ANALYTICAL TECHNIQUES II Medical Laboratory Science Program College of Allied Medical Professions ELECTROCHEMISTRY ■ Study of redox reactions ■ Involves the measurement of current or voltage generated by the activity of specific ion. ■ Uses electrochemical cells ▪ Galvanic Cell – generates current ▪ Electrolytic Cell – requires current Comparison Definition Symbol Unit SI Unit Measuring Instrument Relationship Current Voltage The flow of electric Electrical potential charge across a difference between certain element two points I V A / amps / amperage V / Volts / Voltage 1 ampere = 1coulumb/second 1 volt =1 joule/coulomb Ammeter Voltmeter Current is the effect Voltage is the (voltage being the cause and current cause ). Current is its effect. Voltage cannot flow without can exist without voltage. current. POTENTIOMETRY ■ Measurement of the differences in voltage at a constant current. ■ Expressed in Nernst equation ■ Used in the measurement of pH POTENTIOMETRY ■ Ion-Selective Electrodes (ISE) ■ Potentiometric methods of analysis involve the direct measurement of electrical potential due to the activity of free ions. ■ Ion – selective electrodes (ISEs) are designed to be sensitive toward individual ions. POTENTIOMETRY ■ Ion-Selective Electrodes (ISE) ▪ It is composed of electrochemical transducer capable of responding to one given ion. ▪ Very sensitive and specific to the ion it measures ▪ Practical application: measurement of electrolytes namely Na+, K+, and Cl- POTENTIOMETRY ■ Types of ISE ▪ Inert-metal electrodes (standard H electrode) for Na+ measurements ▪ Metal electrodes (Ag/AgCl electrode) for Cl- measurements ▪ Membrane electrodes (e.g. valinomycin gel electrode) for K+ measurements. POTENTIOMETRY pH Electrode ■ ISE universally used in the laboratory. ■ Determines pH values quickly and accurately ■ Electrode components: ▪ Indicator electrode / Glass electrode ▪ Reference electrode ▪ Liquid junction ▪ Readout device/ meter POTENTIOMETRY Components of a pH meter ■ Indicator electrode/ Glass electrode ▪ Consists of a silver wire coated with AgCl, immersed into an internal solution of KCl, and placed into a tube containing a special glass membrane tip/ ▪ This membrane is only sensitive to Hydrogen ions (H). POTENTIOMETRY Components of a pH meter ■ Indicator electrode / Glass electrode ▪ When the pH electrode is placed into the test solution movement of H+ near the tip of the electrode produces a potential difference between the internal solution and the test solution which is measured as pH and read by voltmeter. POTENTIOMETRY Components of a pH meter ■ Reference electrode ▪ Composed of an internal wire made of either Silver – silver chloride system or ▪ Calomel electrode (mainly mercurious chloride) ▪ A filling solution of potassium chloride, and ▪ A permeable outer casing POTENTIOMETRY Components of a pH meter ■ Liquid junction ▪ Protects the reference system from the medium to be measured without disconnecting the electrical potential between them. ▪ Potassium chloride (KCl) is a commonly used filling solution because K+ and Cl- have nearly the same mobilities. POTENTIOMETRY Components of a pH meter ■ Read-out device/meter (Voltmeter) ▪ Numerically represent the measurements pH pH Meter MECHANISM OF PH METER READINGS ■ The indicator electrode/glass electrode. will be immersed in the solution. ■ The glass electrode is sensitive to H+ ions. ■ If the number of H+ ions in the solution exceeds the number of H+ inside the pH meter probe; the solution will register an acidic / acidic pH. MECHANISM OF PH METER READINGS ■ If the number of H+ ions in the solution is