Chapter 4 bio 300 obe

1,266 views
1,114 views

Published on

Published in: Technology
0 Comments
1 Like
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
1,266
On SlideShare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
38
Comments
0
Likes
1
Embeds 0
No embeds

No notes for slide

Chapter 4 bio 300 obe

  1. 1. 1 Chapter 4: Techniques in Biochemical Analysis
  2. 2. BIO 300 BIOLOGICAL TECHNIQUES AND SKILLS SARINI BINTI AHMAD WAKID FACULTY OF APPLIED SCIENCE Chapter 4: Techniques in Biochemical Analysis 2
  3. 3. CHAPTER 4 Techniques in Biochemical Analysis Chapter 4: Techniques in Biochemical Analysis 3
  4. 4. What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. • Analyze Separate • Identify • Purify Mixture Components Chapter 4: Techniques in Biochemical Analysis • Quantify 4
  5. 5. Chromatography      Chromatography is a method of separating a mixture of molecules depending on their distribution between a mobile phase and a stationary phase. The mobile phase (also known as solvent) may be either liquid or gas. The stationary phase (also known as sorbent) can be either a solid or liquid, a liquid stationary phase is held stationary by a solid. The solid holding the liquid stationary phase is the support or matrix. The molecules in the mixture to be separated are the solutes. Chapter 4: Techniques in Biochemical Analysis 5
  6. 6. Uses for Chromatography Chromatography is used by scientists to: • Analyze – examine a mixture, its components, and their relations to one another • Identify – determine the identity of a mixture or components based on known components • Purify – separate components in order to isolate one of interest for further study • Quantify – determine the amount of the a mixture and/or the components present in the sample Chapter 4: Techniques in Biochemical Analysis 6
  7. 7. Uses for Chromatography Real-life examples of uses for chromatography: • Pharmaceutical Company – determine amount of each chemical found in new product • Hospital – detect blood or alcohol levels in a blood stream patient’s • Law Enforcement – to compare a sample found at a crime scene to samples from suspects • Environmental Agency – determine the level of in the water supply • Manufacturing Plant make a product – to purify a chemical Chapter 4: Techniques in Biochemical Analysis pollutants needed to 7
  8. 8. Definition of Chromatography Detailed Definition: Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass. Terminology: • Differential – showing a difference, distinctive • Affinity – natural attraction or force between things • Mobile Medium – gas or liquid that carries the components (mobile phase) • Stationary Medium – the part of the apparatus that does not move with the sample (stationary phase) Chapter 4: Techniques in Biochemical Analysis 8
  9. 9. Definition of Chromatography Simplified Definition: Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase. Explanation: • • • • Compound is placed on stationary phase Mobile phase passes through the stationary phase Mobile phase solubilizes the components Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases Chapter 4: Techniques in Biochemical Analysis 9
  10. 10. Illustration of Chromatography Stationary Phase Separation Mobile Phase Mixture Components Components Affinity to Stationary Phase Affinity to Mobile Phase Blue ---------------- Insoluble in Mobile Phase Black     Red Yellow Chapter 4: Techniques in           Biochemical Analysis 10
  11. 11. Chapter 4: Techniques in Biochemical Analysis 11
  12. 12. Types of Chromatography • Liquid Chromatography – separates liquid samples liquid solvent (mobile phase) and a column (stationary phase) with a composed of solid beads • Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column of solid beads (stationary phase) composed of a liquid or • Paper Chromatography – separates dried liquid with a liquid solvent (mobile phase) and a phase) samples paper strip (stationary • Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass covered with a thin layer of alumina or silica gel (stationary phase) Chapter 4: Techniques in Biochemical Analysis plate 12
  13. 13. Types of chromatography • • • • Partition chromatography Adsorption chromatography Gel filtration Ion exchange chromatography Chapter 4: Techniques in Biochemical Analysis 13
  14. 14. (A) uses charge, (B) uses pores, and (C) uses covalent bonds to create the differential affinities among the mixture components for the stationary phase. Chapter 4: Techniques in Biochemical Analysis 14
  15. 15. Partition chromatography • The distribution of solutes between two immiscible phases. • The solute will distribute it self between the two phases according to its solubility in each phase, this is called partitioning. Chapter 4: Techniques in Biochemical Analysis 15
  16. 16. Examples of partition chromatography      The two most common types of partition chromatography are thin layer chromatography and paper chromatography. In both cases the stationary phase is a liquid bound to a matrix. In paper chromatography the stationary phase are water molecules bound to a cellulose matrix. In TLC, the stationary phase is the solvent added to the support to form the thin layer so the solvent gets bound to the matrix (support). Partition chromatography is mainly used for separation of molecules of small molecular weight. Chapter 4: Techniques in Biochemical Analysis 16
  17. 17. Paper chromatography • The cellulose support contains a large amount of bound water. • Partitioning occurs between the bound water which is the stationary phase and the solvent which is the mobile phase. Chapter 4: Techniques in Biochemical Analysis 17
  18. 18. Experimental procedure for paper chromatography         A small volume of a solution of a mixture to be separated or identified is placed at a marked spot (origin) on a sheet or strip of paper and allowed to dry. The paper is then placed in a closed chamber and one end is immersed in a suitable solvent. The solvent is drawn (moved) through the paper by capillary action. As the solvent passes the origin, it dissolves the sample and moves the components in the direction of flow. After the solvent front has reached a point near the other end of the paper, the sheet or strip is removed and dried. The spots are then detected and their positions marked. The ratio of the distance moved by a solute to the distance moved by the solvent = Rf. The Rf. is always less than one. Chapter 4: Techniques in Biochemical Analysis 18
