Exp. No 5
Exp. Title: Basic Lab equipments Calibration Spectrophotometer
background
Non-reliable instruments can be a major source of error in all analyses.
Analytical data generated from instruments which are not properly
qualified, nor calibrated with traceable standards are questionable
The use of calibrated instruments is vital in clinical lab to obtain accurate
results in addition to other factors like proper training and use of the
validated assays.
Statement of the Problem
The goal of this experiment is to calibrate an ultraviolet/visible (UV/VIS)
spectrophotometer for use in determining concentrations of a compound in
a solvent. The objectives are to learn how to operate a UV/VIS
spectrophotometer, determine the Amax (the optimal wavelength for
measuring absorbance) of Glucose, produce a calibration curve of amount
of light absorbed (at the Amax) by a solution versus solution concentration,
confirm the applicability of the Beer-Lambert law to the data in calibration
curve, and use the calibration curve to find the concentration of an
unknown solution.
Hypothesis
A spectrophotometer is an optical instrument which measures how the
sample changes the spectrum of the light (e.g. transmission, reflection,
scattering, fluorescence).
A spectrophotometer is divided into seven basic components,
which includes :







Stable source of radiant energy.
An entrance slit to focus the light.
A wavelength selector.
An exit slit to focus the light.
A device to hold the transparent container (cuvette).
A radiant-energy detector and
A device to read out the electrical signal generated by the
detector.
Figure 1 Components: light source, monochromator, sample cell, detector,
and optical system.
Performance of the instrument
In UV/VIS spectroscopy, light in the wavelength range of ultra-violet is
sent through a sample such as a liquid solution. Compounds in the sample
absorb certain wavelengths of the light. The amount (intensity) of light
absorbed is related to the concentration of the component in the sample.
At sufficiently low concentrations of the absorbing component in the
sample, a linear relationship exists between absorbance and concentration
of material. This is expressed by the Beer-Lambert law
A = L C
=
[
]
Where A is the absorbance, e is the extinction coefficient of the material
(compound in the sample), L is the path length through the sample (m), and
[C] is the concentration of the species under consideration (mol/L).
Liquid samples are typically put into a solution cell called a cuvette for
measurement of the absorbance. The standard configuration of a cuvette for
a UV/VIS spectrometer is shown in figure 1
Figure 1: I is intensity of transmitted light, Io is the incident intensity, L is
the path length through the sample, C is the concentration of the species
under consideration (mol/L).
Once a chromogen is proved to follow Beer –Lambert’s law at a specific
wavelength (a linear relationship between absorbance and concentration is
established), it is possible to relate unknown concentrations to a single
standard by a simple proportional equation. Therefore
A standard = C standard
A unknown C unknown
→ C unknown = A unknown x C standard
A standard
The above equation is valid only if the chromogen obeys the Beer’s law and
both standard and unknown are measured in the same cell. The
concentration range over which a chromogen obeys Beer –Lambert’s law
must be determined for each set of analytical conditions. A calibration
graph is drawn with the absorbance versus concentration.
Figure 2 – Relationship of absorbance vs. concentration
An absorbance spectrum (a plot of absorbance as a function of wavelength)
is determined to select the optimal wavelength for analyzing a given
compound. The optimal wavelength (Amax) for measuring absorbance is
that wavelength that is most absorbed by the compound in question.
Fig-3 Absorption spectrum. A graph of absorbance vs. wavelength for a
hypothetical compound. The Amax for this compound is about 500 nm.
Materials
A. Determination of the Amax
In Part A of this exercise you will determine the Amax (the optimal
wavelength for measuring absorbance) of Glucose.
 Materials





Beaker of dH2O.
Tube of Glucose (100 mg/ml)
Cuvettes.
P10-1000 Micropipettor, Yellow and blue tips.
Spectrophotometer
Procedure



Press in the power button (located on the back of the instrument) to
the ON position.
Let the machine warm up for 10 min before you use it.
To determine the Amax of the compound, each team (of four
students) will be assigned a range of 50 nm within the visible range
of the spectrum (400nm to 700 nm). Read the absorbance of the
sample every 10 nm within your team’s assigned range. You will
then write down your data on the sheet near the spectrophotometer
who, each group will then plot a graph of the data to determine the
Amax.

