1
General Aspects of Spectroscopy
Revision
The following questions cover the important concepts that you should have understood in the first
year instrumentation subject.
1.
What is the difference between absorption and emission of radiation?
2.
Why are the peak wavelengths in the absorption and emission spectra of the same species
identical?
3.
In spectroscopic terms, what is the difference between an atom and a molecule? How do their
spectra differ in appearance?
4.
Rank the following regions of the electromagnetic spectrum - ultraviolet, visible and infrared in terms of increasing energy, frequency and wavelength.
5.
How do absorbance and transmittance differ? Which is more useful for analytical purposes?
Why?
1. General aspects of spectroscopy
6.
State Beer’s Law, and explain the meaning of each term. Under what situations does Beer’s
Law not apply?
7.
Explain how you could determine what concentration range for a given species obeyed Beer’s
Law.
8.
Draw a schematic diagram showing the components of a typical absorption
spectrophotometer.
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1. General aspects of spectroscopy
1.1 Radiation sources
All spectroscopic instruments require a radiation source of some type, since it is radiation that is
measured to provide the analytical measurement: spectrum or absorbance. The type of source varies,
particularly between absorption and emission instruments.
EXERCISE 1.1
What is the most important difference between the radiation source in absorption and emission
instruments?
This section will concentrate on matters relating to radiation sources for absorption instruments.
Role of radiation sources (in absorption instruments)
To provide radiation that can be absorbed at specific wavelengths by the analyte, allowing a
comparison of intensity before and after sample
General requirements
The most obvious necessity for a radiation source is that it produces radiation in the wavelength range
that the instrument is designed to operate. Clearly, a source that produces mostly infrared (IR)
radiation would not be much use in a UV-VIS spectrometer.
The majority of radiations sources are continuous: this means that they produce radiation at every
wavelength across the range they are designed to work in.
EXERCISE 1.2
One absorption instrument that you have used does not have a continuous source. Which one is it?
Continuous radiation sources, regardless of wavelength range, have a number of general
characteristics:
•
the intensity should be consistent across the range (normally it will taper off at the extremes of
the range),
•
the intensity should not fluctuate over time
•
the intensity of radiation should be not be too low
•
the intensity of radiation should be not be too high
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1. General aspects of spectroscopy
EXERCISE 1.3
Why are these characteristics regarding source intensity important?
(a)
consistent across the range
(b)
consistent over time
(c)
not too low
(d)
not too high
The consistency across the range of wavelengths is a matter of the choice of emitting substance in the
source, while the amount of intensity is determined the current flowing through the source, a matter
of design. The consistency over time is affected by the voltage provided by the power supply. This can
be affected by local fluctuations in the electricity grid, due to start up or shut down by heavy power
users. Devices to “damp” such disturbances are built into modern instruments.
1.2 Wavelength selectors
As you know, different chemical species absorb at different wavelengths, which makes them able to
be distinguished from other species.
The role of a wavelength selector
To reduce the range of wavelengths reaching the detector to those near the absorption (or emission)
wavelength of the analyte.
Why is it necessary to have a wavelength selector?
A compound which absorbs at 500 nm will do so regardless of whether radiation of other wavelengths
is hitting the detector or not. The amount of 500 nm radiation absorbed is dependent on the
pathlength, concentration and intensity at 500 nm, and is totally unaffected by the other wavelengths.
It would seem, therefore, that a wavelength selector is unnecessary, but clearly it must be needed,
otherwise every spectrometer wouldn’t have one.
There are two basic reasons why a selector is needed, one relating to recording of spectra and the
other about measuring an absorbance value. The spectra one first: a spectrum is a graph of how much
radiation is absorbed at each wavelength. Detectors aren’t picky – they don’t know the difference
between different wavelengths, so if you have no way picking a single wavelength, you could only get
a single measurement. If you tried to plot a graph with one point, you wouldn’t get very far, would
you?
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1. General aspects of spectroscopy
Secondly, the measurement of absorbance for the analyte would be completely wrong if all
wavelengths of light, absorbed and unabsorbed were being detected. This is easiest illustrated with
some simple numbers.
