Spectrophotometry Flashcards
What is electromagnetic radiation?
- Electromagnetic radiation comprises radiant energy extending from cosmic rays (λ 10-9 nm) to radio waves (λ km).
- ‘Visible Light’ – describes radiant energy in the visible portion of spectrum where our eyes work.
- All forms of electromagnetic radiation travel at the speed of light.
speed of light in a vacuum (c): 299,792,458 metres per second.
186,000 miles per second.
What is the equation of wavelegnth?
Wavelength is inversely proportional to a given frequency:
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
What is energy proportional to?
Energy is proportional to frequency
- 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])
What is energy inversely proprotional to?
Therefore, it stands that wavelength (λ) is inversely proportional to E
E = hc / λ
For example: UV radiation at 200 nm possesses greater energy than IR radiation at 750 nm.
What happens when light interacts with matter?
- Be absorbed
- Be transmitted
- Be scattered
- Be reflected
What happens when absorbed light interacts with electrons?
- Electrons in the “ground state” promoted to higher energy orbitals
- Energy absorbed corresponds to difference between orbitals
- Energy is then lost either vibrationally (non-radiative) or radiatively e.g. fluorescence
Why are organic molecules useful in spectrophotemetry?
- Organic molecules often have conjugated double bond systems, so easy for electrons to move around (delocalise).
- Conjugated double bond systems have fairly low energies for absorption.
- Since colour occurs with absorption, many organic conjugatedcompounds are coloured
Why are some chemical coloured?
- lThe wavelength of light absorbed will depend on the chemical structure of the compound in solution.
- Since “white” light is a spectrum, only certain parts of the spectrum will be absorbed. The remainder produces the colour observed.
What is absorbance and how is it calculated?
- 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
- Relationship between concentration and transmittance is non-linear:
- From the transmittance, one can calculate the absorbance (A)
- Convert to absorbance which is the logarithm of the reciprocal
A = log10 1/T
A = 2 – log10 %T
What is the Beer-Lambert Law?
- 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.
What is the equation for Beer-Lambert Law?
A = εcl
Where:
- ε = molar absorptivity (molar extinction coefficient)
- c = concentration (mol/L)
- l = path length (cm).
How can molar absoptivity be used to calculate concentration?
- 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
Where:
ε = molar absorptivity (molar extinction coefficient)
c = concentration (mol/L)
- So that concentration can be calculated from a absorbance reading if the molar absorptivity is known.
Why is absorbance an ideal analysis?
- Measurement is quick and easy
- (relatively) cheap
- Readily integrated into automation
- Reproducible
What is Spectrophotometry?
- The quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.
- 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 are the main types light sources of spectroscopy?
Need to produce light at wavelength where absorbance is measured
Tungsten
- Covers the visible spectral range reasonably well
- Tends to have higher intensity in the red region of the spectrum
- Cheap
Deuterium
- Deuterium: isotopic hydrogen (abundance ~ 1 in 6000 H atoms)
- Deuterium arc light produces mainly UV light (so invisible to the eye)
- Expensive
- Relatively short lifetime
What is needed for optimal analytical performance in Spectrophotometry?
- 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.
How does spectrophotometry achieve optimal analytical performance?
Spectrophotometers use a prism or diffraction grating to isolate a portion of spectrum of white light from the bulb.
What is Collimator and Monochromator?
Collimator
- A lens to produce a near parallel beam of light
- Monochromators only work if beam is collimated
Monochromator
- Coloured filters
- Prism
- Diffraction grating
What is the role of a Prism?
- Light separated by refraction (bending by passing through transparent medium)
- Target wavelength selected by rotating the prism
What is Diffraction Grating?
- Light separated by diffraction (bending of light at the edge of an opaque surface)
What is Holmium Oxide?
- 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 properties of Cuvettes?
- Known path length
- Optically inert
- No absorption in the region of interest
- No internal reflection or scatter
- Smooth, plain walls in optical path
What are are types of Cuvette?
- Polystyrene
- Glass
- Quartz
What are the advantages and disadvantages of Polystyrene Cuvettes?
Advantages
- Cheap
- Disposable
Disadvantages
- Strong absorbance in UV region
- Sensitive to some organic solvents
What are the advantages and disadvantages of Glass Cuvettes?
Advantages
- Intermediate cost
- Scratch resistant
- Chemically resistant
Disadvantages
- Doesn’t have full UV transparency
What are the advantages and disadvantages of Quartz Cuvettes?
Advantages
- Transparent to UV and visible wavelengths
- Chemically resistant
Diasadvantages
- Expensive
- Not disposable
What are types of detectors?
- Photomultiplier tube (very high sensitivity)
- Diode array
What is the use of photomultiplier tube?
(very high sensitivity)
- Exponentially amplify light signal
- Cascade system of “dynodes”
- Convert light to electron beams then to electric current
- Relatively expensive
What is a Diode array’s use?
- Slightly different way to conventional spectrophotometers
- The array contains several light detectors
- Several wavelengths of light can be measured simultaneously
What is Blanking and how is it done?
All components of the cuvette and contents can affect absorbance. Only the absorbance due to the substance being measured is required. Instrument must be “blanked”
How?
- Measure the absorbance before adding the analyte
- Measure absorbance prior to addition of reagents (if producing a coloured product)
- Use a dual beam spectrophotometer
What is aDual-beam spectrophotometer?
- The incident beam is split by a mirror
- Allows correction for drift and power variations in the light source
What are different forms of spectrophotometry?
- Reflectance
- Scatter
- Fluorescence
- Luminometry
What is Reflectance Spectrphotometry?
- Reflectance spectrophotometry is a technique for non-invasive analysis which illuminates the surface to be analyzed with a light of a known spectrum and recording the spectral response of the surfaceof the diffuse light
- 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).
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 analytical sensitivity.
What are features of nephelometry?
- Nephelometric assays are more sensitive (1 - 10mg/L)
- Nephelometry needs more specialist equipment
- Measuring light scattered at 90°
What is Fluorescence spectrophometry?
- Occurs when a molecule absorbs light at one wavelength and re-emits light at a longer wavelength.
- Emitted light is always lost at a longer wavelength (lower energy) than the excitation light
- Stokes shift: difference between λmax of excitation light and emitted fluorescence light.
Where is fluorescence used in the clinical lab?
- lmmunoassay detection systems: e.g. DELFIA, etc.
- In some polarisation methods (FPIA: e.g. for TDMs)
- FISH
- PCR
What are the features of using luminescence?
- This is the generation of light from a chemical reaction (chemiluminescence)
- Extremely sensitive
Siemens Centaur: acridinium ester.
Siemens Immulite platform: adamantyl dioxetane phosphate
What are applications of luminescence?
- Enzymatic reactions (e.g. glucose) – see enzymology lecture.
- Chemical reactions (e.g. creatinine)
- Dye binding (e.g. magnesium, calcium)
- Transition metal complexes (e.g. phosphate)
What are examples of luminescence molecules?
- Glucose – hexokinase coupled with G6PDH with reduction of NADP
- Creatinine – alkaline picrate (Jaffe)
- Magnesium – xylidyl blue
- Calcium – CPC, arsenazo
- Phosphate – Molybdenum.
