Chemistry Unit 1

What is photometry?

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

What is spectrophotometry?

The measurement of the amount of light of a specific wavelength absorbed by a solution when electromagnetic radiation interacts with matter.

What is the relationship between wavelength and energy in the electromagnetic spectrum?

The longer the wavelength, the lower the energy (and the shorter the wavelength, the higher the energy).

What wavelength range is used for UV and visible spectrum spectrophotometry in laboratory applications?

290–800 nm.

State Beer-Lambert’s Law regarding light absorption and concentration.

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

According to Beer-Lambert's Law, what is the relationship between absorbance and path length?

Absorbance is directly proportional to the path length of the radiant energy through the cell.

What condition must be met regarding the cell/cuvette for the Beer-Lambert concentration equation to be valid?

Both standard and unknown must be measured in the exact same cell.

What is the mathematical equation to calculate the concentration of an unknown ($C_u$) using Beer-Lambert's Law?

$C_u = (A_u \times C_s) / A_s$

What is the formula relating absorbance ($A$) to percent transmittance ($\%T$)?

$A = 2 - \log(\%T)$

What happens to absorbance as percent transmittance ($\%T$) increases?

Absorbance decreases.

What is the equation for Beer-Lambert's Law using molar absorptivity ($\varepsilon$)?

$A = \varepsilon b c$

What are the units for molar absorptivity ($\varepsilon$)?

$\text{L/mol}\cdot\text{cm}$

What path length unit is used in the molar absorptivity equation ($A = \varepsilon b c$)?

Centimeters ($\text{cm}$).

What analyte concentration unit is used in the molar absorptivity equation ($A = \varepsilon b c$)?

Moles per liter ($\text{mol/L}$).

What deviation from Beer's Law occurs when solute concentration is extremely high?

It causes a non-linear relationship (deviation from Beer's Law).

How does non-monochromatic incident light affect Beer's Law?

It causes a deviation from Beer's Law because wavelengths are not properly isolated.

What happens to Beer-Lambert linearity if solvent absorption is significant compared to solute absorbance?

It results in a deviation from Beer's Law.

Why does the presence of two or more chemical species absorbing the incident wavelength cause a deviation from Beer's Law?

Multiple absorbing species alter the direct proportionality between single-analyte concentration and absorbance.

How do non-parallel cuvette sides cause a deviation from Beer's Law?

They alter the light path length ($b$) across the sample.

How does an increase in stray light affect monochromicity and Beer's Law adherence?

It decreases monochromicity and causes a deviation from Beer's Law.

Why can measuring a fluorescent solution cause a deviation from Beer's Law?

Fluorescence emits additional light into the detector, falsely altering transmitted light measurements.

What light sources are used in spectrophotometers for the visible range and near infrared?

Tungsten or Tungsten-iodide lamps.

Which light source is used exclusively for the visible range in spectrophotometers?

Quartz-halogen lamp.

Which light source is used for normal UV spectrophotometry ($190\text{--}400\text{ nm}$)?

Hydrogen or deuterium lamp.

Which light source is used specifically for fluorescent UV spectrophotometry?

Mercury lamp.

What are the five most important factors when selecting a spectrophotometer light source?

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

What is the function of a monochromator/wavelength isolator in a spectrophotometer?

To restrict and isolate specific wavelengths of light.

How is the bandpass of a monochromator defined and calculated?

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

At what point on the transmittance peak is bandpass measured?

At $1/2$ the peak transmittance.

What is the primary function of a photodetector in a spectrophotometer?

To convert transmitted radiation energy (photons) into an equivalent amount of electrical energy (electrons).

Which basic photodetector type is suitable for simple spectrophotometers without requiring outside voltage?

Barrier-cell layer detector.

How does a phototube detector differ from a barrier-cell layer detector?

A phototube requires an outside voltage to operate.

How does a photomultiplier tube (PMT) amplify signal?

It converts photons to electrons and amplifies the electric signal through a series of dynodes.

