MLS 220 Chemistry Exam 4 Terms & Definitions Study Set

LECTURE 17: INSTRUMENTATION – LIGHT ENERGY

Light

Type of electromagnetic radiant energy that travels in waves

Wavelength

Distance between two peaks as light travels in wavelike manner

• determines the color seen by the human eye (every wavelength is a particular color)

• expressed in nanometers

Electromagnetic radiation

Spectrum of energy

-long wavelength of radio frequencies to highly energetic gamma rays and x rays

Visible light

380-750 nm

UV light

10-400 nm

Color of light seen by naked eye is _______

Transmitted

• All other wavelengths that are not transmitted are absorbed.

Planks Equation

Shows relationship between wavelength and energy

• Frequency is inversely proportional to wavelength (Ex. ↑Frequency ↓Wavelength )

• Energy of electromagnetic radiation is inversely proportional wavelength ( Ex. ↑Energy ↓Wavelength)

Planks Equation

E = hv = (hc)/λ

E: Energy

h: plank's constant (6.62x10^-27)

V: frequency

c: concentration

Frequency is ____ proportional to wavelength.

inversely

V = C/λ

V= frequency

C= speed of light

λ= wavelength

Energy of electromagnetic radiation is ______ proportional to wavelength

Inversely

Long wavelength =

-Low frequency

-Low energy

Short wavelength =

-high frequency

-high energy

Wavelengths and color observed

380-440: Violet

440-500: Blue

500-580: Green

580-600: Yellow

600-620: Orange

620-750: Red

Beer's law

↑concentration ↑light absorbed

The amount of light absorbed by a solution is directly proportional to the concentration of a solution

The logarithm of the transmitted light of a substance is ___________proportional to its concentration

Inversely

Absorbance equation

A = εbc

A: absorbance

ε: absorptivity

b: length of light path (cm)

c: concentration

Photometry

-Determine concentration of a substance

-Measures intensity of light ->color

Wavelength of light is not taken into account
 Only measures visible light

T or F? Photometry only measures visible light

True

In Photometry, is wavelength taken into account?

No

Spectrophotometry

The measurement of light at selected wavelength

-in ranges of UV, visible, and infrared (colorimetry)

Spectrophotometry uses what type of measurement? Qualitative or quantitative or both?

Qualitative and quantitative

Spectrophotometry components (single beam)

1. Light source

2. Spectral Isolation device (monochromator)

3. Fiberoptics

4. Cuvette

5. Photodetector

6. Read out device

7. Recorder

8. Microprocessor

(Spectrophotometry) Light source include

-Incandescent lamps

-hydrogen lamps

-deuterium lamps

-laser

(Spectrophotometry) Spectral Isolation Device (Monochromator)

Isolated radiant energy of desired wavelengths (ex: 340 nm) while excluding other unwanted wavelengths

-filters

-prisms

-refraction gratings

(Spectrophotometry) Fiberoptics

Improve directional control of the light beam within the instrument using glass fibers

• Bundles of thin, transparent fibers of glass, quartz, or plastic “flexible light pipes

(Spectrophotometry) Cuvette

Small vessel designed to hold liquid in the light

• Square or rectangular → plane-parallel optical surfaces and constant light path
  ❖ Glass, quartz, or plastic
  

(Spectrophotometry) Photodetector

Convert light into measurable electrical signal by detecting photons (electrons are released)

❖ Electrons are released in proportion to the number of photons that strike

(Spectrophotometry) Readout device/computer

amplifies and mathematically manipulates the electrical signal produced and converts it into a convenient format

Absorbance Spectrophotometry

Concentration of an unknown sample is determined by measuring its absorption of light at a particular

- Measures the light absorbed/transmitted by a solution
- Quantitative Measurement
↑ Color Intensity (brightness of color) ↑ Concentration of Substance

Is absorbance Spectrophotometry quantitative or qualitative?

Quantitative

In absorbance spectrophotometry, can the samples be colored?

