After a reagent change, a photometric magnesium method is recalibrated with five calibrators, using point-to-point calibration. Blank-corrected absorbances: 0 mmol/L, 0.000; 0.50 mmol/L, 0.121; 1.00 mmol/L, 0.240; 1.50 mmol/L, 0.418; 2.00 mmol/L, 0.481. The manufacturer states that the method is linear to 2.50 mmol/L, and the zero calibrator reads the same as the reagent blank. With the new curve, the low control (target 0.80 mmol/L) reads 0.80 mmol/L, but the high control (target 1.70 mmol/L) reads 1.47 mmol/L. The most likely cause is:

A faulty 1.50 mmol/L calibrator

Stray light at high absorbances

Loss of linearity above 1.00 mmol/L

Raised absorbance of the reagent blank

Plasma from a 58-year-old man with tumour lysis syndrome is assayed for uric acid. The method is validated only over its linear range, and the patient's absorbance is marked on the calibration curve shown. The correct action is:

Dilute the sample and repeat the assay

Extend the straight line and read it

Re-read the sample in a 2 cm cuvette

Report the result read from the curve

Photometry, Beer's law and standard curves

12 DOH Abu Dhabi (HAAD) lab licensing practice questions on Photometry, Beer's law and standard curves, part of General Laboratory, Quality and Safety. The first 12 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
After a reagent change, a photometric magnesium method is recalibrated with five calibrators, using point-to-point calibration. Blank-corrected absorbances: 0 mmol/L, 0.000; 0.50 mmol/L, 0.121; 1.00 mmol/L, 0.240; 1.50 mmol/L, 0.418; 2.00 mmol/L, 0.481. The manufacturer states that the method is linear to 2.50 mmol/L, and the zero calibrator reads the same as the reagent blank. With the new curve, the low control (target 0.80 mmol/L) reads 0.80 mmol/L, but the high control (target 1.70 mmol/L) reads 1.47 mmol/L. The most likely cause is:
1. AStray light at high absorbances
2. BLoss of linearity above 1.00 mmol/L
3. CA faulty 1.50 mmol/L calibrator
4. DRaised absorbance of the reagent blank
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Answer: C · A faulty 1.50 mmol/L calibrator
Four calibrators lie on a straight line with a slope of about 0.240 per mmol/L, including the top one, but the 1.50 mmol/L calibrator reads 0.418 instead of the expected 0.360, about 16% high. A calibrator reconstituted with too little water, or left open to evaporate, is more concentrated than its label. With point-to-point calibration the analyzer treats 0.418 as 1.50 mmol/L, so samples between 1.00 and 2.00 mmol/L read low: a true 1.70 mmol/L gives about 0.409, which falls on the faulty segment and reads 1.47. The fix is a fresh calibrator and recalibration before patient results are released. Loss of linearity or stray light would bend the top of the curve, but the 2.00 mmol/L calibrator is exactly on the line.
- A: Stray light lowers readings at high absorbance; here a middle calibrator reads high.
- B: The 2.00 mmol/L calibrator lies on the line, so the response is still linear.
- D: The zero calibrator matches the blank, and a blank error would shift every point equally.
Question 2 · Medium
Plasma from a 58-year-old man with tumour lysis syndrome is assayed for uric acid. The method is validated only over its linear range, and the patient's absorbance is marked on the calibration curve shown. The correct action is:
Figure: MedExams
1. AExtend the straight line and read it
2. BRe-read the sample in a 2 cm cuvette
3. CReport the result read from the curve
4. DDilute the sample and repeat the assay
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Answer: D · Dilute the sample and repeat the assay
The calibrators lie on a straight line up to 1,200 µmol/L, but above that the curve bends toward the concentration axis, so Beer's law no longer holds. The patient's absorbance of 0.72 falls on the flat part, where a small change in absorbance stands for a large change in concentration and the result cannot be read reliably. The sample is diluted with the diluent the method specifies so that it reads within the linear range, and the result is multiplied by the dilution factor. Extending the straight line assumes a linearity that the upper calibrators show does not exist.
- A: Extending the line assumes Beer's law holds where the calibrators show it does not.
- B: A longer light path raises the absorbance further into the nonlinear range.
- C: The curved part is outside the validated linear range, so results read there are unreliable.
Question 3 · Easy
The absorbance spectra of NAD+ and NADH at the same concentration are shown. At which wavelength does NADH absorb strongly while NAD+ hardly absorbs at all?
Figure: MedExams
1. A340 nm
2. B405 nm
3. C280 nm
4. D260 nm
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Answer: A · 340 nm
Both coenzymes absorb strongly near 260 nm because of their adenine ring. Only the reduced nicotinamide ring of NADH absorbs in the band centred at 340 nm, where NAD+ absorbs almost nothing. Many enzyme assays, such as LD and coupled ALT and AST methods, therefore follow the rise or fall of absorbance at 340 nm as NADH is formed or used. The molar absorptivity of NADH at 340 nm is 6.22 × 10^3 L/mol/cm.
- B: At 405 nm NADH absorbs very little; that wavelength suits p-nitrophenol.
- C: Both coenzymes still absorb at 280 nm, the wavelength used for protein.
- D: At 260 nm both forms absorb, so NADH cannot be told apart from NAD+.
Question 4 · Hard
A DNA extract is diluted 1:50 and reads A260 0.250 in a 1 cm cuvette. If an A260 of 1.0 equals 50 µg/mL of double-stranded DNA, the concentration of the undiluted extract is:
1. A0.25 µg/mL
2. B500 µg/mL
3. C625 µg/mL
4. D12.5 µg/mL
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Answer: C · 625 µg/mL
The diluted sample contains 0.250 x 50 µg/mL = 12.5 µg/mL of DNA. Multiplying by the dilution factor, 50, gives 625 µg/mL in the extract. Using the RNA factor of 40 µg/mL per A260 unit instead gives 500 µg/mL. Purity is checked with the A260/A280 ratio, which is about 1.8 for clean DNA; a lower ratio suggests protein or phenol contamination.
- A: 0.25 µg/mL divides by the dilution factor instead of multiplying by it.
- B: 500 µg/mL uses the RNA factor of 40 µg/mL per absorbance unit.
- D: 12.5 µg/mL is the concentration in the diluted sample, before the dilution factor.
Question 5 · Medium
NADH has a molar absorptivity of 6.22 x 10^3 L/mol/cm at 340 nm. What absorbance does a 0.10 mmol/L NADH solution give in a 1.0 cm cuvette?
1. A0.0622
2. B0.622
3. C6.22
4. D622
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Answer: B · 0.622
Beer's law gives A = a x b x c, with c in mol/L when a is in L/mol/cm. 0.10 mmol/L is 1.0 x 10^-4 mol/L, so A = 6,220 x 1.0 x 0.00010 = 0.622. Using the concentration in mmol/L without converting gives 622, an impossible absorbance. This absorptivity is the constant that enzyme methods use to turn a change in absorbance at 340 nm into micromoles of NADH.
- A: 0.0622 uses 0.010 mmol/L, a tenfold slip in the other direction.
- C: 6.22 uses 1.0 mmol/L instead of 0.10 mmol/L, a tenfold slip.
- D: 622 leaves the concentration in mmol/L instead of converting it to mol/L.
Question 6 · Medium
A 40 µmol/L solution of a dye has an absorbance of 0.240 in a 0.5 cm cuvette. What is the dye's molar absorptivity?
1. A3,000 L/mol/cm
2. B12,000 L/mol/cm
3. C6,000 L/mol/cm
4. D12 L/mol/cm
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Question 7 · Hard
In the reference cyanmethemoglobin method, blood is diluted 1:251 and reads 0.300 at 540 nm in a 1.000 cm cuvette. The millimolar absorptivity of HiCN is 11.0 L/mmol/cm per heme, and the monomer molar mass is 16,114 g/mol. What is the hemoglobin concentration?
1. A110 g/L (11.0 g/dL)
2. B27.6 g/L (2.76 g/dL)
3. C441 g/L (44.1 g/dL)
4. D0.44 g/L (0.044 g/dL)
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Question 8 · Hard
A calibration line for a photometric method is A = 0.050C + 0.020, with C in mg/L. A sample diluted 1:5 reads 0.270. What result should be reported?
1. A25 mg/L
2. B29 mg/L
3. C27 mg/L
4. D5.0 mg/L
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Question 9 · Medium
A colorimetric method is read at the wavelength where its colored product absorbs most strongly. The main reason is that at this wavelength:
1. AStray light from the monochromator is fully removed
2. BBeer's law holds, whereas it fails at every other wavelength
3. CSensitivity is highest and small wavelength errors matter least
4. DThe reagent blank always has an absorbance of zero
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Question 10 · Medium
A calibrator is described as commutable with patient samples. This means that it:
1. AHas an analyte level near a decision limit
2. BHas a value traceable to a reference method
3. CBehaves like patient samples in the measurement method
4. DStays stable for a set time after reconstitution
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Question 11 · Hard
A photometer is zeroed on water instead of on a reagent blank. The readings are: reagent blank 0.050, 5.0 mmol/L standard 0.350, patient 0.200. What is the patient's concentration?
1. A2.9 mmol/L
2. B3.3 mmol/L
3. C2.5 mmol/L
4. D2.1 mmol/L
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Question 12 · Easy
A solution with an absorbance of 1.0 transmits what percentage of the incident light?
1. A50%
2. B10%
3. C90%
4. D1%
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Photometry, Beer's law and standard curves

