Power point presentation on Osmometry and Nephelometry.pptx

This power point presentation is prepared by scientist B. Hassan of the department of chemical pathology, school of medical laboratory science, Usmanu d
Danfodiyo University Sokoto

1. OSMOMETRY AND NEPHELOMETRY PRINCIPLE AND ITS BIOCHEMICAL APPLICATIONS (MLC 902: DIAGNOSTIC CLINICAL CHEMISTRY) PRESENTATION BY HASSAN, Abubakar Bello (Adm NO.: 24311226001) SUBMITTED TO Dr. JELANI ISMAILA DEPARTMENT OF CHEMICAL PATHOLOGY SCHOOL OF MEDICAL LABORATORY SCIENCE USMANU DANFODIYO UNIVERSITY, SOKOTO August, 2025 8/19/25 1

2. OUTLINE Introduction Osmometry Principle of osmometry Components of osmometer Types of osm ometer Biochemical applications of osmo metry Limitation of osmometry Conclusion References 8/19/25 2

3. INTRODUCTION Osmometry is the measurement of the osmotic strength of a solution (osmolality), colloid, or compound, particularly biological fluids such as plasma, serum, urine and cerebrospinal fluid (CSF) Osmometry is a technique used to measure the concentration of solute particles in a solution, typically expressed as osmolality or osmolarity. It's a valuable tool in various fields, including clinical diagnostics, research, and quality control, particularly in assessing fluid balance and kidney functions Osmolality: is t he concentration of solute particles in a solution expressed as millimoles of solute per kilogram of solvent (mmol/kg). It's a more precise measure than osmolarity because it's temperature-independent while Osmolarity: is t he concentration of solute particles in a solution expressed as millimoles of solute per liter of solution (mmol/L) ( D’Orazio , 2006 ) 8/19/25 3

4. OSMOMETRY Osmometry is the measurement of the osmolality of solutions (osmoles of solute per kilogram of solvent). It is widely used in clinical chemistry, physiology, pharmaceutical sciences, and research The technique relies on the colligative properties of solutions which depend only on the number of solute particles, not their type Osmometer is a device used to measure osmotic pressure, which is a colligative property of solutions, these instrument is essential in various fields including clinical laboratories, research, and quality control for determining the concentration of dissolved substances in solutions Figure 1. Osmometry ( Burtis et al ., 2012 ) 8/19/25 4

5. PRINCIPLE OF OSMOMETRY The working principle of o smometry is based on the colligative properties of solutions which depend on the number of solute particles not their chemical nature. These properties include: Freezing Point Depression: Solutions freeze at a lower temperature than pure solvents. The decrease in freezing point is directly proportional to the number of solute particles Vapor Pressure Depression: The presence of solute particles lowers the vapor pressure of a solution compared to the pure solvent Boiling Point Elevation: Solutes elevate the boiling point of a solution compared to pure solvent Osmotic Pressure Measurement: Directly measuring the pressure needed to prevent solvent movement across a semipermeable membrane The general formula of Osmolality = Number of osmoles of solute​/ Kilogram of solvent ( D’Orazio , 2006 ) 8/19/25 5

6. COMPONENTS OF OSMOMETER Sample Chamber/Cell: Holds sample (20–250 µL) Cooling/Heating System: Controls temperature for freezing/vapor/boiling point measurements Nucleation Mechanism: Stir rod/vibration to initiate freezing (in freezing point osmometer) Temperature Sensor : T hermistor/thermocouple for precise measurement Semipermeable Membrane (in membrane osmometers) : for osmotic pressure measurements Microprocessor and Electronics: C alculates osmolality Calibration System: Standard solutions (100, 290, 1000 mOsm/kg) Display/Interface: Digital output, sometimes linked to LIS (Simons, 2010) 8/19/25 6

