CC1 Lecture: Analytical Techniques/Instrumentation PDF

CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION CLINICAL CHEMISTRY 1 LECTURE: ANALYTICAL TECHNIQUES/INSTRUMENTATION Prepared by: Rahimyar Khan T. Pata, RMT, MD Analytic techniques in the clinical chemistry...

CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION CLINICAL CHEMISTRY 1 LECTURE: ANALYTICAL TECHNIQUES/INSTRUMENTATION Prepared by: Rahimyar Khan T. Pata, RMT, MD Analytic techniques in the clinical chemistry laboratory are essential for diagnosing, monitoring, and treating various diseases. These techniques have evolved significantly, leveraging advancements in technology and scientific understanding. SPECTROPHOTOMETRY ❖ Spectrophotometry is a foundational analytic technique in clinical chemistry, used to measure the concentration of substances by detecting the intensity of light as it passes through a sample. ❖ This discussion will cover the different types of spectrophotometric techniques, with an emphasis on the components of a spectrophotometer. Introduction to Spectrophotometry Principle: Spectrophotometry is based on the principle that each compound absorbs or transmits light over a specific range of wavelengths. According to the Beer-Lambert law, the________ amount of light absorbed by a substance in solution is __________ directly proportional to its concentration. TYPES OF SPECTROPHOTOMETRIC TECHNIQUE TYPE RANGE PRINCIPLE APPLICATION EXAMPLE UV-VISIBLE 200-700 nm Used to measure the Quantification of UV: __________. 200-400 nm concentration of bilirubin and Visible: _____________ 400-700 nm analytes that absorb hemoglobin light in the UV and visible regions. Common applications: analysis of nucleic acids, proteins, and enzymes. ____________________ ATOMIC Primarily UV and Visible regions Measures the Used for determining Measurement of trace ______________________ ABSORPTION concentration of ___________ metals such as metals in blood or elements by detecting the calcium, lead, and iron. urine absorption of light by free atoms in the gaseous state. Each element absorbs light at a specific wavelength. ___________________ INFRARED (IR) 700 nm - 1 mm measures the absorption Used for the __________________________ Analysis of lipids and of infrared light by identification of organic fatty acids molecules, leading to compounds and vibrations in molecular functional groups. bonds. Each molecule has a unique IR spectrum, which acts as a molecular fingerprint FLUORESCENCE/ Measures the intensity of Sensitive detection of Detection of FLUOROMETRY fluorescent light emitted substances at low fluorescently labeled by a substance after it concentrations, such as DNA in molecular absorbs light. vitamins, hormones, diagnostics The emitted light is and nucleic acids. usually at a longer wavelength than the absorbed light. __________________ TURBIDIMETRY Measures the decrease in Quantification of Measurement of serum intensity of light as it proteins, antigens, and ___________ proteins and passes through a sample immune complexes _________________________ immunoglobulins with suspended particles. __________________ NEPHELOMETRY Measures the scattered Quantification of Measurement of serum light at an angle from the proteins, antigens, and proteins and sample. immune complexes immunoglobulins More suited for measuring small particles. Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 1 of 12 CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION PARTS OF SPECTROPHOTOMETER PART FUNCTION OTHER INFO SIGNIFICANCE ________________________ ❖ Provides the initial beam of light that will Types: ❖ The choice of light source is pass through the sample ______________ Tungsten Lamp: critical for accurate LIGHT SOURCE Commonly used for measurements in the desired visible light. wavelength range. Deuterium ____________Lamp: Used for UV light. ________________________ ❖ Selects a specific wavelength of light from Components: ❖ Ensures that only light of the the broad spectrum emitted by the light __________: Prism Refracts light into its desired wavelength reaches MONOCHROMATOR source component wavelengths. the sample. Diffraction _________________: Grating Disperses light into its constituent wavelengths more efficiently than a prism. ________________________ ❖ Holds the sample in the path of the light Materials: ❖ The material and path length beam. _________ Quartz Cuvettes: Used for UV of the cuvette affect the CUVETTE/SAMPLE measurements due to their accuracy and sensitivity of the HOLDER measurement. transparency in the UV range. Glass _______or _________Cuvettes: Plastic Suitable for visible light measurements. ________________________ ❖ Detects the light that passes through the Types: ❖ The detector’s sensitivity and sample and converts it into an electrical Photodiode _____________: Commonly used response time are crucial for DETECTOR/PHOTO signal. for UV-visible accurate measurements, DETECTOR spectrophotometry especially in low- Photomultiplier ________________ Tube(PMT): Used in concentration samples. fluorescence spectrophotometry for detecting low-intensity light ________________________ ❖ Displays the absorbance or transmittance ❖ Modern spectrophotometers readings, often connected to software for often include data processing DIGITAL further analysis. capabilities, allowing for the READOUT automatic calculation of concentrations based on calibration curves. CALIBRATION AND MAINTENANCE OF SPECTROPHOTOMETER a. Calibration Importance: Regular calibration ensures the accuracy and precision of measurements. Methods: o Use of _____________ Standard Solutions: Calibration is performed using solutions of known concentration. o ______________ Baseline Calibration: Setting the instrument to zero absorbance with a blank sample. Frequency: This should be performed daily or before each use, depending on the application. b. Maintenance Cleaning: Regular cleaning of cuvettes, light source, and other optical components is essential to avoid contamination and ensure consistent results. Inspection: Periodic inspection of the light source and detector for any signs of wear or malfunction. Software Updates: Keeping the software up to date ensures compatibility and accuracy in data analysis. Conclusion ❖ Spectrophotometric techniques are vital tools in clinical chemistry, offering a wide range of applications from routine analyte quantification to complex molecular diagnostics. ❖ Understanding the types of spectrophotometric methods and the essential parts of a spectrophotometer is crucial for optimizing their use in the laboratory. Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 2 of 12 CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION CHROMATOGRAPHY ❖ Chromatography is a powerful analytical technique used in clinical chemistry for separating, identifying, and quantifying components in a mixture. ❖ The technique exploits differences in the distribution of components between a stationary phase and a mobile phase. Introduction to Chromatography Principle: Chromatography is based on the differential partitioning between the mobile phase and the stationary phase. As the mobile phase carries the mixture through the stationary phase, different components move at different rates, leading to separation. Applications: Chromatography is used in a wide range of clinical applications, including drug testing, hormone analysis, and the identification of metabolic products. TYPE PRINCIPLE APPLICATION EXAMPLE OTHER INFO ________________________ form of column analysis of amino acids, Determination of TYPES chromatography where peptides, vitamins, and hemoglobin A1c in blood _____________ Reverse phase: non- the mobile phase is a drugs polar stationary phase High-Performance liquid and the separation & polar mobile phase; Liquid Chromatography is based on the commonly used for (HPLC) interactions between the separating compounds sample components and based on their the stationary phase hydrophobicity under high pressure. ____________phase: Normal polar stationary phase & non- polar mobile phase; used for separating polar compounds ________________________ Gas Chromatography separates volatile analysis of volatile Measurement of blood TYPES (GC) compounds based on their substances, such as alcohol levels Gas-Liquid (GLC): distribution between a alcohol, drugs, and liquid stationary phase stationary liquid phase steroids Gas-Solid (GSC): solid and a mobile gas phase stationary phase ________________________ Thin-Layer separation of components identification and Screening for drug Chromatography on a thin layer of comparison of compounds metabolites in urine adsorbent material, such as lipids and drugs usually silica gel, coated on a glass or plastic plate. The mobile phase moves through the stationary phase by capillary action. ________________________ separates ions and polar used for the separation of Separation of hemoglobin molecules based on their proteins, peptides, and variants in clinical Ion-Exchange affinity to the ion nucleotides diagnostics Chromatography exchanger, which is a charged stationary phase ________________________ utilizes the specific used for purifying Purification of binding affinity between proteins, antibodies, and immunoglobulins from Affinity Chromatography an immobilized ligand in enzymes serum the stationary phase and its target molecule in the mobile phase Size-exclusion separates molecules based separation and analysis of Determination of chromatography/ Gel on their size as they pass proteins, polysaccharides, molecular weight Filtration through a porous and nucleic acids distribution in proteins Chromatography stationary phase Larger molecules elute first because they cannot enter the pores and hence travel faster through the column. ________________________ separates compounds separation of small polar Amino acid profiling based on their solubility compounds such as amino Paper Chromatography and adsorption to the acids and sugars paper (stationary phase) as the solvent (mobile phase) moves through the paper by capillary action. PARTS OF A CHROMATOGRAPHY SYSTEM Mobile phase Injector Stationary phase Pump (in HPLC) Column (in column chromatography) Data Acquisition System Detector PART FUNCTION OTHER INFO SIGNIFICANCE _______________________ MOBILE Carries the sample through the stationary The choice of mobile phase PHASE phase affects the separation can be a liquid in HPLC, a gas in GC, or a efficiency and the interaction solvent in TLC and paper of sample components with chromatography. the stationary phase. ________________________ STATIONARY The stationary phase is the medium TYPES: The nature of the stationary PHASE through which the mobile phase and ____________________ Silica Gel : Commonly phase determines the sample components pass. used in TLC and HPLC. separation mechanism, such It can be a solid, a liquid on a solid Ion-exchange resins: Used in as adsorption, partitioning, support, or a gel. ion-exchange chromatography. or ion exchange. ___________________: Porous Beads Used in size- exclusion chromatography. Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 3 of 12 CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION ________________________ houses the stationary phase and provides MATERIALS: Columns can be The column's dimensions the pathway through which the mobile made of stainless steel (HPLC), (length and diameter) and phase and the sample move glass (GC), or plastic (TLC). packing material influence COLUMN Types: the separation resolution and Analytical Columns: Shorter efficiency. columns used for analysis. Preparative Columns: Longer columns used for purification and large-scale separation. ________________________ identifies and quantifies the separated Types: The choice of detector components as they elute from the UV-Visible Detector: depends on the nature of the column Common in HPLC for detecting sample and the sensitivity DETECTOR compounds that absorb UV or required. visible light. Flame Ionization Detector (FID): Used in GC for detecting organic compounds. Refractive Index Detector: Used in HPLC for detecting compounds that do not absorb UV light. ________________________ introduces the sample into the mobile Types: Accurate injection is critical phase at the start of the chromatography Manual Injection: Used in for reproducibility and INJECTOR process simpler systems like TLC. precision in quantitative Autosampler: Common in analysis. HPLC and GC, allows for automated and reproducible sample injection. ________________________ moves the mobile phase through the TYPES The pump’s performance column at a controlled flow rate and Isocratic Pumps: Maintain a affects the separation pressure constant mobile phase efficiency and reproducibility PUMP in HPLC. composition. Gradient Pumps: Vary the mobile phase composition during the run. ________________________ records the detector's output and Components: Accurate data acquisition generates chromatograms, which display Software: Used to control and analysis are essential for DATA ACQUISITION the separated components as peaks the chromatography system identifying and quantifying SYSTEM and analyze data. the components of the Computers/Monitors: sample. Interface for real-time monitoring and data analysis. CALIBRATION AND MAINTENANCE OF CHROMATOGRAPHY SYSTEM a. Calibration Importance: Calibration ensures the accuracy of retention times, peak areas, and concentrations. Methods: o Use of ________________________Solutions: Standard Calibration curves are generated using standards of known concentration. o ________________________Time Retention Calibration: Ensures that peaks correspond to specific analytes. Frequency: Regular calibration is necessary, especially before critical analyses. b. Maintenance Column Care: Regular flushing and proper storage of columns to prevent blockages and degradation. System Checks: Periodic inspection and maintenance of pumps, injectors, and detectors to ensure optimal performance. Cleaning: Routine cleaning of the system to prevent cross-contamination and ensure longevity. Conclusion ❖ Chromatography is an essential analytical tool in clinical chemistry, offering unparalleled separation capabilities for a wide range of compounds. ❖ Understanding the different types of chromatographic techniques and the essential parts of a chromatography system is crucial for achieving accurate, reproducible results. ELECTROPHORESIS ❖ Electrophoresis is a key analytical technique in clinical chemistry and molecular biology, used for separating and analyzing charged particles based on their movement in an electric field. ❖ This discussion will cover various electrophoretic techniques and the essential components of an electrophoresis system. Introduction to Electrophoresis Principle: Electrophoresis separates molecules based on their charge and size. When an electric field is applied, charged molecules migrate toward the electrode with the opposite charge. The rate of migration depends on the molecule’s size, charge, and the medium through which it moves. Applications: Electrophoresis is used for protein and nucleic acid analysis, including the separation of hemoglobin variants, DNA fingerprinting, and protein quantification. TYPES OF ELECTROPHORETIC TECHNIQUES TYPE PRINCIPLE APPLICATION OTHER INFO EXAMPLE ________________________ Uses an agarose gel as the Primarily used for the Procedure: DNA fragment analysis ________________________ medium to separate nucleic separation of DNA and RNA Prepare an agarose in genetic research. ________________________ acids based on size. The gel fragments. gel and pour it into a is a porous matrix that gel casting tray. AGAROSE GEL allows smaller molecules to Load nucleic acid ELECTROPHORESIS migrate faster than larger samples into the (AGE) ones. wells. Apply an electric field and separate the nucleic acids based on size. Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 4 of 12 CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION ________________________ Uses a polyacrylamide gel Protein separation (SDS- Types: Protein quantification ________________________ for the separation of PAGE) and nucleic acid SDS ______-PAGE: Denatures and enzyme activity ________________________ proteins and nucleic acids analysis (native PAGE) proteins and separates studies. based on size and charge. them based on POLYACRYLAMIDE GEL The gel matrix can be molecular weight. ELECTROPHORESIS adjusted to achieve Native ________ PAGE: (PAGE) different resolutions. Separates proteins in their native state, preserving their structure and charge. ________________________ Uses a narrow-bore High-resolution separation TYPES: Analysis of small drug ________________________ capillary tube filled with an of small molecules, peptides, Capillary Zone molecules and genetic electrolyte solution. and nucleic acids. Electrophoresis markers. An electric field is applied (CZE): Separates CAPILLARY across the capillary, causing analytes based on ELECTROPHORESIS ions to migrate based on their charge-to-size their size and