MIDTERM LEC 1.q

Checks if the instrument measures light accurately (wavelength and absorbance)

Makes sure the readings are linear and detects stray light

Wavelength accuracy means the correct light color passes through the device

Quality Assurance for Spectrophotometers Uses special filters to check: (2)

Neutral density filters and dichromate solution test if absorbance is linear

Uses filters or solutions with known absorbance at specific wavelengths

Operator measures and compares results to known values

Best for absorbance check

Absorbance Check Absorbance _ _ cause inaccurate results and bending of the linear plot (deviation from Beer’s law)

It states that the concentration of the unknown substance is directly proportional to the absorbed light (absorbance or optical density) and inversely proportional to the amount of transmitted light (% Transmittance).

It mathematically establishes the relationship between concentration and absorbance.

It is the amount of light absorbed

It is proportional to the inverse log of transmittance.

It is mathematically derived from % T.

Absorbance (A) formula

Absorbance (A) where: A = _ a = _ absorptivity; absorptivity of the compound under standard conditions b = _ of light through the solution c = _ of absorbing molecules/solution

It is the ratio of the radiant energy transmitted (T) divided by the radiant energy incident on the sample (I).

The %T measured by commercial spectrophotometers is the ratio of the sample transmitted beam divided by the blank transmitted beam (sample beam signal/blank beam signal × 100).

In actual practice, the light transmitted by a blank is substituted for _

Blanking Technique Contains reagents only; corrects for reagent color absorbance

Blanking Technique Contains sample without reaction product; corrects for sample interference (ex. hemoglobin)

Blanking Technique Contains serum only; used to measure background absorbance.

process may not be effective in some cases of turbidity, and ultracentrifugation may be necessary to clear the serum or plasma of chylomicrons.

Blanking Technique interfere mainly by increasing light scatter (turbidity).