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History: Classification based on Mobile Phase: The Basic Liquid Chromatograph units : combination of LC and MS , LC-MS Instrumentation LC-MS applications:
It was Mikhail Tswett, a Russian botanist, in 1903 who first invented and named liquid chromatography.
Tswett used a glass column filled with finely divided (calcium carbonate) to separate plant pigments. He observed the separation of colored zones or bands along the column. The development of chromatography was slow and scientists waited to early fifties for the first chromatographic instrument to appear in the market (a gas chromatograph). liquid chromatographic equipment with acceptable performance was only introduced about two decades after gas chromatography.
CHROMATOGRAPHY The separation of a mixture by distribution of its components between a mobile and stationary phase over time. mobile phase = solvent (also called eluent) penetrates or passes through a solid or immiscible stationary phase stationary phase = column packing material -In a chromatographic separation of any type, different components of a sample are transported in a mobile phase (a gas, a liquid, or a supercritical fluid).
Since the stationary phase is the fixed one then those solutes which have:stronger interactions with the stationary phase will tend to move slower (have higher retention times The time a solute spends in a column) than others which have lower or no interactions with the stationary phase will tend to move faster. chromatographic separations are a consequence of differential migration of solutes. maximum interactions between a solute and a stationary phase take place when both have similar characteristics, for example in terms of polarity. when their properties are so different, a solute will not tend to stay and interact with the stationary phase and will thus prefer to stay in the mobile phase and move faster; a polar solvent and a non polar stationary phase
According to the nature of the mobile phase, chromatographic techniques can be :* classified into three classes: a. Liquid chromatography (LC) b. Gas chromatography (GC) c. Supercritical fluid chromatography (SFC)
General classification
Specific method
Stationary phase
Type of equilibrium
Liquid Chromatography(LC) Liquid-liquid or partition Mobile phase: liquid Liquid- bonded phase
Liquid adsorbed on a Partition between solid immiscible Organic species bonded to Partition between liquid and
a Solid surface
Liquid -solid or adsorption solid
Bonded surface
Adsorption
A small volume of the sample is first introduced at the top of the chromatographic column. Elution involves :passing a mobile phase inside the column whereby solutes are carried down the stream but on a differential scale due to interactions with the stationary phase. As the mobile phase continues to flow, solutes continue to move downward the column. Distances between solute bands become greater with time and as solutes start to leave the column they are sequentially detected. The following schematics represent the process at various times:
The dark colors at the center of the solute zones in the above figure represent higher concentrations than are concentrations at the sides . This can be represented schematically as:
Chromatograms The plot of detector signal versus retention time of solutes in a chromatographic column is referred to as a chromatogram. The areas under the peaks in a chromatogram are usually related to solute concentration quantitative analysis. The retention time of a solute is a characteristic property of the solute which reflects its degree of interaction with both stationary and mobile phases. Retention times serve qualitative analysis parameters to identify solutes.
The mobile phase supply system consists of number of reservoirs (200 ml to 1,000 ml in capacity).
-the solvent mixing occurs at high pressure,and then passed to the pump -is the simplest but most expensive. -each solvent requires its own pump.
solvents are premixed at low pressure and then passed to the pump.
The LC Pump
-pneumatic pump, The pneumaticpump can provide extremely high pressures and is relatively inexpensive, -the total air pressure on the piston, diameter (y), is transferred to a piston controlling the liquid pressure, of diameter (x). Because the radii of the pistons differ, there will be a net pressure amplification of
The Sample Valve - liquid samples are usually injected onto the column by a syringe via a injector.
- Sample are placed on an LC column directly with either an internal or external loop sample valve the valve being connected directly to the column.
Column Ovens:
The effect of temperature on LC separations is often not nearly so profound as its effect in GC separations, but can be critical when closely similar substances are being separated. Due to the lesser effect of temperature on solute retentionin LC (compared to that in GC), temperature is not nearly so critical in governing absolute retention time but is often essential in achieving adequate resolution, particularly between closely eluting solutes such as isomers.
Detectors
UV Detector Fixed Wavelength Detector Multi-Wavelength Detectors Diode Array Detector Electrical Conductivity Detector Fluorescence Detector
* combination of LC and MS offers the possibility to take advantage of both LC as a powerful and versatile separation technique and MS as a powerful and sensitive detection and identification technique.
a mass spectrometer is more sensitive and far more specific than all other LC detectors. -It can analyze compounds that lack a suitable chromophore. -It can also identify components in unresolved chromatographic peaks, reducing the need for perfect chromatography.
Mass spectral data complements data fromother LC detectors
Two-dimensional abundance data and three-dimensional mass spectral data from a mass spectrometer
Instrumentation
Mass spectrometers work by :1- ionizing molecules 2- sorting and identifying the ions according to their mass-tocharge (m/z) ratios.
Ion Sources
- ionize the analyte molecules and separate the resultingions from the mobile phase. ionization techniques are: 1a- Electro spray ionization (ESI) 1b- Atmospheric pressure chemical ionization (APCI) 1c- Atmospheric pressure photo ionization (APPI)
1c- Atmospheric pressure photo ionization (APPI) Atmospheric pressure photo Ionization (APPI) for LC/MS is a relatively new technique. As in APCI, a vaporizer converts the LC eluent to the gas phase.
A discharge lamp generates photons in a narrow range of ionization energies.
The range of energies is carefully chosen to ionize as many analyte molecules as possible while minimizing the ionization of solvent molecules. The resulting ions pass through a capillary sampling orifice into the mass analyzer.
2-Mass Analyzers
Although in theory any type of mass analyzer could be used for LC/MS, four types: 2a- Quadrupole 2b- Time-of-flight 2c- Ion trap 2d- Fourier transform-ion cyclotron resonance (FT-ICR or FT-MS)
2a- Quadrupole
A quadrupole mass analyzer consists of four parallel rods arranged in a square. The analyte ions are directed down the center of the square. Voltages applied to the rods generate electromagnetic fields. These fields determine which mass-to-charge ratio of ions can pass through the filter at a given time. Quadrupoles tend to be the simplest and least expensive mass analyzers.
2b- Time-of-flight
In a time-of-flight (TOF) mass analyzer, a uniform electromagnetic force is applied to all ions at the same time, causing them to accelerate down a flight tube. Lighter ions travel faster and arrive at the detector first, so the mass-to-charge ratios of the ions are determined by their arrival times. Time-of-flight mass analyzers have a wide mass range and can be very accurate in their mass measurements.
FT-ICR mass analyzers can perform multiple stages of mass spectrometry without additional mass analyzers.
Applications:LC/MS is suitable for many applications, from pharmaceutical development to environmental analysis. Its ability to detect a wide range of compounds with great sensitivity and specificity has made it popular in a variety of fields.
Full scan mass spectrum of ginsenoside Rb1 showing primarily sodium adduct ions
Figure. MS identification and quantification of individual benzodiazepines from an incompletely resolved mixture