Spectrophotometer Lambert-Beer Law Calibration Curve

1. “DESIGN AND SYNTHESIS OF BRANCHED ZWITTERIONIC GEMINI LIQUIDS MODIFYING
PROPERTIES OF WETTABILITY AND INHIBITORY CORROSION FOR HETEROGENEOUS
RESERVOIRS LITHOLOGY”
BY: Q.I. JORGE FRANCISCO RAMÍREZ PÉREZ

2.  Summary
◦ Electromagnetic spectrum
◦ Color
 Applications
 Spectrophotometer
 Lambert-Beer Law
 Calibration Curve
 Application Example

3. Funcional
grups
Individual
nuclei
Electromagnetic spectrum
OH R
O
R´ R
O 13 C 1 H

4. Color
• In chemistry, the color is the part of the molecule that absorbs energy, this part is called
chromophore.
• The chromophore is a functional part that absorbs
energy in the UV and/or Vis region.
• But can also have a auxocrom, which is a functional
group which alters the absorption of the chromophore.
–OH, -NH2, -Cl, -Br, -CH3
The human eye sees color
complementary to that
absorbed

5. UV-Vis
• UV-visible spectrophotometry is
an analytical technique for
determining the concentration of
a compound in solution. It is
based on molecules that absorb
electromagnetic radiation and in
turn the amount of light absorbed
depends linearly on the
concentration.

6. • In principle, any chemical species that absorbs electromagnetic radiation in the ultraviolet or
visible region is susceptible to be determined by spectrophotometric techniques. The largest
field of application is in the quantitative analysis, spectrophotometry being one of the most
used tools.
Applications
The precision is acceptable, normally uncertainties are obtained
between 1 and 2%, although with certain precautions can be reduced
considerably.

7. Spectrophotometer
• Is the equipment we use to measure the absorption or transmission of light part of a sample.
The spectrum is recorded as
absorbance (A) vs wavelength (λ)
Optical system: through filters and
gratings focuses on beam and
selecting a fixed wavelength.
Sample compartment: This is where the
sample is placed, typically with a
thickness of 1cm path length over which
the beam is incident monochromatic light
Detector: Receives the signal
light intensity transmitted to
each wavelength and
transforms it into an electrical
signal that a computer process.

8. To quantify the concentration of a sample by UV
A = ᶓ b c
Where:
 ᶓ : Molar absorptivity coefficient (L mol-1 cm-1)
 b : Sample length (Cell) (cm)
 c : Concentration of compound in solution (mol L-1)
A is directly proportional to C
Therefore, measurements may be performed at different concentrations of
an analyte for the construction of a calibration curve.

9. Calibration Curve
• This is a reference curve constructed with known amounts of a substance that is used to determine the
amount of the substance present in an unknown sample.
• Selecting the wavelength maximum absorbing substance and using a standard samples of known
concentration can obtain the data of absorbance versus concentration
• Preparation of standards
• Standards of analyte are prepared to cover a suitable range of concentrations, and is measured by the
analytical signal provided thereto.
Solutions of known analyte concentration and increasing

10. Draw a graph of signals versus
concentration of analyte and
calculate the line that "best"
fits the data using a least
squares fit. In this way we
obtain the slope (b) and the
ordinate (a) defining the
origin line.
Calibration Curve
Analytical signal is measured for the
unknown samples and is interpolated in the
calibration curve for analyte concentration
values​​.

11. 0
0.5
1
1.5
2
2.5
190
192
194
196
198
200
202
204
206
208
210
212
214
216
218
220
222
224
226
228
230
Absorbance
Wavelength (nm)
Surfactant absorbance
10 ppm
20 ppm
40 ppm
60 ppm
80 ppm
100 ppm
120 ppm
140 ppm
160 ppm
180 ppm
200 ppm
To 197 nm
Concentratión
(ppm) Absorbance
10 0.2492
20 0.3218
40 0.6366
60 0.9298
80 1.2886
100 1.4608
120 1.6527
140 1.9047
160 2.1076
180 2.131
200 2.2387
y = 0.0111x + 0.2365
R² = 0.9704
0
1
2
3
0 50 100 150 200 250
Absorbance
Concentration (ppm)
Absorbances