EXERCISE 1.4
Let’s assume for the sake of simplicity that we are dealing with a visible absorption instrument, and the
source is generating 100 units of radiation for each 10 nm range (eg 400-410) from 400 to 800 nm.
(a)
How many units of radiation would reach the detector when it is zeroed at the start (no sample
in the cell)?
Now let’s put an absorbing sample into the instrument. Again for the sake of simplicity, let’s assume
that the only wavelengths it absorbs are at 500-510 nm, and it is of a concentration that will absorb
50% of the radiation in the range.
(b)
How many units of 500-510 radiation will reach the detector with the sample out?
(c)
How many units of 500-510 radiation will reach the detector with the sample in?
(d)
What should the % transmittance at 500-510 be?
(e)
What is the total intensity reaching the detector with the sample in (given no wavelength
selector)?
(f)
What is the actual %T that the instrument will display?
Hopefully, this example illustrates the problem. Without the wavelength selector, the detector is
swamped by lots of radiation that has nothing to do with the analyte’s absorption, and simply masks
the effect. This is shown in Figure 1.1.
To use a rather clichéd analogy – needles in haystacks – if there was no wavelength selector, it would
not just trying to find the needle in the haystack, it would be trying to count how many needles were
missing from the haystack! The wavelength selector removes the hay, making the job of seeing how
needles have disappeared (been absorbed) much easier.
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1. General aspects of spectroscopy
radiation not
absorbed
absorption
wavelength
range
radiation
absorbed
FIGURE 1.1 The effect of having no wavelength selector
Types of wavelength selector
Having determined that, yes, we do need one of them, the next thing is to choose which type. The
ideal wavelength selector would allow one wavelength of radiation only to pass to the detector. Given
that the electromagnetic spectrum is continuous, such an ideal is not possible. Thus, wavelength
selectors allow through a range of wavelengths (this is known as the bandpass and is very important
– we will come back to it). How wide that range is depends on the design of the selector, and also the
experimental conditions required.
There are two basic classes of wavelength selector: monochromators and filters. They work very
differently, and achieve a different level of wavelength selection, as described in Table 1.1.
TABLE 1.1 Comparison of monochromators and filters
Monochromator
Filter
Means of selection
Diffraction
Absorption
Wavelength width
Narrow
Wide
Expensive
Cheap
None
Very
Capable of recording spectra
Yes
No
Suitable for quantitative
analysis
Yes
Yes
Cost
Portability
Filters are very simple: they are sheets of plastic or glass that simply absorb wavelengths other than
those required for the analysis. Generally, the range of wavelengths allowed by a filter is relatively
wide. For example, in the visible region, where they are most commonly used, the range is typically
20 nm.
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1. General aspects of spectroscopy
The filter needs to be chosen to maximise its overlap with the absorption peak of the analyte,
otherwise too much of the “haystack” (unabsorbed radiation) will reach the detector. Instruments
based around filters are designed to be simple cheap and portable, many field spectrometers for
environmental monitoring using them.
Monochromators are far more complicated, and comprise a series of optics inside a lightproof box,
which has entry and exit slits which allows the radiation of all wavelengths in and a narrow range of
wavelengths out. Figure 1.2 shows the components of a typical monochromator.
FIGURE 1.2 Typical internal construction of a monochromator
(from Chemicool, www.chemicool.com/definition/wavelength_selectors.html )
The dispersing medium is either a prism or a diffraction grating. Each works by causing the different
wavelengths of radiation to change their direction at different angles depending the wavelength. This
results in a band of single wavelengths which are directed towards the exit slit. Because it is very
narrow, only a small range of wavelengths can actually exit and reach the detector (imagine a paling
fence with one paling missing).
To allow selection of which wavelength leaves the exit slit, the prism or grating rotates causing the
band of radiation to shift, and moving a different wavelength over the exit slit.
A crystalline prism is the simpler option, and its mode of action is familiar to anyone who has seen
white light pass through a piece of glass and be divided into a rainbow spectrum. The problems with
a prism include relatively poor throughput (the total amount of light passes through is significantly less
than the amount that went in) and the difficulty in finding materials that diffract different wavelength
ranges.