7. TYPES OF OSMOMETER Freezing Point Depression Osmometer: A sample is supercooled and then crystallization is induced, the freezing point is determined and osmolality is calculated from the depression, its accurate, reliable and commonly used Vapor Pressure Osmometer: The osmometer compares the dew point (temperature at which vapor condenses) of the sample with a reference one and it is useful in research and industrial laboratories Membrane Osmometer (Colloid Osmometer): The osmotic pressure is determined as the solvent moves across the membrane until equilibrium is reached and it is used mainly for macromolecules like proteins, polymers and colloids Isopiestic (Equilibrium) Osmometer: Here both solutions are placed in a closed chamber; water vapor transfer occurs until equilibrium is reached, and osmolality is deduced Cryoscopic Osmometer (Specialized Freezing Point): A specialized form of freezing point osmometer designed for high precision, often used in biochemistry and pharmaceutical industries for small molecules ( Rajendran and Sundaram, 2018 ) 8/19/25 7

8. BIOCHEMICAL APPLICATIONS OF OSMOMETRY Evaluation of Electrolyte and body fluids : Measurement of serum and urine osmolality aids in diagnosis of disorders such as dehydration, overhydration, hyponatremia and hypernatremia Renal Function Assessment : Urine osmolality testing helps evaluate the kidney’s concentrating and diluting capacity in conditions like diabetes insipidus and syndrome of inappropriate antidiuretic hormone secretion (SIADH) Detection of Osmolal Gap : The difference between measured and calculated osmolality helps detect unmeasured solutes in blood such as ethanol, methanol, ethylene glycol, isopropanol, and mannitol and it is u seful in toxicology screens and poisoning cases Monitoring Therapy : Used to monitor mannitol therapy in patients with cerebral edema and to assesses hyperosmolar therapy in head injury management Cerebrospinal Fluid (CSF) Studies : Helps differentiate between transudates and exudates as well as studying fluid shifts in neurological conditions   ( D’Orazio , 2006 ) 8/19/25 8

9. BIOCHEMICAL APPLICATIONS OF OSMOMETRY CONT’D Drug Formulation and Development : Osmolality determination ensures parenteral solutions (e.g., IV fluids, injections) are iso-osmotic with blood to avoid hemolysis or vascular irritation Cell Culture and Cryopreservation : Osmolality monitoring is essential for maintaining the viability and stability of cell cultures and tissues Protein and Macromolecule Studies : Osmometry helps determine the molecular weight of proteins, polymers and polysaccharides based on colligative properties Food and Beverage Industry : Used in quality control to measure osmolality of soft drinks, milk and nutritional solutions Pharmaceutical Quality Assurance : Verifies osmolality of oral rehydration salts (ORS), intravenous fluids and dialysis solutions ( D’Orazio , 2006 ) 8/19/25 9

10. LIMITATIONS OF OSMOMETRY Volatile solutes interfere with vapor pressure osmometers Lipemic or viscous samples may affect readings with temperature detection in freezing point osmometers Requires regular calibration and skilled operation Small Volume Sensitivity: Although modern osmometers require small sample sizes (20–250 µL), errors in pipetting or incomplete mixing can significantly distort results Calibration Requirements: Ongoing calibration with standard solutions is essential; drift or poor maintenance leads to systematic errors Temperature Sensitivity: Since osmometry depends on colligative properties (especially temperature-related ones like freezing point and vapor pressure), external temperature fluctuations may affect accuracy if the system is not well-insulated Time Consumption: While modern instruments are faster, some methods (e.g., membrane osmometry) remain time-consuming compared to automated chemistry analyzers ( D’Orazio , 2006 ) 8/19/25 10

11. CONCLUSION Osmometry is a vital analytical technique in clinical biochemistry, providing insights on critical diagnostic information on fluid and solute concentration, electrolyte balance, renal function and toxicology. Its principle relies on the measurement of colligative properties, with freezing point remains a cornerstone technique in biochemical and clinical laboratories , depression osmometry being the most widely applied method. Beyond clinical diagnostics, osmometry plays an essential role in pharmaceutical research, food science and molecular studies. 8/19/25 11