charge. ratio. Capillary Gel Electrophoresis (CGE): Utilizes a gel matrix within the capillary for separating larger molecules. ________________________ Separates proteins or other Protein characterization and Procedure: Characterization of ___________________ amphoteric substances separation based on pI. Prepare a gel with a protein isoforms and based on their isoelectric pH gradient. protein profiling point (pI). Load the sample and ISOELECTRIC FOCUSING An electric field is applied apply an electric field. (IEF) across a pH gradient, and proteins migrate to the Proteins migrate to point where their net their respective pI and charge is zero. focus into sharp bands. ________________________ Combines isoelectric Comprehensive protein Procedure: Proteomic studies and ________________________ focusing (IEF) with SDS- analysis and profiling. Perform IEF on a gel identification of protein PAGE to separate proteins strip. biomarkers. in two dimensions: first by Transfer the focused Two-Dimensional pI and then by molecular proteins to an SDS- Electrophoresis (2-DE) weight. PAGE gel for separation based on molecular weight. PARTS OF AN ELECTROPHORESIS SYSTEM Figure: Parts of Gel Electrophoresis Apparatus. Image Source: Cleaver Scientific Ltd. Power supply Buffer solutions Gel Casting and Electrophoresis chambers Staining and Destaining solutions Sample loading equipment Capillary Electrophoresis Components Gel Documentation System PART FUNCTION OTHER INFORMATION SIGNIFICANCE ___________________ Provides the electric field necessary for Features: ensures proper separation of electrophoresis. Voltage and Current samples by controlling the POWER SUPPLY Controls: Allow precise voltage and current applied to control over the electric field the electrophoresis system. strength. Safety Features: Includes automatic shut-off and overcurrent protection. Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 5 of 12 CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION ___________________ GEL CASTING & Holds the gel and provides a space for sample Types: Proper gel casting and ___________________ ELECTROPHORESIS migration. Casting Tray: Used to chamber design are crucial for ___________________ CHAMBERS prepare and solidify the gel. achieving uniform results and Electrophoresis Tank: preventing sample distortion Contains the gel and electrolyte buffer during electrophoresis ___________________ SAMPLE LOADING Used to load samples into the gel wells. Types: Accurate sample loading is ___________________ EQUIPMENT Micropipettes: Used for essential for reproducible precise volume results and effective measurements and sample separation loading. Sample Loading Dyes: Added to samples to track their progress during electrophoresis. ___________________ Captures and analyzes the results of Components: crucial for visualizing and ___________________ electrophoresis Transilluminator: Provides interpreting the results of UV or visible light to electrophoresis GEL DOCUMENTATION visualize the gel. SYSTEM Camera or Imaging System: Captures images of the gel for analysis. Software: Analyzes gel images and quantifies bands. ___________________ Maintains the pH and ionic strength necessary Types: Proper buffer preparation ___________________ for effective electrophoresis. Running Buffers: Used ensures consistent results and during the electrophoresis prevents damage to the gel or BUFFER SOLUTIONS process. samples. Sample Buffers: Contain tracking dyes and sometimes denaturing agents. ___________________ Stains the separated components to make Types: Staining is essential for ___________________ them visible for analysis Coomassie Brilliant Blue: visualizing the separated STAINING AND Commonly used for protein components after DESTAINING staining. electrophoresis. SOLUTIONS Ethidium Bromide: Used for nucleic acid staining. Capillary Electrophoresis System Components Capillary Tubes: Narrow tubes where separation occurs. Injectors: Introduce samples into the capillary. Detectors: Detect separated analytes, often using UV absorbance or fluorescence. Significance: The components of a capillary electrophoresis system are crucial for achieving high-resolution separations and accurate analyses. CALIBRATION AND MAINTENANCE OF ELECTROPHORESIS SYSTEMS a. Calibration Importance: Ensures accuracy and reproducibility of results. Methods: o Use of ____________________________ Molecular Weight Markers: To calibrate and verify the size of separated bands. o Standardization of Buffer Solutions: Ensures consistent pH and ionic strength. Frequency: Regular calibration is required, especially when changing gels or buffers. b. Maintenance Gel Preparation: Regular cleaning of gel casting equipment to prevent contamination. System Checks: Periodic inspection of power supplies and electrophoresis chambers. Buffer Solutions: Frequent replacement and proper storage of buffer solutions to prevent degradation. Cleaning: Regular cleaning of imaging systems and other components to ensure accurate results. Conclusion Electrophoresis is a versatile and essential technique for the separation and analysis of biomolecules in clinical chemistry and molecular biology. Understanding the various electrophoretic techniques and the components of an electrophoresis system is critical for accurate and effective analysis. Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 6 of 12 CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION MASS SPECTROMETRY Mass spectrometry (MS) is a powerful analytical technique used to identify and quantify molecules based on their mass-to-charge ratio (m/z). It plays a crucial role in clinical chemistry, enabling the analysis of complex biological samples, drug testing, and metabolite profiling. INTRODUCTION TO MASS SPECTROMETRY Principle: Mass spectrometry involves ionizing chemical compounds to generate charged particles (ions). These ions are then separated based on their mass-to-charge ratio and detected to provide information about the molecular weight, structure, and concentration of the analytes. Applications: MS is used in clinical diagnostics, drug monitoring, metabolic analysis, and proteomics. TYPES OF MASS SPECTROMETRIC TECHNIQUES TYPE PRINCIPLE APPLICATIONS PROCEDURE EXAMPLE _______________________________ ionizes samples by Identification of small 1. Sample is introduced Analysis of volatile organic bombarding them with organic molecules and into the ionization compounds in ELECTRO IMPACT (EI) high-energy electrons. structural elucidation. chamber. environmental samples This technique is typically 2. High-energy electrons used for volatile and ionize the sample thermally stable molecules, producing compounds ions. 3. Ions are analyzed based on their m/z ratios. _______________________________ uses a laser to ionize Analysis of large 1. Sample is mixed with a Proteomics and analysis of _______________________________ samples embedded in a biomolecules such as matrix and applied to a complex biological matrix material. The proteins, peptides, and target plate. samples MATRIX-ASSISTED LASER matrix absorbs the laser polymers 2. The matrix absorbs the DESORPTION/IONIZATION energy, aiding in the laser energy and (MALDI) ionization of the sample transfers it to the sample, ionizing it. 3. Ions are analyzed based on their m/z ratios. _______________________________ ionizes samples by Analysis of biomolecules, 1. Sample is introduced Quantification of proteins applying a high voltage to including proteins, nucleic into the ion source as a and metabolites in ELECTROSPRAY a liquid sample, producing acids, and metabolites liquid. biological fluids IONIZATION charged droplets that 2. High voltage generates (ESI) evaporate to yield ions a spray of charged droplets. 3. Droplets evaporate, leaving ions that are analyzed based on their m/z ratios. _______________________________ measures the time ions High-resolution analysis of 1. Ions are generated and High-throughput take to travel through a biomolecules and small accelerated by an proteomics and TIME-OF-FLIGHT flight tube to a detector. molecule electric field. metabolomics studies. (TOF) The time of flight is 2. Ions travel through a proportional to the ion's flight tube, with their mass-to-charge ratio time of flight measured. 3. The m/z ratios are calculated based on the time of flight. _______________________________ uses a set of four parallel Quantitative analysis of 1. Ions are generated and Drug testing and rods to filter ions based on small molecules and passed through the quantitative analysis of their m/z ratios. It can targeted proteomics quadrupole. biomarkers QUADRUPOLE operate in either single 2. The quadrupole creates ion monitoring or multiple an electric field that reaction monitoring allows ions of specific modes m/z ratios to pass through to the detector. _______________________________ traps ions in an High-resolution and 1. Ions are trapped in an Detailed analysis of ORBITRAP electrostatic field and accurate mass electrostatic field complex biological measures their oscillation measurements in within an orbitrap. samples and frequency to determine proteomics and 2. The frequency of ion characterization of their m/z ratios metabolomics oscillations is proteins measured to determine the m/z ratio. ______________________________ traps ions in an Structural elucidation and 1. Ions are trapped in a Detailed analysis of ION TRAP electromagnetic field and fragmentation studies three-dimensional or protein structures and sequentially ejects them quadrupole ion trap. fragmentation patterns based on their m/z ratios 2. The trap selectively ejects ions based on their m/z ratios for detection PARTS OF A MASS SPECTROMETRY SYSTEM Ionization source Vacuum System Mass Analyzer Data acquisition and analysis system Detector Sample introduction system Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 7 of 12 CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION PART FUNCTION OTHER INFORMATION SIGNIFICANCE _______________________________ Converts the sample into ions. Types: Proper ionization is critical for Different ionization sources are used Electron Impact (EI): accurate and efficient analysis. IONIZATION SOURCE based on the sample type and analysis Suitable for volatile and small requirement molecules. Matrix-Assisted Laser Desorption/Ionization (MALDI): Ideal for large biomolecules. Electrospray Ionization (ESI): Used for biomolecules in solution. _______________________________ Separates ions based on their mass-to- TYPES The mass analyzer's resolution charge ratio. Quadrupole: Filters ions and accuracy impact the quality of MASS ANALYZER based on m/z ratios. the data. Time-of-Flight (TOF): Measures ion flight time to determine m/z ratios. Orbitrap: Measures ion oscillation frequency for high- resolution analysis. _______________________________ Detects and quantifies the separated Types: Accurate detection is crucial for ions Electron Multiplier: Detects reliable quantification and DETECTOR identification ions by amplifying the signal. Time-to-Digital Converter: Measures the time of flight of ions in TOF MS. _______________________________ Maintains a high vacuum environment Types: Ensures optimal ionization and to prevent ion collisions with air Roughing Pump: Creates a accurate measurement by VACUUM SYSTEM molecules preliminary vacuum. minimizing background High-Vacuum Pump: interference Maintains the high vacuum required for the mass spectrometer. _______________________________ Captures and processes data from the Components: Effective data analysis is essential detector Software: Analyzes mass for interpreting results and DATA ACQUISITION AND spectra, identifies peaks, making accurate conclusions ANALYSIS SYSTEM and quantifies ions. Computer Interface: Allows interaction with the mass spectrometer and data visualization. _______________________________ Introduces the sample into the Types: Consistent sample introduction ionization source. Autosamplers: For ensures reproducibility and SAMPLE INTRODUCTION accuracy of results. SYSTEM automated sample introduction. Manual Injectors: For manual sample loading. CALIBRATION AND MAINTENANCE OF MASS SPECTROMETRY SYSTEMS a. Calibration Importance: Ensures accurate mass measurement and quantification. Methods: o Use of Calibration Standards: Regular calibration with standards of known m/z ratios. o Mass Calibration: Adjusting the mass analyzer to correct any deviations. Frequency: Routine calibration is necessary for maintaining system accuracy. b. Maintenance Ion Source Care: Regular cleaning and maintenance of the ionization source to prevent contamination. Mass Analyzer Checks: Periodic inspection and adjustment to ensure optimal performance. Detector Maintenance: Ensuring the detector is functioning properly and calibrating it as needed. Vacuum System: Regular maintenance to prevent leaks and ensure consistent vacuum levels. Cleaning: Routine cleaning of sample introduction components and other system parts to prevent cross-contamination. Conclusion Mass spectrometry is a versatile and essential analytical tool in clinical chemistry, offering detailed information about molecular composition, structure, and concentration. Understanding the various mass spectrometric techniques and the components of a mass spectrometry system is crucial for effective analysis and accurate results. Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 8 of 12 CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION POTENTIOMETRY Potentiometry is an analytical technique used to measure the _______________________________of a solution relative to a _______________________________electrode. This technique is valuable in clinical chemistry for determining the concentration of ions and assessing the pH of solutions. Introduction to Potentiometry Principle: Potentiometry involves measuring the potential difference between a working electrode and a reference electrode in a solution. This potential difference is related to the concentration of specific ions or the pH of the solution, following the Nernst equation. Applications: Used for pH measurement, ion concentration analysis, and redox potential determination. Types of Potentiometric Techniques TYPE PRINCIPLE APPLICATIONS PROCEDURE EXAMPLE ____________________________ Measures the hydrogen ion Used in clinical 1. Immerse the pH Measurement of blood concentration in a solution, laboratories to measure electrode in the pH to assess acid-base providing the pH value. blood pH, urine pH, and solution. balance pH MEASUREMENT The pH electrode consists of a other bodily fluids 2. The electrode glass membrane sensitive to generates a potential hydrogen ions. proportional to the hydrogen ion concentration. 3. The potential is converted to pH using calibration curves. ____________________________ Measures the concentration of Analysis of electrolyte 1. Place the ion- Determination of _____________________________ specific ions in a solution using levels in blood, urine, and selective electrode in potassium levels in an ion-selective electrode. other biological fluids the solution. serum Ion-Selective Each ISE is designed to 2. The electrode Electrode (ISE) respond to a particular ion, generates a potential Measurement such as sodium, potassium, or related to the ion chloride concentration. 3. The potential is measured and compared to calibration standards to determine ion concentration. ____________________________ Measures the electrode Used in studies of 1. Immerse a redox Assessment of oxidative ____________________________ potential related to the redox oxidation-reduction electrode in the stress in biological reactions in a solution. reactions and in solution. samples The redox potential indicates evaluating the oxidative 2. The electrode Redox Potential Measurement the tendency of a solution to status of biological measures the gain or lose electrons samples potential related to the redox reaction. 3. The potential is used to assess the redox status or the presence of specific redox-active species. ____________________________ Uses potentiometry to Quantitative analysis of 1. Add a titrant to the Determination of the _____________________________ determine the endpoint of a acids, bases, and other sample solution concentration of an titration by monitoring the substances in complex while monitoring the unknown acid in a POTENTIOMETRIC change in potential as a mixtures potential. sample TITRATION reagent is added. 2. The potential changes as the titration progresses. 3. The endpoint is identified by a significant change in potential. PARTS OF A POTENTIOMETRY SYSTEM Electrodes Calibration Standards Potentiometer Data Acquisition and Analysis System Sample Holder Temperature Control PART FUNCTION OTHER INFORMATION SIGNIFICANCE ___________________________ Measure the electrical potential and Types: Accurate electrode performance interact with the solution Reference Electrode: is essential for reliable ELECTRODES Provides a stable reference measurements. potential, such as the Silver/Silver Chloride (Ag/AgCl) electrode. Working Electrode: Detects the potential change in response to the analyte, such as a pH electrode or ion- selective electrode. Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 9 of 12 CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION ____________________________ Measures and displays the potential Features: The potentiometer ensures POTENTIOMETER difference between the working and Voltage Measurement: accurate potential measurement reference electrodes Measures the potential in and data acquisition millivolts. Calibration Controls: Allows calibration with standard solutions. ____________________________ Holds the sample and electrodes Types: Proper sample handling ensures during measurement. Electrode Cells: Designed to consistent results and prevents SAMPLE HOLDER accommodate different types contamination. of electrodes and samples. Beakers and Flasks: Used for holding solutions during titrations or measurements. ___________________________ Provide reference values for Types: Calibration standards are crucial calibration of the potentiometric pH Buffers: Used for for ensuring accuracy and CALIBRATION system calibrating pH electrodes. reliability in measurements. STANDARDS Ion Standards: Used for calibrating ion-selective electrodes. ___________________________ Captures and processes the Components: Effective data analysis ensures ___________________________ potentiometric data. Software: Analyzes potential accurate interpretation and ____________________________ data and provides results, reporting of results DATA ACQUISITION such as pH or ion AND ANALYSIS concentration. SYSTEM Computer Interface: Allows interaction with the potentiometer and data visualization. ______________ Maintains a consistent temperature Types: Temperature control ensures _______________ during measurements, as temperature Thermostatic Baths: Used accurate and reproducible TEMPERATURE can affect electrode performance. to control the temperature of measurements. CONTROL the solution. Temperature Probes: Measure the temperature of the sample. CALIBRATION AND MAINTENANCE OF POTENTIOMETRY SYSTEM a. Calibration Importance: Ensures accuracy and reliability of measurements. Methods: o Regular Calibration: Using standard solutions for pH and ion-selective electrodes. o Adjustment: Calibrating the potentiometer with known reference values. Frequency: Regular calibration is necessary to maintain system performance. b. Maintenance Electrode Care: Regular cleaning and proper storage of electrodes to prevent contamination and deterioration. System Checks: Periodic inspection of the potentiometer and other system components. Calibration Standards: Regular preparation and use of fresh calibration standards to ensure accuracy. Cleaning: Routine cleaning of sample holders and other components to prevent buildup and contamination. Conclusion Potentiometry is an essential analytical technique in clinical chemistry, providing valuable information on ion concentrations, pH levels, and redox potentials. Understanding the different potentiometric techniques and the components of a potentiometry system is crucial for accurate and reliable measurements. AUTOMATION IN CLINICAL CHEMISTRY Automation in clinical chemistry has revolutionized laboratory operations, enhancing efficiency, accuracy, and throughput. Automated techniques streamline processes, reduce manual errors, and increase productivity. Introduction to Automation in Clinical Chemistry Principle: Automation involves using machines and technology to perform laboratory tasks that were traditionally done manually. It includes sample handling, analysis, and data management. Applications: Automated techniques are used for high-throughput testing, routine assays, and complex analyses, such as immunoassays and molecular diagnostics. TYPES OF AUTOMATED TECHNIQUES TECHNIQUE PRINCIPLE APPLICATIONS TYPES PROCEDURE EXAMPLES _______________________ perform assays Used for routine ____________________________Analyzers: Clinical Chemistry 1. Sample Automated ________________________ and tests with clinical tests, Measure analytes in serum, plasma, Loading: clinical AUTOMATED minimal human such as blood or urine. Examples include those Samples are chemistry ANALYZER intervention. chemistry using colorimetric, fluorometric, or automatically analyzers They handle panels, chemiluminescent assays. loaded into the such as the sample electrolyte _________________________ Immunoassay Analyzers: analyzer. Siemens preparation, assays, and liver Used for hormone levels, drug 2. Reagent ADVIA or reagent addition, function tests testing, and infectious disease Addition: Roche Cobas and markers, employing techniques like Reagents are system measurement ELISA or chemiluminescence. dispensed and automatically. mixed with samples. 3. Measurement: Various detection Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 10 of 12 CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION methods (e.g., photometric, fluorometric) are used to determine analyte concentrations. 4. Data Processing: Results are calculated and reported. ___________________________ manage the Used to Types: 1. Sample Automated ___________________________ transport and streamline Robotic Systems: Automated Preparation: track systems processing of workflows, robots handle sample placement, Samples are used in large AUTOMATED samples, especially in pipetting, and mixing. processed, laboratories SAMPLE HANDLERS including high-volume Conveyor Systems: Transport diluted, or to connect aliquoting, laboratories samples between different mixed different mixing, and analytical instruments and automatically. analytical transferring workstations. 