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Affinity chromatography 1


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Affinity chromatography 1

  1. 1. Affinity Chromatography and Ion Exchange Chromatography Shannon E. Spence
  2. 2. What on Earth did scientist do before Chromatography? <ul><li>Extraction </li></ul><ul><li>is based on the difference in solubility material </li></ul><ul><li>is grounded, placed with a solvent which </li></ul><ul><li>dissolves soluble compounds. A second </li></ul><ul><li>extract solvent . The mixture is placed in a </li></ul><ul><li>separatory funnel </li></ul><ul><li>Crystallization </li></ul><ul><li>also based on the difference of solubility. The </li></ul><ul><li>solubility is solved in a fixed volume of solvent. </li></ul><ul><li>The purified compound crystallizes as solution </li></ul><ul><li>cools, evaporates or diffuses </li></ul><ul><li>Distillilation </li></ul><ul><li>separates components based on their volatility </li></ul><ul><li>typically via vaporization-condensation </li></ul><ul><li>method </li></ul><ul><li>Filtration </li></ul><ul><li>separate components of a mixture based on </li></ul><ul><li>their particle size. Used most often to </li></ul><ul><li>separate a liquid from a solid </li></ul> /.../ idealfract.html
  3. 3. What entices the scientists to Chromatography? <ul><li>Just like the previous techniques chromatography is a way to separate two components based on a specific characteristic </li></ul><ul><li>What makes chromatography so useful... </li></ul><ul><li>The results are reproducible with better accuracy than the before mentioned separation techniques </li></ul><ul><li>Chromatography can separate more complex mixtures than the previous techniques </li></ul><ul><li>Chromatography is less time consuming and cheaper </li></ul>
  4. 4. Brief History of Chromatography <ul><li>1903 – Tswett, a Russian botanist coined the term chromatography. He passed plant tissue extracts through a chalk column to separate pigments by differential adsorption chromatogrpahy </li></ul><ul><li>1915 R.M Willstatter, German Chemist win Nobel Prize for similar experiement </li></ul><ul><li>1922 L.S Palmer, American scientist used Tswett’s techniques on various natural products </li></ul><ul><li>1931 Richard Kuhn used chromatography to separate isomers oh polyene pigments; this is the first known acceptance of chromatographic methods </li></ul>
  5. 5. History of the Main techniques <ul><li>1938 Thin Layer chromatography by Russian scientist N.A Izamailov and M.S Shraiber </li></ul><ul><li>1941 Liquid-Liquid partition chromatography developed by Archer John, Porter Martin and Richard Laurence Millington Synge </li></ul><ul><li>1944 Paper Chromatography one of the most important methods in the development of biotechnology </li></ul><ul><li>1945 Gas Chromatography 1 st analytical gas-solid (adsorption) chromatography developed by Fritz Prior </li></ul><ul><li>1950 Gas Liquid Chromatography by Martin and Anthony James; Martin won the Nobel Prize in 1952 </li></ul>British chemist Archer John Porter Martin, co-recipient, with Richard L. M. Synge, of the 1952 Nobel Prize in chemistry, &quot;for their invention of partition chromatography.&quot;
  6. 6. History of the Main Techniques <ul><li>1966 HPLC named by Csaba Horvath, but didn’t become a popular method until 1970s </li></ul><ul><li>1950s Ion-Exchange chromatography declassified this technique </li></ul><ul><li>1970s Ion Chromatography was developed by Hamish Small and co-workers at the Dow Chemical company </li></ul><ul><li>1930s Affinity Chromatography was developed for the study of enzymes and other proteins </li></ul>
  7. 7. Chromatography <ul><li>Applies the principles of the “fractional” separation procedures </li></ul><ul><li>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 </li></ul><ul><li>Can separate complex mixtures composed of many very similar components. </li></ul><ul><li>Chromatography is often coupled with analytical instruments to complete analysis. </li></ul><ul><li>A single chromatographic analysis can isolate, identify , and quantitate multiple components of mixtures </li></ul>
