BE184P Instrumentation for Biological Engineering 2

Name: Celestra, Den Mark O. Student No.2019102287 Program/Year: BE/3 Date:3/10/21

EXERCISE 1.1b SPECTROCHEMICAL ANALYSIS

Objectives:
Familiarize UV-Vis and FTIR spectrometers and calculate concentrations of samples and
unknowns, and identify structures using spectrochemical methods.

Introduction:
Measurements based on light and other forms of electromagnetic radiation are widely used
throughout analytical chemistry. Spectroscopy is an analytical technique that determine the
interactions of radiation and matter. Spectroscopic analytical methods are based on measuring
the amount of radiation produced or absorbed by molecular or atomic species of interest.
Spectroscopic methods are classified according to the region of the electromagnetic spectrum
used or produced in the measurement. The gamma ray, X-ray, ultraviolet (UV), visible, infrared
(IR), microwave, and radio-frequency (RF) regions have been used.
Spectroscopy has played a vital role in the development of modern atomic theory. In addition,
spectrochemical methods have provided perhaps the most widely used tools for the elucidation
of molecular structure as well as the quantitative and qualitative determination of both inorganic
and organic compounds.
In this exercise, students will familiarize the parts and function of the spectrophotometers and
practice solving problems involving measurements made with absorption of UV, visible, and IR
radiation.
BE184P Instrumentation for Biological Engineering 2

Answer the following chronologically. Show complete solution for each problem.

1. In determining unknown concentrations and using a spectrophotometer, a calibration curve is

constructed. It involves at least 5 concentrations of a standard and their absorbance are recorded.
Describe how you would use a serial dilution to prepare 250 mL each of a series of standards with
concentrations of 1.00 × 10–5, 1.00 × 10–4, 1.00 × 10–3, and 1.00 × 10–2 M from a 0.100 M stock solution.

2. A solution in the lab has an absorbance of 0.745 in a 1.0 cm cell, too concentrated for an accurate
measurement. The sample was diluted from 1 to 5 ml and then measured in a 2.0 cm cell. If the
measurement is linear, what is the expected absorbance?

3. If the molar absorptivity of the analyte in Problem #2 is ε = 8.32 L/mole cm, what is the concentration
of the solution?
BE184P Instrumentation for Biological Engineering 2

4. The absorbance of a 0.855 M standard of a complex between magnesium and ASN (mg-ASN) was
collected from 320 to 490 nm in 10 nm steps. Plot the data using Excel and determine the best wavelength
to collect them.
BE184P Instrumentation for Biological Engineering 2

a. What is the molar absorptivity, ε, of the mg-ASN complex if the solution was 0.855 M in a 1.0
cm cell?

b. What is the concentration of the mg-ASN solution that has an absorbance of 0.198 measured
at the same wavelength and in a 0.5 cm cell?
BE184P Instrumentation for Biological Engineering 2

5. Estimate the frequencies of the absorption maxima in the IR spectrum of given compound below. From
these frequencies, assign molecular vibrations of corresponding to each of the bands.
BE184P Instrumentation for Biological Engineering 2
BE184P Instrumentation for Biological Engineering 2

6. Draw the block diagram and label the parts. Briefly describe the functions of each part:

a. UV-Vis spectrophotometer

Light source (Lamp): Deuterium is used for measurement in the UV range, and tungsten lamp is used for
measurement in the visible and near-infrared ranges.

Sample: The sample is enclosed in a glass or quartz cell in order to the light to pass through.

Filter: Used extract light at a specific wavelength.

Monochromator: Used to select appropriate wavelength for the sample itself and has an impact on the
resolution and accuracy of the reading from the instrument.

Mirrors: Are primarily used to bounce the light off from one component to another.

Beam splitter: Used to isolate a single beam from the dispersed light that comes from the
monochromator.

Reference: Used to serve as a “guide” or reference for the sample.

BE184P Instrumentation for Biological Engineering 2

Photodiode: Used as a detector to detect the absorption from UV to VIS region.

Data processing: The translation of the data that was gathered from the photodiode. Most likely a
computer that compiles all the wavelength and data.

b. FTIR spectrometer

IR source: Source of infrared

Moving/Fixed mirror: Reflect the radiation back to the beam splitter. Half of this reflected radiation is
transmitted and half is reflected at the beam splitter.

Beam splitter: Transmits half of the radiation striking it and reflects the other half. Separates one of the
two beams. One is transmitted through the fixed mirror and second is transmitted into the moving mirror.

Sample cell: Sample is directly placed into the IR beam. For solid samples, make sure that the sample is
turned into a fine powder to let the infrared pass through the sample.

Detector: Interferogram is the name of the signal acquired by an FT-IR spectrometer. The detector detects
the interferogram and pass it on into the computer for further analyzation.

Computer: After detecting the interferogram, the computer will analyze the sample using Fourier
transform print the data gathered.