Clinical Biochemistry

1hr exam SAQ 7-8 (75%), ERQ 2 (25%)

Clinical chemistry definition

• The biochemical analysis of body fluids to support the diagnosis and treatment of disease

• Utilizes chemical reactions to identify or quantify levels of chemical compounds in bodily fluids

Instrumentation (Techniques) (20)

• Autoanalysers

• Calibration curves

• Enzymes

• Spectrophotometry and photometry

• Spectrofluorimetry

• Atomic absorption and emission spectrophotometry

• Electrochemistry and ion-selective electrodes

• Electrophoresis and related techniques

• Chromatography and mass spectrometry

• Immunoassays

• Renal function tests

• Electrolyte balance and blood gases

• Liver function, enzymes, and isoenzymes

• Carbohydrate metabolism

• Lipid and lipoprotein metabolism

• Hypothalamus and pituitary axis

• Thyroid function

• Sex hormones

• Adrenal hormones

• Tumour markers

Biochemical Analysis

Characterisation of biological components in a sample using laboratory techniques
Samples:

• Blood

• Serum

• Plasma

• Urine

• Cerebrospinal fluid (CSF)

• Synovial fluid

• Saliva

• Other body fluids or tissues depending on the test

Qualitative vs Quantitative analysis of BA

• Qualitative analysis:

  • Determines whether a biomolecule is present or absent

  • Offers a binary outcome (positive or negative)

  • Example: Testing blood for a particular drug or presence of a biomarker

• Quantitative analysis:

  • Determines the quantity or concentration of a biological molecule in a sample

  • Measures amount or concentration

  • Example: pH, haemoglobin, or glucose concentration in blood

Criteria for Selecting an Analytical Method (11)

• Number of samples to be analysed

• Cost of test and availability of equipment

• Ease and convenience of the method

• Duration of analysis (turnaround time)

• Required level of accuracy and precision

• Expected concentration range of the analyte

• Sensitivity and detection limit of the technique

• Analytical specificity

• Type and physical form of the sample

• Likelihood of interfering substances (e.g. lipaemia, cross-reactivity)

• Operator skills and expertise (=training)

Lipaemia

The presence of a high concentration of lipids (mainly triglycerides) in the blood

Linearity

Ability of a test to provide results directly proportional to analyte concentration in a sample

Limits of Linearity

• Defines a limited range of values between which results are regarded as accurate

• If results exceed the upper limit: dilute sample and retest to obtain accurate value

• If results are below the accepted value: repeat test, report result, inform physician, occasionally repeat using larger sample volumes, or analyse other analytes

• Results outside the linear range require specific corrective actions

• Limit of Detection (LOD)

Limit of Detection (LOD)

• Lowest analyte concentration that can be distinguished with reasonable confidence from blank or background

• Important for comparing analytical procedures, techniques, or instruments

Analytical Specificity

• Ability to measure only the analyte of interest

• Example: Immunoassays rely on Ag-Ab interactions

• Ideally fully specific, but cross-reactivity or interfering substances can occur → obtain detailed patient history to report results confidently

Analytical Sensitivity

• Smallest amount or concentration of an analyte that can be detected

• Related to limit of detection (LOD) for the assay

• Depends on assay generation (1st, 2nd, 3rd, etc.)

Accuracy vs. Precision

• Accuracy:

  • Ability of a test to produce the true value of an analyte in a sample

  • Multiple measurements of the same analyte will distribute around the true value

  • The mean of measurements represents the true value

  • Check: Compare with different methods

  • Poor accuracy → procedural or equipment flaws

• Precision:

  • Ability of a test to reproduce the same result consistently in the same specimen

  • Spread or distribution of results reflects method variability

  • Variability expressed as Standard Deviation (SD)

  • Reproducibility: Check by repeating measurements (trials)

  • Poor precision → poor technique

Accuracy and Precision

• Accurate but Imprecise: Close to true value on average but results are scattered → Random Error

• Precise but not Accurate: Results are consistent but deviate from true value → Systematic Error

• Inaccurate & Imprecise: Scattered and far from true value → poor measurement

• Accurate & Precise: Ideal state, results are consistent and close to true value (difficult to achieve due to multiple variables like lab staff, steps involved)

Improving Accuracy

• Using properly standardized procedures

• Statistically valid comparisons of new methods with established reference methods

• Using samples of known values (controls)