Gratings are more commonly used across the range of instruments examined in this subject. A grating
is a grooved surface, where the grooves are extremely close together. Typically, for a grating in a
UV/VIS absorption instrument, there will be around 1300 grooves/mm (less in the IR). Gratings can
work by transmission or more commonly reflection.
Gratings are no more expensive than prisms, are more compact, and have the major advantage of a
constant dispersion across a range of wavelengths, i.e. the difference in the angle of dispersion
between two wavelengths, for instance, 10 nm apart, is the same at 200 nm as it is at 750 nm. This is
not the case with prisms.
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1. General aspects of spectroscopy
The significance of the exit slit
The exit slit is far more important than simply a place for the light to escape. How wide the exit slit is
determines the range of wavelengths that come through (how many palings are missing, if you like).
This is known as the slit width. In most instruments, the exit slit is adjustable and can open wider or
close to a narrower gap. As the slit width decreases, the range of wavelengths that are passed by the
monochromator decreases (and vice versa).
The actual slit width (typically mm in size) is not important in itself. It is not equal to the range of
wavelengths that pass through it, but it does control that measure. The important measure of the
performance of a monochromator is not the slit width, but rather the spectral bandwidth (or
bandpass). This refers to the wavelength interval of radiation leaving the monochromator through the
exit slit. As an example, if you set the monochromator to 500 nm and the slit width to give a bandpass
of 1 nm, the radiation leaving the exit slit would range from 499.5 to 500.5 nm (actually it isn’t quite
as clear cut as that, but for the purposes of this explanation, it is OK).
Different instruments of the same class (eg UV-VIS) will have different exit slit openings to achieve a
similar set of bandpasses. It is the bandpass that affects the appearance of the spectrum, and this will
be discussed later in this chapter.
Positioning the wavelength selector
In principle, the wavelength selector can go either before or after the sample cell. In reality, it is better
afterwards, but in some instruments, it must go before. The main determining factor of position is the
energy of the beam from the source. If, as in the case of UV radiation, the entire beam – all the
wavelengths – may be too energetic and decompose the sample, the monochromator is placed before
the sample, so that only a fraction of the beam ever hits the sample, as shown in Figure 1.3.
Source
Sample
λ selector
Source
λ selector
Sample
FIGURE 1.3 Wavelength selector placement effect on intensity
If the wavelength selector goes before the sample, then one problem is solved at the expense of
creating another. If the sample compartment is open to the surroundings, here is nothing preventing
light from the surrounds – e.g. laboratory lighting – finding its way to the detector. This is known as
stray light, which is any radiation that reaches the detector that is not from the source
To eliminate this, light-seal doors over the sample compartment are required. Stray light can also
come from the monochromator itself due to scattered radiation reflecting around the interior of the
unit. To reduce this, the inside walls of monochromators are painted in matte black.
In regions such as the infrared, where the energy is much less, the selector can go after the sample,
meaning that stray light is not a problem. The very small amount of radiation from outside the
instrument that is of the same wavelength as the monochromator setting or filter will be there during
the zeroing process and be compensated for (as long as you don’t zero it pointing a torch into the
instrument!).
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1. General aspects of spectroscopy
1.3 Sample holders
The principal requirement of a sample holder is that it doesn’t interfere with the spectrum of the
analyte by absorption or emitting at wavelengths near the analyte peaks.
1.4 Detectors
As mentioned above, most detectors respond only to the total intensity of radiation, and cannot
distinguish between different wavelengths. The ideal detector has the following characteristics, the
reasons for each are fairly obvious:
•
high sensitivity
•
high signal-to-background ratio
•
constant response across the range of wavelengths
•
rapid response
•
linear response (i.e. output is proportional to radiant intensity)
•
minimal response to no radiation (known as dark current)
No real detector achieves all these requirements perfectly, but over time, technology has produced
detectors that go close. The earliest detector was the eyes of the operator. The next stage along the
way employed photographic film. In the 1950s, these made way for electrical and then electronic
systems, where the radiation was converted into an electrical signal, most often current. Therefore,
the actual output of most detectors is measured in amperes (in reality micro- or milliamperes).
