Principles of the Principles of the spectrophotometric methodsspectrophotometric methods

mirka.rovenska@lfmotol.cuni.mirka.rovenska@lfmotol.cuni.czcz

IInteraction of light with matternteraction of light with matter When a beam of light impinges upon a sample:When a beam of light impinges upon a sample:

a) some photons may have no interaction with a sample and bea) some photons may have no interaction with a sample and betransmittedtransmitted

b) some photons may be absorbed by a sampleb) some photons may be absorbed by a sample c) some photons may be scatteredc) some photons may be scattered d) some photons may be reflectedd) some photons may be reflected

The extent of a) – d) depends on the material of the sample and on the The extent of a) – d) depends on the material of the sample and on the wavelength of the radiationwavelength of the radiation

In spectrophotometric measurements, c) and d) should by kept In spectrophotometric measurements, c) and d) should by kept to a to a minimumminimum

The intensity of the transmittedradiation (I) is lower thanthe intensity of the incidentradiation (I0)

incident radiation

reflection

s c a t t e r

transmittedradiation

absorptionof radiation

I0

I

Electromagnetic spectrumElectromagnetic spectrum

λ [m]

10-12 10-10 10-8 10-3 10-1

Absorption of radiationAbsorption of radiation

Molecules of the sample absorb the photons Molecules of the sample absorb the photons of a suitable of a suitable wavelengthwavelength ( ( λλ) and change their energy level (state):) and change their energy level (state):

1) 1) in thein the microwave and far infrared microwave and far infrared region, the photonsregion, the photons have have such a low energy that, if absorbed, can cause only the such a low energy that, if absorbed, can cause only the changes of the changes of the rotationalrotational energy states energy states

2) absorption of2) absorption of photons ofphotons of the the infrared infrared radiationradiation can bring can bring about the changes of the about the changes of the vibrationalvibrational energy states energy states

3) energy of photons of UV and visible light (VIS) is sufficient to 3) energy of photons of UV and visible light (VIS) is sufficient to cause the transition of cause the transition of electronelectron to a higher electronic to a higher electronic energy energy levellevel

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Energy levels of a moleculeEnergy levels of a moleculeen

erg

y

electronic levels

1st excited stateof electron

vibrational levels rotational levels

UV/VIS

IRΔErfar IR

Thus, the change of the electronic state is accompanied by changes of vibrational as well as rotational states!!!

ΔE = ΔEe + ΔEv + ΔEr ; ΔE = hν = hc/λ ΔEe >> ΔEv >> ΔEr

ΔEe ΔEv

ColourColour Only those substances appear coloured that absorb VIS radiationOnly those substances appear coloured that absorb VIS radiation The colour is then determined by the reflected light (theThe colour is then determined by the reflected light (the colour of colour of

thethe substance substance isis complementarycomplementary to that one which has been to that one which has been absorbed): absorbed):

ChromophoresChromophores Absorption of visible light can cause transitions of Absorption of visible light can cause transitions of ππ or n electrons or n electrons

(for (for transition of transition of σσ electrons, UV absorption is necessary electrons, UV absorption is necessary); thus, ); thus, only substances containing only substances containing ππ or n electrons or n electrons can can appear coloured appear coloured

Groups containing unsaturated centres (Groups containing unsaturated centres (ππ electrons) and non- electrons) and non-bonding electrons are called bonding electrons are called chromophores chromophores – e.g..: – e.g..:

A compound will absorb in the visible region (and thus appear coloured) if it contains at least several chromophores (absorption (absorption maximum then moves to a longer maximum then moves to a longer λλ, i.e. from the UV to the visible , i.e. from the UV to the visible region):region):

The compounds that absorb only in the UV region are NOT The compounds that absorb only in the UV region are NOT coloured (saturated hydrocarbons)coloured (saturated hydrocarbons)

Absorption of UV and visible lightAbsorption of UV and visible light

Routinely in biochemistry, absorption of Routinely in biochemistry, absorption of UVUV and and VIS VIS lightlight (that causes electronic transitions) is measured (that causes electronic transitions) is measured

The UV/VIS absorption is the principle of all The UV/VIS absorption is the principle of all the methods that will be discussed from now the methods that will be discussed from now onon

The Beer-Lambert lawThe Beer-Lambert law

This decrease of the radiation intensity can be expressed as: This decrease of the radiation intensity can be expressed as:

T = I/IT = I/I0 0

T is called T is called transmittancetransmittance and varies from 0 to 1 (0 – 100%); it is and varies from 0 to 1 (0 – 100%); it is the the ratio of the transmitted to the incident radiation intensityratio of the transmitted to the incident radiation intensity

