High performance liquid chromatography (HPLC)

HPLC, Pharmacology

1. High performance liquid
chromatography (HPLC)
Capt. Htet Wai Moe
Resident Pharmacology

2. •A form of column chromatography to
separate, identify, and quantify the
compounds.
•Developed in 1970s.
•The most widely used analytical
separation technique.

3. Chromatography
•Chromatography is a technique which
separates components in a mixture due to
the differing time taken for each
component to travel through a
stationary phase when carried through it
by a mobile phase.

4. Basically, all chromatographic systems
consists of two phases.
•Mobile phase - liquid or gaseous and
flows over or through the stationary phase
•Stationary phase - solid, liquid or a
solid/liquid mixture which is immobilized

5. Some chromatography terms
Analyte
•Substance that is to be separated during
chromatography
Immobilized phase
•Stationary phase which is immobilized on
the support particles or on the inner wall of
the column tubing

6. Mobile phase
•Phase which moves in a definite direction.
(liquid/gas/fluid).
•Consists of the sample being separated/
analyzed and the solvent that moves the
sample through the column.
Effluent
•Mobile phase leaving the column.
Some chromatography terms

7. Different types of chromatography methods
• Paper chromatography
• Liquid chromatography
• Gas chromatography
• High performance liquid chromatography

8. High performance liquid chromatography
•HPLC is an extension of conventional
liquid chromatography.
•Powerful tool in analytical techniques
•Columns are tightly packed, and the
eluent is forced through the column under
high pressure(up to 5,000 psi) by a pump.

9. •Allows to use a very smaller particle size
for the column packing material which
gives a much greater surface area for
interactions between the stationary phase
and the molecules flowing through it.
•Allows a much better separation of the
components of the mixture.

10. HPLC Technique
•Utilizes liquid mobile phase to separate
the mixture
•Analytes are first dissolved in a solvent
then through the column under high
pressure of up to 400 atm
•Mixture is resolved into its components in
the column

11. •The total separation time is often 5 or 10
minutes rather than hours or even days
required for some separations by gravity
flow with the larger systems.

12. Components of HPLC
•Pump
•Injector
•Column
•Detector
•Recorder or data system

13. A Flow Scheme for HPLC

15. Pump
•A pump forces the mobile phase through
the column at a much greater velocity than
gravity-flow columns.
•The pump can be pneumatic, syringe-
type, reciprocating, or hydraulic amplifier.

16. Pump (cont.)
•Pneumatic pumps are used for
preoperative purposes.
•The most widely used pump today is the
multihead pump with two or more
reciprocating pistons.

17. •Pumps are designed in order to maintain a
stable flow rate, avoiding pulsations even
when the composition of the mobile phase
varies
•flow range – 0.01-10 ml/min

18. HPLC Pump

19. Injectors
•Inject the liquid sample within range of
0.1- 100 ml of volume under high pressure
•Produce minimum band broadening
•Produce possible flow disturbances
•Volume must be small (0.1-500 uL)

20. A model injector

21. Injector

22. Columns
•Smooth-bore stainless steel or heavy-
walled glass tubing.
•Hundreds of packed columns differing in
size and packing are available from
manufacture.

23. Columns
•E.g. Column packing vary in size (3 to 20
um) with the smaller particles used mostly
for analytical separations and the larger
ones for preparative separation.
•The most common material used for
column packing is silica gel.

24. HPLC columns

26. Detector
•HPLC detectors monitor the elute as it
leaves the column
•Produce an electronic signal proportional
to the concentration of each separated
component

27. Detector
•Crucial in trace analysis
•High sensitivity
•Fast response
•Simplifies quantitation
•Insensitive to changes in type of solvent,
flow rate and temp.

