Unit 3.1. Analytic Techniques: Spectrophotometry & Electrochemistry

• spectrometry

• luminescence

• electroanalytic methods

• chromatography

ENUMERATE

4 basic disciplines within the field of analytic chemistry

ELECTROMAGNETIC RADIATION

Is described as photons of energy traveling in waves.

equate it with light and radiant energy

WAVELENGTH

linear distance between any two equivalent points on a successive wave

nanometer (nm)

unit used for wavelength in visible spectrum

Planck’s formula

relationship between wavelength (λ) and energy (E) is described by

6.62 ×10^-27 erg sec

value of Planck’s constant

wavelength (λ)

distance between two equivalent points on successive waves

visible region: 400-700 nm

ultraviolet region: < 400 nm

infrared region: > 700 nm

WAVELENGTH

visible region: __________

ultraviolet region: __________

infrared region: __________

• violet

• blue

• green

• yellow

• orange

• red

ENUMERATE

colors seen in visible light (chronological order from 400 nm to 700 nm)

inversely proportional

frequency of a wave is ____________ to the wavelength

lower

the longer the wavelength the _________ the energy level

higher

the shorter the wavelength the _________ the energy level

• cosmic

• gamma rays

• X-rays

• ultra-violet

• visible light

• infrared

• microwaves

[Cool Girls eXercise Under Very Intense Moonlight]

ENUMERATE

other types of wavelengths

beer-lambert law

beer’s law aka?

more (conc.) analyte = more absorbance

principle of BEER’S LAW

↑ concentration = ↑ light absorbance

↓ concentration = ↓ light absorbance

[directly proportional]

relationship of CONCENTRATION and ABSORBANCE in beer’s law

↑ concentration = ↓ log of transmitted light

↓ concentration = ↑ log of transmitted light

[inversely proportional - more light is absorbed = less light reaches the detector.]

relationship of CONCENTRATION and TRANSMITTED LIGHT in beer’s law

absorbance

capacity of molecule to absorb light from light source

transmittance

refers to the remaining light that reaches the photodetector

0 %

if ALL LIGHT is blocked or absorbed by the molecule in the cuvette then the % transmittance = ________?

100 %

if NO LIGHT is blocked or absorbed by the molecule in the curvette then the % transmittance = ________?

SPECTROPHOTOMETER

measure the light transmitted by a solution to determine the concentration of the light- absorbing substance in the solution.

• light source

• monochromators

• sample cell / cuvette

• photodetector

• meter / read out device

ENUMERATE

components of spectrophotometer

• Evelyn and Malloy method (560 nm)

• Jendrassik-Gróf method (600 nm)

BILIRUBIN can be read using what methods

Incandescent tungsten or tungsten-iodide lamp

lamp used for VISIBLE & NEAR-INFRARED REGIONS

• Deuterium lamp

• mercury arc lamp

lamp used for UV REGION

Continuum

Which type of light source emits many wavelengths, only emits limited number of discrete lines or bandsof radiation?

• tungsten = visible region

• deuterium = UV region

• xenon arc lamp = visible and UV regions

ENUMERATE

example of continuum light source

line

type of light source that emits only selective wavelength, thus it emits a few discrete lines or bands of radiation

• Mercury and sodium vapor lamps –UV and visible regions

• Hollow cathode lamp - atomic absorption spectroscopy/spectrophotometry

ENUMERATE

example of line light source

MONOCHROMATOR

COMPONENT OF SPECTROPHOTOMETER

selects/isolates a certain wavelength of light coming from the light source

Filters

TYPES OF MONOCHROMATOR

A simple, inexpensive, and useful type of monochromator that isolates only one specific wavelength of light

• interference filter

• absorption filter

give example of monochromator filter

Prism

TYPES OF MONOCHROMATOR

Can be mechanically rotated, allowing only the desired wavelength to pass through an exit slit.

Diffraction Gratings

TYPES OF MONOCHROMATOR

Most commonly used; contain parallel grooves

Spectral Bandwidth (or FWHM) Full Width at Half Peak Maximum

Range wavelength is about ½ peak transmittance.

bandwidth

Total wavelengths seen in monochromator charts

1 cm

size of sample cell/cuvette

• fused silica or quartz

• alumina-silicate glass

• plastic cuvette

ENUMERATE

types of cuvette

• fused silica or quartz = UV region

• alumina-silicate glass = 350 - 2000 nm

• plastic cuvette = visible light

TYPES OF CUVETTE

• fused silica or quartz = _________

• alumina-silicate glass = _________

• plastic cuvette = _________

False

should be optically match for more accurate results

T or F

using two cuvettes with different material type can be utilized

PHOTODETECTOR

COMPONENT OF SPECTROPHOTOMETER

Converts the transmitted radiant energy into an equivalent amount of electrical energy

• barrier-layer cell or photocell

• phototube

• photomultiplier tube (PMT)

• photodiode

ENUMERATE

types of photodetector

Barrier-layer cell or photocell

TYPES OF PHOTODETECTOR

▪ Least expensive ; temperature sensitive

▪ Composed of selenium on a plate of iron

▪ Used mainly in filter photometers

Phototube

TYPES OF PHOTODETECTOR

▪ Contains cathode and anode enclosed in a glass tube

▪ Has photo-sensitive material that gives off electrons when light energy strikes it

Photomultiplier tube (PMT)

TYPES OF PHOTODETECTOR

▪ Most common type

▪ 200 times more sensitive than the phototube

▪ Highly sensitive to UV and visible radiation

Photodiode

TYPES OF PHOTODETECTOR

▪ Not as sensitive as PM tube but with excellent linearity and speed

METER OR READ OUT DEVICE

COMPONENTS OF SPECTROPHOTOMETER

shows the final result after the detector processes the light.

