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FEBRUARY 2024|1 CLINICAL CHEMISTRY I LECTURE (ANALYTICAL TECHNIQUES and INSTRUMENTATIONS) -used in measuring "different analytes" in CC Learning Objectives (ANALYTICAL TECHNIQUES and I...

FEBRUARY 2024|1 CLINICAL CHEMISTRY I LECTURE (ANALYTICAL TECHNIQUES and INSTRUMENTATIONS) -used in measuring "different analytes" in CC Learning Objectives (ANALYTICAL TECHNIQUES and INSTRUMENTATIONS): At the end of this session, the students are expected to: Explain the general principles of each analytic method. Discuss the limitations of each analytic technique. Compare and contrast the various analytic techniques. Discuss existing clinical applications for each analytic technique. Describe the operation and component parts of the following instruments: spectrophotometer, atomic absorption spectrometer, fluorometer, osmometer, ion-selective electrode, and pH electrode. References: Clinical Diagnosis and Management by Laboratory Methods (Henry’s 23rd edition) Clinical Chemistry: Techniques, Principles and Correlations (Michael L. Bishop, et.al 7th edition) This handout is to be used only AS A GUIDE AND NOT AS A SOLE REFERENCE FOR YOUR LECTURE IN CLINICAL CHEMISTRY 1 (ANALYTICAL TECHNIQUES and INSTRUMENTATIONS) PLEASE READ YOUR REFERENCE BOOKS FOR A MORE COMPLETE and DETAILED EXPLANATION OF YOUR TOPICS. ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)  FEBRUARY 2024|2 ANALYTICAL TECHNIQUES in CLINICAL CHEMISTRY Analytic techniques and instrumentation provide the foundation for all measurements made in a modern clinical chemistry laboratory. TERMS to REMEMBER: Energy like: serum, analytes  _________________________ Frequency  Transmitted via ELECTROMAGNETIC WAVES that are characterized by their ________________ and _______________________ Wavelength  __________________________ Wavelength  Distance between TWO SUCCESSIVE PEAKS and is expressed in terms of ___________________ Nanometer (nm) 3 regions:  700nm – INFRARED region PLANCK's Formula The relationship between wavelength and energy (E) is described by ________________  E = hv No need to compute  E – Energy of a photon in JOULES or eV rather just −34 representation to  h – Constant (6.626 x 10 erg sec) their relationship  v – Frequency  ____________________________  NUMBER of VIBRATION of waves per second  The ____________________________________________________________________________ Notes:  NOMINAL WAVELENGTH  Represents the wavelength in nanometer at peak transmittance  A slight error in wavelength adjustments can introduce a significant error in absorbance readings  WAVELENGTH ACCURACY  Wavelength indicated on the control dial being the ACTUAL WAVELENGTH of light that has passed thru the monochromator  __________________________________ – Used to check for WAVELENGTH ACCURACY/WAVELENGTH CALIBRATION  ________________________________________________ – Verifies the ABSORBANCE ACUURACY on LINEARITY COLORIMETRY ________________________________________  Photometric instruments measure light intensity without consideration of wavelength.  Primary analytical utility of spectrophotometry or filter photometry is the isolation of __________________________________ for the purposes of measurement Two types of measurement: a. SPECTROPHOTOMETRIC MEASUREMENT  Measurement of light intensity in a __________________________________________ b. PHOTOMETRIC MEASUREMENT  Measurement of light intensity ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)  FEBRUARY 2024|3 A. SPECTROPHOTOMETRY  Involves measurement of _________________________ by a solution to determine the CONCENTRATION OF THE LIGHT – ABSORBING SUBSTANCE in the solution  Follows the principle of ____________________ ____________________________  States that the concentration of the unknown substance is ______________________________________________________________________________________ _____________________________________________________________  Mathematically establishes the relationship between concentration and absorbance ABSORBANCE (A)  aka __________________________________  Amount of light absorbed  Proportional