Gives basic idea about instrumentation of high performance liquid chromatography

1. Prepared by
M.D.Dwivedi
B.Pharm ɪvth Yr.

2. Modern HPLC essentially consist of following main
components:
 Solvent delivery systems

Pumping systems

Sample Injector systems

HPLC Column(s)

Detector

Data System
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3. Principle Pattern
An Example
Solvent Reservoirs
Controller
Solvent Cabinet
Vacuum Degasser
Binary Pump
Auto sampler
Column
Compartment
Detector
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4. 


Continuously provide eluent (solvent).
Provide accurate mobile phase
compositions.
Includes solvent reservoirs, inlet filter,
and degassing facilities which works in
conjugation.
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5. 

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A good HPLC unit should have
3-4 solvent reservoirs to release
eluent into a mixing chamber at
varying rate.
Inert container for holding the
solvent (mobile phase).
5

6. 


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Type of filter.
Stainless Steel or glass with 10 micron
porosity.
Removes particulates from
solvent.
6

7. 

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Removed dissolved gases (such as
oxygen and nitrogen).
May consist of vacuum pump
system, a distillation system, a
heating and stirring device, or a
system for spearing.
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8. 


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Constant, reproducible, and pulse
free supply of eluent to the HPLC
column.
Flow rate in between 0.1-10 cm3
min-1 .
Operating pressures from 3000
psi to 6000 psi.
8

9. Mainly three types
1. Constant flow reciprocating pump
2. Syringe (or displacement) type
pump.
3. Pneumatic (or constant pressure)
pump.
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10. 






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The term "reciprocating" describes any continuously
repeated backwards and forwards motion.
Widely used (~90% in HPLC system) type of pump.
It gives a pulsating delivery of the eluent.
Pulse damper is used to make the flow pulse free.
Deliver solvent(s) through reciprocating motion
of a piston in a hydraulic chamber.
Solvent is sucked during back stroke and gets
deliver to the column in forward stroke.
Flow rates of eluent can be set by adjusting
piston displacement in each stroke.
10

11. Working:
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12. Advantages:

Small internal volume (35-400 µL)
High output pressures up to 10,000 psi.
 Smart adaptability to gradient elution.
 Constant flow rates independent of
column back pressure, solvent viscosity and
temperature.

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13. 


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Consist of large syringe like chamber
(capacity up to 500 Cm2).
Plunger activated by screw-driven and
hydraulic amplifier machine.
Suitable for small bore column.
13

14. Working:
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15. Advantages:
 Flow is independent of viscosity, back pressure.
 Deliver pulseless flow.
 Provide pressure up to 78,000 psi.
Disadvantages:
 Costly
 Low flow rate (1 to 100 mL/min).
 Limited solvent capacity.
 Inconvenience in frequent refilling i.e. in changing
solvent
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16. 
Gas is used to pressurize the mobile phase
present in a collapsible solvent container.

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17. Working:
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18. Advantages:
 Not very costly.
 Provide pulse free flow.
Disadvantages:
 Produce pressure only up to 2000 psi.
 Not suitable for gradient elution.
 Flow rate depends upon column back pressure,
and viscosity.
 Small capacity for filing of solvent.
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19. Introduce required sample volume accurately
into the HPLC system.
 Introduction of sample without depressurizing
the system.
 Volume of sample must be very small (2 µL to
500 µL).
Types of injection system:
(a) Manual injection(Rheodyne/Valco injectors)
(b) Automatic injection

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20. 



Also know as Rheodyne / Valco injectors.
User manually loads sample into the injector
using a syringe.
Overloading of column causes band
broadening hence volume used must be very
small (2 µL to 500 µL).
Sample should be introduce without
depressing the system.
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21. Working:
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22. 




Also know as Autosampler.
Programmed based sample delivery system.
User loads vials filled with sample solution into the
autosampler tray (100 samples).
Autosampler automatically
1. Measures the appropriate sample volume,
2. Injects the sample,
3. Flushes the injector to be ready for
the next sample, etc., until all sample
vials are processed.
Also controls the sequence of samples for
injection from vials.
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23. 





