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Photometry and atomic absorption

15 min

  • Trace a photometric fault to the lamp, wavelength selector, cuvette, or detector
  • Predict the effect of stray light on a high-absorbance measurement
  • Calculate a concentration from absorbance with the fitted standard curve
  • Match an atomic-absorption interference to its mechanism

Read the full reference

Try first

Try first

A method reads reduced nicotinamide adenine dinucleotide (NADH) at 340 nm. The laboratory switches to disposable cuvettes from a new supplier, and the blank and every specimen now pass much less light to the detector. The lamp energy check with an empty light path is within limits. Which part of the signal path explains the change?

The next section explains it.

The next section explains it.

Right. The next section explains why.

The next section explains it.

Get the idea

Follow the light

A photometer sends light from a source through a wavelength selector and a sample cell to a detector. A signal processor turns the detector's response into a result.1,2 Each symptom points to the part whose job it affects.

PartIts jobFailure clue
SourceSupplies light across the working bandLow or unstable energy
Wavelength selectorIsolates the intended bandWrong peak position, excess bandwidth, stray light
Sample cellHolds the specimen at a set path lengthScratches, bubbles, unequal path length
DetectorConverts light into an electrical signalNoise, saturation, loss of response
Signal processorApplies blanking and calibrationWrong factor, unit or curve fit

Ordinary glass and many plastics absorb ultraviolet light. A 340 nm reading of NADH needs a cell of quartz, fused silica or another material that transmits ultraviolet light.1

Stray light pulls high absorbance down

Stray light is off-band light that reaches the detector from scatter, dust, scratched optics or higher grating orders. At high absorbance little light passes the sample, so the added light is a large share of what the detector receives. Transmittance reads too high and absorbance too low. With 1% stray light, a true absorbance of 2.00 reads about 1.70.1,2 A cutoff filter or a strongly absorbing solution that should pass no light at the test wavelength detects stray light. Holmium oxide or didymium glass checks wavelength accuracy.1

Read the concentration off the fitted line

A validated straight line with an intercept gives the concentration as:

C = (A − intercept) ÷ slope

The single-point ratio, (specimen absorbance ÷ calibrator absorbance) × calibrator concentration, assumes a straight line through the origin. On a line with an intercept, it biases results most at concentrations far from the calibrator.1,3 Read results only inside the validated interval.3

Match the atomic absorption correction to the cause

Atomic absorption measures how much of the lamp's element line free ground-state atoms remove in a flame or graphite furnace.1

  • Chemical. Phosphate binds calcium in a compound that resists atomization. A releasing agent such as lanthanum frees the calcium.
  • Ionization. A hot flame ionizes the atoms. An ionization buffer or a cooler flame corrects it.
  • Background. The matrix absorbs and scatters light at the line. Background correction removes that signal.
  • Physical. Viscosity or dissolved solids change nebulization. Matrix matching, dilution or standard addition corrects it.1,2
References
  1. Bishop ML, Fody EP, Van Siclen C, Mistler JM, Moy M. Clinical Chemistry: Principles, Techniques, and Correlations. 9th ed. Jones & Bartlett Learning; 2023.
  2. Rifai N, Chiu RWK, Young I, Burnham CAD, Wittwer CT, eds. Tietz Textbook of Laboratory Medicine. 7th ed. Elsevier; 2023.
  3. Clinical and Laboratory Standards Institute. Evaluation of Linearity of Quantitative Measurement Procedures. 2nd ed. CLSI guideline EP06. Clinical and Laboratory Standards Institute; 2020. Accessed September 26, 2026. https://clsi.org/shop/standards/ep06/

Watch one

A colorimetric method's validated calibration is a straight line fitted to calibrators from 25 to 400 mg/dL:

A = (0.00250 dL/mg) × C + 0.050

The 200 mg/dL calibrator reads 0.550, and a specimen reads 0.300. What concentration does the line give for the specimen?

  1. Note the model: slope 0.00250 dL/mg, intercept 0.050. The calibrator fits it, since 0.00250 × 200 + 0.050 = 0.550.

    The validated model sets the calculation, and this line has an intercept.

  2. Subtract the intercept: 0.300 − 0.050 = 0.250.

    The intercept is signal present at zero concentration, so it comes off before dividing.

  3. Divide by the slope: 0.250 ÷ 0.00250 dL/mg = 100 mg/dL.

    The slope is absorbance per unit of concentration, so dividing by it returns the concentration.

  4. Check the shortcut: (0.300 ÷ 0.550) × 200 mg/dL = 109 mg/dL, 9% higher than the line's result.

    Comparing with the ratio shows the size of the bias the shortcut would add.

  5. Check the interval: 100 mg/dL lies between 25 and 400 mg/dL.

    A result is read directly only inside the validated interval.

The specimen's concentration is 100 mg/dL.

Your turn

Problem 1 of 3

Stray light reaches the detector while a sample with high absorbance is read. How does it change the reported absorbance?

