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CHROMATOGRAPHY
Brief History and Definition
Liquid chromatography was defined in the early 1900s by the work
of the Russian botanist, Mikhail S. Tswett. His pioneering studies
focused on separating compounds [leaf pigments], extracted from
plants using a solvent, in a column packed with particles.
Tswett coined the name chromatography [from the Greek words
chroma, meaning color, and graph, meaning writingliterally,
colorwriting] to describe his colorful experiment. [Curiously, the
Russian name Tswett means color.] Today, liquid chromatography, in
its various forms, has become one of the most powerful tools in
analytical chemistry.
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Tswett filled an open glass column with particles. Two specific
materials that he found useful were powdered chalk [calcium
carbonate] and alumina. He poured his sample [solvent extract of
homogenized plant leaves] into the column and allowed it to pass
into the particle bed. This was followed by pure solvent. As the
sample passed down through the column by gravity, different colored
bands could be seen separating because some components were moving
faster than others. He related these separated, different-colored
bands to the different compounds that were originally contained in
the sample.
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Column Chromatography
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Separation TechniquesSeparation processes are used to decrease
the complexity of material mixtures. Chromatography and
electrophoresis are representative of this field.
Figure: Separation of black ink on a thin layer chromatography
plate.
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Chromatography (Encyclopedia Britannica):
Technique for separating the components, or solutes, of a
mixture on the basis of the relative amounts of each solute
distributed between a moving fluid stream, called the mobile phase,
and a contiguous stationary phase. The mobile phase may be either a
liquid or a gas, while the stationary phase is either a solid or a
liquid.
Chromatography(IUPAC Compendium of Chemical Terminology):
A physical method of separation in which the components to be
separated are distributed between two phases, one of which is
stationary (stationary phase) while the other (the mobile
phase)moves in a definite direction.
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CHROMATOGRAPHY
Chromatography basically involves the separation of mixtures due
to differences in the distribution coefficient (equilibrium
distribution) of sample components between 2 different phases.
One of these phases is a mobile phase and the other is a
stationary phase.
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Definition:
Different affinity of these 2 components to stationary phase
causes the separation.
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Distribution Coefficient (Equilibrium Distribution )
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Kinds of Chromatography
1. Liquid Column Chromatography
2. Gas Liquid Chromatography
3. Thin-Layer Chromatography
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Schematic Presentation of a Chromatogram
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Skoog and Leary: Principals of Instrumental Analysis, 4th ed.
Suanders, 1992
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The 4 basic liquid chromatography modes are named according to
the mechanism involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)
FOUR BASIC LIQUID CHROMATOGRAPHY
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Normal phase
In this column type, the retention is governed by the
interaction of the polar parts of the stationary phase and solute.
For retention to occur in normal phase, the packing must be more
polar than the mobile phase with respect to the sample
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Structure of silica gel
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Stationary Phase: Alumina
O
AlO O
AlO
OH
AlO
OH
AlO
OH
Al
OH
O
Acidic: -Al-OH
Neutral: -Al-OH + -Al-O-
Basic: -Al-O-
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LIQUID SOLID CHROMATOGRAPHY
30 Si - O - H
+
Normal phase LS Reverse phase LS
Silica Gel
The separation mechanism in LSC is based on the competition of
the components of the mixture sample for the active sites on an
absorbent such as Silica Gel.
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LIQUID SOLID CHROMATOGRAPHY
Si - OH
HEXANE
OH
C-CH3
CH3
CH3- CCH3
CH3
OH
OH
CH3
CH3
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Reverse phase
In this column the packing material is relatively nonpolar and
the solvent is polar with respect to the sample. Retention is the
result of the interaction of the nonpolar components of the solutes
and the nonpolar stationary phase. Typical stationary phases are
nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such
as C18, C8, etc.) and the solvents are polar aqueous-organic
mixtures such as methanol-water or acetonitrile-water.