• Participation in proficiency testing (PT) programs

Ensuring Precision

• Proper inclusion of standards

• Using reference samples or control solutions

• Statistically valid replicate determinations of a single sample

• Duplicate determinations of sufficient numbers of unknown samples

• Measuring day-to-day and between-run precision using control samples

Accuracy and Precision photo

Faulty weighing balance

• May give precise (repeatable) but inaccurate (untrue) results

• What can be done:

  • Perform precision checks (repeat measurements)

  • Compare with known standards or reference methods to detect bias

  • Calibrate the instrument regularly to correct systematic errors

Random Error

• Test values scattered around true mean

• Imprecise, usually >2 SD apart

Systematic Error /Bias

• Constant bias from true value

• Results are grouped closely (within 1 SD) but different from true mean

• Measurement error that skews results consistently to one side

• Causes:

  • Incorrectly calibrated instruments

  • Change in reagent/calibrator lot

  • Inadequate storage of reagents/calibrators

  • Pipettor misadjustments or misalignments causing volume changes

  • Temperature changes in incubators or reaction blocks

  • Procedural differences between operators

• To Overcome:

  • Use carefully standardised procedures

  • Measure a single variable in several different ways and compare results

  • Work blind when possible (e.g., use of codes)

Measurement Error

• Examples:

  • Mistakes in taking readings

  • Faulty equipment

  • Equipment accuracy limits

  • Errors in preparing solutions or dilutions

  • Calibration errors

  • Interfering substances

• To Overcome:

  • Take repeated readings

  • Compare readings between instruments

  • Spike and measure recovery: add a known amount of analyte to the sample and compare the response to the same spike in standard diluent

  • Keep records of batch numbers and measurements for solutions

  • Check controls or standards and construct standard/calibration curves

Validation Procedures

• Use Standard Operating Procedures (SOPs)

• Calibrate assays with certified reference materials containing known analyte amounts, traceable to a national reference lab

• Quality Assurance (QA): System to verify the entire analytical process operates within acceptable limits

• Quality Control (QC): Mechanisms to measure non-conforming method performance

• Importance:

  • Essential for all assays

  • Provides confidence in precision and accuracy of tests

  • Enables early detection of poor assay performance

  • Allows proper corrective actions to minimize the risk of patients receiving incorrect result

• Keep Records:

  • Batch numbers

  • Analysis performance

  • Results

  • Reagent and calibrator temperatures

• Ability to quote assay performance at measurement time allows labs to defend results and gives clinicians confidence in patient care

SOP (Standard Operating Procedure)

A documented set of step-by-step instructions that describes how to perform a specific laboratory or clinical procedure consistently and correctly

Purpose:

• Ensures consistency and reliability of results

• Maintains quality and safety

• Helps train staff and reduce errors

• Provides a reference for audits and compliance

Calibration

• Aligning an instrument to provide accurate, precise, and unbiased measurements consistent with other instruments at a precise point

• Purpose:

  • Ensure accuracy and precision

  • Reduce/eliminate bias across a range of values

  • Maintain consistency between instruments

• Important Notes:

  • Poor standard preparation → inaccurate results

  • Never extrapolate beyond the highest standard - assume linearity only within calibrated range

  • Be consistent in significant figures used

How Calibration is Done

• Select reference standards with known values covering the range of interest

• Measure standards using the instrument to be calibrated

• Plot calibration curve/standard curve (response vs known values)

• Use the curve to determine amount/concentration in test samples

• Correct measurements using the inverse of the calibration curve if necessary

Types of Calibration Curve (photo)

Preparation of a Calibration Curve (9)

1. Choose an appropriate test method

2. Select amount/concentration, range, and number of standards covering expected sample levels

3. Prepare standards carefully (consider volumetric accuracy, standing time, temperature, light protection, include blank/zero standard)

4. Assay standards and test samples simultaneously; take replicate readings

5. For some instruments, periodically check zero and highest standard to ensure stability

6. Draw the standard curve or determine underlying relationship

   • Draw line of best fit for linear curves

   • Quote r² to indicate fit quality

7. Determine unknown sample concentrations from the curve or mathematical relationship (e.g., y=mx+c)

8. Correct for dilution/concentration if test samples were diluted

   • Example: 0.2 mL assayed → multiply result by 5 for value per 1 mL

9. Quote results to an appropriate number of significant figures, reflecting method accuracy and consistency

When to Carry Out Calibration? (8)

