PH 166 Clinical Chemistry 1 Analytical Techniques - PDF

PH 166: CLINICAL CHEMISTRY 1 ANALYTICAL TECHNIQUES AND INSTRUMENTS PART 2 Mr. James C. Chua | February 10, 2025 OUTLINE →​ROYGBIV - short to increasing wavelengths...

PH 166: CLINICAL CHEMISTRY 1 ANALYTICAL TECHNIQUES AND INSTRUMENTS PART 2 Mr. James C. Chua | February 10, 2025 OUTLINE →​ROYGBIV - short to increasing wavelengths →​The shorter the wavelength, the stronger the A.​ Introduction radiation B.​ Spectrophotometry a.​ Components of Spectrophotometer ​ Visible light falls in between, with the color violet at b.​ Flame Emission Spectrophotometry 400-nm and red at 700-nm wavelengths being the c.​ Atomic Absorption Spectrophotometry approximate limits of the visible spectrum. C.​ Light Emission and Scattering Techniques a.​ Fluorometry b.​ Chemiluminescence c.​ Turbidimetry and Nephelometry d.​ Laser D.​ Electrophoresis ​ a.​ Components of Electrophoresis b.​ Electrophoretogram c.​ Power Supply d.​ Buffers e.​ Support Materials f.​ Isoelectric focusing Figure 1. Visible light spectrum. g.​ Electroendosmosis h.​ Capillary electrophoresis COMPONENTS OF SPECTROPHOTOMETER E.​ Review Questions F.​ References INTRODUCTION ​ Optical Instruments: ○​ Instruments that measure light energy, including ​ Spectrophotometers Figure 2. Single-beam spectrophotometer. ​ Flame Emission ​ Atomic Absorption Table 1. Components of a spectrophotometer ​ Analytical determinations measure absorption or Component Functions transmission of light energy to determine concentration of ​ Provide incident light for the system atoms or molecules. ​ Incandescent Tungsten or Tungsten ​ Electromagnetic Energy iodide lamp ○​ For visible and near infrared spectrum ○​ Transmitted via electromagnetic waves Light source ​ Deuterium-discharge lamp and ○​ Waves are measured in nanometers between the Mercury arc lamp: peaks and valleys (wavelength). ○​ For UV spectrum ​ Beer's Law ​ Silicone carbide ○​ The concentration of a substance is: ○​ For infrared spectrum ​ Directly proportional to the amount of light ​ Isolates specific wavelength from the light absorbed source ​ Inversely proportional to the amount of ​ Interference Filter ○​ Based on constructive interference of transmitted light waves ​ Prism Formula 1. Formula for absorbance ○​ Separates white light into a continuous spectrum. Absorbance = 2 log% (Transmittance) Mono- ○​ Advantage: can isolate wavelength by chromator ones (e.g., 635 → 637) ​ Diffraction gratings SPECTROPHOTOMETRY ○​ Bends light and forms wave fronts. ​ Able to measure intensity of color ○​ Light that is in phase reinforce one ​ Measures the light transmitted by a solution to another, if not it cancels out. determine the concentration of the substance in the ○​ It can isolate by tens or hundreds solution ○​ Gratings - like grills, which isolate particular light colors ○​ It functions by converting light into voltage, which ​ Cuvette or analytical cell translates into absorbance value readings. ​ Holds the solution of which the absorption ​ Because the frequency of a wave is inversely is to be measured proportional to the wavelength, it follows that the energy Sample Cell ​ Soft glass cuvette: visible range of electromagnetic radiation is inversely proportional to ​ Quartz cuvette: UV range wavelength. ​ Goal: Be as clear as possible ​ Converts transmitted radiant energy into ​ Color: light that bounces off an object an equivalent amount of electrical energy. ​ How does light travel: in waves that vary in lengths (radiant ​ Photocell (Barrier layer cell) ​ Photo energy/radiation) ○​ Generates electromotive force (no detector ​ Any form of light is radiation external voltage) ​ Visible spectrum ​ Phototube ​ ○​ Similar to photocell but requires #MagkabigkisBenteSais Group 2 | 1 of 6  external voltage be electronically eliminated ​ Photomultiplier tube ​ ​ Deliver a fine spray of sample ○​ Detects and amplifies radiant energy Nebulizer containing the metallic ion to the (200x sensitive) burne ○​ More sensitive ​ Long narrow slit (permits ​ Photo Iodide ​ absorption of incident radiation) ○​ Uses a photosensitive Burner ​ A fuel gas (acetylene) with an positive-negative junction diode to oxidizing agent (compressed air) is produce a photocurrent. burned to produce the flame Entrance slit -​ Monochromator ​ Gratings Exit Slit -​ Photodetector ​ Photomultiplier tube Read-out Device FLAME EMISSION SPECTROPHOTOMETRY ​ Measures light emitted by excited atom ○​ Used to measure sodium, potassium and lithium because they are easy to excite ​ Flame using propane is used to excite the atoms (higher energy state) ​ Excited atoms return to the ground state by