Optical Methods

ooohhh boy this note is so messy, I will sort this out in a few days

 

 

Optical Methods

Contents

•       Principles and instrumentation of uv and visible spectroscopy, and its application to qualitative and quantitative analysis.

•       Spectrofluorimetry - primary and secondary events, quenching, sensitivity and discrimination, instrumentation.

•       An outline of the principles and applications of atomic absorption spectrophotometry and emission flame spectrophotometry

The Electromagnetic Spectrum

Energy of Electromagnetic Radiation

Energy E  =  hν   where h = Plank’s constant

                                            ν = frequency

Frequency  ν =  speed (c) / wavelength (l)

Frequency ν  a  1 / wavelength (l)

Energy  a  1 / wavelength

ie. shorter wavelength = higher energy

Absorption 1

•       Electrons exist in precisely defined energy levels

•       An atom absorbs light of a given wavelength

•       Uses energy to move an electron to higher energy level

–      An outer shell electron

–      Ground state → Excited state

•       Light of a particular energy is absorbed for a particular change in energy level

•       Gives an absorption spectrum

•       Light of a particular energy is absorbed for a particular change in energy level

•       Gives an absorption spectrum

•       Energy difference between electron states is in region of uv & visible light

•       Atoms absorb light in uv-visible range

•       We see reflected (non absorbed light)

•       GS: Ground state

•       E1: 1^st Excited state

•       E2: 2^nd Excited state

•       E3: 3^rd Excited state

Colour

•       Due to Energy levels

•       The internal energy of molecules changes in steps

•       Energy, E = hn

(h = Plank’s constant, n = frequency of light, n = 1 / l , l = wavelength of light)

•       A result of electrons changing energy levels

Atomic & Molecular Absorption Spectra

•       Atomic spectra

–      Transitions between states

–      Eg s₀ → s₁, s₀ → s₂

–      Only light of ‘right’ wavelength is absorbed

–      → Exact energy for a transition (electron jump)

–      Line spectra are obtained

•       Molecular spectra

–      Molecules absorb light of several different wavelengths

–      Electrons in different atoms

–      Vibrations of bonds absorb energy

–      Mixture of signals causes a broadening from a line to a band or peaks around a wavelength

Molecular Absorption Spectrum

•       Molecule absorbs light

•       Electrons from several atoms move to several different energy levels and bonds vibrate

•       Molecule absorbs light of several different wavelengths

•       Absorption spectrum:

•       Spectrum can be a characteristic molecular signature

•       Measured in a spectrophotometer

What colour is chlorophyll?

Does the spectrum show what you would expect?

History of Spectrophotometry – Colorimeter

•       In 1853 a device was invented that shows which light in the spectrum was absorbed

•       Colorimeters were then used to  detect red of haemoglobin or the green of chlorophyll

•       Technique was used to conclusively identify blood at a crime scene

UV- Visible Spectrophotometry

•       A spectrophotometer detects the amount of radiant light energy absorbed by molecules

•       Shine light from near infra red through to ultra violet spectrum through a sample

•       Detect which wavelengths are absorbed 

Spectrophotometer

•       6 basic components:

–      a light source

–      a prism or diffraction grating

–      an aperture or slit

–      a sample cell (of known path length)

–      a detector (a photocell or photoelectric tube)

–       and a digital meter to display the output of the phototube

•       Dual beam or single beam

Basics of a spectrophotometer

Principle 1

•       Incident light is reflected from a diffraction grating.

•       It is split into its component colours or wavelengths, which then diverge.

•       Sections of the projected spectrum can be either blocked or allowed to pass through the slit so that only one wavelength will pass to the other sections of the spectrophotometer.

•       The position of the grating is adjustable so that the region of the spectrum projected on the slit can be changed.

Principle 2

•       Light passes through the slit to the sample tube

•       Light not absorbed by the sample tube travels to the phototube

•       Light creates an electric current µ to the number of photons striking the phototube.

•       If a digital meter is attached to the phototube, the electric current output can be measured and recorded.

•       The scale is usually calibrated in two ways:

–      percent transmittance: 0 to 100 and

–      absorbance, or optical density units 0 to 2.