• Before major or critical measurements

• Before measurements requiring high accuracy

• After major or critical measurements

• When data are unreliable or observations appear questionable

• After incidents that may affect the instrument (e.g., impact, accidents)

• For long-term instrument use, as conditions can change over time

• Per experiment requirements, e.g., when calibration certificates are needed or using a new kit

• As indicated by the manufacturer, following periodic calibration schedules to ensure proper and safe instrument function

Preparing Serial Dilutions

• Linear Dilution Series:

  • Concentrations separated by an equal amount (e.g., 0.0, 0.2, 0.4, 0.6, 0.8, 1.0 µg/mL)

  • Steps:

    1. Prepare a stock solution

    2. Use formula [C1]V1 = [C2]V2 to calculate volumes

    3. Tools like the Tocris dilution calculator can help determine volumes for desired concentrations

       • V1 = volume of stock solution

       • V2 = volume of diluted solution

       • C1 = starting concentration

       • C2 = target diluted concentration

• Logarithmic Serial Dilution:

  • Concentrations separated by a constant proportion rather than equal increments

Clinical Sensitivity

Describes the ability of an assay to detect only patients with a particular disease

Clinical Specificity

Describes the ability of an assay to detect individuals who do not t have a particular disease

Clinical Validation

Examines the probability that an analytically correct result is really possible for that patient

Auto-Validation of Results

• Delta Check: examine any value obtained for an analyte against the previous result

• Range Check: determines whether the result is physiologically possible

• Reference Range: within which 95% of the healthy population fall

Automation

• Steps which were previously performed manually can now be automated

• Scientist can focus on tasks that are not automated

• Increases efficiency and capacity

• Automation has been extended into areas not related to analysis:

  • Processing and transportation of specimens

  • Loading of samples onto analysers

  • Assessing results of the performed tests

Clinical Laboratory Automation

• Integration of robotic transport systems with analytical instruments and pre/post-analytical equipment (e.g., centrifuges, aliquoters, decappers, recappers, sorters, specimen storage/retrieval)

• Computers controlling devices must be interfaced with each other and/or the LIS

• Types of information communicated include:

  • Process control and status of devices/analyzers, specimens, containers, and carriers

  • Patient, order, and result data

  • Specimen flow algorithms and automated decision-making

• Ensures each specimen/aliquot undergoes correct tests in the proper sequence

Why Do We Need Automation?

• Increases the number of tests one person can perform in a given time

• Minimises result variations between operators

• Reduces errors from manual analyses (e.g., equipment variations, pipettes)

• Uses less sample and reagent per test

• Allows scientists to focus on decision-making and result interpretation

• Expands lab services into specialised and technically demanding assays

• Makes performing repeats easier when necessary

• Provides walkaway operations and standardised methodology for accurate and reproducible results

Limitations of Automation

Some basic steps might not be fully automated:

• Specimen preparation and identification

• Labelling (critical)

• Programming of instruments

• Checking Quality Assurance (QA) and Quality Control (QC) – of utmost importance

Analytic Process & Automation

Can be divided into 3 major phases:

• Pre-analytic: Sample processing

  • Steps before the actual chemical analysis

  • Includes:

    • Patient preparation

    • Specimen collection, handling, and transportation

    • Labelling and identification

    • Sample processing (e.g., centrifugation, aliquoting)

  • Critical because errors here can affect all downstream results

• Analytic: Chemical analyses

  • Steps during the actual chemical analysis

  • Includes:

    • Measurement of analytes using chemical or instrumental techniques

    • Use of calibration, standards, and controls

    • Ensuring accuracy, precision, and quality control

  • Most automated phase

• Post-analytic: Data management

  • Steps after the chemical analysis

  • Includes:

    • Data verification and validation

    • Interpretation of results

    • Reporting results to clinicians

    • Archiving and record-keeping

  • Increasingly targeted for automation to improve efficiency and reduce human error

• Research is increasingly focused on automating pre-analytic and post-analytic processes

Auto-validation

Automated process of result validation based on criteria set by scientific and clinical staff

• Post-analytical automation mainly involves it

• Benefits:

  • Improves turnaround time by automatically validating results that meet preset criteria

  • Withholds results that need further attention (e.g., out-of-range, repeats)