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LIQUID-LIQUID CHROMATOGRAPHY
ODPN(oxydipropionylnitrile)
Normal Phase LLC Reverse Phase LLC
NCCH3 CH2 OCH2 CH2 CN(Normal)CH3 (CH2 )16 CH3 (Reverse)
The stationary solid surface is coated with a 2nd liquid (the
Stationary Phase) which is immiscible in the solvent (Mobile)
phase.
Partitioning of the sample between 2 phases delays or retains
some components more than others to effect separation.
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Reverse phase chromatographySilica is alkylated with long chain
hydrocarbon groups, using 18carbons long. This is usually referred
to as C-18 silica.
O
Si
O
O
SiO
OO
O
Si
OO
O
Si
OO
O
SiO O
O
Si
OO
Si
OO
Si
OO
SiO O
OSi
OO
Si
OO
SiO O
O
CH2
CH3
17Si
CH3
CH3
CH2
CH3
17Si
CH3
CH3SiCH3)3
SiCH3)3SiCH3)3
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Size exclusion
In size exclusion the HPLC column is consisted of substances
which have controlled pore sizes and is able to be filtered in an
ordinarily phase according to its molecular size. Small molecules
penetrate into the pores within the packing while larger molecules
only partially penetrate the pores. The large molecules elute
before the smaller molecules.
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Gel-Permeation Chromatography is a mechanical sorting of
molecules based on the size of the molecules in solution. Small
molecules are able to permeate more pores and are, therefore,
retained longer than large molecules.
GEL-PERMEATION CHROMATOGRAPHY
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Ion exchange
In this column type the sample components are separated based
upon attractive ionic forces between molecules carrying charged
groups of opposite charge to those charges on the stationary phase.
Separations are made between a polar mobile liquid, usually water
containing salts or small amounts of alcohols, and a stationary
phase containing either acidic or basic fixed sites.
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MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS
SO3-
SO3-
Na+
COO-
H 3N+
Na+
COOHH 3N
+
pH2
pH4.5
Ion-exchange Resin
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Mechanism of separation in
different forms of HPLC
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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(HPLC)
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HP
igherformance
LiquidChromatography
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HP
ighressure
LiquidChromatography
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HP
ighriced
LiquidChromatography
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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
High Performance Liquid Chromatography (HPLC) is one of the most
widely used techniques for identification, quantification and
purification of mixtures of organic compounds.
In HPLC, as in all chromatographic methods, components of a
mixture are partitioned between an adsorbent (the stationary phase)
and a solvent (the mobile phase).
The stationary phase is made up of very small particles
contained in a steel column. Due to the small particle size (3-5
um), pressure is required to force the mobile phase through the
stationary phase.
There are a wide variety of stationary phases available for
HPLC. In all labs can be used a normal phase (Silica gel), although
reverse phase (silica gel in which a 18 carbon hydrocarbon is
covalently bound to the surface of the silica) columns are
currently one of the most commonly used HPLC stationary phases.
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HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution. HPLC instruments consist
of a reservoir of mobile phase, a pump, an injector, a separation
column, and a detector.
Compounds are separated by injecting a sample mixture onto the
column. The different component in the mixture pass through the
column at differentiates due to differences in their partition
behavior between the mobile phase and the stationary phase. The
mobile phase must be degassed to eliminate the formation of air
bubbles.
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http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
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General Schematic of LC
Source: Skoog, Holler, and Nieman, Principles of Instrumental
Analysis, 5th edition, Saunders College Publishing.
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Chromatography: HPLCHewlettHewlett--PackardPackard
Series 1100 HPLCSeries 1100 HPLC
solventsolvent
pumppump
injectorinjector
columncolumn
detectordetector
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Chromatography: HPLCHPLC ColumnHPLC Column
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Chromatography: HPLCHPLC PumpHPLC Pump
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Chromatography: HPLCHPLC Autosampler and InjectorHPLC
Autosampler and Injector
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Chromatography: HPLCHPLC DetectorHPLC Detector
UV/Visible SpectrophotometerUV/Visible Spectrophotometer
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Chromatography: HPLCHPLC Waste CollectionHPLC Waste
Collection
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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
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http://www.labhut.com/education/flash/introduction07.php
TLC vs High Performance Liquid Chromatography (HPLC)HPLC
Optimization
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Modes of HPLCAdsorption Chromatography is another name for
liquid-solid chromatography.
