify the target sample. The Chromatography  technique used to separate a mixture of compounds in pharmaceutical sciences , analytical analytical Chemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture.

1. High Performance Liquid Chromatography
SUNIL KUMAR
ASSISTANT PROFESSOR
DEPT. OF PHARMACEUTICAL CHEMISTRY
ISF COLLEGE OF PHARMACY
WEBSITE: - WWW.ISFCP.ORG
EMAIL: sunil.medichem@gmail.com
ISF College of Pharmacy, Moga
Ghal Kalan,nGT Road, Moga- 142001, Punjab, INDIA
Internal Quality Assurance Cell - (IQAC)

2. Content 2
1.Introduction
2.HPLC Principle
3.Instrumentation
4.Types of HPLC
5.Pump and sample Injection system
6.Column
7.Detector
8.Method Development
9.Validation
10.HPLC & UHPLC
11.Advantages
12.Applications

3. 3Introduction
Chromatography is an analytical method in which compounds are physically
separated and measured.
The main purpose of chromatography is to separate and quantify the target sample.
The Chromatography technique used to separate a mixture of compounds in
pharmaceutical sciences , analytical analytical Chemistry with the purpose of
identifying, quantifying and purifying the individual components of the mixture.

4. 4
 The principle of separation in normal phase mode and reverse phase mode is
adsorption.
 The component which has more affinity towards the adsorbent, travels slower.
 The component which has less affinity towards the stationary phase travels faster.
Principle

5. 5
Since no 2 components have the same affinity towards the stationary phase, the
components are separated.

6. 6
Component Description
Mobile phase
reservoir
Stores the mobile phase required for analysis
Degasser Degasses the mobile phase
Pump Solvent delivery system, enables the flow of mobile phase
through the system
Injector Sample delivery system, introduces the sample to the system
Column
compartment
Controls the temperature of the column ,if required.
Separates the analyte components.
Detector Detects each component in separated mixture after it elutes
from column
Data processor Converts the data from the detector into results
Waste Collection of the liquid waste
HPLC Component

7. 7In Line-Degasser Unit

8. 8Manual Sampler

9. 9Auto Sampler

10. 10
Mobile Phase
Reservoir Sample
Pump Injector Column
Detector
Collect / Waste
Recorder
HPLC Instrumentation

11. 11
A mobile phase is pumped under pressure from one or several reservoir and flows
through the column at a constant rate.
For normal phase separation eluting power increases with increasing polarity of the
solvent but for reversed phase separation, eluting power decreases with increasing
polarity.
A degasser is needed to remove dissolved air and other gases from the solvent.
Solvent Delivery System

12. 12Solvents used for HPLC
In order of Increasing Polarity:
n-Hexane
Methylene chloride
Chloroform
Tetrahydrofuran
Isopropylalcohol
Acetonitrile
Methanol
Water
 A blend of two (or more solvents) of these is used as
mobile phase.
 The proportion of different solvents in blend act to
adjust the polarity of mobile phase.
 Suitably combined with stationary phase to achieve
separation of a mixture.

13. 13Modes of separation in HPLC
1. Normal phase mode.
2. Reversed phase mode.
3. Reversed phase ion pair chromatography.
4. Affinity chromatography.
5. Size exclusion chromatography

14. 14
Normal phase mode
 Stationary phase-polar in nature.
 Mobile phase-non polar in nature.
 Generally used for separation of
non polar compounds
Reverse phase mode
 Stationary phase-non polar .
 Mobile phase-polar in nature.
 Generally used for separation of polar
compounds

15. 15
Ion Exchange Chromatography
The stationary phase contains ionic groups like NR⁺з, SO⁻з which interact with the
ionic groups of the sample molecules.
This method is suitable for the separation of charged molecules only.
Ion Pair Chromatography
This may be used for the separation of ionic compounds.
Strong acids & basic compounds may be separated by reversed phase mode by
forming ion pairs with suitable counter ions.

