UV-Vis Frequently Asked Questions - Light and Theory

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What is light?

what is light?

Although light is generally said to be a wave, unlike the waves that occur at the surface of a body of water, it does not require a medium. As shown in here light consists of an electric field and a magnetic field that intersect each other at a right angle as they move through a vacuum. The distance between successive peaks of either the electric field or the magnetic field is the wavelength.

 

Why is the wavelength of light important?

why is wavelength important?

To answer this question, we need to know some important characteristics of light in general. Any wave is essentially just a way of shifting energy from one place to another, whether the fairly obvious transfer of energy in waves on the sea or in the much more difficult to imagine waves in light. With waves on water, the energy is transferred by the bulk movement of water molecules; however, a particular water molecule doesn’t travel all the way across the Atlantic, or even all the way across a pond. Depending on the depth of the water, waves follow a roughly circular path from the point of origin. As they move up to the top of the circle, the wave builds to a crest; as they move down again, you get a trough. The energy is transferred by relatively small local movements in the environment. With water waves it is fairly easy to draw diagrams to show this happening with real molecules. With light it is more difficult. The energy in light travels because of local fluctuating changes in electrical and magnetic fields; hence “electromagnetic” radiation. (Upper Left)

If you draw a beam of light in the form of a wave, without worrying too much about what exactly is causing the wave, the distance between two crests is called the wavelength of the light. It could equally well be the distance between two troughs or any other two identical positions on the wave. You have to picture these wave crests as moving from left to right. If you counted the number of crests passing a particular point per second, you have the frequency of the light. It is measured in what used to be called “cycles per second”, but is now called Hertz, Hz. Cycles per second and Hertz mean exactly the same thing. Light has a constant speed through a given substance. For example, it always travels at a speed of approximately 3 x 108 meters per second in a vacuum. This is actually the speed that all electromagnetic radiation travels not just visible light. There is a simple relationship between the wavelength and frequency of a particular color of light and the speed of light. (Lower Left)

This relationship means that if you increase the frequency, you must decrease the wavelength and, of course, the opposite is true. If the wavelength is longer, the frequency is lower. It is really important that you feel comfortable with the relationship between frequency and wavelength. Each particular frequency of light has a particular energy associated with it. The higher the frequency, the higher the energy of the light. Light which has wavelengths of around 380 nm to 435 nm is seen as a sequence of violet colors. Various red colors have wavelengths around 625 nm to 740 nm. Which has the highest energy? The light with the highest energy will be the one with the highest frequency, that will be the one with the smallest wavelength. (Right)

 

How are the different light wavelengths classified?

how are the different light wavelengths classified?

Light consists of certain types of electromagnetic waves. Electromagnetic waves are referred to by different names in accordance with their wavelength, as shown in here "Light" usually refers to electromagnetic waves in the range spanning infrared radiation and ultraviolet radiation, but in some cases, it refers only to visible light. Light with wavelengths in a range of approximately 400 to 800 nm is referred to as "visible light" and is the light that we humans can see with the naked eye. For example, light with a wavelength of 470 nm is blue, light with a wavelength of 540 nm is green, and light with a wavelength of 650 nm is red. Visible light could be described as the kind of light that we humans are familiar with because of our ability to see it.

 

What light wavelength regions are used?

What light wavelength regions are used?

In UV/Vis/NIR spectroscopy the ultraviolet (170 nm to 380 nm), visible (380 nm to 780 nm), and near infrared (780 nm to 3300 nm) are used. A nanometer (nm) is 10-9 meter. Most spectrophotometers are configured as either as UV/Vis instruments that cover the 190 nm to 900 nm (or 1100 nm) wavelength range or UV/Vis/NIR instruments that cover the 175 nm to 3300 nm wavelength range.