less than the number of H+ inside the pH meter probe; the solution will register an alkaline/basic pH ■ If the number of H+ ions in the solution is equal to the number of H+ inside the pH meter probe; the solution will register a neutral pH PRECAUTIONARY MEASURES IN USING THE PH METER: ■ Do not measure the pH of unknown solutions without calibration. Calibrate it using a solution with a pH of 7.0 ■ Wash the electrode after use with clean water and store in 3M KCI solution. ■ Do not touch or wipe the sensor head of the electrode in the electrode cleaning process so as not to affect the reaction time. For periodic cleaning, use the following cleaning, methods: Sample Measured Protein containing samples Sulfide containing samples Grease or organic compound samples General Samples Cleaning Method Immerse the electrode in pepsin or HCl solution for a few hours. Immerse the electrode in thiourea or HCl solution until the diaphragm of the electrode turns white. Apply acetone or alcohol to clean the electrode for a few minutes Immerse the electrodes in 0.1 M NaOH or 0.1M HCl for a few minutes Apply clean water to flush the electrode after the above cleaning steps OTHER TYPES OF ELECTRODES Gas sensing electrodes ▪ Designed to detect specific gases ▪ Examples are ▪ pO2 (Clark electrode) ▪ pCO2 electrode OTHER TYPES OF ELECTRODES Enzyme electrodes ■ Determined by the reaction product of the immobilized enzyme. ■ Examples are: ■ Urease – detects urea ■ Glucose oxidase – detects glucose COULOMETRY ■ Measurement of the amount of electricity (in coulombs) at a fixed potential. ■ Follows Faraday’s Law. ■ Involves electrochemical titration and is used for the measurement of chloride in both sweat and serum. ■ It is now obsolete and is being replaced today by Chloride ISE AMPEROMETRY ■ Measurement of the flow of current produced by redox reactions. ■ Used in the determination of : ▪ Partial pressure of Oxygen (pO2) ▪ Glucose ▪ Chloride ▪ Peroxidase AMPEROMETRY Polarography ▪ Utilizes the principle of amperometry ▪ Measurement of differences in current at a constant voltage ▪ Follows the Ilkovic equation. ▪ Used in the measurement of trace metals , O2, Vitamin C and amino acid concentrations. VOLTAMETRY ■ Anodic stripping Voltammetry ▪ Measurement of current after potential is applied to an electromagnetic cell ■ Used in the measurement of iron and lead. VOLTAMETRY ■ Alternatives for LEAD testing include: ▪ Electrothermal (graphite furnace) ▪ Atomic absorption spectroscopy or preferably, ▪ Inductively Coupled Plasma Mass Spectrometry (ICP-MS) ELECTROPHORESIS ■ Migration of charged solutes or particles in an electrical field ■ Electrophoresis is a separation technique based on the principle that a charged particle in solution will migrate towards one of the electrodes when placed in an electrical field. ELECTROPHORESIS Practical Applications: ▪ Proteomics – large scale study of proteins particularly the structures and functions. ▪ Genomics – study of genome (complete set of DNA or complete set of hereditary information). ELECTROPHORESIS Two Basic Types: 1. Horizontal Electrophoresis ▪ Uses agarose gel ▪ Used in Genomics 2. Vertical Electrophoresis ▪ Uses Sodium Dodecyl Sulfate Polyacrylamide Gel (SDS- PAGE) ▪ Used in Proteomics ELECTROPHORESIS ■ Terminologies ▪ Ions – charged particles ▪ Cations – (+) Charged ions ▪ Anions – (-) Charged ions ▪ Zwitterions – neutrally–charged ions ▪ Electrodes ▪ Cathode – (-) electrode; where reduction occurs ▪ Anode – (+) electrode; where oxidation occurs. ELECTROPHORESIS Terminologies: ▪ Iontophoresis – migration of small charged ions. ▪ Zone Electrophoresis – migration of charged macromolecules. ▪ Electrophoretogram – result of zone electrophoresis and consists of a macromolecule. ELECTROPHORESIS Components: 1. Driving force (electrical power) 2. Support medium (gel) 3. Buffer (fluid) 4. Sample 5. Detecting System ELECTROPHORESIS Components 1. Driving Force (electrical power) ■ Supplies current or voltage. 