Practical 2: Protein analysis

What is Spectrophotometry

It is a qualitative method for studying matter and is based on the absorption of light by a substance

Lamberts Law

States that the absorbance of light passing through a medium is directly proportional to the absorbing species' concentration and the light's path length.

Beers Law

Relates the absorption of light to the properties of the material through which the light is traveling, stating that absorbance is directly proportional to concentration and the path length of the sample.

what is light

form of Electromagnetic Radiation

Properties of Light

Light waves consist of perpendicular, oscillating electric and magnetic fields

Amplitude (γ)

height of the waves electric vector

Wavelength (λ)

distance from peak to peak

Frequency (f)

number of complete oscillations that the waves makes each second

eq of frequency

what are photons

particle aspect of light

energy described in light

The energy of one particle of light (photon) is proportional to its frequency
E = h*f
as frequency (f) increases, energy (E) of light increases

highest to lowest energy wavelength

gamma rays > X rays > UV > visible > IR > microwave > radio waves

Spectrophotometer

Measures absorbance or transmittance of light, as a function of wavelength.

monochromator

prism that splits white light from the lamp into its constituent wavelengths

TRUE OR FALSE
molecules don't absorb light

FALSE, particles or bonds in the molecule can jump to an excited state

transmittance (T)

amount of light making it through the sample (I0/It)

range of transmittance (T)

0 to 100%

eq absorbance

Aλ = log (I0/It)

what is I0

initial light

what is It

transmitted light

what does beer lambert law states

Absorbance is directly proportional to concentration

eq beer lambert law

what is A in beer lambert law

absorbance

what is e in beer lambert law

molar absorptivity or extinction coefficient

TRUE OR FALSE
Absorbance has no units

TRUE

What is the unit of e in beer lambert law

Abs per unit conc. Per unit length (M-1 cm-1)

what is l in beer lambert law

path length of the sample, usually expressed in cm

What is c in beer lambert law?

concentration expressed in mol/L

what happens to the absorbance if the concentration is doubled

the abs is doubled

what is the negative control in the spectrophotometer

reference blank

what is the reference blank

Contains everything found in the sample solution except the substance being analyzed
Calibrates the absorbance reading A=0.000

what is the wavelength of Tyr, Phe and Trp

280 nm

wavelength of peptide bonds

200 nm

what is Biuret Reagent made of

Alkaline solution of Copper sulfate (CuSO4)

how does biuret reagent work

Reacts with NH of peptide bond
less sensitive
Good for solutions with high protein concentration

TRUE OR FALSE
Biuret reagent is not concentration dependent

FALSE , Biuret reagent reacts with peptide bonds, and its colour intensity is directly proportional to the protein concentration.

what happens when copper reacts with the protein in biuret reagent

colour change from blue to violet

what is the wavelength for BSA (Bovine Serum Albumin

540 nm

the absorbance of an NADH solution at 340 nm is 0.19 AU. The extinction coefficient (e) of NADH is 6300 M-1 cm-1. What is the NADH concentration in µM? (path length = 1 cm)

30.2 µM

C = A / (e * l), where C is concentration, A is absorbance, e is the extinction coefficient, and l is the path length.

you have 5% w/v stock solution of BSA you want to make 5 mL of a 3.5% solution. How much of the stock solution do you need?

3.5 mL

What is the name of the reagent used to determine protein concentration?

biuret reagent

f = c/lambda, name each term in this equation

f = frequency
c = speed of light
lambda = wavelength

which of the following correctly orders the EM radiation types from lowest to highest energy?
A) microwave, radio waves, X rays, UV
B) y-rays, radio waves, IR, UV
C) radio waves, visible, UV, IR
D) microwave, IR, visible, UV

D) microwave, IR, visible, UV

relationship between wavelength and frequency

the higher the energy, the lower the wavelength

what pH range does biuret reagent work best

Biuret reagent only works best on alkali solutions