Material: Stainless steel (highly polished
surface).
External diameter: 6.35 mm
Internal diameter: 4-5 mm (usual 4.6 mm)
Length: 10-30 cm (usual 25 cm)
Packing particles size (3 µm, 5µm, 10 µm)
Stainless steel frits or mess discs
(porosity< 2µm) retain packing material.
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24. 
Efficiency or performance of a column may be measured by
fallowing expression :
N = 16(VR/WB)2
H = L/N
…(a)
…(b)
VR = Retention volume of the solute
WB = Volume occupied by a solute
( For efficient column WB < VR )
N = Plate number of the column (dimensionless)
H = Height of the column (mm × µm)
L = Length of the column (cm)

For more efficient column ‘N’ should be larger and
correspondingly ‘H’ gets smaller.
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25. Factors affecting efficiency of column:
 Particle size
 Flow rate
 Thickness of stationary phase
 Mobile phase viscosity
 Diffusion of solute in mobile and stationary
phases
 How well a column is packed
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26. For prolonged life of HPLC columns
 Guard column
 Scavenger column
 Column thermostats
Guard column:
 Also know as pre-column.
 Placed in between injector and analytical column.
 Having same material as in
column but with larger size
particles ~ 30-40 µm.
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27. Scavenger column:
 Place between the pump and injection valve.
 Saturate the aqueous eluent (specially high
or low pH buffers)with silica.
Column thermostats:
 HPLC is performed at ambient temperature in
number of cases.
 Controls temperature of the column for
better resolutions (chromatograms).
 HPLC is performed at ambient temperature
in many cases.
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28. 
On the basis of chromatographic objective
HPLC column can be categorized as follows:
Scale
Chromatographic Objectives
Analytical
Information ( compound identification and
concentration)
Semi-preparative
Data and small amount of purified
compound[<0.5 g]
Preparative
Large amount of purified compound [>0.5 g]
Process (industrial)
Manufacturing quantities ( g to kg)
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29. 



The stationary phase is the substance fixed in
place for the chromatography procedure.
The stationary phase can be a solid, a liquid, or
a bonded phase.
Bonded phase is a stationary phase that is
covalently bonded to the support particles or to
the inside wall of the column tubing.
Chemically-modified silicas, unmodified silica
or cross-linked co-polymers of styrene and
divinyl benzene, commonly used as stationary
phase.
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30. 




Silica particles as the basis of the support.
Sizes 3 µm, 5 µm, and 10 µm (spherical and
regular in shape).
Pore size normally are in the 60 – 100 Å range.
Pore size of 300 Å or larger being used for
larger biomolecules.
Columns are packed using high-pressure to
ensure that they are stable during use.
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31. 
1.
2.
3.
4.
Several types of particles are used in HPLC
column packing.
Micro porous (or diffusive) particle/Porous
microsphere
Perfusion particles
Nonporous (or micropellicular)
Chiral (bounded) stationary phase
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32. Diffusive
Pore
Liquid or ion
exchange film
5 µm
Solid
core
Micropellicular
particles
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Microporus
Particle
Thorough pore
Perfusion Particle
32

33. Microporus (or diffusive) Particles:
 Main surface area is within the pores to interact
with the stationary phase.
 Small particles reduces the diffusion path
length and thereby band broadening.
 Zorbax Rx (Sil) (Silica sol) is a porous
microsphere silica particle with 50% porosity
and a pore size of 100 Å.
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34. Perfusion Particles:
 The particle consist of both small (diffusive)
and large (through) pores in them.
 Diffusive pore provide sorption power.
 Through pore permits the mobile phase to pass
directly through the packing.
 Slightly larger than microporous particles
(~ 12 µm).
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35. Nonporous particles:
 Made from either silica or resin.
 Smaller in size (1.5 - 2.5 µm) with thin porous
layer.
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36. 



Also know as eluent.
Solvent used in HPLC must be of HPLC grade i.e.
Filtered using 0.2 μm filter.
Eluting power of the mobile phase is determined
by its overall polarity, stationary phase polarity
and the nature of the sample components.
For 'normal-phase‘ separations eluting power
increases with increasing polarity of the solvent,
while for 'reverse-phase' separations eluting power
decreases with increasing solvent polarity.
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37. Solvent
Solvent strength e°
parameter, (adsorption)
Solvent strength
parameter, p’
(partition)
UV cut-off (nm)
n-Hexane
0.01
0.1
195
Cyclohexane
0.04
-0.2
200
Tetrachloromethane
0.18
1.6
265
Methylbenzene
0.29
2.4
285
Trichloromethane
0.40
4.1
245
Dichloromethane
0.42
3.1
230
Tetrahydrofuran
0.56
4.0
212
Propanone
0.56
3.9
330
Acetonitrile
0.65
5.8
190
iso-Propanol
0.82
3.9
205
Ethanol
0.88
4.3
205
Methanol
0.95
5.1
205
Ethanoic acid
>1
4.4
255
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>1
10.2
170
37

38. The detector refers to the instrument used for
qualitative and quantitative detection of analytes
after separation.
 Monitors the eluent as it emerges from column.
 Establishing both the identity and concentration of
eluting components in the mobile phase stream.
Characteristics of detectors:
 Adequate sensitivity (10-8 to 10-15 g solute sec-1).
 Desired stability and reproducibility.
 Sort response time
 Minimal internal volume (minimize zone
broadening).