  • Introduces a uniform standard of validation across the laboratory

Types of Analysers

• Continuous Flow Analysers

• Centrifugal Analysers

• Discrete Analysers

Continuous Flow Analysers

• Liquids (reagents, diluents, samples) are pumped through continuous tubing

• Samples introduced sequentially, following the same network and reaction path

• Air bubbles at regular intervals separate and clean tubing

• Sample + reagent → chemical reaction → chromagen solution pumped for spectrophotometric analysis

• Advantages:

  • Uniformity in test performance

  • Run many samples requiring the same procedure

  • Multiple tests on each sample

• Disadvantages:

  • Carry-over problems

  • Wasteful use of continuously flowing reagents

Continuous Flow Analysers (photo)

Centrifugal Analysers

• Use centrifugal force to mix sample aliquot with reagent in spinning rotor

• Sample + reagent passes through detector for quantification

• Features:

  • Single-test batch analysers

  • Sequential analysis

  • Discrete (one compartment per assay)

  • Parallel analysis possible

  • Uses the force generated by centrifugation to transfer and then contain liquids in separate cuvettes for measurement at the perimeter of a spinning rotor

• Advantage: Capable of batch analysis (multiple samples, one test at a time)

Centrifugal Analysers (photo)

Discrete Analysers

• Most widely used automation type; most versatile

• Each sample and reagent in separate cuvette/container

• Can run:

  • Multiple tests on one sample (random access; e.g., LFTs, FBG)

  • Multiple samples, one test at a time (batch analysis; e.g., FBG on 20 patients)

• Random Access:

  • Analysis kept separate in reaction chambers (cuvettes, wells, slides)

  • Minimizes carryover, but increases cost per test due to disposables

• Batch Analysis:

  • Accumulating specimens into one run reduces reagent and personnel costs

  • Cost-effective for specialty tests with small order

Modern Discrete Analyser Capabilities

• Routine and special chemistries, including enzymes, substrates, electrolytes, proteins, drugs of abuse, therapeutic drug monitoring

• Capacity: up to 300 tests/hour

• Continuous access to samples, reagents, cuvettes

• On-board storage: 84 routine samples, 6 STAT samples, 39 fixed cooled calibrator/control positions, 45 refrigerated reagent positions, 600 cuvette storage positions

• Low-volume cuvettes reduce reagent consumption and running costs

• Automatic clot detection ensures result integrity

• Integrated touchscreen workstation enables fast and convenient data management

Laboratory Information Systems (LIS)

• Important development in laboratory automation

• Initially used to generate consolidated laboratory result reports

• Evolved to capture test requests and results and manage laboratory workflow

• Has improved the overall quality of laboratory data

Types of Automation

1. Total Laboratory Automation (TLA):

   • First developed in the 1980s in Japan

   • Conveyor belts carry specimens to workstations

   • Automated pipettors aspirate serum from tubes for testing

   • Can include pre-analytical modules: centrifugation, decapping, aliquoting, labelling

   • Includes transport system for tube delivery to analysers, recapping, sorting, and storage of specimens

2. Modular Automation:

   • Integrates different aspects of automation and analysis into one module

   • Task-targeted automation systems for specific functions

   • Flexible: modules can be added or upgraded as lab needs change

   • Can operate independently or linked with other modules or LIS

   • Often includes sample handling, analysis, and reporting within a single module

Selecting the Analyser

Things to Consider:

• Laboratory’s Workload:

  • Discrete vs large batch testing

  • open vs closed system

  • Single instrument or multiple instruments – ease of use & complexity

  • Back-up options vs outsourcing

• Storage of Reagents:

  • Need for refrigeration or freezing

  • Effect of repeat freeze-thaw cycles

• Random access

• Patient/test orientation

• STAT (emergency) facilities

• Temperature control system (water bath vs Peltier effect)

• Maintenance requirements

• Running costs (excluding reagents, plastic ware, etc.)

Quality Assurance (PHOTO!)

