Instrumentation

Introduction

Have a look at this schematic diagram of a double-beam UV-Vis. spectrophotometer;

Instruments for measuring the absorption of U.V. or visible radiation are made up of the
following components;

1. Sources (UV and visible)

2. Wavelength selector (monochromator)
3. Sample containers
4. Detector
5. Signal processor and readout

Each of these components will be considered in turn.

Instrumental components

Sources of UV radiation

It is important that the power of the radiation source does not change abruptly over it's
wavelength range.

The electrical excitation of deuterium or hydrogen at low pressure produces a continuous UV

spectrum. The mechanism for this involves formation of an excited molecular species, which
breaks up to give two atomic species and an ultraviolet photon. This can be shown as;

D2 + electrical energy  D2*  D' + D'' + hv

Both deuterium and hydrogen lamps emit radiation in the range 160 - 375 nm. Quartz windows
must be used in these lamps, and quartz cuvettes must be used, because glass absorbs radiation of
wavelengths less than 350 nm.

Sources of visible radiation

The tungsten filament lamp is commonly employed as a source of visible light. This type of lamp
is used in the wavelength range of 350 - 2500 nm. The energy emitted by a tungsten filament
lamp is proportional to the fourth power of the operating voltage. This means that for the energy
output to be stable, the voltage to the lamp must be very stable indeed. Electronic voltage
regulators or constant-voltage transformers are used to ensure this stability.

Tungsten/halogen lamps contain a small amount of iodine in a quartz "envelope" which also
contains the tungsten filament. The iodine reacts with gaseous tungsten, formed by sublimation,
producing the volatile compound WI2. When molecules of WI2 hit the filament they decompose,
redepositing tungsten back on the filament. The lifetime of a tungsten/halogen lamp is
approximately double that of an ordinary tungsten filament lamp. Tungsten/halogen lamps are
very efficient, and their output extends well into the ultra-violet. They are used in many modern
spectrophotometers.

Wavelength selector (monochromator)

All monochromators contain the following component parts;

 An entrance slit
 A collimating lens
 A dispersing device (usually a prism or a grating)
 A focusing lens
 An exit slit

Polychromatic radiation (radiation of more than one wavelength) enters the monochromator
through the entrance slit. The beam is collimated, and then strikes the dispersing element at an
angle. The beam is split into its component wavelengths by the grating or prism. By moving the
dispersing element or the exit slit, radiation of only a particular wavelength leaves the
monochromator through the exit slit.

Czerney-Turner grating monochromator

Cuvettes

The containers for the sample and reference solution must be transparent to the radiation which
will pass through them. Quartz or fused silica cuvettes are required for spectroscopy in the UV
region. These cells are also transparent in the visible region. Silicate glasses can be used for the
manufacture of cuvettes for use between 350 and 2000 nm.
Detectors

The photomultiplier tube is a commonly used detector in UV-Vis spectroscopy. It consists of a

photoemissive cathode (a cathode which emits electrons when struck by photons of radiation),
several dynodes (which emit several electrons for each electron striking them) and an anode.

A photon of radiation entering the tube strikes the cathode, causing the emission of several
electrons. These electrons are accelerated towards the first dynode (which is 90V more positive
than the cathode). The electrons strike the first dynode, causing the emission of several electrons
for each incident electron. These electrons are then accelerated towards the second dynode, to
produce more electrons which are accelerated towards dynode three and so on. Eventually, the
electrons are collected at the anode. By this time, each original photon has produced 106 - 107
electrons. The resulting current is amplified and measured.

Photomultipliers are very sensitive to UV and visible radiation. They have fast response times.
Intense light damages photomultipliers; they are limited to measuring low power radiation.

Cross section of a photomultiplier tube

The linear photodiode array is an example of a multichannel photon detector. These detectors
are capable of measuring all elements of a beam of dispersed radiation simultaneously.