to the inverse log of transmittance (reflected light)  Mathematically derived from %T A = abc = 2 – log%T = -logT A – Absorbance a – Molar absorptivity; absorptivity of the compound under standard conditions b – Length of light through the solution c – concentration of absorbing molecules/solution PERCENT TRANSMITTANCE  Ratio of the radiant energy transmitted (T) divided by the radiant energy incident in the sample (I) 𝐼𝑡 %T = 𝑥 100 𝐼𝑜  𝐼𝑡 = transmitted light thru the sample  𝐼𝑜 = intensity of light striking the sample  %T measured by commercial spectrophotometers is the ratio of the sample transmitted beam divided by the blank transmitted beam.  In actual practice, the light transmitted by a blank is substituted for 𝐼𝑜 𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑒𝑎𝑚 𝑖𝑛 𝑙𝑖𝑔ℎ𝑡 %T = 𝑥 100 𝑏𝑙𝑎𝑛𝑘 𝑏𝑒𝑎𝑚 𝑠𝑖𝑔𝑛𝑎𝑙 TYPES OF SPECTROPHOTOMETER (a) ____________________________________  SIMPLEST TYPE of absorption spectrometer  Designed to MAKE ONE MEASUREMENT at a time at _________________________________________  The absorption maximum of the analyte must be _______________________ when used (b)________________________________________  An instrument that splits the monochromatic light into two components:  One beam passes THROUGH THE SAMPLE Other beam passes THROUGH A REFERENCE SOLUTION or BLANK  Corrects for the VARIATION IN THE LIGHT SOURCE INTENSITY  The absorbance of the sample can be recorded directly as the electrical ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)  FEBRUARY 2024|4 output of the sample beam (b.1) Double – beam ________________ – Uses TWO (2) PHOTODETECTORS. For the sample beam and reference beam (b. 2) Double – beam _________________ – Uses ONE PHOTODETECTOR and alternately passes the monochromatic light through the sample cuvet and then reference cuvet using ______________________________________________ Six (6) components of a single or double – beam configuration spectrophotometer 1. Stable source of radiant energy 2. Filter that will isolate a specific region of the electromagnetic spectrum 3. Sample holder 4. Radiation detector 5. Signal processor 6. Read – out device 1. LIGHT/RADIANT SOURCE  Provide the ____________________________ and must generate sufficient radiant energy or power to measure the analyte of interest  An intense beam of light is directed through the monochromator an the sample  To give accurate absorbance measurements thoughout its absorbance range, its response to change in light intensity must be ___________ Two (2) types: 1.1 CONTINUUM SOURCE  Emits radiation that ___________________________  Most WIDELY USED IN THE LABORATORY (a) TUNGSTEN LIGHT BULB  Most commonly used light source in the visible and near infrared region (b) DEUTERIUM LAMP  Routinely used to provide UV radiation in analytic spectrometers (c) XENON DISCHARGE LAMP  Produces a continuous source of radiation which covers both the UV and the Visible range 1.2 LINE SOURCE  Emits LIMITED radiation and wavelength  Emits a few discrete lines and find wide use in AAS, molecular and fluorescent spectroscopy  Examples: (a) Mercury and Sodium vapor lamps – UV and Visible regions (b) Hollow cathode lamp – AAS (c) LASER – Light Amplification by Stimulated Emission of Radiation ALTERNATIVES FOR TUNGSTEN BULB: 1. MERCURY ARC (VISIBLE AND UV) 4. XENON LAMP – UV 2. DEUTERIUM LAMP (165NM) – UV 5. MERST GLOWER – IR 3. HYDROGEN LAMP – UV 6. GLOBAR (SILICONE CARBIDE) – IR Factors for choosing a light source:  Range  Spectral distribution within the range  Source of radiant production  Stability of the radiant energy  Temperature 2 ENTRANCE SLIT  MINIMIZES ___________________________  PREVENTS entrance of ____________________ into the monochromator system STRAY LIGHT  Any wavelength outside the band transmitted by the monochromator  Does not originate from the polychromatic light source  Causes _________________________________  Limits the maximum absorbance that a spectrophotometer can achieve  MOST COMMON CAUSE OF LOSS OF: ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)  FEBRUARY 2024|5 3. MONOCHROMATOR  ISOLATES ______________________________________ BANDPASS  Total