SPECTROPHOTOMETRY

 Spectrophotometry is a method to measure how much a chemical substance absorbs

light by measuring the intensity of light as a beam of light passes through sample
solution.
 Every chemical compound absorbs, transmits, or reflects light (electromagnetic
radiation) over a certain range of wavelength.

 The basic principle is that each compound absorbs or transmits light over a certain range
of wavelength.
 Spectrophotometry is one of the most useful methods of quantitative analysis in various
fields such as chemistry, physics, biochemistry, material and chemical engineering
and clinical applications.
 In biochemistry, for example, it is used to determine enzyme-catalyzed reactions.
 In clinical applications, it is used to examine blood or tissues for clinical diagnosis.
 There are also several variations of the spectrophotometry such as atomic absorption
spectrophotometry and atomic emission spectrophotometry.
 A spectrophotometer is an instrument that measures the amount of photons (the
intensity of light) absorbed after it passes through sample solution. Also, the amount of a
known chemical substance (concentrations) can also be determined by measuring the
intensity of light detected.

 Depending on the range of wavelength of light source, it can be classified into two different
types:
1. Ultraviolet (UV)-visible spectrophotometer: uses light over the ultraviolet range
(185 - 400 nm) and visible range (400 - 700 nm) of electromagnetic radiation
spectrum.
2. Infrared (IR) spectrophotometer: uses light over the infrared range (700 - 15000
nm) of electromagnetic radiation spectrum.
 In visible spectrophotometry, the absorption or the transmission of a certain substance
can be determined by the observed color. For instance, a solution sample that absorbs
light over all visible ranges (i.e., transmits none of visible wavelengths) appears black in
 theory. On the other hand, if all visible wavelengths are transmitted (i.e., absorbs
nothing), the solution sample appears white. If a solution sample absorbs red light (~700
nm), it appears green because green is the complementary color of red. Visible
spectrophotometers, in practice, use a prism to narrow down a certain range of
wavelength (to filter out other wavelengths) so that the particular beam of light is passed
through a solution sample.

Devices and mechanism

 It consists of a light source, a collimator, a monochromator, a wavelength selector, a

cuvette for sample solution, a photoelectric detector, and a digital display or a meter.

 A spectrophotometer consists of two devices; a spectrometer and a photometer.

a) A spectrometer is a device that produces, typically disperses and measures light.
 It produces a desired range of wavelength of light.
1. First a collimator (lens) which transmits a straight beam of light (photons)
that passes through a monochromator (prism).
2. The monochromator splits light into several component wavelengths
(spectrum).
3. The wavelength selector (slit) transmits only the desired wavelengths.
b) A photometer indicates the photoelectric detector that measures the intensity of
light.
  After the desired range of wavelength of light passes through a cuvette which
contains the solution of a sample.
 It is then passed through the photometer which detects the amount of photons
that is absorbed and then sends a signal to a galvanometer or a digital
display.
 There are two kinds of spectrophotometers based on the beams used: single beam and
double beam.
1. Double beam spectrophotometers- it compares the light intensity of the spectrum
from a sample to a reference beam.
 Applications that require stability, speed, and automation rely on double beam
spectrophotometers and are expensive. These typically have similar or better
precision than single beam spectrophotometers.
2. Single beam spectrophotometers- it is cost-effective compared to double beam
variants and have the potential to perform better, as they do not need to expend
energy splitting the beam. However, these devices are less stable than their double
beam counterparts. Moreover, they require more work, as users must provide a
reference to standardize the device before using it.
  A spectrometer is used to produce a variety of wavelengths because different compounds
absorb best at different wavelengths. For example, p-nitrophenol (acid form) has the
maximum absorbance at approximately 320 nm and p-nitrophenolate (basic form) absorb
best at 400nm.

Absorbance of different compounds

 The graph below that measures absorbance and wavelength, an isosbestic point can also be
observed.
 An isosbestic point is the wavelength in which the absorbance of two or more species are the
same. The appearance of an isosbestic point in a reaction demonstrates that an intermediate is
NOT required to form a product from a reactant.
 The amount of photons that goes through the cuvette and into the detector is dependent on
the length of the cuvette and the concentration of the sample.
 Once you know the intensity of light after it passes through the cuvette, you can relate it to
transmittance (T).
 Transmittance is the fraction of light that passes through the sample. It can be calculated
using the equation:

 Where:
It =light intensity after the beam of light passes through the cuvette.
Io =light intensity before the beam of light passes through the cuvette.
 Transmittance is related to absorption by the expression:

 Absorbance (A) stands is the amount of photons that is absorbed.

