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-Crystallization
also based on the difference of solubility. The solubility is solved in a fixed volume of solvent. The purified compound crystallizes as solution cools, evaporates or diffuses
-Distillilation
separates components based on their volatility typically via vaporizationcondensation method
Filtration
separate components of a mixture based on their particle size. Used most often to separate a liquid from a solid
www.chemguide.co.uk/.../idealfract.ht ml
British chemist Archer John Porter Martin, co-recipient, with Richard L. M. Synge, of the 1952 Nobel Prize in chemistry, "for their invention of partition chromatography."
http://www.chemistryexplained.com/MaNa/Martin-Archer-John-Porter.html
library.thinkquest.org
Chromatography
Applies the principles of the fractional separation procedures Non-instrumental analysis which partitions components between two phases usually a mobile phase and a stationary phase, based on the difference in the components physical properties Can separate complex mixtures composed of many very similar components. Chromatography is often coupled with analytical instruments to complete analysis. A single chromatographic analysis can isolate, identify , and quantitate multiple components of mixtures
Principles of Chromatography
Chromatography is used when there is a difference in the retention times of different components Two types of phases 1) Stationary phase 2) mobile phases Properties of Chromatographic Properties 1) immiscible stationary and mobile phases 2) an arrangement where a mixture is depositied at one end of the stationary phase 3) flow of the mobile phase towards the other end of the stationary phase 4) different rates of partitioning for each component 5) means for visualizing the separation of each component
Techniques
Ion Exchange Chromatography (IEC)
separates biomolecules based on the their net surface charge
Terminology
Elution
- washing of the mixture
Stationary Phase
-
Eluent
- additional solvents used for elution
Mobile Phase
- fluid carrying the mixture of analytes
Effluent
- exiting fluid stream
Residency
- time spent on column
Ion-Exchange Chromatography
Usually employed with HPLC Ions are charged molecules - cation positively charged ion - anion negatively charged ion These ions do not separate smoothly under the traditional methods of the liquid and mobile phases of chromatography Requires alteration methods of either the mobile phase or stationary phase are required - mobile phase suppresses the ionic nature of the analyte - stationary phase incorporate ions of the opposite charge to attract and retain analyte
Resin
Common resins are copolymerized styrenes vinylic and aromatic functional groups styrene derivative and divinylbenzene
Creates better stability due to crosslinking of the benzene rings Creates a swelling within the polymer affecting the porosity while taking in the mobile phase liquid Aromatic substitution reaction makes these polymers ideal for charged functional groups
The Ion with the greatest size and charge has the highest affinity
Li+ < Na+ < K+ < Cs+ < Be2+ < Mg2+ < Cu2+ and F- < Cl- < Br- < I-
Applications of IEC
In cell and molecular biology, ion exchange chromatography is used to separate different proteins out of an eluant. areas of research such as the environment, industry, commercial products of organic molecules without UV-vis absorption
Ion Chromatography
The analysis of ionic analytes by separation on ion exchange stationary phases with eluent suppression of excess eluent ions ex) when cations are being exchanged to
effect a separation, variable concentrations of HCl are used as an eluent passing through the analytical anion column without being retained forming largely undissociated species such as water, carbonic acid and bicarbonate ions
AFFINITY CHROMATOGRAPHY
Affinity History
1930s, first developed by Arne Wilhelm Tiselius, won the Nobel Prize in 1948 Used to study enzymes and other proteins Relies on the affinity of various biochemical compounds with specific properties ex) enzymes for their substrates antibodies for their antigens
Affinity chromatography y Separates by specific interactions y Contains a ligand: enzyme substrate receptor hormone antigen antibody (His)6 protein Ni2+
The Sample is injected into the equilibrated affinity chromatography column Only the substance with affinity for the ligand are retained on the column The substance with no affinity to the ligand will elute off The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent
Matrix