16. 16
The pump is one of the most important component of HPLC, since its performance
directly affects retention time, reproducibility and detector sensitivity.
1. Displacement pump
2. Reciprocating pump
3. Pneumatic or constant pressure pump
Pump

17. 17
Displacement Pump
It produce a flow that tends to independent of viscosity and back pressure and also
output is pulse free but possesses limited capacity (250ml).
Reciprocating Pump
It has small internal volume (35-400µl), their high
output pressure(up to 10,000psi) and their constant
flow rates. But it produces a pulsed flow.

18. 18
Pneumatic Or Constant Pressure Pump
1. They are pulse free . Suffer from limited capacity as well as a dependence of flow
rate on solvent viscosity and column back pressure.
2. They are limited to pressure less than 2000psi.

19. 19Sample Injection System
 Loop injection : in which a fixed amount of volume is introduced by making use of
fixed volume loop injector.
 Valve injection: in which, a variable volume is introduced by making use of an injection
valve.
 On column injection: in which, a variable volume is introduced by means of a syringe
through a septum

20. 20Sample Injection System

21. 21
The function of detector in HPLC is to monitor the mobile phase as it merges from the
column.
1. Bulk property detectors: It compares overall changes in a physical property of the
mobile phase with and without an eluting solute e.g. refractive index ,dielectric
constant or density.
2.Solute property detectors: It responds to a physical property of the solute which is
not exhibited by the pure mobile phase. e.g. UV absorbance, fluorescence or
diffusion current.
Detectors

22. 22Types of Detectors
UV / Visible detector
Photodiode array detector (PDA)
Fluorescence detector
Electro-chemical detector (ECD)
Refractive index detector (RID)
Evaporative light scattering detector (ELSD)
Mass spectrometry (MS)

23. 23UV-Visible Detector
They are the work horses of HPLC and constitute about 70% of all detection system
It records compounds that absorb UV or visible light. Absorption occurs above 200
nm if the molecule has at least:.
1.A double bond adjacent to an atom with lone pair of electrons (X=Y-Z:)
2.Bromine, Iodine or Sulphur
3.Ketone group or a nitro group
4.Two conjugated double bonds (X=Y-Z=A)
5.An aromatic ring

24. 24Photodiode Array Detector
It is UV technique but produces a 3D output i.e. On X axis time, Y-axis absorbance
and Z axis wavelength
Chromatogram: On X axis time and Y-axis absorbance
Spectrum: On X axis wavelength and Y-axis absorbance
Allows simultaneous collection of chromatograms at different wavelengths during a
single run. Main application is in the field of method development
Used for peak purity testing

25. 25Photodiode Array Detector

26. 26Fluorescence Detectors
When light is absorbed by a molecule and an electron is promoted to a higher energy
state and while coming back to its ground state it loses energy by emission of a photon,
this process being called fluorescence.

27. 27Electrochemical Detectors
Electrochemical detectors are based on amperometric measurements. Also called
amperometric detectors.
Principle of operation: is the oxidation or reduction of analyte in a flow through
electrolysis cell with a constant applied electrical potential. e.g. oxidation of hydroquinone.

28. 28Refractive Index Detector
The velocity of electromagnetic wave varies as it passes from one medium to another
The ratio of its velocity in vacuum to that in given medium is known as RI of the
medium
The RI detector measures the change in RI of the mobile phase due to the presence of
dissolved analyte
RI detector is very useful for analysis of sugars which have poor UV absorbance or
fluorescence measurements without chemical derivatization
RI detector can not be used in case the solute and mobile phase has the same RI.

29. 29
Refractive Index Detector

30. 30Evaporative Light Scattering Detector (Elsd)
A universal detector. The effluent from column is nebulized and evaporated as it
passes through the drift tube. Analyte particles are detected as they pass thro’
light scattering cell.
Non-volatile analytes and volatile mobile phase.
Compatibility with gradient elution possible. Useful for impurity analyses.

31. 31
1.Nebulization: Inside the nebulizer, the column effluent
passes through a needle, mixes with nitrogen gas, and forms a
dispersion of droplets.
2.Mobile Phase Evaporation: The droplets pass through a
heated “drift tube” where the mobile phase evaporates,
leaving a fine mist of dried sample particles in solvent vapor.
3.Detection: The sample particles pass through a flow cell
where they are hit with a laser light beam. Light scattered by
the sample particles is detected, generating an electrical
signal.