  19. 19. Chromatogram      Once a sample is applied on TLC or paper, it’s called chromatogram. Paper chromatogram can be developed either by ascending or descending solvent flow. Descending chromatography is faster because gravity helps the solvent flow. Disadvantages : it’s difficult to set the apparatus. Ascending is simple and inexpensive compared with descending and usually gives more uniform migration with less diffusion of the sample "spots". Chapter 4: Techniques in Biochemical Analysis 19
  20. 20. Detection of spots  1. 2. 3. 4. Spots in paper chromatograms can be detected in 4 different ways: By their natural color By their fluorescence By their chemical reactions that take place after the paper has been sprayed with various reagents for example: during paper chromatography of amino acids, the chromatograms are sprayed with ninhydrin. By radioactivity Chapter 4: Techniques in Biochemical Analysis 20
  21. 21. Identification of spots • The spots are usually identified by comparing of standards of known Rf values. Chapter 4: Techniques in Biochemical Analysis 21
  22. 22. Thin layer chromatography      Paper chromatography uses paper which can be prepared from cellulose products only. In TLC, any substance that can be finely divided and formed into a uniform layer can be used. Both organic and inorganic substances can be used to form a uniform layer for TLC. Organic substances include: cellulose, polyamide, polyethylene Inorganic: silica gel, aluminum oxide and magnesium silicate Chapter 4: Techniques in Biochemical Analysis 22
  23. 23. TLC • The stationary phase is the solvent used to form a layer of sorbent spread uniformly over the surface of a glass or plastic plate Chapter 4: Techniques in Biochemical Analysis 23
  24. 24. Advantages of TLC over paper chromatography • Greater resolving power because there is less diffusion of spots. • Greater speed of separation • Wide choice of materials as sorbents Chapter 4: Techniques in Biochemical Analysis 24
  25. 25. The separation of compounds by chromatography depends on several factors:  Partition of a solute between a moving solvent phase and a stationary aqueous phase. The solute moves in the direction of a solvent flow at a rate determined by the solubility of the solute in the moving phase. Thus a compound with high mobility is more attracted to the moving organic phase than to the stationary phase. Chapter 4: Techniques in Biochemical Analysis 25
  26. 26. Cont..   Ion exchange effect: any ionized impurities in the support medium will tend to bind or attract oppositely charged ions (solutes) and will therefore reduce the mobility of these solutes. Temperature: Since temperature can effect the solubility of the solute in a given solvent temperature is also an important factor. Chapter 4: Techniques in Biochemical Analysis 26
  27. 27.    The molecular weight of a solute also affects the solubility and hence chromatographic performance. Adsorption of compound (solute) onto support medium: Although the support medium (silica gel) is theoretically inert, this isn't always the case. If a solute tends to bind to the support medium this will slow down its mobility in the solvent system. The composition of the solvent: since some compounds are more soluble in one solvent than in the other, the mixture of solvents used will affect the separation of compounds. Chapter 4: Techniques in Biochemical Analysis 27
  28. 28. Expression of the results  The term "Rf" (relative flow) is used to express the performance of a solute in a given solvent system /support medium. The term Rf value may be defined as the ratio of the distance the compound migrates to the distance the solvent migrates. Rf value is constant for a particular compound, solvent system and insoluble matrix. Rf= Distance of migration of solute Distance moved by solvent Chapter 4: Techniques in Biochemical Analysis 28
  29. 29. Rf values  qualitative results of TLC     expressed as fractions of 1.0 can be expressed from Rf values (eg Rf x 100) no more than two decimal places  due to inaccuracy of physical measurement may not be reproducible    only give an indication of possible nature of unknown complete identification only obtained if spot is eluted and micro-scale physical measurements done (MS, UV, IR) standard references should always be used on same plate for comparison   most sprays produce differential colours of fluorescence colour test provides extra evidence with distance migration Chapter 4: Techniques in Biochemical Analysis 29
  30. 30. Chapter 4: Techniques in Biochemical Analysis 30
  31. 31. Principles of Paper Chromatography • Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity. • Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents. Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase. Chapter 4: Techniques in Biochemical Analysis 31
  32. 32. Paper Chromatography Experiment What Color is that Sharpie? Chapter 4: Techniques in Biochemical Analysis 32
  33. 33. Overview of the Experiment Purpose: To introduce students to the principles and terminology of chromatography and demonstrate separation of the dyes in Sharpie Pens with paper chromatography. Time Required: Prep. time: 10 minutes Experiment time: 45 minutes Chapter 4: Techniques in Biochemical Analysis 33
  34. 34. • • • • • • • • • • • 6 beakers or jars 6 covers or lids Distilled H2O Isopropanol Graduated cylinder 6 strips of filter paper Different colors of Sharpie pens Pencil Ruler Scissors Tape Materials List Chapter 4: Techniques in Biochemical Analysis 34
  35. 35. Preparing the Isopropanol Solutions • Prepare 15 ml of the following isopropanol solutions in appropriately labeled beakers: - 0%, 5%, 10%, 20%, 50%, and 100% Chapter 4: Techniques in Biochemical Analysis 35