A linear photodiode array comprises many small silicon photodiodes formed on a single silicon
chip. There can be between 64 to 4096 sensor elements on a chip, the most common being 1024
photodiodes. For each diode, there is also a storage capacitor and a switch. The individual diode-
capacitor circuits can be sequentially scanned.

In use, the photodiode array is positioned at the focal plane of the monochromator (after the
dispersing element) such that the spectrum falls on the diode array. They are useful for recording
UV-Vis. absorption spectra of samples that are rapidly passing through a sample flow cell, such
as in an HPLC detector.

Charge-Coupled Devices (CCDs) are similar to diode array detectors, but instead of diodes,
they consist of an array of photocapacitors.

Review your learning

You should now have an understanding of the separate components which make up a
spectrophotometer, and how they fit together. Have a look at this schematic of the Hitachi 100-
60 manual double-beam spectrophotometer;

Do you understand what each component does?

Applications of Absorption Spectroscopy (UV, Visible)

1. Detection of Impurities
UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules.
Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw
material. By also measuring the absorbance at specific wavelength, the impurities can be detected.
Benzene appears as a common impurity in cyclohexane. Its presence can be easily detected by its absorption at 255
nm.

2. Structure elucidation of organic compounds.

UV spectroscopy is useful in the structure elucidation of organic molecules, the presence or absence of unsaturation,
the presence of hetero atoms.
From the location of peaks and combination of peaks, it can be concluded that whether the compound is saturated or
unsaturated, hetero atoms are present or not etc.

3. Quantitative analysis
UV absorption spectroscopy can be used for the quantitative determination of compounds that absorb UV radiation.
This determination is based on Beer’s law which is as follows.
A = log I0 / It = log 1/ T = – log T = abc = εbc
Where ε is extinction co-efficient, c is concentration, and b is the length of the cell that is used in UV
spectrophotometer.
Other methods for quantitative analysis are as follows.
a. calibration curve method
b. simultaneous multicomponent method
c. difference spectrophotometric method
d. derivative spectrophotometric method

4. Qualitative analysis
UVabsorption spectroscopy can characterize those types of compounds which absorbs UV radiation. Identification
is done by comparing the absorption spectrum with the spectra of known compounds.
UV absorption spectroscopy is generally used for characterizing aromatic compounds and aromatic olefins.

 Ultraviolet–visible spectroscopy
From Wikipedia, the free encyclopedia

Jump to: navigation, search

Beckman DU640 UV/Vis spectrophotometer.

Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or

UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible
spectral region. This means it uses light in the visible and adjacent (near-UV and near-infrared
(NIR)) ranges. The absorption or reflectance in the visible range directly affects the perceived
color of the chemicals involved. In this region of the electromagnetic spectrum, molecules
undergo electronic transitions. This technique is complementary to fluorescence spectroscopy, in
that fluorescence deals with transitions from the excited state to the ground state, while
absorption measures transitions from the ground state to the excited state.[1]

Contents
[hide]

 1 Applications
 2 Beer-Lambert law
o 2.1 Practical considerations
 2.1.1 Spectral bandwidth
 2.1.2 Wavelength error
 2.1.3 Stray light
 2.1.4 Absorption flattening
 3 Ultraviolet-visible spectrophotometer
 4 See also
 5 Notes

[edit] Applications

An example of a UV/Vis readout

UV/Vis spectroscopy is routinely used in the quantitative determination of solutions of transition

metal ions highly conjugated organic compounds, and biological macromolecules.

 Solutions of transition metal ions can be colored (i.e., absorb visible light)
because d electrons within the metal atoms can be excited from one electronic
state to another. The colour of metal ion solutions is strongly affected by the
presence of other species, such as certain anions or ligands. For instance, the
colour of a dilute solution of copper sulfate is a very light blue; adding ammonia
intensifies the colour and changes the wavelength of maximum absorption (λmax).
  Organic compounds, especially those with a high degree of conjugation, also
absorb light in the UV or visible regions of the electromagnetic spectrum. The
solvents for these determinations are often water for water soluble compounds,
or ethanol for organic-soluble compounds. (Organic solvents may have
significant UV absorption; not all solvents are suitable for use in UV
spectroscopy. Ethanol absorbs very weakly at most wavelengths.) Solvent
polarity and pH can affect the absorption spectrum of an organic compound.
Tyrosine, for example, increases in absorption maxima and molar extinction
coefficient when pH increases from 6 to 13 or when solvent polarity decreases.
 While charge transfer complexes also give rise to colours, the colours are often
too intense to be used for quantitative measurement.

