Chromatography Instrumetation (1).pdf

Semester II
Course Code:Paper 7: Instrumental
Techniques (Physical, Chemical,
Biological)
Dr. Suchita Rawat
(MSc. MPhil PhD)
This Photo by Unknown Author is licensed under CC BY-SA

History of Chromatography
Mikhail Tswett
Chromatography

❑Stationary phase
❑Mobile phase
❑analyte
❑Elute
❑Elution

Based on physical shape
•Planar (paper, thin layer), column (packed, tubular)
Based on interaction of the solute with the stationary phase
1. Adsorption
2. Partition
3. Ion-exchange
4. Size exclusion
5. Affinity Chromatography
6. Reverse phase
Based on nature of mobile phase
•Gas Chromatography (argon, helium)
(Gas Liquid chromatography, Gas- solid chromatography)
Liquid Chromatography (liquid solvents)
(liquid-liquid chromatography/ Liquid solid chromatography)
Based on purpose of separation
•Analytical chromatography, preparative chromatography
Types of Chromatography techniques classification:

Historical background
❑ LC initial setup
❑ Pioneer work in development of High pressure liquid chromatography
❑ UHPLC (ultra high pressure liquid chromatography)
J. Calvin Giddings Horvath and Lipsky
Source :https://images.app.goo.gl/3jfkF18ZALxn95a28
Source: https://images.app.goo.gl/4X22WgzcHsDhHWDb9
H(P)LC

❑ Analyte separation
❑ Kinetics of distribution
This Photo by Unknown Author is licensed under CC BY-SA-NC

Stationary Phases in HPLC
Early
microparticles
irregularly shaped porous silica gel
or alumina of equivalent diameter ≤
10 μm.
high-purity
silica
particles
low in trace metal content, <10 μm,
even <2 μm diameter particles,
Faster separation
small molecules, polypeptides and
many proteins (60–150,200–300, and
1,000–4,000 ˚A)
Source : Horvai 2014

HPLC Column Stationary Phases (according to affinities)
Normal-
Phase
HPLC
(NP-
HPLC)
SP:Polar (silanol
(–Si–OH) groups
cyanopropyl- bonded endcapped
silica
aminopropyl-bonded silica
diol-bonded silica
MP:Non polar with modification of
polar
Interaction with SP: hydrophilic
Reversed-
Phase
HPLC
(RP-
HPLC)
SP: Non Polar
Hydrophilic silanol groups have been
reacted with hydrophobic alkyl
groups (C4,C8,C18)
MP:polar with modification of less
polar
Interaction with SP: hydrophobic
Source :Ham, B. M., & MaHam, A. (2015).

HPLC Column Stationary Phases (according to affinities)
Ion
Exchange
HPLC
(IEX-
HPLC)
SP: (types: cation exchange
chromatography (CEC) sulfate
derivatives and carboxylate
derivatives
anion exchange
chromatography (AEC)
MP: Buffers for CEC ( Buffer A/
Buffer B 1M Nacl pHs between 4
and 7)/ AEC (( Buffer A/ Buffer B
1M Nacl pHs between 7 and 10)
Interaction with SP: charge–
charge coulomb interaction
between

Source: Dr. Deepkumar Joshi Assistant Professor Chemistry department MNSC-Patan

Isocratic
Isocratic
system
HPLC
Gradient
Low pressure
Gradient HPLC/High
Pressure
Gradient HPLC
Number of reservoir tanks / mobile phase

Source: Dr.
Deepkumar Joshi
Assistant Professor
Chemistry
department
MNSC-Patan
Binary: 2 solvent
reservoirs
Ternary: 3 solvent
reservoirs
Quaternary: 4
solvent reservoirs

Pumping Systems
❑ Requirements for Pumps
❑ Types of pumps
Constant pressure
pump (Pneumatic
pumps)
Reciprocating
Pumps
Displacement
Pumps (syringe
type pump)
✓ the generation of pressures of up to
6000 psi
✓ pulse-free output
✓ flow rates ranging from 0.1 to 10
mL/min
✓ flow reproducibilities of 0.5% relative
or better,
✓ resistance to corrosion by a variety
of solvents.