  36. 36. Preparing the Chromatography Strips • Cut 6 strips of filter paper • Draw a line 1 cm above the bottom edge of the strip with the pencil • Label each strip with its corresponding solution • Place a spot from each pen on your starting line Chapter 4: Techniques in Biochemical Analysis 36
  37. 37. Developing the Chromatograms • Place the strips in the beakers • Make sure the solution does not come above your start line • Keep the beakers covered • Let strips develop until the ascending solution front is about 2 cm from the top of the strip • Remove the strips and let them dry Chapter 4: Techniques in Biochemical Analysis 37
  38. 38. Developing the Chromatograms Chapter 4: Techniques in Biochemical Analysis 38
  39. 39. Developing the Chromatograms Chapter 4: Techniques in Biochemical Analysis 39
  40. 40. Chapter 4: Techniques in Biochemical Analysis 40
  41. 41. Observing the Chromatograms 0% 20% 50% 70% 100% Concentration4:of Isopropanol Chapter Techniques in Biochemical Analysis 41
  42. 42. Black Dye 1. Dyes separated – purple and black 2. Not soluble in low concentrations of isopropanol 3. Partially soluble in concentrations of isopropanol >20% 0% 20% 50% 70% Concentration of Isopropanol Chapter 4: Techniques in Biochemical Analysis 100% 42
  43. 43. Blue Dye 1. Dye separated – blue 2. Not very soluble in low concentrations of isopropanol 3. Completely soluble in high concentrations of isopropanol 0% 20% 50% 70% Chapter 4: Techniques in Concentration of Analysis Biochemical Isopropanol 100% 43
  44. 44. Green Dye 1. Dye separated – blue and yellow 2. Blue – Soluble in concentrations of isopropanol >20% 3. Yellow – Soluble in concentrations of isopropanol >0% 0% 20% 50% 70% Chapter 4: Techniques in Concentration of Analysis Biochemical Isopropanol 100% 44
  45. 45. Red Dye 1. Dyes separated – red and yellow 2. Yellow –soluble in low concentrations of isopropanol and less soluble in high concentrations of isopropanol 3. Red – slightly soluble in low concentrations of isopropanol, and more soluble in concentrations of isopropanol >20% 0% 20% 50% 70% Chapter 4: Techniques in Concentration of Isopropanol Biochemical Analysis 100% 45
  46. 46. Alternative Experiments • Test different samples: – Other markers, pens, highlighters – Flower pigments – Food Colors • Test different solvents: – Other alcohols: methanol, ethanol, propanol, butanol • Test different papers: – Coffee filters – Paper towels – Cardstock – Typing paper Chapter 4: Techniques in Biochemical Analysis 46
  47. 47. Alternative Experiments Chapter 4: Techniques in Biochemical Analysis 47
  48. 48. Alternative Experiments Chapter 4: Techniques in Biochemical Analysis 48
  49. 49. Alternative Experiments Chapter 4: Techniques in Biochemical Analysis 49
  50. 50. Chromatography Instruments Chapter 4: Techniques in Biochemical Analysis 50
  51. 51. • Chromatography techniques • Affinity Chromatography (AC) • Hydrophobic Interaction Chromatography (HIC) • Ion Exchange Chromatography (IEC) • Gel Filtration (GF) • Capillary electrochromatography (CEC) Chapter 4: Techniques in Biochemical Analysis 51
  52. 52. Affinity Chromatography Affinity Chromatography Surface bound with Epoxy, aldehyde or aryl ester groups Metal Interaction Chromatography Surface bound with Iminodiacetic acid + Ni2+/Zn2+/Co2+ (Christian G. Huber, Biopolymer Chapter 4: Techniques in Biochemical Analysis Chromatography, Encylcopedia 52 in analytical chemistry, 2000)
  53. 53. Metal Interaction Chromatography (AC) Points to Note: 1. Avoid chelating agents 2. Increasing incubation time 3. Slow gradient elution Chapter 4: Techniques in Biochemical Analysis (www.qiagen.com) 53
  54. 54. Affinity Chromatography Binding Capacity (mg/ml) medium 12mg of histag proteins (MW= 27kDa) Depends on Molecular weight Degree of substitution /ml medium ~15µmol Ni2+ Backpressure ~43psi Change the guard column filter Chapter 4: Techniques in Biochemical Analysis 54 (Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)
  55. 55. Hydrophobic Interaction Chromatography Biopolymer (phenyl agarose - Binding Surface) Driving force for hydrophobic adsorption Water molecules surround the analyte and the binding surface. When a hydrophobic region of a biopolymer binds to the surface of a mildly hydrophobic stationary phase, hydrophilic water molecules are effectively released from the surrounding hydrophobic areas causing a thermodynamically favorable change in entropy. Temperature plays a strong role Ammonium sulfate, by virtue of its good salting-out properties and high solubility in water is used as an eluting buffer Chapter 4: Techniques in Biochemical Analysis Hydrophobic region 55 (Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)
  56. 56. Ion Exchange Chromatography Fractogel matrix is a methacrylate resin upon which polyelectrolyte Chains (or tentacles) have been grafted. (Novagen) Globular Protein Maintenance of conformation while interacting with tentacle ion exchanger Deformation due to interaction with conventional ion exchanger Chapter 4: Techniques in Biochemical Analysis (www.novagen.com) 56
  57. 57. Gel Filtration Chapter 4: Techniques in 57 Biochemical Analysis (http://lsvl.la.asu.edu/resources/mamajis/chromatography/chromatography.html)
  58. 58. Capillary Electrochromatography • • • • CEC is an electrokinetic separation technique Fused-silica capillaries packed with stationary phase Separation based on electroosmotically driven flow Higher selectivity due to the combination of chromatography electrophoresis and Fused silica tube filled with porous methacrylamide-stearyl methacrylatedimethyldiallyl ammonium chloride monolithic polymers, 80 x 0.5mm i.d., 5.5kV. High Plate count ~ 400,000 Height Equivalent to a Theoretical Plate /Plate Count (HETP) H = L/N number of plates N = 16(t/W)2 where L = column length, t = retention time, and W = peak width at baseline Chapter 4: Techniques in Biochemical Analysis (http://www.capital-hplc.co.uk) 58
  59. 59. CEC columns AC, IEC columns CEC column NP, RP columns Chapter 4: Techniques in Biochemical Analysis 59
  60. 60. Schematic of a Multi-dimensional Separation System Chapter 4: Techniques in Biochemical Analysis 60