32. 32Mass Spectrometer (Ms) Detector
LC-MS is a hyphenated technique, combining separation power of HPLC with the
detection power of MS
For using MS there is a need for an interface that will eliminate the solvent and
generate gas phase ions, for MS
For all MS techniques, an analyte is first ionized in the ion source since the MS can
only detect charged species

33. 33Detector Selection
1. Dynamic range
2. Response index or linearity
3. Detector response
4. Detector noise level
5. Detector sensitivity, or minimum detectable concentration.
6. Total system dispersion
7. Pressure sensitivity
8. Flow rate sensitivity.
9. Operating temperature range.

34. 34Chromatographic Column
The column is usually made up of heavy glass or stainless steel tubule to with stand
high pressure.
The columns are usually 10-30cm long and 4-10mm inside diameter containing
stationary phase at particle diameter of 25µm or less.

35. 35Selection of column
Knowledge of the Sample
• Structure of sample components?
• Number of compounds present?
• Sample matrix?
• pKa values of sample components?
• Concentration range?
• Molecular weight range?
• Solubility?
• Other pertinent data?
Goals for the Separation
• Max. resolution of all components?
• Partial resolution?
• Fast analysis?
• Economy (low solvent usage)?
• Column stability and lifetime?
• Preparative method?
• High sensitivity?
• Other goals?

36. 36
Column Selection Chart
Method
Goals
HighEfficiency
HighCapacity
Low
Backpressure
HighResolution
HighSample
Loadability
SuitableforMW
>2000
HighStability
HighSensitivity
FastAnalysis
LowMobile
Phase
Consumption
StabilityatpH
Extremes
FastEqilibration
DefaultColumn
(Goodformost
Applications)
Particle Size
small (3µm) • •
medium (5µm) •
large (10µm) •
Column Length
short (30mm) • • • • •
medium (150mm) •
long (300mm) •
Column ID
narrow (2.1mm) • •
medium (4.6mm) •
wide (22.5mm) •
Surface Area
low (200m2/g) • • •
high (300m2/g) • • •
Pore Size
small (60Å) • •
medium (100Å) •
large (300Å) •
Carbon Load
low (3%) •
medium (10%) •
high (20%) • • •
Bonding Type
monomeric • •
polymeric • • • •
Particle Shape
spherical • • • •
irregular •

37. 37Column Dimension
Short (30-50mm) - short run times, low backpressure
 Long (250-300mm) - higher resolution, long run times
 Narrow ( 2.1mm) - higher detector sensitivity
 Wide (10-22mm) - high sample loading

38. 38
Spherical particles offer reduced back pressures and longer column life when using
viscous mobile phases like 50:50 meoh:h2o.
Particle Shape

39. 39
Smaller particles offer higher efficiency, but also cause higher backpressure.
Choose 3µm particles for resolving complex, multi-component samples.
Otherwise, choose 5 or 10µm packings.
Particle Size

40. 40
High surface area generally provides greater retention, capacity and resolution
for separating complex, multi-component samples.
Low surface area packings generally equilibrate quickly, especially important in
gradient analyses.
Surface Area

41. 41
Pore size larger pores allow larger solute molecules to be retained longer through
maximum exposure to the surface area of the particles.
Pore Size

42. 42Column Types

43. 43
C18 4.6 x 250 mm 5m 300o
A
Stationary Phase
Dimension
Particle Size
Pore Size
Specification of column

44. 44
 This a measure of the sharpness of the
peaks and therefore the efficiency of the
column.
 This can be calculated in various ways,
for example the USP uses the peak width
at the base and the BP at half the height.
Number of theoretical plates (N)
where
Wh = peak width at 1/2 peak
height
Wb = peak width at base
t = retention time of peak

45. 45
 Higher the plate number the more efficient the column.
 The plate number depends on column length - ie the longer the column the
larger the plate number.
HETP= L/N
where
N=number of theoretical plates
L=Length of the column
tr=retention time, and W=peak width.
Van deemter equation
HETP=A+B/u+(Cs+Cm).u
Where HETP= Height Equvalent to theoratical
Plate
A= Eddy Difusion
B= Difusion coefficient
C= Resistance to mass transfer coeficient of the
analyte between mobile and stationary phase
u= Linear velocity

