University of Nebraska Medical Center

Spectrophotometry
Basic Principles of • Basic Principle
Spectrophotometry – Monochromatic light passed through
absorbing solution of fixed depth (cuvette)
Part II – Transmitted light directed to photosensitive
device
– Radiant energy converted to electrical energy
Ricki Otten, MT(ASCP)SC – Instrument adjusted using blank containing all
components of unknown solution except the
substance being measured
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Spectrophotometry Spectrophotometry - Instrument

• Basic Principle • Lamp
– Blank is used to set instrument at: • Monochromator: prism vs diffraction grading
100.0% T

-or-

0.000 absorbance

– Unknown response is measured to determine

concentration of unknown compound
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Spectrophotometry - Instrument Spectral Bandwidth/Bandpass

• Exit Slit • Monochromator – light obtained not truly one
wavelength, a range of wavelengths
• Sample cell or cuvette
• Spectral Bandwidth (Bandpass)
• Photodetector: PMT most often used – Range of wavelengths transmitted to cuvette
– Selected
S l t d wavelength
l th = 450 nm; bbandpass
d = 10 nm
Range of wavelengths through cuvette = 445-455 nm

• Readout device 5 6

CLS 414 Clinical Chemistry

Student Laboratory
Spectrophotometry Principles Part II 1
 Spectral Bandwidth: Spectral Bandwidth:
Affects Linearity and Resolution Affects Linearity
• Narrow bandpass is desirable to increase • Narrow bandpass: increases linearity
– Sensitivity, linearity, resolution

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Spectral Bandwidth:
Spectrophotometry – Sources of Error
Affects Resolution
• Narrow bandpass: increases resolution
and sensitivity
Total error in any given procedure is the
sum of all errors in the entire procedure

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Spectrophotometry – Sources of Error

Spectrophotometry – Sources of Error
• Test sample • Temperature variation; temp not optimal
– Lipemia: false increase absorbance
• pH variation
• Standards & Standardization
– Hemolysis
• False ↑ absorbance at specific wavelengths
– Highest
g purity
y of standards and water
• Interfere with chemical rxn: false ↑ or ↓ – Precise measurement
• Released intracellular components = ↑ in LD, K+, • Preparation of Solutions/Reagents
Mg2+, folate, hemoglobin
– Precise measurement
– Chemically pure water; highest grade
– Icterus – amber to orange due to bilirubin
pigment may interfere at specific wavelengths – Mix contents well
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CLS 414 Clinical Chemistry

Student Laboratory
Spectrophotometry Principles Part II 2
 Spectrophotometry – Sources of Error
• Cuvettes
– Clean, dry, not scratched (use kimwipes)
– Remove air bubbles from solution
• Wavelength selection – not optimal or
calibration error
• Presence of stray light - ↓ABS & ↑%T
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Spectrophotometry – Maintenance & QC
• Purpose of PM & QC
– Verify instrument performance
– Maintain optimal instrument performance
– Ensure precision and accuracy of measurement
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Spectrophotometry – Maintenance & QC
• When is PM performed?
– Routine (daily)
– Monthly
– Bi-annuallyy
• After PM performed – run controls (QC) to
verify instrument is working properly
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CLS 414 Clinical Chemistry
Student Laboratory
Spectrophotometry Principles Part II
Spectrophotometry – Sources of Error
• Incorrect blank used – should be similar to
matrix of sample
• Particulates in solution - ↓ %T, centrifuge
sample to clear sample of particulates
• Clerical errors
– Most common laboratory error
– Always recheck work
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Spectrophotometry – Maintenance & QC
• Preventive Maintenance (PM) consists of:
– Checking specific instrument parameters
• Wavelength Accuracy - Stray Light Detection
• Photometric Linearity - Baseline Stability
• Photometric Accuracy - Temperature
Calibration
– Cleaning waste container
– Cleaning surface and interior components
– Changing tubing, light source, etc.
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Wavelength Accuracy
• Verifies the radiant energy emitted from monochromator exit
slit is the same wavelength selected on the selector dial
• Checked whenever a new lamp is used and then
routinely thereafter
• Methods
– Nickel sulfate solution measurements at 460 and 550 nm
– Didymium filter peaks 585 nm
– Colbalt chloride solution peaks 510 nm
– Potassium dichromate solution peaks 350, 375 & 450 nm
– Transmission stds from NIST: didymium (45%T @ 610 nm)
• Correction: realign exciter lamp w/wavelength selector 18
3
 Linearity of Detector Response Photometric Accuracy
(Photometric Linearity)
• Checks for changes in bandpass & amount of
• Verifies a linear relationship exists between
light energy falling on the photocell
radiant energy absorbed by solution and the
instrument readout (ABS vs conc is linear)
• Method – nickel sulfate (510 nm)
• Method
– Varying concentrations of soln known to follow Beer’s • Correction
– ABS vs. Conc is plotted, straight line = linear response – Realign exciter lamp
– Clean exciter lamp or photocell window
• Correction – Correct faulty slit width
– Recheck dilutions – Replace damaged diffraction grating
– Check for stray light (most common), failing photocell,
incorrect slit width 19 20

Stray Light
Baseline Stability
• Checks for stray light striking detector which will
falsely increase %T and falsely decrease ABS • Baseline stability
– Detects excess baseline drift
• Method:
– Observed at extreme ends of spectrum, where – Method:
detector response or source energy at lowest • Observe ABS (or %T) at a chosen wavelength
– Nickel sulfate (400nm & 700nm) • Look for >2% change within 1 minute
– Sodium nitrite solution (<0.1%T @ 355 nm)

– Correction: change failing exciter lamp

• Correction
– Clean or replace exciter lamp
– Find source of spurious light reflection: check mirrors,
prisms, gratings, dust on optical surfaces 21 22

Temperature
• Ensures cuvette temp is accurate & stable
– Especially important w/enzyme assays

• Method:
– Thermometer or thermistor: NIST certified
– Temperature sensitive dye: cresol-red/Tris
buffer soln

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CLS 414 Clinical Chemistry

Student Laboratory
Spectrophotometry Principles Part II 4