  61. 61. Fast Protein Liquid Chromatograph (FPLC) • No air bubbles (Priming) • Use degassed buffers Injector Module 2 1 Column Inlet 3 Detector 4 Fraction 5 Collector Chapter 4: Techniques in Biochemical Analysis (www.pharmacia.com) 61
  62. 62. Chromatography systems ÄKTAprime: simple automated purification ÄKTAFPLC: high performance purification of proteins & other biomolecules ÄKTApurifier: high performance purification and characterization ÄKTAexplorer: for fast method development and scale-up ÄKTApilot ÄKTAxpress: : for high rapid throughput process tagged development and pilotprotein purification Chapter 4: Techniques in Biochemical scale Analysis 62
  63. 63. High Performance Liquid Chromatography (HPLC)  What is HPLC? Types of Separations Columns and Stationary Phases Mobile Phases and Their Role in Separations Injection in HPLC Detection in HPLC  Variations on Traditional HPLC        Ion Chromatography Size Exclusion Chromatography Chapter 4: Techniques in Biochemical Analysis 63
  64. 64. What is HPLC?  High Performance Liquid Chromatography  High Pressure Liquid Chromatography (usually true]  Hewlett Packard Liquid Chromatography (a joke)  High Priced Liquid Chromatography (no joke)  HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time!  Probably the most widely practiced form of quantitative, analytical chromatography practiced today due to the wide range of molecule types and sizes which can be separated using HPLC or variants of HPLC!! Chapter 4: Techniques in Biochemical Analysis 64
  65. 65. Chapter 4: Techniques in Biochemical Analysis 65
  66. 66. Chapter 4: Techniques in Biochemical Analysis 66
  67. 67. Types of HPLC Separations (partial list)  Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded stationary phase.  Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase.  Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica)  Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support.  Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationary phase. Chapter 4: Techniques in Biochemical Analysis 67
  68. 68. Chapter 4: Techniques in Biochemical Analysis 68
  69. 69. Chapter 4: Techniques in Biochemical Analysis 69
  70. 70. Chapter 4: Techniques in Biochemical Analysis 70
  71. 71. What does the analyst do?    Select the correct type of separation for the analyte(s) of interest, based on the sample type (among other factors). Select an appropriate column (stationary phase) and mobile phase Select an appropriate detector based on whether universal or compound-specific detection is required or available  Optimize the separation using standard mixtures  Analyze the standards and sample Chapter 4: Techniques in Biochemical Analysis 71
  72. 72. Chapter 4: Techniques in Biochemical Analysis 72
  73. 73. Columns and Stationary Phases.  HPLC is largely the domain of packed columns   some research into microbore/capillary columns is going on. Molecules move too slowly to be able to reach and therefore “spend time in” the stationary phase of an open tubular column in HPLC.    In solution, not the gas phase Larger molecules in HPLC vs. GC (generally) Stationary phases are particles which are usually about 1 to 20 µm in average diameter (often irregularly shaped)   In Adsorption chromatography, there is no additional phase on the stationary phase particles (silica, alumina, Fluorosil). In Partition chromatography, the stationary phase is coated on to (often bonded) a solid support (silica, alumina, divinylbenzene resin) Chapter 4: Techniques in Biochemical Analysis 73
  74. 74. Chapter 4: Techniques in Biochemical Analysis 74
  75. 75. Stationary Phases  Polar (“Normal” Phase):    Silica, alumina Cyano, amino or diol terminations on the bonded phase Non-Polar (“Reversed Phase”)   C18 to about C8 terminations on the bonded phase Phenyl and cyano terminations on the bonded phase  Mixtures of functional groups can be used!!  Packed particles in a column require:     Frits at the ends of the column to keep the particles in Filtering of samples to prevent clogging with debris High pressure pumps and check-valves Often a “Guard Column” to protect the analytical column Chapter 4: Techniques in Biochemical Analysis 75
  76. 76. Optimization of Separations in HPLC      Correct choice of column so the above equilibrium has some meaningful (non-infinity, non-zero) equilibrium constants. Correct choice of mobile phase Decision on the type of mobile phase composition  constant composition = isocratic  varying composition = gradient elution Determination if flow rate should be constant  usually it is Decision on heating the column  heating HPLC columns can influence the above equilibrium…. Chapter 4: Techniques in Biochemical Analysis 76
  77. 77. Chapter 4: Techniques in Biochemical Analysis 77
  78. 78. The Mobile Phase in HPLC...  Must do the following:    solvate the analyte molecules and the solvent they are in be suitable for the analyte to transfer “back and forth” between during the separation process Must be:     compatible with the instrument (pumps, seals, fittings, detector, etc) compatible with the stationary phase readily available (often use liters/day) of adequate purity   spectroscopic and trace-composition usually! Not too compressible (causes pump/flow problems)  Free of gases (which cause compressability problems) Chapter 4: Techniques in Biochemical Analysis 78
  79. 79. Typical HPLC Pump (runs to 4,000+ psi) Chapter 4: Techniques in Biochemical Analysis 79
  80. 80. Chapter 4: Techniques in Biochemical Analysis 80
  81. 81. Polarity Index for Mobile Phases…..  The polarity index is a measure of the relative polarity of a solvent. It is used for identifying suitable mobile phase solvents.  The more polar your solvent is, the higher the index.  You want to try to choose a polarity index for your solvent (or solvent mixture) that optimizes the separation of analytes       usually the index is a starting point the polarity of any mixture of solvents to make a mobile phase can be modeled to give a theoretical chromatogram Usually, optimization of solvent composition is experimental A similar number is the Eluent Strength (Eo] Increasing eluent strength or polarity index values mean increasing solvent polarity. Remember, the analyte(s) and samples must be mobile phase and stationary phase compatible! Chapter 4: Techniques in Biochemical Analysis 81
