detail and complete study on the topic of high performance liquid chromatography done by the student fr the student by the student

1. High performance liquid
chromatography ( HPLC)
Ravish Yadav

2. High Performance Liquid
chromatography (HPLC)

3. Instrumentation
• Mobile phase reservoir
• Pumps (reciprocating, displacement, pneumatic) (Self study-30 min 0.5 hr)
• Sample injection systems (Rheodyne injector and autosampler)
• Column types (analytical, guard and preparative columns) and column packing
( porous, pellicular and monolithic),
• Detectors (Concept of solute and bulk property detector - Refractive index
,UV-Vis, Phototodiode array, fluorescence, , Electrochemical, Evaporative Light
Scattering )
• Difference between UPLC and HPLC (Self study-0.5 hr)
• Applications, Advantages and Limitations of HPLC (Self study-0.5 hr)

5. Components of HPLC Instrument
• Mobile Phase Reservoir
• Pump
• Precolumn
• Injector
• Column
• Temperature controller/thermostat
• Detector

6. Mobile phase reservoir
• Reservoir – Holds single solvent or mixture of solvents
used as mobile phase
• When elution carried out using single mobile phase of
constant composition – isocratic elution
• When elution carried out using two or more than two
mobile phases – gradient elution
• Therefore modern HPLC have two or more reservoirs from
which solvent can be introduced into a mixing chamber at
a rate to adjust polarity

7. Pump
Injector
Column
Oven
Detector
Mobile Phase
Simple system with one pump and one solvent reservoir.
If more than one solvent is used, solvents should be premixed.

8. Data
processor
pump
pump
pump
A
B
C
Injector
Column
Oven
Detector
Mixer

9. • Solvent Reservoir
• Inert to water, methanol, ACN etc.
• Usually glass / stainless steel
• Usually 500 to 1000 ml capacity
• Solvents used are HPLC grade

10. • Reservoirs are often equipped with means to
remove
• Filters - Particulate matter, dust etc.
• Degassers - Dissolves gases
• Filters
• Usually 1 – 5 µm microfilters

11. • Degassing can be carried out by
1. By filtration under vacuum
Removes all dissolved air or oxygen
Millipore filters are used
2. By distillation of the mobile phase
3. By ultrasonication
4. By sparging an inert gas of low solubility
Gas flushing system, involves bubbling inert gas of low
solubility through the mobile phase

12. Pumps
• The solvents or mobile phase must be passed
through a column at high pressures at up to 6000
psi(lb/in²) or 414 bar.
• As the particle size of stationary phase is smaller
(5 to 10µ) the resistance to the flow of solvent
will be high.
• That is, smaller the particle size of the stationary
phase the greater is the resistance to the flow of
solvents.
• Hence high pressure is recommended.

13. Requirements for pumps:
• Generation of pressure of about 5000 psi.
• Pulse free output & all materials in the pump
should be chemically resistant to solvents.
• Flow rates ranging from 0.1 to 10 mL/min
• Pumps should be capable of taking the solvent
from a single reservoir or more than one
reservoir containing different solvents
simultaneously.

14. Types of pumps
used in HPLC
SYRINGE OR
DISPLACEMENT
PUMPS
RECIPROCATING
PUMPS
PNEUMATIC OR
CONSTANT
PRESSURE PUMPS

15. Reciprocating Pumps
• Consists of
• Piston provided in syringe type chamber
• Syringe type Chamber is connected to two ball
check valves on two ends
• Two chambers –
• Chamber 1 – collects mobile phase from reservoir,
regulated by ball check valve 1
• Chamber 2 – directs mobile phase from syringe type
chamber into the column, regulated by ball check
valve 2
• The check valves open and close alternately

18. Working
• Piston is drawn back,
• ball checks 1 closes the entry into syringe type
chamber
• Ball check 2 close the entry into chamber 2
• Therefore mobile phase flows into chamber 1 but
not into chamber 2
• When piston direction is reversed,
• Ball check 1 closes entry from reservoir
• Ball check 2 closes entry into column
• Therefore mobile phase from chamber 1 enters
chamber 2

