Lecture 12 Molecular Instrumentation

Lecture 11 Molecular Instrumentation  Centrifuge  Spectrophotometer Spectrometer  Mass Spectrometer  HPLC  NMR  DNA Microarray Lab on a Chip

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Principles and Applications of Ultracentrifugation

Introduction  Centrifuges have been in use since mid-1800  Low-speed (<10,000rpm) - Purify and harvest intact animal and plant cells, chemical precipitates, and micro-organisms.1  High-speed (6000~20,000rpm) - Purify and isolate mitochondria, viruses, chloroplasts, and Golgi membranes etc.1  Ultra-speed (30,000~120,000rpm) - Purify and isolate endoplasmic reticulum, ribosomes, plasmids, DNA, RNA, and proteins.1  Theodor Svedberg – Nobel Prize in Chemistry in 1926

Introduction-cont’d  Preparative & Analytical Ultracentrifuges  Preparative Ultracentrifuge: Used to prepare and purify a sample for subsequent experimental usages.2  Analytical Ultracentrifuge: Used to accurately characterize sample properties such as molecular weight, stoichiometry, conformation, and shape.2  This paper examines the theory and concept behind centrifugation, discusses the important rotor designs, and introduces various applications of ultracentrifuges.

Theory - 1 •

Centrifugal Force = Mω2r where M is the molecular weight, ω is the angular velocity (rad/sec), and r is the radius of rotation • Buoyant Force = Mω2rVρ where V is the volume displaced by the molecule and ρ is the density of the solvent (g/ml) 4. Frictional Force = f(v) = f (dr/dt) where f is the frictional coefficient and dr/dt is the rate of sedimentation for a spherical molecule, f=6πηrm where η is the viscosity of the medium and rm is the radius of the molecule. 1 3 4

Theory - 2 4. Centrifugal Force = Buoyant Force + Frictional Force Mω2r = Mω2rVρ + f (dr/dt) Mω2r - Mω2rVρ = f (dr/dt) M (1-Vρ) ω2r = f (dr/dt) M = [f/(1-Vρ)] × [(dr/dt)/ ω2r] 7. Sedimentation Coefficient (s) s = v/ ω2r = (dr/dt)/ ω2r M = (f)(s)/(1-Vρ) or s = M(1-Vρ)/f since D=RT/f, where D is diffusion constant, R is gas constant, and T is absolute temp, f = RT/D  s = M(1-Vρ)D/RT One unit of 10-13 seconds is defined to be one Svedberg unit. 1 3 4

Theory - 3 1. Centrifugation F = ω2r F is expressed in g’s where g=980 cm/S2 Relative centrifugal force (RCF) = ω2r/980 However, rotor speed is commonly expressed in revolutions per minute (rpm) where ω = π (rpm)/30

or

Therefore, RCF = 11.17r (rpm/1000)2 where r is in cm RCF = 28.38r (rpm/1000)2 where r is in inches. 1 3 4

Design-1 Four main components to a Centrifuge:5 Driver and Speed Control - negative feedback control - prevent overspeeding Temperature Control - thermocouple temperature control Vacuum System - mechanical pump - water-cooled diffusion pump Rotors - Fixed-angle rotor - Swinging-bucket rotor - Near vertical rotor - Vertical rotor - Zonal rotor

Design-2 1. Fixed Angle Rotor:

θ x

Figure 1: Fixed Angle Rotor (angle set between 20 and 45 degrees)4

2. Swinging Bucket Rotor: Start of centrifugation at 90 degrees orientation x

Supernatant

End of centrifugation with pellet at bottom of tube Pellet

Figure 2: Swinging Bucket Rotor4

Design-3 3. Near Vertical Rotor:

4. Vertical Rotor:

θ Near Vertical Rotor with θ = 7~100 16

5. Zonal Rotor:4

Vertical Rotor16

Application-1 Table 1: Summary of Ultracentrifuge Separation Methods 1 Preparative Ultracentrifugation

