lecture 2 questions effect of ph Flashcards
Why are bathochromic shifts associated with increased light absorbance?
bathochromic shifts are to longer wavelength so llower energy
the effect of the auxochrome is to add non bonded electrons n which are already at higher energy than pi or sigma bonds
- the energy required for a n to pi star or n to sigma star transition is therfore lower = longer wavelength
the lone pairs of electrons can also interact with the chromophore groups (aromatic ring) causing resonance stabilised structures see aniline
causes conjugation to be increased which enhances the probability of a higher light absorbance
- reduces energy required for pi to pi star transitions which have higher molar absorptivities (higher absorbance values)
Why are hypsochromic shifts the opposite?
usually occurs when a basic auxochrome becomes ionised and the lone pair no longer interacts with the electrons of the chromophore
- energy gap between levels will be increased shorter wavelength
caused if the lone pair of electrons is no longer availabke
-stabilised by hydrogen bonding or
- if lone pair is used in bonding (becomes ionised, aniline)
Do all weak acids when ionised cause a bathochromic and hypsochromic shift?
only if the ionisation causes a resonance stabilised structure will ionisation cause a bathochromic effect
- oh group in paracetamol is linked to benzene group and can interact more in the O- state rather than the OH
if the OH group is isolated from the chromophore then no effect will be seen
Why are transitions from the ground state?
Absorption always starts at the lowest vibrational energy level of the lowest electronic level at room temp
- due to the probability of molecules in the excited state being so low
- follows boltzmann law
- ratio of number of molecules (N1 and N0) in the 1 and 0 vibrational levels of energy E1 and E0
-requires high temp to excite molecules from the ground state
What transitions are involved from ground state?
most fluorescent molecules exhibit broad and struictureless absorption and emission bands
- each electronic state consists of an almost continuous manifold of vibrational levels
if the energy difference is between the 0 and 1 vibrational levels is only about 500cm-1, the ratio N1/N0 becomes about 0.09
- excitation can then occur from a vibrationally excited level
- explains why absorption spectrum partially overlap the fluorescence spectrum
why can absorbance overlap fluorescence?
The 0-0 transitions is usually the same for absorption and fluorescence
in most cases the absorption spectrum partly overlaps the fluorescemce spectrum
Give emxample of background correction and what to use to for it?
smoke particles, impurities on wall of tube
cant be corrected with a blank
use deuterium lamp background correction
Why is AA more sensitive than FP?
Atomic emission
- intensity is proprtional to number of atoms in excited state
- inc with temp
- dependant on energy gap between ground and excited state
- lots of different emission lines per element
Atomic Absorption
- intensity is proportional to number of atoms in the ground state
- does not inc with temperature
- independant of energy gap between ground and excited state
- less absorption lines than emission (less overlap)
What is limit of detection?
lowest concentration of e.g an impurity which can be detected but not quantitated
real term
The concentration which gives an instrument signal (y) significantly different from the ‘blank’ or ‘background’ signal
what is selectivity or specificity?
expresses how much the results obtaines by the method for a given analyte are influenced by the prescence of foreign substances e.g impurites or excipients
what is the limit of quantitation?
If concentration is above the limit of detection then we can say with some certainty that the analyte is present
If its only slightly above, then precise quantitation may be unjustified
Therefore the limit of quantitation is used
Defined as the concentration which increases the instrument response by an amount equal to ten times the standard deviation of the blank, sB
Regarded as the lower limit for precise quantitative measurements as opposed to qualitative detection
Limit of quantitation = yB + 10SB
what is sensitivity?
defined as the slope of the calibration graph
provided the plot is linear, can be measured at any point on good sensitivity is one with a steep calibration graph
poor sensitivity of one with a shallow calibration graoh
calculate it by calculating the gradient
what is accuracy?
closness of agreement between experimental value and true value
What is precision?
closeness of agreement (degree of scatter) between a series of measurments
what is robustness?
meausure of capacity of assay to remain unaffected by small variations in method
