HPLC- high performance liquid chromatography or high pressure liquid chromatography overall review good animation & GIF for presentation detectors in detail basic instrumentation with detectors

1. Presented By:
Prof. Hiren Thakkar
Sigma Institute of Pharmacy
Vadodara,Gujarat

2. INTRODUCTION
 Chromatography: a technique for the separation of a mixture by passing it
in solution or suspension through a medium in which the components
move at different rates.
 High performance liquid chromatography is a powerful tool in analysis, it
yields high performance and high speed compared to traditional columns
chromatography because of the forcibly pumped mobile phase.
 HPLC stands for “High-performance liquid chromatography”(sometimes
referred to as High-pressure liquid chromatography).
 HPLC is a chromatographic technique that can separate a mixture of
compounds. It is used in biochemistry and analytical chemistry to identify,
quantify and purify the individual components of a mixture.

3.  Stationary phase : The substance on which adsorption of the analyte
(the substance to be separated during chromatography) takes place . It
can be a solid, a gel, or a solid liquid combination
 Mobile phase : solvent which carries the analyte (a liquid or a gas)
 HPLC is a type of liquid chromatography where the sample is forced
through a column that is packed with a stationary phase composed of
irregularly or spherically shaped particles, a porous monolithic layer ,
or a porous membrane by a liquid (mobile phase) at high pressure.

4. PRINCIPLE
 High Performance Liquid Chromatography [HPLC] is principle is
based on adsorption as well as partition chromatography is depending
on the nature of stationary phase, if stationary phase is solid principle is
based on adsorption chromatography and if stationary phase is liquid
principle is based on partition chromatography.
 Adsorption chromatography - The affinity of molecules towards
stationary phase is known as Adsorption chromatography.
 Partition chromatography - The molecule can moves in two phases of
liquid is known as partition chromatography.

5. Continue..
 When a mixture of components are introduced into the column .
Various chemical and/or physical interactions take place between
the sample molecules and the particles of the column packing .
 They travel according to their relative affinities towards the stationary
phase. The component which has more affinity towards the adsorbent,
travels slower. The component which has less affinity towards the
stationary phase travels faster.
 Since no two components have the same affinity towards the stationary
phase, the components are separated

7.  When distribution of solute is equal between two phases , then effective
distribution Kd =1
 Individual zones adjacent to each other in the column where there is
sufficient space for equal distribution of solutes in between stationary
and mobile phase are called theoretical plates
 Length of theoretical plate in the column is called plate height
 More the theoretical plates , more efficient the separation
 2000 or more theoretical plates are ideal for a column
 Components which have more affinity towards mobile phase elute first

8. TYPES OF HPLC SEPARATIONS
 Normal Phase: Separation of polar analytes by partitioning onto a
polar, bonded stationary phase.
 Reversed Phase: Separation of non-polar analytes by partitioning onto
a non-polar, bonded stationary phase.
 Adsorption: In Between Normal and Reversed. Separation of
moderately polar analytes using adsorption onto a pure stationary
phase (e.g. alumina or silica)
 Ion Chromatography: Separation of organic and inorganic ions by their
partitioning onto ionic stationary phases bonded to a solid support.
 Size Exclusion Chromatography: Separation of large molecules based
in the paths they take through a “maze” of tunnels in the stationary
phase.

11. BASED ON PRINCIPLE OF SEPERATION
 Absorption Chromatography
 In the Absorption Chromatography solute molecules bond directly to
the surface of the stationary phase
 the component which has more affinity towards mobile phase elutes
first & the component which has less affinity towards stationary
phase elutes later. No two components have same affinity towards
mobile phase & stationary phase.

12. Ion-exchange chromatography
 Ion exchange chromatography is a process that
allows the separation of ions and polar molecules
based on their charge.
 It can be used for almost any kind of charged
molecule including large proteins, small
nucleotides and amino acids.
 Retention is based on the attraction between solute
ions and charged sites bound to the stationary
phase. Ions of the same charge are excluded.
 The use of a resin (the stationary solid phase) is
used to covalently attach anions or cations onto it.
Solute ions of the opposite charge in the mobile
liquid phase are attracted to the resin by
electrostatic forces.