Analytical Ultracentrifugation

Differential centrifugation - pelleting

Sedimentation velocity – zonal

Density gradient centrifugation - rate zonal

Sedimentation equilibrium - isopycnic

Density gradient centrifugation – isopycnic

Table 2: Summary of Classes of Centrifuges and their Applications4

Application-2 I. Preparative Ultracentrifugation: a. Differential Centrifugation – Pelleting

b. Density Gradient Centrifugation: Rate Zonal

Figure 6: Rate Zonal Centrifugation14 Figure 5: Differential centrifugation – pelleting15

c. Density Gradient Centrifugation: Isopycnic

Figure 7: Isopycnic Centrifugation14

Application-3 Table 3: Different Rotors and Applications4

Application-4 II. Analytical Ultracentrifugation: Table 1: Sedimentation Velocity vs. Sedimentation Equilibrium12 Sedimentation Velocity

Sedimentation Equilibrium

Shape and size

Molecular Weight

Number of species in solution

Stoichiometry

Diffusion constant

Oligomerization

1. Sedimentation Velocity Experiment:

Concentration

Radius Figure 8: Double-sector centerpiece. The sample is placed in one sector and the reference is in the other.9

Figure 9: Concentration vs. radius9

Application-5 II. Analytical Ultracentrifugation: cont’d 1. Sedimentation Equilibrium Experiment:

Figure 10: Schematic representation of sedimentation equilibrium.9

Figure 11: c1 (white circles, 40kD), c2 (triangles, 80kD), and c3(dark circles, c1+c2).9

Newest Technology 1. Absorbance Optical System (Scanning UV/Vis): - scanning monochromator - xenon flashlamp - measure wavelength range of 200 to 800nm - most effective with macromolecules with strong chromophores.11

2. Interference Optical System: - Rayleigh interference optical system - Uses change in refractive index to measure sample concentrations - most effective with macromolecules that lack intense chromophores.11 Figure 12: Absorbance Optical System10

Medical Instrumentation: The Spectrophotometer

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Principles Behind the Spectrophotometer Light Source Refracting Prism Wavelength Selector

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Principles Behind the Spectrophotometer Sample Photoelectric Tube Digital Display

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Principles Behind the Spectrophotometer Beer-Lambert Law states: – There exists a linear relationship between absorbance and concentration of an absorbing species A = log10(I/I0 ) A=Kcl 20

Principles Behind the Spectrophotometer Beer-Lambert Law

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Operation of a Spectrophotometer Setting the Wavelength Cuvettes Sample Holder Take Reading 22

Application and Future of the Spectrophotometer UV/Visible Spectrophotometer Fluorescence Imaging MicroSpectrophotometer Dual Beam Spectrophotometer Multi-channel Spectrophotometer

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Application and Future of the Spectrophotometer 'Star Trek' technology for cancer screening – Portable device using spectrophotometer principles to help diagnose medical conditions in dermatology, specific cancers, skin disorders, etc. – See: http://www.laboratorytalk.com/news/spa/spa100.html

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An Overview of Mass Spectrometry Principles of Operation

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Abstract Mass spectrometry (MS) and its role in cellular &molecular instrumentation Concepts and principles of operation Pertinent operating techniques State of the art commercial product: IonSpec Explorer Mass Spectrometer 26

Introduction Analytical tool – Molecular and structural identification of unknown compounds – Quantifying known materials – Analysis of peptides, oligonucleotides & proteins

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Design/Methods – Operating Principles Operating Principle: molecular weight evaluation – Production of gas-phase ions – Separation by mass to charge ratio (m/z) – Detection in proportion of relative abundance

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Design/Methods – Basic Components Method/device: introduce sample Ionizing source: produce ions Analyzer(s): separate product ions Detectors: detect the various product ions Vacuum: prevent collision of ions Computer: increase processing, analytical power & speed 29

Design/Methods: Tandem MS Block diagram of a typical tandem MS Introduction of sample sample Ionizing source data system control

ions Analyzer 1 parent ions Analyzer 2 daughter ions Detector

signal

data Computer

Mass Spectrum

modified from De Hoffmann, Charette, Stroobant, Mass Spectrometry Principles and Application, 1996, p. 8

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Design/Methods: Ionizing Sources Thermionic emission from heated filament Electron beam strikes sample molecules (g) Collision produces positively charged molecules Molecular ion: XY + e- → XY+ + 2eFragment ions: XY + e- → X+ + Y + 2e-

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Design/Methods: Ionizing Sources Electrospray ionization (ESI) – Ions from solution (sample & organic solvent) – Capillary tube: produce droplets – Strong electric field applied: charge droplets – “Coulombic explosion” => smaller droplets – Allows sensitive analysis of large molecules – Able to be coupled with liquid chromatography (LC) 32