emitting light energy, characteristic for that atom. ○​ Sodium: intense yellow flame ○​ Potassium: violet flame ○​ Calcium: brick red flame ​ Very dangerous ○​ Serum/plasma sample sprayed by aerosol and will be Figure 3. Single-beam atomic absorption spectrophotometer—basic lit on the chamber components. ○​ The color that will be generated will be measured because it wants excited atoms LIGHT EMISSION AND SCATTERING TECHNIQUES ​ Was used for serum electrolyte analysis before, but ​ Instruments that measure light energy anymore because of its inherent danger and impracticality ○​ Fluorometry ○​ Chemiluminescence Table 2. Components of Flame Emission Spectrophotometry (FES). ○​ Turbidity and Nephelometry Component Functions ○​ Laser ​ Atomizer ​ Deliver a fine spray of sample Nebulizer FLUOROMETRY containing the metallic ion to the burner ​ Excitation light ​ A fuel gas (propane) with an ​ Energy emission that occurs when certain compounds Burner oxidizing agent (compressed air) absorb electromagnetic radiation, becomes excited, gives burned to produce the flame off light. ​ Allow only emitted line spectrum ​ Fluorometer Monochromator of specific element to strike the ○​ Measures the concentrations of solution that contain System PMT fluorescing molecules Photosensitive ​ Difference between spectrophotometer ​ Photomultiplier tube detector ○​ Fluorometry measures fluorescent materials; not applicable to all. ATOMIC ABSORPTION SPECTROPHOTOMETRY ○​ Principle of fluorescence: a material absorbs light at ​ Measures light absorbed by ground state atoms a certain wavelength, then it emits light in a different ​ Used to measure concentration of calcium atom (not easily (higher) wavelength. excited) ○​ In-expect mo na ibang kulay ang lalabas. ​ 100 times more sensitive than FES ○​ The wavelength setup would be the only one adjusted. Table 3. Components of Atomic Absorption Spectrophotometry (AAS). ○​ Spectrophotometers are straightforward, you set the Component Functions wavelength of your monochromator to the color that ​ Provide incident light for the you’re expecting. It is the same but with a lower system wavelength. ​ Hallow cathode lamp ○​ Excitation light vs Emitted light ○​ Consists of an evacuated ​ There are certain substances that emit light at a different gas-tight chamber filled wavelength than the original light absorbed. with inert gas (helium or ​ For instance, if the excitation light is violet, then the Light Source argon) lower wavelength light will be emitted (the fluorescence ​ Electrodeless discharge lamp that it will generate), green. ○​ Consists of bulb filled with argon and the Table 4. Components of Filter Fluorometer. element to be tested Component Functions ○​ Radiofrequency generator ​ Gas discharge lamps excites the element ​ Emits short-wavelength ​ Modulates the hollow cathode Light Source high-energy excitation light Beam Chopper light beam ○​ Mercury-vapor lamps ​ Produces an alternating signal to (filter fluorometers) PH 166 | Analytical Techniques and Instruments Part 2 2 of 6  ○​ Xenon-arc lamp ○​ Turbidimetry: light blocked (spectrofluorometers) NEPHELOMETRY Attenuator ​ Controls the light intensity ​ Similar to turbidimetry, except that light scattered by the ​ Selects the wavelength that is best small particles is measured at an angle to the beam Primary Filter incident on the cuvette absorbed by the sample ​ The fluorescing sample emits ​ Light scattering depends on wavelength and particle size Sample Holder fluorescent light in all directions ○​ For macromolecules with a size close to or larger than ​ Passes longer wavelengths of the wavelength of incident light, sensitivity is fluorescent light, preventing increased by measuring the forward light scatter Secondary filter incident light from striking the ​ Monochromatic light obtains uniform scatter and minimizes photodetector sample heating. Photodetector ​ Photomultiplier tube ​ Once light hits a molecule, it will disperse. ​ It measures scattered light, instead of transmitted or Table 5. Advantages and Disadvantages of Filter Fluorometer. absorbed light. Advantage Disadvantage ​ 1000x more sensitive than ​ Quenching interference LASER most spectrophotometry ○​ Reduces intensity of ​ Based on the interaction of radiant energy with suitably fluorescence excited atoms or molecules, which leads to stimulated emission of radiation. ​ Laser light is polarized and coherent and has a narrow spectral width and small cross-sectional area with low divergence. ​ Can serve as the source of incident energy in a spectrometer or nephelometer. ​ Laser spectrometry can also be used for the determination of structure and