  82. 82. Chapter 4: Techniques in Biochemical Analysis 82
  83. 83. Chapter 4: Techniques in Biochemical Analysis 83
  84. 84. Optimization of Mobile Phase Polarity… Changing the mobile phase composition alters the separation. Chapter 4: Techniques in Biochemical Analysis 84
  85. 85. Isocratic versus Gradient Elution  Isocratic elution has a constant mobile phase composition  Can often use one pump!  Mix solvents together ahead of time!  Simpler, no mixing chamber required  Limited flexibility, not used much in research  mostly process chemistry or routine analysis.  Gradient elution has a varying mobile phase composition  Uses multiple pumps whose output is mixed together  often 2-4 pumps (binary to quarternary systems)  Changing mobile phase components changes the polarity index  can be used to subsequently elute compounds that were previously (intentionally) “stuck” on the column  Some additional wear on the stationary phase  Column has to re-equiluibrate to original conditions after each run (takes additional time). Chapter 4: Techniques in Biochemical Analysis 85
  86. 86. Chapter 4: Techniques in Biochemical Analysis 86
  87. 87. Chapter 4: Techniques in Biochemical Analysis 87
  88. 88. Chapter 4: Techniques in Biochemical Analysis 88
  89. 89. Injection in HPLC        Usually 5 to 1000 µL volumes, all directly onto the column  not much worry about capacity since the columns have a large volume (packed). Injector is the last component before the column(s) A source of poor precision in HPLC  errors of 2-3 %RSD are due just to injection  other errors are added to this  due to capillary action and the small dimensions/cavities inside the injector 6-PORT Rotary Valve is the standard manual injector Automatic injectors are available Two positions, load and inject in the typical injector Injection loop internal volume determines injection volume. Chapter 4: Techniques in Biochemical Analysis 89
  90. 90. LOAD (the sample loop) Inject (move the sample loop into the mobile phase flow) Chapter 4: Techniques in Biochemical Analysis 90
  91. 91. Chapter 4: Techniques in Biochemical Analysis 91
  92. 92. Detection in HPLC        Numerous Types (some obscure) Original HPLC Detectors were common laboratory instruments such as spectrophotometers, etc. Must be solvent -compatible, stable, etc. Universal  respond to all analytes Analyte Specific  respond to specific properties of analytes Non-destructive  most Destructive  ELSD, MS and a few others. Chapter 4: Techniques in Biochemical Analysis 92
  93. 93. Chapter 4: Techniques in Biochemical Analysis 93
  94. 94. Standard Absorbance Detector….   Single Beam UV-VIS instrument with a flow-through cell (cuvette) Can use any UV-VIS with a special flow cell   Extra connections lead to band-broadening if UV-VIS is far from HPLC column exit. Usually utilize typical UV-VIS lamps and 254 nm default wavelenth   Can be set to other wavelengths (most) Simple filter detectors no longer widely used   Non-destructive, not-universal    adjustable wavelength units are cost-effective not all compounds absorb light can pass sample through several cells at several different wavelenghts Usually zeroed at the start of each run using an electronic software command. You can have real-time zeroing with a reference cell. Chapter 4: Techniques in Biochemical Analysis 94
  95. 95. Chapter 4: Techniques in Biochemical Analysis 95
  96. 96. SPECTROSCOPY Chapter 4: Techniques in Biochemical Analysis 96
  97. 97. Definition • Spectroscopy - The study of the interaction of electromagnetic radiation with matter Chapter 4: Techniques in Biochemical Analysis 97
  98. 98. Introduction • Spectroscopy is an analytical technique which helps determine structure. • It destroys little or no sample. • The amount of radiation absorbed by the sample is measured as wavelength is varied. Chapter 4: Techniques in Biochemical Analysis 98
  99. 99. Major Types of Spectroscopy     Infrared (IR) spectroscopy measures the bond vibration frequencies in a molecule and is used to determine the functional group. Mass spectrometry (MS) fragments the molecule and measures the masses. Nuclear magnetic resonance (NMR) spectroscopy detects signals from hydrogen atoms and can be used to distinguish isomers. Ultraviolet (UV) spectroscopy uses electron transitions to determine bonding patterns. Chapter 4: Techniques in Biochemical Analysis 99
  100. 100. Introduction of Spectrometric Analyses The study how the chemical compound interacts with different wavelenghts in a given region of electromagnetic radiation is called spectroscopy or spectrochemical analysis. The collection of measurements signals (absorbance) of the compound as a function of electromagnetic radiation is called a spectrum. Chapter 4: Techniques in Biochemical Analysis 100
  101. 101. Energy Absorption The mechanism of absorption energy is different in the Ultraviolet, Infrared, and Nuclear magnetic resonance regions. However, the fundamental process is the absorption of certain amount of energy. The energy required for the transition from a state of lower energy to a state of higher energy is directly related to the frequency of electromagnetic radiation that causes the transition. Chapter 4: Techniques in Biochemical Analysis 101
  102. 102. Spectral Distribution of Radiant Energy Wave Number (cycles/cm) X-Ray UV 200nm Visible 400nm IR Microwave 800nm Wavelength (nm) Chapter 4: Techniques in Biochemical Analysis 102
  103. 103. Electromagnetic Spectrum Chapter 4: Techniques in Biochemical Analysis 103
  104. 104. Electromagnetic Spectrum Chapter 4: Techniques in Biochemical Analysis 104
  105. 105. Electromagnetic Radiation V = Wave Number (cm ) -1 λ = Wave Length C = Velocity of Radiation (constant) = 3 x 1010 cm/sec. υ = Frequency of Radiation (cycles/sec) V = υ 1 = C λ The energy of photon: h (Planck's constant) = 6.62 x 10- (Erg×sec) 27 E = h υh = C λ υ= C Chapter 4: Techniques in Biochemical Analysis λ C = υλ 105
  106. 106. Equation Definitions • E = energy (Joules, ergs) • c = speed of light (constant) • λ = wavelength • h = Planck’s constant • ν = “nu” = frequency (Hz) • nm = 10-9 m • Å = angstrom = 10-10 m Chapter 4: Techniques in Biochemical Analysis 106