  8. 8. Principles of Chromatography <ul><li>Chromatography is used when there is a difference in the retention times of different components </li></ul><ul><li>Two types of phases </li></ul><ul><li>1) Stationary phase </li></ul><ul><li>2) mobile phases </li></ul><ul><li>Properties of Chromatographic Properties </li></ul><ul><li>1) immiscible stationary and mobile phases </li></ul><ul><li>2) an arrangement where a mixture is depositied at one end of the </li></ul><ul><li>stationary phase </li></ul><ul><li>3) flow of the mobile phase towards the other end of the stationary </li></ul><ul><li>phase </li></ul><ul><li>4) different rates of partitioning for each component </li></ul><ul><li>5) means for visualizing the separation of each component </li></ul>
  9. 9. Techniques <ul><li>Ion Exchange Chromatography (IEC) </li></ul><ul><li>separates biomolecules based on the their net surface charge </li></ul><ul><li>Ion Chromatography (IC) </li></ul><ul><li>more general form of IEC allows separation of ions and polar molecules </li></ul><ul><li>based on the charge properties of the molecules </li></ul><ul><li>Affinity Chromatography (AC) </li></ul><ul><li>is the purification of a biomolecule with respect to the specific binding </li></ul><ul><li>of that biomolecule due to the chemical structure </li></ul><ul><li>Gas Chromatography (GC) </li></ul><ul><li>is a technique used to separate organic molecules that are volatile </li></ul><ul><li>Gas-Liquid Chromatography (GLC) </li></ul><ul><li>another name for GC </li></ul>
  10. 10. More Detailed History of IE Chromatography <ul><li>1850 H. Thompson and J.T way treated various clas with ammonium sulfate or carbonate in solution to extract the ammonia and release the calcium </li></ul><ul><li>1927 Zeolite (sodium aluminum silicates) mineral columns were used to remove interfering calcium and magnesium ions from solution to determine sulfate content of water </li></ul><ul><li>1940s Modern Ion-Exchange Chromatography was developed during the wartime Manhattan Project </li></ul><ul><li>- this technique was used to separate and concentrate the radioactive elements needed to make an atomic bomb. The adsorbents would latch onto charged transuranium elements differentially eluting them </li></ul><ul><li>1970s Hamish Small and co-workers of Dow Chemical Company developed ion-chromatography usable for automated analysis </li></ul><ul><li>- IC uses weaker ionic resins for the stationary phase and a neutralizing </li></ul><ul><li>stripper column to remove background eluent ions </li></ul><ul><li>- used for determining low concentrations of ions in water and other </li></ul><ul><li>environmental studies </li></ul>
  11. 11. Terminology <ul><li>Elution </li></ul><ul><li>- washing of the mixture </li></ul><ul><li>Eluent </li></ul><ul><li>- additional solvents used for elution </li></ul><ul><li>Effluent </li></ul><ul><li>- exiting fluid stream </li></ul><ul><li>Residency </li></ul><ul><li>- time spent on column </li></ul><ul><li>Stationary Phase </li></ul><ul><li>- </li></ul><ul><li>Mobile Phase </li></ul><ul><li>- fluid carrying the mixture of analytes </li></ul>
  12. 12. Ion-Exchange Chromatography <ul><li>Usually employed with HPLC </li></ul><ul><li>Ions are charged molecules </li></ul><ul><li>- cation positively charged ion </li></ul><ul><li>- anion negatively charged ion </li></ul><ul><li>These ions do not separate smoothly under the traditional methods of the liquid and mobile phases of chromatography </li></ul><ul><li>Requires alteration methods of either the mobile phase or stationary phase are required </li></ul><ul><li>- mobile phase suppresses the ionic nature of the analyte </li></ul><ul><li>- stationary phase incorporate ions of the opposite charge to attract and </li></ul><ul><li>retain analyte </li></ul>
  13. 13. How do you get those columns to work <ul><li>There is a glass column coated with a resin polymer </li></ul><ul><li>The resin is either positively charged (an acid) or negatively charged (a base) </li></ul><ul><li>An analyte will have ions opposite of the resins charge eluting off the ion of interest </li></ul>
  14. 14. Resin <ul><li>Common resins are copolymerized styrenes </li></ul><ul><li>vinylic and aromatic functional groups </li></ul><ul><li>styrene derivative and divinylbenzene </li></ul><ul><li>Creates better stability due to crosslinking of the benzene rings </li></ul><ul><li>Creates a swelling within the polymer affecting the porosity while taking in the mobile phase liquid </li></ul><ul><li>Aromatic substitution reaction makes these polymers ideal for charged functional groups </li></ul>