The solid phase is usually silica or alumina, which have highly
polar surfaces. The mobile phases are commonly some of the less
polar solvents.
Normal-Phase Chromatography is based on a polar liquid phase
coated or bonded onto a silica support. For historical reasons,
this is called normal phase because the stationary phase is polar
and the mobile phase is typically a nonpolar solvent such as hexane
or isopropylether. In normal phase, the least polar component is
eluted first.
Reversed-Phase Chromatography uses a non-polar stationary phase
(also coated or bonded onto silica or another support) and polar
solvents such as water, acetonitrile or methanol. In reversed-phase
chromatography, the most polar component is eluted first.
Ion-Pair Chromatography is one of the methods used to separate
ions. It uses regular reversed phase columns, and can separate
acids, bases and neutral compounds during the same chromatographic
run.
Ion-Exchange Chromatography is based on the use of ion-exchange
resins as the stationary phase.
Ion Chromatography was developed to separate the ions of strong
acids and bases. The equipment used is different from that of
ion-exchange chromatography.
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CH3|
-0-Si- CH3|CH3
+ HClSilica
CH3|
-Si- CH3|CH3
OH + ClSilica
Could be many differentfunctional groups here
Bonding Phases onto the Silica Support
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Usually half or more of the silanol groups remain unreacted
after bonding with C18. One method used to reduce the effects of
these residual silanol groups is a process called endcapping.
End Capping
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End Capping
After bonding with C18 use small silane molecules such as
trimethylchlorosilane to react some of the remaining OH groups
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CH3|CH2|
-0-Si- CH3|CH2|CH3
Silica
Steric Protection
Hydrolysis may degrade the column by breaking off the bonded
phase.
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Hypersil HyPURITY C18Column: Symmetry C18
Sample: 1. Uracil2. Pyridine 3. Phenol4. Dimethyl phthalate5.
N,N-Dimethylaniline6. 4-Butylbenzoic acid7. Toluene
Mobile Phase: 60% CH3CN40% 50mM KH2PO4, pH 3.2
Comparison of Different C18 Columns
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Common RP Packings
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Other Bonded Phases
Phenyl phases show weak dipole - induced dipole interactions
with polar analytes. Usually this type of bonded phase is used for
separating closely related groups of molecules.
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Other Bonded Phases
The amino-phase is the most polar, and it can also act as weak
anion-exchanger, (protonated at low pH). Amino columns are mainly
used in normal-phase mode, specially for selectiveretention of
aromatic compounds.
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Other Bonded Phases
Diol columns are slightly polar and are used for normal-phase
separations. Diols are useful for samples containing many compounds
with different polarities , and which usually have strong retention
on unmodified silica.
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Non-Silica SupportsThe most common polymer support material for
reversed-phase separation is made of divinylbenzene cross-linked
polystyrene
The main advantage of porous polymers is that they can be used
in the pH range from 1 to 13. (silica supports tend to dissolve at
pH greater than 9).
Because of the surface acidity of silica supports, polymer
supports can be a better choice for separating basic compounds.
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All C18 Columns Are Not Created Equal
Differences in:
Particle SizePore SizeSurface AreaCarbon LoadEnd-CappingSilica
Type*Bonding Density
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RP Column Properties
Hydrophobic Surface Particle Size and Shape Particle Size
Distribution Porosity, Pore Size and Surface Area
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Particle Size
Columns have a distribution of particle sizes
Reported particle diameter is an average Broader distribution
---> broader peaks
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RP Mechanism (Simple)
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Reversed Phase Mechanisms
Classical measures of retention retention factors partition
coefficients Vant Hoff Plots
Give bulk properties only - do not give molecular view of
separation process
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Proposed RP Mechanisms
Hydrophobic Theory Partition Theory Adsorption Theory
See Journal of Chromatography, volume 656.