The Beer-Lambert law states that the absorbance of a solution is directly proportional to the
concentration of the absorbing species in the solution and the path length. Thus, for a fixed path
length, UV/Vis spectroscopy can be used to determine the concentration of the absorber in a
solution. It is necessary to know how quickly the absorbance changes with concentration. This
can be taken from references (tables of molar extinction coefficients), or more accurately,
determined from a calibration curve.

A UV/Vis spectrophotometer may be used as a detector for HPLC. The presence of an analyte
gives a response assumed to be proportional to the concentration. For accurate results, the
instrument's response to the analyte in the unknown should be compared with the response to a
standard; this is very similar to the use of calibration curves. The response (e.g., peak height) for
a particular concentration is known as the response factor.

The wavelengths of absorption peaks can be correlated with the types of bonds in a given
molecule and are valuable in determining the functional groups within a molecule. The
Woodward-Fieser rules, for instance, are a set of empirical observations used to predict λmax, the
wavelength of the most intense UV/Vis absorption, for conjugated organic compounds such as
dienes and ketones. The spectrum alone is not, however, a specific test for any given sample. The
nature of the solvent, the pH of the solution, temperature, high electrolyte concentrations, and the
presence of interfering substances can influence the absorption spectrum. Experimental
variations such as the slit width (effective bandwidth) of the spectrophotometer will also alter the
spectrum. To apply UV/Vis spectroscopy to analysis, these variables must be controlled or
accounted for in order to identify the substances present.[2]

[edit] Beer-Lambert law

Main article: Beer-Lambert law

The method is most often used in a quantitative way to determine concentrations of an absorbing
species in solution, using the Beer-Lambert law:

,
where A is the measured absorbance, I0 is the intensity of the incident light at a given
wavelength, I is the transmitted intensity, L the pathlength through the sample, and c the
concentration of the absorbing species. For each species and wavelength, ε is a constant known
as the molar absorptivity or extinction coefficient. This constant is a fundamental molecular
property in a given solvent, at a particular temperature and pressure, and has units of 1 / M * cm
or often AU / M * cm.

The absorbance and extinction ε are sometimes defined in terms of the natural logarithm instead
of the base-10 logarithm.

The Beer-Lambert Law is useful for characterizing many compounds but does not hold as a
universal relationship for the concentration and absorption of all substances. A 2nd order
polynomial relationship between absorption and concentration is sometimes encountered for very
large, complex molecules such as organic dyes (Xylenol Orange or Neutral Red, for example).

[edit] Practical considerations

The Beer-Lambert law has implicit assumptions that must be met experimentally for it to apply.
For instance, the chemical makeup and physical environment of the sample can alter its
extinction coefficient. The chemical and physical conditions of a test sample therefore must
match reference measurements for conclusions to be valid.

[edit] Spectral bandwidth

A given spectrometer has a spectral bandwidth that characterizes how monochromatic the light
is. If this bandwidth is comparable to the width of the absorption features, then the measured
extinction coefficient will be altered. In most reference measurements, the instrument bandwidth
is kept below the width of the spectral lines. When a new material is being measured, it may be
necessary to test and verify if the bandwidth is sufficiently narrow. Reducing the spectral
bandwidth will reduce the energy passed to the detector and will, therefore, require a longer
measurement time to achieve the same signal to noise ratio.

