A
Appendix
Instrument Instructions
Operation of the Analytical Balances
In measuring the mass of a sample, it is important to protect the original materials (reagents) from
contamination. Therefore, dispense only the amount you need to use; if you should take too much, then
you should discard the excess in a proper manner. Never return the excess to the reagent bottle.
An electronic analytical balance must be zeroed before all measurements. To do this, remove any
objects from the balance pan and close all of the windows on the draft shield, then gently press the TARE
button. After a moment, the display should change to a reading of zero grams (0.0000 g). Place the
sample on to the balance pan, close all of the windows on the draft shield, wait for the balance to stabilize,
and record the mass. It will take several seconds for the balance to reach a stable measurement. When
finished, remove the sample, close all of the balance windows, and re-zero the balance. Also remove any
spilled reagents from the balance to prevent corrosion or contamination of the balance.
It is very tempting to use the TARE button to make the mass of a weighing paper or vessel equal
to zero grams in order to simplify calculations. However, this is a bad habit in this laboratory because
many people will use the same balance. Instead, always zero the balance before weighing the paper or
vessel. Then record the absolute mass of the paper or vessel. Subtract this mass from the mass of the
paper + sample to obtain the mass of the sample. This method is referred to as weighing by difference.
There are many cases in this laboratory when you will be instructed to use a mass of reagent that
is close to some value, for example 0.1 g of zinc. This means that any value within 10% of the indicated
mass will be sufficient. Don’t spend a large amount of time trying to get exactly 0.1000 g of zinc unless
the instructions specifically ask for that level of precision (look at the number of significant digits).
However, it is still important to know the precise mass of what you do use. Thus, all masses should be
recorded to four decimal places (to the nearest 0.0001 g).
Due to the great sensitivity of the analytical balance there are many potential sources of weighing
error. The effects of wind current on the balance reading can be eliminated by closing the draft shields as
discussed above. Heated objects must be allowed to return to room temperature before weighing. A
warm object in the balance will create convection currents that will cause the reading to fluctuate. Lastly,
handling an object with bare hands leaves fingerprints behind. The balance is sensitive enough to detect
the small increase in mass from the fingerprint residue. To avoid this problem, use a strip of paper or
crucible tongs to handle objects that must be reweighed.
Operation of the Spectronic Genesys 10
BASIC OPERATION
1. Turn on the Spectronic Genesys 10 (switch is in the back). Allow 30 minutes to warm up.
2. Make sure the outside of the cuvettes are wiped dry and clean of any fingerprints before placing
them in the instrument.
3. Press the button marked SET NM to set the wavelength.
4. Enter the wavelength using the numeric keypad. Press the button marked SET
complete the entry.
NM
again to
5. Insert a cuvette containing the blank into the sample holder in the well marked B. The clear faces
of the cuvette must face the light path of the instrument. Check that the clear faces are against
the sides of the cuvette well that have holes in them. The grooved or frosted faces should be
against the solid walls of the well.
6. Fill a cuvette with the sample or standard. Insert it into one of the other sample holder wells. If
you have several samples or standards, place them in the other wells. Check the orientation of
the cuvettes so that the light will pass through the clear faces.
7. Close sample cover and press the MEASURE BLANK button. This sets the absorbance to zero.
8. To measure the absorbance of the samples and standards, press the corresponding cell-position
button on the keypad (see figure above). When the button of a cell is pressed, that cuvette is
moved to the measuring position and the absorbance appears on the display.
THEORY OF ABSORPTION MEASUREMENTS
There are two properties of light which are of interest in absorptimetry. One is the quality or
type, which is expressed as the wavelength (λ) or frequency (ν); the second is the quantity or amount, which
is referred to as its intensity. The first property determines the kind of matter with which the light will
interact. The second allows us to make quantitative measurements about the interaction of light and
matter, such as by comparing the intensity of light transmitted through a sample to the intensity with
which the sample was illuminated.