  15. 15. Cation Exchange resins <ul><li>The functional group in cation exchange resins are usually acids </li></ul><ul><li>Sulfonic acids –SO 3 H (strong acid resin) are added to the resin by sulfonation reactions </li></ul><ul><li>Res-(SO 3 H) + M +  Res-(SO 3 M) + H + </li></ul><ul><li>Carboxylic acid –COOH (weak acid resin) </li></ul><ul><li>Res-COOH + M +  Res-COOM + H + </li></ul><ul><li>With both the strong and the weak acid exchange sites an acidic Hydrogen is attached to a functional group chemically bound to the resin </li></ul><ul><li>Cation exchange is good for removing metal ions from an aqueous solution </li></ul>
  16. 16. Anion Exchange Resins <ul><li>The functional groups added to the resin is similar to cation resins but are basic instead of acidic </li></ul><ul><li>Quaternary ammonium a strong base -- CH 2 N(CH 3 ) 3 +OH - </li></ul><ul><ul><li>CH 2 N(CH 3 ) 3 +OH - + B-  Res-CH 2 N(CH 3 ) 3 + Cl - + OH - </li></ul></ul><ul><li>Polyalky amine a weak base -- NH(-R) 2 +OH - </li></ul><ul><li>NH(-R) 2 +OH - + B -  Res-NH(-R) 2 + B- +OH - </li></ul>
  17. 17. To Affinity and Beyond… <ul><li>The rate of ion exchange is controlled by the law of mass action. At equal concentration the greater affinity molecule will control the cation exchange resins in the acid form. </li></ul><ul><li>However if a much higher concentration of strong acid passed through the greater affinity molecule, such as sodium, will form the resin, reversing equilibrium and convert the resin back to an acidic form. </li></ul><ul><li>Generally it is possible to return either ion exchange resin column to a desired starting form by passing a large excess of the desired ion at very high concentration through the resin </li></ul>
  18. 18. What makes it unique… <ul><li>Careful selection of the ionic composition of the eluent, and the gradual adjustment of its strength during elution using a controlled gradient </li></ul><ul><li>The components of a mixture of ions can be induced to separate just as the components of a mixture separated by partitionion chromatography </li></ul><ul><li>The parameters controlling the relative residence of the analyte or other eluent ions is the resin stationary phase or the ionic solution mobile phase </li></ul><ul><li>1) both the relative selectivity of the resin for the ions and their relative concentrations in each phase </li></ul><ul><li>2) In ion exchange, selectivity resides in relative ion-pairing interaction strengths only in the stationary phase </li></ul>
  19. 19. Relative Affinity of Ions <ul><li>The higher the charge the higher the affinity </li></ul><ul><li>Na + < Ca 2+ < Al 3+ and Cl - < SO 4 2- </li></ul><ul><li>The Ion with the greatest size and charge has the highest affinity </li></ul><ul><li>Li + < Na + < K + < Cs + < Be 2+ < Mg 2+ < Cu 2+ and F - < Cl - < Br - < I - </li></ul>
  20. 20. Ion Exchange Chromatography <ul><li>The parameters controlling the residence </li></ul><ul><li>times of analyte or other eluent ions in the </li></ul><ul><li>resin stationary phase or ionic solution </li></ul><ul><li>mobile phase are </li></ul><ul><li>1) both the relative selectivity of the resin for the ions </li></ul><ul><li>2) their relative concentrations in each phase </li></ul>
  21. 21. Applications of IEC <ul><li>In cell and molecular biology, ion exchange chromatography is used to separate different proteins out of an eluant. </li></ul><ul><li>areas of research such as the environment, industry, commercial products of organic molecules without UV-vis absorption </li></ul>
  22. 22. Ion Chromatography <ul><li>The analysis of ionic analytes by separation on ion exchange stationary phases with eluent suppression of excess eluent ions </li></ul><ul><li>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 </li></ul>
  24. 24. Affinity History <ul><li>1930s, first developed by Arne Wilhelm Tiselius, won the Nobel Prize in 1948 </li></ul><ul><li>Used to study enzymes and other proteins </li></ul><ul><li>Relies on the affinity of various biochemical compounds with specific properties </li></ul><ul><li>ex) enzymes for their substrates </li></ul><ul><li>antibodies for their antigens </li></ul>
  25. 25. How do they get those iddy bitty molecules in there?
