4. Spectrophotometry
REFLECTANCE SPECTROPHOTOMETRY
February 2026
WHAT IS SPECTROPHOTOMETRY?
Definition:
A quantitative analytical technique measuring how much light a chemical substance absorbs or transmits at specific wavelengths.
Applicable usually in the ultraviolet, visible, or infrared spectrum.
Determines the concentration of substances by analyzing their interaction with light.
Key Measurements:
Measurement of intensity of light at selected wavelengths.
Beer-Lambert Law:
States that the absorbance (A) of light by a substance dissolved in a solvent is directly proportional to its concentration (c) and the path length (l) that the light travels through the sample.
Expressed as:
A=€lc
A = absorbance
€= molar absorptivity (a constant)
l= path length
c = concentration
SPECTROPHOTOMETRY TERMS
Absorbance (A):
A unitless measure of light absorbed by a sample.
Molar Absorptivity (€/epsilon):
A constant indicating how strongly a substance absorbs light at a specific wavelength.
Path Length (l):
The distance the light travels through the sample, typically 1 cm.
Concentration (c):
The amount of absorbing solute in the solution, measured in mol/L.
Limitations:
Best used for dilute solutions; high concentrations can cause deviations due to electrostatic interactions between molecules.
Monochromatic light is used.
Solutions must be homogeneous.
PARTS OF A SPECTROPHOTOMETER
A spectrophotometer consists of several components:
Light Source:
Provides stable broadband radiation.
Common sources include:
Tungsten lamps for visible light (320 to 1000 nm)
Deuterium lamps for ultraviolet light (190 to 320 nm)
The light source must deliver steady and constant light.
Monochromator:
Isolates a narrow band of wavelengths from the light source.
Typically includes a prism or diffraction grating to disperse the light along with entrance/exit slits to select the desired wavelength.
Extracts interfering wavelengths before they reach the solution.
Collimator:
A lens or mirror that converts diverging light from the source into a parallel (collimated) beam.
Cuvette:
A transparent container that holds the liquid sample.
Made of glass or plastic (for visible range) or quartz/silica (for UV range).
Detector:
A device (e.g., photodiode or photomultiplier tube) that converts transmitted light into an electrical signal, measuring its intensity.
Display/Readout System:
Processes the electrical signal from the detector to display data as absorbance or transmittance.
ABSORBANCE AND TRANSMITTANCE OF LIGHT
Interaction of Solutions with Light:
Solutions contain particles that absorb certain wavelengths and transmit others.
Characteristic colors to the human eye:
Example:
A blue solution appears blue because it absorbs all wavelengths except for blue, which is transmitted.
A red solution appears red due to the absorption of all other wavelengths except for red.
Wavelength:
Defined as the physical distance between two identical points on consecutive waves, which can be measured from crest to crest or trough to trough.
Colorimetric Reaction:
The measurement on a spectrophotometer is based on the reaction between the substance to be measured (e.g., total protein, TP) and a reagent to produce color.
The intensity of color is proportional to the concentration.
Wavelength Ranges:
Violet/blue light has shorter wavelengths (400-500 nm).
Red light has longer wavelengths (around 700 nm).
ABSORBANCE/TRANSMITTANCE
Measurement and Readings:
Absorbance is displayed on an electronic readout as A (to three decimal places).
Instruments can also express data as %T, which stands for percent transmittance.
Absorbance is directly proportional to the concentration of the solution.
Data is plotted on a linear graph, resulting in a straight line (called the line of regression).
%T is the amount of light that passes through the colored solution compared to a blank or standard.
A blank contains all components in the procedure except for the unknown substance, and the machine must be blanked before use.
As the concentration of the colored solution increases, the amount of light absorbed increases, while the percentage of light transmitted decreases.
Transmitted light is not proportional to concentration or color intensity, meaning it won't result in a straight line on a graph.
VISIBLE SPECTRUM
Wavelength (nm) :
Represented color ranges are as follows:
Violet: 380 - 450 nm
Blue: 450 - 485 nm
Cyan: 485 - 500 nm
Green: 500 – 565 nm
Yellow: 565 - 590 nm
Orange: 590 - 625 nm
Red: 625 - 750 nm
Frequency and Photon Energy:
Supplied frequencies in THz and photon energy in eV correlate with the respective wavelengths.
