I, Biomed Lab 4
Photometry and Writing Lab Reports
Objectives
To demonstrate the principles of spectrophotometry To determine The molar extinction coefficient of a weak acid, para-nitrophenol The pKa for the dissociation of p-nitrophenol To learn how to write a GOOD lab report
Introduction
As light passes through a substance, one or more of the following phenomena can occur: reflection, refraction, absorption, interference, diffraction, fluorescence and ionization Spectrophotometric methods are based on absorption. Absorbance is the log10 of % total light (100%) / % of light transmitted. Absorbance (A) = 2 - log %T
If 10% of violet light is transmitted than 90% violet light is absorbed. A = Log 100/10 = 2-1 = 1
All atoms and molecules are capable of absorbing various types of energy. The amount of energy absorbed depends on the structure of the molecules and the number of molecules present.
Visible Light
Today's lab will focus on the visible spectrum of light. Visible light can be broken down into the colors we observe with the human eye. A compound will absorb light at one wavelength, but transmit light at another wavelength. The color of a compound that we see is due to the transmitting wavelengths of the compound. Type Far UV UV Visible Near Infrared Wavelength () absorbed 100-200nm 200-400nm 400-750nm 750-2000nm
Infrared
2000-25,000nm
Visible Light
The molecules that make up Wavelength chlorophyll absorb red light, but transmit green light. That is why we observe most 400-450nm vegetation to be green in color. 450-480
480-490 Color absorbed Violet Blue Green-blue Apparent (transmitted) color Yellow ** Orange Red-orange
490-500 560-575
575-590 590-625
Blue-green Yellow-green
Yellow Orange-red
Red Violet
Blue* Green-blue**
625-750
Red
Green
Jones and Atkins, Chemistry: Molecules, Matter and Change, 2008. 947
Spectrophotometer
% Transmitted
100%
Sample Light source
Monochromatic light
% Absorbed = 100 -%Transmitted
Data values recorded for analysis
Absorbance is different from % Absorbed !
Beer-Lambert Law
Beer-Lambert law states that the absorption of light energy at any wavelength is dependent on the concentration of the absorbing compound (molar or gms/L) and the path length of light ( 1 cm) through the absorbing compound.
However, the highest sensitivity will be observed at wavelength of maximum absorbance
Absorbance coefficient can be expressed as a value a or depending on Units for concentration. Units for is (M-1 x cm-1) and or a is (g/L-1 x cm-1)
A=cl
A=absorbance = absorption coefficient (when c is M) c= concentration l= length of light path
A=abc
A=absorbance a=absorption coefficient (when c is g/L) b= length of light path c=concentration
Introduction
The selective absorption of light is useful to identify compounds and determine their concentration in samples.
During a reaction, products are formed with different absorption characteristics than the reactants.
When the reactant and product differ in their wavelengths of maximum absorption, The rate of a reaction can be followed by measuring the light absorbed by the product at its maximum wavelengh. Example: Dissociation (Ionization) of p-Nitrophenol at alkaline pH
Visible Light and p-nitrophenol
OH
H2O
OH+
+
NO2
into the ionized form on the right (in yellow).
NO2
p-nitrophenol dissociates from the unionized form on the left (in white)
p-nitrophenol absorbs light in the 400-450nm range (violet), but transmits yellow light. That is why we can visibly observe the dissociation reaction of pnitrophenol: the more yellow the solution, the more dissociation to the ionized form.
Visible Light and p-nitrophenol
OH H2O
Unionized! 50% unionized, 50% ionized!
O-
+
H+
Ionized!
NO2
At pH 4 At pH 7.4
NO2
At pH 10
Visible Light and p-nitrophenol
Objective: find the molar absorption coefficient of pnitrophenol. Why is this important?
The absorption coefficient, or molar absorptivity, is the characteristic of a compound that tells us how much light is absorbed at a particular wavelength. So how do we relate the color of a solution, its absorbance, its concentration, and the pathlength of the spectrophotometer to find the extinction coefficient and pKa?
Beer-Lambert Law
Beer's Law can help to measure the pKa of various compounds if the absorption spectra of the species are sufficiently different at various ionization states (at various pHs).
Now we know that p-nitrophenol has different absorption values at different ionization states we can use the information to solve for the pKa.