Design/Methods: Ionizing Sources

Typical layout of an ESI source (from Cambridge University Mass Spectrometry Server, wwwmethods.ch.cam.ac.uk/meth/ms/theory/history.html

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Design/Methods: Ionizing Sources Matrix Assisted Laser Desorption Ionization (MALDI) – – – –

Efficient direct energy transfer Sample co-crystallized with matrix compound Laser radiation: evaporate and form ions Excited cluster made up of matrix molecules and single anayle molecule – Matrix molecules evaporate => High yields of intact analyte – High accuracy and sub-picomolar sensitive – Sample need not be in gaseous phase 34

Design/Methods: Ionizing Sources

Illustration of ion formation via MALDI (from Scripps Center for Mass Spectrometry, http:\\masspec.scripss.edu)

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Design/Methods – Analyzers Separate ions according to m/z ratios Relevant intensity recorded 2 common types of MS – Field aka Sector – Time of Flight (ToF) 36

Design/Methods – Analyzers Field (aka sector) – Magnetic and electric fields applied – Deviation of ion from path – Ions have an initial kinetic energy from momentum generated under an accelerating potential – Under magnetic field B, magnetic force causes ion to subscribe a circular trajectory of radius r – Electric field passes ions further according to their kinetic energy => improved resolution – Ions of m/z sorted according to radius and kinetic energy 37

Design/Methods – Analyzers mv 2 Kinetic energy, Ek =zVs = 2

Equating magnetic force: mv 2 m r 2B2 qvB=  = r z 2Vs Equating electric field force: Diagram of a typical two-sector (electrostatic/magnetic) mass spectrometer (from Cambridge University Mass Spectrometry Server, wwwmethods.ch.cam.ac.uk/meth/ms/theory/history.html

mv 2 m 2Ek qE=  = r z qE 38

Design/Methods – Analyzers Time of Flight (ToF) – Ions accelerated with same potential (same KE) – Ion masses differ => time of flight differs (Smaller or more highly charged ions reach detector first)

mv 2 Kinetic energy, Ek =zVs = 2 d charge, q = ze Total Time of flight, t= v 2

m  d2  m  t 2 t     2 eV  s   z  2Vs e  z  d

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Design/Methods – Detectors Detect charged ion – Intensity, position, and/or timing of arrival

Dynodes/Faraday Cup/Electron multiplier – Ions strikes metal plate, change in charges – Measurable current flow – Electron multiplier: cascaded dynodes

Photomultiplier – Scintillation: Production of photons as ions strike phosphorescent surface 40

Results/Data Main performance characteristics – Upper mass limit ~ highest m/z measurable – Transmission ratio ~ ratio of number of ions reaching the detector to that produced at source – Resolution ~ ability yield distinct signals

Upper mass limit improved – by new ionization sources (ESA, MALDI) – from 650 Da to >12,000 Da 41

Results/Data Improved resolution & sensitivity of mass analyzers and through use of tandem spectrometers

Resolution capabilities of normal mass spectrometers

Resolution capabilities of MALDI Tof-Tof spectrometer

(from the British Mass Spectrometry Society, www.bmss.org.uk) (from the British Mass Spectrometry Society, www.bmss.org.uk)

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Results/Data Overall performance improvements (greater analytical capabilities) by tandem mass spectrometry

A typical result from tandem mass spectrometry (from University of Leeds MRes Informatics, www.bioinf.leeds.ac.uk/mres/home.html )

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Discussion: Fourier Transform MS IonSpec’s Explorer Mass Spectrometer – State of the art commercially available product – Utilizes both ESI & MALDI ionization sources – Uses Fourier transform ion cyclotron resonance mass spectrometry (FTMS) for mass analysis & detection – Quick analysis, extremely high resolution (869,000 Da), high accuracy (0.9 ppm) and good sensitivity (50:1 Sound-noise ratio)

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Discussion: Fourier Transform MS Theoretically unlimited mass limit – Use of ion trap

Speed of analysis, resolution, accuracy – Measures whole frequency spectrum – Frequency measurement most accurate

Picture of IonSpec’s Explorer Mass Spectrometer (from IonSpec, www.ionspec.com)

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Conclusion Mass spectrometry is a valuable tool – Current resolution, sensitivity, accuracy & mass limit capabilities are pretty good => enable much analysis of peptides, oligonucleotides, proteins