13. Ion-pair chromatography
 It is a form of chromatography in which ions in solution can be
"paired“ or neutralized and separated as an ion pair on a reversed-
phase column.
 Ion-pairing agents are usually ionic compounds that contain a
hydrocarbon chain that imparts a certain hydrophobicity so that the
ion pair can be retained on a reversed-phase column.

14. Gel permeation chromatography
 This type of chromatography lacks an
attractive interaction between the stationary
phase and solute.
 The liquid or gaseous phase passes through a
porous gel which separates the molecules
according to its size.
 The pores are normally small and exclude the
larger solute molecules, but allows smaller
molecules to enter the gel, causing them to
flow through a larger volume. This causes the
larger molecules to pass through the column
at a faster rate than the smaller ones.

15. Affinity Chromatography
 This is the most selective type of chromatography employed. It
utilizes the specific interaction between one kind of solute
molecule and a second molecule that is immobilized on a
stationary phase.
 For example, the immobilized molecule may be an antibody to
some specific protein. When solute containing a mixture of
proteins are passed by this molecule, only the specific protein
is reacted to this antibody, binding it to the stationary phase.
This protein is later extracted by changing the ionic strength or
pH.

17. BASED ON ELUTION TECHNIQUE
 1.Isocratic elution
 A separation in which the mobile phase composition remains constant
throughout the procedure is termed isocratic elution
 In isocratic elution, peak width increases with retention time linearly
with the number of theoretical plates. This leads to the disadvantage
that late-eluting peaks get very flat and broad.
 Best for simple separations
 Often used in quality control applications that support and are in close
proximity to a manufacturing process

18.  Gradient elution
 A separation in which the mobile phase composition is changed
during the separation process is described as a gradient elution
 Gradient elution decreases the retention of the later-eluting
components so that they elute faster, giving narrower peaks . This also
improves the peak shape and the peak height
 Best for the analysis of complex samples
 Often used in method development for unknown mixtures
 Linear gradients are most popular

19. BASED ON SCALE OF OPERATION
 1.Analytical HPLC
 No recovery of individual components of substance
 2.Preparative HPLC
 Individual components of substance can be recovered

20. BASED ON TYPE OF ANALYSIS
 1.Qualitative analysis
 Analysis of a substance in order to ascertain the nature of its chemical
constituents
 We can separate individual components but cannot assess the quantity
in this analysis
 2.Quantitaive analysis
 Determining the amounts and proportions of its chemical constituents .
 Quantity of the impurity and individual components can be assessed

21. INSTRUMENTATION

22. Solvent delivery system(mobile phase)
 The mobile phase in HPLC refers to the solvent being continuously applied to
the column or stationary phase
 The mobile phase acts as a carrier to the sample solution
 A sample solution is injected into the mobile phase of an assay through the
injector port
 As a sample solution flows through a column with the mobile phase, the
components of that solution migrate according to the non-covalent
interaction of the compound with the column
 The chemical interaction of the mobile phase and sample , with the column ,
determine the degree of migration and separation of components contained
in the sample
 The solvents or mobile phases used must be passed through the column at
high pressure at about 1000 to 3000 psi. this is because as the particle size of
stationary phase is around 5-10μ, so the resistance to the flow of solvent is
high.

23. Pumps
 The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatograph at a specific flow rate, expressed in
milliliters per min (mL/min).
 Normal flow rates in HPLC are in the 1-to 2-mL/min range.
 Typical pumps can reach pressures in the range of 6000-9000 psi (400-
to 600-bar).
 During the chromatographic experiment, a pump can deliver a constant
mobile phase composition (isocratic) or an increasing mobile phase
composition (gradient).

25. DISPLACEMENT PUMPS
 It consists of large, syringe like
chambers equipped with a plunger
activated by a screw driven mechanism
powered by a stepping motor.
 So it is also called as Screw Driven
Syringe Type Pump.
 Advantages:- It produces a flow that
tends to be independent of viscosity &
back pressure.
 Disadvantages:- It has a limited
solvent capacity(~250) & considerably
inconvenient when solvents must be
changed.