range of wavelengths transmitted  Defines the range of wavelengths transmitted and is calculated as width at more than half the maximum transmittance Types of monochromators: (a) FILTERS  Simple, least expensive, not precise but useful  Usually pass a relatively wide band of radiant energy and have a low transmittance of the selected wavelength  Made by placing semi – transparent silver films on both sides of a dielectric (Magnesium Fluoride)  INTERFERENCE FILTERS  Produces ___________________________ based on the principle of constructive interference of waves wherein light waves enter one side of the filter and are reflected at the second surface. (b) PRISMS  Wedge – shaped pieces of glass, quartz or sodium chloride  A narrow beam of light focused on a prism is refracted as it enters the more dense glass.  Short wavelengths are refracted more than long wavelengths, resulting in dispersion of white light into a continuous spectrum.  The prism can be rotated, allowing only the desired wavelength to pass through an exit slit. (c) DIFFRACTION GRATINGS  MOST COMMONLY USED  Gives better resolution compared to prisms  Consists of many parallel grooves /cutting grooves (15,000 or 30,000 per inch) or slits into an aluminized surface of a flat – piece of crown glass etched onto a polished surface.  Diffraction – the separation of light into component wavelengths based on the principle that wavelengths bend as they pass a sharp corner (d) HOLOGRAPHIC GRATINGS 4. EXIT SLIT  Controls the _______________________________________________________________  Allow only a narrow fraction of the spectrum to reach the sample cuvette  Accurate absorbance measurement requires a bandpass of less than 1/5 the natural bandpass of the spectrophotometer  Spectral purity of the spectrophotometer is reflected by the bandpass, that is, ____________________________________________________________________________ 5. CUVET  Aka: Absorption cell, Analytical cell, sample cell  Holds the solution (whose concentration is to be measured)  Kinds of cuvet: (1) ALUMINA SILICA GLASS  MOST COMMONLY USED  Used in 350 – 2000 nm (2)QUARTZ/PLASTIC  Used for the measurement of solutions requiring visible and ultraviolet spectra (3) BOROSILICATE GLASS (4) SOFT GLASS Key Notes:  Cuvets with scratches on their optical surface → SCATTERED LIGHT, therefore ___________________  Silica cuvettes transmit light effectively at a wavelength of >220 nm  Alkaline solutions should not be left standing in cuvets for prolonged periods  The path of length of cuvet is _________, although much smaller path lengths are used in automated systems.  To increase sensitivity, some cuvets are designed to have a path length of 10cm, increasing the absorbance for a given solution by a factor of 10. ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)  FEBRUARY 2024|6 6. PHOTODETECTOR  ______________________ transmitted light into _________________________  Detects the amount of light that passes through the sample in the cuvet Kinds of detectors: (1) PHOTOCELL/BARRIER CELL/PHOTOVOLTAIC CELL  SIMPLEST, LEAST EXPENSIVE  _________________________________  Used in filter photometers with a wide bandpass  It is a basic phototransducer that is used for detecting and measuring radiation in the visible region  Composed of SELENIUM on a plate of iron covered with transparent layer of silver  Requires _______________________________ but utilized internal electron transfer for current production - _______________________________________- (2) PHOTOTUBE  Contains ____________________ enclosed in a glass case  Has a photosensitive material that gives off electron when light energy strikes it  Requires _____________________ for operation (3) PHOTOMULTIPLIER TUBE (PMT)  MOST COMMONLY USED  __________________ and has a rapid response, can detect very low level of light  Measures both the visible and UV regions  The response begins when incoming photons strike a photocathode.  _____________________________________________________________  These tubes are limited to measuring low power radiation because intense light causes irreversible damage to the photoelectric surface (4) PHOTODIODE  Not as sensitive as the PMT but with __________________________  Measures light at a multitude of wavelengths – detects less amount of light  Lower dynamic range and higher noise compared to PMT  Most useful as ______________________________________ ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)  FEBRUARY 2024|7 7. METER or READ – OUT DEVICE  Displays the output of the detection system  Examples:  Galvanometer  Ammeter  LED (Light – Emitting Diode) display Wavelength (nm) Color absorbed Complementary color 350 – 430 Violet Yellow – Blue 431 – 475 Blue Yellow 476 – 495 Green – Blue Orange 496 – 505 Blue Green Red 506 – 555 Green Purple 556 – 575 Yellow – Green Violet 576 – 600 Yellow Blue 601 – 650 Orange Green – Blue 651 – 700 Red Blue – Green Key notes:  The amount of light absorbed at a particular wavelenght depends on: MOLECULAR and ION TYPE present. This amount may also vary with: CONCENTRATION, pH and TEMPERATURE  The light path must be kept constant to have an absorbance proportional to the concentration  Deviation from the Beer’s law may be caused by changes in instrument functions or chemical reactions.  Instrument deviations is commonly a result of FINITE BAND PASS of the filter or monochromator  Turbidity readings on a spectrophotometer are GREATER IN THE BLUE REGION  An absorbance check is performed using GLASS FILTERS or solutions with known absorbance values for a specific wavelength  The operator simply measures the absorbance of each solution at a specified wavelength and compares the result with the stated values.  The linearity of a spectrophotometer can be determined using OPTICAL FILTERS or solutions that have known absorbance values for a given wavelenght BLANKING TECHNIQUE  Blank solution contains _______________________ to complete the assay  May not be effective in some cases of turbidity and ___________________ may be necessary to clear the serum of plasma of chylomicrons  Lipids interfere mainly by increasing light scatter (Turbidity)  To correct for artifactual absorbance readings “Blanking” procedures or dual – wavelenght methods may be used. B. FLAME EMISSION PHOTOMETRY (FEP/EFP) Measures the light emitted by a _________________________________________________________  Principle: ______________________________________________________________________________________  Light source: ______________________________  Method: INDIRECT INTERNAL STANDARD METHOD  Internal standard used: _________________ → Corrects variations in flame and atomizer characteristics  Application: Measurement of ____________________  FLICKERING LIGHT: Indicates CHANGES IN THE FUEL READING OF THE INSTRUMENT ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)  FEBRUARY 2024|8 C. ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS) Measures the light ___________________________________________________________________________  Principle: Element is __________________________  Light source: __________________________  Interferences: CHEMICAL, MATRIX and IONIZATION  Application: Measurement of __________________  More sensitive than FEP, accurate and very specific  Internal Standard __________________________ SPECIAL COMPONENTS/PARTS:  ATOMIZER/NEBULIZER/GRAPHITE FURNACE  CONVERTS _______________________  CHOPPER  MODULATE ______________________  ANTHANUM/STRONTIUM CHLORIDE  Added to samples to form __________________________________________________ VOLUMETRIC (TITRIMETRIC) Principle: The unknown sample is made to react with a known solution in the presence of an indicator  Examples:  Schales and Schales method (Chloride test)  EDTA titration method (Calcium test) TURBIDIMETRY For measurement of ABUNDANT LARGE PARTICLES (PROTEINS) and BACTERIAL SUSPENSIONS  Principle: It determines the amount of light ____________________________________ by a particulate matter in a turbid solution  Dependent on: ___________________________________________________________________________  Application:  Protein measurement (CSF and Urine)  Detects bacterial growth in broth cultures  Antimicrobial test (Broth method)  Detect Clot formation NEPHELOMETRY For