CHEM - Optical Techniques, Electrochemistry, Chromatography, Electrophoresis, Mass Spectrometry

Many laboratory determinations are made by measuring light that has been what five things?

Transmitted, emitted, absorbed, reflected, or scattered.

What is photometry?

The measurement of the luminous intensity of light or the amount of luminous light falling on a surface from such a source.

What are the two types of photometers?

Filter photometers and spectrophotometers.

What does a filter photometer use to isolate a narrow wavelength range of the spectrum?

Optical filters.

What does a spectrophotometer use to isolate specific wavelengths of light?

A monochromator with prisms or gratings.

What does spectrophotometry involve?

The interaction of electromagnetic radiation with matter.

What is a spectrophotometer used for?

To indirectly determine the amount of a compound present in a solution by shining light of a specific wavelength through the solution and measuring how much was absorbed.

What wavelength range is typically used in laboratory spectrophotometry?

Ultraviolet and visible regions of the spectrum (290-800 nm).

What does Planck's formula describe?

The relationship between wavelength and energy.

What is the relationship between the energy of light and its wavelength?

The energy of light is inversely proportional to the wavelength.

In the equation E = hv = hc/lambda, what does h represent?

Planck's constant.

In the equation E = hv = hc/lambda, what does v represent?

Frequency.

In the equation E = hv = hc/lambda, what does c represent?

The speed of light.

In the equation E = hv = hc/lambda, what does lambda represent?

Wavelength.

What is Io in absorption of electromagnetic radiation?

The original intensity of radiant energy impinging on the cell.

What is Is in absorption of electromagnetic radiation?

The intensity of the transmitted radiant energy.

Why is a blank or reference solution used in absorption measurements?

To eliminate the effects of reflection by the cell wall or solvent, so only the absorption of the compound of interest is measured.

What does a blank or reference solution contain?

Everything but the compound to be measured.

What is the formula for transmittance (T)?

T = Is/I0.

What is the formula for percent transmittance (%T)?

%T = Is/I0 x 100.

What is the formula for absorbance (A) in terms of transmittance?

A = -log(Is/I0).

What is the formula for absorbance (A) in terms of %T?

A = 2 - log %T.

What is Beer-Lambert Law (Beer's Law)?

The concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light.

What is the mathematical relationship of Beer's Law?

A = ebc.

In Beer's Law, what does A represent?

Absorbance.

In Beer's Law, what does e represent?

Molar absorptivity, a constant for a given compound at a given wavelength.

In Beer's Law, what does b represent?

Light path (cell path length in cm), usually 1 cm.

In Beer's Law, what does c represent?

Concentration of the substance of interest (mol/L).

What is the effect on absorbance if the path length through a solution is doubled?

Absorbance is doubled, since twice as many absorbing molecules are present in the radiant energy path.

What is the effect on absorbance as concentration increases?

Absorbance increases and transmittance decreases.

What formula relates an unknown concentration to a single standard?

Cu = (Au / As) x Cs.

In the equation Cu = (Au / As) x Cs, what does Cu represent?

Concentration of the unknown.

In the equation Cu = (Au / As) x Cs, what does Cs represent?

Concentration of the standard.

In the equation Cu = (Au / As) x Cs, what does Au represent?

Absorbance of the unknown.

In the equation Cu = (Au / As) x Cs, what does As represent?

Absorbance of the standard.

What two conditions are required for the proportional equation relating unknown and standard concentrations to be valid?

The sample must obey Beer's Law, and both standard and unknown must be measured in the same cell.

What must be ensured when calculating an unknown using a standard calibration curve?

The data must fall within the standard calibration curve (line of best fit).

Name four conditions that cause deviation from Beer's Law.

Very elevated concentrations, incident light not monochromatic, significant solvent absorption, and non-parallel cell sides.

What are two additional conditions that cause deviation from Beer's Law?

Stray light transmitted by other mechanisms and fluorescence.

If two or more chemical species absorb the wavelength of incident radiant energy with different absorptivities, what happens to Beer's Law?

Beer's Law will not be followed.

What is stray radiation (stray light) in spectrophotometry?

Radiant energy that reaches the detector at wavelengths other than those indicated by the monochromatic setting.

What happens to deviation from Beer's Law as the amount of stray light increases?

Deviation from Beer's Law increases (linearity decreases).

What is an analyte in spectrometric methods?

The chemical of interest.

What is a chromophore?

The part of a molecule responsible for color.

What are the three design approaches for spectrometric methods?

The analyte absorbs at a unique wavelength; the analyte reacts with a reagent to produce a product that absorbs at a unique wavelength; or the analyte is involved in a reaction that produces a chromophore.

What are the four basic components of a spectrophotometer?

Light source, wavelength isolator (filters, prisms, monochromator), detector, and read-out device.

What are the two forms of light sources?

