1. Presented by Clarinda Clare Linus
Diploma in Medical Sciences
January intake 2011

2.  Measure the wavelengths of visible light that
various colores dolutions absorb.
 Understand reason/s as to why each solution
appears as a particular color after gathering
results.
 Learn and appreciate the concepts of
spectrophotometry.

3.  A spectrometer (spectrophotometer,
spectrograph or spectroscope) is an
instrument used to measure properties
of light over a specific portion of
theelectromagnetic spectrum, typically used
in spectroscopic analysis to identify
materials.

5.  Spectrophotometer
 Distilled water
 Colored solution (Red, Blue, Green & Yellow)
 Cuvettes

6.  1. Turn on the power switch, set the desired
wavelength and allow to warm up at least 15
minutes.
 2. Set the transmittance to zero.
 3. Place the blank into the sample compartment
with the vertical line on the cuvette aligned with
the mark on the front edge of the sample
compartment, and close the lid.
 4. Remove the blank from the sample
compartment. Change to "Absorbance" using the
"mode" button.

7.  6. Place a cuvette containing color sample
into the sample compartment, close the lid,
and read the absorbance from the meter.
 7. Record readings and remove sample.
 8. Repeat steps 1 – 7.

8.  Special light filters may be required on some
spectrophotometers if working at certain
wavelengths.
 The machine must be zeroed out after each
sample or if changing the wavelength.
 Failure to allow the spectrophotometer
enough time to warm can result in erroneous
results.
 Make sure cuvettes are free of any particles,
smudges or fingerprints, as these can throw
the machine's calculations off.

9. 4
2 distilled water
red
0 green
distilled water
425
450
yellow
475
500
525
550
575
-2 600
625
650
675
blue
-4

10.  Distilled water – The highest spectrum is at
425nm
 Red – The highest spectrum is at 525nm
 Green – the highest spectrum is at 625 nm
 Yellow – the highest spectrum is at 425nm
 Blue – The highest spectrum is at 425nm

11.  Highest absorbance : Green
 Lowest absorbance : Yellow

12.  According to the result green has the higest
absorbance.
 This is due to the combination of color which
is blue and yellow.

13.  The concentration of colored solute in a
solution is directly proportional to the
intensity of its color,
 which in turn is proportional to the amount of
absorbance of light at the wavelength that the
color absorbs.
 The color, or absorbance, of a solution is also
proportional to the path length that the light
passes through. This is often expressed as
the Beer-Lambert Law, or Beer’s Law:

14.  A=εCl
 Where A is absorbance at a given wavelength
of light,
 ε is the extinction coefficient (amount of
color of the solute per mole)
 C is the concentration of solute in the
solution (doubling the concentration
 doubles the amount of light absorbed)
 and l is the pathlength (if you double the
length of the path of light, you double the
 opportunities for it to be absorbed)

15.  The sequence of events in a modern
spectrophotometer is as follows:
 The light source is imaged upon the sample
 A fraction of the light is transmitted or
reflected from the sample
 The light from the sample is imaged upon the
entrance slit of the monochromator
 The monochromator separates the
wavelengths of light and focuses each of
them onto the photodetector sequentially

16.  Spectrophotometric techniques are used to
measure the concentration of solutes in
solution by measuring the amount of light
that is absorbed by the solution in a cuvette
placed in the spectrophotometer.
Spectrophotometry takes advantage of the
dual nature of light. Namely, light has:
 1. a particle nature which gives rise to the
photoelectric effect
 2. a wave nature which gives rise to the
visible spectrum of light

17.  Absorbance is measured to see the
relationship between concentration of a
compound and its absorbance.