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Hydrophobic Theory
Chromatography of cavities in solvent created by hydrophobic
portion of analyte molecule
Surface Tension Interaction of polar functions with solvent
Stationary phase is passive
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Partition Theory
Analyte distributes between aqueous mobile phase and organic
stationary phase
Correlation between log P and retention organic phase is
attached on one end Does not explain shape selectivity effects
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Adsorption Theory
Analytes land on surface - do not penetrate
Non-polar interactions between analyte hydrophobic portion and
bonded phase
Weak interactions dipole-dipole dipole-induced dipole induced
dipole-induced dipole
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None of these can completely explain all of theobserved
retention in reversed phase HPLC
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Important Reversed Phase Parameters
Solvent (mobile phase ) Strength Choice of Solvent Mobile Phase
pH Silanol Activity
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HPLC Solvents Mobile Phase
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LC Mobile Phase Qualities
High purityReasonable cost (and disposal)Boiling point 20-50 C
above column temperatureLow viscosityLow reactivityImmiscibile with
stationary phaseCompatible with detectorSafety limited flammability
and toxicity
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SOLVENTS
Polar Solvents
Water > Methanol > Acetonitrile > Ethanol >
Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane > Cyclohexane
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HPLC Solvents Properties
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HPLC Solvents Groups
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LC Mobile Phase Selection
k of 2-5 for two or three component mixturek of 0.5-20 for
multicomponent mixture
Match analyte polarity to stationary phase polarityMobile phase
of different polarity
Normal Phase:nonpolar solvent, polar stationary phaseleast polar
component elutes firstincreasing mobile phase polarity decreases
elution time
Reversed Phase:polar solvent (water, MeOH, ACN), nonpolar
stationary phasemost polar component elutes firstincreasing mobile
phase polarity increases elution timemost widely used
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LC Pumping Systems
General Requirements:Generate pressures up to 6000 psiPulse-free
outputFlow rates from 0.1-10 mL/min0.5% or better flow control
reproducibilityCorrosion resistant
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LC Pumping SystemsReciprocating Pumps Pulsed flow must be damped
Small internal volume High output pressures Adaptable for gradient
elution Constant flow rates independent of column back-pressure or
solvent viscosity
Displacement Pumps Flow independent of viscosity and
back-pressure Limited solvent capacity Inconvenient to change
solvents
Pneumatic Pumps Inexpensive Pulse free Limited capacity and
pressure Dependent on solvent viscosity and backpressure Not good
for gradient elution
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HPLC Pump Head
Piston HPLC Pump Head
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Gradient HPLC
High Pressure
Low Pressure
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HPLC Chromatograph injectors
The function of the injector is to place the sample into the
high-pressure flow in as narrow volume as possible so that the
sample enters the column as a homogeneous, low-volume plug. To
minimize spreading of the injected volume during transport to the
column, the shortest possible length of tubing should be used from
the injector to the column.
When an injection is started, an air actuator rotates the
valve:solvent goes directly to the column; and the injector needle
is connected to the syringe. The air pressure lifts the needle and
the vial is moved into position beneath the needle. Then, the
needle is lowered to the vial.
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HPLC columns
The column is one of the most important components of the HPLC
chromatograph because the separation of the sample components is
achieved when those components pass through the column. The high
performance liquid chromatography apparatus is made out of
stainless steel tubes with a diameter of 3 to 5mm and a length
ranging from 10 to 30 cm.
Normally, columns are filled with silica gel because its
particle shape, surface properties, and pore structure help to get
a good separation. Silica is wetted by nearly every potential
mobile phase, is inert to most compounds and has a high surface
activity which can be modified easily with water and other agents.
Silica can be used to separate a wide variety of chemical
compounds, and its chromatographic behavior is generally
predictable and reproducible.
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Skoog and Leary: Principals of Instrumental Analysis,5th ed.