❑ Working: In these pumps
pressure from the gas, cylinder is
delivered to a large piston which
drives the mobile phase. Gas is
used to create pressure. If the
piston in the backward stroke(2nd
nonreturn value closed) mobile
phase moves from the solvent
reservoir if the piston in the
forward stroke (1st nonreturn
value closed) mobile phase
moves to the column.
Constant pressure pump (Pneumatic pumps)

Displacement Pumps (syringe type pump)
❑ Working: working the same
as a constant pressure
pump.
❑ Pressure is driven by the
gear motor
❑ Produces good flow rate
independent of viscosity
and back pressure.
❑ However has a limited
solvent capacity of less
than 250 ml and is
considerably
inconvenient when
solvents must be
changed.

Reciprocating Pumps
❑ Working: Comprises of motor-driven
pistons immersed in a hydraulic
chamber filled forth with oil,
❑ The motor-driven piston moves back
into the hydraulic chamber due to
which pressure is created on the
flexible diaphragm. On a backward
stroke solvent gets sucked from the
solvent reservoir, and the outlet to the
column is closed. In the forward stroke,
the solvent moves to the column, and
the inlet from the solvent reservoir is
closed.
❑ Advantages: Most popular,
inexpensive and produce wide range of
flow rates.

https://www.youtube.com/watch?v=LJ5UJoN2s0s

Sample-Injection Systems (Microvolume sampling valve) Rheodyne
Injector

Columns for HPLC
• Composition (from smooth-bore stainless steel
tubing/heavy-walled glass tubing and polymer
tubing, such as polyetheretherketone (PEEK))
• Price (200=500 dollars)
• Types of columns (analytical vs. precolumn)
• Pre columns (scavenger columns , guard column )
• Column Temperature Control
• Types of Column Packings (pellicular and porous )

✓ Response
✓ Linear response to conc.
✓ Temp. independent Response
✓ Independent of eluent
composition (gradient HPLC)
✓ Tracing lower conc.
✓ HPLC peak should not be
widened
✓ Stable and reproducible signal
✓ Non destructive
Features of Detectors
used in HPLC
Detectors

TYPES OF DETECTORS
Bulk
Property
Detectors
1.Electrical
Conductivity HPLC
Detectors
2.Refractive Index
HPLC detectors
3.Electrochemical
HPLC Detectors
4.Light Scattering
HPLC Detectors
Solute
Property
Detectors
1.Ultraviolet/Visible
Detectors (Fixed Wave
Length Detectors/Variable
Wavelength
Detectors/Diode Array
Detectors)
2.Fluorescence
HPLC Detectors
(Single Wavelength
Excitation Fluorescence
Detector/Multi Wavelength
Fluorescence
Detector/Laser Induced
Fluorescence Detector
(LIFDs))
3.Mass
Spectrometric
HPLC Detectors
4.Infrared Detector
Bulk Property Detectors: Bulk property detectors are
those that measure the changes in solute and mobile
phase in combination. Such detectors show fluctuation
in readings even with slight change in mobile phase
combination. E.g refractive index and conductivity
detectors
Solute Property Detectors: Solute property detectors
are also called as selective detectors because they give
response for a particular physical or chemical property
of the analyte, being ideally independent of the mobile
phase.

Bulk Property Detectors
● Electrical Conductivity HPLC Detectors:
These detectors senses all the ions,
whether they are from a solute, or from
the mobile phase.
● It measures the conductivity of mobile
phase along with the solute which needs to
be backed-off by suitable electronic
adjustments. Thus it is a type of Electrical
Conductivity Detector.
● The measured electronic resistance is
directly proportional to the
concentration of ions present in the
solution
● Refractive Index HPLC detectors: They
are also one of the bulk property detectors
and are based on the change of the
refractive index of the eluent from the
column with respect to pure mobile
phase.
● They are mostly used for detection of non-
ionic compounds that neither fluoresce
nor absorb in the UV region.
● They face the drawback of being less
sensitive, need of temperature control
and less suitability to gradient elution

Bulk Property Detectors
● Electrochemical HPLC
Detectors: they usually measure
the current associated with the
oxidation or reduction of
solutes
● They are sensitive to changes
in the flow rate or composition
of the eluent and require a
working electrode, reference
electrode, and auxiliary
electrode
● Light Scattering HPLC Detectors: Light
scattering HPLC detectors are useful for
large molecular weight molecules like
surfactants, lipids and sugar
● . They are also called as Evaporative
light scattering detector because in this
the beam of light by particles of compound
remaining after evaporation of the mobile
phase.
● acts as universal detector and does not
require a compound to have a
chromophore for detection. They can be
used with gradient elution