Technology will improve further – Current limitations: high cost associated with high performance (tandem) mass spectrometers, slow analytical times due to complex molecules – Future: Further technological advances, more widespread usage (economies of scale) and even higher computer processing power 46

High Performance Liquid Chromatography

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What is HPLC? High Performance Liquid Chromatography (HPLC) is a chromatographic method used to separate molecules from an overall larger mixture – Molecule size range: large proteins and polynucleotides to smaller inorganic molecules – Soluble and insoluble

The term itself was coined to describe the separation of molecules under high pressure in a stainless steel column filled with a matrix 48

HPLC History  HPLC was first developed in the mid 1970’s as an evolution of high pressure liquid chromatography – Before this time, most scientists had to rely on methods such as open-column, paper, and thin-layer chromatogrpahy – These methods were inadequate for the quantification of compounds and the resolution of similar compounds

 By the 1980’s, HPLC became the dominant assay – New techniques improved separation, identification, purification, and quantification far above previous methods – Moreover, computer analysis tools and automation made these HPLC systems even more convenient

 Currently, HPLC is used by a variety of fields including biomedical research, the cosmetics, food, and energy industries and numerous environmental groups

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Background Liquid Chromatography (LC) LC is a chemical separation process that uses a mobile and stationary phase to purify mixed solutions into individual compounds. The separation occurs from the subtle chemical interactions of the mixture with the two phases. This causes different compounds to travel faster or slower than others based on those chemical interactions, which is then recorded by the detector. High Performance Liquid Chromatography (HPLC) As instrumentation for LC became more advanced, it became necessary to use high pressure pumps to force the flow of mixed solutions through the phases. These advances allowed for better detection, higher sensitivity, and faster analysis. This became known as high pressure LC. Later on, when low pressure techniques were also developed, the name was changed to high performance LC.

Figure 15 An example of an HPLC output. A plant extract has been separated to detect ecdysteroids.

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Basic HPLC Theory HPLC works on the principle of absorption followed by elution Two main sections: Mobile Phase and Stationary Phase – Mobile Phase: Liquid carrier for the sample solution; refers to the continuous application of the solvent with sample to the column – Stationary Phase: The solid support structure, contained within the main column, that the mobile phase continuously flows over; sample molecules migrate through and get eluted from the stationary phase

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Basic HPLC Theory  While in contact with the mobile phase, the stationary phase takes away molecules of the sample in a selective manner  The sample molecules form distinct bands in the column and move through the stationary phase according to their absorption properties  Later on in the process, the mobile phase takes the absorbed sample away from the stationary phase in order to separate the various components  The stronger the interaction of the sample with the stationary phase, the longer it takes for the mobile phase to pull it back out, and vice versa 52

Theory Introduction Chromatographic separation is based on chemical properties such as: – – – –

Polarity Size, Molecular Weight Hydrophilicity Functional Groups

The analyte (mixture to be separated) is dissolved into the mobile phase (eluent) and then pumped into a column containing the stationary phase. As the analyte passes through the column, interactions between it and the phases will separate the individual components of the analyte.

Mobile Phase The mobile phase is a solvent that is continuously passing through the column’s stationary phase. It allows the analyte to flow through the column. The properties of the mobile phase are defined by which type of HPLC method is being used. Normally, the chemical properties of the mobile phase are very different from the stationary phase to maximize separation.

Stationary Phase Small porous solid particles are packed tightly inside the column. This packing is called the stationary phase. The size of the particles, size of the pores, and chemical properties on the surface of the particles affect how the analyte and mobile phase will react to it. Particle sizes are usually between 3 to 5 micrometers3.

Separation For example, if the stationary phase consists of large, densely packed particles, then smaller analyte components will travel faster through the column, and will be detected earlier. If the particles have pores in them, then small compound will become trapped inside. Thus, larger components would travel faster in this case.