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39. Sensitivity:
 Expressed as the noise equivalent concentration, i.e. the
solute concentration, Cn, which produces a signal equal
to the detector noise level.
 The lower the value of Cn for a particular solute, the
more sensitive is the detector for that solute.
A linear response:
 The linear range of a detector is the concentration
range over which its response is directly proportional
to the concentration of solute.
Type of response:

Detector is either universal or selective.
 Universal (sense all the constituents of the sample).
 Selective (respond to certain components).
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40. Types of Detectors:
1.
Bulk property detectors
2.
Solute property detectors
Bulk property detectors:
 Measure the difference in some physical property
of the solute present in the mobile-phase in
comparison to the individual mobile-phase.
 Not suitable for gradient elution.
(a) Refractive - index detector
(b) Conductivity detector
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41. Solute property detectors:
 Respond to a particular physical or chemical
characteristic of the solute which should be
ideally and absolutely independent of the
mobile-phase being used.
(a) UV - detectors
(b) Fluorescence Detectors
(c) Electrochemical detectors
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42. Detectors Used in HPLC:
Type
Response
Flow rate Temperature
sensitivity sensitivity
Gradient
elution
UV/Visible absorption 10-4
Selective
No
Low
Yes
Fluorescence
10-5
Selective
No
Low
Yes
IR absorption
10-3
Selective
No
Low
Yes
Refractive index
10-2
Universal
No
± 10-4 oC
No
Conductometric
10-2
Selective
Yes
± 1 oC
No
Amperometric
10-5
Selective
Yes
± 1 oC
_
Mass spectroscopy
10-5
Universal
No
None
Yes
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Limit of
detection
(µg/cm3)
42

43. Also know as ‘RI-Detector’ and ‘Refract meter’.
 Based on refractive index measurement.
 Determine change of refractive index of the eluant
from the column with respect to pure mobile phase.
Types:
(a) Deflection refractometer
(b) Fresnel refractometer

Referactive index (n) =
𝑐
𝑣
c = speed of light in vacuum
v = speed of light in medium
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44. 
The RI of a few commonly used mobile-phase
is stated below :
Mobile-Phase
Benzene
1.501
Decane
1.410
Hexane
1.375
Octane
1.397
Tetrayhydrofuran
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Refractive-Index
1.405
44

45. Working:
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46. Advantages:
 Universal response
 Independent of flow rate.
Disadvantages:
 Less sensitivity
 Temperature dependent, strict temperature
control (±0.001 °C).
 Not suitable for gradient elution.
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47. 



Conductivity measurement of effluent.
Mainly measure inorganic ions and small
organic substances, including organic acids and
amines.
Conductivity detector measures electronic
resistance and measured value is directly
proportional to the concentration of ions
present in the solution.
Employed as a detector in an
ion chromatography.
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48. Working:
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49. 


Based on the principle of absorption of UV
visible light as the effluent from the column is
passed through a small flow cell placed in the
radiation beam.
High sensitivity (detection limit of about 1x10-9
g mL-1 for highly absorbing compounds).
Detector cells are generally 1 mm diameter
tubes with a 10 mm optical path length.
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50. Ultraviolet detector are of fallowing types:
1. Fixed-wavelength detector
2. Variable-wavelength detector
3. Photodiode-array detectors
Fixed-wavelength detector:
 Simplest UV absorption detector.
 Mercury lamp source, optical filters to select a
limited number of wavelengths 220, 250, 254,
280, 313, 334, 365, 436, and 546 nm.
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51. Variable-wavelength detector:
 Deuterium lamp (for UV region) or Tungsten
filament light source (for visible region) a
diffraction grating monochromator for
wavelength selection and a photomultiplier
detector.
 Allow monitoring at any wavelength within
the working range of the detector.
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52. Photodiode-array detectors:
 A photodiode array (PDA) is a linear array of
discrete photodiodes on an integrated circuit
(IC) chip.
 A photodiode is a type of photodetector
capable of converting light into either
current or voltage, depending upon the
mode of operation.
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53. Photodiode-array detectors:
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54. 




Based on filter-fluorimeters or spectrofluorimeters.
Flow cell has a capacity 10-25µL with a narrow
depth (1.07 mm) and large surface area
for excitation-emission collection.
The fluorescent radiation emitted by the sample is
usually measured at 90° to the incident beam.
Simplest detector: mercury excitation source, and
filters (one/more).
Advanced detector: xenon source and a grating
monochromator to isolate emitted fluorescent
radiation.
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57. 