Preparation

•       Choose the wavelength:

–      The diffraction grating is adjusted so that the desired wavelength of light passes through the slit

–      Usually the wavelength of light that is most absorbed by the compound (e.g. 515nm for glucose)

–      Alternatively a scan will give the full spectrum

•       Zero-ing using a blank:

–      The output of the phototube must be adjusted or calibrated to correct for drift in the electronic circuits or contaminants between the source and the detector

–      Dual beam instruments allow simultaneous zeroing or baseline correction

Specs

•       Single beam instruments

Applications

•       Identifying compounds: All solutions of chemical compounds absorb light of specific wavelengths.

•       Determining concentrations: the amount of light absorbed is µ to the conc. of a compound

To analyze water quality, measure glucose levels, monitor bacterial growth, perform protein assays and many applications in the medical, forensic & environmental sciences, physics, and biotechnology.

Proteins

Trp & Tyr have an absorbance peak at 280nm used in protein quantification (Beer Lambert law A= ecl)

Beer Lambert law

•       Relationship between absorbance, at a given wavelength and concentration

•       A= ecl

Where: A = absorbance

e = molar extinction coefficient (M^-1 cm^-1or

                                                                                                                dm³.mol^-1cm^{- 1})

l = length of the light path (usually 1 cm)

c = concentration of the solute (M)

Learn this formula you will use it a lot

Use of Beer Lambert law

•       A= ecl

A solution of NADPH has an absorbance at 340nm of A = 0.622 what is the concentration?

e_(NADPH) = 6220 M^-1 cm^-1

l = 1 cm

c = concentration of the solute (M)

A= ecl  >  c = A/el

c = 0.622/6220 x 1

c = 0.0001 M (or 0.1mM)

Always remember the units

Molecular Absorption Spectrum

•       Spectrum can be a characteristic molecular signature

•       Measured in a spectrophotometer

•          Molecule absorbs light of different wavelengths

•          Electrons from several atoms/ bonds move to several different energy levels and bonds vibrate

Applications

•       Identifying compounds: All solutions of chemical compounds absorb light of specific wavelengths.

•       Determining concentrations: the amount of light absorbed is µ to the conc. of a compound

To analyze water quality, measure glucose levels, monitor bacterial growth, perform protein assays and many applications in the medical, forensic & environmental sciences, physics, and biotechnology.

UV spectrophotometer
(Double beam)

Instruments Measuring in Ultra Violet

2 Light Sources:

•       Tungsten bulb delivers visible light > 320 nm

•       Deuterium lamp delivers uv 160 – 375 nm

•       Automatic switch over at 360 nm

Optics:

•       Must be silica-Glass absorbs uv light.

Light Detection:

•       Two photocells required.

•       ‘Blue’ photocell measures 200 – 500 nm

•       ‘Red’ photocell measures 350 – 800 nm

Sample preparation for UV

Solution or suspension (cloudy) – consider solubility and appropriate solvents

Choose appropriate cuvette (sample holder)

Cuvettes may be:

1. Optically transparent plastic – ok for aqueous samples, not for organic. (480-600 nm) inexpensive and disposable, easily scratched
2. Optical grade Glass – ok for organics (400-900 nm), more scratch-resistant
3. Fused silica or quartz – must be used for wavelength ranges (190-750 nm), very expensive –Care!

Cuvettes

Dual Beam Recording Spectrophotometers

•       Response of the photocells changes with wavelength, cuvettes & buffers may absorb light

•        → spectrophotometer must be re-zeroed against a blank for every wavelength

•       Problem: What happens with a ‘scan’ across wavelengths

•       Double beam allows blank to be continually reset

•       Double beam instrument: the light beam is split into two parts

•       → One part is passed through a blank solvent cell → Other part passes through the sample cell

•       Absorbtion spectra can thus be obtained in a continuous scan

•       Alternative: newer single beam specs record, save and subtract a baseline scan

Transmittance

When light intensity entering the sample (blank) = I₀

and light intensity leaving the sample = I

                I / I₀ = transmittance

Transmittance depends on:

Concentration of the absorbing molecule             (c)

Distance travelled by light through the absorbing substance

-          light path (l)

A constant, characteristic of the absorbing substance  (e)            

Absorbance

Transmittance, I / I₀  =  10^-ecl

               

                log I / I₀  =  -ecl          ie.  - log I / I_o =  ecl

                -log I / I₀  (ie. –log transmittance) is called absorbance (A) a unitless quantity

                                                ie.  A = ecl

This is the origin and derivation of the Beer – Lambert laws.