  107. 107. Spectral Properties, Application and Interactions of Electromagnetic Radiation Wave Number V Energy Kcal/mol 9.4 x 107 9.4 x 103 9.4 x 101 eV 4.9 x 106 4.9 x 102 4.9 x 100 Wavelength λ cm-1 cm 3.3 x 1010 3 x 10-11 3.3 x 106 3.3 x 104 3 x 10-7 3 x 10-5 Frequenc y υ Type Radiation Type spectroscopy Type Quantum Transition Hz 1021 Gamma ray 1017 X-ray 1015 Ultra violet Gamma ray emission Nuclear X-ray absorption, emission Electronic (inner shell) UV absorption Electronic (outer shell) Visible IR absorption 9.4 x 10-1 4.9 x 10-2 3.3 x 102 3 x 10-3 1013 Infrared 9.4 x 10-3 4.9 x 10-4 3.3 x 100 3 x 10-1 1011 Microwave Microwave absorption Radio Nuclear magnetic resonance 9.4 x 10-7 4.9 x 10-8 3.3 x 10-4 3 x 103 107 Chapter 4: Techniques in Biochemical Analysis Molecular vibration Molecular rotation Magnetically induced spin states 107
  108. 108. Chapter 4: Techniques in Biochemical Analysis 108
  109. 109. Spectrum of Radiation Chapter 4: Techniques in Biochemical Analysis 109
  110. 110. Visible Light Chapter 4: Techniques in Biochemical Analysis 110
  111. 111. Visible Light Red R 700 nm Orange O 650 nm Yellow Y 600 nm Green G 550 nm Blue B 500 nm Indigo I 450 nm Violet V 400 nm Chapter 4: Techniques in Biochemical Analysis 111
  112. 112. Dispersion of Polymagnetic Light with a Prism Prism - Spray out the spectrum and choose the certain wavelength (λ) that you want by slit. Infrared Polychromatic Ray PRISM Red Orange Yellow Green monochromatic Ray SLIT Blue Violet Ultraviolet Polychromatic Ray Monochromatic Ray Chapter 4: Techniques in Biochemical Analysis 112
  113. 113. Ultra Violet Spectrometry The absorption of ultraviolet radiation by molecules is dependent upon the electronic structure of the molecule. So the ultraviolet spectrum is called electronic spectrum. Chapter 4: Techniques in Biochemical Analysis 113
  114. 114. INTRODUCTION TO SPECTROPHOTOMETRY Chapter 4: Techniques in Biochemical Analysis 114
  115. 115. Spectrophotometry • Spectrophotometry: An analytical method using several spectra (lights). (State each spectrum used in spectrophotometry.) • Spectrophotometer: An instrument for measuring absorbance that uses a monochromator to select the wavelength. Chapter 4: Techniques in Biochemical Analysis 115
  116. 116. Spectrophotometry -Advantages of spectrophotometers i. relatively inexpensive ii. inexpensive iii. easy to maintain iv. portable Chapter 4: Techniques in Biochemical Analysis 116
  117. 117. BACKGROUND white light is observed, what is actually seen is a mixture of all the colors of light Why do some substances appear colored? When this light passes through a substance, certain energies (or colors) of the light are absorbed while other color(s) are allowed to pass through or are reflected by the substance. If the substance does not absorb any light, it appears white (all light is reflected) or colorless (all light is transmitted). A solution appears a certain color due to the absorbance and transmittance of visible light. For example, a blue solution appears blue because it is absorbing all of the colors except blue. Chapter 4: Techniques in Biochemical Analysis 117
  118. 118. BACKGROUND Chapter 4: Techniques in Biochemical Analysis 118
  119. 119. BACKGROUND • The amount of light absorbed by a solution is dependent on the ability of the compound to absorb light (molar absorptivity), the distance through which the light must pass through the sample (path length) and the molar concentration of the compound in the solution. • If the same compound is being used and the path length is kept constant, then the absorbance is directly proportional to the concentration of the sample. Chapter 4: Techniques in Biochemical Analysis 119
  120. 120. Spectrophotometer • A spectrophotometer is used to provide a source of light of certain energy (wavelength) and to measure the quantity of the light that is absorbed by the sample. Light Bulb Sample Prism Detector Filter Slit Chapter 4: Techniques in Biochemical Analysis 120
  121. 121. Spectrophotometer • The basic operation of the spectrophotometer includes a white light radiation source that passes through a monochromator. The monochromator is either a prism or a diffraction grating that separates the white light into all colors of the visible spectrum. After the light is separated, it passes through a filter (to block out unwanted light, sometimes light of a different color) and a slit (to narrow the beam of light--making it form a rectangle). Next the beam of light passes through the sample that is in the sample holder. The light passes through the sample and the unabsorbed portion strikes a photodetector that produces an electrical signal which is proportional to the intensity of the light. The signal is then converted to a readable output that is used in the analysis of the sample. Light Bulb Sample Prism Detector Filter Slit Chapter 4: Techniques in Biochemical Analysis 121
  122. 122. Spectrophotometer An instrument which can measure the absorbance of a sample at any wavelength. Light Lens Sample Slit Monochromator Detector Chapter 4: Techniques in Biochemical Analysis Slits Quantitative Analysis 122
  123. 123. The process of light being absorbed by a solution concentration 2 concentration 1 blank where Io = I with sample I < Io light source detector Io I b PGCC CHM 103 Sinex Cell with Pathlength, b, containing4:solution in Chapter Techniques Biochemical Analysis As concentration increased, less light was transmitted (more light absorbed). 123
  124. 124. Beer – Lambert Law Light I0 I Glass cell filled with concentration of solution (C) As the cell thickness increases, the transmitted intensity of light of I decreases. Chapter 4: Techniques in Biochemical Analysis 124
  125. 125. R- Transmittance R= I I0 I0 - Original light intensity I- Transmitted light intensity   I I0 % Transmittance = 100 x Absorbance (A) = Log 1 T = Log Log I0 = 2 - Log%T I I is proportional to C (concentration of solution) and is I0 also proportional to L (length of light path Chapter 4: Techniques in Biochemical 125 through the Analysis solution).