  26. 26. So now what…. <ul><li>The Sample is injected into the equilibrated affinity chromatography column </li></ul><ul><li>Only the substance with affinity for the ligand are retained on the column </li></ul><ul><li>The substance with no affinity to the ligand will elute off </li></ul><ul><li>The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent </li></ul>
  27. 27. Specificity of Affinity Chromatography <ul><li>Specificity is based on three aspect of affinity </li></ul><ul><li>1) the matrix </li></ul><ul><li>2) the ligand </li></ul><ul><li>3) the attachment of the ligands to the matrix </li></ul>
  28. 28. Matrix <ul><li>The matrix simply provides a posre structure to increase the surface area to which the molecule can bind </li></ul><ul><li>This has been what kept the Affinity Chromatography from being developed earlier and useful to the scientific community </li></ul><ul><li>The matrix must be activated for the ligand to bind to it but still able to retain it’s own activation towards the target molecule </li></ul>
  29. 29. Matrix <ul><li>Amino, hydroxyl, carbonyl and thio groups located with the matrix serve as ligand binding sites </li></ul><ul><li>Matrix are made up of agarose and other polysaccharides </li></ul><ul><li>The matrix also must be able to withstand the decontamination process of rinsing with sodium hydroxide or urea </li></ul>
  30. 30. Ligand <ul><li>The Ligand binds only to the desired molecule within the solution </li></ul><ul><li>The ligand attaches to the matrix which is made up of an inert substance </li></ul><ul><li>The ligand should only interact with the desired molecule and form a temporary bond </li></ul><ul><li>The ligand/molecule complex will remain in the column, eluting everything else off </li></ul><ul><li>The ligand/molecule complex dissociates by changing the pH </li></ul>
  31. 31. So many ligands so little time <ul><li>The chosen ligand must bind strongly to the molecule of interest </li></ul><ul><li>If the ligand can bind to more than onel molecule in the sample a technique, negative affinity is performed </li></ul><ul><li>- this is the removal of all ligands, leaving the molecule of interest in the column </li></ul><ul><li>- done by adding different ligands to bind to the ligands within the column </li></ul>
  32. 32. Applications <ul><li>Used in Genetic Engineering </li></ul><ul><li>Production of Vaccines </li></ul><ul><li>And Basic Metabolic Research </li></ul>
  33. 33. Gas Chromatography Gas-Liquid Chromatography And Gas-Solid Chromatography
  34. 34. History <ul><li>1945 </li></ul><ul><li>technique developed by Fritz Prior out of post-WWII Europe </li></ul><ul><li>Fritz Prior was a only a graduate student at the time </li></ul><ul><li>1947 </li></ul><ul><li>Prior succeeded in separating O2 and CO2 on a charcoal column </li></ul><ul><li>1950 </li></ul><ul><li>Archer J.P Martin and Anthony James developed Gas-Liquid Partition Chromatography (GLPC) </li></ul><ul><li>this has become the method of choice </li></ul>
  35. 35. Gas Chromatography <ul><li>Gas- Solid Chromatography (GSC) </li></ul><ul><li>is a process of repeated adsorption/desorption of sample from the carrier gas to the solid adsorbent </li></ul><ul><li>Gas-Liquid Partioning Chromatography (GLPC) </li></ul><ul><li>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. </li></ul>
  36. 36. Instrumentation <ul><li>Carrier Gas </li></ul><ul><li>Flow controller </li></ul><ul><li>Injector port </li></ul><ul><li>Column oven </li></ul><ul><li>Column </li></ul><ul><li>Detector </li></ul><ul><li>Recorder </li></ul>
  37. 37. Carrier Gas <ul><li>The carrier gas must be chemically inert. </li></ul><ul><li>Commonly used gases include nitrogen, helium, argon, and carbon dioxide. </li></ul><ul><li>The choice of carrier gas is often dependant upon the type of detector which is used. </li></ul><ul><li>The carrier gas system also contains a molecular sieve to remove water and other impurities. </li></ul>