Quality Control (QC)

• Purpose:

  • Ensure accuracy and reproducibility of laboratory tests

  • Provide early warning of errors so corrective action can prevent major mistakes

  • Maintain continuous record of test precision

  • Monitor analytic process and evaluate method accuracy

  • Evaluate technologist skills

  • Determine analytical errors during analysis

  • Prevent incorrect patient values

• QC Goals:

  • Accuracy

  • Precision

  • Total error of the chemical method

• Ideal Properties of QC Materials:

  • Resemble human serum, plasma, blood, urine, or cerebrospinal fluid

  • Stable for prolonged periods without interfering preservatives

  • Free from communicable diseases (bacteria, viruses, fungi)

  • Known concentration of analytes

  • Easy to store and dispense

  • Affordable

• QC Objectives:

  • Continuous accuracy of results

  • Early warning to take remedies before major mistakes

  • Compare tests at different times using the same control sera

• QC Tools:

  • Procedure manuals

  • Maintenance schedules

  • Calibrations

  • Quality assurance programs

  • Staff training

• QC Results:

  • Acceptable: Within error limits

  • Unacceptable: Excessive errors, out of range

Types of QC Reagents

• Pooled Sera:

  • Analyte levels usually within normal patient range; not suitable for clinically significant levels

  • Stability not validated like true third-party control

  • Increased infection risk (may not be tested for HIV, Hepatitis)

  • Inconsistent long-term supply

• Company/Third-Party Produced Controls:

  • Extended shelf life and stability

  • Enable long-term QC monitoring

  • Detect shifts when reagents or calibrator lots change

Quality Assurance (QA) vs Quality Control (QC)

Aspect	Quality Assurance (QA)	Quality Control (QC)
Definition	Set of activities to ensure quality in processes by which products/tests are developed	Product-oriented activities to ensure quality in finished products/tests
Focus	Prevent defects by improving processes	Identify and correct defects in products/tests
Goal	Ensure defects do not arise	Detect defects before release
How	Establish, implement, and audit quality management systems	Use tools, equipment, assays, and procedures to find defects
Orientation	Proactive; preventive	Reactive; corrective
Process vs Product	Process	Product
Sampling vs Specifications	Specifications	Sampling
Assays vs Documentation	Documentation	Assays
Organisation vs Authorisation	Organisation	Authorisation
Function Type	Staff function	Line function
Defect Handling	Prevent defects	Find and correct defects

Examples of QC vs QA (photo)

Pre-Analytical Errors Prevention

• Patient identification & labelling: Use barcode technology to reduce errors

• Record keeping: Track sample receipt and reporting

• Request form checks: Confirm test tube, name, and requested tests

• Sample adequacy: Ensure sufficient volume

• Sample quality: Check for haemolysis, lipemia, icterus

• Patient history: Record food intake, alcohol, drugs, smoking, stress, sleep, posture; explain instructions for sample collection

• Containers & preservatives: Use correct types to avoid affecting results

• Transport: Ensure proper handling to protect sample integrity

• Processing: Correct separation (e.g., centrifugation) with proper speed, temperature, and operator technique

Analytical Errors Prevention

• Preventive maintenance: Daily/monthly schedules for instruments

• Equipment checks: Monitor water quality, power supply, calibrate balances, glassware, pipettes

• Reagents/kits: Date upon receipt and opening

• Lot validation: Run new reagent lots in parallel with old ones before use

• Standards:

  • Primary standard: Highly purified substance

  • Secondary standard: Concentration determined by comparison with primary standard

• Post-analytic errors: Avoid errors in recording and reporting results

Quality Management (photo)

Photometry

• The measurement of luminous light (luminous intensity) falling on a surface from a light source

• Photometric instruments measure light intensity without considering wavelength (λ)

Electromagnetic Radiation

• Described as photons of energy traveling in waves

• Frequency is inversely proportional to wavelength (λ)

• Energy is inversely proportional to wavelength (shorter wavelength = higher energy)

• Spectrum ranges from short-wavelength, high-energy gamma and X-rays to long-wavelength radio waves

Absorption and Emission

• Spectrophotometers and photometers measure absorption or emission of radiant energy to determine concentration

• Absorption or emission of energy by atoms produces a line spectrum

• Molecules absorb or emit energy over a broad region (band spectrum)

• Solids produce a continuous spectrum

• Absorbance: amount of light absorbed

• Excited electrons return to ground state by emitting a specific wavelength of radiant energy

Light Interaction with Solution

• When a light beam enters a solution:

  • Some light is absorbed

  • The remaining transmitted light reaches the detector and is converted into an electrical signal

• The amount of light absorbed varies according to the concentration of the substance

Light Interaction with Solution (photo)

% Transmittance (T)

• Defined as the ratio of radiant energy transmitted (It) to radiant energy incident on the sample (I₀):
  T = It / I₀

• If all light is absorbed or blocked → 0% T (e.g. a very concentrated sample)