Set the wavelength of the spectrophotometer to the lowest
wavelength in your range assigned to your group.

Place the reference blank cuvette (usually referred to as a “blank”)
into the spectrophotometer (“spec”) and zero the absorbance. (Zero
absorbance = 100% Transmittance).

Remove your blank and place the cuvette containing the -------- into
the spec. Read the absorbance at the first wavelength in your range
and record it in the table provided in the results section.

Repeat the last two steps to read the absorbance of the --------- every
10 nm for the rest of your range.

You must "re-zero" the spec at each wavelength using the blank.

Rinse out your two cuvettes with dH2O and place upside down on a
KimWipe to drain, you will use them again in Part B.
B. The effect of concentration on absorbance
In Part B of this exercise, you will make serial dilutions of Glucose
standard (100mg/ml) and measure their absorbance to see the
relationship between concentration of a compound and its absorbance.
You will then use the Lambert-Beer law to determine the extinction
coefficient for Glucose and determine the concentration of BPB in an
unknown solution.
 Materials






Glucose Standard (100mg/ml)?
Cuvettes
P10-1000 Micropipettes with yellow and blue tips
Spectrophotometer
Test tubes.
Unknown concentration of Glucose solution
 Procedure





Obtain 4 test tubes. Number them 1 to 4.
Use a P100-200 μl to pipette 100 μl of dH2O into each
tube.
Add 100 μl of 100 mg/dl Glucose concentration to tube
1. This tube represents a 1/2 (or a 1:1) dilution. The
concentration of Glucose in this tube is 50 mg/dl, mix
by vortexing for 15 seconds
Add 100 μl of the Tube 1 contents into Tube 2. Mix
using the vortexer. Tube 2 is a 1/4 dilution. (You made a
1/2 dilution of a 1/2 dilution. 1/2 * 1/2 = 1/4).
Transfer 100 μl of Tube 2 into Tube 3. What dilution is





this? You made a 1/2 dilution of a 1:4 dilution. What is
the concentration of Glucose in this tube?
Transfer 100 μl of Tube 3 into Tube 4.
Set the spec. to the Amax wavelength that you
determined in Part A.
Zero the spec using your blank cuvette.( 0% A or 100 %
T)
Read the absorbance of tube 4. Record it in your results
section.
Pour the contents of tube 4 down the sink and read the
absorbance of tubes 3, 2, and 1.







Record them in the results section.
Measure the absorbance of your undiluted Glucose
solution.
Make a graph of absorbance vs. concentration of
Glucose solution.
Obtain a sample of unknown Glucose concentration.
Read the absorbance at the Amax and record it in the
results section of your notebook.
Based on your data, calculate the extinction coefficient
for glucose and determine the concentration of Glucose
in your unknown sample.
When you are finished, pour the dye dilutions into the
sink, rinse out the cuvettes and discard test tubes.
Results (Data)
Exp. No 5
Basic Lab equipments Calibration
Spectrophotometer
Student Name:
Section:
TABLE 1. Record the absorbance values for your assigned wavelengths for
Glucose solution, then plot a graph to determine the Amax of Glucose
Wavelength (nm)
Absorbance
When making graphs, remember the independent variable (the thing you
change, wavelength in this case) goes on the X-axis and the dependent
variable (the thing you measure, absorbance in this case) goes on the Y-axis.
Give your graph a title.
TABLE 2. Determination of the effects of dilution on absorbance of Glucose
solution.
Collect your data by filling out the values in the table
Tube number
Dilution
Undiluted
1
2
3
4
0
1/2
1/4
1/
1/
Concentration
of Glucose
100mg/dl
Absorbance
Graph of the data from Table 2. Absorbance vs. concentration of Glucose.
Be sure to label graph a title.
What was the concentration of Glucose in your unknown solution?
Conclusions
Determine whether or not if the pipettes you used are in the acceptable
range, or require calibration.