identification of samples, as well as for diagnosis. ​ An example of the clinical application of the laser is the Coulter counter, which is used for differential analysis of white blood cells. ​ Light impedance: It is used for automated cell counters. Figure 4. Basic filter fluorometer. For instance, blood cells differ in size. There is a laser. It only allows a single line of cells to pass through. As a cell CHEMILUMINESCENCE passes through, it will block the light. Depending on its ​ A part of the chemical energy generated produces excited size, the machine will detect what passes. Every time a cell intermediates that decay to a ground state with the hits a laser, it will give a reading. They will now figure out emission of photons. how the scatter of the laser is influenced by the type of cell ○​ The emitted radiation is measured with a PM passing through. As each cell passes through the laser, (photomultiplier) tube, and the signal is related to light is impeded. analyte concentration. ​ This is different from fluorescence since no excitation ELECTROPHORESIS radiation is required and no monochromators are needed ​ Separation of molecules according to size and charge. because the chemiluminescence arises from one species. ​ Opposites attract ○​ Negatively charged ion → Positively charged ADVANTAGES electrode ​ Sub-picomolar detection limits ○​ Cations (+) move toward cathode (-) “Cat-Cat” ​ Speed (with flash-type reactions, light is only measured for ○​ Anions (-) move toward anode (+) “An-An” 10 seconds) ○​ Most of the time, we analyze anions ​ Ease of use (most assays are one-step procedures) ​ The migration of charged solutes or particles in an ​ Simple instrumentation electrical field. ○​ Iontophoresis refers to the migration of small ions DISADVANTAGE ○​ Zone electrophoresis is the migration of charged ​ Impurities can cause a background signal that degrades macromolecules in a porous support medium such as the sensitivity and specificity. paper, cellulose acetate, or agarose gel film. ​ Charged particles migrate toward the opposite charged TURBIDIMETRY AND NEPHELOMETRY electrode. TURBIDIMETRY ​ Smaller molecules migrate faster ​ Made with a spectrophotometer to determine the concentration of particulate matter in a sample. COMPONENTS ​ The amount of light blocked by a suspension of particles ​ Driving force (electrical power) depends on concentration and size. ​ Support medium ​ Measuring light scatter at an angle other than at 180° ​ Buffer minimizes error from colored solutions and increases ​ Sample sensitivity. ​ Detecting System ​ It is somehow the same as spectrophotometry in terms of its detection. ○​ Spectrophotometry: light absorbed PH 166 | Analytical Techniques and Instruments Part 2 3 of 6  ​ Separation is performed in narrow-bore fuse silica capillaries REVIEW QUESTIONS 1.​ Isoelectric focusing is the movement of buffer and solvent relative to their flexible support. It detects oligoclonal immunoglobulin bands in CSF, isoenzymes of creatine kinase, acid phosphatase and alkaline phosphatase. Figure 5. Electrophoresis apparatus—basic components. a.​ The first statement is correct while the second statement is incorrect. ELECTROPHORETOGRAM b.​ The second statement is correct while the second ​ It is the result of electrophoresis consisting of separated statement is incorrect. strands of a macromolecule. c.​ Both statements are correct. d.​ Both statements are incorrect POWER SUPPLY 2.​ Stray light in a spectrophotometer places limits on ​ Operating at either constant current or voltage are a.​ Upper range of linearity available commercially. b.​ Sensitivity BUFFERS c.​ Photometric accuracy below 0.1 absorbance units d.​ Ability to measure in the UV range ​ Two buffer properties that affect the charge of ampholytes e.​ Use of a grating monochromator are pH and ionic strength. ​ An ampholyte is a molecule, such as protein, whose net 3.​ Which of the following is/are instruments that measure charge can be either positive or negative. light energy? ​ If the buffer is more acidic than the isoelectric point (pI) a.​ Fluorometry of the ampholyte, it binds H+, becomes positively b.​ Chemiluminescence charged, and migrates toward the cathode. c.​ Turbidity and Nephelometry ​ If the buffer is more basic than the pI, the ampholyte d.​ Laser loses H+, becomes negatively charged, and migrates e.​ All of the above toward the anode. 4.​ Which of the following statement/s is/are TRUE ​ A particle without a net charge will not migrate, regarding the difference between chemiluminescence remaining at the point of application. and fluorescence? I.