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SELF- STUDY TUTORIAL

INTRODUCTION TO SPECTROPHOTOMETRY

Learning Outcomes: on completion of this tutorial/background reading, you should be able to:
demonstrate a basic understanding of the electromagnetic spectrum and the laws of light
absorption by chromophores in order to apply them to practical quantitative analysis using
spectrophotometric and colorimetric assays.

The electromagnetic spectrum (Figure 1) is a continuum of waves of different properties.

Each section of the spectrum can be used for some type of analysis but the areas of chief
interest to biomolecular scientists are the near ultraviolet (180 – 350 nm) and visible
(350 – 800 nm) regions (* Figure 1). Such radiation may be used for chemical quantitative
analysis, eg. spectrophotometric determination of concentration.

The energy of radiation is related to its wavelength by the equation:

h.c
E = ────


where: E = energy,
c = velocity of light
 = wavelength and h = Planck’s constant

Therefore, the energy of the radiation is inversely proportional to its wavelength.

For example: UV and blue light have a shorter wavelength and thus more energy than
red light that has a longer wavelength and thus less energy.

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Figure 1: THE ELECTROMAGNETIC SPECTRUM

Wavelength () Regions of the Interaction Main biological

(m) (nm) EM Spectrum matter applications
---------------- ---------------- ----------------------------------------------- --------------------------
Cosmic rays
 10–12  10–3  Atomic nucleus
-rays Sterilising
 10–11  10–2
----------------------------------------------- ------------------------
 10–10  10–1
X-rays Inner shell electrons X-ray crystallography
 10–9  1 Medical radiography
-------------------- -------------------------- ------------------------
 10–8  10
Ultraviolet (180 – 350 nm) * Chemical quantitative
 10–7  102 Valency electrons analysis: determination
Visible (350 – 800 nm) * of concentration.
 10–6  103 -------------------- -------------------------- ----------------------
Light atoms
 10–5  104  Chemical qualitative
Infrared Molecular vibrations analysis: identification
 10–4  105  of functional groups.
Heavy atoms
 10 –3
 10 6
-------------------- -------------------------- ---------------------
Small molecules
 10–2  107 Microwave  Sterilising
Molecular rotations (Cooking)
 10–1  108 -----------------  (Mobile phones)
Large molecules
 1  109 -------------------------- ----------------------
Nuclear Magnetic Analytical NMR
 10  1010 Radiowave Resonance (NMR) Medical ‘scanners’
 ------------------------- (Wireless
 10 2
 10 11
 communication)

----------- ---------------- ------------------- -------------------------- -----------------------

When a photon strikes an atom or a molecule, one of two things may happen: the photon
may be scattered, or it may be absorbed and its energy transferred to the atom or
molecule, producing an excited state. For a photon to be absorbed, its energy must
match the energy difference between two energy levels of the atom or molecule, ie. the
electron transfers from its ground state to an excited state.

The excited atom or molecule reverts to its ground state by the re-emission – almost
instantaneously and in a random direction – of radiation:
either of the same wavelength in a process known as absorption (or
extinction)
or of a longer wavelength in processes known as fluorescence (and
phosphorescence).

Photons in the visible and UV regions of the spectrum have sufficient energy only to excite
two types of electrons: (i) Lone pairs (requiring a wavelength around 208 nm, ie. fairly high

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energy) and (ii) Double bonds (requiring a wavelength well into the ultraviolet and visible
regions, ie fairly low energy). Hence, visibly coloured compounds often have many double
bonds (usually ‘conjugated’).

BASIC LAWS OF LIGHT ABSORPTION

For a uniform absorbing medium, the proportion of the radiation passing through is called
the TRANSMITTANCE (T),

T = I / I0 where I is the intensity of the transmitted radiation,

and I0 is the intensity of the incident radiation.

The term ABSORBANCE (A) or (more correctly) EXTINCTION (E) is defined as...

A ( = E ) = log 1/T = log I0/I

...and is more commonly used because of its relationship with concentration.

The Beer-Lambert law states that the Absorbance is directly proportional to the
concentration of the absorbing substance and to the thickness of the layer:

A ( = E ) = x.c.d where x is the molar extinction coefficient for the absorbing

substance at wavelength x ,
c is the concentration of the absorbing substance in
mole.dm-3, and
d is the length of the light path in the absorbing
substance in cm.

Transmittance is usually expressed on a range of 0 – 100 %. However, Absorbance has

no units (as it is the log of a ratio) and varies from 0 – infinity (but note well, it is a
logarithmic scale (See Table 2).

Non-Applicability of the Beer-Lambert Law:

Often, the Beer-Lambert law may not be applicable to a system for a number of reasons,
the relationship between Absorbance and concentration sometimes deviating from
linearity:
Firstly, the absorbing substance may ionise or polymerise at higher concentrations, or it
may coagulate to give a turbid suspension that may increase or decrease the apparent
Absorbance.
Secondly, the instrument itself may have some faults. It may be susceptible to stray
radiation as well as only being capable of producing light beams of finite waveband (the
Beer-Lambert law was derived for monochromatic light).