ABSORBANCE SPECTROPHOTOMETRY
Key Measurement:
Measuring the amount of light a sample absorbs at specific wavelengths is crucial.
The interaction of electromagnetic radiation with the sample identifies molecular structures and quantifies concentration (e.g., proteins).
Basis:
Based on Beer-Lambert's Law.
Principle:
Molecules in a sample absorb photons, leading to transitions between energy levels (electronic or vibrational).
Instrumentation:
A spectrometer passes light through the sample (in cuvette) and measures the remaining transmitted light.
Applications:
Commonly used to quantify substances, monitor chemical reactions, and determine molecular structures.
Can be applied to spectral regions including UV, visible, and infrared light.
REFLECTANCE SPECTROPHOTOMETRY
Principle:
Measures the intensity of light reflected from a sample across various wavelengths.
Analyzes spectral signatures based on the surfaces that absorb or scatter light.
Employs quantitative spectrophotometric techniques.
Measurement Method:
Light reflected from the surface of a colorimetric reaction is used to assess the amount of unknown colored product generated.
A beam of light is directed at a flat surface, and the amount of reflected light is recorded.
Detector:
A photodetector measures reflected light directed to it.
Measurement Ratio:
Light ratio reflected by the sample is compared to a reference standard.
Types of Reflection:
Specular Reflection:
Occurs on smooth surfaces where the angle of reflection equals the angle of incidence.
Diffuse Reflection:
Occurs on rough surfaces, causing light to scatter in multiple directions.
REFLECTANCE SPECTROPHOTOMETRY APPLICATIONS
Field Applications:
Evaluates tissue for diseases (measuring hemoglobin or O2).
Measures paints/colors and identifies minerals.
Assesses agricultural quality and food freshness.
Commonly employed in dermatology, gastrointestinal, and surgical fields.
Instrumentation Used:
Requires a light source, fiber optic probe, and a spectrometer.
Output:
Reflectance spectrum indicates the percentage of light reflected at specific wavelengths; dips in spectrum indicate absorption.
Standards Compliance:
Does not adhere to standard linear Beer-Lambert law which limits its approach.
Point-Of-Care Testing (POCT) Applications**:
Glucose monitors, vital therapeutic drug monitoring systems like Vitros, and urinalysis instruments using dry reagents.
DIFFERENCES: ABSORBANCE VS REFLECTANCE SPECTROPHOTOMETRY
Feature | Absorbance Spectrophotometry | Reflectance Spectrophotometry |
|---|---|---|
Light Path | Transmitted through the sample | Reflected/Scattered off the sample |
Typical Samples | Liquids, solutions, gases, thin films | Solids, powders, rough surfaces, coatings |
Information Obtained | Chemical concentration (Beer-Lambert Law) | Surface chemistry, color, material identity |
Sensitivity | High for low-concentration solutions | High for surface-level characterization |
Common Setup | Cuvette, spectrophotometer | Integrating sphere, reflectance probe |
NEPHELOMETRY AND TURBIDIMETRY
Fundamental Concepts:
Light can be absorbed, reflected, scattered, or transmitted.
Nephelometry:
Measurement of scattered light.
Utilizes a nephelometer.
Detectors positioned at 90° to the light source for better sensitivity in dilute solutions.
Applied in quantifying specific proteins like IgG, IgM, IgA, and complement components C3 and C4 in body fluids.
Instrumentation:
A nephelometer contains a light source (laser or tungsten), a collimator, a cuvette, and a photoelectric detector.
Procedure involves a reaction between the assay protein and specific nephelometric antisera forming insoluble complexes that scatter light detectable by photodiode.
Turbidimetry:
Measures the loss of intensity of transmitted light due to scattering.
Detects turbidity of solutions; useful for counting cells, bacterial growth, and protein concentration assays.
Employs a light source and a detector at 180° (or 0°) for direct measure.
Instrumentation for Turbidimetry:
Uses a tungsten lamp as the light source with detection at 180°.
Differences:
Nephelometry is more sensitive, measuring scattered light (90°), while turbidimetry measures transmitted light (180°).
ADVANTAGES AND DISADVANTAGES OF NEPHELOMETRY
Advantages:
Rapid, reproducible, and simple to operate.
Common in high-volume laboratories.
Many applications in immunology (e.g., CRP, RF).
Disadvantages:
High initial costs involved.
