SPECTROPHOTOMETER
Content:
Principle,
schematic representation, important mathematical relations, applications.
A
spectrophotometer measures the relative amounts of light energy passed through
a substance that is absorbed or transmitted. We will use this instrument to
determine how much light of (a) certain wavelength(s) is absorbed by (or
transmitted through) a solution. Transmittance (T) is the ratio of transmitted
light to incident light. Absorbance (A) = – log T. Absorbance is usually the
most useful measure, because there is a linear relationship between absorbance
and concentration of a substance. This relationship is shown by the
Beer-Lambert law:
A=ebc
Where;
e =
extinction coefficient (a proportionality constant that depends on the
absorbing species)
b = path
length of the cuvette. Most standard cuvettes have a 1-cm path and, thus, this
can be ignored
c = concentration.
A
spectrophotometer or calorimeter makes use of the transmission of light through
a solution to determine the concentration of a solute within the solution. A
spectrophotometer differs from a calorimeter in the manner in which light is
separated into its component wavelengths. A spectrophotometer uses a prism to
separate light and a calorimeter uses filters. Both are based on a simple
design, passing light of a known wavelength through a sample and measuring the
amount of light energy that is transmitted. This is accomplished by placing a
photocell on the other side of the sample. All molecules absorb radiant energy
at one wavelength of another. Those that absorb energy from within the visible
spectrum are known as pigments. Proteins and nucleic acids absorb light in the
ultraviolet range. The following figure demonstrates the radiant energy
spectrum with an indication of molecules, which absorb in various regions of
that spectrum. The design of the single-beam spectrophotometer involves a light
source, a prism, a sample holder, and a photocell. Connected to each are the
appropriate electrical or mechanical systems to control the illuminating
intensity, the wave- length, and conversion of energy received at the photocell
into a voltage fluctuation. The voltage fluctuation is then displayed on a
meter scale, is displayed digitally, or is recorded via connection to a
computer for later investigation.
Spectrophotometers
are useful because of the relation of intensity of color in a sample and its relation
to the amount of solute within the sample. For example, if you use a solution
of red food coloring in water, and measure the amount of blue light absorbed
when it passes through the solution, a measurable voltage fluctuation can be
induced in a photocell on the opposite side. If the solution of red dye is now
diluted in half by the addition of water, the color will be approximately ½ as
intense and the voltage generated on the photocell will be approximately half
as great. Thus, there is a relationship between voltage and amount of dye in
the sample. Given the geometry of a spectrophotometer, what is actually
measured at the photocell is the amount of light energy which arrives at the
cell. The voltage meter is reading the amount of light transmitted to the
photocell. We can monitor the transmission level and convert it to a percentage
of the amount transmitted when no dye is present. Thus, if ½ the light is
transmitted; we can say that the solution has a 50% transmittance.
Transmittance
is the relative percentage of light passed through the sample. The conversion
of that information from a percentage transmittance to an inverse log function
known as the absorbance (or optical density). The monochromator selects a
particular wavelength. The sample and a blank are located in cuvettes. The
light from the lamp passes through the cuvette and hits the phototube. The
meter then records the signal from the phototube.
I0 = incident light, has intensity I0
I = light
coming out of the cuvette (that contains light-absorbing substance), has
intensity I.
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SCHEMATIC REPRESENTATION OF SPECTROPHOTOMETER |
Quantitative Aspects of Light
Absorption: The Lambert-Beer Law
Transmittance,
T, is the amount of light that passes through a substance. It is sometimes
called percent transmission:
T = I/I0 %T
= I/I0
I0 is the
intensity of the incident light and I is the transmitted light. The light
absorbed by the substance at a particular wavelength depends on the length of
the light path through the substance. The negative logarithm of the
transmittance, the absorbance A, is directly proportional to the amount of
light absorbed and the length of the light path, and is described by the
Lambert Law:
–log T =
–log I/I0 = A = Kd where d is the length of the solution in the cell and K is a
constant. The negative log of the transmittance is also directly proportional
to the concentration of the absorbing substance, c, and is described by Beer’s
Law:
–log I/I0 = –log T = A = Kc –log T = A = Edc
where E is a physical constant for a light-absorbing substance. A=E cd, d is
usually 1 cm
A =
absorbance (sometimes called the optical density)
E = molar extinction coefficient
c = concentration of the light-absorbing
substance.
