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Spectrophotometer (Spec)An instrument that measures the
amount of light that passes through(is transmitted through) a sample.
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Uses a type of light to detectmolecules in a solution
Light is a type of energy, and
the energy is reported aswavelengths, in nanometers(nm).
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Ultraviolet (UV) Spectrophotometers.Uses ultraviolet light of wave lengthsfrom 2 nm to !" nm.
Visi#le (V$S) Light SpectrumSpectrophotometers.Uses visi#le light (white light) of wavelengths from !" nm to % nm.
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R O Y G B I V
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Shines a #eam of light on a sample. &he molecules in the sample interact
with the light waves in of ! ways'
Absorb the energyReflect the energyTransmit the energy #etween andthrough the atoms and molecules ofthe sample.
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onsider #lue molecules, all thewavelengths of light are a#sor#ed,ecept for the #lue ones.
&he #lue wavelengths aretransmitted or reflected off themolecules. $f these #lue
wavelengths hit a detector (such asin the spectrophotometer or thenerve cells in your eye), they appear#lue.
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*olecules are whatever color oflight that they do not a#sor#.
+reen molecules appear green#ecause they a#sor# mostwavelengths of visi#le light,ecept the green wavelengths.
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&he spectrophotometer measuresthe amount of light transmittedthrough the sample
(&ransmittance). y using an e-uation (eers law),
it converts the transmittancedata to an a#sor#ance value.
hat /ind of data is this0
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&he concentration of an un/nownsample can #e determined #ycomparing the a#sor#ance data tostandards of /nown concentration.
&he data generated with the set of
/nown standards is called astandard curve.
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$nner partsLamp
1rism or grating that directlight of a specific wavelength.
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uter parts'
Sample Holder
Display
Knobs or buttons used to calibrate thespec to measure the designated
molecule.
WavelengthSelection
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Visi#le spectrophotometerontains a tungsten lamp that
produces wwhhiittee lliightght. Ultraviolet
spectrophotometer
ontains a deuterium lamp that produces light in the UVlight part of the spectrum.
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Visi#le Spectrophotometerhite light hits the prism or
grating, it is split into thecolors of the rain#ow (Visi#leSpectrum).
&he wavelength /no# rotatesthe prism3grating, directingdifferent color of light towardthe sample.
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&he wavelength of lightproduced #y the tungsten lamprange from a#out !" nm (Violet
light) to % nm (red light).
&he molecules in the sample
either absorb or Transmit thelight energy of one wavelengthor another.
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&he detector measures theamount of light #eing
transmitted #y the sample andreports that value directly (4transmittance) or converts it to
the amount of light a#sor#ed ina#sor#ance units (au) usingeers Law.
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After collecting data for yourconcentration an a#sorptionspectrum graph is created.
&hese can #e used when
attempting to identify un/nownsu#stances (e.g. S$)
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&he a#sor#ance spectrum is a
graph of a sample5s a#sor#ance
at different wavelengths.
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&he spectrophotometer canmeasure the amount of a#sor#ance
or lac/ of a#sor#ance of differentcolored light for a given
molecule.
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&he concentration of moleculesin a solution affects thesolution5s a#sor#ance.
6emem#er 7 8 is a ratio 9 when wechange one num#er 9 it affects theratio
$f there are more molecules in onesolution than in another, than thereare more molecules to a#sor# thelight.
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:etermines the presence andconcentrations of samples.
:etermines the purity of asample.
Loo/ at the change ofsamples over time.
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A. Measure the absorbance of standardscontaining known concentrations of theanalyte
B. Plot a standard curve with absorbanceon the X axis and analyte concentration onthe Y axis
C. Measure the absorbance of theunknown(s
!. !eter"ine the concentration of "aterialof interest in the unknowns based on thestandard curve
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