2. Support Medium (gel) ■ Provide a matrix that molecules to separate. allows ELECTROPHORESIS Types of Support Medium ■ Paper (Obsolete) ■ Cellulose acetate (replacement paper) – separates particles molecular size. ■ Agarose Gel – separates particles electrical charge (usually used Genomics) for by by for ELECTROPHORESIS Types of Support Medium ■ Starch Gel – separates particles by charge and size. ■ Polyacrylamide Gel – separates particles by charge and size (usually used for proteomics). ELECTROPHORESIS Components 3. Buffer (Fluid) ▪ Provides an ionic solution that allows current to pass through the water. ▪ Most commonly used buffers are: ▪ Barbital (Veronal) Buffer : pH 8.6 ▪ Tris Boris Acid EDTA Buffer/TrisBorate-EDTA: pH 8.7 ELECTROPHORESIS Components 4. Sample ▪ Can be pre-treated serum or DNA ▪ Placed in wells created by comb ▪ Usually combined with a dye (ethidium bromide) for easy visualization of migration. ELECTROPHORESIS Components 5. Detecting System ▪ Converts bands into algorithm to know the exact concentration of substances ▪ It can be a: ▪ Densitometer ▪ Documentation System (Computer Software) ELECTROPHORESIS Factors Affecting the Rate of migration ▪ Net electric charge of the molecule ▪ Size and shape of the molecule ▪ Electric field strength ▪ Nature of the supporting medium ▪ Temperature of operation ELECTROPHORESIS Factors Affecting the Rate of migration ▪ Net electric charge of the molecule ▪ ( - ) migration is cathode to anode ▪ ( + ) migration is anode to cathode ▪ Size and shape of the molecule ▪ Large molecules – travels slowly ▪ Small molecules – travels fast ELECTROPHORESIS Factors Affecting the Rate of Migration ▪ Electric Field Strength ▪ Increasing the strength of the field (increase Current) also increases migration ▪ Nature of the supporting medium ▪ Temperature of operation ELECTROPHORESIS Outline of Steps /Procedure ▪ Preparation of sample ▪ Preparation of gel and well ▪ Submerging of the gel into the buffer ▪ Application of sample and dye to wells ▪ Application of current ▪ Staining of the gel ELECTROPHORESIS Outline of Steps /Procedure ▪ Visualization of the bands and quantitation using: ■ Densitometer or ■ Documentation system ELECTROPHORESIS Possible Stains for Easy Visualization of Electrophoretic Bands ▪ Amino Black ▪ Ponceau S ▪ Oil red O ▪ Sudan Black ▪ Fat Red 7B ▪ Coomassie Brilliant Blue ▪ Silver Nitrate ▪ Nitrotetrazolium Blue ELECTROPHORESIS Laboratory Methodologies / Clinical Applications ■ SERUM PROTEIN ELECTROPHORESIS (SPE) ▪ Proteins in Serum include: ▪ Albumin (most predominant ) ▪ Globulin (alpha, beta, gamma) ▪ SPE can fractionate the different proteins in serum. ELECTROPHORESIS Laboratory Methodologies / Clinical Applications ■ SERUM PROTEIN ELECTROPHORESIS (SPE) ▪ Uses a pH of 8.6 and at this pH; proteins are negatively charged. ▪ Therefore, the migration of protein is from cathode to anode. ELECTROPHORESIS ■ An ampholyte is a molecule, such as protein, whose net charge can be either positive or negative. ■ The pH at which negative and positive charges are equal on a protein is called the isoelectric point. ■ If the buffer is more acidic than the isoelectric point (pl) of the ampholyte, it binds H, becomes positively charged, and migrates toward the