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Table 2: RELATIONSHIP BETWEEN TRANSMITTANCE AND ABSORBANCE

% light Transmittance Absorbance

absorbed % fraction 1/T A = log 1/T
------------ ------------ ------------ ------------ -----------------
0 100 1.00 1 0.00
25 75 0.75 1.3 0.12
50 50 0.50 2 0.30
75 25 0.25 4 0.60
83 17 0.17 6 0.95
90 10 0.10 10 1.00
95 5 0.05 20 1.30
99 1 0.01 100 2.00
99.9 0.1 0.001 1000 3.00
100 0.0 0.000 infinity infinity
------------ ------------ ------------ ------------ -----------------

SOME TERMS AND DEFINITIONS

A CHROMOPHORE is the atom or group of atoms in a chemical compound responsible

for its colour.

ABSORPTION (or EXTINCTION) is the process whereby radiation is absorbed by a

chromophore AND then – almost instantaneously – RE-EMITTED (in a random
direction) as radiation of the SAME wavelength.

FLUORESCENCE is the process whereby radiation is absorbed by a chromophore AND

then – almost instantaneously – RE-EMITTED (in a random direction) PARTLY as
radiation of LONGER wavelength and PARTLY as HEAT.

PHOSPHORESCENCE is very SLOW fluorescence whereby the absorbed radiation is

RE-EMITTED (in a random direction) LONG AFTER the source is switched off.

The WAVELENGTH RANGE is the range of available wavelengths that a

spectrophotometer is designed to generate and/or detect. The greater the range
(especially if involving UV and IR), the more expensive is the instrument!

BANDWIDTH: the light emerging from a monochromator does not consist of a single
wavelength but a group of wavelengths known as bandwidth. The bandwidth is important
because it is indicative of the actual wavelengths involved in a given Absorbance
measurement and determines the resolution of the spectrophotometer: the narrower the
bandwidth, the better the resolution but also the more expensive is the instrument!

RESOLUTION is the ability to distinguish two adjacent absorption peaks.

The SENSITIVITY of a spectrophotometer is its ability to distinguish small differences in

Absorbance. High sensitivity is only achievable with instruments fitted with photomultiplier
tubes (which are expensive!).

The ABSORBANCE RANGE is determined by the sensitivity.

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An ABSORPTION SPECTRUM is a plot of the amount of the radiation (Absorbance or

Extinction) absorbed by a compound as a function of wavelength.

A DIFFERENCE SPECTRUM is the difference between two absorption spectra of two

different compounds or two different forms of the same compound. It may be obtained
indirectly by subtracting one absorption spectrum from another but, in practice, it is
usually obtained directly by placing one compound in the reference cuvette.

SPECTROPHOTOMETRIC AND COLORIMETRIC ASSAYS

SPECTROPHOTOMETRY: the measurement of the INTENSITY of the radiation at

selected wavelengths in the electromagnetic spectrum.

COLORIMETRY (definition I): the measurement of the INTENSITY of the COLOUR of a

substance or solution. In reality, this is simply a sub-division
of spectrophotometry (as defined above).
(definition II): the photometric assay of COLOUR developed by a
chemical reaction (eg: the assay of a non-chromophoric
solute using a colorimetric procedure involving reaction with
a reagent to give a coloured product).

Colorimetric assays (colorimetry as in definition II) are extremely important and widely-
used techniques in ANALYTICAL BIOCHEMISTRY. Substances, which do not possess
significant extinction coefficients in the visible region of the spectrum (and this includes
very many compounds of biological interest), can be made to react quantitatively with
some other reagent to give COLOURED products. This property is used as a basis for
estimating such substances. Moreover, if the method has been well designed in terms of
the wavelength at which the chromophoric product absorbs, the assay can be performed
in the presence of other compounds (a situation frequently encountered in biological
extracts) without the need for purification.

The colour is produced under STANDARD conditions from known quantities (or
concentrations) of the substance and the Absorbances (Extinctions) of these samples are
measured, using a REAGENT BLANK in the reference cuvette. A reagent blank contains
all of the reagents except the substance being estimated. The Absorbance reading is set
to ZERO using this blank. The measured Absorbances are plotted against the quantity
(or concentration) of the test substance producing the colour. This graph is known as the
CALIBRATION CURVE. Then, UNKNOWN quantities (or concentrations) of the
substance may be reacted to produce the colour under the same standard conditions, the
Absorbances measured and, using the calibration curve, the quantity (or concentration)
thus estimated.

It is often possible to produce comparatively high Absorbances with relatively small

amounts of substance (most biomolecules are present at relatively low concentrations)
thereby making colorimetric assays sensitive enough for many biochemical analyses.

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Spectrophotometric assays (including colorimetric assays as defined under colorimetry

– definition I) are also of considerable importance but have much more specialized uses in
biochemical analyses. They are naturally much easier to perform in that measurements of
Absorbance can be made directly but there are very few biomolecules which absorb light
in the visible region of the spectrum, and those which absorb in the UV region often have
Absorption peaks close to other interfering compounds. However, the general principles of
these assays and the precautions to be taken (as described below) are very similar to
those for colorimetric assays where the colour is generated by reaction with a colour-
forming reagent.