• It is important to always run a blank to account for background absorbance

• Absorbance (A) = –log(T) = –log(It / I₀)

• The difference in transmitted light between the blank and the sample is due only to the compound being measured

Beer’s Law (Beer-Lambert Law)

• Establishes the relationship between analyte concentration and absorbance

• States that absorbance (A) is directly proportional to concentration (c) and path length (L)

• Mathematically:
  A = ε × L × c

  • ε = molar absorptivity (ability of a molecule to absorb a specific wavelength)

  • L = path length of light through the solution

  • c = concentration of the absorbing molecule

• The amount of light absorbed at a given wavelength depends on:

  • Type of molecules or ions present

  • Concentration

  • pH and temperature

Absorbance and Concentration

• Unknown concentrations are determined using a calibration curve plotting absorbance vs. concentration for known standards

• This allows for accurate quantitative analysis and instrument calibration

Spectrophotometry

• When light passes through an object, part of it is reflected, absorbed, and transmitted

• Purpose: Measures the amount of light transmitted by a solution to determine the concentration of the light-absorbing substance

• Principle: Measures light intensity as a function of wavelength by:

  • Diffracting the light beam into a spectrum of wavelengths

  • Directing it onto a sample

  • Receiving and detecting the transmitted or reflected light

  • Measuring the intensity and displaying it as a graph on the detector/display device

Spectrophotometer Components

• Light Source

• Monochromator

• Sample Cell (Cuvette)

• Photodetector

Light Source

• Provides the initial beam of radiant energy

• Important factors:

  • Range/Bandwidth (spectral distribution)

  • Brightness

  • Stability (bulb condition)

  • Temperature

  • Lifetime

• Common Light Sources

  Source	Region	Notes
  Tungsten Lamp	Visible & IR	- Produces 320–2500 nm range- Often paired with heat-absorbing filter to block IR- Glass construction (400–800 nm)
  Advantages: Cheap, non-toxic, good for small-area lighting
  Disadvantages: Inefficient (90% energy lost as heat), unsuitable for large areas
  Deuterium Lamp	UV	- Continuous emission down to 165 nm- Common for UV work (160–325 nm)- Quartz construction (does not block UV)
  Mercury or Xenon Lamp	UV-Vis	- Mercury provides line spectra; Xenon provides continuous spectra
  Laser	Variable - Xray/ UV/ IR	- High power, narrow bandwidth, coherent, tunable wavelength, very expensive
  Plasma/Furnace Sources	Elemental studies	Used for atomic absorption or emission spectroscopy

Monochromator

• Purpose: To isolate individual wavelengths of light and direct only one onto the sample

• Reason: Each compound absorbs optimally at a specific wavelength (e.g. Urea: 260–280 nm; Creatinine: 280–320 nm)

• Function:

  • Isolation of wavelength depends on the entrance and exit slit width

  • Bandpass (bandwidth): Defines the range of wavelengths transmitted

  • Slit width determines resolution and signal-to-noise ratio

    • Large slit width: More light energy → higher signal-to-noise ratio → lower resolution

    • Small slit width: Less energy → lower signal-to-noise ratio → better resolution

• Wide slit → more energy, lower resolution

• Narrow slit → less energy, higher resolution

• Diffraction gratings = most accurate and commonly used

• Coloured-glass filters = cheapest, least precise

• Interference filters = narrow, efficient, precise

• Prism = adjustable, produces continuous spectrum

Types of Monochromators

• Coloured-Glass Filters

• Interference Filters

• Prism

• Diffraction Gratings

Coloured-Glass Filters - Monochromators

• Simple filters that transmit a wide band of wavelengths and absorb others

• Features:

  • Least expensive and simple to use

  • Pass a broad range of wavelengths with low precision

  • Application: Used in colorimeters for measuring color intensity

Interference Filters - Monochromators

• Based on constructive interference of light waves

• Construction: Two glass plates, each mirrored on one side, separated by a transparent spacer equal to half the target wavelength

• Features:

  • Produces monochromatic light with a narrow bandwidth

  • Provides high efficiency and better wavelength selectivity

Prism - Monochromators

• Light is refracted as it enters and exits the prism, separating white light into its spectral components

• Operation:

  • A narrow light beam enters the prism and disperses into a continuous spectrum

  • Rotation of the prism allows selection of the desired wavelength through the exit slit