Beer-Lambert Law

states that the absorbance of a sample (A), at a particular wavelength, is proportional to the concentration (c) of the sample (M or mol dm^-3) and the path length of the light (l) through the sample (cm)

A = e c l

also expressed as A = -log₁₀ (I/Io) =log(Io/I)

 e is the molar extinction coefficient and is a measure of how strongly the compound absorbs at that wavelength
(units of M^-1cm^-1 or dm³.mol^-1cm^-1)

Beer-Lambert Law A = e c l

A linear relationship between A & c
Molar extinction coefficient e is the slope of line
 l =1 cm
(think of y = mx + c   :   A = ec + 0)

Important Practical Point

Remember that absorbance A = -log transmittance.

When A = 1,  transmittance = 0.1 ie. 10%

When A = 2,  transmittance = 0.01 ie. 1%

When A = 3,  transmittance = 0.001 ie. 0.1%

Many digital instruments will give A = of 2.5 or 3. NB. Here A is much less than 1% of the incident light → very inaccurate

Don’t use absorbances higher than about 1.5

Ideal range: 0.1<A<1

Quantification by UV-Vis

•       Beer Lambert Law: A = e c l

–      Last lecture, but need to know e

•       Comparison of unknown to standard concentrations

–      Plot a standard curve

•       Derivatisation & Colorimetric reactions

–      Non absorbing substance is reacted with reagent which converts it/is converted to a derivative which absorbs light

–      Requires a standard curve

Practical points about colorimetric assays

•       Standards treated exactly the same way as the test sample/s

•       If a test sample gives an absorbance higher than the highest value of the calibration curve it should not be diluted to make it fit the curve.

–      there might not be enough derivatisation reagent present to react all the sample if a sample of higher concentration than the highest standard is used

–      Further dilution might alter ‘blank’

–      Results in a false low measurement.

Examples of substances measured by colorimetric reactions

Many modern Biomedical tests are colorimetric assays:

•       Trinder’s assay detects glucose by oxidation of a colourless  dye → A₅₁₅ ↑ (pink)

•       Reducing sugars eg. Glucose and fructose, derivatised using dinitro salicylic acid (DNSA). The derivative absorbs light maximally at 540nm.

•       Protein reacted with Folin reagent forms a derivative which absorbs light maximally at 750nm.

                Non derivatised protein absorbs light at 280nm, but the Folin assay allows measurement in the visible range, and is more sensitive.

•       Other protein assays include: Bradford, Biuret, BCA & Lowry – all have a colour change proportional to [Protein]

•       Amino acids form a derivative with ninhydrin which absorbs maximally at 570nm.

•       Phosphate forms a derivative with ammonium molybdate which absorbs light maximally at 600nm.

Clinical Chemistry Analyser

•       Roche Cobas 8000

•       up to 8,400 tests per hour with a total of 270 reagents

•       Multiple analytes from 1 patient sample

•       Provides a complete clinical chemistry profile

•       Based on spectrophotometer

BCA Assay

•       Determination of unknown protein concentration

•       Colorimetric assay

•       Use standard curve

•       Protein causes a blue/violet colouration

•       Measure at l = 562 nm

Standard Curve

•       Use protein standards: dilutions of a known 1mg/ml BSA solution to plot a standard curve

Calculate the concentrations of the unknowns from standard curve using the absorbances measured

Enzyme assays in a spectrophotometer

S + E                       ES               P + E

1)      Production of the product

2)      Consumption of substrate

e.g. NADH or other chromophore

Commonly by determination of Absorbance values using a spectrophotometer

A real example: Glucose-6 Phosphate Dehydrogenase

Follow production of the NADPH molecule

Absorbance = ε.c.l

(ε = molar extinction coefficient, c = concentration, 1 = 1cm)

Concentration = Absorbance/ ε

Concentration/min = Absorbance/ min. ε

Vo = slope/ ε

Slope: 0.00348abs/sec

For Glucose-6 Phosphate Dehydrogenase assays, ε = 11.5 dm³.mol ^-1.cm ^-1  Vo = 0.00348abs/sec/11.5 dm3.mol -1.cm -1 

Vo = 0.000302608 mol/sec

Glucose-6 Phosphate Dehydrogenase and Glucose-6 Phosphate Dehydrogenase with Aurintricarboxylic Acid Ammonium salt (ATA) an enzyme inhibitor

Turbidimetry and Nephelometry

•       Spectrophotometers can be used to measure light scatter from particles.