26. RECIPROCATING PUMPS
 Consists of a small motor driven piston which moves rapidly back and
forth in a hydraulic chamber that may vary from 35-400μL in volume
 On the back stroke , the separation column valve is closed , and the piston
pulls in solvent from the mobile phase reservoir
 On the forward stroke, the pump pushes solvent out of the column from the
reservoir
 A wide range of flow rates can be attained by altering the piston stroke
volume during each cycle , or by altering the stroke frequency.
 Dual and triple head pump consists of identical piston chamber units which
operate at 180 or 120 degrees out of phase(this system is significantly
smoother because one pump is filling while the other is in the delivery cycle.

28. Syringe type pump
 These are most suitable for
small bore columns because
this pump delivers only a finite
volume of mobile phase before
it has to be refilled. These
pumps have a volume between
250 to 500mL
 The pump operates by a
motorized lead screw that
delivers mobile phase to the
column at a constant rate .The
rate of solvent delivery is
controlled by changing the
voltage on the motor.

29. Constant pressure pump
 In these types of pumps , the mobile
phase is driven through the column
with the use of pressure from the gas
cylinder
 A low-pressure gas source is needed to
generate high liquid pressures
 The valving arrangement allows the
rapid refill of the solvent chamber whose
capacity is about 70mL
 This provides continuous phase flow
rates

30. Injector:
 The injector serves to introduce the liquid sample into the flow stream of the mobile phase
for analysis.
 It is equipped with six port valves so that a sample can be injected into the flow path at
continuous pressure
 For a manual injector, the knob is manually operated to deliver the sample to the column
 The knob is set to LOAD position for sample injection using a syringe , the sample is injected
into the sample loop , which is separated from the flow path
 The knob is turned to INJECT position and the eluent travels through the loop from the
pump and delivers the sample to the column
 Typical sample volumes for manual injector are 5-to 20-microliters (μL).
 The injector must also be able to withstand the high pressures of the liquid system.
 An autos ampler is the automatic version for when the user has many samples to analyze or
when manual injection is not practical. It can continuously Inject variable volume a of 1 μL –
1 mL

32. Column
 Considered the “heart of the chromatograph” the column’s stationary phase separates the
sample components of interest using various physical and chemical parameters.
 It is usually made of stainless steel to withstand high pressure caused by the pump to move the
mobile phase through the column packing other material include PEEK and glass
 The small particles inside the column are called the “packing” what cause the high back
pressure at normal flow rates.
 Column packing is usually silica gel because of its particle shape , surface properties , and pore
structure give us a good separation
 Other material used include alumina, a polystyrene-divinyl benzene synthetic or an ion-
exchange resin – Pellicular particle: original, Spherical, nonporous beads, proteins and large
biomolecules separation (dp: 5 μm) – Porous particle: common used, dp: 3 ~ 10 μm. Narrow size
distribution, porous microparticle coated with thin organic film
 The dimensions of the analytical column are usually -straight, Length(5 ~ 25 cm), diameter of
column(3 ~ 5 mm), diameter of particle(35 μm). Number (40 k ~ 70 k plates/m)

33. Guard column is used to remove particular matter and contamination, it protect the analytical column
and contains similar packing its temperature is controlled at < 150 °C, 0.1 °C
As mention before , columns are divided into different types according to their functions (see types of
HPLC)

34. Detector:
 A chromatography detector is a device used in high performance liquid
chromatography (HPLC) to detect components of the mixture being
eluted off the chromatography column.
 The detector senses the presence of the individual components as they
leave (elute) the column.
 The detectors converts a change in effluents into an electric signal that
is recorded by data system.

35. IDEAL PROPERTIES OF A DETECTOR
 High sensitivity.
 Good stability and reproducibility.
 A linear response to solute.
 Negligible base line noise.
 Should be inexpensive.
 Capable of providing information on the identity of the solute.
 A short response time independent of flow-rate.
 The detector should be non-destructive.
 Responses independent of mobile phase composition.