In considering the first of these properties it is initially useful to view the quality of light in terms
of frequency (ν) in units of cycles per second. The energy content of light can be described in terms of its
frequency by the equation E=hν , where h=Plank’s constant, 6.63×10-34 J•s. The energy of a beam of light
can be regarded as being divided into discrete units, or quanta, called photons. According to quantum
theory, radiation is absorbed by matter only when the energy content of the photon corresponds to some
energy requirement of the matter with which it comes in contact. Light is absorbed when its energy is
equal to that needed to excite a vibration, rotation, or electronic transition in the atoms and molecules of
the illuminated matter.
Recognizing that the frequency and wavelength of light are related through the speed of light:
c = λν, where c is 2.998×108 m/s. The quality of light can be expressed equally as the frequency or
wavelength. When working in the visible and ultraviolet portion of the spectrum, it is customary to
express the type of light as wavelength in units of nanometers (nm). Thus, you will see wavelength used
throughout this manual and on the spectrophotometers in the laboratory.
Quantitative measurements of light are described by a few simple physical laws that allow us to
express the extent to which the intensity of light is decreased upon passing through a sample. The effects
of the thickness and concentration of a solution upon the intensity of light passing through it are intuitive
to anyone who has estimated the strength of a cup of coffee by looking at how far in to the cup one can
see. Two laws, and their corresponding mathematical expressions, can be used to describe the
phenomena that permit quantitative measurements of light absorption.
The first law expresses the relationship between the thickness of an absorbing substance and the
fraction of incident light that will be absorbed. 1 This law states that if P0 is the radiant power (i.e.,
intensity) of light incident upon a sample of thickness b, the radiant power (P) of light transmitted by the
sample is given by the equation
log
P
 k1b
P0
where b is in cm and k1 is and constant that depends on the concentration, identity of the absorbing
material, etc. Notice that this equation does not include a term for the concentration, so it is not quite
ready to be used for quantitative absorptimetry.
1
This law was first formulated by Bouguer in 1729 and restated by Lambert in 1768.
The second law 2 relates the intensities of light incident upon and transmitted through a solution
of fixed thickness to the concentration of the solution:
log
P
 k 2 c
P0
where k2 is another numerical constant and c is the concentration of the solution.
Combining these two equations gives a single expression that relates the intensity of light to both
the thickness of the solution and its concentration. This expression is commonly known as the BeerLambert Law:
log
P
 abc
P0
which is the fundamental equation of quantitative absorptimetry. The terms used to write the BeerLambert law shown in the above equation are not necessarily the most convenient or practical for making
measurements. The symbol b represents the pathlength or thickness of the absorbing sample and is
always expressed in centimeters. The concentration of the sample is given by c. The quantity a is a
constant whose value depends on the identity of the absorbing species, the wavelength of light,
temperature, and so on. When c is expressed in molarity (moles per liter), the value of a is called the molar
absorptivity and represented by ε.
The ratio of the transmitted and incident radiant power may be expressed in two different ways.
The first is transmittance, T, where
T 
P
P0
The second is known as absorbance, A, where
A   log T or A   log
P
P0
This last equation allows us to write the Beer-Lambert Law in a most useful form:
A  bc
Unlike the prior expression, here the absorbance is directly proportional to the concentration of the
absorbing species. In this equation the molar absorptivity is used indicating that the concentration is in
molarity. The molar absorptivity is then equal to the absorbance of a 1 cm thick sample of 1 M solution.
2
The second law was formulated independently by Beer and Bernard in 1852, although is typically attributed
to Beer.
PRACTICAL ABSORPTION MEASUREMENTS
A spectrophotometer for use in the visible region of the spectrum is a simple instrument. Figure
A-1 is a block diagram of a spectrometer similar in design to the Spectronic Genesys 10 that will be used
in this course. It is called a single-beam spectrophotometer because only one beam of light goes through
the instrument. Spectrophotometers with more complex optical systems are also in widespread use.
Figure A-1: Simple single-beam spectrophotometer. Courtesy of Professor emeritus Stephen Brewer, Department of Chemistry,
Eastern Michigan University.
Any spectrophotometer consists of five basic parts: light source, wavelength dispersion device,
sample cell, detector, and transducer. In the instrument that we will use, the light source is a tungsten light
bulb regulated so that it emits light of constant intensity over the visible spectrum. As in most modern
spectrophotometers, a monochromator containing a diffraction grating is the wavelength dispersion
device. The diffraction grating spreads the component wavelengths of light spatially so that by placing a
narrow slit in one portion of the dispersed spectrum, a small band of wavelengths may be selected.