  126. 126. A ∝ CL = ECL by definition and it is called the Beer - Lambert Law. A = ECL A = ECL E = Molar Extinction Coefficient ---- Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution Chapter 4: Techniques in Biochemical Analysis 126
  127. 127. E = Absorbance x Liter Moles x cm UNITS A = ECL A = No unit (numerical number only) E = Liter Cm x Mole L = Cm C = Moles/Liter A = ECL = ( Liter Cm x Mole )x Chapter 4: Techniques in Biochemical Analysis Mole Liter x Cm 127
  128. 128. The BLANK     The blank contains all substances expect the analyte. Is used to set the absorbance to zero: Ablank = 0 This removes any absorption of light due to these substances and the cell. All measured absorbance is due to analyte. PGCC CHM 103 Sinex Chapter 4: Techniques in Biochemical Analysis 128
  129. 129. Beer’s Law A = abc where a – molar absorptivity, b – pathlength, and c – molar concentration See the Beer’s Law Simulator PGCC CHM 103 Sinex Chapter 4: Techniques in Biochemical Analysis 129
  130. 130. Spectrophotometer The spectrophotometer displays this quantity in one of two ways: (1) Absorbance -- a number between 0 and 2 (2) Transmittance -- a number between 0 and 100%. The sample for a spectral analysis is prepared by pouring it into a cuvette which looks similar to a small test tube. A cuvette is made using a special optical quality glass that will itself absorb a minimal amount of the light. It is also marked with an indexing line so that it can be positioned in the light beam the same way each time to avoid variation due to the differences in the composition of the glass Chapter 4: Techniques in Biochemical Analysis 130
  131. 131. Fundamentals of Spectrophotometry Absorption of Light Beer’s Law  The relative amount of a certain wavelength of light absorbed (A) that passes through a sample is dependent on: distance the light must pass through the sample (cell path length - b) amount of absorbing chemicals in the sample (analyte concentration – c) ability of the sample to absorb light (molar absorptivity - ε) Increasing [Fe2+] Chapter 4: Techniques in Biochemical Analysis Absorbance is directly proportional to concentration of Fe+2 131
  132. 132. Fundamentals of Spectrophotometry Absorption of Light 3.) Beer’s Law   Absorbance is useful since it is directly related to the analyte concentration, cell pathlength and molar absorptivity. This relationship is known as Beer’s Law A = abc where: Beer’s Law allows compounds to be quantified by their ability to absorb light, Relates directly to concentration (c) A = absorbance (no units) α = molar absorptivity (L/mole-cm) b = cell pathlength (cm) c = concentration of analyte (mol/L) Chapter 4: Techniques in Biochemical Analysis 132
  133. 133. Fundamentals of Spectrophotometry Absorption of Light 4.) Absorption Spectrum  By choosing different wavelengths of light (λA vs. λB) different compounds can be measured λA λB Chapter 4: Techniques in Biochemical Analysis 133
  134. 134. Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design  An instrument used to make absorbance or transmittance measurements is known as a spectrophotometer Chapter 4: Techniques in Biochemical Analysis 134
  135. 135. Single Beam Spectrophotometer Chapter 4: Techniques in Biochemical Analysis 135
  136. 136. Dual Beam Spectrophotometer Chapter 4: Techniques in Biochemical Analysis 136
  137. 137. Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design  Light Source: provides the light to be passed through the sample Tungsten Lamp: visible light (320-2500 nm) Low pressure (vacuum) Tungsten Filament - - based on black body radiation: heat solid filament to glowing, light emitted will be characteristic of temperature more than nature of solid filament Deuterium Lamp: ultraviolet Light (160-375 nm) In presence of arc, some of the electrical energy is absorbed by D2 (or H2) which results in the disassociation of the gas and release of light D2 + Eelect  D*2  D’ + D’’ + hν (light produced) Excited state Chapter 4: Techniques in Biochemical Analysis 137
  138. 138. Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design  Wavelength Selector (monochromator): used to select a given wavelength of light from the light source Prism: - Filter: Chapter 4: Techniques in Biochemical Analysis 138
  139. 139. Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design  Wavelength Selector (monochromator): used to select a given wavelength of light from the light source Reflection or Diffraction Grating: Chapter 4: Techniques in Biochemical Analysis 139
  140. 140. Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design  Sample Cell: sample container of fixed length (b). - Usually round or square cuvet Made of material that does not absorb light in the wavelength range of interest 1. Glass – visible region 2. Quartz – ultraviolet 3. NaCl, KBr – Infrared region Chapter 4: Techniques in Biochemical Analysis 140
  141. 141. Cuvettes (sample holder) • Polystyrene – 340-800 nm • Methacrylate – 280-800 nm • Glass – 350-1000 nm • Suprasil Quartz – 160-2500 nm Chapter 4: Techniques in Biochemical Analysis 141