Lamps and lasers.

What type of lamp is used for the visible range and near infrared?

Tungsten or tungsten-iodide.

What type of lamp is used for UV (190-400 nm)?

Hydrogen or deuterium.

What type of lamp is used for UV?

Mercury.

What does LASER stand for?

Light Amplification by Stimulated Emission of Radiation.

What are the characteristics of lasers as light sources?

They provide intense radiation of a narrow wavelength; expensive; typically used for fluorescence and specialized testing.

What are the most important factors for a light source?

Range, spectral distribution within the range, source of radiant production, stability of radiant energy, and temperature.

Must a light source be exactly aligned when replaced?

Yes, it must be exactly aligned when replaced.

What are the three devices used to separate white light into a specific band?

Filter, prism, and grating.

What are absorbance filters made of?

One or more layers of coloured glass.

How do absorbance filters work?

By absorbing all other wavelengths that are not of interest.

What is another name for absorbance filters?

Wratten Absorption Filter.

What is the band pass of absorbance filters?

35-50 nm.

What are the advantages and disadvantages of absorbance filters?

They are simple and inexpensive but not precise due to wide band pass.

What are interference filters made of?

Semi-transparent silver films on both sides of a thin transparent layer of magnesium fluoride.

What is another name for interference filters?

Fabry-Perot.

What are interference filters used for?

For a "fixed wavelength."

What principle do interference filters use to produce monochromatic light?

Constructive interference of waves.

What type of band pass do interference filters have?

A narrow band pass.

How does a prism separate white light?

A narrow beam of light focused on a prism is refracted as it enters the denser glass; short wavelengths are refracted more than long wavelengths, resulting in dispersion of white light into a continuous spectrum.

What type of prism is used for visible light?

Glass prism.

What type of prism is used for UV?

Quartz prism.

What is the major disadvantage of using a prism?

It gives a nonlinear separation of bands.

Why must several wavelengths be measured for proper wavelength calibration with a prism?

Because of the non-linear refraction of light.

What is the advantage of a prism?

Inexpensive and offers a good means of separating light into bands.

What are the two types of gratings?

Transmittance (made of glass) and reflection (made of aluminum).

What is the advantage of gratings over prisms?

They offer linear dispersion of light over the entire UV and visible range.

How are modern holographically produced gratings made?

Using a laser, making them very accurate and minimizing stray radiant energy.

What does a diffraction grating consist of?

A highly polished reflecting surface with many equally spaced parallel grooves (15,000 or 30,000 per inch) with sharp corners.

What determines the resolution of a diffraction grating?

The number of grooves.

How is the wavelength reflected by a grating altered?

By changing the angle at which the radiation strikes the grating.

What are the two types of slits in a spectrophotometer?

Entrance slit and exit slit.

What is the function of the entrance slit?

Focuses the light on the grating or prism.

What is the function of the exit slit?

Isolates the selected narrow band of wavelength.

What happens when the exit slit width is increased?

The band of emerging light is broadened, which increases intensity but decreases spectral purity.

What is the bandpass of a monochromator?

The range of wavelengths transmitted, calculated as width at over half the maximum transmittance.

If bandpass = 10 nm and the wavelength setting is 455 nm, what is the range of transmitted light?

450 nm to 460 nm (455 +/- 5 nm).

At what point is bandpass measured?

At one half the peak transmittance.

What does bandpass describe?

The purity of the light passing through the sample.

What are the three factors that determine the bandpass of an instrument?

The light intensity output of the source, the efficiency of the system to isolate wavelength bands, and the sensitivity of the detector.

What is the trade-off of a narrow band pass?

It offers greater resolution and sensitivity but there is a decrease in intensity.

What is another name for a sample holder in spectrophotometry?

A cuvette or cell.

What materials can cuvettes be made of?

Quartz (UV), glass, or transparent plastic (visible).

Why are quartz or fused silica cuvettes necessary for ultraviolet radiation?

Because glass can absorb UV light.

What is the advantage of square cuvettes over round cuvettes?

There is less error from the lens effect, orientation in the spectrophotometer, and refraction.

What should be done with cuvettes that have scratches on their optical surface?

They should be discarded.

What is the error for every 5 degree deviation in orientation for round cuvettes?

0.25%.

What happens to sensitivity as sample size increases?

Sensitivity increases.

What is the normal cell path length for a cuvette?

1 cm.

When is a longer path length required?

For very dilute samples.

What is the purpose of the detector in a spectrophotometer?

To convert the transmitted radiation energy (photon) into an equivalent amount of electric energy (electron).

What are the four types of detectors?

Barrier layer cell, silicon photodiode, phototube, and photomultiplier tube.

What is a barrier layer cell good for?

Basic, simple spectrophotometers.

What are the layers of a barrier layer cell?

Cathode (silver), selenium (semiconductor), and anode (iron or copper).