Typically, the band is 10 to 20 nanometers wide which closely approximates monochromatic light. This
narrow band of light with a radiant power P0 then impinges upon the sample cell. The cell that we will use
is a square cuvette with flat sides. Light of radiant intensity P emerges from the cell. This transmitted
light is detected by a photoelectric device which emits a current proportional to the radiant power striking
it. The final part of the instrument is the transducer. Simply, the transducer converts the detector current
into a voltage, then into a form that we can read (e.g., displacement of a needle, digital numeric display,
etc.)
In practical measurements it is exceedingly difficult to simultaneously measure the quantities P0
and P. Consequently, two measurements are usually made: one for the sample to quantify the transmitted
radiant power (P) and a second for the blank to quantify the incident radiant power (P0). A blank contains
all the sample constituents except the analyte (i.e., the substance whose absorbance is being measured).
The handling of the blank and sample should be as similar as possible: same reagents, same type of
cuvette, same preparation, etc. Prepared in this way, a blank may be used to remove any contribution to
the absorbance that the cuvette or other reagents may have, such that the absorbance of the blank is
defined as A=0.
It is often the case that there is more than one absorbing species in a sample. Fortunately, the
Beer-Lambert Law is additive for multiple species. That is to say, the total absorbance of two or more
absorbing species is the sum of the individual absorbances:
Atotal  A1  A2  A3    An
Atotal  1bc1   2bc 2     n bc n
If the individual absorptivities (ε) of the various species can be determined, it is often possible to calculate
the concentration of each species in the mixture.
Operation of the Thermo Orion pH Meter
CALIBRATION
Calibration must be performed once per day before using the meter for any measurements.
1. Check that the pH electrode is connected to the meter and the meter is on. Press the
button if the meter is off.
POWER
2. Check that the meter is in pH mode (a ▼ will point to pH at the bottom of the display). Press
the MODE button if it is not.
3. If the meter has been recently calibrated, skip to step 15. If the meter has not been calibrated,
proceed with step 4.
4. Remove the glass electrode from the storage solution and rinse the end of it with distilled
water. Gently blot the electrode dry with a Kim-wipe.
5. Place the electrode in the pH 7 buffer. The buffer should be gently stirred.
6. Press the 2ND then CAL buttons to enter the calibration mode. The date and time of the last
calibration will be displayed briefly, and then the meter will display the pH. Wait for the
measurement to stabilize.
7. When READY is displayed next to the reading it will begin to flash and the meter will beep.
Press YES to accept the first calibration point. (The meter will automatically switch to buffer
two as indicated by P2 on the display.)
8. Remove the pH electrode from the buffer. Rinse the electrode with distilled water and gently
blot dry with a Kim-wipe.
9. Place the electrode in the pH 4 buffer and wait for the measurement to stabilize.
10. When READY is displayed next to the reading it will begin to flash and the meter will beep.
Press YES to accept the second calibration point. (The meter will automatically switch to
buffer two as indicated by P3 on the display.)
11. Remove the pH electrode from the buffer. Rinse the electrode with distilled water and gently
blot dry with a Kim-wipe.
12. Place the electrode in the pH 10 buffer and wait for the measurement to stabilize.
13. When READY is displayed next to the reading it will begin to flash and the meter will beep.
Press YES to accept the third calibration point.
14. The meter will automatically switch to measure mode.
15. Remove the pH meter from the buffer. Rinse the electrode with distilled water and gently blot
dry with a Kim-wipe.
The pH meter is now prepared to make measurements.
MEASUREMENT
To make a pH measurement, place the electrode in the sample solution so that the bulb of the glass
electrode is immersed. Gently stir the solution. The pH meter will continuously read the pH.
When finished with the pH meter, always rinse the electrode and replace it into the storage solution.
Operation of the Orion 720Aplus Meter
CHLORIDE DETERMINATI ON
1. Check that the electrodes are properly connected.
Plug the chloride/reference combination electrode into Input 1.