People are very familiar with the visible light region, since these are the wavelengths that the human eye is able to see. The diagram “The Visible Spectrum” above shows an approximation to the spectrum of visible light. The color order in the visible region is easy to remember using the mnemonic Roy G Biv. So, from long wavelength to short the colors are red, orange, yellow, green, blue, indigo, violet. Now we must place the visible light region into the rest of the electromagnetic spectrum. This electromagnetic spectrum doesn’t stop with the colors you can see. It is perfectly possible to have wavelengths shorter than violet light or longer than red light. The figure above shows ultraviolet and the infrared as the extremes, but this can be extended even further into x- rays and radio waves, amongst others. The diagram above shows the approximate positions of some of these on the spectrum.

Don’t worry too much about the exact boundaries between the various sorts of electromagnetic radiation, because there are no boundaries. Just as with visible light, one sort of radiation merges into the next. Just be aware of the general pattern. Also, be aware that the energy associated with the various kinds of radiation increases as the frequency increases (or wavelength decreases).

 

How does “absorption” spectroscopy work?

How does “absorption” spectroscopy work?

Let us consider the absorption of light by matter. This is closely related to quantum mechanics. Solving the equations of quantum mechanics that relate to the electrons in an atom gives a model, like that shown here in which the electrons have discrete energy states. E0 is called the "ground state" and E1, E2, etc., are called "excited states". In order for an electron to switch from E0 to E1, light with an energy of (E1 - E0) must strike the electron. This is the "absorption" of light. Electrons have particular energy levels, and rays of ultraviolet and visible light have the energy to change the energy states of the electrons.

Because the higher energy state, E1, is unstable, the electron soon returns to the ground state, E0. The energy discharged when the electron returns from E1 to E0 (E1 - E0) is converted to heat. If, for some reason, it is not converted to heat, the energy is discharged as light. The phenomenon of light emission is well known as fluorescence or phosphorescence.

In relation to quantitative measurement performed with spectroscopy, the consequence of this phenomenon is that there is a large amount of absorption if a large number of target molecules exist in a solution, and only a small amount of absorption if there is only a small number of target molecules. Obtaining the quantity, and thereby the concentration, of a substance from the degree of absorption is the fundamental principle behind quantitative measurement.

 

What type of information does a spectrophotometer produce?

What type of information does a spectrophotometer produce?

UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and certain biological macromolecules. Measurement is usually carried out in solution. 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 color of metal ion solutions is strongly affected by the presence of other species, such as certain anions or ligands. For instance, the color of a dilute solution of copper sulfate is a very light blue; adding ammonia intensifies the color 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. Some organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at lower ultra-violet wavelengths. Solvent polarity and pH can affect the absorption spectrum of certain organic compounds.

The output of a spectrophotometer is a spectrum (usually). A spectrum (plural spectra) is a graph displaying either values for transmitted, absorbed, or reflected light (Y axis) vs. various light wavelength values (X axis).

 

What are the different data collection modes of a typical spectrophotometer?

What are the different data collection modes of a typical spectrophotometer?

There are four common modes of data collection on a typical UV/Vis/NIR spectrophotometer. Below is the main menu for Shimadzu’s instruments.

1) Scan Mode - The most commonly used mode is wavelength scanning. Here the wavelength is scanned while the ordinate value is recorded to produce a spectrum as seen above. Instruments scan from longest wavelength to shortest. This is to reduce the sample’s exposure to high energy UV light which could cause photo-decomposition.

2) Quant Mode - Quantization is usually performed on a major peak of the sample. Data can be collected as either peak height or area. Beer’s law states that the sample absorbance is directly proportional to concentration. Standards are measured first and can consist of either one or many different concentrations. Concentrations of the standard are then analyzed and graphed using a least squares statistical analysis (seen above). Unknown samples can be calculated from the line fitting equation. If only a single standard is used, linearity is assumed.

3) Photometric Mode - Sometimes a wavelength scan is not required, but multiple discontinuous wavelength data needs to be collected. Photometric mode allows for a table of wavelengths to be defined and collected. This process is usually performed to save time since this function is quicker than a wavelength scan.

4) Time Drive Mode - This mode collects ordinate data from a fixed wavelength as a function of time. Frequently used for kinetic analysis to investigate samples changes over time.