Susceptible to interfering substances (e.g., microbial contamination).
Specimen turbidity/lipemia may exceed preset limits, potentially requiring clear agents or ultracentrifugation.
PREPARATION OF STANDARD CURVE
Definition:
A graph plotting absorption (A) or %T on the y-axis against increasing concentrations of the standard on the x-axis.
Construction:
If Beer-Lambert’ law is adhered to, the resulting line should be straight.
Derived from %T/A readings of known concentration solutions (standards) plotted graphically.
Procedure:
Prepare 5-6 standards along the expected concentration range.
Ensure maximum absorbance occurs for the desired wavelength, assuring high sensitivity.
Calibrate/blank the spectrophotometer using only the solvent, excluding unknowns.
Measure absorbance of each standard from lowest to highest concentration, recording data in duplicates or triplicates for precision.
CREATING AND USING THE STANDARD CURVE
Graph Plotting:
Create a scatter plot and add a trendline (linear regression line), ensuring the majority of points fall close to this line.
Validate R² (coefficient of determination) should ideally be 0.95
Analysis of Unknowns:
Ensure unknowns fall within the range of standards.
Requirement for New Curves:
New curves are necessary when using new reagent lots, new methodologies, or after major instrument maintenance (e.g., bulb changes).
STANDARD CURVE CONSIDERATIONS
Clinical Practice Update:
Current standard curve preparation is automated and not conducted manually.
Quality Control Check:
Must run QC to ensure accuracy.
Common Error Sources:
Bottom of y-axis does not start at 0.
Compressed axes not labeled correctly.
Incorrect regression line placement; consider small point marks for data representation.
Graphing Advice:
Always draw a straight line amidst the majority of points.
QUALITY CONTROL IN SPECTROPHOTOMETRY
Purpose:
QC ensures accuracy and reliability of diagnostic tests through:
Calibration and performance verification.
Calibration Frequency:
Instruments require regular calibration; established SOPs dictate daily checks and monthly intervals for wavelength accuracy and linearity.
Use of Certified Reference Materials:
Standard materials like potassium dichromate for UV range accuracy or holmium oxide checks.
Internal QC:
Executed daily; if results exceed +/- 2SD, troubleshooting is essential.
Instrument Features:
High-quality instruments automatically compensate for lamp fluctuations.
Lab Preparation:
Ensure environmental controls such as humidity and temperature to prevent detector drifts.
Regular compliance with regulatory guidelines (e.g., Accreditation Canada, IQMH) for records and validations.
CALIBRATION PROCEDURE
Steps:
Warm up the spectrophotometer.
Select the desired wavelength.
Determine if a dedicated blank is required.
Zero the spectrophotometer to display all zeros.
Use dedicated filters, e.g., Holmium oxide for wavelength accuracy, and record readings.
If data aligns with expected values (certificate of calibration), proceed; otherwise, troubleshoot.
Calibrate with potassium dichromate to validate absorbance accuracy and linearity.
DETERMINING PATIENT RESULTS
Preparation Steps:
Prepare standards with known increasing analyte concentrations.
Set up QC samples with known expected values.
Prepare samples using known solvent.
If patient results exceed the highest standard, dilute samples.
Zero the spectrophotometer with the blank.
Measure absorbance for each standard, QC, and unknown patient samples at desired wavelength, from lowest to highest concentration.
Plot the graph to establish a line of regression (best fit) and generate an equation.
Use the calibration curve to estimate the concentration (c) for measured absorbance.
UNKNOWN RESULTS CALCULATION
Example Calculation:
Total Protein concentration is expressed as g/L (g/dL).
Formula:
Total protein (g/L) or (g/dL)=0.375×800.440
Result:
Total protein = 68 g/L (or 6.8 g/dL)
Where 0.375 = absorbance of the unknown, 0.440 = absorbance of the calibrator, and 80 g/L = concentration of the calibrator.
CHROMATOGRAPHY
Definition:
Mixtures of solutes dissolved in a solvent are separated based on differential distribution between stationary and mobile phases.
Purpose:
Used to separate, identify, and purify individual components within a mixture.
The solvent (mobile phase) carries solute mixture through the stationary phase.
Concentrations of solutes plotted versus time or distance result in peaks indicating various analytes' presence.
Types of Chromatography:
Broadly classified by the phase type in the carrier (stationary vs mobile).