measuring the amount of ANTIGEN – ANTIBODY COMPLEXES (PROTEINS)  Principle: It determines the amount of ________________________by a particulate matter suspended in a turbid solution  Light scattering depends on: _________________________________________________________  Light scattered by particles is measured at an angle, typically 15 – 90 degrees to the beam incident on the cuvet  Most antigen – antibody complexes have a diameter of 250 – 1500 nm, and the wavelength used are 320 – 650 nm, thus light is scattered FORWARD (RAYLEIGH – DEBYE TYPE)  The detector (PMT) output is proportional to the concentration Components of nephelometer:  Light source  Sample cuvet  Collimator  Stray light trap  Monochromator  Photodetector ELECTROPHORESIS  Migration of CHARGED PARTICLES in an electric field  Separates PROTEINS based on their electric charge densities  The ACIDIC and BASIC AMINO ACIDS determines the net charge on a protein, hence its electrophoretic mobility  During electrophoresis, proteins are NEGATIVELY CHARGE ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)  FEBRUARY 2024|9 (ANIONS) and they move towards the ANODE Important terms to remember:  __________________________  Has a net charge of that can either be positive or negative depending on the pH condition  __________________________  Movement of buffer ions and solvent relative to the fixed support  __________________________  Migration of small charged ions  __________________________  Migration of charged macromolecules Components of electrophoresis:  Electrical power  Support medium  Buffer: BARBITAL (pH 8.6)  Sample  Detecting system Factors affecting rate of migration: 1. Net electric charge of the molecule 2. Size and shape of the molecule 3. Electric field strength 4. Nature of the supporting medium 5. Temperature of the operation Supporting media: 1. CELLULOSE ACETATE 2. AGAROSE GEL 3. POLYACRYLAMIDE GEL Stains for visualization of Fractions (Bands) 1. Amido Black 2. Ponceau S 3. Oil Red O 4. Sudan Black 5. Fat Red 7B 6. Coomasie Blue 7. Gold/Silver Stain DENSITOMETRY Measures the _____________________________________________ SCANS and QUANTITATES electrophoretic pattern ISOELECTRIC FOCUSING  Separates molecules by MIGRATION THROUGH _______________________________  Ideal for separating proteins of IDENTICAL SIZES but DIFFERENT ______________________  Proteins move in the electric field until they reach a pH equal to their isoelectric point Advantages: 1. The ability to RESOLVE MIXTURE OF PROTEINS 2. Detects ISOENZYMES 3. Identify GENETIC VARIANT OF PROTEINS 4. Detects CSF OLIGOCLONAL BANDING CAPILLARY ELECTROPHORESIS  Sample molecules are separated by __________________________________  Utilizes _____________________________________ of specimens  Positively charged ions in the specimen emerge early at the capillary outlet because of EOF and the ion movements are in the same directions  Negatively charged ions in the specimen move towards the capillary outlet but a t a slower rate  Application:  Separation, quantitation and determination of molecular weights of proteins and peptides  Analysis of PCR products, organic and inorganic substance and drugs ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)  F E B R U A R Y 2 0 2 4 | 10 CHROMATOGRAPHY Separation of _____________________________________ in a solution by specific differences in the ____________________________________________ of the different constituents Bases of separation: 1. Rate of diffusion 8. Ionic attraction 2. Solubility of the solute 9. Differential distribution between 2 3. Nature of the solvent immiscible liquids 4. Sample volatility/solubility 10. Selective separation of substances 5. Distribution between 2 liquids 11. Differences in the absorption and 6. Molecular size desorption of solutes 7. Hydrophobicity of molecule TWO FORMS OF CHROMATOGRAPHY A. PLANAR 1. PAPER CHROMATOGRAPHY  Used for the fractionation of sugar and amino acid  Sorbent (Stationary phase) – WHATMAN PAPER 2. THIN LAYER CHROMATOGRAPHY (TLC)  ___________________________________________________________  Sample components are identified by comparison with