Suanders, 1998
Reverse Phase HPLC
-
Al
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c
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C
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a
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-
0
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-
Alltech Chromatography Sourcebook, 2004-04 catalog
-
xa
b
c
a b c
c b a
0
0
Time
Time
Normal Phase (SiO2)
Reverse Phase (C18)
Normal Phase (SiO2) TLC
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(TLC vs Normal Phase and
Reverse Phase HPLC)
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RP-HPLC Stationary Phase
Skoog and Leary: Principals of Instrumental Analysis, 5th ed.
Suanders, 1998
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http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
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Characteristics of PerformanceThe performance criteria affecting
quality of the result include:
Accuracy Precision (repeatability and reproducibility)
Sensitivity (LOD and LOQ) Selectivity Linearity Dynamic range
Stability
The performance criteria for the economics include:
Cost of purchase, installation and maintenance Analysis time
Safety aspects Running costs supplies, gases, consumables Training
Sample throughput
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HPLC Detectors No universal or versatile detector ...?!
Types
General respond to mobil phase bulk properties which vary in the
presence of solutes (e.g. refractive index)
Specific respond to some properties of the solute (not possessed
by the mobil phase (e.g. UV adsorption)
Hyphenated detector LC-MS
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Common LC Detectors
Bulk property detectors
Refractive index detector Conductivity detector Light scattering
detector.
Analyte property detectors
9 UV detector9 Fluorescence detector9 Amperometric detectorMass
spectrometric detectorCombination detectors
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Refractive Index Detector1. The RI detector is one of the few
universal detector available in LC 2. Principle:
The RI detectors measure a bulk property of the mobile phase
leaving the column: its ability to refract to bend light (i.e., its
refractive index). This property changes as the composition of the
mobile phase changes, such as when solutes from the column. By
detecting this change, the presence of solutes can be detected.
3. Detector Design:
i. One of simplest of RI detectors is the deflection RI
detector
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ii. In this detector, light is created by a source and passed
through flow-cells containing mobile phase eluting from the column
(sample stream) and a reference stream (usually mobile phase with
no solute in it). The light passing through these flow/cells is
passed through a second time using a mirror and passed to a
detector where its intensity is measured.
iii. When the refractive index of liquid in the sample and
reference flow-cell are the same, little or no bending of light
occurs at the interface between the low-cells. This allows the
largest amount of light possible to reach the detector.
iv. As solute elute from the column, the refractive index of the
liquid in the sample flow-cell will be different that that in the
reference flow-cell and light will be bent as it passes between
them. This changes the amount of light reaching the detector,
producing a response.
-
4. Applications:
RI detector are universal applicable to the detection of any
solute in LC. This makes them useful in preliminary work in LC
where the nature or properties of a compound may not be known yet.
They also the detector of choice for work with carbonhydrates or in
the separation of polymer by size-exclusion chromatography.
Some disadvantages: (1) they do not have very good limits of
detection, (2) they can not used with gradient elution, where the
composition of the mobile phase is changing with time. (3) The
temperature of the system must also be controlled to avoid baseline
fluctuations with these detectors.
5. SensitivityThe response of a RI detector is approximately the
same for all compounds.
6. Limit of Detection: 10-5 to 10-6 M
7. Linearity/ Dynamic Range: The response of a RI is usually
linear over a 104-fold range in concentration.
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Refractive Index Detector
Plus:1) Measures a bulk property2) Nearly Universal (different
RI than mobile phase)3) Comparable response for different
analytes4) Detects species with no chromophores
Minus:1) Temperature dependent2) Poor sensitivity (LOD 100 ng)3)
No gradient elution
HPLC Bulk Property Detectors
-
2. Principle:
i. This detector measures the ability of a solution to conduct a
current when placed in an electrical field. This ability depends on
the number of ions or ionic compounds present in the solution.
ii. The relationship between the current, electric field and
conductivity of the solution is shown as follows:
I = C E
I = CurrentC = conductivityE = electric field strength
Conductivity Detector1. A conductivity detector is an example of
a universal detector for ionic
compound.
