Spectrophotometry –

I. Definition

Spectrophotometry is a quantitative measurement technique used to determine the amount of

light absorbed by a solution. The amount of light absorbed correlates with the concentration of a
specific solute (analyte) in the solution.

It is widely used in clinical biochemistry, hematology, pharmacology, microbiology, and

environmental science.

II. Scientific Basis: Beer-Lambert Law

The foundation of spectrophotometry lies in the Beer-Lambert Law, which combines two
related laws:

1. Beer’s Law (Absorbance ∝ Concentration)

As the concentration of the absorbing species increases, the amount of light absorbed increases.

2. Lambert’s Law (Absorbance ∝ Path Length)

As the path length (distance the light travels through the sample) increases, absorbance also
increases.

Mathematical Equation:

A=ε×c×lA = \varepsilon \times c \times l

Where:

• A = Absorbance (unitless)
• ε = Molar absorptivity/extinction coefficient (L mol⁻¹ cm⁻¹) – constant for each substance
at a specific wavelength
• c = Concentration (mol/L)
• l = Path length of light through the sample (cm)

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 III. Components of a Spectrophotometer

Component Function
Light Source Provides continuous radiation. UV: Deuterium lamp, Visible: Tungsten lamp
Monochromator Selects a narrow band of wavelengths using prisms or diffraction gratings
Slit Controls the amount of light entering the cuvette; affects resolution
Cuvette Holds the sample; quartz (for UV) or glass (for visible). Path length = 1 cm
Sample Holder Mechanism to insert/remove cuvettes
Converts light into an electrical signal (usually photodiode or
Detector
photomultiplier)
Amplifier Boosts the weak signal from the detector
Digital Display Displays the absorbance or % transmittance

IV. Types of Spectrophotometry

Wavelength
Type Used For
Range
UV Spectrophotometry 200–400 nm DNA, RNA, protein quantification
Visible Colorimetric biochemical tests (glucose,
400–700 nm
Spectrophotometry cholesterol)
Organic compounds, functional groups
IR Spectrophotometry 700 nm – 1 mm
identification

V. Standard Operating Procedure (SOP)

1. Warm up the instrument (15–30 minutes recommended).

2. Select wavelength appropriate for the analyte.
3. Zero the instrument with a blank (reagent without sample).
4. Measure standard solution to check calibration.
5. Insert test solution and record absorbance.
6. Run control samples (normal & abnormal).
7. Plot standard curve if applicable (for unknowns).
8. Calculate concentration using Beer-Lambert’s Law or standard curve.

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 VI. Applications in BMLT Labs

Biochemistry:

• Glucose: GOD-POD method at 505 nm

• Urea: DAM method at 540 nm
• Creatinine: Jaffe reaction at 520–530 nm
• Cholesterol: CHOD-PAP method at 505 nm
• Total Protein: Biuret method at 540 nm
• Bilirubin: Diazo reaction at 546 nm

Hematology:

• Hemoglobin (Cyanmethemoglobin method) at 540 nm

Microbiology:

• Optical Density (OD) to monitor bacterial growth at 600 nm

Urinalysis:

• Colorimetric estimation of protein, sugar, ketones

VII. Wavelength Accuracy and Calibration

Test Wavelength (nm)

Hemoglobin (cyanmethemoglobin) 540
Glucose (GOD-POD) 505
Creatinine (Jaffe) 520–530
Urea (DAM) 540
Total Protein (Biuret) 540
Cholesterol (CHOD-PAP) 505

Calibration Tips:

• Use didymium or holmium oxide filters for wavelength calibration.

• Run daily quality control (QC) samples to ensure accuracy.

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 VIII. Common Errors and Troubleshooting

Problem Cause Solution

Fluctuating Air bubbles in cuvette, unstable power Remove bubbles, stabilize
absorbance supply power
Clean with alcohol, handle
High baseline Dirty cuvette or fingerprints
properly
Recheck settings, use fresh
Incorrect readings Wrong wavelength, expired reagents
reagents
Light source failure Lamp burned out Replace lamp
No zero reading Blank not inserted properly Reinsert and recalibrate

IX. Interpretation and Data Analysis

Standard Curve Method

• Plot absorbance (Y-axis) vs. concentration (X-axis) of standards.

• Use the straight-line equation (y = mx + c) to calculate unknown concentration.

X. Advantages

• Rapid and reliable

• Non-destructive
• Highly sensitive (detects microgram levels)
• Applicable to both colored and colorless substances (in UV)

XI. Limitations

• Sample must be clear (no turbidity or precipitate)

• Reagents must be color-stable
• Calibration is required for accuracy
• Not useful if analyte doesn't absorb light in UV-visible range

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 XII. Advanced Variants of Spectrophotometry

Type Description Application

Splits light into sample &
Double Beam More accurate
reference paths
Na+, K+, Ca++
Flame Photometry Measures emission, not absorption
detection
Atomic Absorption Measures absorption by free Heavy metals,
Spectrophotometry (AAS) atoms minerals
Infrared radiation, measures
IR Spectrophotometry Organic chemistry
functional groups

Why Do We Use a Spectrophotometer Instead of a

Colorimeter?
Though both instruments are used for measuring light absorption by colored solutions, a
spectrophotometer is a more advanced and accurate version compared to a colorimeter.