INSTRUMENTATION

For the different sorts of spectrophotometers and colorimeters used at Coventry

University, you should consult the Appendices at the end of the Molecular/Analytical
Biochemistry Unit booklet where you will also find the operating instructions.

The diagrams on the next page show the basic layouts of some spectrophotometric
equipment:
Colorimeter
Spectrophotometer
Fluorimeter. (US spelling, Fluorometer)
Further explanation will be given in the laboratory classes.

SPECTROPHOTOMETRIC EQUIPMENT

a) Colourimeter

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b) Spectrophotometer

i) Single beam machine

The monochromator is usually a diffraction grating, rather than a prism.

The detector is either a photomultiplier tube or a diode array, the latter is more sensitive at
the red end of the spectrum (>600nm).

Single beam machines require the blank to be placed in the light beam first, the machine
set to zero, then the test solution is measured in the light beam.

Double beam machines pass the light through two cuvettes simultantously (or almost) and
subtract the blank reading from the test. There are a number of differences of detail in
these machines, but often the light is either split into two and each half sent through one of
the cuvettes to two separate detectors, or the same beam is “chopped” and sent rapidly
alternately through one cuvette then the other. The Perkin Elmer machines in the
Biochemistry lab use this principle; the light is chopped 32 times / second and the blank
value subtracted automatically each time from the test.

ii) Double beam machine – split light beam

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Note two light sources. A tungsten (W) lamp for visible light, and a deuterium (D2) lamp for ultra
violet.

c) Fluorimeter (U.S. = Fluorometer)

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COLORIMETRIC ASSAYS – POINTS TO REMEMBER

REPRODUCIBILITY IS FAR MORE IMPORTANT THAN THE BEER-LAMBERT

RELATIONSHIP

It is the REPRODUCIBILITY of the assay in the investigator's hands that determines

the ACCURACY and PRECISION of the final data, not necessarily what the instructions
state. Both accuracy and precision may be good if (a) the investigator is competent and
meticulous, and (b) all the equipment used is identical to that in the protocol and also in
proper working order.
For MAXIMUM REPRODUCIBILITY, all references, standards and "unknowns" should
be treated IDENTICALLY. In other words, all samples should be prepared at roughly the
same time, with the same reagents, employing the same pipettes, using the same
spectrophotometer and even the same operator, etc, etc.
The calibration curve MAY VARY between batches of reagents and standards and,
hence, a NEW calibration curve should always be produced unless there is evidence to
suggest that the previously obtained curve is still valid. In other words, all calibration
curves cannot be used as "standard curves".

METICULOUS ATTENTION MUST BE PAID TO DETAILS AND LIMITATIONS OF THE

METHOD

The apparatus (test-tubes, pipettes, etc) must be CLEAN and DRY; volumes, timings
and temperatures must be accurately measured and adhered to.
The cuvettes should ideally be OPTICALLY MATCHED (checked by reading them
against each other in the spectrophotometer).
The reference cuvette should be a true REAGENT BLANK, that is, contain all the
reagents other than the substance being assayed in the same concentrations as the test
cuvette.
The reference cuvette and standards require, if anything, MORE CARE than any other
because any error in these will be reflected in all measurements.

FOR THE PRODUCTION OF AN ACCURATE CALIBRATION CURVE:

A MINIMUM OF FIVE POINTS are required to plot a straight line and MORE THAN
FIVE for a curve.
Normally, all estimations should be performed at least in DUPLICATE and individual
values, NOT mean values, should be plotted.
The Absorbance obtained for a reagent blank is as important BUT NOT MORE SO
than that for any other standard. In other words, a best-fit line should be drawn using all
the points, not necessarily the best-fit line through the origin and the other points.
Because of the LOGARITHMIC nature of the Absorbance scale, calibration curves are
most accurate over the range A = 0.0 to 1.0 (sometimes only up to 0.6), particularly on
basic colorimeters and spectrophotometers. However, it is better to use a calibration curve
with data that extend beyond this rather than extrapolate a curve beyond this range from
inadequate data.

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CHOICE OF WAVELENGTH TO USE FOR A PARTICULAR PHOTOMETRIC ASSAY:

A chromophore has the COMPLEMENTARY colour to that which it absorbs; eg: a blue
compound appears blue because it absorbs red light; thus, it must be estimated in the red
region of the spectrum!
Colorimetric assays are most sensitive at the WAVELENGTH OF THE ABSORPTION
PEAK (max) of the difference spectrum of the chromophore produced minus reagent
blank. Thus, ideally, this spectrum should be obtained before commencing the assay on
any one spectrophotometer. This enables accurate estimations to be performed even on
poorly calibrated instruments.
If a lower sensitivity can be tolerated, colorimetric assays do not necessarily need to be
performed at max for a number of reasons, eg. interference from other chromophoric
substances, inadequate range of wavelength adjustment on a particular
spectrophotometer, etc.

References

Reed, R., Weyer, J., Jones, A., Holmes, D. (2007) Practical Skills in Biomolecular
Sciences. 3rd ed. Benjamin Cummings.

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