1.4 Reagent Preparation, Laboratory Water, Glassware, Pipettes, and Centrifuges

Reagent Preparation, Laboratory Water, Glassware, Pipettes, and Centrifuges

The Clinical Chemistry blueprint assigns 5% to reagent preparation and laboratory mathematics. Reagent quality sets the ceiling on analytical quality: an assay run with the wrong chemical grade, contaminated water, or a misread pipette will fail quality control no matter how good the analyzer is. This section covers chemical grades and standards, laboratory water, solution preparation with hydrates, glassware and pipettes, balances and desiccants, and centrifuge calculations. Dilution, molarity, and normality calculations are worked in the quality control and laboratory mathematics section.

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1. Chemical Grades and Reference Materials

• A primary standard is a highly purified chemical that can be weighed directly to make a solution of exactly known concentration. It must be stable, non-hygroscopic, and of known composition.
• A secondary standard has its concentration determined by comparison with a primary standard.
• Standard reference materials (SRMs) from the US National Institute of Standards and Technology (NIST) and other certified reference materials give calibration traceability (see the calibration hierarchy in the quality control section).
• Every reagent container is labeled with its name, concentration, date received, date opened or prepared, expiration date, storage conditions, hazard warnings, and the preparer's initials.

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2. Laboratory Water

Water Quality Categories

The Clinical and Laboratory Standards Institute (CLSI) replaced the older Type I, II, and III classification with categories based on intended use:

• Clinical laboratory reagent water (CLRW): The default water for reagent preparation and most testing. Typical specifications are resistivity of at least 10 MΩ·cm at 25 °C, total organic carbon below 500 ppb, microbial content below 10 CFU/mL, and filtration through a 0.22 µm filter.
• Special reagent water (SRW): Water with extra specifications for a particular method, such as low organics for HPLC or low trace metals for metal analysis.
• Instrument feed water: Water meeting an analyzer manufacturer's specifications.
• Water for autoclaves and washers, and commercially packaged purified water, have their own specifications.

Older texts still cited by reviewers use Type I water (highest purity, at least 10 MΩ·cm; for trace metals, enzymes, electrolytes, and buffers), Type II (at least 1 MΩ·cm; general chemistry, hematology, and microbiology media), and Type III (at least 0.1 MΩ·cm; glassware washing).

Purification Methods

Resistivity is monitored continuously or daily, and bacterial counts are checked on a schedule. Falling resistivity means the deionizer cartridges are exhausted.

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3. Preparing Solutions and Hydrates

• Percent solutions: w/v (grams per 100 mL), v/v (mL per 100 mL), and w/w (grams per 100 g). Physiologic saline is 0.85% to 0.9% NaCl (w/v), or 8.5 to 9 g/L.
• Molarity is moles of solute per liter of solution. Normality is equivalents per liter. Molality is moles per kilogram of solvent, which does not change with temperature.
• Hydrates: When the available chemical is a hydrate, use the hydrate's molecular weight, or scale an anhydrous mass by the ratio of molecular weights:

$\text{g of hydrate} = \text{g of anhydrous compound} \times \frac{\text{MW of hydrate}}{\text{MW of anhydrous compound}}$

• Worked example: Prepare 500 mL of 0.1 M copper sulfate from CuSO4·5H2O (MW 249.7). Grams = 0.1 mol/L × 0.5 L × 249.7 g/mol = 12.48 g. If you used the anhydrous MW (159.6) by mistake, you would weigh only 7.98 g and the solution would be about 36% too weak.
• Terms: Hygroscopic substances absorb moisture from the air; deliquescent substances absorb so much that they dissolve (for example, sodium hydroxide pellets); efflorescent substances lose their water of hydration to the air. Store hygroscopic chemicals and primary standards in a desiccator with a drying agent such as silica gel or anhydrous calcium chloride.

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4. Glassware, Plasticware, and Pipettes

Glass and Plastic Types

• Borosilicate glass (such as Pyrex and Kimax) resists heat and most chemicals and is the standard for laboratory glassware.
• Low-actinic (amber or red) glassware protects light-sensitive analytes such as bilirubin and vitamin A.
• Soda-lime (flint) glass is inexpensive but less resistant to heat and chemicals. Boron-free alkali-resistant glass is used for strong alkaline solutions.
• Plastics: Polypropylene and polyethylene resist most chemicals; polystyrene is clear but cracks with many solvents; polycarbonate is strong; fluorocarbon (Teflon) is nearly inert.
• Class A volumetric glassware meets the strictest tolerance standards (traceable to NIST) and is used for preparing standards. Graduated cylinders are much less accurate than volumetric flasks.

Pipette Terminology

• TD (to deliver): Calibrated to deliver the stated volume when drained.
• TC (to contain): Holds the stated volume; the contents must be rinsed out completely with diluent. The Sahli hemoglobin pipette is a TC pipette.
• Blow-out: Marked by an etched or frosted band (or double ring) near the top; the last drop is expelled.
• Self-draining: The last drop is allowed to remain in the tip.

Pipette Types

• Read the bottom of the meniscus at eye level for clear liquids. Hold pipettes vertically while drawing and delivering.
• Reverse pipetting (aspirate past the first stop, dispense to the first stop) improves accuracy with viscous or foaming liquids.
• Pipettes are calibrated gravimetrically by weighing delivered distilled water and correcting for its density at the measured temperature.
• Glassware is cleaned with laboratory detergent, rinsed thoroughly with tap water, then rinsed with reagent-grade water. Glassware for trace-metal work is soaked in dilute nitric acid. Chromic acid (dichromate) cleaning solution is now avoided because hexavalent chromium is toxic and carcinogenic.

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5. Balances and Centrifuges

• Analytical balances weigh to 0.0001 g (0.1 mg) inside a draft shield. They are leveled, kept away from vibration and air currents, and checked with certified weights. Top-loading balances are used for less exacting work.
• Relative centrifugal force (RCF) depends on both speed and rotor radius:

$\text{RCF } (\times g) = 1.118 \times 10^{-5} \times r \text{ (cm)} \times (\text{rpm})^2$

• Worked example: A rotor radius of 10 cm at 3,000 rpm gives RCF = 1.118 × 10^-5 × 10 × 9,000,000 ≈ 1,006 × g. Procedures should state RCF, not rpm, because the same rpm produces different forces in rotors of different radii.
• Centrifuges are balanced with tubes of equal mass opposite each other, the lid is kept closed until the rotor stops, and speed is verified with a tachometer as part of maintenance.