1.5 Instrument configurations
The development of spectroscopic instruments over the last 50 years has led to obviously better
performance, but also a number of different arrangements of the internal components.
Scanning or non-scanning
The distinction here is the ability to create a spectrum. A scanning instrument is able to
“automatically” change wavelength to measure intensities at enough points to create a spectrum. This
indicates two things about the instrument:
•
it is able to measure the intensity at wavelengths that are very close together
•
it is able to vary the wavelength without human assistance
EXERCISE 1.5
Answer True or False to the following regarding the type of wavelength selector and justify your
answer:
(a)
An instrument using a filter cannot be a scanning instrument.
(b)
An instrument using a monochromator must be a scanning instrument.
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1. General aspects of spectroscopy
The means by which different wavelengths can be measured without human assistance is traditionally
a stepping motor which rotates the grating or prism by very small angles. More recently, other ways
to produce spectrum have been devised, as will be seen below.
EXERCISE 1.6
What type of instrument – scanning or non-scanning – is the filter photometer being displayed?
Single or double beam
Absorption instruments require a measurement of the intensity of radiation going into the sample, as
well as how much gets through. Before the advent of computers, there were two instrumental designs
that allowed this “before” measurement to be made:
•
single beam – only one sample holder; the “before” measurement is taken using a blank at the
start; if the wavelength is changed, the instrument needs re-zeroing
•
double beam – two sample holders and a split optical system, allowing the “before” intensity to
be measured continually (see Figure 1.4); it is really double-path, not double-beam, but that is
the term that is used
EXERCISE 1.7
Which of these configurations allows scanning? Why?
Rotating chopper (see below)
Mirror
Source
Detector
Mirror
Semi-transparent mirror
Design of Chopper
transparent sector
mirrored sector
FIGURE 1.4 Configuration of a double-beam instrument (position of wavelength selector not shown)
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1. General aspects of spectroscopy
You might have thought that a double-beam configuration would have two of everything: beams,
sources, detectors etc, but it would cost so much, and still not be adequate because there would be
no way that the two systems could be made exactly equal.
Therefore, the two-path setup is the better (but not perfect) configuration. The source beam interacts
with the chopper, which rotates a few times a second, alternately presenting a straight-through path
and a mirror reflection to the beam. This sends it either to one cell or the other, so the readout device
simply takes the ratio of the alternating intensities (after/before/after/before/after etc).
Unfortunately, there are some problems with the double-path setup. The extra optics (mirrors) mean
that less radiation goes through the system (known as throughput) and they also make the whole
system more fragile, because there are even more bits and pieces which can get out of alignment.
Further, it is necessary to have two cells to record measurements (sample and reference) and they
must perform identically (matched), which can be difficult (in other words, expensive) to achieve.
A single-beam instrument does not have these problems or requirements, but without a computer,
nor can it record spectra (regardless of whether it has a monochromator and stepping motor) since
each time the wavelength changes, the instrument must be re-zeroed. This means taking the sample
out and putting the reference back in.
EXERCISE 1.8
Why is it necessary to re-zero the instrument when the wavelength changes?
The advent of desktop computers revolutionised spectrometer design as it allowed the entire
spectrum baseline (the “before” measurement for all wavelengths) to be measured at the start and
stored in the computer’s memory. Therefore, a single-beam instrument gained the capacity to be a
scanning instrument.
Double-beam spectrometers with a computer are made (we have one – Cary 5000), but really you
need one or the other, not both. The power of the computer allows you store and manipulate spectra,
so clearly having one is an advantage, but why then have the problems associated with the doublebeam setup?
A representative of the Cary instrument company, when asked this question, answered that “it
guaranteed that there would be no drift problems”, meaning that the single-beam setup will be
inaccurate if the measured baseline at the start is not the real baseline at the end of the scan. Given
a scan normally takes no more than two minutes, an instrument with drift problems in that space of
time is a worry for other reasons!
EXERCISE 1.9
What type of instrument is the filter photometer?