Comparison Table: Spectrophotometer vs. Colorimeter

Feature Spectrophotometer Colorimeter

Wide range (UV + Visible: ~190– Limited to visible range (~400–700
Wavelength Range
1100 nm) nm)
Wavelength
Monochromator (highly precise) Uses colored filters
Selection
Accuracy High (up to 0.001 absorbance units) Moderate (less sensitive)
Can detect very small concentration Less precise, especially at low
Precision
differences concentrations
Deuterium + Tungsten lamps (UV +
Light Source Only tungsten lamp (Vis)
Vis)
Measurement
Both %T and Absorbance (A) Usually only Absorbance or %T
Mode
Cost More expensive Cheaper
Research, Clinical labs, Molecular
Applications Basic clinical testing, fieldwork
analysis
More sophisticated (can auto-
Calibration Manual or basic calibration
calibrate)

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 Why Spectrophotometer is Preferred Over Colorimeter: Key Points

1. Broader Wavelength Capability

• Spectrophotometers can work in UV and visible ranges.

• Useful for colorless analytes (e.g., DNA at 260 nm) — not possible with a colorimeter.

2. Better Accuracy and Sensitivity

• Can detect very small changes in absorbance.

• Crucial for low-concentration samples (e.g., trace enzymes, hormones).

3. More Reliable Wavelength Selection

• Monochromator allows precise wavelength adjustment.

• In contrast, colorimeters rely on filters which have fixed bands and lower resolution.

4. Higher Versatility

• Used in biochemistry, molecular biology, environmental science, microbiology, etc.

• Accepts both solid and liquid samples (in some models).

5. Data Analysis & Automation

• Digital spectrophotometers can store, print, and calculate data automatically.

• Enables standard curve plotting, multi-wavelength analysis, etc.

Limitations of Colorimeter

• Cannot measure colorless substances.

• Less sensitive to small concentration changes.
• Only measures in the visible spectrum.
• Prone to filter degradation or mismatch.

Example Where Spectrophotometer is Essential

• Measuring DNA concentration at 260 nm using UV light.

• A colorimeter cannot detect this because it lacks UV capability.

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 When Colorimeters May Be Used Instead

• For routine, low-cost tests like hemoglobin estimation or sugar testing in small clinics.
• In field testing where portability and simplicity are priorities.

Conclusion

Spectrophotometers are used instead of colorimeters when:

• Higher accuracy and sensitivity are needed

• The analyte absorbs in the UV range
• More precise wavelength control is required
• You're working with low concentrations, advanced biochemical tests, or research
applications

Spectrophotometry MCQs

1. The principle of spectrophotometry is based on:

A) Emission of light
B) Absorption of specific wavelengths of light
C) Reflection of light
D) Refraction of light

Answer: B
Explanation:
Spectrophotometry works on the absorption of light by a substance at specific wavelengths. The
absorbance is measured to determine the concentration of the analyte.

2. Spectrophotometry follows which law?

A) Charles’ Law
B) Beer-Lambert Law
C) Newton’s Law
D) Henry’s Law

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 Answer: B
Explanation:
Beer-Lambert Law states that the absorbance of a solution is directly proportional to the
concentration of the solute and the path length.

3. In the Beer-Lambert law, what does "A = εcl" represent?

A) A = acceleration
B) A = absorption capacity
C) A = absorbance
D) A = atomic number

Answer: C
Explanation:
In this equation:

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

This law underlies spectrophotometric analysis.

4. What is the unit of absorbance?

A) nm
B) AU (Absorbance Unit)
C) mol/L
D) No unit

Answer: D
Explanation:
Absorbance is a dimensionless quantity. It has no unit because it is a logarithmic ratio of
intensities.

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5. Which of the following is used as a light source for UV spectrophotometry?

A) Tungsten lamp
B) Halogen lamp
C) Deuterium lamp
D) Fluorescent lamp

Answer: C
Explanation:
Deuterium lamps emit continuous ultraviolet light and are used in UV region (190–400 nm) of
spectrophotometers.

6. The visible range of the electromagnetic spectrum is:

A) 200–400 nm
B) 400–700 nm
C) 100–300 nm
D) 700–1000 nm

Answer: B
Explanation:
The visible range for humans is 400–700 nm. Spectrophotometers operating in this range
analyze colored solutions.

