Clinical Chemistry: Unit 3 - Analytical Techniques PDF

CAGAYAN STATE UNIVERSITY ANDREWS CAMPUS, TUGUEGARAO CITY, CAGAYAN COLLEGE OF ALLIED HEALTH SCIENCES BACHELOR OF SCIENCE IN PUBLIC HEALTH CLINICAL CHEMISTRY: UNIT 3: ANALYTICAL TECHNIQUES REAN STAR PAULINE C. CORTINA, RMT INSTRUCTOR, CSU-CAHS OUTLINE §...

CAGAYAN STATE UNIVERSITY ANDREWS CAMPUS, TUGUEGARAO CITY, CAGAYAN COLLEGE OF ALLIED HEALTH SCIENCES BACHELOR OF SCIENCE IN PUBLIC HEALTH CLINICAL CHEMISTRY: UNIT 3: ANALYTICAL TECHNIQUES REAN STAR PAULINE C. CORTINA, RMT INSTRUCTOR, CSU-CAHS OUTLINE § Spectophotometry § Electrochemistry § Osmometry § Electrophoresis § Chromatography and Mass Spectrophotometry  SPECTROPHOTOMETRY § Electromagnetic radiation is described as photons of energy traveling in waves can take several forms, the most recognizable being light and radiant energy other types - gamma rays and X-rays, microwaves, radiofrequency radiation, and ultraviolet radiation § Wavelength is the linear distance between any two equivalent points on a successive wave unit used in the visible spectrum is nm  The relationship between wavelength (λ) and energy (E) is described by Planck’s formula. E = hv where h is the Planck’s constant (6.62 X 10 -27 erg sec) and v is frequency Frequency - the number of oscillations of the waveform in a second Wavelength Visible region: 400 - 700 nm Ultraviolet region: < 400 nm Infrared region: > 700 nm BEER’S LAW Beer ’s law states that the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light.  ε = molar absorptivity b = is the length of light path through the solution c = is the concentration of absorbing T = radiant energy transmitted / molecules transmitted light I = radiant energy incident on the sample / incident light Spectrophotometer - is used to measure the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution. Components of a Spectrophotometer 1. Light Source 2. Monochromator 3. Sample Cell or Cuvet 4. Photodetector 5. Meter or read-out device Components of a Spectrophotometer 1. LIGHT SOURCE Provides polychromatic light Incandescent tungsten or tungsten-iodide lamp – visible and near-infrared regions Deuterium lamp and mercury arc lamp – UV region  Components of a Spectrophotometer 1. LIGHT SOURCE 2 types: A. Continuum Wide applications in the laboratory Emits limited number of discrete lines or bands of radiation Examples: Tungsten (visible region) , deuterium (UV region), xenon (visible and UV regions)  Components of a Spectrophotometer 1. LIGHT SOURCE 2 types: B. Line Emits a few discrete lines or bands of radiation Examples: Mercury and sodium vapor lamps – UV and visible regions Hollow cathode lamp - atomic absorption spectroscopy / spectrophotometry  Components of a Spectrophotometer 2. MONOCHROMATOR Isolates individual wavelengths of light Characteristics: Nominal wavelength Spectral bandwidth (or FWHM) ***FWHM – Full Width at Half Peak Maximum Bandpass  Components of a Spectrophotometer 2. MONOCHROMATOR Types: A. Filters Isolate monochromatic light Simple, inexpensive, and useful Interference and absorption filters B. Prism Can be rotated, allowing only the desired wavelength to pass through an exit slit C. Diffraction gratings Most commonly used; contain parallel grooves  Components of a Spectrophotometer 3. SAMPLE CELL OR CUVET may be round or square and must be made of material that is transparent to radiation used to hold samples; path length is 1 cm Types: A. Fused silica or quarts – UV region B. Alumina-silicate glass – 350-2000 nm C. Plastic cuvet – visible region Double-beam spectrophotometers – two cuvets (for the sample and for the solvent)  Components of a Spectrophotometer 4. PHOTODETECTOR Converts the transmitted radiant energy into an equivalent amount of electrical energy Types: A. Barrier-layer cell or photocell Least expensive; temperature sensitive Composed of selenium on a plate of iron Used mainly in filter photometers B. Phototube Contains cathode and anode enclosed in a glass tube has photosensitive material that gives off electrons when light energy strikes it  Components of a Spectrophotometer 4. PHOTODETECTOR Types: C. Photomultiplier tube (PMT) Most common type 200 times more sensitive than the phototube Highly sensitive to UV and visible radiation D. Photodiode Not as sensitive as PM tube but with excellent linearity and speed  Components of a Spectrophotometer 5. METER OR READ-OUT DEVICE Displays output of the detection system Examples: Digital meters, d’Arsonval meters, recorders, light- emitting diodes (LEDs), cathode-ray tubes (CRTs), and liquid crystal displays (LCDs). Single-Beam