  38. 38. Sample injection port <ul><li>For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a &quot;plug&quot; of vapour </li></ul><ul><li>- slow injection of large samples causes band broadening and loss of resolution. </li></ul><ul><li>The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. </li></ul><ul><li>The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample. </li></ul><ul><li>For packed columns, sample size ranges from tenths of a microliter up to 20 microliters. </li></ul><ul><li>Capillary columns, on the other hand, need much less sample, typically around 10 -3 mL. For capillary GC, split/splitless injection is used. </li></ul>
  39. 39. Sample injection port <ul><li>The injector can be used in one of two modes; split or split less. </li></ul><ul><li>The injector contains a heated chamber containing a glass liner into which the sample is injected through the septum. </li></ul><ul><li>The carrier gas enters the chamber and can leave by three routes (when the injector is in split mode). </li></ul><ul><li>The sample vaporizes to form a mixture of carrier gas, vaporized solvent and vaporized solutes </li></ul><ul><li>A proportion of this mixture passes onto the column, but most exits through the split outlet. </li></ul><ul><li>The septum purge outlet prevents septum bleed components from entering the column </li></ul>
  40. 40. Columns <ul><li>There are two general types of column, </li></ul><ul><li>1) packed </li></ul><ul><li>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. </li></ul><ul><li>2) capillary (also known as open tubular </li></ul><ul><li>Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; </li></ul><ul><li>a) wall-coated open tubular (WCOT) </li></ul><ul><li>consist of a capillary tube whose walls are coated with liquid stationary phase </li></ul><ul><li>b) support-coated open tubular (SCOT). </li></ul><ul><li>the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, which the </li></ul><ul><li>stationary phase has been adsorbed. </li></ul><ul><li>SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns . </li></ul>
  41. 41. Column <ul><li>In 1979, a new type of WCOT column was devised - the Fused Silica Open Tubular (FSOT) column </li></ul><ul><li>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. </li></ul>
  42. 42. Column Temperature <ul><li>For precise work, column temperature must be controlled to within tenths of a degree. </li></ul><ul><li>The optimum column temperature is dependant upon the boiling point of the sample. </li></ul><ul><li>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. </li></ul><ul><li>Minimal temperatures give good resolution, but increase elution times. </li></ul><ul><li>If a sample has a wide boiling range, then temperature programming can be useful. </li></ul><ul><li>The column temperature is increased (either continuously or in steps) as separation proceeds </li></ul>
  43. 43. Detectors <ul><li>There are many detectors which can be used in gas chromatography. </li></ul><ul><li>Different detectors will give different types of selectivity. </li></ul><ul><li>A non-selective detector responds to all compounds except the carrier gas </li></ul><ul><li>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. </li></ul><ul><li>Detectors can also be grouped into concentration dependant detectors and mass flow dependant detectors . </li></ul><ul><li>The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample </li></ul><ul><li>Dilution of with make-up gas will lower the detectors response. </li></ul><ul><li>Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. </li></ul><ul><li>The response of a mass flow dependant detector is unaffected by make-up gas. </li></ul>
  44. 44. Flame Ionization Detector (FID) <ul><li>The effluent from the column is mixed with hydrogen and air, and ignited. </li></ul><ul><li>Organic compounds burning in the flame produce ions and electrons which can conduct electricity through the flame. </li></ul><ul><li>A large electrical potential is applied at the burner tip, and a collector electrode is located above the flame. </li></ul><ul><li>The current resulting from the pyrolysis of any organic compounds is measured. </li></ul><ul><li>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. </li></ul><ul><li>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. </li></ul><ul><li>It is also robust and easy to use, but unfortunately, it destroys the sample. </li></ul>