 

How does a spectrophotometer measure a sample?

How does a spectrophotometer measure a sample?

For each wavelength of light passing through the spectrometer, the intensity of the light passing through the sample cell is measured. The intensity of light entering the sample is usually referred to as I0. The intensity of the light passing through the sample cell and emerging on the other side is usually designated I1. If we now calculate the percent of the ratio these two values (I1/I0)*100 we get a percent transmission (%T) value. The %T value is a measure of the light that passes through the sample to strike the detector. If I1 is less than I0, then obviously the sample has absorbed some of the incident light. A simple bit of mathematics is then done in the computer to convert this into something called the absorbance of the sample, given the symbol, A (sometimes abs). The absorbance is a measure of the amount of light that disappears (interacts) with the sample. The Beer-Lambert Law (above), is the relationship between absorbance as a function of sample concentration c, the sample extinction coefficient a, the  sample path length L, I0, and I1 is given by:

For most inexpensive spectrophotometers you will come across, the absorbance ranges from 0 to 3, but it can go higher than that for more expense instruments. The UV-3600 Plus and Solid Spec 3700 can measure up to 8 and sometimes higher. An absorbance of 0 means that no light of that particular wavelength has been absorbed by the sample. The intensity of light entering and exiting the sample are both the same, so the ratio I1/I0 is 1, so -log10 of 1 is zero. An absorbance of 1 happens when 90% of the light at that wavelength has been absorbed, which means that the intensity is 10% of what it would otherwise be. In that case, I1/I0 is 10/100 = 0.1) and -log10 of 0.1 is 1.

Absorbance is a logarithmic scale similar to the Richter Scale used for earthquakes. An increase of 1 unit on the absorbance scale translates into a factor of 10 decrease on the %T scale. Another name people tend to use for a logarithmic relationship (factor of 10 change) is “order of magnitude”. Thus an instrument that can measure to 3 absorbance units has a range of 3 orders of magnitude. Remember also, as the %T value decreases the absorbance value increases. Above is a simple table that summarizes the relationship between %T and absorbance.

 

What is “molar absorptivity”?

So, what about ξ (sometimes designated a) in the Beer’s law equation? ξ is the molar absorptivity, also known as the extinction coefficient of the sample. It is a unique physical constant of the chemistry of the sample that relates to the sample’s ability to absorb light at a given wavelength. Like path length (b) and sample concentration (c), ξ is also directly proportional to Absorbance.

To begin we will rearrange the equation:

A = ξbc

to

ξ = A / bc

2) Quant Mode - Quantization is usually performed on a major peak of the sample. Data can be collected as either peak height or area. Beer’s law states that the sample absorbance is directly proportional to concentration. Standards are measured first and can consist of either one or many different concentrations. Concentrations of the standard are then analyzed and graphed using a least squares statistical analysis (seen above). Unknown samples can be calculated from the line fitting equation. If only a single standard is used, linearity is assumed.

In words, this relationship can be stated as “ξ” is a measure of the amount of light absorbed per unit of concentration” at a defined wavelength. Molar absorptivity is a constant for a particular substance, so if the concentration of the solution is halved so is the absorbance, which is exactly what you would expect. A compound with a high molar absorptivity is very effective at absorbing light at the stated wavelength, and hence low concentrations of a compound with a high molar absorptivity can be detected at lower concentrations. In addition, the absorbance value at a given wavelength can be calculated if you know the molar absorptivity, path length, and concentration.

 

How do you use the Beer-Lambert Law to perform quantitative analysis?

A = -log10 (I1/I0) = a*b*c

A = a*b*c

Let’s look at the Beer-Lambert law and explore its significance. This is important because people who use the law often don’t understand it, even though the equation representing the law is straightforward. So far we have considered only the amount of light entering and exiting the sample. There are three other important factors related to the sample that define the absorbance.