Spectrophotometer Double-Beam Spectrophotometer  TWO INSTRUMENT DESIGN Double beam in space – uses 2 photodetectors Double-beam in time – uses 1 photodetector; chopper is used to pass the monochromatic radiation through the sample cuvet and then to the reference cuvet Chopper – a device that rotates or breaks up radiation beams  Quality Assurance in Spectrophotometry Wavelength or photometric accuracy - implies that a photometer is measuring at the wavelength that it is set to - Special glass-type optical filters – didymium glass (600 nm) ; holmium oxide (360 nm) Absorbance check - using glass filters or solutions that have known absorbance values for a specific wavelength  Linearity - the ability of a photometric system to yield a linear relationship between the radiant power incident upon its detector and the concentration - optical filters or solutions Stray light - any light that impinges upon the detector that does not originate from a polychromatic light source - Special cutoff filters  ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS) Atomic Absorption Spectrophotometer Measures concentration by detecting the absorption of electromagnetic radiation by atoms rather than by molecule Light source = hollow-cathode lamp; electrodeless discharge lamp Chopper = modulate the light beam Photodetector = PM tube Sensitive and precise Application: to measure concentrations of trace metals Single-beam atomic absorption spectrophotometer – basic components  ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS) Flameless AAS - uses an electric furnace to break chemical bonds (electrothermal atomization)  FLUOROMETRY Filter fluorometers =measure the concentrations of solutions that contain fluorescing molecules. Light source = mercury (filter fluorometers) and xenon arc (spectrofluorometers) Photodetector = PM tube  FLUOROMETRY Advantages Specificity Sensitivity (1000 times more sensitive than most spectrophotometric methods) Disadvantage Sensitive to environmental changes Quenching - decrease in fluorescence due to these changes  CHEMILUMINESCENCE is different from fluorescence in that no excitation radiation is required and no monochromators are needed oxidation reactions of luminol, acridinium esters, and dioxetanes characterized by a rapid increase in intensity of emitted light followed by a gradual decay Advantages: Subpicomolar detection limits, speed, ease of use, and simple instrumentation Disadvantage Impurities can degrade sensitivity and specificity  TURBIDIMETRY AND NEPHELOMETRY Principle: Turbidimetry and nephelometry are based on the scattering of radiation by particles in suspension. Applications: measurement of antigen–antibody reactions, prealbumin, and other serum proteins Nephelometry is the measurement of the light scattered by a particulate solution. 3 types of light scattering: Rayleigh theory Mie theory Rayleigh-Debye theory  TURBIDIMETRY AND NEPHELOMETRY Three types of light scattering: Rayleigh theory - If the wavelength (λ) of light > the diameter (d) of the particle (d < 0.1λ), the light scatter is symmetrical around the particle. Minimum light scatter occurs at 90 degrees to the incident beam. Mie theory - If the wavelength of light < the particle diameter (d > 0.1λ), then the light scatters forward. Rayleigh–Debye theory - If the wavelength of light is approximately the same as the particle size, more light scatters in the forward direction than in other directions.  TURBIDIMETRY AND NEPHELOMETRY Turbidimetry determines the amount of light blocked by a suspension of particles Applications: It is used in microbiology analyzers, coagulation analyzers, and is used to quantify protein concentration in biologic fluids such as urine and CSF.  LASER APPLICATIONS based on the interaction of radiant energy with excited atoms or molecules ca n s e r ve a s s o u rc e o f i n c i d e nt e n e rg y i n a spectrometer or nephelometer Applications: Hematology and flow cytometer analyzers for the differential analysis of white blood cells  ELECTROCHEMISTRY involves measurement of the current or voltage generated by the activity of specific ions POTENTIOMETRY measurement of potential (voltage) between two electrodes in a solution Reference electrode – electrode with a constant voltage Calomel and Silver/silver chloride Indicator electrode – measuring electrode Concentration of ions – difference between 2 electrodes Nernst equation  ELECTROCHEMISTRY POTENTIOMETRY Ion-Selective Electrode (ISE) is very sensitive and selective for the ion it measures consists of a membrane separating a reference solution and a reference electrode from