  45. 45. Applications <ul><li>Main purpose is to separate and analyze multiple component mixtures: </li></ul><ul><li>Essential oils </li></ul><ul><li>Hydrocarbons </li></ul><ul><li>solvent </li></ul><ul><li>Biomedical </li></ul><ul><li>Biochemical </li></ul><ul><li>Physics </li></ul>
  46. 46. Mass Spectrometry <ul><li>Creates ions from molecules </li></ul><ul><li>It analyzes those ions, providing information about it’s molecular weight and chemical structure based on the fragmentation patterns </li></ul>
  47. 47. Instrumentation <ul><li>Sample introduction/separation </li></ul><ul><li>Ionization method </li></ul><ul><li>- Electron Impact ionization </li></ul><ul><li>Ion separation method </li></ul><ul><li>- Low (unit) resolution – 1 Dalton </li></ul><ul><li>- High resolution – 0.0001 Dalton </li></ul><ul><li>Ion Detector </li></ul>
  48. 48. Operation of Instrument <ul><li>Computer System </li></ul><ul><li>Sample Introduction </li></ul><ul><li>Ionization Method </li></ul><ul><li>Ion-Separation </li></ul><ul><li>Detector </li></ul>
  49. 49. History <ul><li>1886 Eugene Goldstein </li></ul><ul><li>observed “rays” which travelled through channels of a perforated cathode. These rays would travel towards an anode </li></ul><ul><li>1899 William Wien </li></ul><ul><li>discovered the “rays” could be deflected by either a strong electrical field or a strong magnetic field </li></ul><ul><li>constructed a device which could separate the positive rays by their mass to charge ratio (m/z) </li></ul><ul><li>1918 and 1919 Arthur Jeffrey Dempster and F.W Aston (respectively) </li></ul><ul><li>created the modern day Mass spectrometer </li></ul>
  50. 50. History of Modern Day MS <ul><li>1929 Walker Bleakney </li></ul><ul><li>developed Electron Impact mass spectrometry </li></ul><ul><li>hard impact technique </li></ul><ul><li>1987 Franz Hillenchamp and Michael Karas </li></ul><ul><li>developed Matrix Assisted Laser Desorption/Ionization </li></ul><ul><li>used in the identification of biomolecules </li></ul><ul><li>2002 John Bennett Fenn </li></ul><ul><li>developed ESI </li></ul>
  51. 51. Mass Spectrometry <ul><li>Mass spectrometry is an analytical tool used for measuring the molecular mass of a sample. </li></ul><ul><li>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 </li></ul><ul><li>within a 4 Daltons (Da) or atomic mass units (amu). </li></ul><ul><li>This is sufficient to allow minor mass changes to be detected </li></ul><ul><li>the substitution of one amino acid for another, or a post-translational modification. </li></ul><ul><li>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. </li></ul><ul><li>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. </li></ul><ul><li>This procedure is useful for the structural elucidation of organic compounds and for peptide or oligonucleotide sequencing. </li></ul>
  52. 52. Applications <ul><li>Biotechnology: the analysis of proteins, peptides, oligonucleotides </li></ul><ul><li>Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism </li></ul><ul><li>Clinical: neonatal screening, haemoglobin analysis, drug testing </li></ul><ul><li>Environmental: PAHs, PCBs, water quality, food contamination </li></ul><ul><li>Geological: oil composition </li></ul><ul><li>Physics: identification of space particles </li></ul>
  53. 53. Great Scott, it can be used in Biochemistry too! <ul><li>Accurate molecular weight measurements: sample confirmation, to determine the purity of a sample, to verify amino acid substitutions, to detect post-translational modifications, to calculate the number of disulphide bridges </li></ul><ul><li>Reaction monitoring: to monitor enzyme reactions, chemical modification, protein digestion </li></ul><ul><li>Amino acid sequencing: sequence confirmation, de novo characterization of peptides, identification of proteins by database searching with a sequence &quot;tag&quot; from a proteolytic fragment </li></ul><ul><li>Oligonucleotide sequencing: the characterization or quality control of oligonucleotides </li></ul><ul><li>Protein structure: protein folding monitored by H/D exchange, protein-ligand complex formation under physiological conditions, macromolecular structure determination </li></ul>