SINGLE-BEAM SPECTOPHOTOMETER

● This the simplest type of absorption spectrophotometer

● Designed to make one measurement at a time at one specified wavelength

Chopper

is a rotating device that alternates the light beam between the sample and the reference cuvette.

Double-Beam in Space

2 detectors, 2 light paths, simultaneous measurement.

Double-beam in time

1 detector, 1 light path, uses a chopper to alternate between the sample and the reference.

• didymium glass

• holmium oxide

ENUMERATE

special glass-type optical filter used for QA of Wavelength or photometric accuracy

• didymium glass = 600 nm

• holmium oxide = 300 nm

QA FOR PHOTOMETRIC ACCURACY

• didymium glass = __________

• holmium oxide = __________

Wavelength or photometric accuracy

This QA checks whether the spectrophotometer is using/measuring the correct wavelength that you selected.

Absorbance check

This QA checks whether the instrument gives the correct absorbance value.

Linearity

this QA checks whether the absorbance measured by the analyzer is proportional to the concentration of the analyte that we measure.

stary light

refers to the unwanted light that enters the detector

special cutoff filter

instrument used to check the presence of stray light

cuvette

most common cause of STRAY LIGHT

colorimeter

measures the intensity of light absorbed by a colored solution

spectrometer

an optical instrument used for visual identification if atomic emission lines; separate light into its component wavelengths

spectrometer

provides information about the intensity of radiation as a function of wavelength or frequency

ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS)

Measures concentration by detecting the absorption of electromagnetic radiation by atoms rather than by molecule

• hollow-cathode lamp;

• electrodeless discharge lamp

light source used in AAS

trace metals

(i.e. lead, mercury, cadmium, manganese, cobalt)

AAS is used to measure what?

flame

in AAS the chopper comes in the form of _________

FLAMELESS ASS

type of AAS that uses an electric furnace to break chemical bonds (electrothermal atomization)

tiny graphite cylinder

in AAS what is used to hold the sample. thus when an electric current passes this material, its wall causes evaporation of the solvent.

False

large droplets = drained ; fine droplets = atoms (processed)

T or F

small droplets are automatically drained in the draining chamber of AAS

FLUOROMETRY

this is intended for substances with fluorescing property

attenuator

In FLUOROMETRY, what controls the light intensity as it passes through the light filter. same as chopper

the amount of fluorescence production by certain analyte is directly proportional to the concentration of analyte of interest

working principle of FLUOROMETRY

• mercury(filter fluorometers)

• xenon arc

light source used in FLUOROMETRY

• prism

• grater

monochromator used in FLUOROMETRY

High degree of Specificity

(1000× more sensitive than ordinary spectrophotometry.)

ADVANTAGE of fluorometry

Sensitive to environmental changes

DISADVANTAGE of fluorometry

Quenching phenomenon

terminology used to describe the decrease in fluorescence due to the changes

CHEMILUMINESCENE

emission of light from a chemical reaction

luminol ; acridinium esters ; dioxetanes

Chemiluminescence reactions are oxidation reactions of _______, ________, and ________ characterized by a rapid increase in intensity of emitted light followed by a gradual breakdown/decay

Impurities can degrade sensitivity and specificity

DISADVANTAGE of chemiluminescence

fast and easy to use

ADVANTAGE of chemiluminescence

TURBIDITY AND NEPHELOMETRY

the measurement of the amount of light that is blocked

Nephelometry

the measurement of the light scattered by a particulate solution.

• Rayleigh theory

• Mie theory

• Rayleigh–Debye theory

ENUMERATE

three types of light scatters

rayleigh theory

iden the type of light scatter

mie thoery

iden the type of light scatter

rayleigh-debye theory

iden the type of light scatter

• Mercury arc lamp

• Tungsten lamp

• LED

• Laser

light source for NEPHELOMETER

• microbiology analyzers (AST)

• coagulation analyzers

• protein conc quantification in biologic fluids

applications of TURBIDITY

light amplification by stimulated emission of radiation

LASER means?

LASER APPLICATIONS

Based on the interaction of radiant energy with excited atoms or molecules.

• hematology

• flow cytometer

(differential analysis of WBC)

applications of LASER

reflectometer

a filter photometer that measure the quantity of light reflected by a liquid sample

• specular (polished surface)

• diffuse (non-polished surface)

ENUMERATE

2 types of reflectance

• urine dipstick

• dry slide chemical analysis (vitros analyzer)

applications of REFLECTOMETRY

POTENTIOMETRY

measures voltage (electrical potential) between two electrodes.

ELECTROCHEMISTRY

Involves measurement of the current or voltage generated by the activity of specific ions

reference electrode

2 ELECTRODE IN POTENTIOMETRY

Electrode with a constant voltage

indicator electrode

measuring electrode