Spectrophotometery Flashcards
Spectrophotometry
-determining the concentration of an analyte in solution from the intensity of it’s color
-measuring the radiant transmitted when monochromatic light is directed through a solution with the analyte
-transmittance is dependent on concentration
-measuring in blood and fluids
-when light passes through cuvette it can be reflected, absorbed and transmitted
-clinical chemistry depends on radiant light including the emitted light
how does a spectrophotometer work
- measures the amount of light that is absorbed or transmitted by the sample - quantitative measure of the concentration of matter in the sample – i.e. amount of bacteria r
Spectrometer:
-COLLIMATOR- a lens that transmits a straight beam of light (photons).
-The light passes through a MONOCHROMATOR (PRISM) splitting it into many wavelengths.
-the SLIT selects and transmits the wavelength you want measured & this wavelength of light is shined onto a sample
Photometer: detects number of photons or intensity of light transmitted or absorbed AFTER the wavelength of light passes through the sample in cuvette and converts into an electrical signal on a display
the more concentrated a solution (darker) is the higher the absorbance of light and lower the transmittance
what is the light source in the spec
-incandescent and hot (400-700nm) usually tungsten or quartz halogen in visible range
-light transmittance needs to be 100%
-if in the UV range (200-360nm) - hydrogen, deuterium
-the laser givens focused non divergent beam of light
Monochromator
splits into the individual wavelengths
-reduces light stray
-makes light rays parallel
-uses lenses and mirrors to collimate, redirect, and focus light . There are many in automated systems to get the proper wavelength for each test
- disperse light through grating and prisms
-wave lengths are isolated by angle of diffraction (grating) or angle of refraction
Sample Cell or Cuvette
glass: for visible portion of spectrum
silica (quartz): for UV range
plastic: for both visible and UV ranges with 1-cm light paths that must be kept constant
Au480 has semi permanent cuvettes with weekly maintenance - with weekly photocal measurements
purpose of blank - sets the baseline for 100% transmittance which will help measure the increase in absorbance
Photodetector
-converts radiant light to electrical signal with phototubes and photomultiplier tubes. They have rapid response time due to sensitivity
Readout device
measurements in %T or absorbance
Principles of Measurement
-must measure only substance of interest
-initially set at 100%T with reference blank which is the same as the sample but without the solute to measure
-decrease in %T is the increase in absorbance which is related to concentration
Transmittance
-measuring the light transmitted by solution in light path as %T
-the greater the concentration the lower the transmittance as the light is absorbed by the solution
Absorbance
-amount of light absorbed by the solution in the light path
-absorbance is not directly measured however there is an inverse and logarithmic relationship between the A and T where A = 2-log%T ****
100%T = 0% Abs
0%T = 100% (∞)Abs
as %T increases, A decreases
Wavelength selection
determine absorption spectrum of substance of interest
-Ability of substance to absorb light dependent on its chemical nature
-Each substance absorbs characteristic levels of radiant energy or wavelengths and transmits other wavelengths
choose wavelength at peak of maximum absorbance
the ideal wavelength for the measurement is a wavelength that produces the max difference in absorbance readings with a small change in concentration of the compound being measured
Beer-lamberts’ law
Amount of light transmitted depends on:
Nature of substance
Wavelength
Amount of substance in light path
At a given wavelength T= 10^–abc
T = Transmittance
a= constant dependent on chemical nature of light absorbing substance
b= dept of the solution which light passes (light path = 1cm)
c= concentration of the light absorbing substance
Beer’s Law
A = abc
-amount of light absorbing depends on concentration and depth of solution through which light passes.
A= Absorbance
a = molar absorptivity - fraction of wavelength usually a constant
b= length of light path through solution-1 cm)
c = concentration of absorbing molecules [M]
The graph is linear and can be used to determine concentration of unknown
Standardization
-standards act as reference points (known concentration) and establish a relationship between absorbance (signal) and concentration
Application in automation
Hexokinase is the enzyme in the reagent that catalyzes the reaction of glucose
-measuring NADP to NADPH. The change in absorbance at 340nm is proportional to the amount of glucose present in the patient sample