cathode. ELECTROPHORESIS ■ If the buffer is more basic than the pl, the ampholyte loses H, becomes negatively charged and migrates toward the anode. ■ A particle without a net charge will not migrate, remaining at the point of application. ELECTROPHORESIS ■ Migration of Serum Proteins (fastest to slowest ) ▪ Albumin (lightest, most anodal ) ▪ α1 , Globulin ▪ α2 , Globulin ▪ β Globulin ▪ γ Globulin (most cathodal) MIGRATION OF SERUM PROTEINS ( FASTEST TO SLOWEST ) _ + albumin α1 α2 β γ “tall spike” MULTIPLE MYELOMA Decreasing albumin Increasing ά2, β globulin HYPOALBUMINEMIA A. LIVER DISEASE B. KIDNEY DAMAGE C. MALNUTRITION ELECTROPHORESIS Laboratory Methodologies / Clinical Applications ■ PLASMA PROTEIN ELECTROPHORESIS (PPE) ▪ Proteins in Plasma include : ▪ Albumin (most predominant) ▪ Globulin (alpha, beta , gamma) ▪ Fibrinogen (show an extra band between beta and gamma region) MIGRATION OF PLASMA PROTEINS ( FASTEST TO SLOWEST ) _ + albumin α1 α2 β Fibrinogen γ ELECTROPHORESIS Laboratory Methodologies / Clinical Applications ■ LIPOPROTEIN ELECTROPHORESIS (LPE) ▪ Lipoproteins are composed of a lipid and a protein molecule. ▪ They are the main transporters of lipids in the body. LIPOPROTEIN ELECTROPHORESIS (LPE) Lipoprotein Lipid Content Protein Content TAG γ - Globulin 2.LDL (Low Density Lipoprotein) Cholesterol β - Globulin 3.VLDL (Very Low Density Lipoprotein) TAG Pre- β –Globulin 1.Chylomicrons 4.HDL (High Density Cholesterol Lipoprotein) α- Globulin LIPOPROTEINS ELECTROPHORESIS ■ Chylomicrons – gamma lipoprotein ■ LDL – Beta lipoprotein ■ VLDL – Pre- Beta lipoprotein ■ HDL – Alpha lipoprotein LIPOPROTEIN ELECTROPHORESIS ▪ Lipid staining dyes include ( Oil Red O, Fat Red 7B, Sudan Black, Scharlach Red ) LPP Protein Migration 1. Chylomicrons Gamma- globulin Gamma globulin 2. VLDL Pre-beta globulin Pre-beta globulin 3. LDL Beta Globulin Beta globulin 4. HDL Alpha Globulin Alpha Globulin LIPOPROTEIN ELECTROPHORESIS _ + HDL VLDL LDL Chylo ELECTROPHORESIS Laboratory Methodologies / Clinical Applications ■ CEREBROSPINAL FLUID (CSF) ELECTROPHORESIS ▪ CSF is the third major fluid in the body ▪ CSF Electrophoresis is performed to diagnose multiple sclerosis CEREBROSPINAL FLUID CONSTITUENTS ■ Normal CSF Protein constituents: ▪ Major protein is albumin ▪ Next to albumin is prealbumin (now called transthyretin ) ☺ ▪ Alpha-globulins include Ceruloplasmin and Haptoglobin CEREBROSPINAL FLUID CONSTITUENTS ■ Normal CSF Protein constituents: ▪ Beta-globulin include Transferrin and TAU (CHO deficient transferrin found only in CSF) ☺ ▪ Gamma globulins: Major globulins is IgG with Some IgA ▪ Proteins not found in CSF are IgM, beta-lipoprotein and fibrinogen. NORMAL CSF ELECTROPHORESIS _ + TTR Albumin α1 α2 β1 β2 γ ELECTROPHORESIS Laboratory Methodologies / Clinical Applications ■ CEREBROSPINAL FLUID (CSF) ELECTROPHORESIS ▪ CSF Electrophoresis: must be done in conjunction with serum electrophoresis. ▪ Difference: CSF contains double bands at the beta region ( Because of TAU transferrin ) CEREBROSPINAL FLUID (CSF) ELECTROPHORESIS ■ Clinical Significance – Diagnosis of multiple sclerosis. ■ Multiple sclerosis is a demyelinating disease which is characterized by destruction of the myelin sheath. ■ The protein fraction increased in this kind of disease is called myelin basic protein. CEREBROSPINAL FLUID (CSF) ELECTROPHORESIS ■ Clinical Significance – Diagnosis of multiple sclerosis. ■ Multiple sclerosis is a demyelinating disease which is characterized by destruction of the myelin sheath. ■ The protein fraction increased in this kind of disease is called myelin basic protein. ELECTROPHORESIS