Study sheet: Laboratory Techniques and Safety

📋 Course Outline

1. Laboratory Water and Solutions
2. Laboratory Safety and Conduct
3. Glassware Selection and Identification
4. Glassware Rinsing and Maintenance
5. Pipette and Pipette-Filler Handling
6. Micropipette Use and Precision
7. Dilutions and Calibration Series
8. Spectrophotometer Measurements
9. Microscope Operation and Magnification
10. Graphs and Linear Regression

📖 1. Laboratory Water and Solutions

🔑 Key Concepts & Definitions

• Distilled water : Obtained by boiling followed by condensation, which removes the vast majority of minerals, impurities, and microorganisms.
• Solute : A substance dissolved in a solution.
• Solvent : A liquid that dilutes or extracts other substances without chemically changing them or undergoing change itself.
• Fume hood : A ventilated enclosure that draws in laboratory air through an adjustable opening, exhausts it outside, and operates under negative pressure to protect users from inhaled chemical pollutants.
• Demineralized or ultrapure water : Water from which dissolved minerals and ions have been removed and that is supplied in 5L or 10L containers.

💡 Memory Hook

Solute dissolves; solvent dilutes or extracts.

📖 2. Laboratory Safety and Conduct

★ Must-know

• A fastened cotton lab coat, safety goggles for chemistry practicals, trousers covering the legs, closed-toe shoes, and tied-back long hair are required or recommended laboratory attire, while contact lenses are strongly discouraged and tights and long skirts are prohibited.

• Eating, drinking, smoking, using a phone, and mouth pipetting are prohibited in the practical workroom, and hands must be washed at the end of the session and immediately after contamination.

• During a session, containers must remain identified, solutions must be poured into a beaker before pipetting, bottles must be closed immediately after use, and equipment must be handled carefully.

• After a session, solutions must not be returned to original bottles, waste must be disposed of appropriately, unknown solutions must not remain on benches, equipment must be cleaned and reset, and the workbench must be checked by a supervisor before leaving.

Further detail

📌 Before a session, benches and fume hoods must be clear, both exits must remain accessible and unlocked, and all bottles must be labelled.

💡 Memory Hook

Before: clear and label; during: identify and protect; after: dispose and clean.

📖 3. Glassware Selection and Identification

🔑 Key Concepts & Definitions

• Volumetric flask : Used to prepare a solution of precise concentration from a solid by dissolution or from a stock solution by dilution.
• Burette : Allows the dropwise addition of a reagent whose initial volume is unknown.

★ Must-know

📌 Beakers, Erlenmeyer flasks, and graduated cylinders are low-precision glassware used for approximate volumes, whereas volumetric flasks, graduated pipettes, volumetric pipettes, and burettes measure volumes precisely.

📌 During volume reading, the bottom of the meniscus must align with the graduation mark while the eye is level with the mark to avoid parallax.

Further detail

• A graduated pipette withdraws precise variable volumes from 0.1 to 50 mL.

• A two-mark volumetric pipette withdraws a precise fixed volume from 1 to 50 mL, whereas the listed available sizes range from 1 to 100 mL.

📌 Glassware and small laboratory materials must be labelled with the relevant identity, concentration, sample, medium, temperature, time, date, or operator information required by the procedure.

💡 Memory Hook

Beakers approximate; volumetric glassware measures precisely.

📖 4. Glassware Rinsing and Maintenance

★ Must-know

• 🔄 Container rinsing consists of: removing marker annotations, collecting remaining liquid, rinsing several times with tap water, lightly rinsing with distilled water, placing the container upside down or on a drying rack

• 🔄 A used pipette is rinsed by:

  1. rinsing the tip
  2. filling it with distilled water
  3. allowing it to drain
  4. emptying it
  5. repeating the process twice

Further detail

📌 Alcohol may accelerate drying only after proper water washing because it can dehydrate residues and cause red blood cells to adhere irreversibly to the pipette.

• 🔄 After use, a burette is:
  1. emptied into the appropriate waste container
  2. lightly rinsed into the waste beaker
  3. filled with distilled water
  4. emptied into a rinse-water beaker
  5. discarded down the sink

💡 Memory Hook

Remove labels, collect residues, rinse with tap water, finish with distilled water.

📖 5. Pipette and Pipette-Filler Handling

📝 Essential Points

• 🔄 A pipette filler bulb is operated by:
  1. pressing A to expel air
  2. pressing S gradually to draw liquid
  3. pressing E to expel liquid

📌 A pipette must never be used by mouth or left hanging from the pipette filler, and it must be held either with the upper hand and filler or with the lower hand between the index and middle fingers.

💡 Memory Hook

A removes air, S suctions, E expels.

📖 6. Micropipette Use and Precision

🔑 Key Concepts & Definitions

• Micropipette : A precision instrument used to dispense variable micro-volumes, with the models described covering volumes from 2 to 1000 µL.