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Test Results From Column  The elution profile shows the results from a BIOAdvantage HL C18 4.6 x 150mm column with Hodges Peptide test mix, a challenging peptide mixture that is used to quantify HPLC peptide column performance  Shows very little noise and good resolution, despite the similarity of the compounds  Good reproducibility of results

Fig 8: Hodges Peptide test mix with peak identities: 1. H2N-RGAGGLGLGK-amide 2. AC-RGGGGLGLGK-amide 3. AC-RGAGGLGLGK-amide 4. AC-RGVGGLGLGK-amide 5. AC-RGWGLGLGK-amide

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Stationary Phase Types  Liquid-solid – Based on polarity; polar compounds bind more strongly

 Liquid-liquid

 Normal phase – Stationary phase is polar, mobile phase is non-polar; non-polar (hydrophobic) compounds elute faster

– Same as liquid-solid but better for  Reverse phase medium polarity entities – Stationary phase is non Size-exclusion polar (n-alkyl ligands), – Based on molecular size; larger mobile phase is polar; polar molecules are excluded and elute (hydrophilic) compounds first elute faster

 Ion-exchange

– Selective exchange of ions in sample with counterions in phase; sample is retained by replacing its ions with the counterions

 Affinity – Use immobilized biochemicals that have a specific affinity for the compound of interest; other compounds elute faster 55

Mobile Phase Types  Isocratic – Compounds are eluted with a mobile phase of constant composition – Advantages: Simple and inexpensive – Disadvantages: Questionable resolution and extended retention of certain compounds

 Gradient – Uses increasingly stronger organic solvents in order to elute different compounds; strength increased in a stepwise or linear manner – Same results as Isocratic but better bandwidth of elution profile peaks

 Polytyptic – Mixed-mode; uses many different techniques in the same column – The columns contain rigid, macroporous hydrophobic resins covalently bonded to a hydrophilic organic layer. By changing the mobile phase, the mode of separation is thereby changed which allows the user to achieve the desired selectivity in the separations 56

Applications: Peptide/Protein HPLC using Reverse Phase Chromatography (RPC)  Five main categories of HPLC applications: – Purification – Identification – Preparation (combination of purification and identification) – Quantification – Chemical Separation

 Myriads of molecules and compounds that can be studied by simply changing mobile and stationary phase conditions

 RPC: the most common form of HPLC  A common and vital step in the process of synthetic peptide production and the purification of natural peptide sequences – Uses analytical columns that are more suited towards identification and quantification, but preparative in nature due to the low number of active proteins in tissue – Biological activity is usually maintained, as well as secondary and tertiary structure of the protein

 Most commercially available RPC columns use C8 (octyl chain) or C18 (octadecyl chain) phases to retain hydrophobic peptide residues 57

RPC HPLC of Proteins/Peptides  The BIOAdvantage column manufactured by Thomson Instruments is one of the more reputable RPC columns on the market  Specifications – – – – – –

Silica based (ultra clean silica) 3 or 5 μm particle size 100Å or 300Å pore size C8 or C18 phases 0.17 to 4.6mm interior diameter 30 to 250mm in length

 High resolution with equal selectivity between all model variations: allows user to maintain elution order throughout different experiments => better reproducibility  Modular design: can be used in almost any machine through the use of a column-specific, adapter-like guard column 58

Theory Types of HPLC Adsorption An adsorbent stationary phase is used to separate compounds with repeated adsorption-desorption processes. There are two types of adsorption HPLC: –

Normal Phase: a polar stationary phase with a nonpolar mobile phase

–

Reverse Phase: a nonpolar stationary phase with a polar mobile phase

Ion-Exchange Ionically charged stationary phase is used to attract oppositely charged particles in the analyte.

Detection The detection of compounds inside the column are recorded with respect to time. A voltage output is correlated to the detection peak. Fig 2 shows a sample output2. The capacity factor, k’, gives the peak of the eluting compounds relative to the unretained solute. t t k' R 0 t0 Resolution tells how close the spacing between two peaks can be. Rs 

t R 2  t R1 0.5( w1  w2 )

Size Exclusion Smaller molecules are separated from the larger ones in the analyte by packing pores of a specific size inside the column.

59 Fig. 2

Instrumentation HPLC System Figure 3 shows the general setup for an HPLC system. The sequence of events are as follows: 1. The mobile phase is fed into the pump, which pushes it through the tightly packed column. 2. As the mobile phase is flowing through the column, the analyte is injected via the injection valve. 3. The loop shown in Figure 3 is used to maintain consistency in flow and pressure as the analyte enters the column. 4. The detector’s output is then sent to the data station. 5. The user controls all configurations through the interface, which can be manual or computer based.

Fig. 3 HPLC system setup1

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Instrumentation Pump The pump maintains the constant flow of eluent and analyte into the column. Standard HPLC pump requirements are1 : – –

Flow rate range: from 0.01 to 10 ml/min Pressure range: from 1 to 5,000 psi

–

Pressure pulsations: less than 1% for normal and reversed phase mode less than 0.2% for size exclusion mode.