The matrix simply provides a posre structure to increase the surface area to which the molecule can bind This has been what kept the Affinity Chromatography from being developed earlier and useful to the scientific community The matrix must be activated for the ligand to bind to it but still able to retain its own activation towards the target molecule
Matrix
Amino, hydroxyl, carbonyl and thio groups located with the matrix serve as ligand binding sites Matrix are made up of agarose and other polysaccharides The matrix also must be able to withstand the decontamination process of rinsing with sodium hydroxide or urea
Ligand
The Ligand binds only to the desired molecule within the solution The ligand attaches to the matrix which is made up of an inert substance The ligand should only interact with the desired molecule and form a temporary bond The ligand/molecule complex will remain in the column, eluting everything else off The ligand/molecule complex dissociates by changing the pH
Applications
Used in Genetic Engineering Production of Vaccines And Basic Metabolic Research
Gas Chromatography
Gas-Liquid Chromatography And Gas-Solid Chromatography
History
1945 technique developed by Fritz Prior out of post-WWII Europe Fritz Prior was a only a graduate student at the time 1947 Prior succeeded in separating O2 and CO2 on a charcoal column 1950 Archer J.P Martin and Anthony James developed Gas-Liquid Partition Chromatography (GLPC) this has become the method of choice
Gas Chromatography
Gas- Solid Chromatography (GSC) is a process of repeated adsorption/desorption of sample from the carrier gas to the solid adsorbent Gas-Liquid Partioning Chromatography (GLPC) involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.
Instrumentation
Carrier Gas Flow controller Injector port Column oven Column Detector Recorder
http://teaching.shu.ac.uk/hwb/chemistry/tutor ials/chrom/gaschrm.htm
Carrier Gas
The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of detector which is used. The carrier gas system also contains a molecular sieve to remove water and other impurities.
Columns
There are two general types of column, 1) packed contain a finely divided, inert, solid support material (commonly based on diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm. 2) capillary (also known as open tubular Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; a) wall-coated open tubular (WCOT) consist of a capillary tube whose walls are coated with liquid stationary phase b) support-coated open tubular (SCOT). the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, which the stationary phase has been adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns.
Column
In 1979, a new type of WCOT column was devised the Fused Silica Open Tubular (FSOT) column These have much thinner walls than the glass capillary columns, and are given strength by the polyimide coating. These columns are flexible and can be wound into coils. They have the advantages of physical strength, flexibility and low reactivity.
Column Temperature
For precise work, column temperature must be controlled to within tenths of a degree. The optimum column temperature is dependant upon the boiling point of the sample. As a rule of thumb, a temperature slightly above the average boiling point of the sample results in an elution time of 2 - 30 minutes. Minimal temperatures give good resolution, but increase elution times. If a sample has a wide boiling range, then temperature programming can be useful. The column temperature is increased (either continuously or in steps) as separation proceeds
Detectors
There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound. Detectors can also be grouped into concentration dependant detectors and mass flow dependant detectors. The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response. Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas.
The effluent from the column is mixed with hydrogen and air, and ignited. Organic compounds burning in the flame produce ions and electrons which can conduct electricity through the flame. A large electrical potential is applied at the burner tip, and a collector electrode is located above the flame. The current resulting from the pyrolysis of any organic compounds is measured. FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detector's response. The FID is a useful general detector for the analysis of organic compounds; it has high sensitivity, a large linear response range, and low noise. It is also robust and easy to use, but unfortunately, it destroys the sample.