Samples must be naturally colored or capable of being colored

In absorbance spectrophotometry. Color intensity is _________ proportional to the concentration of the substance

Directly

Absorbance (A)

Expression of the amount of light absorbed by a solution

Percent Transmittance (%T)

Amount of light that passes through a colored solution compared with the amount of light that passes through a blank or standard solution

Blank

contains all of the reagents used in the procedure w/o the unknown substance

Absorbance equation

A = 2 - log₁₀ %T

Amount of light absorbed by a solution

Standard curve

Graphical representation of the relationship between concentration and absorbance or %transmittance

• Beer’s Law

Reflectance spectrophotometry

Light reflected from the surface of a sample is used to measure the amount of unknown colored product (quantitative)

Sample concentration ↑ Intensity of the color ↑ Amount of light reflected ↓
(Amount of light absorbed ↑)

specular reflectance:

The amount of light reflected by a colored reaction product on a reflective surface composed of paper or plastic (smooth or mirror like surface)

Reflectance density:

The amount of light absorbed by the colored reaction product on the smooth surface

-inversely proportional to the light intensity reflected by the sample

Reflectance spectrophotometry relationship

Sample concentration ↑ intensity of the color ↑ amount of light reflected ↓ (amount of light absorbed ↑)

Fluorescent spectrophotometry

Uses a beam of light, typically UV light, to excite electronsin molecules causing them to emit light of a different wavelength

Fluorimetry

The measurement of emitted fluorescence

Fluorescence

When a molecule absorbs light at one wavelength and remits light at a longer wavelength

Fluorphore

An atom or molecule that fluoresces

-frequently used as tags/labels in immunoassays and flow cytometry

Which is more sensitive and specific? Absorption spectrophotometry or fluorescent spectrophotometry?

Fluorescent spectrophotometry

-emitted fluorescent signal comes directly

From sample

FLUORESCENT SPECTROPHOTOMETRY
▪ Disadvantages

▪ Sensitive to environmental changes
▪ pH, temperatures, contaminants
▪ Loss of excitation due to environmental factors → quenching

Fluorometer

Measures fluorescent intensity

How is fluorometer different than spectrophotometer?

Fluorometer measures the fluorescent intensity of light source, sample cell, and detector.

Light source in fluorescent spectrophotometry must be

High intensity lamp or laser producing light in the UV and short wavelength

Nephelometry

Scattered light towards a detector not the direct path of transmitted light

-used to assay larger particles at lower concentration

Turbidimetry

measures the reduction of light transmitted through a homogenous solution due to the light's scattering (solution becomes turbid)

-used to assay smaller particles at higher concentrations

Luminometer

Measures light emission via chemiluminescence, bioluminescence, and electrochemiluminescence

Chemiluminescence

Chemical reaction that produces light without producing heat

Ex: luminol, glow sticks, etc.

Bioluminescence

Form of chemiluminescence found in biological system

Ex: firefly glow

Electrochemiluminescence

Differs from chemiluminescence -> occurs at the surface of an electrode

Summary of Chapter 17

Chapter 18

Electrochemistry

chemical energy is converted to an electric current (a flow of electrons) in a galvanic cell

In Electrochemistry, what is chemical energy converted to?

An electrical current or flow of electrons in galvanic cell

Galvanic cell

two half cells ->each contain a metal in a solution of one of its salts.

Components

 Two half-cells
 Salt Bridge
- Can be liquid or solid
- Helps keep the positive/negative balance between the two cells
 Two Electrodes
- Cathode (-) and Anode (+)
 Voltmeter

OILRIG

Oxidation is Loss of Electrons

Reduction Is Gain of Electrons

Anode

-Positive pole

-oxidation reaction

-loss of electrons

Cathode

-negative pole

-reduction reaction

-Gain of electrons

What does electrochemistry measure?

-ions

-drugs

-hormones

-metals

-gases

techniques used to measure electrochemistry

1. potentiometry

2.amperometry

3.conductometry

4.coulometry

Potentiometry

Measurement of voltage potential difference between two electrodes immersed in a
solution under zero current conditions

o Direct measurement of electrical potential due to the activity of free ions
- H+, Na+, K+
- Nernst equation relates the measured electrical potential
to the concentration (or activity) of ions
o Voltage also sometimes referred to as potential or electromotive force (EMF)
o Difference is measured using a voltage or pH meter

Two electrodes utilized in potentiometry

1. indicator electrode

2. reference electrode

*Need both need the the reference to measure against the reference solution

1. indicator electrode

measure analyte in your sample

ex. ion selective electrode

reference electrode

used as fixed reference (qc)

-most common: silver or silver chloride like a

-ph electrode is 7 the reference compared to your solution

ELECTROCHEMISTRY & CHEMICAL SENSORS POTENTIOMETRY

Ion – Selective Electrode (ISE) (indicator electrode)

o Membranes selectively permeable to certain ions (H+, K+, Na+, Mg2+)
o Interaction with the membrane creates a potential difference
• Polymer membranes → most prominent class of potentiometric ISE used in chemical analyzers
• Glass membranes (silica dioxide) → pH electrode
• Glass membrane permits interaction with H+ ions

PH

Measure of H+ ions (protons)

pH = log(1/[H+]) = -log[H+]**

what is attached to ISE probe?