•

The most popular type is the reciprocating piston pump (fig. 4). This pump allows for an unlimited supply of eluent. However, because of its nature, a pulse damper or dual reciprocating piston pump must be used to counter the pulse effects (fig. 5).

Injector The injector allows the analyte to enter the column without interrupting the mobile phase flow, because the detector is sensitive to changes in flow rate and pressure.

Fig. 4 Reciprocating piston pump

Column Columns are normally 5 to 25 cm in length with an inner diameter of about 4 mm3. They are usually made of stainless steel because it is relatively inert and can withstand high pressures. Columns typically have an internal pressure of 24 to 100 bar2. The stationary phase packing material is dependent on the HPLC method and analyte composition. The most common packing material is silica.

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HPLC Systems  All HPLC systems have the same basic components; the only system innovation deals with the detector (RI, UV, MS, Near-IR, NMR, LS) and the computer system  Different molecules can be studied by changing the specific combination of the mobile phase, stationary phase, and detector Delivers solvent at Simple jar to hold solvent uniform rate; high pressure b/c SP is small, tight particles Injection Solvent Pump Port Reservoir

Protects column from dirt/contaminants and adapts it to other HPLC systems

Loads sample and injects it into mobile phase Guard Column

Column Detector Computer Holds stationary Phase

Back Pressure Regulator

Waste Reservoir

Simple jar to hold waste Modern HPLC System

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Instrumentation Detector There are many different types of detectors that can be used with HPLC. The key characteristics of all are: – – – – – –

Low drift and noise High sensitivity Fast response Linearity Low dead volume Insensitivity to changes in conditions

Detectors are usually attached to a separate cell at the end of the column. The following are the different types:

Refractive Index (RI) RI detector is the only universal HPLC detector. It simply measures the change in refractive index of the solution flowing inside the column.

Photodiode Array (PDA) The PDA is similar to the UV detector but it allows for further qualitative analysis by being able to detect absorption at specific wavelengths.

Fluorescent A beam of xenon or deuterium radiation is emitted inside the flow cell to detect fluorescence of conjugated systems of various compounds.

Nuclear Magnetic Resonance (NMR), Mass Spectrometry (MS) The latest advancements in HPLC deal with combining the separating process of HPLC with analytical detection methods of NMR and MS. The combination gives faster identification of molecular species in one single test5.

UV Absorbance The absorbance of UV light is detected based on the Beer-Lambert law: A = ecb where A = absorbance, b = length of cell, and e = molar absorptivity2.

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Applications Uses for HPLC

Chemical Detection in Food

HPLC is used in a wide variety of fields, Here, food dyes were analyzed (fig. 6) and then including pharmaceutical, petrochemical, and electronic industries. The following are detected in everyday foods, such as orange soda (fig 7)7. just a few examples of HPLC applications.

Genetic Sampling HPLC can be used to separate and analyze various DNA components. Figure 5 shows data from a mixture of purine bases, nucleosides, and nucleotides using on-line reverse phase (ODS) HPLC with anion exchange HPLC6. Fig. 6 Food dye detection

Fig. 5 Genetic material mixture Peaks: 1 – 8 = bases and nucleosides 9 – 21 = nucleotides

Fig. 7 Food dye detection in orange soda

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Conclusion HPLC has proven to be the most powerful of all chromatographic techniques. The benefits of HPLC are: – Separation and detection of compounds that would be difficult or impossible to detect in other types of separation methods – Can separate a wide range of compounds in a single run – Versatility in choosing mobile and stationary phase types – Fast and highly efficient – Affordable

In addition to these benefits of using HPLC, advancement in HPLC technology are continuously being made. Future focus will be on optimizing HPLC-NMRMS systems.