Applications
Main purpose is to separate and analyze multiple component mixtures: Essential oils Hydrocarbons solvent Biomedical Biochemical Physics
Mass Spectrometry
Creates ions from molecules It analyzes those ions, providing information about its molecular weight and chemical structure based on the fragmentation patterns
http://www.chem.arizona.edu/massspec/intro_html/intro.html
Instrumentation
Sample introduction/separation Ionization method - Electron Impact ionization Ion separation method - Low (unit) resolution 1 Dalton - High resolution 0.0001 Dalton Ion Detector
Operation of Instrument
Computer System Sample Introduction Ionization Method Ion-Separation Detector
History
1886 Eugene Goldstein observed rays which travelled through channels of a perforated cathode. These rays would travel towards an anode 1899 William Wien discovered the rays could be deflected by either a strong electrical field or a strong magnetic field constructed a device which could separate the positive rays by their mass to charge ratio (m/z) 1918 and 1919 Arthur Jeffrey Dempster and F.W Aston (respectively) created the modern day Mass spectrometer
Mass Spectrometry
Mass spectrometry is an analytical tool used for measuring the molecular mass of a sample. For large samples such as macromolecules, molecular masses can be measured to within an accuracy of 0.01% of the total molecular mass of the sample within a 4 Daltons (Da) or atomic mass units (amu). This is sufficient to allow minor mass changes to be detected the substitution of one amino acid for another, or a posttranslational modification. For small organic molecules the molecular mass can be measured to within an accuracy of5 ppm or less, which is often sufficient to confirm the molecular formula of a compound, and is also a standard requirement for publication in a chemical journal. Structural information can be generated using certain types of mass spectrometers, usually those with multiple analyzers which are known as tandem mass spectrometers. This is achieved by fragmenting the sample inside the instrument and analyzing the products generated. This procedure is useful for the structural elucidation of organic compounds and for peptide or oligonucleotide sequencing.
Applications
Biotechnology: the analysis of proteins, peptides, oligonucleotides Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism Clinical: neonatal screening, haemoglobin analysis, drug testing Environmental: PAHs, PCBs, water quality, food contamination Geological: oil composition Physics: identification of space particles
Ionization Source
The sample has to be introduced into the ionization source of the instrument. Once inside the ionization source, the sample molecules are ionized, because ions are easier to manipulate than neutral molecules. These ions are extracted into the analyzer region of the mass spectrometer where they are separated according to their mass (m) -to-charge (z) ratios (m/z) . The separated ions are detected and this signal sent to a data system where the m/z ratios are stored together with their relative abundance for presentation in the format of a m/z spectrum .
Analyzer
The analyzer and detector of the mass spectrometer, and often the ionization source too, are maintained under high vacuum to give the ions a reasonable chance of travelling from one end of the instrument to the other without any hindrance from air molecules. The entire operation of the mass spectrometer, and often the sample introduction process also, is under complete data system control on modern mass spectrometers.
Sample Introduction
The method of sample introduction to the ionization source often depends on the ionization method being used, as well as the type and complexity of the sample. The sample can be inserted directly into the ionization source, or can undergo some type of chromatography in route to the ionization source. This method of sample introduction usually involves the mass spectrometer being coupled directly to a high pressure liquid chromatography (HPLC), gas chromatography (GC) or capillary electrophoresis (CE) separation column. The sample is separated into a series of components which then enter the mass spectrometer sequentially for individual analysis.
Ionization Methods
Many ionization methods are available and each has its own advantages and disadvantages The ionization method to be used should depend on the type of sample under investigation and the mass spectrometer available. Ionization methods include the following: 1. Atmospheric Pressure Chemical Ionization (APCI) 2. Chemical Ionization (CI) 3. Electron Impact (EI) 4. Electrospray Ionization (ESI) 5, Fast Atom Bombardment (FAB) 6. Field Desorption / Field Ionization (FD/FI) 7. Matrix Assisted Laser Desorption Ionization (MALDI) 8. Thermospray Ionization (TSP) The ionization methods used for the majority of biochemical analyses are Electrospray Ionization (ESI) and , and Matrix Assisted Laser Desorption Ionization With most ionization methods there is the possibility of creating both positively and negatively charged sample ions, depending on the proton affinity of the sample, therefore beginning an analysis, the user would need to determine if the ions are cations or anions.