-
3. Detector Design
4. Applications: for any compound that is ionic or weakly ionic.
It is widely used in ion chromatography.
5. Sensitivity:The response of a conductivity detector depends
on the charge and size
of the compound of interest. Small, highly charged compounds
tend to produce larger response that large, less charged
compound.
6. Limit of Detection: 10-6 M7. Linearity/Dynamic range:
104-fold
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Plus:1) Measures a bulk property2) Nearly Universal (must be
ionic)3) Common for Ion Exchange LC4) Detects species with no
chromophores5) Simple, robust
Minus:1) Fair sensitivity (LOD 1 ng)2) No salts or buffers in
mobile phase3) Gradients a challenge
HPLC Bulk Property Detectors
Conductivity Detector
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Evaporative Light Scattering Detector
-
Plus:1) Measures a bulk property2) Nearly Universal (must be
non-volatile)3) Does not detect liquids (gradients are ok)4)
Detects species with no chromophores
Minus:1) Signal not linear with concentration2) Fair sensitivity
(LOD 1 ng)3) No salts or buffers in mobile phase
Evaporative Light Scattering Detector
HPLC Bulk Property Detectors
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Absorbance Detector (UV/Vis)1. The absorbance detector is the
most common type of detector in LC.
2. Principle:
Absorbance detector measures the ability of solutes to absorb
light at a particular wavelength range. This absorbance is
described by the Beer-Lambert Law.
A = l cwhere: A = Absorbance of light at a given wavelength
= Molar absorption coefficient of the solutel = path length of
the flow-cellc = concentration of solute
-
3. Detector design:i. There are three types of UV-Vis absorbance
detector: fixed wavelength detectors, variable and diode array
detector. They are generally based on the following type of
design:
ii. In a fixed wavelength detector, absorbance of only one given
wavelength is monitored by the system at all time. The wavelength
is usually 254 nm.
A fixed wavelength detector is the simplest and cheapest of
types of detector, but is limited in terms of it flexibility and
the types of compounds it can used to monitor.
-
iii. In a variable wavelength detector, a single wavelength is
monitoredat any given time, but any wavelength in a wide spectral
range can be selected.
The wavelengths that can be monitored can vary from 190 nm to
900nm. The ability to use one instrument for more than one
wavelength is achieved by adding in more advanced optics to the
system.
Diode Array Detector
-
HPLC UV/VIS Spectrophotometer
-
5. Sensitivity:
The response of an absorbance detector depends on the molar
absorption coefficient. The larger this value is, the larger the
response of the detector
6. Limit of detector: 10-8 M
7. Linearity/Dynamic range: 105-fold range
4. Applications:
Absorbance detector can be used to detect any compound absorbing
at the wavelength monitored. Absorbance detector can be sued with
gradient elution.
-
HPLC Specific Property Detectors
Multi-wavelength UV-Vis Absorption Detector
Plus:1) Measures a specific property2) Nearly Universal (must
have chromophore)3) Most common of all detectors (~75%)4) Potential
to provide qualitative info.5) Simple, robust
Minus:1) Fair sensitivity (LOD 1 ng)2) Expensive with PDA,
limited with Hg Lamp3) Misses some important analytes
-
F = I (1-e- l c) = I l c (at low concentration)
F = Fluorescence intensityI = intensity of the excitation light
= Fluorescence quantum yield = Molar absorption coefficient of the
solutel = path length of the flow-cellc = concentration of
solute
2. Principle:
Fluorescence Detector
1. A fluorescence detector is an example of a selective
detector, with limits of detection smaller than those by either RI
or absorbance monitors.
-
4. Applications:
It can be used to detect any compound absorbing and emitting
lightat the given excitation and emission wavelength.
