Pipetting in analytical chemistry laboratories presents fundamentally different challenges than in biological or clinical settings. While a biology lab largely manipulates aqueous buffers, proteins, and DNA at neutral pH, a chemistry lab routinely handles volatile organic solvents, corrosive acids, viscous oils, and surfactants.
These fluid properties drastically affect accuracy. A micropipette that is perfectly accurate for water may undershoot by 5% with methanol due to evaporation and low surface tension. Furthermore, chemical compatibility is a major concern; the wrong plastic tip can leach plasticizers into an HPLC sample, creating “ghost peaks” that ruin trace analysis.
This guide helps laboratory managers and analysts choose the correct liquid handling tool based on application, fluid physics, and chemical resistance, rather than relying on a generic product catalog.
Quick Selector for Chemistry & Analytical Labs
Application Table
Select your tool based on the fluid type and the downstream analytical method.
Decision Logic
- If highest accuracy is required at a fixed volume (e.g., 10.00 mL for a stock solution) → Volumetric Glass Pipette (Class A).
- If handling variable volumes of aqueous solutions → Air Displacement Micropipette.
- If handling volatile (high vapor pressure) or viscous liquids → Positive Displacement Pipette or Glass Syringe.
- If large volumes (1–25 mL) are needed for non-quantitative work → Serological Pipette (Plastic/Glass).
- If evaporation is critical (e.g., small volume of methanol) → Minimize dwell time and use Positive Displacement.
Main Types of Pipettes Used in Chemistry Labs
Volumetric Pipettes (Class A vs Class B)
For preparing calibration curves and standard solutions, the glass volumetric pipette remains the non-negotiable standard in analytical chemistry.
- Class A vs. Class B: Class A glassware meets tighter ASTM/ISO tolerance limits (e.g., ±0.02 mL for a 10 mL pipette) compared to Class B (±0.04 mL). Class A is required for regulated work (GLP/GMP).
- TD vs. TC: Most volumetric pipettes are marked TD (To Deliver). This means they are calibrated to deliver the stated volume by gravity drainage. They should not be blown out unless explicitly marked with a double ring or “Blow Out”.
- Traceability: Modern Class A pipettes often come with individual batch certificates and serial numbers, essential for audit trails.
Graduated Pipettes (Mohr, Serological)
These are cylindrical tubes with graduation marks along the length.
- Accuracy Trade-offs: They are significantly less accurate than volumetric pipettes (typically ±1–2% vs ±0.1%).
- Analytical Use Cases: Acceptable for adding reagents “in excess” (where the exact volume doesn’t affect the calculation) or for general solvent additions in synthesis. They are not suitable for preparing standard solutions for quantitative analysis (HPLC/GC).
- Meniscus Reading: Requires care; the graduation lines must be read at eye level, aligned with the bottom of the meniscus for clear liquids.
Micropipettes in Analytical Chemistry
Variable volume micropipettes (air displacement) are ubiquitous but frequently misused in chemistry.
- Appropriate Use: Excellent for aqueous samples, buffers, and dilute acids where density $\approx$ 1.0 g/mL.
- The Error Source: They rely on an “air piston.” If the liquid has a high vapor pressure (e.g., acetone, hexane), the vapor expands into the air space, forcing liquid out of the tip (dripping). Conversely, if the liquid is cold, the air contracts, aspiring too much sample.
- Solvent Limits: Standard polypropylene (PP) tips may swell or soften with aggressive solvents like chloroform or THF, altering the tip geometry and volume.
Positive Displacement Pipettes
These are the problem-solvers for “difficult” liquids in the chemistry lab.
- Mechanism: A disposable capillary piston makes direct physical contact with the liquid. There is no air cushion.
- Organic Solvents: Because there is no air gap to expand, volatile solvents (DCM, Acetone) are pipetted with high accuracy and no dripping.
- Viscosity: The piston physically wipes the inside of the capillary clean, ensuring accurate delivery of oils, syrups, or concentrated acids that would otherwise cling to a standard tip.
Material Compatibility and Chemical Resistance
In analytical chemistry, the interaction between the solvent and the pipette material is a critical source of contamination.
Glass vs. Polypropylene
- Glass (Borosilicate): Inert to almost all organics and acids (except HF). It is the safest choice for trace organic analysis to avoid leachables.
- Polypropylene (PP): The standard material for micropipette tips. Generally resistant to acids and bases but has limited resistance to aromatic and halogenated hydrocarbons.
Organic Solvent Compatibility Table
Critical Note for HPLC/MS: Cheap plastic tips often contain “slip agents” (Oleamide, DiHEMDA) to help them eject from the mold. These compounds wash out into organic solvents (like Methanol) and appear as contaminant peaks in Mass Spectrometry. For trace analysis, always use high-quality, “low-retention” tips certified free of plasticizers.
