Laboratory apparatus and equipment

 

Classification of laboratory apparatus

Classifying apparatus based on the type of material:

Glassware: graduated cylinders, side arm, Erlenmeyer flask, beaker, funnels, test tubes, watch glasses, fractionating column, west condenser, separator funnel, stopper, round bottomed flask, three-way adapter (distilling head), Claisen adapter, vacuum adapter, thermometer adapter, drying tube, Hickman head, water condenser, air condenser, conical vial and spin vane.

Ceramics: crucible, mortar and pestle, Buchner funnel, pipe clay triangle, tiles

Rubber: dropper, rubber tubing, rubber corks, pipette filler

Plastic: wash bottle, funnel, test tube rack

Platinum (and its alloys): platinum loop, tongs. Platinum offers high electrical conductivity and resistance to chemical attack.

Quartz: cuvette

Aluminium: TLC paper

Nichrome wire loop is also called a smear loop, micro streaker and inoculation wand. It is easy to sterilize reuse because nichrome wire resists deterioration with repeated heat/ cooling cycles (easy to heat and easy to cool). The loop of wire at the tip may be made of 24 nichrome, platinum or tungsten.

 

Classifying apparatus based on the function

Measurement category: this function involves measuring quality, volume, temperature and density. Examples are burette, pipette, measuring cylinder

Reaction category: apparatus used for chemical reaction e.g. test tube, flask, evaporating dish, and crucible….etc.

Vessel category: chemical reagent bottle used for holding or keeping solid, liquid, gas chemical reagent.

Separation category: apparatus used for filtration, skimming, extraction, evaporation, firing, crystallization and fractional distillation, including funnel, evaporating dish, flask, condenser, beaker and so on.

Solid clamping category: apparatus used for fixing and clamping various chemical laboratory instruments, including iron clamp, iron ring, iron stand and funnel stand.

Heating category: examples are test tube, flask, beaker, evaporating dish and crucible.

Matching category: For example, glass tube, glass valve, rubber tube and rubber plug which are used for assemble and chemical laboratory instruments connection.

LABORATORY EQUIPMENT

Equipment	Function
Spectrophotometer	Measures the interaction between electromagnetic radiation and the sample
Colorimeter	measures the intensity of color and relates the intensity to the concentration of the sample
Vortex	Mixing laboratory samples in test tubes, well plates, or flasks.
Magnetic stirrer	used to make a stir bar immersed in a liquid, quickly spin, or stirring or mixing a solution
Centrifuge	Rotates at high speed and separates substances of different densities.
Biological Safety Cabinet	enclosed, ventilated laboratory workspace for safely working with materials contaminated with pathogens
Mass balance	measure an object’s mass to a very high degree of precision
Hot plate	used to heat and stir substances
Deionizer	Removals contaminant such as minerals in food
Incubator	grow and maintain microbiological cultures
Oven	uses dry heat to sterilize
Autoclave	sterilize equipment
Heating mantle	Heating
Water bath	device that maintains water at a constant temperature and is used in the microbiological laboratory for incubations
pH meter	Ph  measurement
Colony counter	Count colonies growing on media
Microscope	Perform magnification and resolution
Vacuum pump	Change the pressure in a contained space to create a full or partial vacuum
Anaerobic jar	Culture anaerobic bacteria.
TLC	Separate non-volatile mixtures.
Bunsen burner	Produces a single open gas flame, which is used for heating and sterilization.
Laboratory refrigeration	Maintenance and storage of stock culture between sub culturing periods. Storage of sterile media to prevent dehydration Repository for thermo-labile solutions, antibiotics and serums

 

 

We shall focus on the following equipment:

a)      Biological Safety Cabinet

The primary purpose of biosafety cabinets is to protect the laboratory personnel and the environment from the pathogenic microorganism. These cabinets are provided with HEPA-filters (High Efficiency Particulate Air) that decontaminate the air moving out of the cabinet.

It is an enclosed, ventilated laboratory workspace. It is mainly used for safely working with materials contaminated with pathogens (disease causing micro-organisms). It is usually in a negative pressure for containment purpose.

Types of BSC

Classification	Biosafety level	Application
Class I	1,2,3	low to moderate risk biological agents
Class II	1,2,3	low to moderate risk biological agents
Class III	4	high risk biological agents

 

**Biosafety levels

Biosafety is the prevention of risks (human health and safety, environment conservation) associated with the laboratory activities occur either from the samples or the procedural requirements.

 Thus, biosafety levels are designed to identify various protective measures that are to be taken in a laboratory setting to protect the researchers, the environment, and the microorganisms. These levels are defined by the Central for Disease Control and Prevention (CDC), where each of these levels is outlined with specific practices and safety requirements.

Biosafety level 1 (BSL1)

Lowest safety level, and the precautions required are limited and not extensive.