3. Detector design
-
5. Sensitivity:
F = I (1- e- l c) = I l c (at low concentration)
6. Limit of detection: 10-10 M
7. Linearity/Dynamic Range: 103 to 104-fold
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HPLC Specific Property Detectors
Plus:1) Measures a specific property2) Highly Selective (must
fluoresce)3) Second most common of all detectors (~15%)4) High
sensitivity (LOD 0.01 ng)5) Can interrogate very small volumes
Minus:
1) Not Universal2) Limited Applications
Fluorescence Detector
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Electrochemical detector1. It can be used to detect an compound
which can undergo an electrochemical reaction2. Principle:i. This
detector measure the ability of a solute to undergo either
oxidation
(i.e., loss of electrons) or reduction (i.e. gain of
electrons)
Oxidation: A A+ + e-
Reduction: A + e- A-
ii. One way in which such a reaction can be monitored is by
measuring the change in current under a constant electric field.
Another way is to measure the change in the electric field produced
when a constant current is present.
3. Detector Design
-
4. Applications:
Electrochemical detectors can be used to detect any solute that
can undergo oxidation or reduction.
Detection by reduction: aldehydes, ketones, nitriles, conjugated
acids
Detection by oxidation: phenols, peroxides, purines, diols
5. Sensitivity: It depends on the extent of oxidation or
reduction that occurs at given potential of the electrode.
6. Limit of detection: 10-11 M
7. Linearity/Dynamic Range: 106-fold
8. Disadvantages: destructive detector
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HPLC Specific Property Detectors
Plus:1) Measures a specific property2) Highly Selective (depends
on reduction potential)3) High sensitivity (LOD 0.01 ng)
Minus:
1) Not Universal2) Must have electrolyte in mobile phase3)
Mobile phase must be aqueous4) Gradients not possible
Electrochemical Detector
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MassMass spectrometryspectrometryMass spectrometry probably is
the most versatile and comprehensive analytical technique currently
used by chemists and biochemists.
It measures the masses of individual molecules, fragments of
molecules and atoms.
It provides ultrahigh detection sensitivity requiring only a few
picomoles of a compound to obtain characteristic information
regarding the structure and the molecular weight of a compound.
In all cases, energy is transferred to the compound molecules to
effect ionization, causing the formation of the molecular ion of
the compound.
The molecular ion fragments into a variety of fragment ions and
the resulting fragmentation pattern constitutes the mass
spectrum.
The mass spectrum of each compound is unique and can be used as
chemical fingerprint together with its retention time to
characterize the compound.
-
The first essential step in mass spectrometry analysis to
convert the analyte molecules by ionization into gas phase ionic
species.
The excess energy transferred to the molecules leads
fragmentation.A mass analyzer separates the molecular ion and
fragment ions according to their mass/charge (m/z) ratio.
Data are recorded and then converted into a mass spectrum.These
steps are carried out under high vacuum [10-2 10-6 Pascal
(Pa)].
(1 Pa = 0.0075 torr; 1 atm = 1.013 x 105 Pa)
General concepts General concepts ofof mass mass
spectrometryspectrometry
-
Basic components of a mass spectrometerBasic components of a
mass spectrometer
GC or LC
Ionization methods
1. Electron-impact ionization (GC/MS)
2. Chemical ionization (GC/MS)
3. Atmospheric pressure electrospray ionization (LC/MS)
-
Skoog and Leary: Principals of Instrumental Analysis, 4th ed.
Suanders, 1992
-
van Deemter Equation
H = A + B/u +Cu
HPLC HPLC -- Column EfficiencyColumn Efficiency
Skoog and Leary: Principals of Instrumental Analysis, 5th ed.
Suanders, 1998
-
HPLC HPLC -- Column EfficiencyColumn Efficiency
H = H = AA + B/+ B/uu + C+ Cuu
A = 2A = 2ddpp
1.1. depends on particle size distribution, the depends on
particle size distribution, the narrower the distribution the
smaller the l narrower the distribution the smaller the l
2.2. ddpp = particle size= particle size3.3. Independent of
mobile phase flow rateIndependent of mobile phase flow rate4.4.
Also known as eddy diffusionAlso known as eddy diffusion
Skoog and Leary: Principals of Instrumental Analysis, 5th ed.