  142. 142. Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design  Light Detector: measures the amount of light passing through the sample. - Usually works by converting light signal into electrical signal Photomultiplier tube Process: a) light hits photoemissive cathode and e- is emitted. b) an emitted e- is attracted to electrode #1 (dynode 1), which is 90V more positive. Causes several more e- to be emitted. c) these e- are attracted to dynode 2, which is 90V more positive then dynode 1, emitting more e-. d) process continues until e- are collected at anode after amplification at 9 dynodes. e) overall voltage between anode and cathode is 900V. Chapter 4: Techniques in f) one photon produces 106 – 107 electrons.142 Biochemical Analysis g) current is amplified and measured
  143. 143. Applications of Spectrophotometry Quantitative Applications • Usually using UV-Vis • IR can be used - Environmental applications; analysis waters & waste waters - Clinical applications: analysis of glucose - Industrial analysis; analysis of iron content in food - Forensic applications: Determination of blood alcohol Chapter 4: Techniques in Biochemical Analysis 143
  144. 144. Advantage of spectrophotometer over colorimeter    can be used to profile printers & scanners, measure colors "in the wild", measure your illumination colorimeter measures only 3 points on the specturm (RGB), while a spectrophotometer measures many points across the entire spectrum colorimeters use a single type of light (such as incandescent or pulsed xenon) Spectrophotometers can compensate for this shift, making spectrophotometers a superior choice for accurate, repeatable color measurement. Chapter 4: Techniques in Biochemical Analysis 144
  145. 145. Sample Cells UV Spectrophotometer Quartz (crystalline silica) Visible Spectrophotometer Glass Chapter 4: Techniques in Biochemical Analysis 145
  146. 146. Light Sources UV Spectrophotometer 1. Hydrogen Gas Lamp 2. Mercury Lamp Visible Spectrophotometer 1. Tungsten Lamp Chapter 4: Techniques in Biochemical Analysis 146
  147. 147. Chemical Structure & UV Absorption Chromophoric Group ---- The groupings of the molecules which contain the electronic system which is giving rise to absorption in the ultra-violet region. Chapter 4: Techniques in Biochemical Analysis 147
  148. 148. UV Spectrometer Application Protein Amino Acids (aromatic) Pantothenic Acid Glucose Determination Enzyme Activity (Hexokinase) Chapter 4: Techniques in Biochemical Analysis 148
  149. 149. Flurometric Application Thiamin (365 nm, 435 nm) Riboflavin Vitamin A Vitamin C Chapter 4: Techniques in Biochemical Analysis 149
  150. 150. Visible Spectrometer Application Niacin Pyridoxine Vitamin B12 Metal Determination (Fe) Fat-quality Determination (TBA) Enzyme Activity (glucose oxidase) Chapter 4: Techniques in Biochemical Analysis 150
  151. 151. Major Types of Light Spectroscopy  Absorption spectroscopy     Atomic emission spectroscopy     Samples fluoresce when they emit at higher λ than what they absorb Measures solvent interactions, distances, molecular shape, and motion Circular Dichroism spectroscopy    Measures light emitted from burned sample Elemental analysis Fluorescence spectroscopy   Measures amount of light absorbed Most common, non-destructive Concentration, pH measures, purity, ID Absorption of circular polarized light Chiral compound identification Transmission spect. (colorimetry) Chapter 4: Techniques in Biochemical Analysis 151
  152. 152. Introduction  Atomic absorption is the absorption of light by free atoms. An atomic absorption spectrophotometer is an instrument that uses this principle to analyze the concentration of metals in solution. The substances in a solution are suctioned into an excited phase where they undergo vaporization, and are broken down into small fragmented atoms by discharge, flame or plasma. Chapter 4: Techniques in Biochemical Analysis 152
  153. 153. Atomic Emission Spectroscopy  By exposing these atoms to such temperatures they are able to “jump” to high energy levels and in return, emit light. The versatility of atomic absorption an analytical technique (Instrumental technique) has led to the development of commercial instruments. In all, a total of 68 metals can be analyzed. Chapter 4: Techniques in Biochemical Analysis 153
  154. 154. Advantages of AA        Determination of 68 metals Ability to make ppb determinations on major components of a sample Precision of measurements by flame are better than 1% rsd. There are few other instrumental methods that offer this precision so easily. AA analysis is subject to little interference. Most interference that occurs have been well studied and documented. Sample preparation is simple (often involving only dissolution in an acid) Instrument easy to tune and operate Chapter 4: Techniques in Biochemical Analysis 154
  155. 155. Atomic emission spectrometer Chapter 4: Techniques in Biochemical Analysis 155
  156. 156. NEXT CLASS: Chapter 5 DNA Technology THANK YOU Chapter 4: Techniques in Biochemical Analysis 156

×