Connect the temperature probe to the ATC/DIN plug.
2. Make sure the meter is on, if not plug it in.
3. Remove the electrodes from the storage solution. Rinse the electrodes with distilled water and
gently blot dry with a Kim-wipe.
4. If the meter has been recently calibrated, skip to step 16. If the meter needs to be calibrated,
press mode until the concentration mode indicator CON appears on the display.
5. Press calibrate. CALIBRATE will be displayed as well as the time and date of the last
calibration.
6. When ENTER NO. STDS prompt appears, enter the number of standards to be used, 2, and
then press yes.
7. Immerse the electrodes in the 10.0 mM chloride standard.
8. Briefly swirl the beaker to mix, but do not stir or mix during the measurement.
9. When the READY ENTER VALUE prompt appears, enter the concentration, 10.0, and press
yes.
10. Remove the electrodes from the solution, rinse electrodes, and blot dry with a Kim-wipe.
11. Immerse in 25.0 mM standard and briefly swirl.
12. When the READY ENTER VALUE prompt appears, enter the concentration, 25.0, and
press yes.
13. The electrode slope is calculated and displayed.
14. Meter advances to MEASURE mode.
15. Rinse electrodes and blot dry with a Kim-wipe.
16. Immerse the electrodes in the sample solution, and briefly swirl the solution, but do not stir
during the measurement, be sure the electrode is immersed in the solution.
17. Wait for the system to equilibrate; MEAS will appear on the screen while the system is
equilibrating, when the system is stable and ready it will display RDY. The system displays the
concentration of chloride in millimolar, mM.
18. Rinse electrodes and gently blot dry with a Kim-wipe.
19. Repeat for the other 3 samples.
20. After the last measurement, rinse the electrodes and return them to the electrode storage
solution.
THEORY OF ELECTRODE MEASUREMENTS
Since the 1920s it was known that a potential or voltage could be developed across a glass
membrane if the solutions on either side of it had different hydrogen ion concentrations. Advances in
electronic instrumentation soon made it possible to exploit that potential to make measurements of
hydrogen ion concentrations or pH. The glass electrode was the first of the ion-selective electrodes.
Generally, ion-selective electrodes are devices that respond to concentration changes in one species of ion
(such as chloride or hydronium), with little interference from other species in the solution.
To understand the functioning of a pH or other ion-selective electrode, it is necessary to look at
some basic principles of electronics. It is essential to appreciate that any electrometric measurement
determines the potential difference between two points. The potential of a single point is not
experimentally measurable without the use of a reference point. That is to say, measurements of potential
are always relative to some standard.
The requirements for measuring a potential indicate that two electrodes will be needed to make a
pH measurement. The first of these is a glass electrode. The thin glass bulb typically contains a solution
of Ag/AgCl and is connected to the meter by a silver wire. The reference electrode often also contains a
silver/silver chloride electrode immersed in a solution of KCl. Unlike the glass electrode, the reference
must be in direct ionic contact with the solution being measured. This is accomplished by allowing a very
slow leakage of the KCl solution into the test solution.
The pH electrode used in this laboratory contains both the glass and reference electrode in one
body. Similarly, the chloride electrode is composed of a reference electrode and an electrode sensitive to
the presence of chloride.
The potential registered by a pH meter is the sum of all of the potentials in the circuit.
E=Eº´ref + Ej – Eglass
Since the reference potential (Eº´ref) and junction 3 potential (Ej) are constant, the measured potential is
dependent only on changes in the potential at the glass electrode. At 25ºC ,
Eglass = Eºglass + 0.059 log aH
where aH is the activity of hydrogen ions, which is a function of concentration. Remembering that
–log[H+]=pH, it can be stated
Eglass=constant + 0.059 pH
Thus, the relationship of measured potential to pH is linear. This linear relationship is one reason why the
pH scale has remained in use and why the pH meter is nearly ubiquitous in chemistry laboratories.
3
The junction potential is actually composed of the sum of all of the small potentials that occur at the
interface of two different materials, such as the junction between the silver wire and solution within the
electrode or even at a point where two types of wire are joined.