standards on the same plate  Extraction of the drug is _________________________  When all drug spots including the standards have migrated with the solvent front, it is caused by INCORRECT AQUEOUS to NON – AQUEOUS SOLVENT MIXTURE  Sorbent: Thin plastic plates impregnated with a layer of silica gel or alumina B. COLUMN 1. GAS CHROMATOGRAPHY (GC)  Used for the separation of  Useful for compounds that are __________________________ or can be easily converted into a volatile form  Samples are introduced into the GC column using a hypodermic syringe or an automated sample → Specimen are vaporized and swept onto the column (Elution order of volatiles are based on their boiling point  Mobile phase: Nitrogen, Helium, Hydrogen and Argon (Inert gasses) MASS SPECTROSCOPY (MS)  Based on _________________________________ of molecules using a suitable source of energy  Can also detect structural information and determination of molecular weight  BEFORE A COMPOUND CAN BE DETECTED BY MS, IT MUST BE SEPAREATED FIRST BY GC TANDEM MASS SPECTROSCOPY (MS/MS)  Can detect 20 inborn errors of metabolism form a single blood spot GAS CHROMATOGRAPHY – MASS SPECTROSCOPY (GC –MS)  ______________________________________________________  Also used for xenobiotics, anabolic steroids and pesticides  Quantitative measurement of drug can be performed by selective ion monitoring  Uses an electrode beam to split the drug emerging from the column into its component ions – drugs are detected by means of the presence of decomposition fragments which arises after degradation of the analytes  The position of the parent – molecule – ion and degradation products give rise to fingerprint patterns that which will provide the final identity of the drug of interest 2. LIQUID CHROMATOGRAPHY  Based on the distribution of solutes between a liquid phase and a stationary phase  HPLC – Most widely used HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)  Uses pressure for fast separations, controlled temperature, in – line detectors and gradient elution technique  Use: Fractionation of drugs, hormones, lipids, carbohydrates and proteins. Separation and quantitation of various hemoglobin associated with specific disease LIQUID CHROMATOGRAPHY – MASS SPECTROSCOPY (LC – MS)  Detecting ________________________________ in the body fluid  Utilized to __________________________________________ → Complementary method to GC – MS ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)  F E B R U A R Y 2 0 2 4 | 11  Also used in therapeutic drug monitoring, toxicology and studies of drug metabolites  Requires interface methods to convert non – volatile to volatile compounds  Interface methods: ELECTROSPRAY (ES) and ATMOSPHERIC PRESSURE CHEMICAL IONIZATION (APCI) Separation mechanism in liquid Chromatography: 1. Gel/Gel Permeation/Gel Filtration/Size Exclusion/ Molecular Sieve Chromatography 2. Ion exchange chromatography 3. Partition Chromatography (Liquid – Liquid Chromatography) 4. Affinity Chromatography 5. Adsorption Chromatography FLUOROMETRY/MOLECULAR LUMINESCENCE  Measures the amount of light intensity over a _______________________________  Principle: Determines the light emitted by a molecule after excitation by electromagnetic radiation  Light source: Mercury arc or Xenon lamp  Light detector: Photomultiplier Tube or Phototube  Application: Measures porpyrins, magnesium, calcium and catecholamines  Uses _______________________________________ (Either: Filters, Prisms or Gratings)  The wavelength that is best absorbed by the solution to be measured is selected by the primary filter  The incident light is prevented from striking the photodetector by the secondary filter  About 1000x more sensitive than spectrophotometer – Emitted radiation is measured directly  Affected by QUENCHING – pH, Temperature changes, Chemical contaminants, UV light changes CHEMILUMINESCENCE  Emission of light created from a chemical or electrochemical reaction and not from absorption of electromagnetic energy  Principle: The chemical reaction yields an electronically excited compound that emits light as it returns to its ground state or that it transfers