How much more sensitive is a PMT compared to a standard phototube?

$200\times$ more sensitive.

What type of photodetector is made of a photosensitive semiconductor and measures light at a multitude of wavelengths?

Photodiode (silicon).

Which photodetector type is predominantly used in medical analytical laboratories?

Photomultiplier tube (PMT).

Which photodetector type is primarily used in research laboratories?

Photodiodes.

What is the structural setup of a single-beam spectrophotometer?

A single beam position shared sequentially by the reference and sample cuvette.

How does a double-beam-in-space spectrophotometer split and detect light?

A single light source is split into two equal-intensity beams measured simultaneously by two separate matched detectors.

How does a double-beam-in-time spectrophotometer split and detect light?

A rotating chopper or mirror alternates the light beam between reference and sample cuvettes onto a single detector as light pulses.

How is spectrophotometer linearity demonstrated during quality control?

When changes in concentration produce a straight-line calibration curve.

What is wavelength accuracy in spectrophotometer QC?

Ensuring the wavelength indicated on the dial matches the actual wavelength of light passed by the monochromator.

How is spectrophotometer wavelength accuracy checked?

Using standard absorbing solutions or filters with maximum absorbance at a known wavelength.

What is stray light in spectrophotometry?

Any light reaching the detector that lies outside the bandpass transmitted by the monochromator.

How is stray light detected during spectrophotometer quality control?

Using cutoff filters.

What is the principle of refractivity?

When light passes between media of different densities, its direction and velocity are altered.

Name two clinical laboratory applications of refractivity.

Measuring total serum protein and urine specific gravity.

What is the principle of reflectance photometry?

Measuring light reflected from a colorimetric reaction on a testing strip to determine analyte concentration.

Does reflectance photometry follow Beer-Lambert's Law?

No, it has a non-linear relationship between color intensity and analyte concentration.

List three clinical applications/instruments that utilize reflectance photometry.

Glucometers, urinalysis test strip readers, and Vitros chemistry analyzers.

What is the fundamental principle of Atomic Absorption Spectrophotometry (AAS)?

Ground-state free atoms absorb light energy at specific wavelengths corresponding to their element.

What clinical analytes are measured using Atomic Absorption Spectrophotometry?

Trace or toxic metals.

What component in AAS produces light specific to the analyte being tested?

Hollow Cathode Lamp (HCL).

What material forms the filament of a Hollow Cathode Lamp in AAS?

The same metal element that is being tested in the sample.

What is the function of the chopper in Atomic Absorption Spectrophotometry?

To pulse the light coming from the Hollow Cathode Lamp before it hits the sample.

How are free ground-state atoms generated from a liquid sample in AAS?

The sample is nebulized into a flame.

Why does the detector in an AAS instrument ignore light emitted by excited sample atoms returning to the ground state?

The detector is electronically tuned to detect only pulsed light (from the chopper), ignoring unpulsed emission from the flame.

What is the purpose of the monochromator in an AAS setup?

It is placed before the detector to isolate the specific analytical wavelength of interest.

List the 8 major components of an Atomic Absorption Spectrophotometer in order of light path.

Hollow Cathode Lamp, Chopper, Flame/Nebulizer, Entrance slit, Monochromator, Exit slit, Detector, Meter.

What is Zeeman magnetic correction in AAS?

Using a strong magnetic field to split analyte absorbance from background absorbance.

How is net analyte absorbance calculated using Zeeman background correction in AAS?

Background absorbance is subtracted from total absorbance.

In AAS, how do closely absorbing atomic species or molecular absorption affect measured absorbance?

They cause spectral interference, resulting in falsely increased absorbance.

How does salt/oxide scattering affect absorbance results in AAS?

It causes spectral interference, resulting in falsely increased absorbance.

How does background emission affect measured absorbance in AAS?

It causes spectral interference, resulting in falsely decreased absorbance.

What is non-specific non-spectral interference in AAS, and how does it affect results?