The term 'electrochemical detector' in HPLC
normally refers to amperometric or coulometric
detectors.
Measure the current associated with the oxidation
or reduction of solutes.
Complete removal of oxygen is almost difficult,
therefore, electrochemical detection is normally
based upon the oxidation of the solute.
Amperometric detector is presently
considered to be the best
electrochemical detector.
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58. Working electrode:
 Commonly made of glassy carbon, is the electrode
at which the electro active solute species is
monitored.
Reference electrode:
 Usually a Ag-AgCl electrode, gives a stable,
reproducible voltage to which the potential of the
working electrode is referred.
Auxiliary electrode:
 Current-carrying electrode and usually made of
stainless steel.
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59. Auxiliary
Electrode
Potentiostat
Reference
Electrode
From Column
Working Electrode
To waste
Electro Chemical Detector
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60. Advantages:
 Very small internal cell-volume,
 High degree of sensitivity,
 More limited range of applications, and
 Excellent for trace analyses as UV-detector
lacks adequate sensitivity.
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61. Used for both qualitative and quantitative analyses
of environmental, pharmaceutical, industrial,
forensic, clinical, and consumer product samples.
A few typical examples:
 Isolation of natural pharmaceutically active
compounds
 Control of microbiological processes
 Assay of cephalosporins
 Assay of frusemide
 Assay of theophylline
 Assay of corticosteroids
 Assay of dichlorphenamide
 Assay of human insulin

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62. Isolation of natural pharmaceutically active
compounds
Category of Natural
Products
Constituents
Used as
Alkaloids
Morphine; Codeine
Analgesic, Antitussive
Glycoside
Digitalis glycosides
Sennosides
Cardiovascular
diseases, Laxatives
Chromatographic Conditions :
Column : Size-25 cm × 4.6 mm ID
Adsorbent : Lichrosorb RP-8
Mobile-phase : Water/Acetonitrile-Gradient Elution
Detector : UV 254 nm
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63. Control of microbiological processes:
 Determine kinetics of the microbiological
process
 Monitoring of the on-going process
 Isolation and purification of active ingredients
 Purity control of active constituents
 Monitoring derivatization reactions
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64. Controlled analysis of a microbiological
process during Penicillin Production
Chromatographic conditions:
Column : Size-25 cm × 4.6 mm ID
Adsorbent : Lichrosorb-NH2 (10 μm)
Mobile-phase : 0.005 M H2SO4
buffer (pH4.4)/acetonitrile (50:50)
Flow rate : 3 ml min-1
Detector : UV-220 nm

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65. Assay of Cephalosporins:
 Several commercially available cephalosporin
antibiotics have been adequately separated by
HPLC methods under the following
experimental parameters
Column : ODS-SIL-X-II
Mobile-phase : 0.95 M Ammonium
Carbonate/Methanol (95 : 5)
Detector : UV-220 nm
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66. Assay of Theophylline:

Theophylline contains other related substances as
impurities, namely : theobromine, caffeine and βhydroxypropyltheophylline
Chromatographic conditions:
Sample size : 10 μL
Column : size – 250 × 4.6 mm ID
Adsorbent : Lichrosorb (R) RP-8, 10 μm
Mobile-phase : 0.02 M KH2PO4 Buffer
(pH 3.5)/Acetonitrile (95 : 5)
Detector : UV-254 nm
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67. Some other applications:
Field
Typical Mixture
Pharmaceuticals
Antibiotics, Sedatives, Steroids
Biochemicals
Amino acids, Proteins,
Carbohydrates, Lipids
Food Products
Additives, Artificial Sweeteners,
Anti - oxidents
Polluants
Pesticides, Herbicides, PCBs
Forensic Chemistry
Drugs, Poisons,
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68. 1.
2.
3.
4.
5.
6.
7.
8.
Kar Ashutosh “Pharmaceutical Drug Analysis”, Revised Second Edition,
New Age Internal (P) Limited Publishers, page no. 452-474.
Jeffery G.H., Bassett J., Mendham J. , Denney R. C., “Vogel's Textbook Of
Quantitative Chemical Analysis”, Fifth edition 1989, Longman Scientific
& Technical, Page no. 220-229.
Patnaik Pradyot, ”Dean’s Analytical Chemistry Hand Book”, Second
Edition, McGRAW-HILL, Page no. 5.60-5.91.
Kealey D. , Haines P. J. , “Instant Notes Analytical Chemistry” , Frist
edition 2002, BIOS Scientific Publishers Limited, Page no. 155-173.
Harvey David, “Modern Analytical Chemistry”, McGraw-Hill Higher
Education, Page no. 578-589.
Lee David C. and Webb Michael L. , “Pharmaceutical Analysis” , Frist
Published 2003, Blackwell Publishing Ltd, Page no. 44-49.
Kamboj P.C. , “Pharmaceutical Analysis volume ɪɪ Instrumental Methods”
First Edition 2010, Vallabh Publication , Delhi, Page no. 239-280.
WWW.Google.co.in
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69. “Optimism is the faith that leads to
achievement, nothing can be done
without hope and confidence”
—- Helen Keller
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