​ In fluorescence, there is no excitation SUPPORT MATERIALS radiation required and no monochromators ​ Cellulose acetate are needed because it arises from one ○​ Cellulose acetylated with acetic anhydride species. ​ Agarose gel II.​ In chemiluminescence, there are no ○​ Purified fraction of agar monochromators needed because it arises ​ Polyacrylamide gel from multiple species having light colors of ○​ Separates proteins with more fraction than cellulose similar wavelengths. acetate/agarose III.​ Chemiluminescence does not require ​ Starch and gel excitation radiation and monochromators because it arises from one species. ISOELECTRIC FOCUSING IV.​ Fluorescence requires excitation radiation ​ Used in isoenzyme analysis monochromators ​ Movement of buffer and solvent relative to their fixed a.​ I, II, III, and IV support. b.​ Two of the statements are correct ​ Isoelectric Point (pI) is the pH, at which a protein will have c.​ None of the choices is correct a net charge of zero. d.​ III only ​ Object with no charge in electrophoresis: It will stop 5.​ What determines the direction an ampholyte will moving migrate during electrophoresis? ​ Charged proteins migrate through a support medium that a.​ Size of ampholyte has a continuous pH gradient (layering). b.​ The relationship between the buffer's pH and the ​ Individual proteins move in the electric field until they reach ampholyte's isoelectric point (pI) a pH equal to their isoelectric point, at which point they c.​ The strength of the electrical current have no charge and cease to move. d.​ The type of support medium ​ Detects: 6.​ What is the SI unit for measuring concentration in a ○​ oligoclonal immunoglobulin bands in CSF solution? ○​ isoenzymes of creatine kinase a.​ Molarity (M) ○​ acid phosphatase and alkaline phosphatase b.​ Milligrams per deciliter (mg/dL) c.​ Microgram per milliliter (µg/mL) ELECTROENDOSMOSIS d.​ Grams per liter (g/L) ​ Movement of buffer and solvent relative to their fixed 7.​ What type of pipette would be used to deliver small support. volumes accurately typically in a microliter range? a.​ Graduated pipette CAPILLARY ELECTROPHORESIS b.​ Volumetric pipette c.​ Micropipette PH 166 | Analytical Techniques and Instruments Part 2 4 of 6  d.​ Serological pipette (Difficult to synthesize or purify) 14.​ C 8.​ Which clinical laboratory supply is essential for 15.​ B accurately measuring and dispensing liquid in a reproducible manner? a.​ Burette b.​ Beaker c.​ Petri dish REFERENCES Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2017). Clinical chemistry : d.​ Watch glass techniques, principles, correlations (8th ed.). Wolters Kluwer 9.​ What is the primary purpose of a reagent blank Health/Lippincott Williams & Wilkins. ‌ (meaning: it is just a reagent; it does not contain anything; you use it to set the zero value in your Chua, J. (2025). Analytical Techniques and Instrumentation [Slides]. spectrophotometer) in clinical chemistry? https://drive.google.com/file/d/1lqocMN6ps2ZPbVkmMTKGkg7fUra7rX hB/view. a.​ To calibrate the analytical instrument b.​ To adjust for interference of the sample matrix c.​ To clean the laboratory equipment d.​ To measure the concentration of the sample 10.​ Which of the following techniques measure light energy to determine the concentrations of atoms or molecules in the sample? a.​ Electrophoresis b.​ Spectrophotometry c.​ Potentiometry d.​ Amperometry 11.​ Which of the following is a characteristic feature of a standard reference material, whether primary or secondary? a.​ It has an uncertain concentration b.​ It is used to calibrate instruments c.​ It is produced by non-official sources d.​ It does not require maintenance or evaluation 12.​ What type of glassware is most suitable for heating liquids in a laboratory? a.​ Beaker b.​ Test tube c.​ Erlenmeyer flask d.​ Volumetric flask 13.​ Which of the following is not a common characteristic of high quality reagent with chemicals? a.​ High purity b.​ Minimal impurities c.​ Available in large quantities d.​ Consistent between batches 14.​ Which type of glassware would be best for preparing a solution of known concentration by diluting a more concentrated stock solution? a.​ Burette b.​ Beaker c.​ Volumetric flask d.​ Pipette 15.​ Which of the following is considered a high purity grade that is suitable for preparation of standard solutions in clinical chemistry? a.​ Technical b.​ Analytical Reagent c.​ Pharmaceutical d.​ Industrial ANSWER KEY 1.​ B 6.​ A 2.​ A 7.​ C 3.​ E 8.​ A 4.​ B 9.​ B 5.​ B (Analytical instrument: will use standard) 10.​ B 11.​ B 12.​ B 13.​ C PH 166 | Analytical Techniques and Instruments Part 2 5 of 6 PH 166 | Analytical Techniques and Instruments Part 2 6 of 6