2. Spectroscopy
It is the branch of science which deals with the interaction
of electromagnetic radiation with matter is called
spectroscopy.
Or
It is the branch of science which deals with the study of
interaction of matter with light.
3. Uv spectroscopy
The interaction of electromagnetic radiation with matter when
source is uv is called uv spectroscopy
UV / Vis spectrophotometer
• UV / Vis spectrophotometer uses visible light and ultraviolet to analyze
the chemical structure of substance. A spectrophotometer is a special
type of spectrometer, which is used to measure the intensity of light, and
the intensity is proportional to the wavelength.
• UV / Vis spectrophotometer has two types, single-beam and dual-beam
4. Spectrophotometry and its
Applications in Microbiology
Certain covalent bonds in molecules are able to absorb energy at
particular wavelengths ranging from infrared to ultraviolet. This
absorbance is readily detected by using some kind of spectrometer
which sends light of a specific wavelength through the sample; if the
chemical absorbs energy, then the light arriving at the detector is
less intense than the incident light that was shown on the sample.
For most biological applications, the substance is in solution, the
wavelength is that of visible light (350-700 nm) and the instrument
used is a spectrophotometer.
5.
6. Frequently, these measurements are made to determine the quantity of some
chemical because fortunately, Beer’s Law states that the amount of light absorbed
is proportional to the amount of the substance doing the absorbance, in other
words
A = 0lc where
A is the absorbance
0 is the extinction or molar absorptivity coefficient, a characteristic of the
molecule being measured which tells how much light is absorbed per mol of
molecules
l is the pathlength, or the distance the light travels though the sample; a sample is
placed into a cuvette which are designed to have a path length of 1 cm,
conveniently
c is the concentration.
Thus, A, the absorbance, is directly proportional to c, the concentration.
7. Sometimes it is convenient to consider the flip side, that is, how much of the
light makes it through to the detector instead of how much is absorbed. This
is called Percent Transmittance which is defined mathematically as %T = 100 x
It / I0 where It is the light transmitted through the sample and reaches the
detector and I0 is the incident light.
The mathematical relationship between Absorbance and Percent
Transmittance is:
A = 2 - log (%T)
While the analysis of biochemicals is important in microbiology, many times
we are interested in using spectrophotometry as an easy way to determine the
relative numbers of bacteria in a sample. This creates complications because
Beer’s Law applies to molecules in solution, whereas bacteria are particles in
suspension. Fortunately, Beer’s Law has been shown to apply in this case
(known as the Beer-Lambert Law). Both %T and A are frequently used. I prefer
to use A because then there is a direct relationship between the absorbance
units and the number of bacteria, so it is intuitively more comfortable.
8. Identification of unknown
biomolecules by spectroscopy
The uv-vis spectrum of biomolecules reveals much about
its molecular structure. Therefore a spectral analysis is one
of the first experimental measurement made on unknown
biomolecules. Natural molecules often contain
chromophoric (color-producing) functional groups that
have characteristics spectra. For example the spectra of
biomolecules FMN, FMNH2, NAD, NADH.
9.
10. The procedure for obtaining the uv-vis spectrum begins with
the preparation of a solution of the specie under study. A
standard solution should be prepared in a appropriate
solvent. An aliquot of the solution is transferred to a cuvette
and placed in a sample chamber of spectrophotometer . A
cuvette containing solvent is placed in the reference holder.
The spectrum is scanned over a desired wavelength range and
an absorption coefficient is calculated for each major λmax.