the solution to be analyzed Glass aluminum silicate Valinomycin gel Organic liquid ion exhangers Gas electrodes Enzyme electrodes  ELECTROCHEMISTRY POTENTIOMETRY Ion-Selective Electrode (ISE) 2 types based on sample preparation: A. Direct ISE B. Indirect ISE  ELECTROCHEMISTRY POTENTIOMETRY pH electrodes Used to measure hydrogen ion activity Buffer: has known hydrogen ion concentration Internal reference electrode: silver/silver chloride External reference electrode: Calomel electrode / mercurous chloride Gas-sensing electrodes Are designed to detect specific gases pCO2 electrode – Severinghaus electrode pO2 electrode – Clarke electrode  ELECTROCHEMISTRY POTENTIOMETRY Enzyme electrodes Used for enzymatic methods Urease – urea / BUN Glucose oxidase – glucose  ELECTROCHEMISTRY COULOMETRY measures the quantity of electricity (in coulombs) needed to convert an analyte to a different oxidation state Application: to measure chloride ion in serum, plasma, CSF, and sweat samples Sweat chloride determination – Coulometric titration  ELECTROCHEMISTRY AMPEROMETRY is the measurement of the current flow produced by an oxidation–reduction reaction Application: to measure chloride ion in serum, plasma, CSF, and sweat samples; pO2 electrode in blood gas analyzers  ELECTROCHEMISTRY VOLTAMMETRY is a method in which a potential is applied to an electrochemical cell and the resulting current is measured Anodic stripping voltammetry – used to measure heavy metals such as lead  ELECTROPHORESIS is the separation of charged compounds based on their electrical charge Cations (+) move toward the cathode (-), and anions (-) toward the anode (+) Support media: paper, cellulose acetate, agarose gel, polyacrylamide gel, starch gel Proteins in serum, urine, CSF and other body fluids  ELECTROPHORESIS Electroendosmosis/Endosmosis - is the movement of buffer ions and solvent relative to the fixed support Iontophoresis - is the migration of small charged ions Zone electrophoresis - is the migration of charged macromolecules Electrophoretogram - the result of zone electrophoresis; consists of sharply separated zones of macromolecule  5 components: Power supply Buffer Support medium Sample Detecting system  ELECTROPHORESIS Stains for Visualization of Fractions 1. Amido Black 2. Ponceau S 3. Oil Red O 4. Sudan Black B 5. Fat Red 7B 6. Coomassie Blue 7. Gold/Silver stain- very sensitive even in nanogram quantities of proteins ELECTROPHORESIS  ELECTROPHORESIS Application: Electrophoresis is considered the most useful single technique for ALP (alkaline phosphatase) isoenzyme analysis. (origin)INTESTINAL PLACENTAL BONE LIVER(anode) For CK (Creatine kinase) isoenzyme analysis  OSMOMETRY O s m o m e t r y i s t h e m e a s u re m e nt o f t h e osmolality of an aqueous solution such as serum, plasma, or urine. Osmotically active particles – glucose, urea nitrogen, sodium O s m o m ete rs – m e a s u re s f re ez i n g p o i nt depression or vapor pressure depression; milliosmolal per kilogram (mOsm/kg)  OSMOMETRY Freezing point osmometer Freezing point depression is proportional to the number of solute particles.  CHROMATOGRAPHY is a separation method based on different interactions of the specimen compounds with the mobile phase and with the stationary phase as the compounds travel through a support medium Basic components: Mobile phase (gas or liquid) – carries the sample Stationary phase (solid or liquid) – which mobile phase flows Column – holds the stationary phase Eluate – separated components  CHROMATOGRAPHY Applications: Paper Chromatography fractionation of sugar and amino acids Thin Layer Chromatography a semi-quantitative drug screening test  CHROMATOGRAPHY Applications: Gas chromatography (GC) for compounds that are naturally volatile or can be easily converted into a volatile form various organic molecules, including many drugs Liquid Chromatography HPLC is the most widely used liquid chromatography  REFERENCES Anderson, Shauna C. et al., (2007). Clinical Chemistry: Concepts and Application, International edition Bishop, Michael et al. (2023). Clinical Chemistry Principles, Techniques, and Correlation, 9th ed. Jones & Bartlett Learning Henry, John Bernard. (2011). Henry’s Clinical Diagnosis and Management by Laboratory Methods 22nd ed. Philadelphia: W.B Saunders Nucum, Zenaida et al. (2005). Laboratory Manual for Biochemistry Philippines: C&E Publishing Inc Patton et al., (2010) Laboratory Manual Seeley’s Essentials of Anatomy and Physiology 7th ed. New York: McGraw Hill Co. Teitz, Norbert et al., (2008) Tietz Fundamentals of Clinical Chemistry Philadelphia: WB Saunders