References 1. Yuri Kazadevich, “Textbook on High Performance Liquid Chromatography”, http://hplc.chem.shu.edu/HPLC/index.html, last modified January, 2002. 2. Sandie Lindsay, “High Performance Liquid Chromatography, Second Edition”, John Wiley & Sons, Inc, New York, 1992. 3. John G. Dorsey, "Liquid chromatography", in AccessScience@McGraw−Hill, http://www.accessscience.com/server−java/Arknoid/science/AS/Encyclopedia/3/38/Est_386200_frameset.html, last modified: June 3, 2002. 4. Richard A. Henry, “A Perspective on High-Performance Liquid Chromatography”, American Laboratory, June 2002, 25-31. 5. I. D. Wilson, "HPLC−combined spectroscopic techniques", in AccessScience@McGraw−Hill, http://www.accessscience.com/server−java/Arknoid/science/AS/ResUpdates/2002/YB_020515_frameset.html, last modified: March 25, 2002. 6. Richard L. Patience, and Elizabeth S. Penny, HPLC in Endocrinology, Advances in Chromatography 1987; 27: 38-69. 7. Harold T. McKone, “An Introduction to High Performance Liquid Chromatography: Separation of Some FD&C Dyes”, Journal of Chemcial Education, 1980, 57:321-2.

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Nuclear Magnetic Resonance An Introduction to Spectroscopy

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Introduction Was discovered by Bloch and Purcell in 1946 Is used as a cross-disciplinary tool Relies on nuclear spins excited by external magnetic fields Is rich in measurable characteristics (strength, frequency, decay) that convey details of structure and environmental interaction 67

Nuclear Magnetic Resonance  Main magnetic field – Align all spins with main field – Larmor frequency

ω0 = γB0

Fig. 1. Equilibrium condition due to main magnetic field

 Radiofrequency pulse – Excite all nuclei – Tilts spins by 90° – Spins precess back to equilibrium state

Fig. 2. Magnetization after a 90° RF pulse

D. Nishimura, Principles of Magnetic Resonance Imaging, MRSRL Press, 1996.

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Applications in NMR Spectroscopy  Elements can be identified and their atomic and molecular structure elucidated by studying the spectral lines  Use magnetic fields to excite material; then measure change in magnetic moment

Fig. 3.

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P NMR Spectrum

 Chemical shift D. Nishimura, Principles of Magnetic Resonance Imaging, MRSRL Press, 1996.

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Spectroscopy Instrumentation Continuous wave Fourier transform Operation:

Fig. 4. Free Induction Decay

– – – –

Select sample Pulse sample Measure FID Find Fourier transform of FID – Make prediction

Fig. 5. Fourier transformed FID

J. Edwards, “Principles of NMR,” Process NMR Associates LLC, 1999.

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Foxboro Process NMR Analyzer Seven components: – – – – – – –

Central computer Switching control unit Shimming control unit Power supply Magnet Probe Heater control unit Fig. 6. Foxboro Process NMR

Analyzer, Model NMRB, Style C

Foxboro Company, The, “I/A Series: Process NMR Analyzer, Model NMRB, Style C,” Datasheet, The Foxboro Company (Foxboro, MA), 2000. 71

Research Involving Spectroscopy  Chemistry Department: – Molecular dynamics – Inter-molecular recognition – Protein organization

 Kennedy Krieger Institute – – – –

fMRI and NMR spectroscopy Cellular function in brain Metabolite levels and fluxes Relation to diseas

Fig. 7. Human spectroscopic data for several slices of the brain

A. Horska, W.E. Kaufmann, L.J. Brant, S. Naidu, J.C. Harris, and P.B. Barker, “In vivo quantitative proton MRIS study of brain development from childhood to adolescence,” Journal of Magnetic Resonance Imaging, vol. 15(2), pp. 137 – 43, 2002. 72

Current Issues in Spectroscopy  Technology – Stronger signal – Higher bandwidth – Larger external magnetic fields – Hybridized magnets

 Research – Improve signal quality, acquisition speed and SNR – Hardware – Procedures

Fig. 8. Department of Energy’s new 900 MHz magnet (largest in the world)

S. Maloof, US Department of Energy, “World’s largest, most powerful NMR spectrometer,” US Department of Energy Research News, Apr. 2002. 73

Summary Strong correlations between NMR spectra and molecular organization Reasonable predictions for structure and function of unknown substances Growing understanding of organ function in medical studies Developing technology spanning many fields and applications 74

