Curs HPLC
Curs HPLC
Curs HPLC
Column Chromatography
Separation Techniques
Separation processes are used to decrease the complexity of material
mixtures. Chromatography and electrophoresis are representative of this
field.
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.
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.
Kinds of Chromatography
3. Thin-Layer Chromatography
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
OH
OH
OH
OH
Al
Al
Al
Al
Al
Acidic: -Al-OH
Neutral: -Al-OH + -Al-OBasic: -Al-O-
Normal phase LS
Reverse phase LS
Si - O - H
Silica Gel
HEXANE
Si - OH
CH3
OH CH
3
C-CH3
CH3
CH3- C
CH3
CH3
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.
MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS
pH2
SO3
Na
H3N
COOH
Ion-exchange Resin
SO3
H3N
Na
+
-
COO
pH4.5
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.
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.
LIQUID-LIQUID CHROMATOGRAPHY
ODPN(oxydipropionylnitrile)
Normal Phase LLC
Reverse Phase LLC
NCCH3CH2OCH2CH2CN(Normal)
CH3(CH2)16CH3 (Reverse)
Mechanism of
separation in
different
forms of
HPLC
High
Performance
L iquid
C hromatography
High
Pressure
L iquid
C hromatography
High
Priced
L iquid
C hromatography
http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html
General Schematic of LC
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
Chromatography: HPLC
Hewlett-Packard
Series 1100 HPLC
solvent
pump
injector
column
detector
Chromatography: HPLC
HPLC Column
Chromatography: HPLC
HPLC Pump
Chromatography: HPLC
HPLC Autosampler and Injector
Chromatography: HPLC
HPLC Detector
UV/Visible Spectrophotometer
Chromatography: HPLC
HPLC Waste Collection
http://www.labhut.com/education/flash/introduction07.php
Modes of HPLC
Silica
Silica
CH3
|
-0-Si- CH3
|
CH3
OH
Cl
CH3
|
-Si- CH3
|
CH3
HCl
Could be many different
functional groups here
Common RP Packings
End Capping
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
After bonding with C18 use small silane
molecules such as trimethylchlorosilane to
react some of the remaining OH groups
Steric Protection
CH3
|
Silica
Hydrolysis may degrade the
column by breaking off the
bonded phase.
CH2
|
-0-Si- CH3
|
CH2
|
CH3
Sample:
Mobile Phase:
60% CH3CN
40% 50mM KH2PO4, pH
3.2
1. Uracil
2. Pyridine
3. Phenol
4. Dimethyl phthalate
5. N,N-Dimethylaniline
6. 4-Butylbenzoic acid
7. Toluene
Non-Silica Supports
The most common polymer support material for reversed-phase
separation is made of divinylbenzene cross-linked polystyrene
RP Column Properties
Hydrophobic Surface
Particle Size and Shape
Particle Size Distribution
Porosity, Pore Size and Surface Area
Particle Size
Columns have a distribution of particle
sizes
Reported particle diameter is an average
Broader distribution ---> broader peaks
RP Mechanism (Simple)
Proposed RP Mechanisms
Hydrophobic Theory
Partition Theory
Adsorption Theory
See Journal of Chromatography, volume 656.
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
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
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
HPLC Solvents
Mobile Phase
Water is a solvent for polar molecules and the most common solvent used by living things; all the
ions and proteins in a cell are dissolved in water within a cell. Solvents find various applications
in chemical,pharmaceutical, oil and gas industries, including in chemical syntheses and
purification processes.
CLASIFICAREA SOLVENILOR
Solveni : - disociani
- nedisociani (indifereni)
Solveni : - polari (apa > metanol > acetonitril > etanol > )
- nepolari (n-decan > n-hexane > n-pentane > toluen, )
Solveni : - protici: - protogeni (HF, HCN, )
- protofili (apa, amoniac, piridin, )
- amfiprotici (apa, alcooli, )
- aprotici (ineri) (benzene, tetraclorur de carbon, )
Reversed Phase:
polar solvent (water, MeOH, ACN), nonpolar stationary phase
most polar component elutes first
increasing mobile phase polarity increases elution time
most widely used
a
a
c
Time
b
Reverse Phase (C18)
c
c
x
0
b
Time
LC Pumping Systems
Reciprocating Pumps
Displacement Pumps
Pneumatic Pumps
Inexpensive
Pulse free
Limited capacity and pressure
Dependent on solvent viscosity and backpressure
Not good for gradient elution
H = A + B/u +Cu
Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998
H = A + B/u + Cu
B/u = 2DM/u
= constant depending
on quality of packing
2.
3.
http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html
HPLC Detectors
No universal or versatile detector ...?!
Types
General respond to mobile 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 mobile phase (e.g. UV
adsorption)
Hyphenated detector LC-MS
Characteristics of Performance
The performance criteria affecting quality of the result include:
Accuracy
Precision (repeatability and reproducibility)
Sensitivity (LOD and LOQ)
Selectivity
Linearity
Dynamic range
Stability
Common LC Detectors
Bulk property detectors
Refractive index detector
Conductivity detector
Light scattering detector.
Analyte property detectors
UV detector
Fluorescence detector
Amperometric detector
Mass spectrometric detector
Combination detectors
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 detectors 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 sizeexclusion 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. Sensitivity
The 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 104fold range in concentration.
Conductivity Detector
1. A conductivity detector is an example of a universal detector for ionic
compound.
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=CE
I = Current
C = conductivity
E = electric field strength
3. Detector Design
A = l c
where: A = Absorbance of light at a given wavelength
Molar absorption coefficient of the solute
l = path length of the flow-cell
c = 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:
Diode Array
Detector
4. Applications:
Absorbance detector can be used to detect any compound absorbing at the
wavelength monitored. Absorbance detector can be sued with gradient elution.
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
Fluorescence Detector
1.
2. Principle:
F = I (1-e- l c) = I l c (at low concentration)
F = Fluorescence intensity
I = intensity of the excitation light
= Fluorescence quantum yield
Molar absorption coefficient of the
solute
l = path length of the flow-cell
c = concentration of solute
3. Detector design
4. Applications:
It can be used to detect any compound absorbing and emitting light
at the given excitation and emission wavelength.
5. Sensitivity:
Electrochemical detector
1. It can be used to detect an compound which can undergo an
electrochemical reaction
2. 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+ + eReduction: A + e- Aii. 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.
Mass spectrometry
Mass spectrometry probably is the most versatile and comprehensive
analytical technique currently used by chemists and biochemists.
The molecular ion fragments into a variety of fragment ions and the
resulting fragmentation pattern constitutes the mass spectrum.
GC or LC
Ionization methods
1. Electron-impact ionization (GC/MS)
2. Chemical ionization (GC/MS)
3. Atmospheric pressure electrospray ionization (LC/MS)
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
RP-HPLC - Example
Computer
Computer
Mass
Mass
Spectrometer
Spectrometer
UV
UV
Detector
Detector
Solvents
Solvents
Ion
Ion source
source
Column
Column
Pumps
Pumps
Interface
Interface
Sampler
Sampler
Injection
Injection port
port
Rough
Rough pump
pump