p h meter and colorimeter

1. Course: BSc Biotech Sem-I
Subject: Biophysics and Bioinstrumentation
Unit - 3

2. 1

3.  A colorimeter is a light-sensitive instrument that
measures how much color is absorbed by an object
or substance. It determines color based on the red,
blue, and green components of light absorbed by
the object or sample.
 When light passes through a medium, part of the
light is absorbed, and as a result, there is a
decrease in how much of the light reflected by the
medium.
 A colorimeter measures that change so users can
analyze the concentration of a particular substance
in that medium.

4.  The device works on the basis of Beer-
Lambert's law, which states that the absorption
of light transmitted through a medium is
directly proportional to the concentration of the
medium.
 It means that the concentration of a dissolved
substance, or solute, is proportional to the
amount of light that it absorbs. A common
application of a colorimeter is therefore to
determine the concentration of a known solute
in a given solution.

5.  A colorimeter is an instrument which compares
the amount of light getting through a solution
with the amount which can get through a
sample of pure solvent.
 Substances absorb light for a variety of reasons.
Pigments absorb light at different wavelengths.
A cloudy solution will simply scatter/block the
passage of light.
 The % transmission or the % absorbance is
recorded.

6.  At its most basic, a colorimeter works by passing a
specific wavelength of light through a solution, and then
measuring the light that comes through on the other side.
 In most cases, the more concentrated the solution is, the
more light will be absorbed, which can be seen in the
difference between the light at its origin and after it has
passed through the solution.
 To find the concentration of an unknown sample, several
samples of the solution in which the concentration is
known are first prepared and tested.
 These are then plotted on a graph with the concentration
at one axis and the absorbance on the other to create a
calibration curve; when the unknown sample is tested,
the result is compared to the known samples on the curve
to determine the concentration.

7.  The essential parts of a
colorimeter are:
 a light source, which is
usually an ordinary
filament lamp
 an aperture which can be
adjusted
 a detector which
measures the light which
has passed through the
solution
 a set of filters in different
colors
 filters are used to select the
wavelength of light which
the solution absorbs the
most.
 Solutions are usually
placed in glass or plastic
cuvettes.
(1) Wavelength selection,
(2) Printer button
(3) Concentration factor
adjustment,
(4) UV mode selector (Deuterium
lamp)
(5) Readout
(6) Sample compartment
(7) Zero control (100% T),
(8) Sensitivity switch. 7
2

8.  Advantages:
-Can be specific to one chemical species
-Good for process quality control for non-chemistry
personnel
-Can be inexpensive per analysis
 Disadvantages:
-Similar colors from interfering substances can produce
errors in results
-More precise analysis can require tighter wavelength
band width (more expensive)
-Matrix interferences can produce bad results in
uncontrolled situations
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9.  Besides being valuable for basic research in chemistry
laboratories, colorimeters have many practical
applications.
 For instance, they are used to test for water quality, by
screening for chemicals such as chlorine, fluoride,
cyanide, dissolved oxygen, iron, molybdenum, zinc
and hydrazine.
 They are also used to determine the concentrations of
plant nutrients (such as phosphorus, nitrate and
ammonia) in the soil or hemoglobin in the blood and to
identify substandard and counterfeit drugs.
 In addition, they are used by the food industry and by
manufacturers of paints and textiles.
9

10.  A common application of a colorimeter is
therefore to determine the concentration of a
known solute in a given solution.
 In biology, a colorimeter can be used to
monitor the growth of a bacterial or yeast
culture. As the culture grows, the medium in
which it is growing becomes increasingly
cloudy and absorbs more light.
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11. 3

12.  A pH meter is an electronic device used for
measuring the pH of a liquid (though special
probes are sometimes used to measure the pH of
semi-solid substances).
 A typical pH meter consists of a special measuring
probe (a glass electrode) connected to an electronic
meter that measures and displays the pH reading.
 The probe is a key part of a pH meter, it is a rod
like structure usually made up of glass. At the
bottom of the probe there is a bulb, the bulb is a
sensitive part of a probe that contains the sensor.

13.  The probe is a key part of a pH meter, it is a rod
like structure usually made up of glass.
 At the bottom of the probe there is a bulb, the bulb
is a sensitive part of a probe that contains the
sensor.
 Never touch the bulb by hand and clean it with the
help of an absorbent tissue paper with very soft
hands, being careful not to rub the tissue against
the glass bulb in order to avoid creating static. To
measure the pH of a solution, the probe is dipped
into the solution. The probe is fitted in an arm
known as the probe arm.