19. • When piston is withdrawn again
• Fresh mobile phase enters chamber 1
• But mobile phase from chamber 2 cannot enter
column, because no force to push it
• When piston direction is reversed again
• Mobile phase from chamber 1 enters chamber 2
and pushes the already existing mobile phase in
chamber 2 into the column

20. 20
Plunger Reciprocating Pump
motor and cam
plunger
plunger seal
check valvepump head
5 - 50µL
out
in
Mobile phase
check valve

21. 21
21
Plunger Reciprocating Pump
motor and cam
plunger
plunger seal
check valvepump head
5 - 50µL
out
in
Mobile phase
check valve

22. • Therefore
• When mobile phase enters chamber 1, no M.P
goes into column
• When it goes into column no MP enters
Chamber 1
• Therefore we get pulsed flow in reciprocating
column

23. • Disadvantage can be overcome
• Reducing time of filling
• Use of dual piston pump
• Dual – Piston Reciprocating Pumps
• Produces pulse free flow
• One is filling and other is pumping

24. Advantages
1. Internal volume is less – changeover from
one MP to another is fast
2. High output pressure
3. Constant flow rate of mobile phase

25. Syringe Pumps
• Constant flow rate pump
• Non-pulsating flow
• Low flow rates (1 to 100 mL/min)
• Isocratic flow only
• Refill required when reservoir (~50mL)
expended

26. Syringe type/Displacement type
pumps
– syringe-like chambers activated by screw-driven
mechanism powered by a stepper motor
– advantages: output is pulse free
– disadvantage: limited solvent capacity (~20 mL) and
inconvenience when solvents need to be changed

27. 27

29. Pneumatic Pumps
• Consists of collapsible bag containing MP, housed in
metallic cylinder
• Compressed gas is passed into metallic cylinder
• This squeezes MP through the outlet
• Advantage – Simple, cheap, pulse free flow
• Disadvantages – Flow depends on viscosity and back
pressure in column
• Not used for gradient elution

30. Precolumn/Guard column
• Placed between pump and injection system
• Also called guard column
• Broader than analytical columns
• Chemically identical to stationary phase in
analytical column
• Particle size is bigger
• Only MP is passed
• short length of 2 to 10 cm

31. • Rationale
• Impurities get adsorbed, so do not
contaminate analytical column, hence do not
interfere with separation
• MP becomes saturated with stationary phase,
so no stripping of analytical column

32. Sample Injection system
• the most useful and widely used sampling device
for modern LC is the microsampling injector valve
• samples can be introduced reproducibly into
pressurized columns without significant
interruption of flow, even at elevated
temperatures.
• Rheodyne injector consists of six-port Rheodyne
valve

33. Load position
• sample fills an external loop

34. Inject
• A clockwise rotation of the valve rotor places
the sample-filled loop into the mobile-phase
stream, with subsequent injection of the
sample onto the top of the column through a
low-volume, cleanly swept channel.

37. Automatic injectors
• Most of the autosamplers have a piston
metering syringe type pump to suck the
preestablished sample volume into a line and
than transfer it to the relatively large loop
(~100 ml) in a standard six-port valve.

38. COLUMN TYPES (ANALYTICAL, GUARD AND
PREPARATIVE COLUMNS) AND COLUMN
PACKING ( POROUS, PELLICULAR AND
MONOLITHIC)

39. CLASSIFACTION OF CLOUMN
column
Main column Guard column
Analytical column Preparative column
Standard column
Narrow bore
Short fast column
Micro preparative
Preparative column
Macro preparative
39
A) BASED ON APPLICATION

40. Columns
• HPLC column is made up of glass or stainless steel
• Glass columns can bear pressure upto 1000 psi
• Stainless steel coulmn can bear pressure from
2000 to 6000 psi
• Length ranges from 15 – 150 cm

41. Analytical columns
• Usual length – 10 to 30 cm
• Straight
• Occasionally coiled – results in loss in efficiency
• Inside diameter – 4 to 10 mm
• Particle size of packing – 5 to 10 µm
• Most commonly used column
• Length – 25 cm
• Inside diameter 4.6 mm
• Particle size 5 µm
• 40000 – 60000 plates/meter