These factors are:

  1. the path length of the sample (represented by b)
  2. the concentration of the sample (represented by c)
  3. the extinction coefficient of the sample (represented by a). Sometimes a is also called the molar absorptivity.

The extinction coefficient is a physical property of the molecular bonding (chemical structure) of the sample compound. The same molecule will always have the same value for a at the specified wavelength. For example, a weakly absorbing peak (n to pi* bonding) may have a a value of only 1000; whereas, a strongly absorbing peak (pi to pi* bonding) can have values of 600,000 Values for a can range from several 100 to 1,000,000. The important feature of the a value is that it is a constant for the unique chemistry of the sample and will only change when the chemistry changes.

The reason why we prefer to express the law as absorbance (rather than %T) with this equation... A = a*b*c

is because absorbance is directly proportional to the other parameters, as long as the law is obeyed. The take home message is that

  1. if I double the path length of the sample, I double the absorbance value and
  2. if I double the concentration of the sample, I double the absorbance value.

Concentration and path length have a linear proportion relationship with the absorbance value.

 

What is the difference between “amount” and “concentration”?

It is vital in chemistry to understand the difference between amount of a sample and the concentration of a sample. An amount is a quantity of something; a concentration is an amount of something in a volume of something else. So 10 grams of sugar is an amount, while 10 grams of sugar dissolved in 100 milliliters of water is concentration. Amounts in chemistry are usually expressed as moles; whereas concentrations use the term molar. A mole of something is the molecular weight for the compound expressed as grams. So if the molecular weight of a compound is 245, then one mole of that com- pound would be 245 grams. A one molar solution would be 245 grams dissolved in one liter of solvent (water).

 

Why is quantitative analysis always done in Absorbance rather than %Transmission?

Why is quantitative analysis always done in Absorbance rather than %Transmission?

An example, now, suppose we have a solution of copper sulphate (which appears blue because it has an absorption maximum at 600 nm). We look at the way in which the intensity of the light changes as it passes through the solution in a 1 cm cuvette. We will look at the reduction every 0.2 cm as shown in the diagram above. Beer’s Law says that the fraction of the light absorbed by each layer of solution is the same. For our illustration, we will suppose that this fraction is 0.5 for each 0.2 cm “layer” and calculate the data table (upper right).

This data can be graphed to better see the relationship. For %T the plot displays a curve (upper left). However, for absorbance the relationship is a straight line (lower left). This is a crucial difference from %T. Scientists love straight lines rather than curves. Why? Because it is easier to mathematically model a straight line as opposed to a curve.

The equation A = abc tells us that absorbance depends on the total quantity of the absorbing compound in the light path through the cuvette (lower right). If we plot absorbance at the peak wavelength against concentration, we get a straight line passing through the origin (0,0). The linear relationship between concentration and absorbance is both simple and straightforward, which is why we prefer to express the Beer-Lambert law using absorbance as a measure of the absorption rather than %T. From this, it is clear that one would use absorbance as the scale for any UV/Vis/NIR application that involved quantitation. With absorbance we can use spectroscopy to measure how much of something is present.

 

What types of chemistry can a UV/Vis spectrophotometer measure?

What types of chemistry can a UV/Vis spectrophotometer measure?

Here we will consider the electron transitions of the UV/Vis spectral region. This high energy light, compared to near infra-red, has enough energy to cause electronic transitions. These types of transitions move electrons from low energy levels, in an atom or molecule, to higher energy levels.

First, we will explain what happens when organic compounds absorb UV or visible light, and why the wavelength of light absorbed varies from compound to compound. Put simply the energy from the light is imparted to the electrons involved in chemical bonding. This causes the electrons to be promoted (moved) to a higher energy level; thereby, absorbing the light energy. These energy levels are associated with the bonding and anti-bonding orbitals of conjugated double and triple bonded carbon atoms or aromatic benzene type ring systems in organic molecules. Remember that the diagram above isn’t intended to be to scale, it just shows the relative placing of the different orbitals. When light passes through the compound, energy from the light is used to promote an electron from a lower energy bonding or non- bonding orbital into higher energy empty anti- bonding orbitals. The possible electron jumps that light can cause are seen in this slides figure (upper right).

