PH 166 Analytical Techniques and Instruments PDF

PH 166: CLINICAL CHEMISTRY 1 ANALYTICAL TECHNIQUES AND INSTRUMENTS PART 1 Mr. James C. Chua | February 6, 2025 ​ Exemption: United States Pharmacopeia (USP) and OUTLINE...

PH 166: CLINICAL CHEMISTRY 1 ANALYTICAL TECHNIQUES AND INSTRUMENTS PART 1 Mr. James C. Chua | February 6, 2025 ​ Exemption: United States Pharmacopeia (USP) and OUTLINE National Formulary grade (NF) classify chemicals as safe A.​ Clinical Chemistry Laboratory for medical use and consumption B.​ Basic Principles and Practices a.​ Units of Measure Classification of Chemicals b.​ Reagents ​ Ultra-pure Chemicals i.​ Chemicals ii.​ Reference Materials ○​ Highest purity iii.​ Water Specifications ○​ Suitable for tests that are incredibly sensitive, thus c.​ Clinical Laboratory Supplies requiring extremely pure chemicals d.​ Basic Separation Techniques ○​ Suitable for techniques that require extremely pure C.​ References chemicals (e.g. AAS, EIA, MDx) ○​ Carry designations of HPLC or chromatographic CLINICAL CHEMISTRY LABORATORY ​ Analytical Grade ○​ Suitable for most analytical procedures and routine ​ Concerned with the analysis of biochemical byproducts in tests biological fluids ○​ Not the highest purity ○​ Pure Blood Chemistry ○​ Can be used as standard grade reference materials or ​ Lipids, carbohydrates, and protein lab use ○​ Enzymology ○​ Carry designations as AR or ACS and For Laboratory ​ Enzymes Use or ACS standard-Grade Reference materials ○​ Endocrinology ​ Chemically-pure ​ Hormones ○​ Impurity limitations are not stated ○​ Toxicology ○​ Not for analytical procedures and laboratories ​ Drugs of abuse, heavy metals, poison, etc. ○​ Not recommended for clinical laboratories BASIC PRINCIPLES AND PRACTICES ○​ Not uniform in production ○​ Not to be mistaken as Ultra-pure chemicals UNITS OF MEASUREMENT ​ United States Pharmacopeia (USP) and National Quantitative Laboratory Results Formulary grade (NF) ​ Components of quantitative laboratory results ○​ Usually for making drugs ○​ Number ○​ Safe for human consumption ○​ Unit (SI) ○​ Exemption to the classifications according to purity ​ e.g. 15 mL ○​ Purity standards are based on the criterion of not ​ Universal way of reporting measurements to ensure being injurious to man reproducibility/replicability ​ Technical or Commercial Grade ​ Most results are masses of substances ○​ Used for manufacturing ○​ E.g. mg/dL, g/dL, meq/L, and IU ○​ Similar to chemically-pure but of lower grade ​ Substance concentration ○​ Considers the atomic masses of substances REFERENCE MATERIALS ○​ E.g. moles ​ Primary standard ​ Mass of substance ○​ Substance of exact known concentration and purity ○​ e.g. mg/dL, g/dL, g/L, mEq/L, and IU ○​ Produced by a highly purified chemical that can be measured directly REAGENTS ​ Secondary standard ​ Includes chemicals, reference materials and water ○​ Substance of lower purity with concentration specifications determined by comparison with a primary standard ​ Commercially prepared ​ Can be chemical reference materials WATER SPECIFICATIONS ​ Must have the following labels: ​ Tap water is unsuitable for laboratory applications ○​ Required storage temperatures ​ Distilled ○​ Volume ○​ Purified by distillation to remove almost all organic ○​ Expiration date materials. ○​ Lot. no. ​ Deionized ​ In clinical chemistry, reagents must be quantified ○​ Water purified by ion exchange (anion or cation ○​ ↑ Intensity of color = ↑ concentration of analyte exchange resin) with some/all ions removed ​ Spectrophotometer ○​ Removes dissolved solids and dissolved gases ○​ Measures intensity of color ​ Reverse osmosis (RO) water ○​ Blank is used to correct for the original color of analyte ○​ Uses pressure to force water through a semi ○​ Concentration of analyte is calculated based on the permeable membrane