Compounds with T (in the VIS region) approaching 100%...transparent;Compounds with T (in the VIS region) approaching 100%...transparent; 0%......opaque0%......opaque

due to absorption, the intensity of due to absorption, the intensity of the transmitted light is lower thanthe transmitted light is lower than the intensity of the incident lightthe intensity of the incident light

incident radiation

reflection

s c a t t e r

transmittedradiation

absorptionof radiation

I0

I

l

Absorbance is then defined as: Absorbance is then defined as: A = - log T = log IA = - log T = log I00/I/I

The Beer-Lambert law states that the absorbance (at a given The Beer-Lambert law states that the absorbance (at a given λλ) is ) is directly proportional to the thickness of the absorbing layer (l) and directly proportional to the thickness of the absorbing layer (l) and to the molar concentration (c) of the absorbing substanceto the molar concentration (c) of the absorbing substance: :

A = A = εελλ c lc l εε…molar absorption coefficient [dm…molar absorption coefficient [dm33molmol-1-1cmcm-1-1] = [M] = [M-1-1cmcm-1-1]]

The law is only true for monochromatic light !!! The law is only true for monochromatic light !!!

ε ε depends on depends on λλ and this dependenceand this dependence characterizes the substancecharacterizes the substance

Absorption spectrumAbsorption spectrum Spectrum is the dependence of Spectrum is the dependence of the intensity of radiation on its the intensity of radiation on its

wavelength or frequency (wavelength or frequency (νν = 1/ = 1/λλ)) Absorption spectrum can be acquired by analysis of radiation Absorption spectrum can be acquired by analysis of radiation

(emitted from the source) that has passed through the analyzed (emitted from the source) that has passed through the analyzed substance (by comparing the intensities of the incident and substance (by comparing the intensities of the incident and transmitted light)transmitted light)

Absorption spectrum of a given substance is often depicted as the Absorption spectrum of a given substance is often depicted as the dependence of absorbance or dependence of absorbance or εε on the wavelength on the wavelength of the radiationof the radiation

Spectrophotometry Spectrophotometry deals withdeals with acquiring and analyzing the acquiring and analyzing the absorption spectraabsorption spectra

KMnO4

A

Characteristics of an absorption bandCharacteristics of an absorption band

Absorption band is characterized by the:Absorption band is characterized by the: wwavelengthavelength(s)(s) λλmaxmax of its peak of its peak(s) (s) – – usually, usually, AA is measured at this is measured at this λλmaxmax by the corresponding by the corresponding εεmaxmax

λ [nm]

Abso

rbance

(re

lati

ve u

nit

s)

λ1max λ2

max

Example 1: the Beer-Lambert lawExample 1: the Beer-Lambert law

If we know If we know εε (for the compound and given (for the compound and given λλ) and the thickness ) and the thickness of the absorbing layer (i.e. the width of a cuvette), we can of the absorbing layer (i.e. the width of a cuvette), we can calculate the concentration of the absorbing compound (using calculate the concentration of the absorbing compound (using the B-L law) from the absorbance measuredthe B-L law) from the absorbance measured

E.g.: How many grams of vitamin DE.g.: How many grams of vitamin D22 are are solved solved in 1 liter of in 1 liter of solution, if solution, if itsits absorbance measured in the 2-cm wide cuvette is absorbance measured in the 2-cm wide cuvette is AA264264 = 0,4 and = 0,4 and εε for vit. D for vit. D22 at this at this λλ is 18,4 [dm is 18,4 [dm33molmol-1-1cmcm-1-1]. M]. Mrr of of vitaminevitamine D D22 is 396. is 396.

A = A = εελλ.c.l .c.l c = A / ( c = A / (εελλ.l) = 0,4 / (18,4 . 2) = 0,01 M.l) = 0,4 / (18,4 . 2) = 0,01 M

m = c . M . V = 0,01 . 396 . 1 = 4,3 gm = c . M . V = 0,01 . 396 . 1 = 4,3 g

Calibration graphCalibration graph

In practice, it can often be more precise to determine the In practice, it can often be more precise to determine the concentration of the absorbing compound not by calculation concentration of the absorbing compound not by calculation according to the Beer-Lambert law, but by construction of the according to the Beer-Lambert law, but by construction of the calibration graph:calibration graph:

we prepare a series of standards of known concentration of the absorbing compound, measure absorbance for each of them and plot the absorbance values against their individual concentrations

we measure absorbance of the „unknown“ sample; itswe measure absorbance of the „unknown“ sample; its concentration can then be read from the calibration graph

Example 2: determination of Example 2: determination of concentration using calibration graphconcentration using calibration graph

To determine the protein conc. in the sample, the Lowry protein assay can be To determine the protein conc. in the sample, the Lowry protein assay can be used: by the reaction of proteins with the reagent, a coloured complex is used: by the reaction of proteins with the reagent, a coloured complex is formed that absorbsformed that absorbs light light at at λλ= 750 nm= 750 nm

We prepare several samples ofWe prepare several samples of a pure protein, e.g. a pure protein, e.g. BSA (bovine serum BSA (bovine serum albumin) so that the final conc. of BSA is 5, 10, 20, 40, 60, and 80 µg/ml. albumin) so that the final conc. of BSA is 5, 10, 20, 40, 60, and 80 µg/ml. The The rreagent is added.eagent is added.