• Output: Continuous spectrum with adjustable wavelength selection

Diffraction Gratings  - Monochromators

• Most commonly used monochromator type

• Construction: A polished surface etched with many fine parallel grooves (15,000–30,000 per inch)

• Principle:

  • Light is diffracted (bent) when it passes by sharp edges

  • Constructive interference (in-phase waves) reinforces certain wavelengths → produces clear spectral lines

  • Destructive interference (out-of-phase waves) cancels out others

• Output: Complete and highly resolved spectra

• Note: Accessory filters are often added to reduce stray light caused by multiple diffraction orders

Sample Cell (Cuvette)

• Shape: Can be round or square

• Key Point: The light path must remain constant since absorbance is directly proportional to concentration

• Round cuvettes:

  • Must have an etched mark indicating the correct position for use

• Square cuvettes:

  • Have plane-parallel optical surfaces and a constant light path

  • Frosted sides to ensure correct positioning

  • Advantages:

    • Reduced lens effect and refraction errors

    • Less dependent on orientation

    • Most commonly used type

• Condition:

  • Cuvettes with scratches scatter light and should be discarded

• Material:

  • Glass cuvettes: Cheap and disposable; suitable for visible range but absorb UV light

  • Quartz cuvettes: Reusable and essential for UV applications

Photodetector

• Purpose: Converts transmitted radiant energy into an equivalent electrical signal

• Choice of detector: Depends on the wavelength being studied

• Single-beam spectrophotometer: 100% transmittance control must be adjusted each time the wavelength is changed

• Types of detectors:

  • Potentiometric recorders

  • Photocell

  • Phototube

  • Photomultiplier (PM) tube

  • Amplifiers & Ammeters

• Requirements:

  • High sensitivity to detect low radiant energy

  • Short response time

  • Long-term stability

  • Electrical signal easily amplified for readout

Photomultiplier (PM) Tube - Photodetector

• Purpose: Detects and amplifies radiant energy

• Advantages:

  • 200× more sensitive than other detectors

  • Ideal for extremely low light levels or very short light flashes

• Current signal is proportional to light intensity

• Analog signal is converted to voltage, then to digital via an A/D converter

Single-beam Spectrophotometer

One light path for both reference and sample

• Light passes through collimating lens → entrance slit → filter → sample → shutter → photovoltaic cell → readout

• Requires blank for correction between reference and sample absorbance

• Advantages:

  • Less expensive

  • High energy throughput → high sensitivity

  • Easy to operate

• Disadvantages:

  • Instability from electronic, voltage, or mechanical fluctuations

  • Must be zeroed or calibrated regularly → human error

  • Variations in light intensity → errors in %T

  • Non-monochromatic light → deviation from Beer’s law

  • %T and A not “true” values

  • Not designed for spectral data

Double-beam Spectrophotometer

• Splits light path into reference and working segments

• Automatically corrects for variations in light intensity

• Advantages:

  • High speed, stability, flexibility

  • Reduces errors from single-beam fluctuations

  • More reproducible measurements

  • Typically simpler operation

  • Wavelengths easily selected

• Disadvantages:

  • Changes in wavelength may cause variations

Measurement Options

• Qualitative Analysis

• Quantitative Analysis

• Typical Plate-based Spectrophotometric Assay

• Enzyme Assays

Qualitative Analysis

• Visible and UV spectrophotometers can identify classes of compounds in pure or biological samples

• Done by plotting absorption spectrum curves

• Absorption in different regions gives structural hints

  Absorption Range (nm)	Structure / Type of Compounds
  220–280	Aliphatic or alicyclic hydrocarbons or derivatives
  220–250	Compounds with two unsaturated linkages in conjugation, benzene derivatives
  250–330	Compounds with more than two conjugated double bonds
  450–500	Beta-carotene (11 conjugated double bonds, precursor of Vitamin A)
  250–330	Vitamin K1

Quantitative Analysis

• Substrates: UA, Urea, Creatinine, LFTs, Lipid profile, etc

• DNA

• Protein (measured at 280 nm, depends on tyrosine and tryptophan content)

Typical Plate-based Spectrophotometric Assay

• Often kit-based, specific for particular analyte (e.g., ELISAs, small molecules, lipids)

