Identfication of unknown from its λmax and determination of its concentration
by Beer’s Law
Abstract
Spectrophotometry is a science that deals with the absorbance of light by various
compounds . The electronic structure of a compound is what determines the amount of
light and the energy (the wavelength) of the light absorbed by it. Therefore, each
compound has a characteristic light absorbance that can be measured by Beer-Lambert’s
Law (A= €M c l) where A is the Absorbance and is directly proportional to the
concentration of the substance (c) and the path length (l) through which light travels; €M
is the proportionality constant called the absorptivity. In this lab, we measured the
wavelength of maximum absorbance (λmax) for molecules such as NAD+, NADH,
oxidized and reduced cytochrome c, ATP and CTP by using a valuable tool called
spectrophotometer. Comparing the λmax value of an unknown substance with these
values, we identified the substance. The concentration of the unknown was also
calculated by using its plot of absorbance vs wavelength. From our experiment, we
identified the unknown to be CTP at pH 2 having λmax value of 280nm whereas its
concentration determined from its standard curve was 0.03mM.
Result
Table 1. Wavelength of maximum absorbance for different molecules.
Compound
Wavelength (λmax) (nm)
NAD+
260
NADH
260
340
Oxidized cytochrome c
400
Reduced cytochrome c
400
525
550
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ATP at pH 2
CTP at pH 2
CTP at pH 7
Unknown
255
280
270
280
Table 2. Absorbance vs concentration for the unknown molecule
Concentration (mM)
Absorbance
0.01
0.1563
0.02
0.2803
0.04
0.5931
0.06
0.8567
0.08
1.1252
Fig 3. Standard curve for the unknown. Five different dilutions of 0.1 mM CTP were
made with 0.1mM HCl used as the diluent. Absorbance for the corresponding dilutions
was then calculated.
Slope = € (molar absorption coefficient) = 14.24 mM-1cm-1
Absorbance of unknown at 280nm = 0.23
Concentration of unknown read from Fig 1. = 0.02mM
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Discussion
It is clear from the results that the oxidized NAD i.e. NAD+ shows one major
peak whereas its reduced form (NADH) shows an additional peak too (Table 1). While
identifying each peak, the
λmax
for NAD+ was found to be 260nm which is also the same
for ATP. This indicates that the absorbance observed in oxidized form of NAD (NAD+)
is due to its adenine moiety. However, NADH shows two peaks, one at 260nm and the
other at 340nm. The first peak relates to its adenine moiety whereas the second peak is
due to its nicotinamide moiety as the nicotinamide gets reduced in NADH (Figure 1).
Likewise, oxidized cytochrome c peak was observed at 400nm and a small plateau-like
peak was also observed at around 525nm. In case of reduced form of cytochrome c,
three peaks were observed, one at 400nm which was very strong and broad whereas the
other two were small peaks observed at 520nm and 545nm (Figure 2).
The λmax values were also calculated for the nucleotides ATP and CTP. They
showed peaks at expected wavelengths. It was noticed that only one λmax value existed
for ATP at 255nm wavelength. On the other hand, the λmax value for CTP changed
depending upon pH. It is striking to see CTP absorb light at higher wavelength when the
pH was lowered to 2 from pH 7 (Table1). Therefore, we can conclude that the absorbance
of CTP depends largely on the pH of the solution.
By comparing the observed λmax values for different compounds and similar
values for the given unknown, it was identified to be CTP at pH 2 since at pH 2, CTP has
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λmax at 280nm same as the λmax for unknown. The molar absorption coefficient(€M) of
the unknown was found to be 14.24 mM-1cm-1 by using its standard curve (Table 2, Fig
1.) This value is slightly lower than the published value 13mM-1cm-1 which could be due
to some pipetting error.
Thus, the calculated experimental λmax values for NAD+, NADH, oxidized
cytochrome c, reduced cytochrome c, ATP and CTP matched well with the literature
values. This confirmed that each molecule has its characteristic absorption property, and
has its characteristic λmax values. It was also found that a standard curve ( plot of
absorbance vs concentration) of a specific substance allows for calculation of the
concentration of the substance at any given absorbance. For example, in this lab, with a
characteristic λmax value the unknown was identified to be CTP at pH 2 and with its
standard curve its concentration was easily determined.
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