We measureWe measure the the absorbance absorbance values values of these standards and plot them against of these standards and plot them against conc. of BSAconc. of BSA (protein) (protein)::

Then, we Then, we add the reagent toadd the reagent to the sample in which we want to determine the the sample in which we want to determine the protein concentration, measure its Aprotein concentration, measure its A750750 and read the corresponding and read the corresponding protein protein conc. from the calibration graph (e.g. for A = 0,3, the conc. is 50 µg/mlconc. from the calibration graph (e.g. for A = 0,3, the conc. is 50 µg/ml))

y = 0,0054x + 0,0248

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

0,5

0 10 20 30 40 50 60 70 80 90

konc. proteinu [µg/ml]

A

protein concentration

Example 3: monitoring enzymatic reactionExample 3: monitoring enzymatic reactionss Using spectrophotometry, we can monitor the increase ofUsing spectrophotometry, we can monitor the increase of the the

product concentration or product concentration or the the decrease ofdecrease of the the substrate substrate concconcentrationentration, respectively, respectively

Often, reactions using NADOften, reactions using NAD++/NADH+H/NADH+H++ as coenzymes are as coenzymes are monitored, based on the difference between the absorption monitored, based on the difference between the absorption spectra of these two coenzymes: spectra of these two coenzymes:

E.g: determination of acetoacetate and E.g: determination of acetoacetate and -hydroxybutyrate: their -hydroxybutyrate: their ratio in arterial blood reflects the intramitochondrial redox state ratio in arterial blood reflects the intramitochondrial redox state and is used to assess the energy state of the liver after and is used to assess the energy state of the liver after transplantation or failure: transplantation or failure:

mol

ární

[M-1cm

-1]

The increase/decrease of NADH concentrationper a fixed period is measured at 340 nm andcompared with calibration

-hydroxybutyrate+ NADH + H+

acetoacetate+ NAD+

mola

r abso

rpti

on c

oeffi

cient

wavelength [nm]

How can we measure absorbance?How can we measure absorbance? Most often by a Most often by a spectrophotometerspectrophotometer that consists of: that consists of:

The source usually emits The source usually emits aa polychromatic radiation ( polychromatic radiation (containscontains various various λλ) from which monochromator (prism or diffraction grating + slit) separates ) from which monochromator (prism or diffraction grating + slit) separates the the monochromatic light (one monochromatic light (one λλ) ) As a source of As a source of the visible lightthe visible light, the tungsten lamp is used; the deuterium lamp can serve as a source of UV , the tungsten lamp is used; the deuterium lamp can serve as a source of UV sourceprism

monochromator

sample

detector

readoutsystem

PC

slit

SpectrophotometersSpectrophotometers

Before we start to analyze the samples, we have to measure the A Before we start to analyze the samples, we have to measure the A value of „blank“, i.e. solution containing value of „blank“, i.e. solution containing the samethe same components as the components as the samples (the same buffer, coenzymes…) except for the absorbing samples (the same buffer, coenzymes…) except for the absorbing substance; substance; itsits absorbance value must be subtracted from the A values absorbance value must be subtracted from the A values of the samplesof the samples

The solvent should not absorb at the wavelength used for measurement The solvent should not absorb at the wavelength used for measurement The solutions must not be turbid, must not contain bubbles…The solutions must not be turbid, must not contain bubbles… The error of measurement is acceptable for A values ranging from 0,1 The error of measurement is acceptable for A values ranging from 0,1

to 1; if A to 1; if A >> 1, we have to dilute the sample and measure A once again! 1, we have to dilute the sample and measure A once again!

CuvettesCuvettes

Cuvettes must by made of a material that does not absorb the Cuvettes must by made of a material that does not absorb the radiation used for measurement radiation used for measurement cuvettes used for cuvettes used for measurement in the VIS region can be made of glass, cuvettes measurement in the VIS region can be made of glass, cuvettes used for measurement in the UV region must be quartzused for measurement in the UV region must be quartz

Measuring absorbance in microplatesMeasuring absorbance in microplates

If we want to measure A values of more samples at once, we can If we want to measure A values of more samples at once, we can pipette all the samples into the wells of a microplate that can be pipette all the samples into the wells of a microplate that can be analyzed in the microplate reader: analyzed in the microplate reader:

http://www.moleculardevices.com/pages/instruments/readers_main.html

ELISA – sandwich assayELISA – sandwich assay ELISA=enzyme-linked immunosorbent ELISA=enzyme-linked immunosorbent

assayassay Wells of the plate are coated with the Wells of the plate are coated with the

antigen (Ag) antigen (Ag) We add the sample in which the (primary) We add the sample in which the (primary)

antibody (Ab) against the antigen is to be antibody (Ab) against the antigen is to be determined; if the antibody is present, it determined; if the antibody is present, it binds the antigen binds the antigen