Laboratory Methodologies / Clinical Applications ■ HEMOGLOBIN ELECTROPHORESIS ▪ Hemoglobin is the main pigment responsible for the red color of the red blood cells. ▪ Hemoglobin electrophoresis can detect normal and abnormal hemoglobin in the blood. HEMOGLOBIN ELECTROPHORESIS ▪ If alkaline cellulose acetate is used; the pH is 8.4 and the migration is from cathode to anode: _ + H I A1 F S D G A2 C E O HEMOGLOBIN ELECTROPHORESIS ▪ To confirm the presence of Hemoglobin C and S; an acidic buffer can be used (e.g. Acid Citrate agar with a pH of 6-6.2) and the migration is from anode to cathode. _ + C S A D G E F ELECTROPHORESIS Laboratory Methodologies Applications / Clinical ■DNA TESTING ▪ DNA Testing is usually carried out for: ▪ Paternity Testing ▪ Forensic Cases ELECTROPHORESIS Laboratory Methodologies / Clinical Applications ■ HIV TESTING ▪ HIV Testing can be carried out in a variety of ways but the GOLD Standard test for HIV is called Western Blot and can be performed using electrophoresis. WESTERN BLOT ▪ GOLD STANDARD DIAGNOSIS OF HIV. TEST Markers: ▪ p24 – must marker ▪ Gp41 – must marker ▪ Gp120 – good marker ▪ Gp160 – good marker FOR THE WESTERN BLOT Interpretation: ■ HIV ( + ) ▪ p24 and gp41 are both present ■ HIV ( - ) ▪ p24 and gp41 are absent ■ Indeterminate ▪ Only one marker is present (either p24 or gp41) repeat the test after 6 months ELECTROPHORESIS Laboratory Methodologies / Clinical Applications ■ FRACTION OF CK AND LD ISOENZYMES ▪ CK AND LD ENZYMES have specific isoenzymes and are both used mainly for the diagnosis of myocardial infarction. •CK Isoenzymes _ + CK - BB CK - MB CK - Macro CK - MM CK - MI •LD Isoenzymes _ + LD1 LD2 LD3 LD4 LD5 ISOELECTRIC FOCUSING (IEF) ■ Modification of electrophoresis ■ Charged proteins migrate through a support medium with continuous pH gradient ■ Proteins move in the electric field until they reach a pH equal to their isoelectric point. ISOELECTRIC FOCUSING (IEF) ■ Charged proteins migrate through a support medium with continuous pH gradient ■ The pH gradient is created by adding acid to anodic area and base to cathodic area. ISOELECTRIC FOCUSING (IEF) ■ A solution of ampholytes with different isoelectric points (pIs) is placed between two electrodes. ▪ Ampholytes close to the anode carry a net positive charge. ▪ Ampholytes close to the cathode carry a net negative charge. ISOELECTRIC FOCUSING (IEF) ■ When an electric voltage is applied : ▪ Each ampholyte will migrate to the area where the pH is equal to its isoelectric point. ▪ This is responsible for the separation of sample into its individual components. ISOELECTRIC FOCUSING (IEF) ■ Useful for the separation of proteins with identical sizes but different charges (e.g. enzymes and isoenzymes) ■ Advantages : ▪ Resolve mixture of proteins ▪ Detect isoenzymes (ACP; CK; ALP ) in serum ▪ Identify genetic variants of proteins (alpha-1-antitrypsin) ▪ Detect CSF oligoclonal banding CAPILLARY ELECTROPHORESIS ■ Uses narrow-bore fused silica capillaries which are filled with buffer ▪ Capillary is filled with buffer ▪ Sample is then loaded ▪ Electric field is then applied ▪ Sample are separated into component or fragments. ▪ Detection is done at the other end of the capillary. CAPILLARY ELECTROPHORESIS ■ Sample molecules are separated by ELECTRO-OSMOTIC FLOW (EOF) ■ EOF refers to the bulk flow of liquid toward the cathode upon application of electrophoretic field. CAPILLARY ELECTROPHORESIS ■ Cations-> migrate fast because both the EOF and cations electrophoretic attraction are both towards the cathode. ■ Anions move toward the capillary outlet by EOF in a slower rate because