The equation below, derived form the Henderson Hasselbach equation, may be used for this purpose.
pKa = 7.4 log
a (HA + A-) a (HA)
a (A-) a (HA + A-)
Finding the pKa of p-nitrophenol using spectrophotometry
OH O-
H2O
+
H+
NO2
At pH 4 At pH 7.4
NO2
At pH 10
AHA
Absorbance value (from spec) at pH 4
A (HA + A-)
Absorbance value (from spec) at pH 7.4
AAAbsorbance value (from spec) at pH 10
Finding the pKa of p-nitrophenol using spectrophotometry
Follow directions CAREFULLY read and ahead!
First, find the max using the pH 10 sample Then, for each of the pH buffers + p-nitrophenol samples, read and record the absorbance values.
Using Beer's Law, calculate the molar extinction coefficient. Using the equation below, calculate the pKa of p-nitrophenol at pH 7.4.
Activity II: Writing Lab Reports
Writing a lab report
Why are lab reports important? They are a written record of the purpose, methods, your observations and results, and conclusions during an experiment make sure to take good notes! Every student, NOT group, writes their OWN lab report. NEVER EVER "fudge data" because you think it's the wrong answer! Your data is what YOU ACTUALLY observed in lab, not what you THINK you SHOULD HAVE observed.
Your lab reports will be written in Microsoft Word and any graphs will be created in Microsoft Excel.
Writing a lab report
Title of the Lab Report: - Make sure that it is appropriate for the lab. - Should not be too long; it is the basic topic of the experiment - Think about this: would someone understand what the lab was about based on the title? Introduction - Include background information ONLY about the basic topic of the experiment. Short and precise!
- For the Photometry lab, briefly discuss the visible light spectrum, p-nitrophenol, Beer's Law, etc. DO NOT add "filler". More is not always better.
- Ask yourself again: if someone were to read the introduction of your lab report, would they understand what the lab was about? Did you discuss anything that would NOT be conducted in the lab? Take this out!
Writing a lab report
Observations - Write all observations exactly as observed from the lab keep an accurate and NEAT record of your work!!
- This includes solution concentrations, tables, photographs, images, *unexpected
results
- There is usually no need for written material here, only RAW data - Example: for lab 4, the charts from pages 2 and 3, and any calculated values go here! Results - Your results compare all observations obtained from the whole lab. - How do YOUR results compare with controls or known standards? - Example: for lab 4, how does your pKa value for p-nitrophenol compare with the known value? - Include your Excel graphs here. For lab 4, we will create them in lab.
Writing a lab report
Discussion - The Discussion is meant to actually discuss the results you obtained from the lab. - Similar to the ending paragraphs of an essay, highlight some of the information from the introduction here. How is the introduction related to the discussion?
- You can use other sources* to support your results, but make sure the information actually supports your statements.
- Example: the pKa value for p-nitrophenol I obtained from lab was close to 6, but the known value is closer to 7. Based on (this source), the reason these values differ could be attributed to... - The discussion should have more information than the conclusion.
Writing a lab report
Conclusion - Main goals: 1) Was the original hypothesis supported or rejected? 2) Were the original objectives of the experiment met? 3) If someone read your work, would they feel as though you proved your point? Does the conclusion REALLY conclude the lab report? Too much filler? - The conclusion does not need supplementary material, only enough for to make the points of the main goals.
Writing a lab report
Sources of Error - There is always SOME room for experimental error. Why are your observations different than what you should have gotten? How is your experimental technique? Did you follow directions?? - Discuss some areas that need improvement in your technique.
- Sometimes we are very successful in getting the desired result. In that case, there is no need to go into detail about sources of error.
Writing a lab report
References - There are ALWAYS sources from which the information in your lab report came from.
- This is not called a "Bibliography" or "Works Cited"; we refer to the list of sources as "References" - Accepted: Textbooks, class notes, peer-reviewed journal articles. - Unaccepted: WEBSITES, other than (check with teaching fellows) www.sciencedirect.com www.pubmed.org Websites must be peer-reviewed! www.acs.org www.ncbi.nlm.nih.gov
Writing a lab report
References
- You MUST reference a sentence even if you DO NOT use the exact quote. If you paraphrase some ideas, you STILL MUST REFERENCE! - RULE: If you didn't come up with it, don't take credit for it! Acceptable ways to reference a sentence: Direct quote: "Information is interpreted more easily and more uniformly if it is placed where most readers expect to find it." (Gopen and Swan, "The Science of Scientific Writing", American Scientist, (Nov-Dec 1990), Vol. 78, 550-558) Paraphrase: Readers are able to understand scientific articles easily and uniformly if information is placed in appropriate paragraphs. (Gopen and Swan, "The Science of Scientific Writing", American Scientist, (Nov-Dec 1990), Vol. 78, 550-558)