[edit] Wavelength error

In liquids, the extinction coefficient usually changes slowly with wavelength. A peak of the
absorbance curve (a wavelength where the absorbance reaches a maximum) is where the rate of
change in absorbance with wavelength is smallest. Measurements are usually made at a peak to
minimize errors produced by errors in wavelength in the instrument, that is errors due to having a
different extinction coefficient than assumed.

[edit] Stray light

See also: Stray light

Another important factor is the purity of the light used. The most important factor affecting this
is the stray light level of the monochromator [3] . The detector used is broadband, it responds to
all the light that reaches it. If a significant amount of the light passed through the sample contains
wavelengths that have much lower extinction coefficients than the nominal one, the instrument
will report an incorrectly low absorbance. Any instrument will reach a point where an increase in
sample concentration will not result in an increase in the reported absorbance, because the
detector is simply responding to the stray light. In practice the concentration of the sample or the
optical path length must be adjusted to place the unknown absorbance within a range that is valid
for the instrument. Sometimes an empirical calibration function is developed, using known
concentrations of the sample, to allow measurements into the region where the instrument is
becoming non-linear.

As a rough guide, an instrument with a single monochromator would typically have a stray light
level corresponding to about 3 AU, which would make measurements above about 2 AU
problematic. A more complex instrument with a double monochromator would have a stray light
level corresponding to about 6 AU, which would therefore allow measuring a much wider
absorbance range.

[edit] Absorption flattening

At sufficiently high concentrations, the absorption bands will saturate and show absorption
flattening. The absorption peak appears to flatten because close to 100% of the light is already
being absorbed. The concentration at which this occurs depends on the particular compound
being measured. One test that can be used to test for this effect is to vary the path length of the
measurement. In the Beer-Lambert law, varying concentration and path length has an equivalent
effect—diluting a solution by a factor of 10 has the same effect as shortening the path length by a
factor of 10. If cells of different path lengths are available, testing if this relationship holds true is
one way to judge if absorption flattening is occurring.

Solutions that are not homogeneous can show deviations from the Beer-Lambert law because of
the phenomenon of absorption flattening. This can happen, for instance, where the absorbing
substance is located within suspended particles.[4] The deviations will be most noticeable under
conditions of low concentration and high absorbance. The reference describes a way to correct
for this deviation.

[edit] Ultraviolet-visible spectrophotometer

See also: Spectrophotometry

The instrument used in ultraviolet-visible spectroscopy is called a UV/Vis spectrophotometer.

It measures the intensity of light passing through a sample (I), and compares it to the intensity of
light before it passes through the sample (Io). The ratio I / Io is called the transmittance, and is
usually expressed as a percentage (%T). The absorbance, A, is based on the transmittance:

A = − log(%T / 100%)

The UV-visible spectrophotometer can also be configured to measure reflectance. In this case,
the spectrophotometer measures the intensity of light reflected from a sample (I), and compares
it to the intensity of light reflected from a reference material (Io)(such as a white tile). The ratio
I / Io is called the reflectance, and is usually expressed as a percentage (%R).

The basic parts of a spectrophotometer are a light source, a holder for the sample, a diffraction
grating in a monochromator or a prism to separate the different wavelengths of light, and a
detector. The radiation source is often a Tungsten filament (300-2500 nm), a deuterium arc lamp,
which is continuous over the ultraviolet region (190-400 nm), Xenon arc lamps, which is
continuous from 160-2,000 nm; or more recently, light emitting diodes (LED) [5] for the visible
wavelengths. The detector is typically a photomultiplier tube, a photodiode, a photodiode array
or a charge-coupled device(CCD). Single photodiode detectors and photomultiplier tubes are
used with scanning monochromators, which filter the light so that only light of a single
wavelength reaches the detector at one time. The scanning monochromator moves the diffraction
grating to "step-through" each wavelength so that it's intensity may be measured as a function of
wavelength. Fixed monochromators are used with CCDs and photodiode arrays. As both of these
devices consist of many detectors grouped into one or two dimensional arrays, they are able to
collect light of different wavelengths on different pixels or groups of pixels simultaneously.