Solute Property Detectors
● Ultraviolet/Visible Detectors: The most
common HPLC detectors used are UV
detectors because of the fact that most of
the compounds absorb in UV or visible
region.
● The basis of working for optical detectors
is the change in intensity when a beam of
electromagnetic radiation passes through
the detector flow cell.
● Fixed Wave Length Detectors: Such type
of detectors does not allow change in
wavelength of the radiation
● . Low pressure mercury lamp is used for
very intense light at 253.7nm or 254nm.
● Variable Wavelength Detectors:
Variable wavelength detectors can be
adjusted to work on any wavelength
over full UV- visible region.
● Diode Array Detectors: In diode array
detector, the sample is subjected to
light of all wavelengths generated
by the lamp at once.
● DAD helps to see the response of the
analyte at different wavelengths in
only single run and thus saves time
and energy

FORENSIC APPLICATION OF HPLC
Drugs Analysis
Sports Doping drugs
Toxicological analysis
Drug facilitated sexual assault
Alcohol analysis

Problem No. 1: No Peaks/Very Small Peaks
Problem Probable Cause
1.Detector lamp off.
2.Loose/broken wire
between detector and
integrator or recorder.
3.No mobile phase flow.
4.No Sample/deteriorated
sample/ wrong sample.

1.Pump off.
2.Flow
interrupted/obstructe
d.
3.Leak.
4.Air trapped in
pump head.
(Revealed by
pressure
fluctuations.)
Problem No. 2: No Flow

Problem No. 3: Variable Retention Times
1.Leak.
2.Change in mobile phase
composition. (Small changes can
lead to large changes in retention
times.)
3.Air trapped in pump.
(Retention times increase and
decrease at random times.)
4.Column temperature
fluctuations (especially evident
in ion exchange systems).

Problem No. 4: Loss of Resolution
1.Mobile phase
contaminated/deteriorated
(causing retention times and/or
selectivity to change).
2.Obstructed guard or analytical
column.

Problem No. 5: Split Peaks
1.Contamination on
guard or analytical
column inlet.
2.Partially blocked
frit.
3.Small (uneven)
void at column
inlet.
4.Sample solvent
incompatible with
mobile phase.

Problem No. 6: Peaks Tail on Initial and Later Injections
1.Sample reacting with
active sites.
2.Wrong mobile phase pH.
3.Wrong column type.
4.Small (uneven) void at
column inlet.

Problem No. 7: Tailing Peaks
1.Guard or analytical
column
contaminated/worn out.
2.Mobile phase
contaminated/deteriorated.
3.Interfering components
in sample.

Problem No. 8: Fronting Peaks
1.Column
overloaded.
2.Sample solvent
incompatible with
mobile phase.

Problem No. 11: Rounded Peaks
1.Detector operating
outside linear
dynamic range.
2.Column
overloaded.
3.Sample-column
interaction.

Problem No. 12: Broad Peaks
1.Mobile phase composition changed.
2.Mobile phase flow rate too low.
3.Leak (especially between column and detector).
4.Detector settings incorrect.
5.Extra-column effects:a. Column overloaded/ b. Detector response time or cell
volume too large./
c. Tubing between column and detector too long or I.D. too large.
d. Recorder response time toohigh.
6.Buffer concentration too low.
7.Guard column contaminated/worn out.
8.Column contaminated/worn out.

Problem No. 13: Negative Peak(s)
1.Refractive index of solute less
than that of mobile phase (RI
detector).
2.Sample solvent and mobile phase
differ greatly in composition
(vacancy peaks).
3.Mobile phase more absorptive
than sample components to UV
wavelength.

Problem No. 14: Ghost Peak
1.Contamination in injector or
column.
2.Late eluting peak (usually
broad) present in sample.

References
● Horvai, George. "Gary D. Christian, Purnendu (Sandy) Dasgupta and Kevin
Schug: Analytical chemistry." (2014): 5255-5256..
● Ham, B. M., & MaHam, A. (2015). Analytical chemistry: a chemist and
laboratory technician's toolkit. John Wiley & Sons.
● Sunil, A., Anju, G., & Rajat, V. (2018). HPLC Detectors, Their Types and Use:
A Review. Organic & Medicinal Chemistry International Journal, 6(5), 143-
146. (research article)
● Skoog, D. A., Holler, F. J., & Crouch, S. R. (2017). Principles of instrumental
analysis. Cengage learning.

Chromatography Instrumetation (1).pdf

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