•       Can be used to estimate the amount of particulate material eg bacteria in growth medium.

•       The method is known as turbidimetry.

•       A purpose built instrument called a nephelometer can be used for exactly the same purpose.

A simple turbidimetry assay for aggregation

Conclusion

•       UV-Vis is a key tool in many areas of bioscience study and diagnostics

•       Quantitative data can be obtained from the use of:

–      Beer-Lambert Law A = e c l

–      Colorimetric assays

•       For more info and worked examples see following slides – a similar calculation to the NAD+/NADH calculation will be in the exam

Special uses of spectrophotometers

  For example it is possible to measure substances with

similar but different absorbtion spectra when mixed in the

same sample.

An important example is the co-factor nicotinamide adenine

dinucleotide (NAD⁺) and its reduced form, NADH.

These two substances have identical absorbtion spectra

below 300nm, but NADH has an absorbance peak at

340nm, where NAD⁺ does not absorb.

By measuring at 340nm the concentration of NADH can be

measured in the presence of NAD⁺.

   By measuring at 260nm and 340nm the concentrations of

NAD⁺ and NADH in a mixture can be measured, as in the

example below.

   Protein (absorbance max 280nm) and nucleic acid

(absorbance max 260nm) can be measured in a mixture by

a similar method. This is not quite so easy since protein

has some absorbance at 260 and nucleic acid at 280nm,

so an algorithm ( a formula specifically for this calculation)

has to be used in this case. 

NAD⁺ and NADH measurement in a mixture

Molar absorbance of NAD⁺ at 260nm = 18,000

Molar absorbance of NADH at 260nm = 18,000

Molar absorbance of NAD⁺ at 340nm =   0

Molar absorbance of NADH at 340nm = 6220

A mixture of NAD⁺ and NADH measured in a cell of 1cm

light path gave the following absorbances:

A at 260nm = 1.4

A at 340 nm = 0.25

Using the Beer – Lambert law

 A = Ecl       (or c = A / El)

[NAD⁺] + [NADH] = 1.4 / 18,000 x 1 M  = 7.7 x 10^-5M

[NADH] = 0.25 / 6220 x 1 M  = 4.0 x 10^-5M

Therefore [NAD⁺] = 7.7 x 10^-5 –  4.0 x 10^-5M

                                                       = 3.7 x 10^-5 M             

Linked assays

Because NADH can be measured in the presence of NAD,

enzyme reactions which use NAD / NADH as co-factors

(dehydrogenases / reductases) can be measured by

following production of NADH from NAD at 340nm.

The method of enzyme measurement is so simple and so

useful that it is used to measure enzymes which do not use

NAD / NADH as co-factors by linking them to enzymes

which do. This type of enzyme assay is called a linked

assay.    

Measurement of metabolite concentration using NAD / NADH

   The dehydrogenase assay and linked assay methods can

also be used to measure metabolite concentrations in

unpurified mixed samples, such as plasma, or tissue

homogenates.

The methods can only measure concentrations within quite

narrow limits, but most samples can be diluted into the

measurement range.

The methods are specific because they use enzyme active

sites to identify the material to be measured. The sample

can therefore be a constituent of an impure mixture.

Electrons, Energy & Light – Revision

n  Electrons (e^-) exist in defined energy levels

n  Light has energy E = hn

(h = Plank’s constant, n = frequency of light)

n  Atoms & bonds absorb light of given wavelengths

n  Use energy to move an e^- to higher energy level

n  Energy is also lost from high energy e^-

Fluorescence 1

1. Light absorbed
2. → e- jumps from ground state to a vibrational energy level of excited state
3. But e- falls to base level of the excited state within 10^-12 seconds
4. e- then falls back to the ground state within 10^-8 seconds
5. Absorbed energy is released from molecule
6. This may be:
1. kinetic energy (heat)
2. emitted as a quantum of light (less common)

→ Re-emitted light is fluorescence

Re-emitted light is fluorescence.

