Clinical Chemistry Lecture - Lesson 3: Spectrophotometry

photometry

described as the measurement of radiant energy absorbed or given off by a molecule after illumination by a light source

photometry

this still remains as the primary technique most often utilized in today's chemistry laboratories

electromagnetic radiation

radiant energy which includes short gamma rays to long wavelength radio waves. These are photons of energy travelling in a wavelike manner.

electromagnetic energy

the shorter the wavelength the higher the?

cosmic rays, gamma rays, x-rays, visible rays, infrared rays, ultraviolet rays; tv, radio waves, microwaves

enumerate the 7 types of EMR

theory of light waves

light is a photon or discrete packet of energy travelling in waves.

peak or trough

light ray will have a?

wavelength of light

is the distance between two successive peaks/crest which gives the light it's characteristic color (if the wavelength is in the visible region)

amplitude

is the other term used to define a light wave and is the distance between the peak and the trough

nanometer (nm)

is the standard international (SI) units for expressing the wavelength of light

1×10^-9

the measurement of nanometer

millimicron (mu)

the old term used for nn

angstrom (a)

oldest term use for measurement

visible, invisible

what are the 2 kinds of spectra/wavelength

visible light

this spectra is the one visible to the human naked eye

340-700/780

what are the ranges of visible light?

ultraviolet, infrared

what are the two types of invisible light?

ultraviolet (UV)

any light below 340 nm

infrared (IR)

any light above 780 nm

colorimetry

this is the term associated with spectrophotometry

absorbed, concentration

based on law of colorimetry(principle), it involves the measurement of the amount of light _____ by a solution and relating that absorption to the solution _____.

quality of light, intensity of light

2 primary considerations with the law of colorimetry (principle)

beer’s law

this states that the Absorbance (A) or the optical density (O.D) of the colored solution is equal to the product of the concentration (C) of the color producing substance

proportional, transmitted light

so according to beer’s law, the concentration of the substance is directly _____ to the amount of light absorbed and inversely proportional to the logarithm of the _____.

visual, photoelectric colorimetry

what are the 2 types of colorimetry?

colored filters

visual colorimetry, use ordinary _____ for wider spectrum

endpoint

visual colorimetry, uses the eye in determining the?

crude, subjective

visual colorimetry is a very _____ and _____ form of measurement

intensity of the color

visual colorimetry, this _____ must be proportionate to the concentration of the color producing substances

simplest colorimetric, unknown, standards

visual colorimetry is the most _____ analysis using a simple device that involves the comparison of an _____ with a series of _____

standard substance

has a known exact value or purity associated with the substance

calibrator

is used to adjust substance

duboscq colorimeter

give one example of a refined type of visual colorimetry

light source, 2 glass-bottomed cup, 2 movable platforms, optical system, 2 identical scales

what are the 5 essential parts of the duboscq colorimetry?

spectrophotometry, filter photometry

what are the 2 types of photoelectric colorimetry?

narrower wavelength, prism/diffraction gratings, continuous spectrum

in spectrophotometry, it measures light intensity in such a _____ using a devices like a _____ to disperse the source of light into a _____.

amount of light absorbed

in spectrophotometry, quantitation of a substance is accomplished by measuring the _____ by the solution after appropriate treatment

sensitivity, rapid measurements, visual colorimeter

in spectrophotometry, it gives a relatively high _____ with greater ease of _____ can be made compared to _____

proper reagents, analytical separation

in spectrophotometry, high specificity can be obtained by reacting the substance of interest with _____ therefore producing different colors or by _____ prior to color formation reactions

highly sensitive, ease with which rapid movements are made, highly specific

in spectrophotometry, what are it’s 3 advantages?

amount of light absorbed, logarithm

according to beer’s law, the concentration of substance is directly proportional to _____ or inversely proportional to the _____ of transmitted light.

decrease

an increase absorbance reading means a _____ transmittance of light

concentration of substance

what equates to the amount of light absorbed?

light source, entrance slit, monochromator (lens system), exit slit, analytical cuvette, detector/photodetector, meter/data read-out device

what are the 7 essential components of a spectrophotometry?

light source

provides radiant energy in the form of visible or invisible light that may pass through the monochromator to be separated into discrete wavelengths

analytical cell

the light of proper wavelength will be made incident on the _____ holding the solution whose absorption is to be measured

tungsten lamp, tungsten iodine lamp, deuterium discharge lamp, quartz halide, infrared energy source, hg vapor lamp, hollow cathode lamp

what are the 7 types of light source?