28. The most widely used detection methods
•Spectrophotometers
•Fluorometers
•Electrochemical detectors
•Mass spectrometer
•Refractive index detector

29. Detectors used in HPLC
Type Principle Detection
limit
Comments
Spectro-
photometer
Measure
absorbance of
light
<1 ng Analyte must
absorb UV or
visible light
FluorometersMeasures
fluorescence
pg to ngAnalyte must
fluoresce
Electro-
chemical
detectors
Electrochemically
measures
oxidized/ reduce
analyte
pg to ngUseful for
catecholamine
s

30. Detectors used in HPLC
Type Principle Detection
limit
Comments
Mass
spectrometer
Detects ions after
separation by
mass-to-charge
ration
fg to ngAnalyte must
be converted
to ionized
form
RefractometerMeasure change
in refractive index
1 µg Detection of
most
compounds
but relatively
low sensitivity

31. Depending on the relative polarity of the
solvent and stationary phase, there are two
variants in use in HPLC
1.Normal phase HPLC
• Utilize polar adsorbent surface and non-
polar eluent
• Polar substance in the mixture sticks to polar
adsorbent than non-polar
• Non-polar ones will pass more quickly
through the column

32. 2. Reversed phase HPLC
•Utilize non-polar adsorbent surface and
polar eluent
•Attraction between non-polar compound in
the mixture and non-polar adsorbent

33. 2. Reversed phase HPLC (cont.)
•Polar molecules will travel through the
column more quickly because there is
strong attraction between polar solvent
and polar molecules when pass through
the column
•Reversed phase HPLC is the most
commonly used form of HPLC

34. Solvents used in mobile phase
•hexane, heptane, cyclohexane, carbon
tetrachloride, benzene, toluene, diethyl
ether, chloroform etc.
Adsorbents used in stationary phase
•silica gel, alumina, celite, cellulose powder,
ion-exchange, cellulose, starch

35. Retention time
•The time taken for a particular compound
to travel through the column to the
detector
•From the time at which the sample is
injected to the point at which the display
shows a maximum peak height for that
compound.

37. Types of chromatic separation
•Adsorption Chromatography
•Ion- exchange Chromatography
•Size Exclusion Chromatography

38. Adsorption Chromatography
•Competition for adsorption sites occurs
between the molecules of the mixture to
be separated and the molecules of the
mobile phase
•Mobile phase can be either a single
solvent or two or more solvents depend on
the analytes to be desorbed
•Speed of migration of the component
along the column depend on adsorptive
affinity

39. Ion- exchange Chromatography
•Molecules can be separated by their ionic
charges in a process known as Ion-
exchange Chromatography.
•Ion-exchange resins are used as the
column packing materials.
•This method is used for separation of ionic
species, such as amino acids.

41. •Known as gel permeation chromatography
or gel filtration chromatography.
•Packing material with very small pore is
used.
•Precisely controlled pore size materials in
the column
•Large molecules, such as polymers are
physically prevented from passing through
the column
Size Exclusion Chromatography

42. Applications
HPLC is used for
•Chemistry and biochemistry research
analyzing complex mixtures,
•Purifying chemical compounds
•Quality control to ensure the purity of raw
materials
•Analyzing air and water pollutants,

43. Applications (cont.)
•Monitoring materials that may jeopardize
occupational safety or health
•Monitoring pesticide levels in the
environment.
•To survey food and drug products,
•To identify confiscated narcotics
•To determine the amount of such chemical
compounds found in new drugs in
pharmaceutics

44. HPLC as compared with the classical
technique
•Small diameter, reusable stainless steel
columns
•Column packing with very small particles
•Control flow of mobile phase
•Precise sample introduction

45. HPLC as compared with the classical
technique (cont.)
•Good pumping system
•Special continuous flow detectors- can
handle small flow rates and detect very
small amounts
•Rapid analysis
•High resolution

46. Disadvantages of HPLC
•Cost
•Complexity
•Low sensitivity for some compounds
•Irreversibly adsorbed compounds not
detected
•Co-elution difficult to detect

47. Summary
•The modern day technique is greatly
enhanced in terms of selectivity,
resolution, through miniaturization and the
use of very elaborate stationary phases.
•Therefore HPLC is widely used for
separation of molecules in biological,
pharmaceutical, food, environmental and
industrial process

48. References:
•Harold Varley practical clinical chemistry
•http://scimedia.com
•http://www.forumsci.co.il/HPLC

49. THANK YOU!