  54. 54. Mass spectrometers can be divided into three fundamental parts. <ul><li>Ionization source </li></ul><ul><li>Analyzer </li></ul><ul><li>Detector </li></ul>
  55. 55. Ionization Source <ul><li>The sample has to be introduced into the ionization source of the instrument. </li></ul><ul><li>Once inside the ionization source, the sample molecules are ionized, because ions are easier to manipulate than neutral molecules. </li></ul><ul><li>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) . </li></ul><ul><li>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 . </li></ul>
  56. 56. Analyzer <ul><li>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. </li></ul><ul><li>The entire operation of the mass spectrometer, and often the sample introduction process also, is under complete data system control on modern mass spectrometers. </li></ul>
  57. 57. Sample Introduction <ul><li>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. </li></ul><ul><li>The sample can be inserted directly into the ionization source, or can undergo some type of chromatography in route to the ionization source. </li></ul><ul><li>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. </li></ul><ul><li>The sample is separated into a series of components which then enter the mass spectrometer sequentially for individual analysis. </li></ul>
  58. 58. Ionization Methods <ul><li>Many ionization methods are available and each has its own advantages and disadvantages </li></ul><ul><li>The ionization method to be used should depend on the type of sample under investigation and the mass spectrometer available. </li></ul><ul><li>Ionization methods include the following : </li></ul><ul><li>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) </li></ul><ul><li>The ionization methods used for the majority of biochemical analyses are Electrospray Ionization (ESI) and , and Matrix Assisted Laser Desorption Ionization </li></ul><ul><li>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. </li></ul>
  59. 59. Ionization Methods Soft method  very high  mass to  500,000  Daltons Sample mixed  in solid  matrix Peptides  Proteins  Nucleotides Matrix Assisted Laser Desorption  (MALDI) Soft method  but harder  than ESI or  MALDI to  6,000  Daltons Sample mixed  in viscous  matrix Carbohydrates  Organometallics  Peptides  nonvolatile Fast Atom Bombardment (FAB) Soft method  ions often  multiply  charged to  200,000  Daltons Liquid  Chromatography  or syringe Peptides  Proteins  nonvolatile Electrospray (ESI) Soft method  molecular ion  peak [M+H]+ to  1,000  Daltons GC or  liquid/solid  probe Relatively  small  volatile Chemical Ionization (CI) Hard method  versatile  provides  structure info to  1,000  Daltons GC or  liquid/solid  probe Relatively  small  volatile Electron  Impact (EI) Method Highlights Mass Range Sample Introduction Typical Analytes Ionization method
  60. 60. Electrospray Ionization (ESI) <ul><li>ESI is one of the Atmospheric Pressure Ionization (API) techniques and is well-suited to the analysis of polar molecules ranging from less than 100 Da to more than 1,000,000 Da in molecular mass. </li></ul><ul><li>During standard electrospray ionization the sample is dissolved in a polar, volatile solvent and pumped through a narrow, stainless steel capillary at a flow rate of between 1 µL/min and 1 mL/min. </li></ul>
  61. 61. Electrospray Ionization (ESI) <ul><li>A high voltage of 3 or 4 kV is applied to the tip of the capillary, which is situated within the ionization source of the mass spectrometer, and as a consequence of this strong electric field, the sample emerging from the tip is dispersed into an aerosol of highly charged droplets, a process that is aided by a co-axially introduced nebulizing gas flowing around the outside of the capillary. </li></ul><ul><li>This gas, usually nitrogen, helps to direct the spray emerging from the capillary tip towards the mass spectrometer. The charged droplets diminish in size by solvent evaporation, assisted by a warm flow of nitrogen known as the drying gas which passes across the front of the ionization source. </li></ul>