7. In spectrophotometry, the function of the monochromator is to:

A) Convert light into electricity

B) Split light into different components
C) Select a specific wavelength of light
D) Store data

Answer: C
Explanation:
A monochromator selects a narrow wavelength band of light to ensure precise absorbance
readings.

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8. What kind of cuvette is suitable for UV light measurement?

A) Plastic
B) Glass
C) Quartz
D) Steel

Answer: C
Explanation:
Quartz cuvettes are transparent to UV light and are essential for accurate UV
spectrophotometric analysis.

9. The standard path length used in spectrophotometry cuvettes is:

A) 0.5 cm
B) 1.0 cm
C) 1.5 cm
D) 2.0 cm

Answer: B
Explanation:
1 cm is the standard path length for cuvettes in most spectrophotometric measurements. This is
considered during calculations.

10. Which part of the spectrophotometer converts light signal into an electrical signal?

A) Cuvette
B) Light source
C) Monochromator
D) Photodetector

Answer: D
Explanation:
The photodetector detects the transmitted light and converts it into an electrical signal for data
interpretation.

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11. What is the function of the blank solution in spectrophotometry?

A) Cleans the cuvette

B) Increases absorbance
C) Sets the baseline (zero absorbance)
D) Used as a sample

Answer: C
Explanation:
A blank solution is used to zero the instrument, correcting for any background absorbance
from solvents or reagents.

12. Spectrophotometry is primarily used to:

A) Measure body temperature

B) Detect radioactivity
C) Determine concentration of solutes
D) Count red blood cells

Answer: C
Explanation:
It is widely used in clinical labs to quantify the concentration of biomolecules like glucose,
urea, creatinine, etc.

13. The graph between absorbance and concentration is:

A) Curved
B) Zigzag
C) Linear
D) Parabolic

Answer: C
Explanation:
According to Beer’s Law, the relationship between absorbance and concentration is linear,
making it ideal for calibration curves.

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14. Which of the following materials is NOT suitable for UV spectrophotometry cuvettes?

A) Quartz
B) Silica
C) Plastic
D) None of the above

Answer: C
Explanation:
Plastic cuvettes absorb UV light and are therefore unsuitable for UV measurements. Quartz
and silica are ideal.

15. What is the wavelength of maximum absorption called in spectrophotometry?

A) Lambda min
B) Lambda zero
C) Lambda max
D) Lambda absorb

Answer: C
Explanation:
Lambda max (λmax) is the wavelength at which a substance absorbs the most light — used
to improve sensitivity and accuracy.

16. Which of the following is NOT a part of a basic spectrophotometer?

A) Light source
B) Monochromator
C) Cuvette
D) Incubator

Answer: D
Explanation:
An incubator is unrelated to spectrophotometry. The core components are: light source,
monochromator, cuvette, detector, and display.

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17. The role of a filter or monochromator is to:

A) Amplify the signal

B) Adjust temperature
C) Select a specific wavelength
D) Block all radiation

Answer: C
Explanation:
The monochromator or optical filter isolates a specific wavelength suitable for the analyte,
improving measurement accuracy.

18. The optical density (OD) measured by a spectrophotometer is equivalent to:

A) Concentration
B) Reflectance
C) Absorbance
D) pH

Answer: C
Explanation:
Optical Density (OD) is another term for absorbance, which indicates how much light is
absorbed by a sample.

19. Which of the following is a common application of spectrophotometry in clinical labs?

A) Measuring hemoglobin concentration

B) Counting RBCs
C) Estimating DNA length
D) Culturing bacteria

Answer: A
Explanation:
Spectrophotometry is routinely used to measure hemoglobin, bilirubin, glucose, urea, and
many more analytes in blood and urine.

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20. If a sample absorbs more light, what will be the value of transmittance?

A) High
B) Low
C) Same
D) Zero

Answer: B
Explanation:
If more light is absorbed by the sample, then less light is transmitted, resulting in a low
transmittance value.

21. What is transmittance (T) in spectrophotometry?

A) Ratio of absorbed light to total light

B) Ratio of transmitted light to incident light
C) Difference between incident and reflected light
D) None of the above

Answer: B
Explanation:
Transmittance = (I / I₀), where I is the intensity of transmitted light, and I₀ is the intensity of
incident light.

22. Spectrophotometry differs from colorimetry primarily because:

A) It uses only colored solutions

B) It allows narrow band wavelength selection
C) It is based on reflection
D) It measures conductivity

Answer: B
Explanation:
Unlike colorimeters, spectrophotometers use monochromators to select precise, narrow
wavelengths, increasing accuracy and allowing UV measurements.

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23. The best cuvette cleaning method is:

A) Rinse with water and wipe dry

B) Use detergent and cotton
C) Rinse with distilled water and air dry
D) Wash with hot water only

Answer: C
Explanation:
To avoid scratches and residues that affect readings, cuvettes should be rinsed with distilled
water and allowed to air dry.

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