Laboratories at this level provide general space in which work is done with viable agents that are not associated with disease in healthy adults

Biosafety level 2 (BSL2)

Laboratories at this level are used for tasks involving microbial agents of moderate potential hazards to the laboratory personnel, the environment, and the agent. However, if accidentally inhaled, swallowed, or exposed to the skin, they pose danger.

Biosafety level 3 (BSL3)

At this level, the agents worked on may cause severe or potentially lethal disease through inhalation or aerosol formation, to the personnel, and may even contaminate the environment.

Working in such laboratories requires laboratory personnel with specific training in handling pathogenic and potentially lethal agents.

Biosafety level 4 is the highest level that is employed while working with dangerous infectious agents that present a high individual as well as environmental risk in the form of life-threatening disease, aerosol transmission, or unknown risk of transmission.

The BSL-4 laboratories are often used while handling and manipulating Risk Group 4 pathogens that is extremely dangerous, with no known vaccines or therapies, and require extreme precautions during work.

The BSL-4 laboratories are of two types; cabinet laboratory where all the work is performed in a Class III biosafety cabinet or similar physical containment with very carefully formulated precautions and suit laboratory where all the laboratory personnel are required to wear full-body, air-supplied suits protective gears in the form of PPEs.

Class I

Class I provide personnel protection but no product protection and environmental. They are either connected to the building exhaust system or recirculating filtered exhaust back into the laboratory.

Class II

Class II cabinets provide both kinds of protection of the samples and of the environment. The makeup air is also HEPA‐filtered (High Efficiency Particulate Air). These are further classified as Type A1, Type A2, Type B1, Type B2.

Class III

Class III cabinet, generally only installed in maximum containment laboratories. Specifically it was designed for work with highly contaminant (BSL-4) pathogenic agents, providing maximum protection. The enclosure is gas‐tight. All materials enter and leave through a double‐door autoclave.

 

Type of tests done for BSCs:

·         Down flow velocity.

·         Inflow velocity.

·         Airflow smoke patterns.

·         Filter leak.

·         Light intensity.

·         Vibration.

·         Noise level

·         UV light integrity

a)      SPECTROSCOPY

Spectroscopy is the study of the interaction between light and matter where the absorption and emission of light or other radiation by the matter are studied and measured. A spectrometer is a scientific instrument that is used to measure the variation or differences in various properties caused by an object over a particular range. A spectrophotometer is a particular type of spectrometer that measures the interaction (absorption, reflection, scattering) of electromagnetic radiation from a sample or the emission (fluorescence, phosphorescence, electroluminescence) of electromagnetic radiation by various sample.

Electromagnetic spectrum

 

Courtesy of Chandra X-ray observation

The electromagnetic spectrum is made up of all electromagnetic radiation. Electromagnetic radiation travels in waves: thus it is characterized by frequency (number of cycles of a wave to pass some point in a second) and wavelength (distance between one wave amplitude to the next). From the Radio waves to Gamma, the wavelength decreases as the frequency increases and the vice versa is true.

**Difference between colorimeter and spectrophotometer

 

Colorimeter	Spectrophotometer
Measures concentration of a colored solution	Measures the interaction of electromagnetic radiation as it passes through the sample
Uses fixed wavelength in visible range	Wavelength is used in a wider range (UV,IR..)
Colorful samples	Colorless samples
Quantifies the color by measuring the three primary color components (red, green and blue)	Measures the amount of light that passes through the sample

 

Types of spectroscopy

1. Absorption spectroscopy

2. Astronomical spectroscopy

3. Atomic absorption spectroscopy

4. Circular dichroism spectroscopy

5. Electrochemical impedance spectroscopy (EIS)

6. Electron spin resonance (ESR) spectroscopy

7. Emission spectroscopy

8. Energy dispersive spectroscopy

9. Fluorescence spectroscopy

10. Fourier-transform infrared (FTIR) spectroscopy

11. Gamma-ray spectroscopy

12. Infrared (IR) spectroscopy/ Vibrational spectroscopy

13. Magnetic resonance spectroscopy

14. Mass spectroscopy

15. Molecular spectroscopy

16. Mossbauer spectroscopy

17. Nuclear magnetic resonance (NMR) spectroscopy

18. Photoelectron spectroscopy

19. Raman spectroscopy

20. UV spectroscopy

21. Ultraviolet and visible (UV/Vis) spectroscopy

22. X-ray photoelectron spectroscopy

At this level, we shall focus on UV spectroscopy and UV/Vis spectroscopy which use absorption spectroscopy.

 

Absorption spectrophotometer mechanism

Its main components are:

·         Energy source

·         Monochromator: breaks the polychromatic radiation into component wavelength (or) bands of wavelengths. It can be made up of:

A prism disperses polychromatic light from the source into its constituent wavelengths.

Gratings are often used in the monochromators of spectrophotometers operating ultraviolet, visible and infrared regions.