36. The detectors used in HPLC are of majorly
two types:
 Selective detectors (solute property) : These detectors respond to a particular physical or
chemical property of the solute, being ideally independent of the mobile phase. They are as
follow:
 Absorbance detectors
 Fluorescence detectors
 Electrochemical detectors
 Mass spectrometric detectors
 Universal detectors (bulk property): measure the difference in some physical property of the
solute in the mobile phase compared to the mobile phase alone. They are generally universal
in application but tend to have poor sensitivity and limited range. Such detectors are usually
affected by even small changes in the mobile-phase composition which precludes the use of
techniques such as gradient elution. They are as follow:
 Refractive index detectors
 Evaporating light scattering detectors

37. CLASSIFICATION OF HPLC DETECTORS
 UV/VIS
- Fixed Wavelength
- Variable Wavelength
- Diode array (PDA)
 Refractive index
-Deflection Detector
-Refractive Detector (Fresnel refractometer)
 Fluorescence Detector
 Electrochemical Detector
 Conductivity Detector
 Evaporative light scattering (ELSD)
 Mass detector (LC-MS)
 IR detector
 Optical rotation detector (chiral detectors)

38. ULTRAVIOLET/VISIBLE
SPECTROSCOPIC DETECTORS
 Measures the ability of solutes to
absorb light at a particular
wavelength(s) in the ultraviolet (UV)
or visible (Vis) wavelength range.
 When light of a certain wavelength is
directed at a flow cell, the substance
inside the flow cell absorbs the light. As
a result, the intensity of the light that
leaves the flow cell is less than that of
the light that enters it. An absorbance
detector measures the extent to which
the light intensity decreases (i.e., the
absorbance).

39.  Three Common types of UV/Vis Absorbance Detectors
  Fixed wavelength detectors
  Variable wavelength detectors
  Photodiode array detectors
 Fixed Wavelength Detector absorbance of only one given wavelength is
monitored by the system at all times (usually 254 nm)
Simplest and cheapest of the UV/VIS detectors
Limited in flexibility
Limited in types of compounds that can be monitored
 Variable Wavelength Detector a single wavelength is monitored at any
given time, but any wavelength in a wide spectral range can be selected
Wavelengths vary from 190-900 nm.
More expensive, requires more advanced optics
More versatile, used for a wider range of compounds
More

40. Photo Diode Array Detector
 operates by simultaneously monitoring
 absorbance of solutes at several different wavelengths.
 Light from the broad emission source such as a deuterium lamp is collimated by an
achromatic lens system so that the total light passes through the detector cell onto a
holographic grating. In this way, the sample is subjected to light of all wavelengths
generated by the lamp. The dispersed light from the grating is allowed to fall on to a
diode array. The array may contain many hundreds of diodes and the output from each
diode is regularly sampled by a computer and stored on a hard disc.

41. FLUORESCENCE DETECTOR
 It is based on the fluorescent radiation
emitted by some compounds.
 The excitation source passes through
the flow cell to a photo detector while
a monochromatic measures the
emission wavelengths.
 More sensitive and specific.
 The disadvantage is that most
compounds are not fluorescent in
nature.
 Fluorescence is a type of luminescence
in which the light energy is released in
the form of a photon in nanoseconds
to microseconds.

42. REFRACTIVE INDEX DETECTOR
 Measures the overall ability of the mobile phase and its solutes to refract
or bend light.
 Refractive index detector measures the molecule’s ability to deflect light
in a flowing mobile phase in a flow cell relative to a static mobile phase
contained in a reference cell.
 The amount of defection is proportional to the concentration of the solute
in the mobile phase.

43. ELECTROCHEMICAL DETECTORS
 It is based on the measurement of the current resulting from an oxidation/ reduction reaction of the analyte
at a suitable electrode.
 The level of current is directly proportional to the analyte concentration.
 Three electrodes are employed which are:
 Working electrode
 Auxiliary electrode
 Reference electrode

44. ELECTRICAL CONDUCTIVITY DETECTOR
 Used in analytical applications of
ion-exchange chromatography for
the detection of ionic compounds.
 Detector measures the ability of
the mobile phase to conduct a
current when placed in a flow-cell
between two electrodes.
 Conductivity detectors measures
electronic resistance and measured
value is directly proportional to
the concentration of ions present in
the solution

45. EVAPORATIVE LIGHT SCATTERING
DETECTOR (ELSD)
 Detection is based on the scattering of a beam of light by particles of compound
remaining after evaporation of the mobile phase. It is a universal detector and does
not required a compound to have a chromophore for detection
 There are three steps involved in detection:
 Nebulization
 Mobile phase evaporation
 Detection
 The flow from the column is nebulized with a stream of inert gas (Nebulize means to
convert a liquid into a fine spray or mist). The mobile phase, whichmust be volatile,
is evaporated, leaving tiny particles of the analytes (you can see why this detection
method will not work with volatile compounds). The particles are passed through a
laser beam and they scatter the laser light. The scattered light is measured at right
angles to the laser beam by a photodiode detector.