46. 46
 Method development usually requires selecting the method requirements and
deciding on what type of instrumentation to required and why.
 The wide variety of equipment, columns, eluent and operational parameters
involved makes hplc method development .
Method Development

47. 47
1. A suitable method for particular analyte in the specific matrix is not available.
2. Existing methods may be too error or they may be unreliable (have poor accuracy
or precision)
3. Existing methods may be too expensive, time consuming.
There are several reasons for developing new methods of analysis

48. 48
Consideration must be given to the following
 Sample preparation
 Types of chromatography
 Column selection
 Detector selection
 Selection of mobile phase composition
Selection of HPLC Method

49. 49
It is the procedure which proves that a method yields the expected results with
reliability, adequate precision and accuracy.
Validation

50. 50
Specificity
Linearity
Range
Accuracy
Validation parameter
Precision
Limit of detection
Limit of quantitation
Robustness

51. 51
It is accessed by the following factors:
Theoretical plates (Efficiency) (N)
Capacity factor (k’)
Separation (Relative Retention)
Resolution (R)
Tailing factor (T)
Relative Standard Deviation (%RSD)
System Suitability

52. 52Capacity factor k’
It is the ratio of number of molecules of solutes in the stationary phase to the number
of molecules in the mobile phase.

53. 53
This describes the relative position of two adjacent peaks. ldeally, it is calculated using the
capacity factor because the peaks' separation depends on the components‘ interaction
with the stationary phase.
Therefore considering peaks A and B.
Separation factor

54. 54
This is not only a measure of the separation between two peaks, but also the efficiency of
the column. It is expressed as the ratio of the distance between the two peak maxima to
the mean value of the peak width at base.
R = 2 (Va-Vb) / Wb1+Wb2 (R>2)
Peak Resolution (R)

55. 55
This is a measure for the asymmetry of the peak.
Tailing factor (T)

56. 56
For an HPLC system this would involve the reproducibility of a number of replicate
injections of an analytical solution.
 If a relative standard deviation of <2% is required then five replicate injections
should be used.
 If a relative standard deviation of >2% is required then six replicate injections should
be used.
Relative Standard Deviation

57. 57
1. Separations fast and efficient (high resolution power)
2. Continuous monitoring of the column effluent
3. It can be applied to the separation and analysis of very complex mixtures
4. Accurate quantitative measurements.
5. Repetitive and reproducible analysis using the same column.
6. Adsorption, partition, ion exchange and exclusion column separations are excellently
made.
7. Both aqueous and non aqueous samples can be analyzed
8. A variety of solvents and column packings are available
9. It provides a means for determination of multiple components in a single analysis.
Advantages of HPLC

58. 58
 Reducing the particle diameter of a packing material, column length.
The back pressure required for the use of the small particle column.
Monolithic columns provide lower flow resistance than conventional columns.
Columns can be operated at high flow rates.
By using smaller particles, speed and peak capacity can be extended to new limits.
 It provides good resolution.
HPLC vs UPHLC

59. 59Applications in Pharmaceutical Chemistry
1. Clinical diagnosis of diseases, disorders.
2. In scientific research.
3. In pharmaceutical labs for analysis.
4. In food industry for quality control.
5. For standards control.
6. For separation of similar molecules.
7. Drug Discovery programs.

60. 60
 Snyder, Lloyd R., Joseph J. Kirkland, and Joseph L. Glajch. Practical HPLC method
development. John Wiley & Sons, 2012.
 Hart, David J., and K. John Scott. "Development and evaluation of an HPLC method
for the analysis of carotenoids in foods, and the measurement of the carotenoid content
of vegetables and fruits commonly consumed in the UK." Food Chemistry 54.1 (1995):
101-111.
 Hertog, Michael GL, Peter CH Hollman, and Dini P. Venema. "Optimization of a
quantitative HPLC determination of potentially anticarcinogenic flavonoids in
vegetables and fruits." Journal of Agricultural and Food Chemistry 40, no. 9 (1992):
1591-1598.
References

61. 61
1. Discussion session
2. Questions and feedback
3. Assignment

62. 62
Thank you