During a semiannual performance check of a photometer used for NADH methods at 340 nm, certified absorbance standards of 0.500, 1.000, 1.500 and 2.000 read 0.486, 0.946, 1.338 and 1.609. The blank reads 0.000 in a new cuvette. A holmium oxide filter shows its peaks within 0.5 nm of their certified wavelengths. A sharp cut-off filter that transmits no light below about 400 nm reads 1.5% transmittance at the 340 nm setting. The most likely cause of the linearity failure is:

Stray light reaching the detector

An error in wavelength calibration

A dirty or scratched cuvette window

Fading of the absorbance standards

Stray light reaching the detector of a spectrophotometer mainly causes:

Falsely low absorbance at high concentrations

Falsely high absorbance at low concentrations

A raised absorbance reading for the blank

A shift in the wavelength of peak absorbance

Basic equipment, spectrophotometry and photometry

8 ASCP MLS practice questions on Basic equipment, spectrophotometry and photometry, part of Laboratory Operations. The first 8 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

Question 1 · Hard
During a semiannual performance check of a photometer used for NADH methods at 340 nm, certified absorbance standards of 0.500, 1.000, 1.500 and 2.000 read 0.486, 0.946, 1.338 and 1.609. The blank reads 0.000 in a new cuvette. A holmium oxide filter shows its peaks within 0.5 nm of their certified wavelengths. A sharp cut-off filter that transmits no light below about 400 nm reads 1.5% transmittance at the 340 nm setting. The most likely cause of the linearity failure is:
1. AAn error in wavelength calibration
2. BA dirty or scratched cuvette window
3. CFading of the absorbance standards
4. DStray light reaching the detector
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Answer: D · Stray light reaching the detector
The error grows with absorbance, from about 3% low at 0.500 to 20% low at 2.000, a negative deviation from Beer's law that is typical of stray light. When a sample absorbs most of the intended light, a small amount of unwanted light reaching the detector becomes a large share of the signal, so the measured transmittance is too high and the absorbance too low. The cut-off filter confirms this: it should pass no light at 340 nm, yet the detector sees 1.5% transmittance. The holmium oxide peaks rule out a wavelength error; the optics should be cleaned or serviced, and results limited to the range that passes the linearity check until then.
- A: The holmium oxide peaks are in the right places, so the wavelength scale is correct.
- B: A dirty cuvette adds absorbance, and the new cuvette blanks at zero.
- C: Fading would lower every standard by about the same percentage, not more at high absorbance.
Question 2 · Hard
A 4.0 mmol/L lactate standard has an absorbance of 0.260. A sample from a septic patient, read under the same conditions, transmits 30% of the light. What is the patient's lactate?
1. A4.6 mmol/L
2. B10.8 mmol/L
3. C2.0 mmol/L
4. D8.0 mmol/L
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Answer: D · 8.0 mmol/L
First convert the sample's transmittance to absorbance: A = 2 - log 30 = 2 - 1.477 = 0.523. By Beer's law, concentration of the unknown = (A unknown ÷ A standard) × concentration of the standard = (0.523 ÷ 0.260) × 4.0 = 8.0 mmol/L. %T cannot go into the ratio directly, because it is not proportional to concentration. A lactate this high in sepsis is a sign of poor tissue perfusion.
- A: 4.6 mmol/L uses 0.30, the transmittance, as if it were absorbance.
- B: 10.8 mmol/L uses 0.70, the fraction absorbed, as the absorbance.
- C: 2.0 mmol/L inverts the ratio of the two absorbances.
Question 3 · Hard
In an endpoint iron method, the photometer is zeroed on a reagent blank and a 100 µg/dL [17.9 µmol/L] standard reads 0.400. An icteric patient's test reads 0.560, and a serum blank (the patient's serum without the color reagent) reads 0.080. What is the iron result?
1. A83 µg/dL [14.9 µmol/L]
2. B120 µg/dL [21.5 µmol/L]
3. C140 µg/dL [25.1 µmol/L]
4. D160 µg/dL [28.6 µmol/L]
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Answer: B · 120 µg/dL [21.5 µmol/L]