47. MASS SPECTROMETER AS AN
HPLC DETECTOR
 Liquid chromatography–mass spectrometry
(LC-MS, or HPLC-MS) is an analytical
chemistry technique that combines the
physical separation capabilities of liquid
chromatography (or HPLC) with the mass
analysis capabilities of mass spectrometry
(MS).
 It is a method that combines separation
power of HPLC with detection power of
Mass spectrometry.
 Mass spectrometry (MS) is a powerful
analytical tool that can supply both
structural information about compounds
and quantitative data relating to mass.

48. INFRARED SPECTROPHOTOMETRIC DETECTOR
 Infrared (IR) detectors have been
used to a limited extent only for the
analysis of non-polar lipids, with
the specific absorbancefor the
carbonyl function between 1650 and
1860 cm-1 (or at about 5.75 microns)
being the spectral region of value.
 The wavelength scanning is provided
by semicircular filter wedges, the
wavelength range being from 4000-
690 cm-1 . It is not very sensitive and
have serious drawback that most
mobile phase solvents absorb
strongly in the IR region

49. CHIRAL DETECTORS
 Chiral detectors are used for detection of optically active compounds such as amino acids,
sugars, terpenes and other compounds containing an asymmetric carbon.
 There are two chiral detection techniques, polarimetry or optical rotary dispersion (ORD)
and circular dichorism (CD).
 ORD detectors are based on differences in refractive index and CD detectors differentiate
enantiomers by measuring differences between the absorption of light and left-handed
circularly polarized light due to existence of a chiral chromophores.

50. RECORDERS AND INTEGRATORS
 Recorders are used to record responses obtained from the detectors after
amplification, if necessary.
 They record the baseline & all the peaks obtained with respect tot time.
 Retention time can be found out from this recordings, but area under curve cannot
be determined.
 The Integrators are improved versions of recorders with some data processing
capabilities.
 hey can record the individual peaks with retention time, height, width of peaks, peak
area, percentage area, etc.
 Integrators provides more information on peaks than recorders.
 In recent days computers and printers are used for recording and processing the
obtained data & for controlling several operations.

51. Applications
 HPLC is one of the most widely applied analytical separation
techniques.
 Pharmaceutical:
 Tablet dissolution of pharmaceutical dosages.
 Shelf life determinations of pharmaceutical products.
 Pharmaceutical quality control.

52.  Environmental:
•Phenols in Drinking Water.
•Identification of diphenhydramine in sediment samples.
•Biomonitoring of PAH pollution in high-altitude mountain lakes
through the analysis of fish bile.
•Estrogens in coastal waters - The sewage source.
•Toxicity of tetracyclines and tetracycline degradation products to
•environmentally relevant bacteria.
•Assessment of TNT toxicity in sediment..

53. Forensics
 Identification of anabolic steroids in serum, urine, sweat and hair.
 Forensic analysis of textile dyes.
 Determination of cocaine and metabolites in meconium.
 Simultaneous quantification of psychotherapeutic drugs in human
plasma.

54.  Clinical
 Quantification of DEET in Human Urine.
 Analysis of antibiotics.
 Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis.
 Detection of endogenous neuropeptides in brain extracellular fluids.
 Food and Flavor
 Ensuring soft drink consistency and quality.
 Analysis of vicinal diketones in beer.
 Sugar analysis in fruit juices.
 Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits.
 Trace analysis of military high explosives in agricultural crops.
 Stability of aspartame in the presence of glucose and vanillin

55. ADVANTAGES OF HPLC:
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. HPLC is more versatile than GLC in some respects, because it has the advantage of
not being restricted to volatile and thermally stable solute and the choice of mobile and
stationary phases is much wider in HPLC
8. Both aqueous and non aqueous samples can be analyzed with little or no sample pre
treatment
9. A variety of solvents and column packing are available, providing a high degree of
selectivity for specific analyses.
10. It provides a means for determination of multiple components in a single analysis.

57. Thank You