Diagram of a single-beam UV/Vis spectrophotometer.

A spectrophotometer can be either single beam or double beam. In a single beam instrument
(such as the Spectronic 20), all of the light passes through the sample cell. Io must be measured
by removing the sample. This was the earliest design, but is still in common use in both teaching
and industrial labs.

In a double-beam instrument, the light is split into two beams before it reaches the sample. One
beam is used as the reference; the other beam passes through the sample. The reference beam
intensity is taken as 100% Transmission (or 0 Absorbance), and the measurement displayed is
the ratio of the two beam intensities. Some double-beam instruments have two detectors
(photodiodes), and the sample and reference beam are measured at the same time. In other
instruments, the two beams pass through a beam chopper, which blocks one beam at a time. The
detector alternates between measuring the sample beam and the reference beam in synchronism
with the chopper. There may also be one or more dark intervals in the chopper cycle. In this case
the measured beam intensities may be corrected by subtracting the intensity measured in the dark
interval before the ratio is taken.

Samples for UV/Vis spectrophotometry are most often liquids, although the absorbance of gases
and even of solids can also be measured. Samples are typically placed in a transparent cell,
known as a cuvette. Cuvettes are typically rectangular in shape, commonly with an internal width
of 1 cm. (This width becomes the path length, L, in the Beer-Lambert law.) Test tubes can also
be used as cuvettes in some instruments. The type of sample container used must allow radiation
to pass over the spectral region of interest. The most widely applicable cuvettes are made of high
quality fused silica or quartz glass because these are transparent throughout the UV, visible and
near infrared regions. Glass and plastic cuvettes are also common, although glass and most
plastics absorb in the UV, which limits their usefulness to visible wavelengths.[6]

Specialized instruments have also been made. These include attaching spectrophotometers to
telescopes to measure the spectra of astronomical features. UV-visible microspectrophotometers
consist of a UV-visible microscope integrated with a UV-visible spectrophotometer. These are
commonly used for measuring thin film thickness in semiconductor manufacturing, materials
science research, measuring the energy content of coal and petroleum source rock, and in
forensic laboratories for the analysis of microscopic amounts of trace evidence as well as
questioned documents.

A complete spectrum of the absorption at all wavelengths of interest can often be produced
directly by a more sophisticated spectrophotometer. In simpler instruments the absorption is
determined one wavelength at a time and then compiled into a spectrum by the operator. By
removing the concentration dependence, the extinction coefficient (ε) can be determined as a
function of wavelength.

Figure 1 (graphics1.jpg)

Figure 2: Illustration of a double beam UV-vis instrument.Figure 2 (graphics2.jpg)

Figure 3: Illustration of a simultaneous UV-vis instrument.Figure 3 (graphics3.jpg)

What information can be obtained from UV-vis spectra?

UV-vis spectroscopic data can give qualitative and quantitative information of a given
compound or molecule. Irrespective of whether quantitative or qualitative information is
required it is important to use a reference cell to zero the instrument for the solvent the
compound is in. For quantitative information on the compound, calibrating the
instrument using known concentrations of the compound in question in a solution with
the same solvent as the unknown sample would be required. If the information needed
is just proof that a compound is in the sample being analyzed, a calibration curve will
not be necessary; however, if a degradation study or reaction is being performed, and
concentration of the compound in solution is required, thus a calibration curve is
needed.