The length of the arrow is proportional to the amount of energy needed to promote the electron between energy levels. So, if you have a bigger energy jump, you will absorb light with a higher frequency, which is the same as saying that you will absorb light with a lower wavelength.

Real world analogy: The promotion of an electron to a higher energy level is just like throwing a ball into the air. The energy from the muscles of your arm (light) is imparted (promotion) to the ball (electron) causing it to rise against the force of gravity (ground state). The ball then reaches a certain height (excited state) that is determined by the amount of effort (light wavelength) you used in the throw. This is the process of light absorption in a nutshell, except! The ball (electron) does not just hang out at the higher energy levels, it falls back to the ground level from where it was originally promoted. The same thing happens to the promoted electron when the light energy is shut off. The electron drops back down to the ground state. The potential energy in the falling electron is released as heat or it can be re-emitted as light (fluorescence or phosphorescence). This re- emitted light can be studied by luminescence spectroscopy.

The most important electron orbital jumps for UV/Vis spectroscopy are:
1) From pi bonding orbitals to pi anti- bonding orbitals (π to π*)
2) From non-bonding orbitals to pi anti- bonding orbitals (n to π*)
3) From non-bonding orbitals to sigma anti- bonding orbitals (n to σ*)

That means that in order to absorb light in the region from 200 to 800 nm (which is where the spectra are measured), the molecule must contain either pi bonds Double bond or aromatic ring systems) or atoms with non- bonding orbitals. Remember that a non- bonding orbital is a lone electron pair on, say, oxygen, nitrogen or a halogen. Groups in a molecule which absorb light are known as chromophores. In addition to organic molecules, individual inorganic at- oms as well as metallic ionic complexes can absorb as well. Inorganic complexes tend to yield sharper spectral peaks with narrow half band widths. An example of a rare earth inorganic compound, holmium oxide, is shown at bottom left.

 

What is the relationship between absorption spectroscopy and polyaromatic ring structure?

What is the relationship between absorption spectroscopy and polyaromatic ring structure?

There are many organic compounds that have conjugated double bond systems (hereafter referred to as “conjugated systems”), in which every other bond is a double bond. These conjugated systems have a large influence on peak wavelengths and absorption intensities.

The top figure shows the structures of benzene, naphthalene, and anthracene. The bottom figure shows the absorption spectra obtained by dissolving these compounds in ethanol and analyzing the resulting solutions. The concentrations were adjusted so that the absorption intensities of the components were roughly the same. It can be seen in the figure that peak wavelengths tend to be shifted toward the long wavelength region as the conjugated system gets larger.

 Why, then, does the peak wavelength tend to be shifted toward the long wavelength region as the size of the conjugated system increases? Let us consider the relationship between the energy of light and the movement of electrons.

Light exhibits properties of both waves and particles (photons). The energy of one photon is expressed as hc/λ, where h is Planck’s constant, c is the speed of light, and λ is the wavelength. Absorption in the ultraviolet and visible regions is related to the transition of electrons. “Transition” refers to the switching of an electron from one state of motion to another. The state of motion of the π electrons in the conjugated system changes more easily than that of the σ electrons that form the molecular frameworks. If a photon collides with a π electron, that π electron readily changes to a different state of motion. This is true even if the photon has only a small amount of energy. The π electrons in relatively large conjugated systems are more easily affected by low-energy photons. Transition expresses the way that the energy of photons is absorbed by electrons. If a photon has a relatively small amount of energy, the value of hc/λ for that photon is relatively small, and therefore the value of λ is relatively large. λ is observed as the absorption wavelength and so, if there is a conjugated system, peaks tend to appear in regions where λ is large, i.e., the long wavelength region.

 

How does pi orbital conjugation influence peak wavelength?

How does pi orbital conjugation influence peak wavelength?