Plasma from a 58-year-old man with tumour lysis syndrome is assayed for uric acid. The method is validated only over its linear range, and the patient's absorbance is marked on the calibration curve shown. The correct action is:

The absorbance spectra of NAD+ and NADH at the same concentration are shown. At which wavelength does NADH absorb strongly while NAD+ hardly absorbs at all?

A DNA extract is diluted 1:50 and reads A260 0.250 in a 1 cm cuvette. If an A260 of 1.0 equals 50 µg/mL of double-stranded DNA, the concentration of the undiluted extract is:

NADH has a molar absorptivity of 6.22 x 10^3 L/mol/cm at 340 nm. What absorbance does a 0.10 mmol/L NADH solution give in a 1.0 cm cuvette?

A 40 µmol/L solution of a dye has an absorbance of 0.240 in a 0.5 cm cuvette. What is the dye's molar absorptivity?

In the reference cyanmethemoglobin method, blood is diluted 1:251 and reads 0.300 at 540 nm in a 1.000 cm cuvette. The millimolar absorptivity of HiCN is 11.0 L/mmol/cm per heme, and the monomer molar mass is 16,114 g/mol. What is the hemoglobin concentration?

A calibration line for a photometric method is A = 0.050C + 0.020, with C in mg/L. A sample diluted 1:5 reads 0.270. What result should be reported?

A colorimetric method is read at the wavelength where its colored product absorbs most strongly. The main reason is that at this wavelength:

A calibrator is described as commutable with patient samples. This means that it:

A photometer is zeroed on water instead of on a reagent blank. The readings are: reagent blank 0.050, 5.0 mmol/L standard 0.350, patient 0.200. What is the patient's concentration?

A solution with an absorbance of 1.0 transmits what percentage of the incident light?