Gas Chromatography (GC): Solute phase in gaseous state.
Liquid Chromatography (LC): Solute phase in solution state.
Further sub-categorized into flat and column methods for LC, and primarily column methods for GC.
PRINCIPLE OF CHROMATOGRAPHY
Separation Process:
Components with higher affinity for stationary phase move slower; those more attracted to mobile phase move faster, leading to separation.
Categories:
Liquid Chromatography (non-volatile substances) separates ions/molecules in solution.
Gas Chromatography analyzes volatile components in gas phase.
Components of a Chromatographic System:
Stationary Phase: Fixed substance (e.g., paper, silica, resin).
Mobile Phase: Solvent or gas moving through the stationary phase, carrying the sample.
Eluent: The solvent used in the separation process.
Eluate: Mixture (solvent + separated solutes) exiting the system.
GAS CHROMATOGRAPHY
System Components:
Gas flow regulator, carrier gas (inert gases like He, N$2$, H$2$), injector port, column, detector, waste, oven, and computer/data analysis interface.
LIQUID CHROMATOGRAPHY
System Components:
Mobile phase is a liquid solvent, sample introduced via delivery pump, flows through a column (stationary phase), and separated components detected and converted to electrical signals.
DIFFERENCES BETWEEN GC AND LC
Mobile Phase:
GC utilizes inert gases (e.g., He, N$_2$) while LC uses liquids (e.g., water, organic solvents).
Sample Suitability:
GC is ideal for volatile and thermally stable compounds.
LC analyzes non-volatile substances, particularly high molecular weight compounds.
Temperature Requirements:
GC operates at elevated temperatures (>100 °C) to vaporize samples, while LC operates at lower temperatures with high pressure (5000-18000 psi) to move liquids through columns.
APPLICATIONS OF GAS AND LIQUID CHROMATOGRAPHY
Gas Chromatography (GC) Applications:
Toxicology and blood alcohol testing, detecting volatile poisons, and identifying drugs of abuse.
Vital for newborn screening for diseases through dried blood spots.
Liquid Chromatography (LC) Applications:
Used for therapeutic drug monitoring, assessing Vitamin D levels, and analysis of steroid hormones.
Assessing metabolic disorders using small blood samples in newborns.
MASS SPECTROMETRY (MS)
Overview:
Incorporates mass spectrometry with either GC or LC to identify and weigh chemicals.
Combination Benefits:
Chromatography determines retention time while mass spectrometry gives exact mass-to-charge ratios.
Essential for accuracy in medical labs.
GC-MS Applications:
Ideal for small stable molecules; can scan 300,000+ compounds to identify toxins or doping substances.
LC-MS Applications:
Suitable for larger, more fragile molecules that GC could damage; retains full molecular structure for analysis.
Useful for screening multiple metabolic diseases with a single blood drop, distinguishing between Vitamin D forms for precise clinical evaluations.
ELECTROCHEMISTRY
Definition:
A branch of chemistry focused on the relationship between electrical potential difference and chemical change, particularly the movement of electrons in oxidation-reduction (redox) reactions.
Key Components:
Redox Reactions: Involves electron transfer, where one substance is oxidized (loses electrons) and another is reduced (gains electrons).
Electrochemical Cell: Generates electricity from spontaneous redox reactions or uses electricity to drive non-spontaneous reactions.
Components include anode (oxidation site), cathode (reduction site), and electrolyte (ion-conducting medium).
Clinical Application Example:
Glucose testing in handheld glucose meters using glucose oxidase to oxidize glucose, generating an electrical signal proportional to concentration.
POTENTIOMETRY
Definition:
An electroanalytical technique measuring the electrical potential voltage of an electrochemical cell under negligible current flow to determine solute concentration.
Instrumentation:
Comprises an indicator electrode sensitive to analyte concentration and a reference electrode (e.g., silver-silver chloride).
High impedance potentiometer/voltmeter setup.
Principle:
Utilizes the Nernst equation linking potential to analyte concentration/activity.
Potentiometric Titration:
Measures potential as a function of titrant volume instead of using color indicators; effective in colorful or turbid solutions.
Applications:
Commonly used for detecting ions such as Na⁺, K⁺, and Cl⁻.
Capable of detecting low concentrations of specific ions without consuming the analyte.