14. A typical modern pH probe is a combination electrode, which
combines both the glass and reference electrodes into one body.
The combination electrode consists of the following parts (see
the drawing):
1. a sensing part of electrode, a bulb made from a specific glass
2. internal electrode, usually silver chloride electrode or calomel
electrode
3. internal solution, usually a pH=7 buffered solution of 0.1
mol/L KCl for pH electrodes
4. when using the silver chloride electrode, a small amount of
AgCl can precipitate inside the glass electrode
5. reference electrode, usually the same type as 2
6. reference internal solution, usually 0.1 mol/L KCl
7. junction with studied solution, usually made from ceramics
or capillary with asbestos or quartz fiber.
8. body of electrode, made from non-conductive glass or
plastics.
4

15. Key parts of a pH meter: (1) Solution being
tested; (2) Glass electrode, consisting of (3) a
thin layer of silica glass containing metal
salts, inside which there is a potassium
chloride solution (4) and an internal
electrode (5) made from silver/silver
chloride. (6) Hydrogen ions formed in the
test solution interact with the outer surface of
the glass. (7) Hydrogen ions formed in the
potassium chloride solution interact with the
inside surface of the glass. (8) The meter
measures the difference in voltage between
the two sides of the glass and converts this
"potential difference" into a pH reading. (9)
Reference electrode acts as a baseline or
reference for the measurement—or you can
think of it as simply completing the circuit.
5

16.  Between measurements any glass and
membrane electrodes should be kept in the
solution of its own ion (Ex. pH glass electrode
should be kept in 0.1 mol/L HCl or 0.1 mol/L
H2SO4). It is necessary to prevent the glass
membrane from drying out.
 Occasionally (about once a month), the probe
may be cleaned using pH-electrode cleaning
solution; generally a 0.1 M solution of
hydrochloric acid (HCl) is used, having a pH of
one.

17.  For very precise work the pH meter should be
calibrated before each measurement. For normal
use calibration should be performed at the
beginning of each day.
 The reason for this is that the glass electrode does
not give a reproducible e.m.f. over longer periods
of time.
 Calibration should be performed with at least two
standard buffer solutions that span the range of
pH values to be measured. For general purposes
buffers at pH 4.00 and pH 10.00 are acceptable.

18.  The pH meter has one control (calibrate) to set the
meter reading equal to the value of the first standard
buffer and a second control which is used to adjust the
meter reading to the value of the second buffer. A third
control allows the temperature to be set.
 However, for more precise measurements, a three
buffer solution calibration is preferred. As pH 7 is
essentially, a "zero point" calibration.
 After each single measurement, the probe is rinsed
with distilled water or deionized water to remove any
traces of the solution being measured, blotted with a
scientific wipe to absorb any remaining water which
could dilute the sample and thus alter the reading, and
then quickly immersed in another solution.

19.  First, Nobel-Prize winning German chemist
Fritz Haber (1868–1934) and his student
Zygmunt Klemensiewicz (1886–1963)
developed the glass electrode idea in 1909.
 The modern, electronic pH meter was invented
about a quarter century later, around 1934/5,
when American chemist Arnold Beckman
(1900–2004) figured out how to hook up a glass
electrode to an amplifier and voltmeter to make
a much more sensitive instrument.

20. Photo: How do you measure the pH of soils on Mars? Simple!
You build a pH meter into a robotic space probe. The Mars
Phoenix Lander space probe (left) used this built-in, mini
chemical laboratory (right) to measure different aspects of the
Martian soil, including acidity and metal concentrations. Photos
by courtesy of NASA Jet Propulsion Laboratory (NASA-JPL).
6 7

21. READING IMAGES
 Principles and techniques of
biochemistry and molecular
biology by Keith Wilson, John
Walker. – 7th ed.
 http://www.explainthatstuff.com
/how-ph-meters-work.html
 http://en.wikipedia.org/wiki/P
H_meter
 http://www.seafriends.org.nz/d
da/ph.htm
 http://www.fondriest.com/pdf/t
hermo_colorimeter_theory.pdf
1 & 2:
http://www.fondriest.co
m/pdf/thermo_colorimet
er_theory.pdf
3:
http://en.wikipedia.org/
wiki/PH_meter
4-7:
http://www.explainthatst
uff.com/how-ph-meters-
work.html