2. Transport: instruments between Samples are different stages moved to of testing different stations for further analysis. _________________________ perform precise Used for sample Types: 1. Pipetting: Tecan _________________________ liquid handling preparation, Single ________-Channel Pipettors: Automated Freedom EVO tasks, such as reagent addition, Dispense one sample at a time. systems and Hamilton dispensing and and mixing in _________- Multi Channel Pipettors: aspirate and Microlab AUTOMATED transferring various assays Handle multiple samples dispense STAR systems PIPETTING liquids between liquids with SYSTEMS simultaneously, increasing containers throughput. high precision. 2. Mixing: Some systems include mixing functions to ensure homogeneous samples. _________________________ These systems Used for high- Types: 1. Liquid Labcyte Echo ___________________________ automate throughput _______________________________Systems: Deck-based Transfer: liquid complex liquid screening, Include multiple modules for Automated handlers and AUTOMATED handling tasks, sample various liquid handling tasks. systems Beckman LIQUID including sample preparation, and transfer liquids Coulter HANDLING ___________________ Workstation Systems: Integrate preparation, assay liquid handling with other with high Biomek SYSTEMS reagent development laboratory functions, such as accuracy. systems dispensing, and incubation or shaking. 2. Plate plate formatting Preparation: Systems prepare microplates for assays, including dispensing reagents and samples. 3. _________________________ Manage and Ensure accurate Types: 1. Data Laboratory _________________________ process data data recording, LIS Integration: Connects Collection: information generated by result analysis, automated systems with laboratory Automated systems like AUTOMATED DATA automated and integration databases and electronic health systems collect Sunquest or MANAGEMENT analyzers and with laboratory records. and store data Cerner SYSTEMS other systems, information Data Analysis Software: Analyzes from various including result systems (LIS) and interprets results, including assays. reporting and statistical analysis and trend 2. Result quality control monitoring. Reporting: Generates reports and integrates with LIS for patient results. Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 11 of 12 CC1 LECTURE: ANALYTIC TECHNIQUES/INSTRUMENTATION PARTS OF AN AUTOMATION SYSTEM PART FUNCTION COMPONENTS SIGNIFICANCE __________________________ Automatically loads samples into Carousel or Rack: Holds and organizes Ensures efficient and accurate SAMPLE LOADER the analyzer or processing system sample tubes. sample handling Robotic Arm: Transfers samples to the appropriate location. ____________________________ Dispenses reagents into samples or Reagent Bottles or Cartridges: Contain Accurate reagent dispensing is REAGENT assay wells reagents crucial for consistent assay DISPENSING UNIT Dispensing Mechanism: Controls the results volume and timing of reagent addition. __________________________ Where samples and reagents mix Mixing Mechanism: Ensures thorough Proper reaction conditions are REACTION and react mixing of reagents and samples essential for accurate CHAMBER Temperature Control: Maintains measurement optimal conditions for reactions _________________________ Measures the results of the assays Optical Detectors: Includes Accurate detection is critical for DETECTION photometers, fluorometers, or reliable assay results SYSTEM chemiluminescence detectors Signal Processing: Converts detected signals into quantitative data ___________________________ Analyzes and processes data from Computer System: Runs software for Ensures accurate interpretation DATA PROCESSOR the detection system data analysis of results and integration with Software: Performs calculations, laboratory systems statistical analysis, and generates reports __________________________ Allows users to control and Touchscreen or Computer User-friendly interfaces ensure monitor the automation system Interface: For system operation efficient system operation and CONTROL INTERFACE and monitoring. troubleshooting User Controls: Includes settings for calibration, maintenance, and operation. Calibration and Maintenance of Automation Systems a. Calibration Importance: Ensures accuracy and precision of measurements. Methods: o Calibration Standards: Regular calibration with known standards for analyzers and pipettors. o Routine Checks: Calibration of detection systems and reagent dispensers. Frequency: Regular calibration is essential for maintaining system performance and reliability. b. Maintenance System Checks: Periodic inspection of all system components, including sample loaders, reagent dispensers, and detection systems. Cleaning: Routine cleaning of sample holders, reaction chambers, and other components to prevent contamination. Troubleshooting: Addressing any issues or malfunctions promptly to ensure smooth operation. Software Updates: Regular updates to software and data management systems for improved functionality and security. Conclusion Automated techniques in clinical chemistry significantly enhance laboratory efficiency, accuracy, and throughput. Understanding the different types of automated techniques and the components of an automation system is crucial for optimizing laboratory operations and ensuring high-quality results REFERENCES Clinical Chemistry: Principles and Correlations (9th edition) by Bishop et al. Tietz Fundamentals of Clinical Chemistry (6th edition) by Burtis et al. END OF HANDOUT Prepared by: Rahimyar Khan T. Pata, RMT, MD Prepared by: Asst. Prof Rahimyar Khan T. Pata, RMT, MD Page 12 of 12