12. SOME CITED REFERENCES D’Orazio, P. (2006). "Osmometry: principles and clinical applications." Clinical Chemistry , 52(12): 2253–2262. Burtis, C. A., Ashwood, E. R., Bruns, D. E. (2012). Tietz Textbook of Clinical Chemistry and Molecular Diagnostics . 5th Edition. Elsevier Saunders. Simons, T. J. B. (2010). "Clinical osmometry." Journal of Clinical Pathology , 63(5): 397–402. Rajendran, R., and Sundaram, V. (2018). Textbook of Medical Biochemistry . Jaypee Brothers Medical Publishers, Pp 236 8/19/25 12

13. THANK YOU FOR LISTENING 8/19/25 13

14. OUTLINE Introduction Nephelometry Principle of Nephelometry Components of Nephelometer Types of Nephelometry Biochemical applications of Nephel o metry Limitation of Nephelometry Conclusion References 8/19/25 14

15. INTRODUCTION Nephelometry is an analytical technique used to determine the concentration of particles in solution by measuring light scattering. It is highly sensitive and widely applied in clinical chemistry, immunology and biochemical research particularly for proteins quantifications, immunoglobulins, complement components and immune complexes Unlike turbidimetry (which measures reduction in transmitted light), nephelometry focuses on the light scattered by suspended particles at an angle (commonly 90°) making it more sensitive for low-concentration analytes (Dati et al ., 2001) 8/19/25 15

16. NEPHELOMETRY Nephelometry is an analytical photometric technique based on the measurement of light scattered by particles in suspension. The amount of scattered light is proportional to the size, number and the concentration of the particles in the medium In clinical chemistry and immunology, nephelometry refers to a quantitative immunoassay method that measures the intensity of scattered light produced when antigen-antibody complexes form in solution The degree of light scattering corresponds to the concentration of specific proteins or analytes, making nephelometry a sensitive and rapid tool for measuring serum proteins, immunoglobulins, complement components, C-reactive protein (CRP) and other biomarkers of disease N ephelometer is an instrument used to measure the concentration of suspended particles in a fluid (liquid or gas) by detecting the scattering of light. It essentially measures how cloudy or turbid a sample is by passing a light beam through it and measuring the amount of light scattered at a particular angle ( Burtis et al ., 2012 ) Figure 2. Nephelometer 8/19/25 16

17. PRINCIPLE OF NEPHELOMETRY A beam of light is passed through the sample or a solution containing suspended particles, and the amount of light scattered by the suspended particles is measured by a detector The intensity of the scattered light is directly proportional to the concentration of particles in the solution, size and refractive index of the particles and the wavelength of the incident light Nephelometers typically measure scattered light at an angle (commonly 90°) relative to the incident beam, By comparing the measured light intensity against standard calibrators of known concentration, the concentration of the analyte in the test sample can be determined ( Skooge et al ., 1998 ) 8/19/25 17

18. COMPONENTS OF NEPHELOMETER Light Source : Provides a stable, intense, monochromatic beam, common sources: tungsten-halogen lamps, LEDs, or laser light is preferred in modern nephelometers due to its high intensity and coherence, improving sensitivity Collimating System (Lenses and Filters): Directs and focuses the light into a narrow beam before it passes through the sample, optical filters or monochromators may be used to select a specific wavelength Sample Holder/Cuvette : Transparent container that holds the test solution, designed to minimize reflection or absorption and to allow optimal scattering detection Detector(s) : Positioned at a fixed angle (commonly 90°) to the incident light to capture scattered light, may use photomultiplier tubes (PMTs) or photodiodes for high sensitivity and some instruments have multiple detectors at different angles for advanced measurements 8/19/25 18

19. COMPONENTS OF NEPHELOMETER CONT’D Reference Detector (optional) : Measures the intensity of incident (unscattered) light to normalize results and correct for fluctuations in the light source Signal Processing Unit / Electronics : Converts light intensity into an electrical signal, amplifies, digitizes, and processes the signal to calculate particle concentration Microprocessor and Software : Controls instrument functions, performs calculations, and applies calibration curves, provides readout of analyte concentration. Calibration System : Uses standard solutions of known protein or particle concentration., essential for accuracy and reproducibility. Display/Output Interface : Digital screen for displaying results, many modern nephelometers interface with Laboratory Information Systems (LIS) for automatic data transfer. 8/19/25 19