 With the amount of absorbance known from the above equation, you can determine the
unknown concentration of the sample by using Beer-Lambert Law.
 The figure below illustrates transmittance of light through a sample. The length l is
used for Beer-Lambert Law.
Beer-Lambert Law

 Also known as Beer's Law.it states that there is a linear relationship between the absorbance
and the concentration of a sample. Hence, Beer's Law can only be applied when there is a
linear relationship. Beer's Law is written as:

 Where:

A=measure of absorbance (no units).

ϵ=molar extinction coefficient or molar absorptivity (or absorption coefficient).

L=path length.

C=concentration.

 The molar extinction coefficient is given as a constant and varies for each molecule. Since
absorbance does not carry any units, the units for ϵ must cancel out the units of length and

concentration. As a result, ϵ has the units: L·mol-1·cm-1. The path length is measured in
centimeters. Because a standard spectrometer uses a cuvette that is 1 cm in width, l is always
assumed to equal 1 cm. Since absorption, ϵ, and path length are known, we can calculate the
concentration c of the sample.
QUESTIONS

1. The absorption coefficient of a glycogen-iodine complex is 0.20 at light of 450 nm. What
is the concentration when the transmission is 40 % in a cuvette of 2 cm?.

2. Guanosine has a maximum absorbance of 275 nm. ϵ275=8400M−1cm−1 and the path

length is 1 cm. Using a spectrophotometer, you find the A275=0.70. What is the
concentration of guanosine?
3.
 a. There is a substance in a solution (4 g/liter). The length of cuvette is 2 cm and only
50% of the certain light beam is transmitted. What is the absorption coefficient?
b. How much is the beam of light is transmitted when 8 g/liter?

CALORIMETER
 A colorimeter is an electrically powered instrument which measures the concentration of
analytes in coloured solutions.
 It is a simple version of a photometer. The difference in the quality of its filters makes it
less sensitive.
 The colorimeter is used for clinical chemistry, namely for determining hemoglobin
concentrations.
 Colorimeters may be manual or semi-automated. Absorbance readings are done with
needle or digital readouts. The haemoglobinometer is a portable colorimeter designed to
provide direct, accurate haemoglobin concentration readings in g/dl or g/l.

OPERATING PRINCIPLE
 A colorimeter uses filters to produce light of a single wavelength selected according to
the colour of the solution being measured.
 The coloured light passes through the sample and the amount of light emerging is
measured on a scale of absorbance. The absorbance is directly proportional to the
concentration of the coloured compound in the solution according to Beer-Lambert law.
 It can usually measure reliably between 0 and 0.7 absorbance units.
 Calibration factors are higher for colorimeters than for photometers as they are less
sensitive.
 Haemoglobinometers measure the concentration of haemoglobin in blood. Most require
dilution of blood before hemoglobin measurement. Some models use a device for
collecting blood without dilution; these devices are single use and disposable, thus
increasing the cost of haemoglobin estimation.
COMPONENTS
 The basic components of colorimeters are similar to those of a photometer.
 The light source may be a diode lamp emitting monochromatic light. Alternatively light
produced by a tungsten or halogen lamp may be filtered to achieve the required
wavelength.
 The components are:
 Display window.
 ON/OFF button.
 Cuvette chamber.
 Test button.
 Reference button.
 Various modes selection button, e.g., Absorbance/%Transmittance, Kinetics.

OPERATION OF THE COLORIMETER

 Connect the unit to the power supply and switch ON.

 Allow 15 minutes for the instrument’s optical and electronic systems to warm up.
 Select the correct wavelength for the compound to be tested e.g. 540 nm for
haemoglobincyanide.
 Select “absorbance” using the Mode button.
 Arrange all the required solutions in a test rack: blank (reagent containing no sample);
standard of known concentration and test solutions (samples).
  Carefully clean the cuvette using lint-free soft tissue or lens paper to avoid scratches.
Always hold by the opaque ground side.
 Transfer the blank solution into the cuvette and place it into the sample compartment with
the clear sides facing the light path.
 Close the chamber and set the display to zero using the SET BLANK control.
 Remove the cuvette from the compartment and pour the solution back into its original test
tube.
 Pour the standard solution into the cuvette and read the absorbance.
 Repeat step 9.
 Read the test solutions in the same fashion.
 Using a table of values obtained from a calibration curve derived from the instrument,
read the concentration of the test samples against the absorbance.
 After use, switch off the power supply and cover the equipment to protect it from dust.
 Rinse the cuvette with distilled water, drain dry and wrap in soft material. Store carefully
into a small box to prevent scratches and dust.