Electrical read out

how is pH meter used?

1. Immersed pH in solution #1 of known pH

2.Immerse pH electrode in solution #2 of known pH

3.A slope is calculated by the pH meter based on the two values assigned to the known pH standards.

Amperometry

measures current flow through electrochemical cells between two electrodes while a constant external voltage is applied

Clark Electrode->platinum cathode is surrounded by_____

tubular silver anode

Clark Electrode” → pO2 electrode

Platinum cathode surrounded by tubular silver anode

Oxygen diffuses through polymeric membrane to internal electrolyte solution, reduced at the
platinum cathode

Measures the amount of current flow in a circuit that is related to the amount of oxygen being
reduced at the cathode

Cathode (Red Cat)
• Negative Pole
• Reduction Reaction
• Gain of Electrons

Conductometry

Measurement of electrolytic conductivity

• Electrolytes in solution carry a current by migration of ions
  - Current is proportional to concentration of the ions

Current is ___________to concentration of the ions ( for conductometry)

proportional

Coulemetry measures

the amount of charge(in coulombs) passing between two electrodes at a FIXED POTENTIAL (based on amperometry) as a result of chemical reaction

Coulometry

Technique in which the charge required to completely electrolyze a sample is measured; measures amount of current passing between two electrodes in an electrochemical cell
- Determines the amount of analyte consumed or produced by measuring the amount of electricity (in coulombs)
- Based on a chemical reaction

Number of coulombs = direct measurement of quantitative oxidation or reduction of an electroactive species at one
of the electrodes
 The more of an analyte is present, the higher charge measured
 Produces an insoluble product

Number of coulombs

Direct measurement of quantitative oxidation or reduction of an electroactive species at one of the electrodes
 The more of an analyte is present, the higher charge measured
 Produces an insoluble product

LECTURE 19:
INSTRUMENTATION – CHROMATOGRAPHY

Chromatography

• Physical separation technique that separates solutes dissolved in a common solvent

• Particles in the mobile phase with higher affinity for the stationary phase migrate slower than those with a lower affinity (migrate faster)
  

  • A Mobile and Stationary Phase
    

Mobile Phase

phase that carries the sample through a stationary phase; usually gas or liquid

Stationary Phase

a fixed phase through/over which the mobile phase (sample) passes; usually solid

How are the analytes separated ?

• Affinity, Size Exclusion

Where is the Stationary Phase ?

• Column, Planar (flat)

Retention Factor (R _f )

Distance leading edge of component moves/ Total distance solvent front moves

• Find Unknown Components

Planar Chromatography

• Stationary phase is planar (flat)

  • Paper layered with stationary phase (paper chromatography)

  • Solid phase on glass plate support (thin layer chromatography)

Column Chromatography

• Stationary phase coated onto particles which are placed in or coated onto a tube or column

• Two Main Types

  • Gas Chromatography

  • Liquid Chromatography

Gas Chromatography

Mobile phase = carrier gas (helium, hydrogen, or nitrogen)

• Moves volatile sample through the column stationary phase

• Solutes separated based on differences in vapor pressure and interactions with the stationary phase

• More volatile/Less interactive solute will elute faster than a less volatile/more interactive one

Gas Chromatography - Components

oCarrier gas

oSample injector

• Heated

oColumn/Oven

• Components separated based on their abilities to bind to stationary phase

oTemperature control

oDetector

oComputer

Packed Column

Filled with support materials that are used uncoated

• Gas-Solid Chromatography (GSC)

Capillary Column

Non-volatile liquid is coated or chemically bonded with the support particles or directly onto the wall

• Gas-Liquid Chromatography (GLC)

Resolution

How well that analytes are separated by chromatography

• Measured betweentwo peaks on chromatogram

• Peaks characterized based on retention time and width

Column - Liquid Chromatography (LC)

Liquid mobile phase

Components similar to GC but LC requires a solvent reservoir and pump

• Columns much smaller diameter

  • Capillary columns <1 mm à fused silica

• Detectors: Fluorometer, UV Vis Spectrophotometer, electrochemical

High Performance Liquid Chromatography (HPLC)

Uses pressure for faster separations