they are attracted to the anode but is repelled by the cathode. ■ Neutral ions are all carried by EOF but are not separated from each other. ■ Uses nanoliter of samples. CAPILLARY ELECTROPHORESIS ■ Useful for: ▪ Analysis of organic and inorganic substances ▪ Analysis of pharmaceutical drugs and drugs of abuse ▪ Analysis of Polymerase Chain Reaction (PCR) products. ▪ Detection, quantitation and determination of molecular weight of proteins and peptides ▪ Separation of serum proteins and hemoglobin variants TWO DIMENSIONAL ELECTROPHORESIS ■ Uses two different electrophoretic dimensions to separate proteins from complex matrices such as serum or tissue ▪ First dimension – proteins resolved according to their isoelectric points ▪ Second dimension – proteins are separated according to their relative size or molecular weight using SDS- PAGE. FLOW CYTOMETRY ■ A flow cytometry measure multiple properties of cells suspended in a moving fluid medium. ■ As each particle passes single-file through a laser light source, it produces a characteristic light pattern that is measured by multiple detectors for scattered light (forward and 90 degrees) and fluorescent light (if the cell is stained with a fluorochrome). FLOW CYTOMETRY ■ The cell suspension aliquots are introduced into the flow chamber using air pressure. ■ As cells pass through the flow chamber; a low pressure sheath fluid surrounds them. ■ The fluid stream creates a laminar flow forcing the specimen to the center and results in a single file alignment of the individual cells. ■ This is called Hydrodynamic Focusing FLOW CYTOMETRY ■ Flow Cytometry is used to count and sort cells, as well as viral particles, DNA, fragments, bacteria and latex beads. ■ It is a core component of hematology cell counters and the technology used to differentiate white blood cells. ■ As of the present, flow cytometry can also be used for counting urine sediments (Urine Flow Cytometry). FLOW CYTOMETRY ■ A Laser beam passes through each cell that causes light to scatter. ▪ Forward light scatter is detected by forward scatter photodetector and is proportional to cell size ▪ 90° or right angle scatter is detected by the right angle photodetector and corresponds to cell granularity and nuclear irregularity. FLOW CYTOMETRY ■ A Laser beam passes through each cell that causes light to scatter. ▪ Light scatter from cells labeled with fluorochromes is detected by two fluorescence detectors PHOTO Waste REFLECTOMETRY ■ Measurement of analytes in biologic fluids. ■ It uses an instrument called reflectometer. ■ A reflectometer is a filter photometer that measures the quantity of light reflected by a liquid sample that has been dispensed onto a grainy or fibrous solid support. REFLECTOMETRY ■ Two Clinical Applications: – Urine Dipstick/ Reagent Strip Analysis – Dry Slide Chemical Analysis ■ Kodak Ectachem (now VITROS ) REFLECTOMETRY ■ Two Types ▪ Specular reflectance ▪ Occurs on a polished surface (e.g. mirror) ▪ Diffuse reflectance ▪ Occurs on a non-polished surface (e.g. grainy or fibrous pad ) REFLECTOMETRY ■ Components : ▪ Light Source – tungsten-quartz halide or halogen lamp ▪ Monochromator – isolates wavelength ▪ Slit – directs light to the strip pad ▪ Solid state photodiodes – are used a detector which detect reflected radiant energy. ▪ Computer or microprocessor converts reflectance signals into direct read-out concentrations. CONDUCTANCE ■ Works on the principle of electrical conductivity ▪ This refers to the ability of a solution to carry electrical current. ▪ Used for : ▪ Measurement of blood urea ▪ Components of detectors in High Performance Liquid