readings of both the blank and the unknown solution ○​ Does not remove gases of analyte ​ Ultrafiltration and nanofiltered water, UV oxidation, sterilization or ozone treatment CHEMICALS ○​ Targets microorganisms ​ Has different classifications of purity or process of ​ Reagent grade water purification #MagkabigkisBenteSais Group 2 | 1 of 4  ○​ Usually the requirement in laboratories ○​ Kimax® / Pyrex® (borosilicate) ○​ Obtained by initial filtration, followed by RO, ​ Has high heat resistance deionization and a 0.2 mm filter ○​ Corex® (aluminosilicate) ​ Has high heat resistance​ Grades of Water ○​ High silica ​ Categorization of water purity ○​ Vycor® (acid or alkali resistant) ​ Type I ○​ Low actinic (amber-colored) ○​ Has the most stringent requirements and generally ○​ Flint glass (lime soda) suitable for routine laboratory use ​ Plasticware - usually disposable laboratory supplies ○​ For test methods requiring minimum interference ​ Examples of commonly used resins: ​ Trace metal analysis by FES and AAS ○​ Polystyrene ○​ Gas, pH, enzyme, and electrolyte analysis ○​ Polyethylene ​ Type II ○​ Polypropylene ○​ For analytical preparations ○​ Tygon® ○​ Reagent, QC and standard preparation ○​ Telon® ​ Type III/autoclave wash water ○​ Glassware washing Laboratory vessels ○​ Not acceptable for analysis and reagent preparation ​ Class A volumetric flask ○​ Calibrated to hold one exact amount of liquid (TC) CLINICAL LABORATORY SUPPLIES ○​ Ideal for diluting ○​ Allows for mixing THERMOMETERS ○​ Add water first before acid ​ Warming components is necessary for analytical ○​ For clear liquid, use the lower meniscus procedures and is accomplished by circulating water/ice ○​ For colored liquid, use the higher meniscus baths or heating/cooling metal blocks ​ Either part of instrument of placed in device for temperature maintenance ​ All must be calibrated to ascertain accuracy Classification of Thermometers ​ Liquid-in-glass ○​ Use of a colored liquid or mercury encased in plastic/glass material with a bulb at one end a Figure 2. Class A volumetric flask (DDBioLab, N.D.) graduated stem ○​ Measure between 20˚C and 400˚C ​ Erlenmeyer flasks and Griffin beaker ○​ 3 types: ○​ Hold different volumes ​ Total Immersion ○​ Used in reagent preparation ​ Used for refrigeration applications ​ Partial Immersion ​ Used for measuring temperatures in units such as heating blocks and water baths and should be immersed to the proper height as indicated by the continuous line etched on the thermometer stem ​ Surface thermometer ​ Check temperatures on flat surfaces (refrigerators, incubators, or heating oven) Figure 3. Erlenmeyer flask (Stellar Scientific, N.D.) ​ Electronic thermometer or thermistor probe ○​ Fast reading with millisecond response time ○​ Probe converts heat into electricity ​ Digital thermometer ○​ Converts heat to electrical energy ○​ Increase in heat → increase in electricity converted → the higher the reading Figure 4. Griffin beaker (Sigma Aldrich, N.D.) ​ Graduated cylinder ○​ Used to measure volumes of liquid ○​ Accurate (the smaller the measurements, the more accurate) Figure 1. Liquid-in-glass thermometers GLASSWARES and PLASTICWARE ​ Glassware - laboratory supplies consisted of some type of glass PH 166 | Analytical Techniques and Instruments Part 1 2 of 4  Figure 7. Ostwald-Folin pipette (Laboratory Info, N.D.) Figure 5. Graduated cylinder (Eisco Industrial, N.D.) ○​ Mechanical or automatic pipette ​ Micropipette - delivers amount 1mL ​ Glass or plastic utensils used to transfer liquids ​ Air displacement - the piston does not come in ​ Always vertical contact with the liquid ​ Classification of Pipettes according to design or ​ Positive displacement - the piston moves in the calibration marks tip and comes in contact with the liquid ○​ To contain (TC) ​ Used for viscous samples ​ Uses mercury as calibrating medium ​ Proper use requires rinsing of the pipette with the final solution after contents are delivered into the diluent (rinsing technique) Figure 8. Macropipette (Auxilab, N.D.) ○​ To deliver (TD) ​ Used for non-viscous samples ○​ Micropipette ​ Uses distilled water as calibrating medium ​ Blowout pipette - with etched ring or two small, close continuous rings; the last drop of fluid needs to be blown ​ Self-draining pipette - without marking; drains completely ​ Classification of pipette according to use Figure 9. Micropipette (Teclab, N.D.) ○​ Measuring or graduated pipette Burets ​ Graduated uniformly along its length ​ Used in titration ​ Designed to deliver any amount within its ​ For delivering exact volumes of liquid capacity ​ Used if precision is not essential ​ Serological pipette - has graduation marks to the tip (generally a blowout pipette) ​ Mohr pipette - no graduation marks to the tip (self-draining) ○​ Volumetric or Transfer pipette ​ Designed to transfer one volume ​ Ostwald-Folin pipette - for viscous fluids Figure 10. Burette (Brittanica, N.D.) (blowout pipette): smaller in size ​ Volumetric pipette - for aqueous solutions (self-draining); larger in size Syringes ​ Pasteur pipette - no calibration marks; for ​ Used for very small amounts of liquid transferring fluids without consideration of volume Figure 11. Syringes (Endure Medical, N.D.) Figure 6. Mohr, Serological, and Volumetric pipettes (Chemistry Stock Exchange, N.D.) DESICCATORS AND DESICCANT ​ Use hygroscopic substances that take up water/moisture on exposure to air ​ Desiccators - used for drying PH 166 | Analytical Techniques and Instruments Part 1 3 of 4  7.​ Glass or plastic utensils used to transfer liquids are called _____. a.​ Burets b.​ Pipettes c.​ Erlenmeyer flasks d.​ Thermometers 8.​ It is referred to as the process in which a centrifugal force is used to separate solid matter from a liquid suspension. Figure 12. Desiccator (MRC Lab, N.D.) a.​ Filtration b.​ Titration BALANCES c.​ Centrifugation ​ For weighing d.​ Weighing ​ Depends on sensitivity 9.​ Which of the following use/s hygroscopic substances ​ According to operating ranges: that take up water/moisture on exposure to air? ○​ Precision balance (2 µg) a.​ Desiccators and desiccant ○​ Analytic balance (0.001 g) b.​ Pipettes ​ Commonly used c.​ Balances ○​ Microbalances (0.1 µg) d.​ Thermometers ​ Used in clean rooms 10.​ TRUE OR FALSE: Technical or commercial grade is similar to chemically-pure but of higher grade. ANSWER KEY 1.​ A 6.​ FALSE 2.​ FALSE 7.​ B 3.​ B 8.​ C 4.​ D 9.​ A 5.​ TRUE 10.​ FALSE REFERENCES Figure 13. Analytical balance (Infitek, N.D.) ​ Chua, J. (2025). Analytical Techniques and Instruments Part 1. BASIC SEPARATION TECHNIQUES ​ Centrifugation ○​ Must be balanced (right across each other) ○​ Process in which a centrifugal force is used to separate solid matter from a liquid suspension (solid matter settles below) ​ Filtration REVIEW QUESTIONS 1.​ Which classification of chemicals is suitable for tests that are incredibly sensitive? a.​ Ultra-pure chemicals b.​ Analytical grade c.​ Chemically pure d.​ Technical or commercial grade 2.​ TRUE OR FALSE: Tap water is suitable for laboratory applications. 3.​ Which pipette classification uses mercury as calibrating medium and is used for viscous samples? a.​ To deliver (TD) b.​ To contain (TC) c.​ A and B d.​ None of the above 4.​ Which volumetric or transfer pipette has no calibration marks? a.​ Serological pipette b.​ Ostwald-Folin pipette c.​ Volumetric pipette d.​ Pasteur pipette 5.​ TRUE OR FALSE: In measuring clear liquids, the lower meniscus is used. 6.​ TRUE OR FALSE: When using a blowout pipette, the last drop of fluid does not need to be blown as it drains completely. PH 166 | Analytical Techniques and Instruments Part 1 4 of 4