43. Preparative Column
• Aim – to isolate compounds
• Length – 15 – 50 cm
• Diameter – 10 – 40 mm
• Packing size 5 to 60 µm
• Flow rate – 5 to 100 ml/min
• Load – 10 to 1000 mg

44. ANALYTICAL COLUMN
STANDARD COLUMN
• Internal diameter 4 – 5 mm and length 10 – 30 cm.
• Size of stationary phase is 3 – 5 µm in diameter.
• Used for the estimation of drugs, metabolites,
pharmaceutical preparation and body fluids like plasma.
NARROW BORE COLUMN
• Internal diameter is 2 – 4 mm.
• Require high pressure to propel mobile phase.
• Used for the high resolution analytical work of compounds
with very high Rt.
44

45. SHORT FAST COLUMN
• Length of column is 3 – 6 cm.
• Used for the substances which have good affinity
towards the stationary phase.
• Analysis time is also less (1- 4 min for gradient elution
& 15 – 120 sec for isocratic elution).
PREPARATIVE COLUMN
• Used for analytical separation i.e. to isolate or purify
sample in the range of 10-100 mg from complex
mixture.
– Length – 25- 100 cm
– Internal diameter – 6 mm or more.
45

46. TYPES OF PREPARATIVE COLUMN
Micro preparative or semi preparative column
• Modified version of analytical column
• Uses same packaging and meant for purifying
sample less then 100 mg.
Preparative column
• Inner diameter – 25 mm .
• Stationary phase diameter – 15- 100 µm
Macro Preparative Column
• Column length – 20 – 30 cm
• Inner diameter – 600 mm
46

47. • Scale up factor
• = Dp

48. • Three types of column packing material
• Porous support
• Pellicular support
• Bonded phases
Types of packing

49. Types of packing
Porous support
• Porous, silica-based packings - Diffusive pores dominate a typical porous
packing
• porous microparticulate packings - 5 to 10 µm
• major surface area of the particle is contained within these pores.
• In a porous particle, solutes transfer from the moving mobile phase outside of
the particles into the stagnant mobile phase within the pores in order to
interact with the stationary phase.
• solute molecule must diffuse out of the particle and continue its journey down
the column. Such a mass transfer occurs many times as the differential
separation process proceeds and the solute is eluted from the column.
• While the solute spends its time in the diffusive pores, the mobile phase in
which it was located originally moves down the column ahead of the solute.
• This slow rate of mass transfer into and out of the porous particle is a major
source of band broadening in HPLC

50. • use of smaller particles shortens the pathlength
of this diffusion process, improves mass transfer,
and provides better efficiency.
• Manufacturers can now produce small diameter
particles with fairly narrow particle size
distributions down to 1.5-mm average diameter,
although 3–3.5 mm and 5-mm particles are still
the norm.
• with the improvement in efficiency, was the
decrease in column permeability; that is, an
increase in column back pressure.

51. • totally porous silica particles give considerable
improvements in column efficiency, sample
capacity, and speed of analysis.

52. • Pellicular Packing
• Thin layer of stationary phase coated on glass beads
• also referred to as superficially porous packings or porous
layered beads,
• good efficiency relative to the large porous particles (with
diameters of 100 mm or so),
• poor sample capacity - due to their small specific surface
areas
• transition from large porous particles and pellicular
materials to small porous particles occurred in the early
1970s when microparticulate silica gel (dp , 10 mm)

53. • Monoliths
• Monoliths are columns that are cast as continuous
homogeneous phases rather than packed as individual
particles.
• Monolithic columns have great potential in offering a stable,
easily replaced column for both analytical and preparative
separations.
• Both silica-based and polymer-based monoliths have been
studied extensively.
• These columns are solid rods of silica monolith
• Rods are prepared by a polymerization process either in situ

54. • There are two important characteristics for current silica
monolith columns:
• they have the efficiency equivalent to about a 3–5 mm
silica particle
• pressure drop is about 40–50% lower than a 5-mm silica
particle.
• Thus, columns can be coupled in a serial manner thereby
generating higher plate counts for more difficult
separations.
• The polymeric monolith columns also have made their
mark on separation science. These columns consist of a
continuous crosslinked, porous monolithic polymer usually
polymethacrylates or methyacrylate copolymerizates.

