Spec Flashcards
What is the principle of spec?
To measure the intensity of light energy absorbed by an analyte solution at a selected wavelength.
What is the visible spectrum?
390 - 770nm
What is the UV spectrum?
~100 - 400nm
What is spec in terms of analytical?
it is a quantitative and qualitative analytical tool.
What are the 2 basic concepts of spec?
- That light absorption is exponentially related to the no. of molecules in the absorbing solute (conc.
- Light absorption is exponentially related to the the length of the light path through the absorbing solution.
What is the intensity of the transmitted light defined as?
T = I/Io
What are some of the errors of the incident light of the spec?
- Some of the incident light may be reflected by the surface of the cell or absorbed by the cell or solvent.
Elimination = use of a reference cell identical to the sample cell except compound of interest is blank
What is Abs defined by?
A = -log IS / IR = -log T
What are the 5 components of a spec?
- light source
- monochromator (spectral isolation
- cuvette
- photodetector
- readout device
What is a type of light source that is used in a spec?
Incandescent lamps ex: tungsten
What is the role of the monochromator?
To isolate radiant energy of a desired wavelength -
What is an example of a monochromator?
Prisms, diffraction gratings and or filters
What is the main type of cuvette required for readings below 340nm?
Quartz
What is the role of photodetectors?
To convert light into an electrical signal - proportional to the number of protons striking its surface.
What is a multiplier tube and which part of the spec is is associated with?
This produces rapid response times, very sensitive and slow to fatigue.
- Photodetector
What are the 2 types of specs?
Single beam and double beam
What do double beam instruments allow for?
‘real time’ referencing
What is a double beam in time spec composed of?
Beam is split in two, but measured by the same detector.
What is a double beam in space spec composed of?
Beam is split into two paths and measured by matched detectors.
Split between reference and sample
What is the Beer Lambert Law?
Demonstrates that the conc of a substance is directly proportional to the amount of light absorbed or inversely proportional to the log of the transmitted light.
Beer lambert equation?
A = ecl
What is an absorption spectrum?
This is the recording of the absorbance of a molecule as a function of wavelength.
What is the absorption maximum?
This is the wavelength of highest light absorption.
How can the linear limitation (abs vs conc) be used to calculate the conc of an unknown target?
- Calib curve
A single purified substance can be quantified using Beer Lambert - given absorptivity is known at lambda max
What is molar absorptivity?
This is the abs of a solution with a conc of 1molL-1 of the compound in a light path of 1 cm = M-1cm-1
What is the equation used to find the conc of an unknown solution?
(abs of unknown/ abs of standard) x glucose standard
Why are reference blanks important?
- calibration
- document the baseline response of the environment - instrument sample system.
- documented the influence of the cuvette
- using the same solvent, any abs, fluorescent or scattering due to the solvent phase itself are documented.
Why are control samples important?
- quality control, ensure that the results obtained are reliable
Why are calibrating solutions important?
Minimise uncertainty by ensuring the accuracy of testing equipment.
calibration quantifies and controls errors or uncertainties within measurement processes to an acceptable level
What can an assay design be based on?
- the analyte absorbing at a unique wavelength
- the analyte reacting with a reagent producing a new compound absorbing at a unique wavelength.
- analyte is involved in a reaction producing a chromophore
What is a chromophore?
This is the part of a molecule that is responsible for colour - absorbing light.
What is the Biuret Reagent composed of?
- potassium hydroxide
- copper sulfate
- potassium tartrate
What is the principle of the colorimetric biuret assay?
Presence of peptides, copper ions form a violet coloured coordination complexes in an alkaline solution - more peptides present = more intense the colour
What is an example of a dye used in the formation of coloured compounds and chromophores?
Quinoneimine dye - abs max @500nm
What has two peaks ? NAD or NADH
NADH
What is fluoresence?
This is the emission of light by a substance that has absorbed light or other electromagnetic radiation.
What does the emitted light of fluorescence have?
The emitted light has a longer wavelength and lower energy, than the absorbed radiation.
What is the stokes shift?
This is the difference between the abs exc and abs em.
What are the 2 spectrums which fluorescent molecules have?
- absorption
- emission
What is the principle components of fluroescence specs?
- 2 monochromators
What is an example of an intrinsic fluorescent molecule?
aspirin
pollutants
What are the 2 advantages of fluorescence spec?
enhanced sensitivity and specificity
What is a disadvantage of fluorescence spec?
- not all cpds show intrinsic fluorescence, limited application but can couple to fluorescent dyes or fluorophores.
- ‘quenching’ - oxygen can interfere with measurements