The table here gives the peak wavelengths and the molar absorption coefficients of various organic compounds. The molar absorption coefficient is a measurement of how strongly a substance absorbs light. The larger its value, the greater the absorption. With larger conjugated systems, the absorption peak wavelengths tend to be shifted toward the long wavelength region and the absorption peaks tend to be larger.

 

What is the relationship between absorption spectroscopy and sigma orbital bonding?

What is the relationship between absorption spectroscopy and sigma orbital bonding?

The top figure shows the structures of food dyes New Coccine (Red No. 102) and Brilliant Blue FCF (Blue No. 1) and the bottom figure shows their absorption spectra. Food dyes tend to have large conjugated systems, like those shown in figure, and therefore their peak wavelengths tend to be shifted toward the long wavelength region, with peaks appearing in the visible region (400 to 700 nm). Therefore they are recognized as colors. Incidentally, the color that we see is the color that is not absorbed by the substance (which is called the “complementary color”). As shown in the figure, New Coccine absorbs blue and green light in the range 450 to 550 nm, and so the complementary color, red, is seen by the human eye. Brilliant Blue FCF absorbs yellow light in the range 560 to 650 nm and so blue is seen by the human eye.

 

What is the influence of functional groups?

What are the influence of functional groups?

Absorption peaks are also influenced by functional groups. The top figure shows the absorption spectra of benzene, phenol, which consists of a hydroxyl group bonded to a benzene ring, and p-nitrophenol, which consists of a hydroxyl group and a nitro group bonded to a benzene ring. The functional groups influence the conjugated systems, causing the absorption peaks to appear at longer wavelengths than the peak wavelength of benzene, although they do not go beyond 400 nm and enter the visible region. The color of organic compounds, then, is influenced more strongly by the size of the conjugate system.

 

Does molecular size matter if it is not pi electrons?

Does molecular size matter if it is not pi electrons?

The figures here show the absorption spectra of prednisolone, which is used as a pharmaceutical, and benzene. Although prednisolone has a large molecular framework, its conjugated system is small and so its peak wavelengths are not shifted greatly toward the long wavelength region, and its peaks appear at roughly the same position as those of benzene.

 

Electronic theory of Near Infra-Red (NIR) chemistry?

Electronic theory of Near Infra-Red (NIR) chemistry?

Electronic theory of Near Infra-Red (NIR) chemistry?

As can be seen from the graphic at the top, both the mid infra-red and near infra-red spectral regions are at longer wavelengths (lower energy) than UV/Visible light. This energy is sufficiently lower that electron promotions do not result from it. But while electrons are not promoted, chemical bonds can be affected.

The energy diagram above shows the rather large energy differences between bonding electronic transitions verses the bond deforming vibrational and rotational energy levels caused by mid infra-red frequencies. As such, infra-red spectroscopy yields information on what atoms are bonded to other atoms in a molecule. This is very useful for materials identification and characterization.

 

What types of chemistry can a Near Infra-Red (NIR) spectrophotometer measure?

What types of chemistry can a Near Infra-Red (NIR) spectrophotometer measure?

The near infra-red spectral region is nothing more than an “overtone mirror” of the mid infra-red region. The near infra-red region contains exactly the same information as the mid infra-red region. So why use the NIR region if there’s no new information there? Overtones are typically much less intense in amplitude than their primary counterparts. This is another way of saying that their extinction coefficients (molar absorptivity) is less. So, from Beer’s Law this means that the path lengths used for NIR spectroscopy can be longer. Typical MIR path lengths are on the order of microns, whereas, the path lengths for NIR spectroscopy are in the range of millimeters. This longer path length can be useful in certain modes of materials characterization.

One interesting fact of NIR spectroscopy is its great sensitivity to small amounts of water, or in general any -OH group. As seen in the spectrum on the right, the -OH group has three bands in the NIR at around 1450 nm, 1900 nm, and 2600 nm. This can pose a problem for any sample with a significant -OH group component.

For Research Use Only. Not for use in diagnostic procedures.

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