References 

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E. Atalar, course notes for 580.473: Magnetic Resonance Imaging in Medicine, Johns Hopkins University, Department of Biomedical Engineering, Baltimore, MD, unpublished, 2003. F. Bloch, “Nuclear induction,” Physical Review, vol. 70, pp. 460 – 473, 1946. P. Bottomley, “Instrumentation for MRI and MRS,” course notes for 580.473: Magnetic Resonance Imaging in Medicine, Johns Hopkins University, Department of Biomedical Engineering, Baltimore, MD, unpublished, 2003. P. Bottomley, “Introduction to magnetic resonance spectroscopy in biomedicine,” course notes for 580.473: Magnetic Resonance Imaging in Medicine, Johns Hopkins University, Department of Biomedical Engineering, Baltimore, MD, unpublished, 2003. P. Bottomley, “Spatially localized magnetic resonance spectroscopy,” course notes for 580.473: Magnetic Resonance Imaging in Medicine, Johns Hopkins University, Department of Biomedical Engineering, Baltimore, MD, unpublished, 2003. Bruker-Biospin, NMR – Products – Systems, http://www.brukerbiospin.com/nmr/products/systems.html, 2003. Committee for High Field NMR, The, “A New Millenium Resource,” report, unpublished, 2000. J. Edwards, “Principles of NMR,” Process NMR Associates LLC, 1999. Foxboro Company, The, “I/A Series: Process NMR Analyzer, Model NMRB, Style C,” Datasheet, The Foxboro Company (Foxboro, MA), 2000.

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A. Garroway, P. Grannell, and P. Mansfield, “Image formation in NMR by a selective irradiative pulse,” Journal of Physics C: Solid State Physics, vol. 7, ppl. L457 – L462, 1974. E. Haacke, R. Brown, M. Thompson, and R. Venkatesan, Magnetic Resonance Imaging: Principles and Sequence Design, Wiley, 1999. A. Horska, W.E. Kaufmann, L.J. Brant, S. Naidu, J.C. Harris, and P.B. Barker, “In vivo quantitative proton MRIS study of brain development from childhood to adolescence,” Journal of Magnetic Resonance Imaging, vol. 15(2), pp. 137 – 43, 2002. P. Lauterbur, “Image formation by induced local interaction: Examples employing nuclear magnetic resonance,” Nature, vol. 242, pp. 190 – 191, 1973. S. Maloof, US Department of Energy, “World’s largest, most powerful NMR spectrometer,” US Department of Energy Research News, Apr. 2002. D. Nishimura, Principles of Magnetic Resonance Imaging, MRSRL Press, 1996. E. Purcell, H. Torrey, and R. Pound, “Resonance absorption by nuclear magnetic moments in a solid,” Physical Review, vol. 69, pp. 37 – 38, 1946. J.R. Tolman, H.M. Al-Hashimi, L.E. Kay, and J.H. Prestegard, “Structural and dynamic analysis of residual dipolar coupling data for proteins,” Journal of the American Chemistry Society, no. 123, pp. 1416 – 1424, 2001. R. Weiss, “Two developers of MRI awarded Nobel Prize,” Washington Post, Oct. 7, 2003, p. A02, 2003.

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Introduction What is LC Technology?  Lab-on-a-Chip (LC) is a new technology developed from microelectronics and telecommunications that incorporates microarray, microfluidic and microelectromechanical systems  LC has made possible rapid miniaturization and low-cost production of biological laboratory analysis and diagnostic testing

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Introduction LC Technology Applications  Integrated into portable devices for high performance of multiple applications and tens-ofthousands of laboratory tests  Examples of applications – Identification and replication of specific sequences of DNA – Diagnosis of diseases – Detection of biotoxins and explosives

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Introduction: Main Processes/Characteristics Two main processes performed on LC microchips – Chemical separation – Chemical analysis

Main characteristics of LC microsystems: – – – – –

Reduced processing time Maximized sensitivity from minimal reagent input Low false positive rate Minimal power requirements Flexibility and reusability 78

Basic Principles Chemical Separation Two major categories: – Gas or Liquid Chromatograph – powerful and widely-used separation techniques – Electrokinetics – used for separation of charged particles • Electrophoresis • Electroosmosis – less often used

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Basic Principles Chromatography Micro-distances on LC microsystem minimize diffusion distances and maximize the use of gas and liquid chromatography

Liquid chromatography – Use open tubes etched in spiral configuration – Some use particle-packed columns • Difficult to pack and may prevent uniform particle densities • Etched columns help resolve problems with packing and particle densities

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Basic Principles Electrokinetics Electroosmosis – Uses continuous flow and an electrical potential to separate charged particles – Electrical potential minimizes back flow of particles

Electrophoresis – Used most often in LC microsystems – Separates larger molecules such as DNA or other proteins – Often used in conjunction with PCR – Electric field separates particles based on size and polarizability 81