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Principles and uses of :

Colorimeter, Spectrophotometer,
Centrifuge, pH meter, Water bath,
Hot air oven, Incubator
Prince Sir ( MLT )
P. G. in Biochemistry

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1. COLORIMETER
✅ Principle : Based on Beer-Lambert’s Law:

Beer’s Law: Absorbance is directly proportional to the concentration of the solution.

Lambert’s Law: Absorbance is directly proportional to the path length of the cuvette.

📌 Combined law:

A=ε×c×l
Where:
•A = Absorbance
•ε = Molar absorptivity
•c = Concentration
•l = Path length

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✅ Working:
1.A light source emits white light.
2.Light passes through a filter (selects specific wavelength).
3.The sample solution absorbs part of the light.
4.Photodetector measures the light that passes through.
5.Output is in Optical Density (OD) or Absorbance.

✅ Uses:
•Estimation of Hemoglobin, Blood glucose, Proteins.
•Used in biochemistry labs for routine analysis.
•Suitable for colored solutions only.

✅ Key Features:
•Wavelength range: 400–700 nm (Visible range)
•Less accurate than spectrophotometer.
•Cheaper and easier to operate.

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2. SPECTROPHOTOMETER
✅ Principle:
•Also based on Beer-Lambert’s Law.
•Measures absorbance or transmittance of light by a solution at specific wavelengths (UV
or visible).📌 Measures both:
•UV region (190–400 nm)
•Visible region (400–700 nm)

✅ Working:
1.A light source emits UV/visible light.
2.Light passes through monochromator (selects exact wavelength).
3.The light then passes through the sample in a cuvette.
4.Detector measures the transmitted light.
5.Output shows Absorbance vs. Wavelength.
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✅ Types:
•Single Beam: Measures sample and blank separately.
•Double Beam: Splits beam for simultaneous measurement.

✅ Uses:
•DNA, RNA quantification
•Enzyme assays
•Estimation of bilirubin, urea, creatinine, cholesterol
•Used in clinical biochemistry, microbiology, molecular biology

✅ Key Features:
•More accurate and sensitive than colorimeter.
•Can detect both colorless and colored substances.
•Can scan across wavelengths (spectral scan).
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3. CENTRIFUGE
✅ Principle:
•Based on sedimentation by centrifugal force:
Particles of different mass and density settle at different rates under high-speed spinning.

📌 Related Centrifugal Force (RCF):

RCF = 1.118 × 10⁻⁵ × r × (RPM)²
Where:
•r = Radius of rotation
•RPM = Speed in revolutions per minute

✅ Working:
1.Tubes with samples are placed in rotor.
2.Rotor spins rapidly → creates centrifugal force.
3.Heavier particles settle at the bottom (pellet).
4.Lighter components stay as supernatant.

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✅ Types of Centrifuge:

•Small bench centrifuge: For basic separation.

•High-speed centrifuge: For protein purification.
•Refrigerated centrifuge: For temperature-sensitive samples.
•Ultracentrifuge: Used in molecular biology for viruses, DNA, etc.

✅ Uses:
•Separation of serum/plasma from blood.
•Isolation of cells, bacteria, organelles.
•Urine, CSF, and semen sample analysis.
•Diagnostic tests like HbA1c, ESR, etc.

✅ Safety Tips:
•Balance tubes before spinning.
•Use closed caps to avoid aerosol.
•Do not open lid until rotor stops.

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⚖️4. pH Meter
✅ Principle:
Measures hydrogen ion concentration using a glass electrode and a reference electrode
to produce a voltage that corresponds to the pH.
📌 Formula: pH = –log[H⁺]
✅ Working:
•The electrode is dipped into the solution.
•It senses H⁺ ions and generates a potential difference.
•The meter converts the signal into a digital pH reading.
✅ Uses:
•Checking acidity or alkalinity of samples
•Media and buffer preparation
•Analysis of biological fluids like urine, saliva, etc.
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5. Water Bath
✅ Principle:
Uses heated water to provide uniform and constant temperature for incubating or
processing samples.
✅ Working:
•Water is heated using an electric heater.
•The set temperature is maintained using a thermostat.
•Samples are placed in tubes or flasks and submerged.
✅ Uses:
•Incubation of reagents
•Enzyme reactions
•Melting agar or warming reagents
•Common in biochemistry and microbiology labs
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🔥 6. Hot Air Oven
✅ Principle:
Uses dry heat sterilization by circulating hot air within a closed chamber to kill
microorganisms.
📌 Sterilization standard:
160°C for 2 hours or 180°C for 30 minutes.
✅ Working:
•Air is heated using heating coils.
•A fan circulates hot air throughout the chamber.
•The heat penetrates glassware or instruments and sterilizes them.
✅ Uses:
•Sterilizing glassware (e.g., test tubes, Petri dishes), metal instruments, oils, and powders
•Not suitable for rubber, plastic, or culture media
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🌱 7. Incubator
✅ Principle:
Provides and maintains optimal environmental conditions (like temperature and humidity) for the growth of
microorganisms or cell cultures.
📌 Standard incubation temperature: 37°C (human body temp)

✅ Working:
•Temperature is set using a thermostat.
•The chamber maintains consistent conditions for microbial or cellular growth.
•Some incubators also control CO₂ or humidity.
✅ Uses:
•Bacterial and fungal culture
•Cell culture and tissue culture
•Antibiotic sensitivity testing
•Vaccine research

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Equipment Principle Primary Use Special Notes
Colorimeter Beer-Lambert’s Law (Visible light) Colored solution analysis Less accurate, low cost

Spectrophoto Absorbance in UV/Visible Detects both colorless and

Beer-Lambert’s Law (UV-Vis)
meter light colored substances
Separation of sample Requires balancing of
Centrifuge Sedimentation by centrifugal force
components tubes
Measuring
pH Meter Electrochemical voltage detection Needs frequent calibration
acidity/alkalinity (pH)

Water Bath Heated water for constant temp Reactions, incubation Temperature limit ~100°C

Sterilizing glassware and

Hot Air Oven Dry heat sterilization Not for plastics or media
heat-stable items
Growth of microbes or Mostly used at 37°C for
Incubator Controlled environment
cells bacteria

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