Suanders, 1998
-
HPLC HPLC -- Column EfficiencyColumn Efficiencyparticle
sizeparticle size
Skoog and Leary: Principals of Instrumental Analysis, 5th ed.
Suanders, 1998
-
HPLC Column EfficiencyHPLC Column Efficiency
Longitudinal Diffusion (B)Longitudinal Diffusion (B)
H = A + H = A + B/B/uu + C+ Cuu
B/u = 2B/u = 2DDMM/u/u
1. = constant depending on quality of packing
2. DM is the mobile phase diffusion coefficient
3. Inversely related to mobile phase flow rate
-
HPLC Column EfficiencyHPLC Column Efficiency
Mass Transfer Mass Transfer (Cs + Cm)
H = A + B/u + (Cs + Cm)u
CS = fS(k)df2 / DS
CM = fM(k)dp2 / DM
DDMM is the mobile phase diffusion is the mobile phase diffusion
coefficientcoefficient
DDSS is the stationary phase is the stationary phase diffusion
coefficientdiffusion coefficient
ddff is film thicknessis film thickness ddpp is particle sizeis
particle size Directly related to mobile phase Directly related to
mobile phase
flow rateflow rate
Skoog and Leary: Principals of Instrumental Analysis, 5th ed.
Suanders, 1998
-
Flow Chart I: Small Molecules1. m. wt.
2. solubility
-
1. m. wt.
2. solubility
Flow Chart II: Large Molecules
-
Uses of HPLC This technique is used for chemistry and
biochemistry research
analyzing complex mixtures, purifying chemical compounds,
developing processes for synthesizing chemical compounds, isolating
natural products, or predicting physical properties. It is also
used in quality control to ensure the purity of raw materials, to
control and improve process yields, to quantify assays of final
products, or to evaluate product stability and monitor
degradation.
In addition, it is used for analyzing air and water pollutants,
for monitoring materials that may jeopardize occupational safety or
health, and for monitoring pesticide levels in the environment.
Federal and state regulatory agencies use HPLC to survey food and
drug products, for identifying confiscated narcotics or to check
for adherence to label claims.
-
Area of application:
Separation and purification of substances and Analysis
Chemistry Biomedical and Clinical Pharmaceutics Agriculture and
Food Enviromental Veterinary
-
RP-HPLC - Example
Alltech Chromatography Sourcebook, 2004-04 catalog
-
RP-HPLC - Example
Alltech Chromatography Sourcebook, 2004-04 catalog
-
RP-HPLC Gradient Elution
Alltech Chromatography Sourcebook, 2004-04 catalog
-
Chromatogram of Organic Compounds from Fermented Cabbage
-
Chromatogram of Orange Juice Compounds
-
Liquid chromatograph/mass spectrometerLiquid chromatograph/mass
spectrometer
MassSpectrometer
MassSpectrometer
LiquidChromatograph
LiquidChromatograph
Rough pumpRough pump
UVDetector
UVDetector
SamplerInjection port
SamplerInjection port
ColumnColumn
SolventsSolvents
Ion sourceIon source
PumpsPumps InterfaceInterface
ComputerComputer
Separation TechniquesNormal phase Stationary Phase:
AluminaReverse phase Reverse phase chromatographySize exclusion
GEL-PERMEATION CHROMATOGRAPHY Ion exchange MECHANISM OF
ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS Mechanism of separation
in different forms of HPLCHHHCommon RP PackingsRP Column
PropertiesParticle SizeRP Mechanism (Simple)Reversed Phase
MechanismsProposed RP MechanismsHydrophobic TheoryPartition
TheoryAdsorption TheoryImportant Reversed Phase ParametersHPLC
Solvents Mobile PhaseHPLC Solvents PropertiesHPLC Solvents
GroupsHPLC Pump HeadGradient HPLCHPLC Chromatograph injectors HPLC
columnsCharacteristics of PerformanceHPLC DetectorsHPLC UV/VIS
SpectrophotometerFlow Chart I: Small MoleculesFlow Chart II: Large
MoleculesUses of HPLC