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1. General aspects of spectroscopy
Dispersive or non-dispersive
Dispersive means “dividing up”, so in this context, it means a component (eg grating) that divides up
the radiation by wavelength. Some instruments can still do useful work without this (and not by simply
using a filter).
The simpler type of non-dispersive (ND) instrument has no wavelength selector at all. These are
commonly employed for pollution monitoring in harsh environments, such as chimneys and on tops of
buildings, where a more delicate instrument would not survive. They achieve some measure of
selectivity by clever use of reference materials but are non-scanning. They are mostly in the infrared
region, and we will return to them in that chapter.
In the last two decades, instruments using a mathematical technique known as the Fourier transform,
have been developed to produce spectra without the need for a dispersing element. FT instruments
have the best of both worlds:
•
they are very fast (no mechanical stepping through wavelengths)
•
simpler internal configuration (better throughput)
Like the other ND instruments, they are mostly employed in IR spectrometers, and we will return to
them there.
A third class of instrument uses a detector that is it itself capable of distinguishing between different
wavelengths of radiation. At this stage, the only region of the spectrum where such a detector works
is the X-ray region. We will look at this in the Advanced Instrumental Techniques subject.
EXERCISE 1.10
What type of instrument is the filter photometer?
Single- or multi-channel
The channel here refers to the number of detectors, so you’d think it would have made more sense to
call them single- or multi-detector! Because scanning using a stepping-motor monochromator takes
time and the optics can become misaligned, another approach is to have numerous detectors, each
responsible for a specific wavelength (or range of wavelengths).
No, these detectors still can’t tell the difference, so a dispersing medium is still needed. However, the
exit slit and motor parts of the monochromator are thrown out. Instead, the dispersed radiation
travels to the detectors, fixed in place so that only a certain wavelength hits each one.
Some of these instruments are not capable of producing a spectrum, only numerous wavelength
measurements (for quantitative analysis, one analyte per detector) while others have a continuous
bank of very small detectors which cover the entire range and do allow scanning (see Figure 1.5).
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1. General aspects of spectroscopy
Array
Radiation
Sample
Dispersing
of
source
cell
element
detectors
FIGURE 1.5 Schematic diagram of multi-channel instrument
EXERCISE 1.11
What type of instrument is the filter photometer?
Transmission or reflectance
The forms of absorption spectroscopy (this distinction does not apply to emission instruments) which
you are familiar with involve a semi-transparent liquid or solid, and absorbance measurements are
derived from how much radiation passes through the sample. Not all materials will transmit radiation.
Some are sufficiently opaque that unabsorbed light is reflected, rather than transmitted. In such cases,
conventional transmission spectroscopy will not be suitable.
EXERCISE 1.12
Give examples of materials which transmission spectroscopy would be unsuitable for.
Reflectance spectroscopy measures the radiation that bounces off the surface of an opaque material.
This may be a direct reflection by a mirror-like surface (called specular) or scattered at a variety of
angles by a rough surface (called diffuse). The basic principles still apply: certain wavelengths will be
absorbed, and an absorption spectrum obtained (see Figure 1.6).
Transmission
Reflectance
Iout
Iin
Iout
Iin
FIGURE 1.6 Transmission and reflectance measurements
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1. General aspects of spectroscopy
The technique is used in both the UV/VIS and IR regions, and most conventional transmission
instruments can be modified for reflectance measurements by the addition of a special module that
replaces the normal sample holder.
EXERCISE 1.13
What type of instrument is the filter photometer?
EXERCISE 1.14
Classify the instruments in the laboratory listed on the provided sheet.
1.6 Factors affecting the appearance of the spectrum
A spectrum is no more than a graph representing the absorption/emission characteristics of the
compound. How close a representation it is depends on the instrument and some of its performance
settings. The two most important are the:
•
scan speed
•
bandpass
These both affect the resolution of the scan: the ability of the instrument to separate peaks which are
very close together in wavelength. This is most important in more complex spectra, e.g. IR, high
temperature emission. In UV/VIS spectra, where the peaks are few in number and broad, this is less
of a problem.