Correct. Unwanted light adds to the small transmitted signal, so apparent transmittance is too high and calculated absorbance too low. The error grows as absorbance rises.

Incorrect. Extra light at the detector raises apparent transmittance, and A = −log₁₀(T) then falls. Scatter from turbidity or bubbles is the cause that raises apparent absorbance.

Incorrect. The effect is largest when little light passes the sample, because the unwanted light is then a large share of the detected signal. With 1% stray light, a true absorbance of 2.00 reads about 1.70.

Hint
  1. Stray light adds to the light the detector receives.
  2. At high absorbance, how much light passes the sample to begin with?

Review Departures from linear response

Problem 2 of 3

A flame atomic absorption method for serum calcium reads low on specimens with high phosphate. Background absorption at the calcium line is within limits. Which correction fits?

Phosphate holds calcium in a refractory compound that does not break into free atoms, so less calcium absorbs the lamp's line. Lanthanum binds the phosphate and frees the calcium.

Background correction removes matrix absorption and scatter at the line. The calcium stays bound to phosphate, and the result stays low.

Used a background correction for an atomization failure

Phosphate binds calcium in a refractory compound that does not break down into free atoms, so the result reads low. Background correction removes matrix absorption and scatter at the analytical line and leaves the calcium bound. A releasing agent such as lanthanum, or a hotter atomization condition, corrects this chemical interference.

An ionization buffer protects atoms that a hot flame ionizes. The calcium here never becomes free atoms, because phosphate holds it.

Hint
  1. Background correction, an ionization buffer and a releasing agent each act on a different problem.
  2. Ask whether the calcium is still bound to something when it reaches the flame.

Review Atomic absorption and elemental analysis

Problem 3 of 3

A method's validated calibration is a straight line fitted to calibrators from 20 to 300 mg/dL: A = (0.00300 dL/mg) × C + 0.060. The 200 mg/dL calibrator reads 0.660. A specimen reads 0.420. What concentration does the line give for the specimen?

Show the answer

120 mg/dL

C = (0.420 − 0.060) ÷ 0.00300 dL/mg = 0.360 ÷ 0.00300 dL/mg = 120 mg/dL. The calibrator ratio, (0.420 ÷ 0.660) × 200 mg/dL, gives 127 mg/dL because it ignores the intercept.

Review From transmitted light to concentration

Use it

  • The chemistry analyzer's scheduled photometer checks run at 340 nm before the day shift.
  • Lamp energy is within the manufacturer's limits.
  • The holmium oxide wavelength check passes.
  • Linearity standards read, as expected absorbance to measured absorbance: 0.500 to 0.495, 1.000 to 0.981, 1.500 to 1.438, 2.000 to 1.826.
  • Both control levels are within limits.
  • Specimens waiting for testing include several with high values.
Decision 1 of 3

Which part of the signal path do you look at first?

Lamp energy is within limits. A failing lamp shows as low or unstable energy.

The low standards read close to expected, and the error grows with absorbance. Stray light and excess bandwidth both bend high readings down, and both belong to the wavelength selector and its optics. Its peak position already passed.

Unequal path length gives a proportional bias at every level. Here the lowest standard reads almost true and only the high ones fall short.

A wrong factor scales every result by the same proportion. These linearity standards are read as absorbance, and their error grows with absorbance.

Review Signal path

Decision 2 of 3

A cutoff solution that should pass no light at 340 nm shows light reaching the detector. How does this fault change the result for a specimen that reads at high absorbance?

The effect is largest at high absorbance, where little light passes the sample and the stray light is a large share of what the detector receives.

Stray light adds light at the detector. Apparent transmittance rises, and absorbance, calculated from transmittance, falls.

Predicted that stray light raises measured absorbance

Unwanted light adds to the small transmitted signal, so apparent transmittance is too high and calculated absorbance too low, most of all at high true absorbance. A high-concentration specimen then reads falsely low.

Stray light raises apparent transmittance, so absorbance and the concentration calculated from it read low. The 2.000 standard reading 1.826 shows the size of the error at the top of the range.

Review Departures from linear response

Decision 3 of 3

What do you do with the waiting specimens that have high values?

This photometer reads high absorbance low, so high results from it would be reported below their true values. An analyzer that passes its checks gives results that can be released, and this one returns to service after repair and a passing repeat check.

Controls sit at fixed concentrations and may never reach the absorbance where this fault shows. The 1.500 and 2.000 standards read low, and specimens reading there would read low too.

A correction made up at the bench has no validation behind it. The fault is repaired and the photometer checked again before it measures patient specimens.

Review Departures from linear response

The clue that settles this case is the shape of the linearity error:

  • The 0.500 standard reads within 1% of expected.
  • The 2.000 standard reads about 9% low.
  • Lamp energy and wavelength position both pass.

An error that grows with absorbance, with the lamp and the peak position in order, points to stray light in the wavelength selector's optics. The cutoff solution confirms it, and high specimens are tested on a photometer that passes its checks.

Keep

Sources checked