11. UV visible
spectroscopy in
separation analysis
EVALUATION OF A SECOND DERIVATIVE UV/VISIBLE
SPECTROSCOPY TECHNIQUE FOR NITRATE AND TOTAL
NITROGEN ANALYSIS OF WASTEWATER SAMPLES
12. Nitrogen
Nitrogen is an essential nutrient for autotrophic and
heterotrophic organisms and is often limited in aquatic
ecosystems. Conversely, large inputs of nitrogen can cause
excessive phytoplankton and macrophyte production, and the
subsequent death and decay of these organisms can result in a
depletion of dissolved oxygen. Because nitrogen com-pounds
are biochemically transformable through nitrification,
denitrification, and mineralization, wastewater treatment systems
are frequently designed to convert nitrogen compounds to more
environmentally benign forms (i.e., N2 gas) that will not cause
problems associated with nutrient enrichment and oxygen
depletion. Nitrogen occurs in various inorganic and organic
forms, including nitrite (NO‾), nitrate (NO3‾ ), ammonium
(NH+4) and organic nitrogen (organic N).
13. MATERIALS AND METHODS
The procedure is based on the second derivative of the
absorption spectrum for NO3‾ , which has a peak at 224 nm
that is proportional to the NO3‾ concentration The second
derivative peak, unlike the absorption peak at 203 nm, is not
influenced by other common constituents in fresh waters (i.e.,
organic matter). The principle of the modification to analyze for
total N is to digest and oxidize all nitrogenous compounds to
NO3‾ by auto claving. The NO3‾ concentration of the digested
sample is then equivalent to total N. Organic N can be
determined by analyzing the sample for NO3‾, NO2‾ , and
NH+4 and subtracting these values from total N.
14. Analysis of NO3‾
NO3‾ standards of concentration ranging from 0.1 to 3.0 mg were
prepared by dilution from a 1000 mg. NIST-traceable stock
solution of NO3‾ . Ultrapure water blanks, standards and samples
were scanned on a Perkin-Elmer Lambda-40 UV/VIS
spectrophotometer interfaced to a microcomputer, with ultrapure
water used as a blank in the spectrophotometer reference cell.
Standards an samples were scanned between 190 and 250 nm in
quartz cells with a 10-mm optical path length (Crumpton et al.,
1992). Scans were conducted at 120 nm/min, and the second
derivative value of the NO3‾ peak at 224 nm was measured and
recorded. A standard curve was developed by linear regression of
the second derivative peak vs concentration of the standards. The
standard curve was then used to quantify all samples.
15. This method allows analysis of N concentrations up to 3 mg with
out dilution, as the N curve is nonlinear beyond this range. Samples containing
more than 3 mg N were diluted to a concentration within the linear range.
Analysis of total N/organic N
Organic-N standards of concentrations ranging from 0.1 to 3.0 mg were
prepared from reagent-grade urea. Standards or samples (initially 10 ml)
were placed in Teflon-lined screwcap vials for digestion by autoclave.
Oxidizing reagent was prepared by dissolving 6.0 g of low N potassium
persulfate in 100 ml of 1.5 N sodium hydroxide solution. The oxidizing
reagent (1.5 ml) was added to each vial with sample or standard, and
the vials were tightly capped and shaken. After autoclaving, samples
were cooled and acidified to below pH 2 with concentrated sulfuric acid.
16. Wastewater samples
Water samples were collected from a constructed wetland system treating
domestic wastewater to secondary standards and from a conventional
secondary wastewater treatment system in order to obtain samples of
varying N concentrations. Samples were placed in plastic vials and were
stored at 48C or frozen until analyzed. All sample filtration was
conducted in the field or immediately upon return to the laboratory. For
analysis by several methods,
samples were shaken and split into separate vials immediately before
analysis. All sample and standard concentrations were reported as NO3‾ ,
NH+4, and organic N.
17. CONCLUSIONS
The second derivative spectroscopy method was found to be
accurate, based on comparison with quality control check samples
of known concentrations. The method also compared favorably
with currently accepted methods for analysis of NO3‾ and total N
in wastewater samples. The second derivative spectroscopy
method is a simple, rapid technique for both NO3‾ and total N
analysis. The method eliminates many of the disadvantages
associated with other analysis techniques, and accuracy and
precision are not compromised. The analysis technique can be
used to analyze domestic wastewater and other freshwater
samples containing a range of inorganic and organic N
concentrations.