The scan speed is the simpler of the two. In conventional instruments, the rate at which the motor
adjusting the position of the dispersing elements in the monochromator is operating will affect the
ability of the detector to respond to small changes in the sample absorption. If the scan speed is too
rapid, minor “shoulder” peaks on the side of larger peaks may be missed. However advances in
detector design – their response time – have meant that this is no longer an issue in most situations.
The choice of bandpass is basically a trade-off between intensity (brightness) and resolution. A broader
slit width allows more radiation (a greater spectral bandwidth) through at any one time. This achieves
more reliable results at high absorbances since the detector is receiving a greater signal. This is better
for quantitative analysis, as the crucial absorbance value will be slightly more accurate.
Closing the slit width to allow less radiation through (a smaller spectral bandwidth) means that the
light is more monochromatic in nature and allows better resolution of peaks. Basically, the graph
which is the spectrum has more points at narrow bandpasses.
GENERAL RULE
Quantitative analysis ⇒ Wide bandpass
Qualitative analysis ⇒ Narrow bandpass
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1. General aspects of spectroscopy
EXERCISE 1.15
To illustrate the effect on resolution of opening the bandpass, join every second dot on the
spectrum below (you never thought you’d do this in a chemistry class!!). This simulates the
doubling of the bandpass.
What You Need To Be Able To Do
•
define important terminology
•
explain the role of each component in spectrometers
•
explain specific aspects of component performance (eg position of the wavelength selector)
•
compare the different configuration options for spectrometers
•
identify the configuration type of real instruments
•
describe instrumental factors that affect spectrum appearance
Revision Questions
1.
List THREE general requirements for a radiation source.
2.
Explain why ONE of these in Q1 is important.
3.
Draw a diagram showing the components of a typical monochromator. Explain what happens
when a change in wavelength occurs.
4.
What is the difference between slit width and bandpass? Which is more important? Why?
5.
For the three basic types of spectroscopic instruments (single-beam non-scanning, single-beam
with computer, double-beam no computer), explain how the “intensity in” measurement is
made (remember transmittance is intensity out ÷ intensity in).
6.
Which configuration is better for a scanning instrument: single-beam with computer or doublebeam?
7.
The UV-VIS instrument you are using has a range of bandpasses available from 0.2 to 4 nm.
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1. General aspects of spectroscopy
(a) Choose and justify a bandpass appropriate for the recording of a spectrum to help identify a
compound.
8.
9.
10.
11.
12.
(b) Choose and justify a bandpass appropriate for the measurement of absorbances at a fixed
wavelength.
What is stray light? Give ONE example.
What is a multi-channel instrument? Give an example of a multi-channel instrument in our
laboratory. What is one advantage of such an instrument?
Give TWO examples of samples which could only be analysed by reflectance spectroscopy.
Why are non-dispersive scanning instruments better than their dispersive equivalents?
What does the presence of a light-seal panel on a spectrometer tell you about the internal
configuration of the instrument?
Answers on next page
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1. General aspects of spectroscopy
Answers to Revision Questions
1. see page 3
2. see answers to Exercise 1.3
3. see Figure 1.2. A wavelength change means the grating rotates.
4. Slit width is the physical dimension of the exit slit, while bandpass is the range of wavelengths
allowed through. Bandpass is more important because it is a direct measure of the resolution of the
instrument.
5. Single-beam non-scanning: instrument zeroed at start, must be manually re-zeroed each time
wavelength is changed.
Single-beam with computer: full spectrum of blank recorded at start and stored in memory
Double-beam: reference beam measures "before" intensity at any wavelength
6. Single-beam with computer has less optics, doesn't require matched cells
7. (a) 0.2 nm, because more detail will be shown in spectrum
(b) 4 nm, because more light to detector means it measures differences more accurately
8. see page 8
9. One with multiple detectors. Hewlett-Packard UV/VIS. Time: no mechanical movement in the
monochromator to create a scan - the spectrum is created instantly at all wavelengths.
10. see Exercise 1.13
11. No moving parts to be knocked out of alignment, no slow mechanical scanning.
12. If there is a light-seal door, it means the monochromator is before the sample.
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