1. Prepare standards of known concentrations

2. Add samples to wells

3. Add reactants (assay-dependent)

4. Color develops based on analyte concentration

5. Read at appropriate wavelength

6. Create standard curve

7. Calculate sample concentrations using standard curve

Enzyme Assays

• Examples: LDH, GPT, GGT, GOT, CK, ALP

• Substrate or product absorbs light in visible or UV range

• Lactate Dehydrogenase (LDH)

  • Reaction: Lactate + NAD → Pyruvate + NADH + H⁺

  • NADH absorbs at 340 nm; NAD does not

  • Forward reaction monitored by measuring increase in absorbance at 540 nm

Spectrophotometer QC – Maintenance / Routine Checks

• Washing, blanking, water check

• Temperature check

• Alignment of the needle

• Cuvette dispenser

• Bulb lifetime

• Piping (contamination, kinks, solids)

• Waste disposal and container washing

• Washing of pipes

Spectrophotometer QA

Periodic Checks (Monthly or more)

• Wavelength accuracy: Using standard absorbing solutions or filters with known absorbance maxima

• Stray light: Caused by scratches or dust in the light path, λ outside the transmitted band

• Linearity: High concentrations may not be linear; dilution and calibration curve needed

• Filters: Checked and replaced as needed

• Spectrophotometers should be able to automatically dilute samples when necessary

Fluorescence Spectroscopy

Based on the ability of certain molecules (fluorophores) to absorb light at one wavelength (excitation) and emit light at a longer wavelength (emission)

Fluorescence

• Some molecules absorb energy from photons and move to an excited state

• When they return to the ground state, they release a photon as fluorescence emission

Fluorophores

Molecules that can be excited by light of a particular wavelength, absorb photons, and emit light at a different wavelength

• Rigid planar structure

• Highly conjugated system with alternating single and double bonds

• Condensed fused-ring system containing one or more heteroatoms

• Presence of electron-donating groups (e.g., –OH, –NH₂)

• Presence of electron-accepting groups conjugated with the donating groups

Biological Fluorophores

• Endogenous Fluorophores

• Exogenous Fluorophores

Endogenous Fluorophores

• Amino acids

• Structural proteins

• Enzymes and co-enzymes

• Vitamins

• Lipids

• Porphyrins

• Certain metals

Exogenous Fluorophores

• Cyanine dyes

• Drugs, both medicinal and illegal

• Pollutants

• Photosensitizers

• Molecular markers, e.g., GFP

Applications of fluorophores

• Immunofluorescence

• Cytogenetics: FISH

• Flow cytometry

• Fluorescent immunoassay

• DNA sequencing

• Determination of fluorescent drugs in low-dose formulations in the presence of non-fluorescent excipients

• Carrying out limit tests where the impurity is fluorescent

• Studying the binding of drugs to components in complex formulations

• Widely used in bioanalysis for measuring small amounts of drug and studying drug-protein binding

Immunofluorescence - Applications of fluorophores

• Important immunochemical technique that utilizes fluorescence-labelled antibodies to detect specific target antigens

• Used widely in both scientific research and clinical laboratories

• Allows for excellent sensitivity and amplification of signal

• Analysis of samples labelled with fluorescence-labelled antibodies must be performed using a fluorescence microscope or other type of fluorescence imaging

Cytogenetics: FISH - Applications of fluorophores

= Fluorescence in situ hybridization

• Involves unwinding of the double helix structure and binding of fluorescently labeled DNA probes to specific sequences in sample DNA

• The fluorescent probes are nucleic acids labeled with fluorescent groups and can bind to specific DNA or RNA sequences

• This allows visualization of where and when a specific DNA sequence exists in cells

Flow cytometry - Applications of fluorophores

• Operates on the principles of light scattering, excitation, and emission

• Fluorescently tagged cell components are excited when they pass through a laser beam, producing light of different wavelengths

• The fluorescence is used to analyze cellular properties such as size, granularity, and protein expression

Fluorescent immunoassay - Applications of fluorophores

• Utilizes fluorescent molecules as labels to measure antigen concentrations

• Time-resolved immunoassay methods allow overcoming background fluorescence

DNA sequencing - Applications of fluorophores

Fluorescent dyes label nucleotides in sequencing reactions:

• red for thymine

• green for adenine

• blue for cytosine

• yellow for guanine

Fluorescence process

• Stage 1: Excitation

• Stage 2: Excited state lifetime

• Stage 3: Fluorescence emission

Stage 1: Excitation

Stage 2: Excited state lifetime