Any factor affecting sample nebulization, resulting in falsely decreased absorbance.

What is chemical interference in AAS?

Anions failing to dissociate completely from cations in the flame, reducing ground-state free atoms.

Give an example of chemical interference in AAS and its corrective agent.

Formation of calcium phosphate; minimized by adding lanthanum.

What is ionization interference in AAS?

Atoms in the flame becoming ionized instead of remaining in the unexcited ground state.

How can ionization interference be corrected in AAS?

By operating at a lower flame temperature or adding an excess of a more easily ionized element.

What is matrix interference in AAS?

Differences in physical/chemical composition between the standard and the test sample.

What is the principle of fluorometry?

Measuring light of a longer wavelength emitted by a fluorophore when excited by light of a shorter wavelength.

How does fluorometry sensitivity compare to absorption spectrophotometry?

Fluorometry is significantly more sensitive.

What is the relationship between fluorescence intensity and analyte concentration?

Fluorescence intensity is directly proportional to concentration.

In a fluorometer, what is the geometric positioning of the emission detector relative to the excitation source?

Placed at a $90^\circ$ angle.

What is Rayleigh scattering in fluorometry?

Light scattering where excitation and emission wavelengths are identical.

What is Raman scattering in fluorometry?

Light emitted at a longer wavelength by solvent molecules rather than the target analyte.

What term describes a loss of fluorescence signal due to energy transfer or environmental factors?

Quenching (or bleaching).

What is chemiluminescence?

Excitation of an analyte by a chemical reaction to produce emitted light.

What is bioluminescence?

Excitation of an analyte by a biochemical reaction/system to produce emitted light.

What is electrochemiluminescence?

Excitation of an analyte by an electrochemical reaction to produce emitted light.

What is luminometry?

Measuring light emitted by an analyte when excited by a chemical reaction.

List three limitations of luminometry.

Light leaks, light piping, and high background luminescence from reagents/vessels.

What is potentiometry?

Measuring the potential difference between two electrodes under zero current flow.

What three components are required for a potentiometric measuring system?

An indicator electrode, a reference electrode, and a high-impedance measuring device.

Name two standard reference electrodes used in potentiometry.

$\text{Ag/AgCl}$ electrode and Mercury/Calomel ($\text{HgCl}$) electrode.

What is the principle of an Ion-Selective Electrode (ISE)?

It develops an electrical potential proportional to the activity of one specific ion.

What membrane material is used in a sodium ($\text{Na}^+$) ISE?

Glass membrane.

What ionophore membrane material is used in a potassium ($\text{K}^+$) ISE?

Valinomycin.

What membrane material is used in a chloride ($\text{Cl}^-$) ISE?

Polymer membrane.

What membrane material is used in a calcium ($\text{Ca}^{2+}$) ISE?

Dioctyl phenyl phosphonate.

What is measured using a standard glass pH electrode?

Hydrogen ion ($\text{H}^+$) activity/concentration.

How does a Severinghaus electrode measure $p\text{CO}_2$?

It converts $p\text{CO}_2$-induced pH changes across a gas-permeable membrane into an electrical signal.

What electrical parameter is measured in amperometry?

Electrical current.

What specific electrode uses amperometry to measure $p\text{O}_2$?

Clark electrode.

What is coulometry?

Applying a constant current and measuring the time required to complete an electrochemical reaction.

What clinical test uses coulometry?

Sweat chloride test (for diagnosing cystic fibrosis).

In sweat chloride coulometry, what reaction is measured?

The time required for all $\text{Cl}^-$ ions in $10\ \mu\text{L}$ of sweat to bind free $\text{Ag}^+$ ions.

What is the principle of electrophoresis?

Using an electrical field to separate electroactive colloidal particles across an electrolyte solution.

During electrophoresis, to which electrode do negatively charged particles migrate?

The positive anode.

During electrophoresis, to which electrode do positively charged particles migrate?

The negative cathode.