Sample cell

 The sample compartment provides a light-tight environment that limits

the addition of stray radiation.
 Samples are normally in the liquid or solution state, and are placed in
cells constructed with UV/Vis transparent materials, such as quartz, glass,
and plastic.
 A quartz or fused-silica cell is required when working at a wavelength
<300 nm where other materials show a significant absorption.
 The most common pathlength is 1 cm (10 mm), although cells with
shorter (as little as 0.1 cm) and longer pathlengths (up to 10 cm) are
available. Longer pathlength cells are useful when analyzing a very dilute
solution, or for gas samples
Application
QUALITATIVE ANALYSIS
Should be used in combination with other methods:
 Measuring boiling pont
 TG, TGA

QUANTITATIVE ANALYSIS
The most frequently encountered quantitative analytical methods,
because:
 Many organic and inorganic compounds have strong absorption bands
in the UV/Vis region of the electromagnetic spectrum.
 if an analyte does not absorb UV/Vis radiation—or if its absorbance is
too weak—we often can react it with another species that is strongly
absorbing.
 Applied to: Environmental aapplications; Clinical applications;
Industrial Analysis; Forensic applications
DEVELOPING A QUANTITATIVE METHOD FOR
A SINGLE COMPONENTS
the conditions under which Beer’s law is obeyed must be established
1. Determining the best wavelength for the analysis, corresponding to a
maximum absorption, because it provides greater sensitivity and is less
susceptible to instrumental limitations.
2. Choosing an appropriate slit width. Usually we set the slits to be as
wide as possible because this increases the throughput of source
radiation, while also being narrow enough to avoid instrumental
limitations to Beer’s law. F
3. Constructing a calibration curve to determine the range of
concentrations for which Beer’s law is valid.
4. Considering the effect of potential interferents and establishing an
appropriate blank.
Single analyte
 The most common methods are a normal calibration curve using
external standards
 The method of standard additions. A single point standardization is also
possible
Quantitative analysis of mixtures
Suppose, we need to determine the concentration of two analytes, X and
Y, in a sample.
 If each analyte has a wavelength where the other analyte does not
absorb, then we can proceed using the approach in single analyte.
 If UV/Vis absorption bands are so broad that it frequently is not possible
to find suitable wavelengths, then:
 To obtain results with good accuracy and precision the two wavelengths
should be selected so that X > Y at one wavelength and X < y at the
other wavelength.
 When the choice of wavelengths is not obvious, one method for locating
the optimum wavelengths is to plot X / Y as function of wave-length, and
determine the wavelengths where X / Y reaches maximum and
minimum values.
 When the analyte’s spectra overlap severely, such that X  Y at all
wavelength
CSX and CSY are the standard solutions of
X and Y
CX and CY are the concentration of X, Y in
the mixture
ASX and ASY are the absorbance values
for standard solutions of components X
and Y at any wavelength

Slope: CY/CSY ; y-intercept: CX/CSX.

Visible absorption spectra for 0.0250 M Cr3+, 0.0750 M Co2+, and for a mixture of Cr
3+ and Co2+ . The two wavelengths used for analyzing the mixture of Cr3+ and Co2+
are shown by the dashed lines.
Qualitative Applications (mainly for IR )
Method: Comparing its spectrum against a library of reference spectra,
calculates the cumulative difference between the sample’s spectrum and a
reference
spectrum
D is the cumulative difference
Asample is the sample’s absorbance at wavelength or wavenumber i,
Areference is the absorbance of the reference compound at the same
wavelength or wavenumber,
n is the number of digitized points in the spectra.

The cumulative difference is calculated for each reference spectrum. The

reference compound with the smallest value of D provides the closest
match to the unknown compound.
The accuracy of spectral searching is limited by:
 the number and type of compounds included in the library,
 the effect of the sample’s matrix on the spectrum.