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.

1. Dr.A.DINESH KARTHIK
ASSOCIATE PROFESSOR & HEAD,
P G & RESEARCH DEPT. OF CHEMISTRY
SHANMUGA INDUSTRIES ARTS & SCIENCE
COLLEGE, TIRUVANNAMALAI-606603.
dineshkarthik2008@gmail.com.

2. High Performance Liquid
Chromatography
 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.
Dr.A.DINESH KARTHIK

3. You’ve Got a Problem to Solve
I need a quantitative
separation of
carbohydrates in some
of our products
as soon as possible.
I’ll need a separation
technique.
I’ll get
on it!
Dr.A.DINESH KARTHIK

4. Separation Techniques
I have two separation techniques in my lab,
High Performance Liquid Chromatography
and Gas Chromatography. Which should I use?
Dr.A.DINESH KARTHIK

5. How can We Analyze the
Sample?
Carbohydrates
1. fructose
2. Glucose
3. Saccharose
Zorbax NH2 (4.6 x 250 mm)
70/30 Acetonitrile/Water
1 mL/min Detect=Refractive Index
1
2
3
4
5
mAU
time
6
Dr.A.DINESH KARTHIK

6. Instrumentation
 Solvent Reservoirs
 Pump
 Sample Injector
 Column(s)
 Detector
 Data System
Dr.A.DINESH KARTHIK

7. Dr.A.DINESH KARTHIK

8. HPLC system
Dr.A.DINESH KARTHIK

9. Mobile Phase Reservoirs
 Inert container with inert lines leading to
the pump are required.
 Reservoir filters (2-10 mm) at reservoir
end of solvent delivery lines
 Degassed solvent
- Vacuum filtration
- Sparge with inert gas (N2 or He)
- Ultrasonic under vacuum
 Elevate above pumps Dr.A.DINESH KARTHIK

10. Separations
Separation in based upon differential
migration between the stationary and
mobile phases.
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.
Injector
Detector
Column
Solvents
Mixer
Pumps
High Performance Liquid Chromatograph
Waste
Dr.A.DINESH KARTHIK

11. Separations
Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
Dr.A.DINESH KARTHIK

12. Separations
Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

13. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

14. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

15. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

16. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

17. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

18. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

19. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

20. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

21. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

22. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

23. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

24. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

25. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

26. Separations
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Dr.A.DINESH KARTHIK

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

28. HPLC Analysis Parameters
Mobile Phases
Flow Rate
Composition
Injection Volume
Column
Oven Temperature
Wavelength
Time Constant
Dr.A.DINESH KARTHIK

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

30. Isocratic elution: A separation that
employs a single solvent or solvent mixture
of constant composition.
Gradient elution: Here two or more solvent
systems that differ significantly in polarity
are employed. After elution is begun; the
ratio of the solvents is varied in a
programmed way, sometimes continuously
and sometimes in a series of steps.
Separation efficiency is greatly enhanced
by gradient elution.
Dr.A.DINESH KARTHIK

31. FOUR TYPES OF LIQUID
CHROMATOGRAPHY
 Partition chromatography
 Adsorption, or liquid-solid
 chromatography
 Ion exchange chromatography
 Size exclusion, or gel, chromatography
Dr.A.DINESH KARTHIK

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

33. Picture of HPLC instrument
Dr.A.DINESH KARTHIK

34. HPLC Chromatograph injectors
 The function of the injector is to place the sample
into the high-pressure flow in as narrow volume as
possible so that the sample enters the column as a
homogeneous, low-volume plug. To minimize
spreading of the injected volume during transport to
the column, the shortest possible length of tubing
should be used from the injector to the column.
 When an injection is started, an air actuator rotates
the valve: solvent goes directly to the column; and
the injector needle is connected to the syringe. The
air pressure lifts the needle and the vial is moved
into position beneath the needle. Then, the needle is
lowered to the vial.
Dr.A.DINESH KARTHIK

35. 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 and has a high
surface activity which can be
modified easily with water
and other agents. Silica can
be used to separate a wide
variety of chemical
compounds, and its
chromatographic behavior is
generally predictable and
reproducible.
Dr.A.DINESH KARTHIK

36. Picture of an HPLC column
Dr.A.DINESH KARTHIK

37. WHAT AFFECTS SYSTEM
Column Parameters
 Column Material
 Deactivation
 Stationary Phase
 Coating Material
Instrument
Parameters
 Temperature
 Flow
 Signal
 Sample Sensitivity
 Detector
Dr.A.DINESH KARTHIK

38. WHAT AFFECTS SYSTEM
Sample Parameters
 Concentration
 Matrix
 Solvent Effect
 Sample Effect
Dr.A.DINESH KARTHIK

39. Several column types
(can be classified as )
 Normal phase
 Reverse phase
 Size exclusion
 Ion exchange
Dr.A.DINESH KARTHIK

40. 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
Dr.A.DINESH KARTHIK

41. 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.
Dr.A.DINESH KARTHIK

42. 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. Dr.A.DINESH KARTHIK

43. 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.
Dr.A.DINESH KARTHIK

44. Selectivity Factor
 K’ values tell us where bands elute
relative to the void volume. These
values are unaffected by such variables
as flow rate and column dimensions.
The value tell us where two peaks elute
relative to each other. This is referred
to as the selectivity factor or separation
factor (now and then as the chemistry
factor).
Dr.A.DINESH KARTHIK

45. Types of Liquid Column
Chromatography
(LCC)
 LLC (Liquid
Liquid)
 LSC (Liquid Solid -
adsorption)
 SEC (Size Exclusion)
 GLC GSC
 SFC (Supercritical
Fluid)
Dr.A.DINESH KARTHIK

46. Types of Detectors
 Absorbance (UV
with Filters, UV with
Monochromators)
 IR Absorbance
 Fluorescence
 Refractive-Index
 Evaporative Light
Scattering Detector
(ELSD)
 Electrochemical
 Mass-
Spectrometric
 Photo-Diode Array
Dr.A.DINESH KARTHIK

47. EVALUATION PARAMETERS
 EFFICIENCY
 RESOLUTION
 INERTNESS
 RETENTION INDEX
 COLUMN BLEED
 CAPACITY FACTOR
Dr.A.DINESH KARTHIK

48. HPLC Applications
Chemical
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
Dr.A.DINESH KARTHIK

49. 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, for monitoring materials that may jeopardize
occupational safety or health, and for monitoring
pesticide levels in the environment. Federal and state
regulatory agencies use HPLC to survey food and drug
products, for identifying confiscated narcotics or to
check for adherence to label claims.
Dr.A.DINESH KARTHIK

51. Dr.A.DINESH KARTHIK
dineshkarthik2008@gmail.com