Chromatography (HPLC); cell counters, Capillary electrophoresis and Gas Chromatography (GC) IMPEDANCE ▪ Electrical impedance measurement is based on the change in the electrical resistance across an aperture when a particle in conductive liquid passes through the aperture. ▪ Employed in some HEMATOLOGY analyzers (e.g. COULTER COUNTER ) which is used to perform: ▪ Complete Blood Count to enumerate erythrocytes (RBC’s); leukocytes (WBC’s) and thrombocytes (platelets) IMPEDANCE ■ Principle: ▪ As a cell passes through an aperture; the cells partially occlude it. ▪ As a result; electrical impedance increase producing a voltage pulse. ▪ The size of this voltage pulse is proportional to cell size. IMPEDANCE ▪ Once blood is aspirated it would be separated into two basic separate volume for measurements. ▪ One volume is mixed with the diluent and is delivered to the cell bath where erythrocyte and platelet counts are performed. ▪ Particles measuring 2 to 20 fL are counted as platelets. ▪ Particles measuring greater than 36 fL are counted as erythrocytes IMPEDANCE ▪ The other blood volume is mixed with the diluent and a cytochemical-lytic reagent that lyses only the red blood cells ▪ Particles greater than 35 fL are recorded and counted as leukocytes CHROMATOGRAPHY CHROMATOGRAPHY ■ Involves the separation of the soluble components in a solution by specific differences in physical and chemical characteristics of the different constituents. ■ Is a technique for separating mixtures into their components in order to analyze, identify, purify and/or quantify the mixture or components. CHROMATOGRAPHY • Quantify CHROMATOGRAPHY ■ Chromatography is used by scientist to: ▪ Analyze – examine a mixture, its components and their relations to one another ▪ Identify – determine the identity of a mixture or components ▪ Purify – separate components in order to isolate one of interest for further study ▪ Quantify – determine the amount of the mixture and/or the components present in the sample. PRACTICAL APPLICATIONS OF CHROMATOGRAPHY ■ Pharmaceutical Company ▪ Determine amount of each chemical found in drugs. ▪ Gold standard for drug test ▪ Gas Chromatography Mass Spectrometry ■ Hospital ▪ Detect drug or alcohol levels in patient’s samples ■ Law Enforcement ▪ Compare a sample found in a crime scene to samples for suspects (forensic) ■ Environment Agency ▪ Determine the level of pollutants in water ■ Manufacturing Plant ▪ Purify a chemical needed to make a product CHROMATOGRAPHY ■ PRINCIPLE ▪ Separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary absorbing medium through which they pass. CHROMATOGRAPHY ■ TERMINOLOGIES ▪ 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 – can be solid or liquid; the part of the apparatus that does not move with the sample (stationary phase) 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 FORMS OF CHROMATOGRAPHY A. PLANAR 1. Paper Chromatography ▪ Uses Sorbent-Whatman paper as the stationary phase and solvent as mobile phase. ▪ Used in the fractionation of sugar and amino acids. FORMS OF CHROMATOGRAPHY A. PLANAR 2. Thin Layer Chromatography ▪ The stationary phase that is used for TLC can be sorbent thin plastic plates impregnated with a layer of silica gel or alumina. ▪ The mobile phase is a solvent. ▪ Sample components are identified by comparison with standards on the same plate. ▪ It is used for urine drug screening (semiquantitative screening test). FORMS OF CHROMATOGRAPHY B. COLUMN ▪ 1. Gas Chromatography ( GC ) ▪ Separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or solid beads (stationary phase) ▪ Mobile phase: nitrogen, helium, hydrogen