  62. 62. Electrospray Ionization (ESI) <ul><li>Eventually charged sample ion s , free from solvent, are released from the droplets, some of which pass through a sampling cone or orifice into an intermediate vacuum region, and from there through a small aperture into the analyzer of the mass spectrometer, which is held under high vacuum. The lens voltages are optimized individually for each sample. </li></ul>
  63. 63. Electron Impact (EI) <ul><li>The gas molecules exiting the GC are bombarded by a high-energy electron beam (70eV) </li></ul><ul><li>An electron will strike the molecule, supplying enough energy to remove an electron from that molecule </li></ul><ul><li>Will produce a singly charges ion containing one unpaired electron </li></ul><ul><li>The instability on this molecule causes it to fragment into smaller pieces </li></ul>
  64. 64. Analyzers <ul><li>Analysis and Separation of Sample Ions </li></ul><ul><li>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-to-charge (m/z) ratios. </li></ul><ul><li>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 . </li></ul>
  65. 65. Analyzers <ul><li>These mass analyzers have different features </li></ul><ul><li>Including the m/z range that can be covered, </li></ul><ul><li>the mass accuracy, and the achievable resolution. </li></ul><ul><li>The compatibility of different analyzers with different ionization methods varies. </li></ul><ul><li>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 . </li></ul>
  66. 66. Analyzers <ul><li>Tandem (MS-MS) mass spectrometers are instruments that have more than one analyzer and so can be used for structural and sequencing studies. </li></ul><ul><li>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. </li></ul><ul><li>More popular tandem mass spectrometers include those of the quadrupole-quadrupole, magnetic sector-quadrupole , and more recently, the quadrupole-time-of-flight geometries . </li></ul>
  67. 67. Detectors <ul><li>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 . </li></ul><ul><li>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. </li></ul><ul><li>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. </li></ul>
  68. 68. Key Terminology <ul><li>Molecular Ion (M.+) </li></ul><ul><li>is the charged molecule which remains intact, usually is the molecular weight of molecule </li></ul><ul><li>Reference Spectra </li></ul><ul><li>mass spectral patterns which are reproducible </li></ul><ul><li>Base peak </li></ul><ul><li>100% abundance </li></ul>
  69. 69. Interpreting Spectra <ul><li>Ex) Methanol </li></ul>
  70. 70. <ul><li>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. </li></ul><ul><li>Protonated molecular ions are expected when the sample is analyzed under positive ionization conditions. </li></ul>
  71. 71. References <ul><li>Robinson, Skelly Frame, Frame II, Undergraduate Instrumental Analysis, Chromatography pg, 721-851 </li></ul><ul><li>Robinson, Skelly Frame, Frame II, 6 th Edition, Undergraduate Instrumental Analysis. Mass Spectrometry, pg. 613-721 </li></ul><ul><li>Matthews, PhD, Fred, Organic Spectroscopy, Spring 2007 Lecture notes, Mass Spectroscopy and GLC </li></ul><ul><li>Brennan, PhD, Carrie, Instrumental Analysis, Spring 2007 Lecture Notes, Chromatography </li></ul><ul><li>Brennan, PhD, Carrie, Quantitative Analysis, Fall 2006 Lecture Notes, Chromatography </li></ul><ul><li>Silberberg, Chemistry 3 rd Edition, pg 75 </li></ul><ul><li>Silverstrin, Webster, Kiemle, Spectrometric Identification of Organic Compounds, 7 th Edition, Chapter 1 Mass Spectrometry </li></ul><ul><li>McMurry, Organic Chemistry, 6 th Edition, Chapter 12 </li></ul><ul><li> , accessed June 16, 2007 </li></ul><ul><li> accessed July 3,2007 </li></ul><ul><li> accessed July 3, 2007 </li></ul><ul><li> accessed July 3, 2007 </li></ul>