About 10% of molecules which absorb light also fluoresce

→ Fluorescence spectroscopy / fluorimetry / spectrofluorimetry

Fluorescent Biomolecules

•       Drugs

–      Aspirin, morphine,barbiturates, tetracyclins

•       Vitamins

–      Vitamins A, B6 & E, riboflavins, nicotinamide

•       Polutants

–      Napthalene, anthracene, benzopyrene

•       Proteins

–      Due to tryptophans in the polypeptide chain

Characteristics of fluorescence 1
Stokes Shift

•       Not all the light energy absorbed is re-emitted

•       Some lost from the loss of vibrational energy

•       Therefore:

–      emitted light has less energy than that absorbed

–      ie. it is emitted at a longer wavelength

•       Difference between wavelengths of maximum absorbance and of maximum emission is the Stokes shift

•       Stokes shift =   λ_maxF - λ_maxA

Characteristics of fluorescence 2 Quantum Efficiency

•       Not all absorbed light is re-emitted as fluorescence

•       → Some energy is always lost as heat

•       Less light is emitted as was absorbed

•       Ratio of no. of photons absorbed to no. of photons fluoresced = quantum efficiency

•       Stokes shift and quantum efficiency are characteristics of a fluorescent molecule.

Measurement of Fluorescence 1

•       Photons fluoresced  a  conc. of fluorescent material

•       Relationship between fluorescent intensity and concentration is linear (not log., like absorbance)

•       Fluorescence can be used to measure concentration

•       Fluorescence can be used over a wide concentration range

•       → very versatile technique when a molecule fluoresces

•       Performed in a fluorimeter

•       Fluorescent light is emitted from molecules → can be measured against a black background

•       More efficient to measure a small quantity of light against a black background than to measure a small quantity of light absorbed from a bright beam

•       Thus: measurement of fluorescence more sensitive than absorbance.

•       eg. about 100mg of adrenalin can be measured by absorbance methods, but about 100 pg can be measured by fluorescence.  

Absorbance v Fluorescence

Fluorimeter (or fluorometer)

•       Measures fluorescence

•       Has two monochromators (filters), to select the wavelengths of absorbed light (excitation light), and emitted light (fluorescence)

•       Fluorescent light is emitted in all directions equally

•       Fluorimeters measure fluorescent light against a black background at right angles to the excitation beam

•       Detector is a photomultiplier tube.

Thioflavin T (TfT) fluoresces when bound to aggregated proteins

Here its used to measure Amyloidb

Advantages of Fluorescence Measurement

•       Measurement of fluorescence is more sensitive than absorbance measurement

•       → usually about 1000x more sensitive for same compound

•       Sensitivity is good enough to measure concs. of material released from cells and tissue

•       Linear relationship between conc. and fluorescence → method used over a wide conc. Range

•       Many cyclic organic molecules with conjugated double bonds fluoresce inc. important biological molecules eg…

–      tryptophan (in proteins)

–      nucleic acid bases (in NAD and ATP as well as DNA and RNA).

Disadvantages of Fluorescent Measurement

•       Not all molecules which absorb light fluoresce

•       Fluorescence varies with pH and temperature, therefore assays must be buffered and temperature controlled

•       Fluorescence is easily affected by quenching ie. absorbance of light by chemicals or coloured compounds present in samples as contaminants.

Fluorescent Labels

•       Fluorescent molecules can be attached to other molecules by chemical linkers to act as labels

•       Fluorescent labelled antibodies can be used to pinpoint specific antigens in cells can be used to view living cells.

•       Specific cells can be labelled with fluorescent antibodies and collected using a fluorescence activated cell sorter (FACS).

•       This allows detection, measurement and collection of very specific cell types, and is very important in diagnosis eg of leukaemias, and immunological research.

•       DNA is now sequenced using fluorescence labelled bases

•       Proteins can be engineered with fluorescent reporters eg green fluorescent protein

Green Fluorescent Protein

GFP = A jelly fish protein

Acts as a reporter when GFP DNA is fused to cDNA in transfections

Atomic Spectroscopy

•       Atomic absorption spectrophotometry (flame absorption spectrophotometry)

•       Atoms dispersed in flame

•       Measures light absorbed at specific wavelengths by atoms eg Cu, Zn, Pb

•       Relies on Beer Lambert law

•       Flame atomic emission spectrophotometry (flame photometry)

•       Measures light from metal atoms in a flame

•       Commonly K⁺ ,Na⁺  & Ca⁺ in biological fluids

Atomic absorption spectrophotometry

Flame atomic emission spectrophotometry

•       For more info on atomic spectroscopy go to: http://www.resonancepub.com/atomicspec.htm