and argon (inert gas) ▪ Used for the separation of steroids, barbiturates, blood, alcohol, and lipids. FORMS OF CHROMATOGRAPHY B. COLUMN 1. Types of Gas Chromatography ( GC ) a. Gas Solid Chromatography (GSC) ▪ Separation occurs by difference in solute partitioning between the gaseous mobile phase and the solid stationary phase. b. Gas Liquid Chromatography (GLC) ▪ Separation occurs by the differences in solute partitioning between the gaseous mobile phase and the liquid stationary phase . FORMS OF CHROMATOGRAPHY B. COLUMN 2. Liquid Chromatography ▪ Separates liquid samples with a liquid solvent (mobile phase ) and a column composed of solid beads (stationary phase ). ▪ The most widely used technique is called High Performance Liquid Chromatography ( HPLC ) FORMS OF CHROMATOGRAPHY B. COLUMN ▪ High Performance Liquid Chromatography (HPLC) ▪ Used for the fractionation of drugs, hormones, lipids, carbohydrates and proteins ☺ ▪ Uses pressure for fast separation ▪ The separation of samples is governed by selective distribution of the solutes between the mobile and the stationary phase. FORMS OF CHROMATOGRAPHY B. COLUMN ▪ High Performance Liquid Chromatography ( HPLC ) ▪ Mobile phase uses solvents like acetonitrile, methanol, ethanol, isopropanol and water. ▪ Types : ▪ Isocratic elution – strength of solvent remains constant during separation ▪ Gradient elution – strength of solvent continually increases during separation FORMS OF CHROMATOGRAPHY B. COLUMN ▪ High Performance Liquid Chromatography (HPLC ) ▪ Stationary phase is a column made up of organic material bonded to silica. ▪ Types : ▪ Normal Phase – liquid chromatography – polar stationary phase ; non-polar mobile phase ▪ Reversed Phase – liquid chromatography – non-polar stationary phase; polar mobile phase FORMS OF CHROMATOGRAPHY B. COLUMN ▪ High Performance Liquid Chromatography (HPLC ) ▪ A solvent reservoir contains pump that pushes the mobile phase through the column ▪ The sample is then introduced through a loop injector ▪ The sample and the mobile phase is then introduced to the column (stationary phase) ▪ The solutes are then introduced to the detector in order that each was eluted. MASS SPECTROMETRY ■ Based on the fragmentation and ionization of molecules using a suitable source of energy ■ It involves three distinct processes: ▪ 1. Conversion of the parent molecule into a steam of ions (usually singly charged positive ions); ▪ 2. Separation of the ions by mass/charge ratio (identification) ▪ 3. Counting of the number of ions of each type or measurement of current produced when the ion strike a transducer (quantification). ▪ The number of ions relates proportionally to concentration. MASS SPECTROMETRY ■ Before a compound can be detected and quantified by MS, it must be separated by GC or HPLC. ▪ GC – MS ▪ LC – MS ■ Mass spectrometry is high quality technique for identification of : ▪ Drugs or drug metabolites (toxicology) ▪ Amino acid composition of proteins ▪ Steroid hormones like testosterone and fat soluble vitamin like Vitamin D MASS SPECTROMETRY GAS-CHROMATROGRAPHY MASS SPECTROMETRY ( GCMS ) ■ It is the gold standard for drug test ■ It uses an electron beam to split the drug emerging from the column into its component ions. ■ Quantitative measurement of drug can be performed by selective ion monitoring. MASS SPECTROMETRY ■ TANDEM MASS SPECTROMETRY ▪ A GC or HLPC is connected to two mass spectrometers (GC/MS/MS ) or (HPLC/MS/MS ) ▪ Ions of different mass continue to be fragmented in the second spectrometer. ▪ Used in the performance of 20 inborn errors of metabolism from a single blood spot.
