Spectrophotometry Flashcards
What is the meaning of wavelength?
All forms of electromagnetic radiation travel at the speed of light.
speed of light in a vacuum (c): 299,792,458metres per second.
186,000 miles per second.
Wavelength is inversely proportional to a given frequency (oscillation of the photon):
v = c / λ
v = frequency of light (cycles per second) c = speed of light in a vacuum (3 x 1010 cm/s) λ = wavelength in cm
The relationship between the energy of photons and their frequency is proportional.
It makes sense that the more times a wave wobbles in a second, then the more energy required to make that happen:
E = hv
E = energy (ergs) h = Plank’s constant (6.62 x 1027 erg s) v = frequency of light (cycles per second [Hz])
How is absorbance calculated?
Must know the intensity and tansmision effect of incidence light.
Transmission light is the proportion of incidence light that has travelled through. Absorbance is calculated from intensity
Incident light with intensity (I0)
Light reaching the detector with intensity (I)
Transmittance (the fraction of incident light reaching the detector):
T = I /I0
%T = I x 100 / I0
From the transmittance, one can calculate the absorbance (A).
Relationship between concentration and transmittance is non-linear:
Convert to absorbance which is the logarithm of the reciprocal of T to give a proportional linear relationship (libear to about 1-2 absorbable units):
A = log10 1/T
What are the absorbance laws?
Light is absorbed when a photon collides with a molecule.
Therefore, it is not surprising that the amount of light absorbed depends on the concentration of the compound in solution.
Beer-Lambert Law: Concentration of a substance is directly proportional to the amount of light absorbed.
A = εcl
Where:
ε = molar absorptivity (molar extinction coefficient)
c = concentration (mol/L)
l = path length (cm) - generally 1 cm
How does the beer lambert law relate concentration and absorbance?
A proportionality constant for any given compound at any given wavelength of light.
ε = A / cl
Since most cuvettes have a path length of 1 cm, the above equation can be simplified to:
ε = A / c
We can then rearrange to calculate concentration:
c = A / ε
So that concentration can be calculated from a absorbance reading if
the molar absorptivity is known.
For example, Measuring concentration of glucose.
Multiply by 1000 to convert units.
Must account for adjustment by dilution too, divide total by initial sample concentration.
What is spectrophotometry?
Spectrophotometry: the quantitative measurement of how much a chemical substance absorbs light by passing a beam of light through the sample using a spectrophotometer.
The light passing through the solution is detected by the photo-detector, generating an electrical current proportional to the intensity of the light, which is then converted into a reading.
What is the light source used in spectrophotometry?
Need to produce light at wavelength where absorbance is measured. Two main types for general spectroscopy:
- Tungsten
- Deuterium
Tungsten:
Covers the visible spectral range reasonably well
Tends to have higher intensity at the red end of the spectrum
Cheap
Deuterium:
isotopic hydrogen (abundance ~ 1 in 6000 H atoms)
Deuterium arc light produces mainly UV light (so invisible to the eye)
Shorter half life but produce intense light n the UV spectrum - 200-300 no (where proteins fall)
Expensive
How is monochromatic light achieved?
For optimal analytical performance:
- incident light beam is parallel and of a constant wavelength
(monochromatic) - Incident light beam is of the wavelength which gives the maximum
absorption (minimum transmission) of the light.
To achieve monochromatic light, spectrophotometers use a prism or diffraction grating to isolate a portion of spectrum of white light from the bulb. Monochromators only work if beam is collimated, these include:
- prisms (Light separated by refraction (bending by passing through transparent medium) - rotate to achieve target wavelength of interest. Target wavelength selected by rotating the prism)
- coloured filters (
- diffraction gratings (Light separated by diffraction (bending of light at the edge of an opaque surface) Target wavelength selected by varying angle of incident light and passing through slits).
How is wavelength calibration performed?
Since monochromators are essentially mechanical they are subject to variation
The wavelength on the instrument should correspond to the wavelength of light being measured
Wavelengths need to be calibrated (albeit rarely - 3 to 6 months, usually by manufacturing engineers)
Holmium oxide calibrators:
Holmium (atomic mass 165) is a rare earth lanthanide element
It forms an oxide Ho2O3
This has a complex absorption spectra with sharp, well defined peaks across the UV/visible range
What are the properties of the cuvette?
Known path length Optically inert No absorption in the region of interest No internal reflection or scatter Smooth, plain walls in optical path
There are three common types:
polystyrene
- cheap and disposable
BUT
- have strong absorbance in UV region and are sensitive to some organic solvents.
Glass:
- intermediate cost, scratch resistance and chemically resistant
BUT
- don’t have full UV transparency
Quartz:
- transparent to UV and visible wavelengths, chemically resistant
BUT
- expensive and non-disposable
What are the properties of the detector?
Photomultiplier tube (very high sensitivity)
- Exponentially amplify light signal
- Cascade system of “dynodes”
- Convert light to electron beams then to electric current
- Relatively expensive, but exponentially increase signal
Transmitted light through sample, and hits photocathode, generally in a photomultiplier dinode set up. Photon generates an electron, cascade of dinodes that exponentially increase signal.
Why is blanking important and how is it achieved?
All components of the cuvette and contents can affect absorbance
We measure a change. Want to measure only the sample and nothing else!
Only the absorbance due to the substance being measured is required
Instrument must be “blanked”
How?
1: measure the absorbance before adding the analyte
2: measure absorbance prior to addition of reagents (if producing a coloured product)
3: use a dual beam spectrophotometer
What is a dual beam spectrophotometer and how does it achieve blanking?
The incident beam is split by a mirror, so it falls on sample and blank reference cell (assesses in real time drift in incidence light).
Allows correction for drift and power variations in the light source
What is reflectance spectrophotometry?
Diffuse light reflected from a surface is measured
A comparison is made with a reference surface.
Used in dry-reagent chemistry systems (e.g. Vitros), and in some POCT applications (e.g. bilirubinometer - measure transcutaneous bilirubin - not as accurate as serum, but non invasive and transportable).
What is immunoturbidimetry?
Antibodies bind to the analyte (usually a protein)
Form aggregates which scatter light
The amount of scatter is proportional to the amount of analyte (antigen)
Sensitivity is of the order of 100mg/L
Attaching antibodies to latex or polystyrene particles can increase aggregation and therefore analytical sensitivity.
What is nephelometry?
Nephelometric assays are more sensitive (1 - 10mg/L), proportion of scattered light is more than the amount of transmitted light.
Nephelometry needs more specialist equipment
Measuring light scattered at 90°