After the unbound components have been After the unbound components have been washed away, the secondary antibody, washed away, the secondary antibody, specific to the primary antibody, is addedspecific to the primary antibody, is added

The secondary antibody is enzyme-linked The secondary antibody is enzyme-linked and after addition of a substrate (S), the and after addition of a substrate (S), the enzyme (E) catalyzes formation of a enzyme (E) catalyzes formation of a coloured product that is measured using coloured product that is measured using spectrophotometerspectrophotometer

ELISA can also be performed other way ELISA can also be performed other way round: the wells are coated with the round: the wells are coated with the antibody that binds antibody that binds the antigenthe antigen present in present in the sample; then, another, enzyme-linked the sample; then, another, enzyme-linked antibody against the antigen is added antibody against the antigen is added

positive sample negat. control

Ag

AbagainstAg

secon-dary Ab

wash → measurement of A

AbagainstAg

Ag

enzyme-linked Ab

E

S

colouredproduct

E

Example: HIV infection screeningExample: HIV infection screening Presence of anti-HIV antibodies in patient's serum is assessed:Presence of anti-HIV antibodies in patient's serum is assessed:

the wells are coated with the antigen – viral protein (e.g. protein of the wells are coated with the antigen – viral protein (e.g. protein of the viral envelope…commercially available) and patient'sthe viral envelope…commercially available) and patient's serum is serum is added; after infection, the serum contains antibodies against the added; after infection, the serum contains antibodies against the viral proteins and these antibodies bind the antigen in the wellviral proteins and these antibodies bind the antigen in the well

after washing, the enzyme-linked secondary antibody is added that after washing, the enzyme-linked secondary antibody is added that binds the patient's antibodies; the enzyme then catalyzes binds the patient's antibodies; the enzyme then catalyzes formation of a coloured productformation of a coloured product

HIVprotein

AbagainstHIV

secondaryAb

positive negative

Turbidimetry and nephelometryTurbidimetry and nephelometry Turbidimetry measures the decrease of the intensity of the Turbidimetry measures the decrease of the intensity of the

TRANSMITTED light compared to the intensity of the incident light TRANSMITTED light compared to the intensity of the incident light that has been caused by scatter on the particles of a SUSPENSION that has been caused by scatter on the particles of a SUSPENSION (e.g. precipitate) (e.g. precipitate)

Turbidance is defined by analogy with absorbance: Turbidance is defined by analogy with absorbance:

Under certain conditions and when using the monochromatic Under certain conditions and when using the monochromatic light, turbidance is directly proportional to the concentration of light, turbidance is directly proportional to the concentration of suspended particles and thickness of the layer (analogy of the B-L suspended particles and thickness of the layer (analogy of the B-L law)law)

Turbidance can be measured by spectrophotometers; the Turbidance can be measured by spectrophotometers; the detector is thus detector is thus aligned with the cell and the source of radiation:aligned with the cell and the source of radiation:

S = log IS = log I00/I/I

detectorI0

transmittedradiation

cuvettesource

Nephelometry measures the intensity of the Nephelometry measures the intensity of the SCATTERED radiation (scattering is again caused by SCATTERED radiation (scattering is again caused by the suspended particles) that is emitted from the the suspended particles) that is emitted from the cuvette in the direction cuvette in the direction perpendicular to the path of perpendicular to the path of incident radiationincident radiation::

The intensity is measured byThe intensity is measured by nephelometers nephelometers::

sourceI0

cuvette

scaterred radiation

detector

ApplicationApplicationss of turbidimetry and of turbidimetry and nephelometrynephelometry

These methods can be used to assess clinically relevant proteins in These methods can be used to assess clinically relevant proteins in blood:blood: an antibody against the protein (antigen) is added and an antibody against the protein (antigen) is added and

formation of immunocomplexes antigen-antibody is monitored formation of immunocomplexes antigen-antibody is monitored e.g.: e.g.:

quantification of CRP (C-reactive protein): its concentration quantification of CRP (C-reactive protein): its concentration increases with all invasive bacterial infections, but rarely with viral infections it‘s determination is useful to the clinician in evaluating acute phase response

quantification of IgG, IgA a IgM: can be helpful in differential quantification of IgG, IgA a IgM: can be helpful in differential diagnosis of chronic liver diseases and in diagnosis of diagnosis of chronic liver diseases and in diagnosis of immuno-deficiencies and autoimmune diseasesimmuno-deficiencies and autoimmune diseases