★ Must-know

📌 The micropipette with the largest capacity that can deliver the required volume should be selected; for 100 µL, the P100 is chosen over the P200 and P1000 for greater precision.

• 🔄 Micropipetting consists of:
  1. attaching a tip
  2. pressing to the first stop
  3. immersing the tip a few millimeters
  4. gently releasing the plunger
  5. dispensing with the first and second stops
  6. ejecting the tip
  7. returning the micropipette to its stand

Further detail

📌 During sterile handling, only the sterile tip may enter the tube, while the tip cone must remain outside and be wiped with an alcohol-soaked wipe before and after the procedure.

💡 Memory Hook

Choose, aspirate, dispense, eject, return.

📖 7. Dilutions and Calibration Series

🔑 Key Concepts & Definitions

• Dilution : Reduces the concentration of a stock solution by adding solvent while conserving the amount of solute, so the final concentration depends on the final volume.

📝 Essential Points

📐 Formula — Conservation of solute in a dilution is expressed as $n = C_sV_s = C_dV_d$.

📐 Formula — The dilution factor is expressed as $DF = \frac{C_{stock}}{C_{diluted}} = \frac{V_{diluted}}{V_{stock}}$.

• 🔄 Preparing a dilution consists of:

  1. calculating the stock volume
  2. measuring it with a suitable pipette
  3. transferring it to a volumetric flask
  4. filling to the graduation mark with solvent
  5. stoppering the flask
  6. mixing by repeated inversion

• A calibration series generally includes a blank at zero concentration and at least five calibration points distributed across the studied concentration range.

💡 Memory Hook

Conserved solute with increased volume causes lower concentration.

📖 8. Spectrophotometer Measurements

📝 Essential Points

📐 Formula — Absorbance is calculated as $A = \log\left(\frac{I_0}{I}\right)$.

📌 Plastic cuvettes are used for non-corrosive solutions in the visible range from 400 nm to 800 nm, glass cuvettes are used for corrosive solutions and certain organic solvents in the visible range, and quartz cuvettes are used in the ultraviolet range from 200 nm to 400 nm.

• 🔄 In absorbance mode,:

  1. set the wavelength
  2. zero with the blank
  3. place the sample cuvette
  4. read absorbance
  5. rerun the blank to check drift

• A calibration curve of absorbance versus concentration is a straight line passing through the origin, and the concentration of an unknown can be determined from its measured absorbance.

💡 Memory Hook

Choose cuvette, set wavelength, blank, measure, recheck blank.

📖 9. Microscope Operation and Magnification

★ Must-know

• 🔄 Microscope focusing consists of:

  1. using the lowest-power objective
  2. obtaining an initial image with coarse focus
  3. sharpening with fine focus
  4. switching to a higher objective
  5. readjusting with fine focus

• Total magnification is calculated by multiplying the eyepiece value by the objective value.

📌 The 100x objective is used only with immersion oil for very small structures, and after oil is applied the observer must not switch back to a lower-power objective.

Further detail

• The microscope settings give total magnifications of 40x, 100x, 400x, and 1000x for objectives x4, x10, x40, and x100 respectively.

💡 Memory Hook

Low power, coarse focus, fine focus, higher power, immersion oil.

📖 10. Graphs and Linear Regression

★ Must-know

• 🔄 A graph is prepared by:
  1. drawing and scaling labelled axes
  2. plotting small plus-sign points
  3. drawing a best-fit line or smooth curve
  4. adding captions or a legend
  5. writing an explicit title

📌 A graph title must express the result variable as a function of the studied variable, corresponding mathematically to $y = f(x)$.

📐 Formula — A linear regression line is written as $y = ax + b$, where $a$ is the slope and $b$ is the intercept, and the line passes through the centroid of the points.

• Least-squares regression minimizes the sum of the squared differences between observed y-values and calculated y-values.

📌 A correlation coefficient r close to 1 or -1 indicates that a line is a suitable model, whereas a value close to 0 indicates a more circular scatter for which determining a line is not justified.

Further detail

📐 Formula — The slope between two points is calculated as $a = \frac{y_B-y_A}{x_B-x_A}$.

💡 Memory Hook

Measured pairs produce a fitted line whose slope and correlation assess the relationship.

📊 Synthesis Tables

Laboratory Glassware Precision

Equipment	Main use	Precision
Beaker	Holding liquid or intermediate sampling	Low
Erlenmeyer flask	Collecting liquid or containing potentially violent reactions	Low
Graduated cylinder	Approximate volume measurement	Low
Volumetric flask	Preparing a precise final volume	High
Graduated or volumetric pipette	Transferring variable or fixed precise volumes	High
Burette	Dropwise addition of a reagent	High