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Introduction of LC
Introduction of LC
Introduction of LC
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Agenda
Introduction Overview of LC Overview of MS Sample Inlet Ionization Techniques Mass Analyzers Quadrupole Operations
Agenda
Detector Vacuum Method Optimization for LCMS Data Interpretation Applications Of LCMS Software- ANALYST References
Introduction
Mass Spectrometry is a powerful analytical technique for identifying the unknown compounds by determining their molecular weights, for qualitative & quantitative determination. The basis in MS is the production of ions, that are subsequently separated or filtered according to their mass-to-charge ratio(m/z) and detected. It is the most sensitive method of molecular analysis.
Glossary
Ions AMU Isotopes High molecular weight ions Fragment ions
Atom
nucleus: neutrons and protons surrounded by equal number of electrons charge 0
Ion
nucleus: neutrons and protons surrounded by 1 or more or less electrons or protons + charge = number of surplus protons - charge = number of surplus electrons
Atoms or molecules must be converted into ions in order to be analyzed by mass spectroscopy
Isotopes
Naturally occurring More neutrons Higher amu but no change in charge Atoms in the periodic table with higher numbers of protons have more isotopes associated with them Lead has 82 protons and either 122,124, 125,or126 neutrons
N a tu ra l O c c u ra n c e o f L e a d (P b )
60 50 40
%
Introduction to LC
Liquid chromatography (LC) is an analytical technique of separation & identification of the sample which are dissolved in a solvent. Discovered by Russian botanist Mikhail Tswett in 1903. He separated various plant pigments by passing solutions through a glass column containing calcium carbonate. Chroma meaning color + graphein meaning to write
Separation
Solvent
A+B
Packed column
A B A B
detector
t0 t1 t2
A t3
B t4
Signal
A Time, t
Chromatography
Chromatogram - Detector signal vs. retention time or volume
Detector Signal
time or volume
History of LC
British Chemists A.T.P.Martin and R.L.M. Synge developed partition chromatography in 1942; ( Nobel prize in 1954) Since 1940s, chemists used gravity-fed silica columns to purify organic materials In late 1960s, LC turned high performance with small particle columns that require high pressure pumps Development of online detectors allow HPLC to become sensitive and quantitative technique for diverse applications
Types of Chromatography
Adsorption Chromatography
Normal Phase or Liquid-solid Chromatography (NP,LSC) -Separation based on adsorption/desorption of the analyte onto a polar surface Reversed Phase Chromatography (RPC) Separation based on analytes partition coefficients between the mobile phase and the bonded stationary phase.
Partition Chromatography
Separation is mainly dependent upon the relative solubility of the compounds (Solutes) in the liquid attached to the support (Stationary phase) and in the liquid flowing through the stationary phase (Mobile phase) If the affinity of the solute for the stationary phase is high, the solute does not elute. Ex: Fat dissolves in oil but not in water. If oil is the stationary phase, a fatty molecule will only elute with an oily mobile phase. Therefore, one goes from a weak solvent (water) to a strong solvent (oil).
Adsorption Chromatography
Solute interacts directly by adsorption with the stationary phase. Solute and solvent compete for adsorption sites. Ex. stationary phases: Silica, Alumina, Charcoal, Cellulose Non-polar molecules interact weakly with polar adsorbents. Polar molecules interact strongly with polar adsorbents.
+++ - + + + + -
Flow
Chromatography
HPLC is a physical separation technique in which a sample dissolved liquid is injected into a column packed with small particles and is separated into is constituents components. The most important and widely used analytical technique for the quantitative analysis of organics and biomolecules. Applicable to many sample types - Most useful for pharmaceuticals, biomolecules, and labile organics.
HPLC: Principle
High Performance Liquid Chromatography is a separation technique utilizing differences in distribution coefficient of compounds between two phases, stationary phase and mobile phase. Stationary Phase: Typically Designates a thin layer created on surface of fine silica particles. Mobile Phase : Designates the liquid flowing over the particles.
A peak is characterised by the retention time (selectivity) and its shape (efficiency)
t0
Solvent peak
Solute A
Selectivity Parameters ()
K1
K2
is measure of differential retention time of two analytes and must be > 1.0 for
peak separation.
Solute A
Solute B
Resolution
Resolution (measure of the separation between two consecutive peaks) depends on the systems selectivity and efficiency
R = t/Wb2
HPLC:Diagrammatic presentation
Reciprocating Pump
Most HPLC pumps are reciprocating A motor driven cam drives the piston to deliver solvent through the . outlet check valve.
using 2 or more pumps (high pressure mixing) or solenoidactuated proportioning valves (Low-pressure mixing)
Unlimited capacity - but
pulsed flow.
The loop is filled from the syringe The loop is inserted between the pump and the mobile phase flows from & the column so that the mobile phase pump to column. sweeps the sample onto the column.
Columns
Usually stainless steel : 1 - 5 mm diameter, ~5 mm packing. Very efficient but limited length
Packing - usually silica. More usual, to use similar chemical species actually bonded to the silica (longer column life), e.g. R can be nonpolar (C8 or C18 hydrocarbon chain) or polar (amine, nitrile etc.).
Me Si O Si R Me
Advantage of HPLC
Fast technique High Resolution Various Types of columns Used Wide range of Samples Versatile Non destructive (detector dependent) Good for quantitation Variety of separation mechanisms Scalable from Entry Level Operates at near ambient temperature
Applications of LC
Field of Application Separation
Antibiotics, Sedatives, Steroids, Analgesics Amino acids, Proteins, Carbohydrates, Lipids Artificial Sweeteners, Antioxidants, Preservatives Condensed Aromatics, Surfactants, Propellants, Dyes Drugs, Poisons, Blood Alcohol, narcotics Bile Acids, Drug Metabolites, Urine Extracts, Estrogens Pesticides, Herbicides, Phenols, PCBs Pharmaceuticals Biochemical Food Products Industrial Chemicals Forensic Chemistry Clinical Medicine Pollutants