its energy to another compound, which then produces emission  Use: Immunoassays  Photodetector: Photomultiplier tube (LUMINOMETER)  More sensitive than fluorescence  In this method, no excitation radiation is requires and no monochromator are needed because the chemiluminescence arises from one species  This involves oxidation of an organic compound (Dioxetane, Luminol, Acridium Ester) by an oxidant (hydrogen peroxide, hyochlorite or oygen). These oxidation reactions may occur in the presence of catalysts, such as enzymes (ALP, HRP or microperoxidase). Metal ions (Cu2+ or Fe3+ phthalocyanine complex) and hemin  The excited products formed in the oxidation reaction produces chemiluminisence on return to the singlet state  A typical signal from a chemiluminescent compound rises rapidly with time and reaches a maximum when reagent and analyte are completely mixed OSMOMETRY  Measurement of OSMOLALITY of an aqueous solution such as serum, plasma or urine  Principle: Based on measuring changes in the colligative properties of solutions that occur owing to variations in particle concentrations  Colligative properties: Osmotic pressure, Boiling point, Freezing point and vapor pressure  Osmotic particles: Glucose, Urea and Sodium  When an active osmotic particels are added to a solution, osmolality increases and four other properties of te solution are affected As the osmolality of a solution increases, the following reaction occurs:  Osmotic pressure increases  Boiling point is elevated  Freezing point is depressed  Vapor pressure depressed Freezing point depression osmometry  Most commonly used method for measuring the changes in the colligative properties of a solution ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)  F E B R U A R Y 2 0 2 4 | 12  Based on the principle that addition of solute molecules lowers the temperature at which a solution freezes.  A 1.0 mOsm/kg solution has a freezong point depression at 0.00186⁰C when compared with pure solvent (usually water)  Blood plasma, with an osmolality of about 285 mOsm/kg, has a freeezing point of about -53 ⁰ C ELECTROCHEMISTRY TECHNIQUES  Measurement of CURRENT and VOLTAGE generated by the activity of a specification 1. POTENTIOMETRY  Measurement of ___________________________________ due to the activity of free ions  Follows the ____________________________________  Change in the voltage indicates activity of each analyte  Measurement of differences in voltage (potential) at a constant current  Reference electrodes: CALOMEL and SILVER – SILVER CHLORIDE  Use: pH and pCO2 tests ION SELECTIVE ELECTRODE  Electrochemical transducer capable of responding to one given ion  VERY SENSITIVE and SELECTIVE for the ion it measures.  Measures the activity of one ion much more than the other ions present in the sample.  Ionic selectivity depends on the membrane/barrier composition used  ISE analyzers measure the electrolyte dissolved in the fluid phase of the sample in mmol/L of plasma water  It is a sensitive method but NOT SPECIFIC → does not discriminate between ions causing voltage differences between the measuring electrode and the standard electrode  Those that uses undiluted samples are not subjected to pseudohyponatremia caused by hyperlipemic samples 2. COULOMETRY  Measurement of the ___________________________________  Electrochemical titration in which the titrant is electrochemically generated and the endpoint is detected by amperometry  Follows the ________________________________  Use: Chloride test (CSF, Serum and Sweat)  Interferences: Bromide, Cyanide and Cysteine 3. AMPEROMETRY  Measurement of the ____________________________________________  Use; pO2, glucose, chloride and peroxidase determinations POLAROGRAPHY  Measurement of differences in current at a constant voltage  Follows the _______________________________________ 4. VOLTAMMETRY  Measurement of current after which a potential is applied to an electrochemical cell  Allows the sample to be preconcentrated, thus utilizing minimal analyte ____________________________________ – For lead and iron testing ANALYTICAL TECHNIQUES TJ Rivera Jaula, RMT, MLS(ASCPi)