W1 UV-Vis Spectroscopy of Biomolecules

Question 1

wavelength definition

Answer 1

distance between 2 consecutive points in a wave that are in phase with each other

Question 2

wave number definition

Answer 2

number of waves that can fit in one cm

shorter wavelengths > more energetic waves and higher wavenumber

Question 3

period definition

Answer 3

time required for a wave to go through a distance of wavelength

measured in seconds

1 hertz = 1/s > hertz is a unit of frequency

Question 4

definition of spectra

Answer 4

tells us which waves resonate with our system and get absorbed by it

Question 5

resonance definition

Answer 5

when excitation of large systems at their characteristic resonant frequency leads to optimal energy absorption and increasing amplitude of oscillation

Question 6

3 observations for Lambert beer law

Answer 6

1. light intensity passing through a cuvette is reduced for higher concentrations
2. an equal fraction is absorbed for each successive layer
3. this fraction is independent from initial light intensity

Question 7

extinction coefficient definition

Answer 7

measure of how much light a substance absorbs at a specific wavelength

independent of concentration, varies with wavelength

(imagine small little blocks blocking the path of light)

Question 8

absorbance definition

Answer 8

measure of amount of light absorbed by a solution

directly proportional to concentration

Question 9

why does the LB law not work for high concentrations

Answer 9

high concentration > more interaction between sample particles and photons > more scattering of photons > stray photons do not follow expected path > reach detector without passing through the sample

Question 10

how is problem of stray light solved

Answer 10

increase intensity transmitted either by:

1. increasing power of lamp
2. reducing the absorbance (reduce concentration or reduce path length)

Question 11

can carbohydrates and lipids be measures in UV-Vis spectrophotometer

Answer 11

no as they only absorb far below 200nm > colourless and cannot be absorbed as light from lamp usually >200nm

Question 12

do proteins have absorption in the visible region

Answer 12

usually completely transparent unless they contain chromophores such as flavins, hemes or metals etc

Question 13

absorption band of proteins

Answer 13

absorption below 220nm very intense; caused by amide bonds

band absorption depends on secondary structure > cannot be used to determine concentration

aromatic a.a present > protein absorbs at 280nm

absorption at <220nm is always much higher than at 280nm

Question 14

absorption band for nucleic acids

Answer 14

transparent in visible region but absorb in the UV, 260nm

Question 15

units for extinction coefficient

Answer 15

either inverse concentration * inverse length

or

area/mol

Question 16

absorption bands for common amino acids

Answer 16

Phe has max around 260nm but extinction coefficient at 280nm almost 0

if protein has cys and if they form disulphide bonds > absorb at 280nm but contribution is very low

Question 17

how to determine protein contamination of nucleic acids

Answer 17

level of protein contamination determined by ratio of absorbance 260/280

more contamination > lower ratio

1.8 ratio > pure, 1.6 or lower > some contaminants

Question 18

what are the two main proteins used to determine concentration of amino acids

Answer 18

tryptophan and tyrosine