Method
1. Turn on
the spectrophotometer and allow 10 minutes for the instrument to warm up before
use.
2. Adjust
the wavelength to that specified for the procedure you are using.
3. Be sure
the cover is closed on the cuvette holder and use the left knob on the front
panel to adjust the dark current so that the meter is reading 0 transmittance.
At this point, you are simply adjusting the internal electronics of the
instrument to blank out any residual currents. This adjusts the lower limit of
measurements. It establishes that no light is equivalent to 0 transmittance or
infinite absorbance.
4. Insert a
clean cuvette containing the blank into the holder. Be sure that the tube is
clean, free of fingerprints, and that the painted line marker on the tube is
aligned with the mark on the tube holder. Close the top of the tube holder. The
blank for this exercise is the solution containing no dopachrome, but all other
chemicals. The amount of solution placed in the cuvette is not important, but
is usually about 5 mL. It should approximately reach the bottom of the logo
printed on the side of the cuvette.
5. Adjust the meter to read 100%
transmittance, using the right knob on the front of the instrument. This
adjusts the instrument to read the upper limit of the measurements and
establishes that your blank will produce a reading of 100% transmittance (0
absorbance).
6. Remove
the blank from the instrument and recheck that your 0 transmittance value has
not changed. If it does, wait a few minutes for the instrument to stabilize and
read steps 1–5. Periodically throughout the exercise, check that the
calibration of the instrument is stable by reinserting the blank and checking
that the 0 and 100% T values are maintained.
7. To read a sample, simply insert a cuvette
holding your test solution and close the cover. Read the transmittance value
directly on the scale.
8. Record
the percent transmittance of your solution, remove the test tube cuvette, and
continue to read and record any other solutions you may have. It is possible to
read the absorbance directly, but with an analog meter (as opposed to a digital
readout); absorbance estimations are less accurate and more difficult than
reading transmittance. Absorbance can be easily calculated from the
transmittance value. Be sure that you note which value you measure!
Absorption Spectrum
Analysis of
pigments often requires a slightly different use of the spectrophotometer. In
the use of the instrument for determination of concentration (Beer- Lambert
Law), the wavelength was preset and left at a single value throughout the use
of the instrument. This value is often given by the procedure being employed,
but can be determined by an analysis of the absorption of a solution as the
wavelength is varied. The easiest means of accomplishing this is to use either
a dual-beam spectrophotometer or a computer-controlled instrument. In either
event, the baseline must be continuously reread as the wavelength is altered.
To use a single-beam spectrophotometer, the machine is adjusted to 0 first,
with the blank solution, and then the sample is inserted and read. The
wavelength is then adjusted up or down by some determined interval, the 0 is
checked, the blank reinserted and adjusted, and the sample reinserted and read.
This procedure continues until all wavelengths to be scanned have been read. In
this procedure, the sample remains the same, but the wavelength is adjusted.
Compounds have differing absorption coefficients for each wavelength. Thus,
each time the wavelength is altered, the instrument must be recalibrated. A
dual-beam spectrophotometer divides the light into 2 paths. One beam is used to
pass through a blank, while the remaining beam passes through the sample. Thus,
the machine can monitor the difference between the 2 as the wavelength is
altered. These instruments usually come with a motor-driven mechanism for
altering the wavelength or scanning the sample. The newer version of this
procedure is the use of an instrument, which scans a blank and places the
digitized information in its computer memory. It then rescans a sample and
compares the information from the sample scan to the information obtained from
the blank scan. Since the information is digitized (as opposed to an analog
meter reading), manipulation of the data is possible. These instruments usually
have direct ports for connection to personal computers, and often have built-in
temperature controls as well. This latter option would allow measurement of
changes in absorption due to temperature changes (known as hyperchromicity).
These, in turn, can be used to monitor viscosity changes, which are related to
the degree of molecular polymerization with the sample. For instruments with
this capability, the voltage meter scale has given way to a CRT display,
complete with graphics and built-in functions for statistical analysis. A
temperature-controlled UV spectrophotometer capable of reading several samples
at preprogrammed time intervals is invaluable for enzyme kinetic analysis. An
example of this type of instrument is the Beckman DU-70.
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