Spectrophotometry, Light Properties, and Analytical Techniques in Chemistry

What is spectrophotometry?

A technique that uses properties of light for qualitative and quantitative measurements.

What does wavelength measure?

The distance between identical sites on consecutive waves, such as the distance between two crests.

What is electromagnetic radiation composed of?

Photons of energy traveling in waves.

How is radiation related to wavelength?

Radiation is inversely proportional to wavelength; as wavelength increases, frequency decreases.

What is the wavelength range for ultraviolet (UV) light?

190 to approximately 390 nanometers.

What is the wavelength range for visible light?

390 to 750 nanometers.

What is the significance of wavelengths longer than 800 nanometers?

They are classified as infrared and are perceived as heat instead of light.

What does Beer's Law state?

The concentration of a substance is directly proportional to the amount of light absorbed.

What is the formula for absorbance in spectrophotometry?

A = ℇ x b x c, where A is absorbance, ℇ is molar absorptivity, b is the length of the light path, and c is concentration.

What does percent transmittance indicate?

The proportion of light that penetrates the solution; inversely related to concentration.

What is the role of the aperture in a spectrophotometer?

It is a small opening that limits the amount of light entering the instrument.

What is a diffraction grating?

An optical component that disperses light into a linear spectrum by diffracting it at multiple angles.

How does a prism differ from a diffraction grating?

A prism produces a nonlinear spectrum by separating light based on differing indices of refraction.

What is the purpose of a double-beamed photometer?

To correct for variation in light source intensity.

What does turbidimetry measure?

The concentration of particulate matter in a sample by determining the amount of light blocked by particles.

What is nephelometry?

A technique that measures light scattered by suspended particles at a 90-degree angle from the light path.

What factors are checked for spectrophotometer quality assurance?

Wavelength accuracy, stray light, and linearity.

How can a spectrophotometer's wavelength be calibrated?

Using a didymium filter or holmium oxide glass to verify wavelength settings.

What is atomic absorption spectrophotometry?

A method that measures concentration by detecting the absorption of electromagnetic radiation by atoms.

What is bi-chromatic analysis?

A technique where absorbance readings are taken at two wavelengths to correct for spectral interference.

What is fluorometry?

A method based on light absorbed and emitted from a sample (fluorescence), using two monochromators.

What is flow cytometry?

A technique for determining different cell types by their surface antigens using fluorochromes attached to specific antibodies.

What is fluorescence polarization?

A method similar to fluorometry but with two polarizing filters to allow radiant energy to be polarized in a single plane.

What does a polarizing filter do?

It allows the passage of light that is in one plane and blocks all others.

What happens when two polarizers are parallel?

Light that is in the same plane as excitation light is passed to the detector.

What occurs when two polarizers are perpendicular?

Emitted light that is in the same plane as excitation light is blocked.

How does the rotation of large molecules affect polarized light emission?

Large molecules emit polarized light along the same plane as the incident light.

What type of light do small molecules emit?

Small molecules emit depolarized light because they rotate out of the plane of polarization.

Why are fluorometric assays preferred over spectrophotometric methods?

Fluorometric assays are more specific and sensitive.

What do ion-selective electrodes measure?

They measure the activity of specific ions in solution.

What is the role of the reference electrode in ion-selective electrodes?

It provides a constant potential for measurement of the analyte in solution.

What is the effect of voltage applied to an ion-selective electrode?

It changes the membrane's electrochemical properties, resulting in a potential change.

What is electrophoresis used for?

It is used for separation and analysis of biomacromolecules based on size and charge.

What is cataphoresis?

The electrophoresis of positively charged particles.

What is anaphoresis?

The electrophoresis of negatively charged particles.

What happens to proteins in electrophoresis?

Proteins migrate towards the anode (positive end) because they are negatively charged.

What is the purpose of using a buffer in electrophoresis?

The buffer acts as a carrier for ions and helps maintain pH.

What do anions and cations do in an electrophoresis setup?

Anions migrate towards the anode, while cations migrate towards the cathode.

What is osmomery used for?

It measures the osmolality of solutions to determine the concentration of solute.

What is the most common method of osmolality measurement in clinical labs?

Freezing point depression osmometry.

What principle does chromatography rely on?

Differential solubility of substances in different solvents.

What is thin layer chromatography used for?

To isolate nonvolatile mixtures, measured in Rf values.

What is high-performance liquid chromatography (HPLC)?

A method that uses pressure for fast separations of compounds.

What does mass spectrometry measure?

The mass to charge ratio of molecules present in a sample.

What is gas chromatography used for?

To separate mixtures of compounds that are volatile or can be made volatile.

What is the purpose of matrix-assisted laser desorption ionization (MALDI-TOF)?

To analyze molecules such as peptides and proteins by measuring the time it takes for ions to reach the detector.