55. • show higher permeability and lower flow
resistance than conventional liquid
chromatography columns

56. • Bonded-Phase Supports :
• molecules, comprising the stationary phase, i.e., the surfaces
of the silica particles, are covalently bonded to a silica-based
support particle.
• most popular bonded-phase, siloxanes, are formed by heating
the silica particles-in dilute acid for a day or two so as to
generate the reactive silonal group :
• which is subsequently treated with an organochlorosilane :

57. • These bonded phases are
• fairly stable between the pH range 2 to 9 and upto temperatures of
about 80 °C.
• nature of the R group of the silane solely determines the surface
polarity of the bonded phase.
• common bonded phase is made with a linear C18 hydrocarbon, also
known as ODS (octadecyl silane)
• Column are called as bondapack columns
• When R = C18H37 – C18 column
R = C8H17 – C8 column

59. • The procedure chosen for column packing depends
chiefly on the
• Mechanical strength of the packing
• particle size.
• Particles of diameter >20 pm can usually be dry packed
• particles with diameters < 20 pm slurry packing
techniques are used in which the particles are
suspended in a suitable solvent and the suspension (or
slurry) driven into the column under pressure.

60. Detectors
• Liquid chromatographic detectors are of two basic types.
• Bulk property detectors respond to a mobile-phase bulk property,
• measure the difference in some physical property of the solute in
the mobile phase compared to the mobile phase alone
• Example - refractive index, dielectric constant, or density.
• Solute property detectors respond to some property of solutes,
• respond to a particular physical or chemical property of the solute,
being ideally independent of the mobile phase
• Example - UV absorbance, fluorescence, or diffusion current
• Detectors to be studied in detail
• (Refractive index ,UV-Vis, Phototodiode array, fluorescence, ,
Electrochemical, Evaporative Light Scattering)

61. Refractive index detectors
• bulk property detector are based on the change of refractive index
of the eluent from the column with respect to pure mobile phase.
• disadvantages –
• lack of high sensitivity,
• lack of suitability for gradient elution,
• need for strict temperature control to operate at their highest
sensitivity.
• A pulseless pump, or a reciprocating pump equipped with a pulse
dampener
• limitations may be overcome by the use of differential systems in
which the column eluent is compared with a reference flow of pure
mobile phase.

62. • The two chief types of RI detector are as follows.
1. Deflection refractometer
• measures the deflection of a beam of monochromatic
light by a double prism in which the reference and sample
cells are separated by a diagonal glass divide.
• When both cells contain solvent of the same composition,
no deflection of the light beam occurs;
• if, however, the composition of the column mobile phase
is changed because of the presence of a solute, then the
altered refractive index causes the beam to be deflected.
• The magnitude of this deflection is dependent on the
concentration of the solute in the mobile phase.
Refractive index detectors

63. 2. Fresnel refractometer
• measures the change in the fractions of reflected and
transmitted light at a glass-liquid interface as the refractive
index of the liquid changes.
• In this detector both the column mobile phase and a reference
flow of solvent are passed through small cells on the back
surface of a prism.
• When the two liquids are identical there is no difference
between the two beams reaching the photocell, but when the
mobile phase containing solute passes through the cell there
is a change in the amount of light transmitted to the photocell,
and a signal is produced.
• The smaller cell volume (about 3 µl) in this detector makes it
more suitable for high-efficiency columns
• for sensitive operation, the cell windows must be kept
scrupulously clean.
Refractive index detectors

65. Ultraviolet detectors
• solute property detector, most widely used in HPLC,
• based on the principle of absorption of UV visible light as the effluent from the column
is passed through a small flow cell held in the radiation beam.
• It is characterized by high sensitivity (detection limit of about 1 x 10-'g mL-' for highly
absorbing compounds)
• since it is a solute property detector, it is relatively insensitive to changes of temperature
and flow rate.
• The detector is generally suitable for gradient
• The presence of air bubbles in the mobile phase can greatly impair the detector signal,
this effect can be minimized by degassing the mobile phase
• Both single and double beam instruments are commercially available.
• single- or dual-wavelength instruments (254 and/or 280 nm),
• variable-wavelength detectors covering the range 210-800 nm

