Clinical Biochem week 2 flashcards

Photometry

• “light measurement”

• any method measuring light intensity

• classified by the manner light is measured

• Compares an unknown [sample] to a known [standard]

absorbance/transmittance

• [SAMPLE] determined by measuring amount of light absorbed/transmitted

• compared to the amt of light absorbed/transmitted by STANDARD solution

Colorimetry

• when the chemical reaction occurs, there is a color change

Emission

• luminescence, chemiluminescence

• sample absorbs energy (light, heat) → emits light

• [SAMPLE] directly proportional to light emission

• applications: luminescence methods

  • sample absorbs energy

Scatter

• particle in solution scatter light

• [SAMPLE] proportional to light scattered

• Applications: turbidimetry; Nephelometry

Reflectance

• light strikes a sample on a reflective surface

• [SAMPLE] inversely proportional to light reflected

  • higher [ ], less light reflected

• applications: reflectance methods

Wavelength

• distance between 2 peaks in nm = 10^-9nm

amplitude

• light wave height → light intensity

Light dispersion (spectrum)

• white light bends and wavelength’s separate

  • different refractive indices

• longer wavelength ROYGBiV — shorter wavelength

• visible wavelength = 380nm - 760nm

Photometers

• measure light intensity absorbed / transmitted by a sample solution

Spectrophotometer

• measures monochromatic light absorbed/transmitted by a solution

• Light source: tungsten, hydrogen/Deuterium

• monochromator: filters, diffraction gratings

• Cuvet: Quartz

Transmittance (%T)

• monochromatic light strikes a solution

• some light absorbed, remainder transmitted through a solution

  • %T = light transmitted

    • light striking sample x 100

• %T v. [ ] is non-linear relationship

Absorbance (A)

• measure of monochromatic light absorbed by sample

  • molecules absorb light at specific wavelengths

  • monochromatic light ensures accuracy

• A is directly proportional to concentration

  • not measured directly by a photometer

• A= 2-log%T

Beer’s Law

• A directly proportional to [ ]

Lambert’s Law

• A = # of light absorbing molecules in lightpath

Beer-Lambert Law

• A= abc

  • A absorbance

  • a = molar absorptivity

  • b = lightpath (cm)

  • c = concentration

Using Beer’s Law for results

• standard curve

  • absorbance vs. concentration

  • construct curve using “best fit” line from >1 STANDARD

  • must go through origin

  • upper linearity to [Highest STANDARD]

• Calculation

  • 1 standard

  • upper linearity must be established

Analytical sampels

• reagent blank

  • corrects light absorbed by reagent from final sample absorbance reading

• standards (calibrator)

• sample

Light source

• routine

  • visible and near UV (340nm) and IR wavelength

    • tungsten, quartz-halogen

• special

  • strict UV

    • hydrogen, deuterium

  • pure monochromatic light

    • lasers

monochromator

• produces monochromatic light (1 color)

• Bandpass (Bandwidth)

  • amount of light directed at sample

  • wavelength range produced

  • 550 nm selected 525-575 nm light produced

    • bandwidth = 50 nm

  • describes light purity

    • narrow = more pure

    • wide = less pure

Monochromator types

• Filters (wider bandwidth)

  • transmit wanted λ and absorb unwanted λs

• Diffraction gratings (narrow)

  • mirror-like material with etched parallel grooves

  • Source light strikes the grating → each groove produces a linear spectrum

  • more grooves → smaller bandwidth

Sample cuvet/cells

• material

  • UV work → quartz glass or special plastic

• pathlength = 1 cm

• cuvet chamber shielded

  • stray light

    • light reaching detector at a λ other than selected

    • all light not passing through sample

    • causes false decrease in absorbance

Photodetector

• detects light, converts to electrical signal and sends to a readout device

Photomultiplier tube (PMT)

detects and amplifies initial signal up to a million times

photodiodes

silicon chips convert light to electrical signal

can measure many λs simultaneously

Reflectance photometry

• Reflectometry

  • monochromatic light directed at a flat surface at a 45° angle

    • reagents impregnated on flat test surface → reacted with sample

    • indirectly proportional

• Detector

  • positioned at 90° angle to test surface

Chromophores

• some light gets absorbed by chromophores on test surface

• unabsorbed light reflected

  • measured by detector as “reflection density”

• Reflection density indirectly proportional to [ ] and non-linear

• Algorithim linearizes (correction factor makes it linear)

Atomic absorption spectrophotometry

• Analytes

  • calcium, magnesium, trace metals

  • sensitive and specific

• Principle

  • Ground state atoms (sample) in the flame absorb specific light λ

    • [ ] directly proportional to absorbance

AAS

• analogous to absorption spectrophotometry (atoms vs. molecules)

• Hollow cathode lamp → analyte-specific light source

• flame: sample holder

Hollow cathode lamp

• specific light λ

• passes through flame containing sample atoms

• ground-state atoms specifically absorb λ

Sample handling

• sample chamber

  • atomizer

    • vaporizes sample, mixes with fuel/oxidant

    • Acetylene/air

• flame

  • reduces atoms to ground state

Flameless AAS graphite furnace

• sample

  • carbon rod well or metal strips

  • heat sample to atomize

• Principle (same as Flame AAS)

  • hollow cathode light source passed through atomized sample

  • ground state atoms absorb specific light λ