Spectrophotometric Determination of Nucleic Acid Concentration Flashcards
The range of all possible EM radiation
Electromagnetic waves
the spectrum of different wavelengths that eyes recognize as white
light
Electromagnetic waves are of _____ and waves
photons
Part of the EM spectrum that can be seen by the naked eye
visible spectrum
which waves are in the right of the visible light in the spectrum, these are characterized to have longer wavelengths
infrared, microwave, and radiowaves
t or f
shorter wavelengths are less harmful than longer wavelengths
F (high frequency, shorter wavelength = more harmful)
which waves are found at the left of visible light, these are characterized to have a higher frequency
gamma ray, x-ray, and ultra violet rays
Should we be more wary of waves that are higher or lower in frequency?
higher frequency (shorter waves)
an optical instrument for measuring the intensity of light in any specific wavelength related to solution concentrations
spectrophotometer
In what section of the lab are spectrophotometers used to measure the concentration of an analyte
clinical chemistry
T or F
spectrophotometers enable for a qualitative analysis of the substance only
F (quali and quanti)
Before measuring the analyte using a spectrophotometer, what do you measure first for control purposes?
blank / reference cell
What should be the result of the blank or reference cell?
zero
100% transmittance and 0% absorbance
the measure of the capacity of a substance to absorb light of a specific wavelength
absorbance / optical density
the measure of how much light passes through a substance
Transmittance
what formula is this for?
transmittance
what formula is this for?
Absorbance
A mathematic relationship that is the basis of analysis in spectrophotometry
beer’s law
T or F
In beer’s law, the concentration of the substance is inversely proportional to the amount of light absorbed
F (proportional)
directly proportional
T or F
Beer’s law states that the concentration of the substance is inversely proportional to the logarithm of transmitted lights
T
this produces intense reproducible constant beam of light, where everything starts
light source
T or F
White light contains all of the wavelengths
T
What is the type of light source for UV region
deutrium lamp
Deutrium (high-pressure hydrogen) lamp
what is the most common light source for visible and infrared rays regions
Tungsten - halogen
What is the component of the spectrophotometer that makes it so that all of the light beams are parallel to each other
collimator
This is a system for isolating radiant energy of a desired wavelength and excluding that of other waves
monochromator / diffraction grating
T or F
When you change the light source, the machine should be calibrated
T
Why should you recalibrate the machine when you change the light source?
because the angle of the light shining in the monochromator also changes
T or F
the monochromator flashes all of the wavelengths
F (you can select the specific wavelength of your chosen analyte)
T or F
prisms achieve dispersion due to the difference in the material refracted index according to its wavelength
T
What does diffraction grating use to split the light?
difference of diffraction direction due to interference
prism or diffraction grating?
can be made to spread colors over a larger angle
diffraction grating
prism or diffraction grating?
more sensitive to the color of the light
prism
A small vessel used to hold a liquid sample to be analyzed in a spectrophotometer
Cuvettes
What shapes do cuvettes come in?
round, square, or rectangular
What materials are cuvettes constructed from?
glass, silica, or plastic
What type of cuvette is used for UV range
quarts
Why do we discard the cuvette if it becomes scratched?
the scratch can affect the spectrophotometer reading
the light bounces of the scratches :((
what type of cuvette for visible range
glass
converts light energy (transmitted by a solution) to an electrical signal
photodetectors
simplest photodetectors
photocell and phototube
commonly used photodetector for measuring light intensity in the UV and visible region, it’s rapid and very sensitive
photomultiplier tube (PMT)
An electrical energy coming out from a detector is displayed on some type of digital display or readout system
readout device
in what forms can a readout be
chart, digital display, or a computer printout
What are the 2 applications of spectrophotometer
- Measurement of concentration
- Detection of impurities
Why does nucleic acids absorb uv light
due to the presence of heterocyclic rings in the bases
what is the wavelength of maximum absorption for both DNA and RNA
260 nm
which characteristic is used to quantify the amount of nucleic acids
amount of absorbed light
Wavelength of maximum absorption for detecting proteins and salts
- 280nm - proteins
- 230nm - salts