20. TYPES OF NEPHELOMETRY End-point Nephelometry : Antigen and antibody are allowed to react until they reach equilibrium and the scattered light intensity is measured at that end-point and it is suitable for stable immune complexes Kinetic Nephelometry : Measures the rate of formation of antigen-antibody complexes by monitoring scattered light over time, it also provides faster results and is less affected by antigen excess Rate Nephelometry (Turbidimetric Inhibition Immunoassay - TINIA) : Used when small analytes cannot form large lattices, i nstead, competition occurs between sample antigen and reagent antigen and it is c ommonly use in drug monitoring and hormone assays High-sensitivity Nephelometry : Employs enhanced optics (e.g., laser nephelometry) to detect very low concentrations of proteins such as C-reactive protein (CRP) at high sensitivity Laser Nephelometry : Uses a laser as a light source, which provides high-intensity, monochromatic and coherent light improving accuracy and reproducibility 8/19/25 20

21. BIOCHEMICAL APPLICATIONS OF NEPHELOMETRY Quantification of Plasma Proteins : Immunoglobulins (IgG, IgA, IgM, IgE, IgD), complement components (C3, C4), acute-phase proteins (CRP, haptoglobin, transferrin and α1-antitrypsin) Detection of Immune Complexes : Used in autoimmune diseases, chronic infections and inflammatory disorders Diagnosis and Monitoring of Diseases : Hypogammaglobulinemia, multiple myeloma, immunodeficiencies and monitoring of inflammatory status via CRP levels Drug Monitoring (TINIA method) : Measurement of drugs and hormones that are too small to form large antigen-antibody complexes Protein Purity and Concentration Determination in biochemical research Particle Size Studies in colloid and polymer research Environmental Science: water quality testing (detection of turbidity and suspended solids) Pharmaceutical Industry : characterization of drug formulations, especially those containing proteins or nanoparticles ( Wild, 2013 ) 8/19/25 21

22. LIMITATIONS OF NEPHELOMETRY Interference from Sample Turbidity : Hemolysis, lipemia, or particulate matter can cause non-specific light scattering, leading to false readings. Prozone (Antigen Excess) Effect : In antigen-antibody reactions, very high antigen concentrations may prevent lattice formation, giving falsely low results. Sensitivity to Particle Size : Very small molecules or particles that do not scatter light efficiently cannot be measured directly. Light Source and Angle Dependency : Measurements can vary depending on detection angle and wavelength, requiring careful calibration. Instrument Cost and Maintenance : Nephelometers are more expensive than simple spectrophotometers and require regular calibration. Limited Dynamic Range : Accurate only within certain concentration ranges; dilution may be necessary for very high concentrations. Standardization Issues : Variability between instruments and methods can complicate inter-laboratory comparisons. Operator Expertise : Requires skilled personnel to avoid errors in sample preparation and interpretation. 8/19/25 22

23. CONCLUSION Nephelometry is a sensitive and reliable technique for protein quantification and immunoassays in clinical and research settings. The performance of a nephelometer depends heavily on its components: a stable light source, precise optics, sensitive detectors,and robust calibration. While limitations such as antigen excess and sample interference exist, modern laser-based and high-sensitivity nephelometers have improved accuracy and extended applications in both diagnostics and biotechnology. 8/19/25 23

24. SOME CITED REFERENCES Dati, F., Schumann, G., Thomas, L. (2001). "Nephelometric and turbidimetric methods for protein analysis." Journal of Clinical Chemistry and Clinical Biochemistry , 39(6): 549–555. Skoog, D. A., Holler, F. J., Nieman, T. A. (1998). Principles of Instrumental Analysis; Harcourt Brace & Co, Pp 378 Burtis, C. A., Ashwood, E. R., Bruns, D. E. (2012). Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 5th Edition. Elsevier , Pp 157. Wild, D. (2013). The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA, and Related Techniques. 4th Edition. Elsevier, Pp 206 8/19/25 24

25. THANK YOU FOR LISTENING 8/19/25 25