Experiment # 4

Determination of lamda max of unknown compound

spectrophotometrically
Objective
Determination of wavelength of maximum absorbance ( lamda max for unknown compounds)
Introduction
A spectrophotometer is an instrument that measures the amount of photons (the intensity of light) abs
orbed after it passes through sample solution. With the spectrophotometer, the amount of a known che
mical substance (concentrations) can also be determined by measuring the intensity of light detected.
Depending on the range of wavelength of light source, it can be classified into two different types:
UV-visible spectrophotometer uses light over the ultraviolet range (185 - 400 nm) and visible range
(400 - 700 nm) of electromagnetic radiation spectrum.
IR spectrophotometer uses light over the infrared range (700 - 15000 nm) of electromagnetic radiati
on spectrum.
In visible spectrophotometry, the absorption or the transmission of a certain substance can be determi
ned by the observed color. For instance, a solution sample that absorbs light over all visible ranges (i.
e., transmits none of visible wavelengths) appears black in theory. On the other hand, if all visible wa
velengths are transmitted (i.e., absorbs nothing), the solution sample appears white. If a solution samp
le absorbs red light (~700 nm), it appears green because green is the complementary color of red. Visi
ble spectrophotometers, use a prism to narrow down a certain range of wavelength (to filter out other
wavelengths) so that the particular beam of light is passed through a solution sample.
Beer-Lambert Law
Beer-Lambert Law (also known as Beer's Law) states that there is a linear relationship betwe
en the absorbance and the concentration of a sample. For this reason, Beer's Law can only be
applied when there is a linear relationship. Beer's Law is written as:
A= Ɛo×lc
 A is the measure of absorbance (no units),
 epsilon is the molar extinction coefficient or molar absorptivity (or absorption coefficient)
 l is the path length
 c is the concentration.
The molar extinction coefficient is given as a constant and varies for each molecule. Absorba
nce does not carry any units. Since absorption, epsilon, and path length are known, we can cal
culate the concentration c of the sample.
Apparatus
 test tubes, test tube holder, measuring cylinder, pipette, Erlenmeyer flask, spectrophotometer
Chemicals Required
 Unknown Compound
 Water
Procedure
1.Preparation of 100 mL of a stock standard solution of 0.001M of Unknown compound.Transfer qua
ntitatively to a 100 mL volumetric flask and fill to the mark with water. This is the stock solution.
2.Prepare six standards in 100 mL volumetric flask with concentrations of 0.0004 M (solution no.1), 0.
0002 M (solution no.2), 0.0001 M (solution no.3) and 0.00005 M (solution no.4) by diluting the stock
solution prepared in Step 1 and this is termed as serial dilution.
3. Rinse one of the cuvettes with distilled water and fill it with water. Put the cuvette in the sample
Compartment. This is the reference solution. Set the wavelength to 400 nm, then set the Absorbance t
o zero.
4.Rinse a second cuvette once with distilled water and once with standard solution no.1, then fill it wit
h standard solution no.1 (0.001M solution). Place the cell in the sample compartment, measure the Ab
sorbance at 410.
5. Repeat this procedure (steps 3 and 4 above) for the two cuvettes at wavelengths 420, 430,440,
450…….600 nm. First setting A = 0 for the cuvette with water, then measuring A for the cuvette with
0.001 M solution , recording the absorbance at each wavelength. Record in data table.
6. Prepare a graph of absorbance A vs. wavelength λ and determine λmax (maximum wavelength).
Fig # 4.1 UV-Visible spectrophotometry

Reference
:https://www.lpdlabservices.co.uk/analytical_techniques/chemical_analysis/uv_spectrop
hotometry.jpg

 Part A ( Unknown Compound A)

Observations and Calculations
The absorption spectrum of unknown compound
Sr.No. Wavelength (nm) Absorbance
1 410 0.056
2 420 0.033
3 430 0.037
4 440 0.077
5 450 0.237
6 460 0.239
7 470 0.401
8 480 0.585
9 490 0.940
10 500 1.173
11 510 1.616
12 520 1.751
13 530 2.10
14 540 1.81
15 525 2.019
16 560 1.287
17 570 1.168
18 580 0.686
19 590 0.311
20 600 0.217

Graphical representation of absorbance at Single Concentrations

 0.2 Absorbance spectrum
0.18
0.16
0.14
0.12
absorbance

0.1
0.08
0.06
0.04
0.02
0
220 320 420 520 620 720
wavelength (nm)

Graphical representation of Absorbance of solutions a different concentrations

λmax=525nm
 Result
After determining the absorbance at different concentration by a spectrophotometer and then plotting
a graph we came to know that the lamda max value of unknown compound is 525nm, which is then
matched with literature and we came to know that the unknown compound in KMnO 4 which have
lamda max value of 525mn.