·         Transport vessels (cuvettes), to hold the sample. Cuvettes meant for the visible region are made up of either ordinary glass (or) sometimes Quartz.

·         A Photosensitive detector and an associated readout system. Most detectors depend on the photoelectric effect. The current is then proportional to the light intensity and therefore a measure of it. These electronic signals are interpreted into a digital output.

 

UV spectroscopy

UV spectroscopy is a type of absorption spectroscopy where UV lights are absorbed by the electrons in the sample that causes them to excite to a high energy state.

Principle

UV rays pass to the sample and absorbed by the electrons, which increases the energy of the system.

Excitation forms an absorption spectrum that can be detected by the detectors in the spectrometer.

The amount of photon (radiation) absorbed results in an absorption spectrum which can then be measured in terms of absorbance.

Uses of UV spectroscopy

Detection of impurities in organic substances

Quantitative determination of compounds

 

Ultraviolet and visible (UV/Vis) spectroscopy

Ultraviolet and visible spectroscopy is an absorption spectroscopy technique which uses the radiation in the UV range and the adjacent visible range of the electromagnetic radiation.

Principle

·         The incident light in this spectrometer is in the range of UV and visible spectrum of the electromagnetic spectrum.

·         When the photon of sufficient energy reaches an object, the energy is absorbed by the electrons causing them to bump into a higher energy state.

·         Materials produce an absorption spectrum which is a range of absorbance resulting from the radiation absorbed by the material at different frequencies.

·         Thus, the absorption spectrum of materials depends on the atomic and molecular composition of that material.

 

Uses of UV/Vis spectroscopy

Qualitative identification of certain classes (proteins and nucleic acids) of compounds both in the pure state and in biological mixtures

Quantification of biological samples either directly or via colorimetric assays

 

a)      Autoclave

The autoclave is a sealed device (similar to a pressure cooker) that kills microorganisms using saturated steam under pressure / performs sterilization.

Principle

The use of moist heat facilitates the killing of all microorganisms, including heat-resistant endospore which is achieved by heating the materials inside the device at temperatures above the boiling point of water.  At 121°C, the time of autoclaving to achieve sterilization is generally considered to be 15-20 min, depending on the volume of the load.  Air should be evacuated so that the chamber fills with steam.

An autoclave machine consists of a pressure chamber, a lid, and an electrical heater.

Types of autoclave

·         Gravity displacement type autoclave: It is the most common type used in laboratories and is available in various sizes and dimensions.

·         Vertical type (small volume capacity)

·         Horizontal autoclave (large volume capacity)

·         Positive pressure displacement type autoclave

·         Negative pressure (vacuum) displacement type.

 

Sterilization control

Techniques employed to determine effectiveness of sterility:

        i.            Use of biological indicator such as spores of Bacillus stearothermophilus are the best indicator because they are resistant to steam.

      ii.            Use of autoclave tapes: Adhesive-backed paper tape with heat-sensitive, chemical indicator marking that changes color or display-diagonal stripes, the words “sterile” or “autoclaved” when exposed to effective sterilization temperature (121°C) are used to check the efficacy of autoclaves.

    iii.            Use of a thermocouple, a temperature measuring device that records the temperature by a potentiometer

    iv.            Use of Browne’s tube that has a heat-sensitive red dye that turns green after being exposed to a certain temperature for a definite period of time

 

b)      Thin Layer Chromatography

Thin layer chromatography (TLC) is a chromatographic technique used to separate the components of a mixture using a thin stationary phase supported by an inert backing. It is an analytical tool widely used because of its simplicity, relative low cost, high sensitivity, and speed of separation.

Principle

TLC functions on the same principle as all chromatography:

A compound will have different affinities for the mobile and stationary phases, and this affects the speed at which it migrates. The goal of TLC is to obtain well defined, well separated spots.

 

Stationary phase

Silica gel and alumina are among the most common stationary phases, but others are available as well.

Glass plates are chemically inert and best withstand reactive stains and heat, but are brittle and can be difficult to cut.

Aluminum and plastic plates can be cut with scissors, but aluminum may not withstand strongly acidic or oxidizing stains, and plastic does not withstand the high heat required to develop many stains. Aluminum and plastic plates are also flexible, which may result in flaking of the stationary phase.

N/B: Never under any circumstances touch the face of a TLC plate with your fingers as contamination from skin oils or residues on gloves can obscure results. Instead, always handle them by the edges, or with forceps.

 

Mobile phase

Proper solvent selection as mobile phase in TLC is important as one takes note of the chemical properties of the analytes. A common starting solvent is 1:1 hexane: ethyl acetate.

 

Applications of TLC

·         Analysis of drug residues and antibiotics in food and environmental samples

·         Identification and quantification of colors, ingredients, preservatives, and sweetening agents in food and cosmetic products

·         Quality control and purity testing of pharmaceutical formulations