To make a calibration curve, at least three concentrations of the compound will be

needed, but five concentrations would be most ideal for a more accurate curve. The
concentrations should start at just above the estimated concentration of the unknown
sample and should go down to about an order of magnitude lower than the highest
concentration. The calibration solutions should be spaced relatively equally apart, and
they should be made as accurately as possible using digital pipettes and volumetric
flasks instead of graduated cylinders and beakers. An example of absorbance spectra
of calibration solutions of Rose Bengal (4,5,6,7-tetrachloro-2',4',5',7'-
tetraiodofluorescein, Figure 4) can be seen in Figure 5. To make a calibration curve, the
value for the absorbances of each of the spectral curves at the highest absorbing
wavelength, is plotted in a graph similar to that in Figure 6 of absorbance versus
concentration. The correlation coefficient of an acceptable calibration is 0.9 or better. If
the correlation coefficient is lower than that, try making the solutions again as the
problem may be human error. However, if after making the solutions a few times the
calibration is still poor, something may be wrong with the instrument; for example, the
lamps may be going bad.

Figure 4: The molecular structure of Rose Bengal (4,5,6,7-tetrachloro-2',4',5',7'-

tetraiodofluorescein).Figure 4 (FigB4.jpg)

Figure 5: UV-vis spectra of different concentrations of Rose Bengal.Figure 5

(FigB11.jpg)

Figure 6: Calibration curve of Rose Bengal. Equation of line: y = 0.0977x – 0.1492 (R2 =
0.996)Figure 6 (FigB21.jpg)

Limitations of UV-visible spectroscopy

Sample
UV-vis spectroscopy works well on liquids and solutions, but if the sample is more of a
suspension of solid particles in liquid, the sample will scatter the light more than absorb
the light and the data will be very skewed. Most UV-vis instruments can analyze solid
samples or suspensions with a diffraction apparatus (Figure 7), but this is not common.
UV-vis instruments generally analyze liquids and solutions most efficiently.

Figure 7: Schematic representation of the apparatus for collecting UV-vis spectra from
solid materials.Figure 7 (Spec.jpg)

Calibration and reference

A blank reference will be needed at the very beginning of the analysis of the solvent to
be used (water, hexanes, etc), and if concentration analysis needs to be performed,
calibration solutions need to be made accurately (e.g., Figure 6). If the solutions are not
made accurately enough, the actual concentration of the sample in question will not be
accurately determined.

Choice of solvent or container

Every solvent has a UV-vis absorbance cutoff wavelength. The solvent cutoff is the
wavelengthbelow which the solvent itself absorbs all of the light. So when choosing a
solvent be aware of its absorbance cutoff and where the compound under investigation
is thought to absorb. If they are close, chose a different solvent. Table 1 provides an
example of solvent cutoffs.

Table 1: UV absorbance cutoffs of various common solvents.

Solvent

UV absorbance cutoff (nm)

Acetone
 329

Benzene

278

Dimethylformamide (DMF)

267

Ethanol

205

Toluene

285

Water
 180

The material the cuvette (the sample holder) is made from will also have a UV-vis
absorbance cutoff. Glass will absorb all of the light higher in energy starting at about
300 nm, so if the sample absorbs in the UV, a quartz cuvette will be more practical as
the absorbance cutoff is around 160 nm for quartz (Table 2).

Table 2: Three different types of cuvettes commonly used, with different usable
wavelengths.

Material

Wavelength range (nm)

Glass

380 - 780

Plastic

380 - 780
 Fused quartz

below 380

Concentration of solution

To obtain reliable data, the peak of absorbance of a given compound needs to be at

least three times higher in intensity than the background noise of the instrument.
Obviously using higher concentrations of the compound in solution can combat this.
Also, if the sample is very small and diluting it would not give an acceptable signal,
there are cuvettes that hold smaller sample sizes than the 2.5 mL of a standard
cuvettes. Some cuvettes are made to hold only 100 μL, which would allow for a small
sample to be analyzed without having to dilute it to a larger volume, lowering the signal
to noise ratio.

Bibliography

* D. A. Skoog, F. J. Holler, S. R. Crouch, Principles of Instrumental Analysis, 6th Ed.,

Thomson Brooks/Cole (2007).

* J. P. Sibilia, Materials Characterization and Chemical Analysis, 2nd Ed., Wiley-

VCH, New York (1996).

* D. C. Harris, Quantitative Chemical Analysis, 7th Ed., Freeman, New York(2007).