Ionization Methods
Ionization method Typical Analytes Sample Introductio n GC or liquid/solid probe Mass Range to 1,000 Daltons Method Highlights Hard method versatile provides structure info Soft method molecular ion peak [M+H]+ Soft method ions often multiply charged Soft method but harder than ESI or MALDI Soft method Electron Im Relatively small pact (EI) volatile
GC or liquid/solid probe
to 1,000 Daltons
Fast Atom Carbohydrat Sample mixed Bombardme es Organometa in viscous nt (FAB) matrix llics Peptides nonvolatile Matrix Assisted Peptides Proteins Sample mixed
to 500,000
Analyzers
Analysis and Separation of Sample Ions The main function of the mass analyzer is to separate the ions formed in the ionization source of the mass spectrometer according to their mass-tocharge (m/z) ratios. There are a number of mass analyzers, the more common known mass analyzers are quadrupoles , time-of-flight (TOF) analyzers, magnetic sectors , Fourier transform and quadrupole ion traps .
Analyzers
These mass analyzers have different features Including the m/z range that can be covered, the mass accuracy, and the achievable resolution. The compatibility of different analyzers with different ionization methods varies. For example, all of the analyzers listed above can be used in conjunction with electrospray ionization, whereas MALDI is not usually coupled to a quadrupole analyzer.
Analyzers
Tandem (MS-MS) mass spectrometers are instruments that have more than one analyzer and so can be used for structural and sequencing studies. Two, three and four analyzers have all been incorporated into commercially available tandem instruments, and the analyzers do not necessarily have to be of the same type, in which case the instrument is a hybrid one. More popular tandem mass spectrometers include those of the quadrupole-quadrupole, magnetic sector-quadrupole , and more recently, the quadrupole-time-of-flight geometries.
Detectors
The detector monitors the ion current, amplifies it and the signal is then transmitted to the data system where it is recorded in the form of mass spectra . The m/z values of the ions are plotted against their intensities to show the number of components in the sample the molecular mass of each component, and the relative abundance of the various components in the sample. The type of detector is supplied to suit the type of analyzer; the more common ones are the photomultiplier, the electron multiplier and the micro-channel plate detectors.
Key Terminology
Molecular Ion (M.+)
is the charged molecule which remains intact, usually is the molecular weight of molecule
Reference Spectra
mass spectral patterns which are reproducible
Base peak
100% abundance
Interpreting Spectra
Ex) Methanol
Samples (M) with molecular masses up to 1200 Da give rise to singly charged molecular-related ions, usually protonated molecular ions of the formula (M+H)+ in positive ionization mode, and deprotonated molecular ions of the formula (M-H)- in negative ionization mode. Protonated molecular ions are expected when the sample is analyzed under positive ionization conditions.
References
Robinson, Skelly Frame, Frame II, Undergraduate Instrumental Analysis, Chromatography pg, 721-851 Robinson, Skelly Frame, Frame II, 6th Edition, Undergraduate Instrumental Analysis. Mass Spectrometry, pg. 613-721 Matthews, PhD, Fred, Organic Spectroscopy, Spring 2007 Lecture notes, Mass Spectroscopy and GLC Brennan, PhD, Carrie, Instrumental Analysis, Spring 2007 Lecture Notes, Chromatography Brennan, PhD, Carrie, Quantitative Analysis, Fall 2006 Lecture Notes, Chromatography Silberberg, Chemistry 3rd Edition, pg 75 Silverstrin, Webster, Kiemle, Spectrometric Identification of Organic Compounds, 7th Edition, Chapter 1 Mass Spectrometry McMurry, Organic Chemistry, 6th Edition, Chapter 12 http://pubs.acs.org/hotartcl/tcaw/98/sep/creat.html, accessed June 16, 2007 www.chem.arizona.edu/massspec/inter_html/inter.html accessed July 3,2007 www.astbury.leeds.ac accessed July 3, 2007 www.chemistry.wustl.edu accessed July 3, 2007