tungsten lamp

provides energy wavelength between 340-780 nm (visible range)

tungsten iodine lamp

for visible and UV measurements

deuterium discharge lamp

wavelength below 300-320 nm

needs power supply, more expensive, common use for UV

deuterium discharge lamp 3 disadvantages

minimizes stray light from the light source, prevent scattered light from entering the monochromator system

what are the 2 functions of the enter slit?

stray light

any wavelength of light across/outside the band transmitted and selected by the monochromator

stray light

this light originates from light source

band pass/width

certain range of wavelengths allowed to pass by the monochromator from the light source to travel into the sample

reflection of light from scratches on optical surfaces, dust particles, reflection from within measurement/instruments, entrance of extraneous room light, high-order spectra produced by diffraction gratings

what are the 5 most common causes of stray light?

beer's Law

if a stray light passes through the cuvette, what will not be followed thus follows an error in absorbance.

higher range of stray light

significant error maybe produced in the

cut-off filters

use of _____ to eliminate all radiation at wavelength beyond the one of interest

Didymium, Holmium Oxide

give 2 examples of cut-off filters

200-400 nm

check stray light in the near UV region by inserting filter of how much range?

blocked

No light will be transmitted if the light path is?

cut-off, not transmitted

light is _____ above/below a certain wavelength

stray light

if the instrument reading is greater than 0% t, what is still present?

nickel sulfate, sodium nitrite, acetone

what are these certain liquid chemicals that absorb strongly to short wavelength?

0, absence of stray light

using of black object to block light path, would have what transmittance percentage? and by the results it means?

single color

monochromator is derived from the Greek word which means?

wavelength selector

monochromator is also known as a?

prism, grating

wavelength selector which isolate specific wavelength of light by the use of?

isolate

monochromator’s main function is to _____ individual wavelength of light

prisms, diffraction gratings, color graters, interference filters

what are the 4 types of devices that monochromator uses?

interference filters

produce monochromatic light-constructive interference of waves

prism

triangular wedge-shaped piece of glass made out of materials that allows transmission of light

quartz, sodium chloride, potassium bromide

what are prisms made of?

short wavelengths

refracts more and resulting to the dispersion of light into continuous spectrum

glass prism

prism used in visible spectrum

quartz fused silica prism

prism used in UV spectrum

sodium chloride, potassium bromide

prism used in IR spectrum

allows greater degree of wavelength/spectral line separation than filters

what is the advantage of prism?

variation of refractive index

prisms disperses white light into a continuous spectrum of colors based on _____ of the different wavelength

short wavelength

the wavelength refracted the most

red end of the spectrum

refracted the least

blue or violet end

refracted the most

grating, diffraction gratings

is a device that has small groves cut into it at such an angle that each groove behaves like small prism

3000 or more

gratings may have how many small grooves/millimeter cut into the grating surface?

bent

gratings separates white light into its various color components based on the principle that wavelength are _____ as they pass a sharp corner

reflected off, loss of energy

light is _____ the grating rather than transmitted through the prism, thereby reduces the _____

higher line wavelength resolution

gratings provides much _____ than is possible with prism and respond to all optical wavelengths

prism, gratings

these 2 things allow greater degree of wavelength (spectral line) separation than the filters

provides a linear spectrum and therefore maintain a constant bandpass which is simple, can be used on regions of spectrum where light energy is absorbed by glass prism

what are the 2 advantages of gratings over prisms?

colored filters

allows a wider range of wavelength to pass through

20-60 nm

colored filters have a wider band widths of?

less expensive, less sensitive interference filters

colored filters advantage and disadvantage is?

filter photometer

colored filters are devices used in _____ to obtain spectral purity instead of using prism/grating

exit slit

delivers the light of desired wavelength simply by adjusting the wavelength dial to dictate that wavelength

amount of light

exit slit controls the _____ that passes into the cuvette

analytical cuvette

is an optical cell used to hold solution in the instrument where absorption is to be measured

sample holder, absorption cell

analytical cuvette are also known as?

soft, borosilicate glass, quartz or plastic

analytical cuvette are made of?

soft glass cuvette

is used for solutions that are acidic and do not etch glass