66. • Diode array (multichannel) detector,
• polychromatic light is passed through the flow cell.
• The emerging radiation is diffracted by a grating and then falls on to an
array of photodiodes, each photodiode receiving a different narrow
wavelength band.
• A microprocessor scans the array of diodes many times a second and the
spectrum so obtained may be displayed
• An important feature of the multichannel detector is that it can be
programmed to give changes in detection wavelength at specified points
in the chromatogram; this facility can be used to 'clean up' a
chromatogram
• e.g. by discriminating against interfering peaks due to compounds in the
sample which are not of interest to the analyst.
Ultraviolet detectors

67. • Diode permits qualitative information to
be obtained beyond simple
identification by retention time.
• There are two major advantages of
diode array detection.
• allows for the best wavelength(s) to be
selected for actual analysis. This is
particularly important when no
information is available on molar
absorptivities at different wavelengths.
• The second major advantage is related
to the problem of peak purity.
• Often, the peak shape in itself does not
reveal that it actually corresponds to
two (or even more) components. In
such a case, absorbance rationing at
several wavelengths is particularly
helpful in deciding whether the peak
represents a single compound or, is in
fact, a composite peak.

69. Fluorescence detectors
• enable fluorescent compounds (solutes) present in the mobile
phase to be detected
• passing the column effluent through a cell irradiated with
ultraviolet light and measuring any resultant fluorescent
radiation.
• Radiation from a Xenon-radiation or a Deuterium-source is
focused on the flow cell through a filter.
• The fluorescent radiation emitted by the sample is usually
measured at 90° to the incident beam. The second filter picks up
a suitable wavelength and avoids all scattered light to reach
ultimately the photomultiplier detector.

70. • Although only a small proportion of inorganic and organic
compounds are naturally fluorescent, many biologically
active compounds (e.g. drugs) and environmental
contaminants (e.g. polycyclic aromatic hydrocarbons) are
fluorescent
• Because both the excitation wavelength and the detected
wavelength can be varied, the detector can be made
selective.
• The application of fluorescence detectors has been
extended by means of pre- and post-column derivatization
of non-fluorescent or weakly fluorescing compounds
Fluorescence detectors

72. Electrochemical detectors.
• refers to amperometric or coulometric detectors - measure the current
associated with the oxidation or reduction of solutes.
• serious interference (large background current) caused by reduction of oxygen
in the mobile phase. Complete removal of oxygen is difficult so that
electrochemical detection is usually based on oxidation of the solute.
• Examples of compounds are phenols, aromatic amines, heterocyclic nitrogen
compounds, ketones, and aldehydes.
• detectors are selective and selectivity may be further increased by adjusting
the potential applied to the detector to discriminate between different
electroactive species.
• an anode becomes a stronger oxidising agent as its electrode potential
becomes more positive.
• requires conducting mobile phases, e.g. containing inorganic salts or mixtures
of water with water-miscible organic solvents

73. • Amperometric detector is widely used electrochemical detector, having
the advantages of high sensitivity and very small internal cell volume.
• Three electrodes are used:
1. the working electrode - made of glassy carbon - the electrode at which
the electroactive solute species is monitored;
2. the reference electrode, usually a silver-silver chloride electrode, gives
a stable, reproducible voltage to which the potential of the working
electrode is referred;
3. the auxiliary electrode is the current-carrying electrode and usually
made of stainless steel.
• amperometric detectors have a more limited range of applications,
used for trace analyses where the ultraviolet detector does not have
sufficient sensitivity.
Electrochemical detectors.

76. Evaporative Light Scattering Detector
• Column effluent is passed into nebulizer
• Converted to fine mist
• At controlled temperature evaporation of mobile phase
takes place, leading to formation of particles of analyte
• Analyte particles then pass through laser beam
• The scattered light is detected at right angles by silicon
photodiode