Accuracy, Evaporation, and Technique
Temperature Equilibration
Liquid density changes with temperature. Pipettes are calibrated (usually) at 20°C or 25°C. If a solvent is stored in the fridge (4°C) and pipetted immediately, the volume delivered will be incorrect due to density differences and the cooling effect on the air cushion (in micropipettes). Rule: Allow all liquids and pipettes to equilibrate to room temperature before analysis.
Minimizing Evaporation Loss
Volatile solvents begin evaporating immediately.
- Speed: Work quickly with volatiles.
- Pre-wetting: Aspirate and dispense the solvent 3 times before the final draw. This saturates the air cushion with solvent vapor, establishing equilibrium and preventing the “drip” effect.
Meniscus Reading
For glass pipettes, reading the meniscus correctly is the largest source of operator error.
- Clear Liquids: Read the bottom of the curve.
- Opaque/Dark Liquids: Read the top of the liquid line (if the meniscus is not visible).
- Parallax Error: The eye must be exactly level with the graduation mark.
Common Mistakes in Chemistry Pipetting
- Using Air Displacement for Volatiles: Using a standard micropipette for acetone or chloroform. The liquid drips out, and the delivered volume is consistently low.
- Fix: Use a positive displacement pipette or pre-wet the tip 5 times.
- Blowing Out “TD” Glass Pipettes: Forcing the last drop out of a volumetric pipette marked “TD”. This adds extra volume that was intended to remain in the tip due to surface tension.
- Fix: Touch the tip to the vessel wall, let it drain, and wait 3 seconds. Leave the final drop.
- Ignoring Temperature: Pipetting cold enzyme solutions or solvents. The air inside the pipette contracts, aspiring more volume than set.
- “Hand Warming” Effect: Holding the micropipette body too tightly for too long warms the internal air piston, causing volume expansion and inaccurate delivery.
- Fix: Place the pipette on the stand between samples.
- Carryover in Trace Analysis: Assuming a tip is clean because it “looks” empty. Organic solvents can form a film.
- Fix: Use fresh tips for every standard in a calibration curve.
Standardization in Analytical Laboratories
To maintain data integrity (DI) and reproducibility, labs must standardize their liquid handling strategy.
- Tip + Pipette = One System: ISO 8655 guidelines state that a pipette is only calibrated for the specific tip type used during calibration. Switching from “Brand A” tips to cheaper “Brand B” tips invalidates the calibration.
- Validation Checks: Analytical labs should perform “gravimetric checks” (weighing water) on micropipettes every 3–6 months. For volumes <20 µL, a 6-place microbalance is now required by ISO standards.
- Defining Tolerances: Establish internal SOPs. For example, a “Class A” glass pipette might have a tolerance of ±0.2%, while a micropipette set to 1000 µL might have a tolerance of ±1.0%. Ensure the tool matches the method’s required uncertainty.
Frequently Asked Questions
Can micropipettes be used for organic solvents?
Yes, but with caveats. You must use high-quality PP tips (to avoid leaching) and use the “pre-wetting” technique to saturate the air cushion. For highly volatile solvents (DCM, Chloroform), positive displacement pipettes are significantly better.
What is the difference between TD and TC pipettes?
TD (To Deliver): Calibrated to dispense the specific volume. The small amount left in the tip is accounted for. (Most common in labs).
TC (To Contain): Holds the exact volume inside. To get that volume out, you must wash it out with solvent. (Rare, used for viscous fluids or specific clinical tests).
When is glass preferred over plastic?
Glass is preferred for preparation of primary standards, handling strong organics (THF, DCM) that dissolve plastic, and any application requiring Class A volumetric certification.
How accurate are graduated pipettes compared to volumetric?
Graduated (Mohr/Serological) pipettes are generally ±1% to ±2% accurate. Volumetric Class A pipettes are ±0.1% to ±0.2% accurate. Never use a graduated pipette for quantitative analysis (e.g., preparing an HPLC standard).
What is the best pipette for HPLC sample prep?
For volumes >1 mL, use Class A volumetric glass. For volumes <1 mL, use a positive displacement pipette or a micropipette with “Low Retention” and “Certified Plasticizer-Free” tips to avoid ghost peaks.
Key Takeaways
- Application is King: Do not use the same pipette for aqueous buffers and volatile organic solvents.
- Watch the Vapor Pressure: Volatiles cause dripping in air-displacement pipettes. Use positive displacement or rigorous pre-wetting.
- Respect the “TD” Mark: Gravity drain volumetric glass; do not blow it out unless marked.
- Beware of Plasticizers: Standard pipette tips can leach chemicals into HPLC/GC samples. Use high-grade consumables.
- Temperature Matters: Equilibration of liquids to room temperature is essential for accurate density and volume transfer.
- System Integrity: Calibration applies to the pipette and the specific tip brand used. Do not mix and match without verification.
- Class A for Standards: Volumetric glass remains the standard for creating calibration curves.
- Viscosity requires force: Viscous liquids (oils, acids) require positive displacement to ensure the full volume is wiped from the tip.