The serum blank measures the absorbance of the sample's own color, here mostly bilirubin, which does not come from the iron reaction. Corrected absorbance = 0.560 - 0.080 = 0.480. Iron = (0.480 ÷ 0.400) × 100 = 120 µg/dL [21.5 µmol/L]. Serum blanks are used for icteric, hemolyzed or lipemic samples in endpoint methods.
- A: 83 µg/dL inverts the ratio of standard to sample absorbance.
- C: 140 µg/dL leaves out the serum-blank correction.
- D: 160 µg/dL adds the serum blank instead of subtracting it.
Question 4 · Medium
Stray light reaching the detector of a spectrophotometer mainly causes:
1. AFalsely high absorbance at low concentrations
2. BA raised absorbance reading for the blank
3. CFalsely low absorbance at high concentrations
4. DA shift in the wavelength of peak absorbance
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Answer: C · Falsely low absorbance at high concentrations
Stray light is any light reaching the detector that lies outside the selected wavelength band or has not passed through the sample. It adds to the transmitted light, so measured transmittance is too high and absorbance too low. The effect is greatest when little light gets through, at high absorbance, so the standard curve bends toward the concentration axis. It is checked with sharp-cutoff filters that should block all light at the test wavelength.
- A: The error is greatest at high absorbance, where little light passes the sample.
- B: Stray light lowers absorbance readings; it does not raise the blank.
- D: Stray light does not move the absorbance peak; wavelength calibration errors do.
Question 5 · Easy
Which material is used to check the wavelength accuracy of a spectrophotometer?
1. ASharp-cutoff glass filter
2. BHolmium oxide glass filter
3. CPotassium dichromate solution
4. DNeutral-density glass filter
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Answer: B · Holmium oxide glass filter
Holmium oxide glass has narrow absorbance peaks at known wavelengths, for example near 361 and 536 nm. Scanning it and comparing the peak positions with the certified values shows whether the monochromator is set correctly. Didymium glass is an older alternative with broader peaks. Wavelength checks are part of routine instrument function verification and are repeated after service.
- A: Sharp-cutoff filters are used to detect stray light.
- C: Potassium dichromate solutions check absorbance accuracy, not wavelength.
- D: Neutral-density filters check absorbance (photometric) accuracy and linearity.
Question 6 · Medium
A solution transmits 25% of the incident light. What is its absorbance?
1. A1.398
2. B0.750
3. C0.602
4. D0.250
Answer and explanation in LearnFree account
Question 7 · Hard
After a service visit, the calibration slope of a 540 nm method falls by 15%; the curve is still linear and the blank reads zero. A holmium oxide filter now shows its 536 nm peak at 541 nm. The most likely cause is:
1. ADirty or scratched cuvettes
2. BCalibrator deterioration
3. CA wavelength calibration error
4. DStray light in the optics
Answer and explanation in LearnFree account
Question 8 · Medium
If a method follows Beer's law, which change doubles the absorbance of a colored solution?
1. ADoubling the intensity of the incident light
2. BHalving the concentration of the analyte
3. CDoubling the percent transmittance
4. DDoubling the light path length through the cuvette
Answer and explanation in LearnFree account

Basic equipment, spectrophotometry and photometry

A 4.0 mmol/L lactate standard has an absorbance of 0.260. A sample from a septic patient, read under the same conditions, transmits 30% of the light. What is the patient's lactate?

In an endpoint iron method, the photometer is zeroed on a reagent blank and a 100 µg/dL [17.9 µmol/L] standard reads 0.400. An icteric patient's test reads 0.560, and a serum blank (the patient's serum without the color reagent) reads 0.080. What is the iron result?

Stray light reaching the detector of a spectrophotometer mainly causes:

Which material is used to check the wavelength accuracy of a spectrophotometer?

A solution transmits 25% of the incident light. What is its absorbance?

After a service visit, the calibration slope of a 540 nm method falls by 15%; the curve is still linear and the blank reads zero. A holmium oxide filter now shows its 536 nm peak at 541 nm. The most likely cause is:

If a method follows Beer's law, which change doubles the absorbance of a colored solution?