ROUTINE MAINTENANCE

Frequency: Daily

1. Any spill on, or around the instrument should be cleaned immediately.

2. At the end of the day, turn off the instrument or disconnect the power source or the
battery terminals as appropriate.
3. Keep the cuvette chamber empty and closed when not in use.
4. Cover the instrument after use. Store appropriately, protected from dust.

Frequency: As needed
1. Replace blown fuses and bulbs according to the manufacturer’s instructions.
2. If the equipment is faulty, consult a qualified biomedical engineer.
Frequency: Every six months
1. Inspect the instrument visually to verify the integrity of its components according to the
manufacturer’s specifications.
2. Verify that the buttons or control switches and mechanical closures are mounted firmly
and that their labels are clear.
3. Ensure that all the accessories are clean and intact.
4. Check the adjustment and condition of nuts, bolts and screws.
5. Make sure the electrical connections do not have cracks or ruptures. Test that these are
joined correctly.
6. If applicable:
a) Verify that cables securing devices and terminals are free from dust, grime or
corrosion.
b) Verify that cables are not showing signs of splicior of being worn out.
c) Examine that the grounding system (internal and external) is meeting the electric
code requirements.

7. Make sure the circuit switches or interrupters, fuse box and indicators are free from dust,
corrosion and grime.
8. Check lamp alignment if recommended by the manufacturer.
General maintenance

 .Cuvette use and maintenance Cuvettes must be rigorously clean for accurate
measurements.
1. Always hold cuvettes by their opaque, non-optical walls.
2. Unless specified by the operator’s manual, do not perform any measurements
without performing a blank determination.
3. Use a single cuvette or a set of matched cuvettes for proper performance of the
instrument. Note: Absorbance of cuvettes should not exceed 0.01 when measuring
distilled water. To avoid incorrect results, a cuvette exceeding this limit should
not be used as part of a set unless it is matched with one with the same absorbance
reading when measuring distilled water.
4. Remove bubbles present in the solution by gently tapping the cuvette with the
finger.
5. Ensure that there is a high enough level of solution in the cuvette (above the light
beam) so that the reflection of light from the surface does not interfere with the
reading.
 6. All solutions used and the specimen to be measured should be clear. If the mixed
reagent solution and specimen is turbid, the measurement must be repeated
after checking and confirming the cuvette’s transparency and cleanliness.
7. If a kinetic measurement is performed over a long period of time, seal the cuvette
to avoid evaporation causing erroneously high readings. When performing
readings on a series of specimens, read just the zero every 5 to 10 measurements
by reading the blank solution to avoid a drift of the zero.
8. Do not leave the cuvette in the instrument.
9. If using semi-micro or micro-cuvettes, ensure correct positioning in the light path
to avoid false readings due to partially reflected light.
10. Store in a dust-free box to prevent damage as scratched or damaged cuvettes can
lead to incorrect measurements.

Optical filters use and maintenance

 Handle removable filters by the circumference to avoid contamination.

 Keep spare filters in a dust-free box to insure protection from breakage or scratches.
 Ensure that a filter is in its slot when the lamp is turned ON to avoid damage to the
photocell. Store filters in the appropriate storage box when the instrument is not in use.
 When the instrument is cool and turned OFF, clean the filters and optical window with
lens tissue as instructed by the manufacturer.

Light source use and maintenance

1. Turn OFF the lamp after each use to maximize its life span. Some manufacturers
recommend keeping a record log of the instrument lamp use.
2. Check lamp periodically. Replace if it is the cause of instability in the absorption signal.

Lamp alignment
 The following are procedures to align new lamps. Realign the new lamp as
follows:
1. Place a clean cuvette filled with distilled water in position in the
instrument.
2. Set the meter to a mid-scale reading, e.g. at 50% transmission.
3. Move each optical component slightly in turn and check if the reading was
affected.
4. If needed, adjust the lamp alignment for maximum
transmission.
5. Alternatively, place a white card in front of the photocell
(some instruments will allow this). Observe the image of
the lamp on the card. It should be vertical and in focus.
If not, adjust the lamp alignment until the best image
is obtained.