Instrumental Analysis Laboratory Manual: CHM 311 at La Salle University - Prof. Michael Pr, Lab Reports of Analytical Chemistry

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Lab Manual

Instrumental Analysis Laboratory

La Salle University

Department of Chemistry and Biochemistry

Michael J. Prushan

Table of Content

• Laboratory Syllabus……………………………………………………..
• Laboratory Notebooks and Final Laboratory Reports…………………..
• Atomic Absorption Analysis of Iron Content of Food………………….
• UV-VIS Spectrophotometry ……………………………………………
• Fluorescence Spectrophotometry ………………………………………
• Fourier Transform Infrared Absorption Spectrophotometry………..…
• Cyclic Voltammetry…………………………………………………….
• GC-MS…………………………………………………………………
• HPLC……………………………………………………………………

INDEPENDENT PROJECT

After completion of the final “prepared” laboratory experiment, the lab will consist of you and your partner(s) proposing, designing, researching, performing, and writing up an extended project. You should start thinking of your project topic early in the semester so we can talk about its feasibility and equipment requirements. Two months before the date of the beginning of the project, you will have to provide a detailed preliminary proposal, including chemicals, procedures and instruments needed to complete the project. You should start thinking of your project topic early in the semester so we can talk about its feasibility and equipment requirements. The purpose of this project is to get you away from the “handout” type of lab and get you to perform a “real” lab where you must design the experiment and decide the scope of the problem. There are many issues involved in an analytical analysis in addition to merely running an instrument. At the end of the project, you will write up a scientific paper on your project.. As we get closer to the date of beginning the projects, there will be more information provided that covers the schedule and expectations.

Please note that in order to pass the course, you must receive a passing grade in both the lecture and the laboratory parts of the course.

CHM 311

Instrumental Analysis Laboratory Notebooks and Final Laboratory Reports Fall 2002

One of the main activities that you will perform as a scientist will be to write up your experimental results for other scientists either in peer-review journals or for corporate reports. It is essential that you learn to write clear and concise scientific reports. Therefore, there will be a strong emphasis in this course to get you accustomed to thinking and writing like a scientist.

Purpose of the Laboratory Notebook

The laboratory notebook is your record of what you did during an experiment. It is your account of the purpose, method, procedure, and all data you acquired. Laboratory notebooks cannot be too detailed and are more than likely not detailed enough. In reality, no experiment is just done once, but repeated many times over the course of weeks or months or even years. The laboratory notebook is there to go back to when you do repeat something so you know how you did it the previous time. If your initial record of the experiment is not complete, it will be hard to repeat the experiment and impossible to compare the results. Whether you enter industrial, government, or academic research, a well-kept laboratory notebook is very important.

Writing in the Laboratory Notebook

Each person has his or her own style of keeping a laboratory notebook. It is your responsibility to maintain your laboratory notebook and it will not be reviewed or graded by the instructor. A few general guidelines for keeping a notebook:

a) Don’t hesitate to put down too much detail. Even things that seem obvious can be forgotten later if they are not written down.

b) Try to use complete sentences. Invariably, a cryptic note in the margin of the notebook cannot be deciphered later and important information will be lost.

c) Write everything in the notebook! Don’t write a weight measurement on a paper towel and stick that in your notebook to transfer later. Record observations and data immediately. Having tables made before hand for measurements will save time and insure that you did not forget an important measurement.

d) Errors you make in the notebook should be crossed out but still legible in case what you crossed out was actually correct. Don’t erase, white out, or tear pages out of your notebook. Keep your notebook as an honest account of the experiment; mistakes do occur.

e) Number the pages in the notebook sequentially and save room at the beginning for a table of contents.

discussion. This serves as a minimum that should be discussed. Be sure that you understand the questions and answers, as these are usually important points of the experiment. This section contributes the most to the grade received on the laboratory report.

Format of the Independent Project Report

The format of the report on the independent project will take the form of a manuscript from the journal Analytical Chemistry. More details will be available on this report as the project gets started. You will have an opportunity to submit the article for an initial review and have a chance to make corrections.

CHL311 Instrumental Analysis Laboratory

Atomic Absorption Laboratory

Atomic Absorption Analysis of Iron Content of Food

Introduction

This experiment will use atomic absorption spectroscopy to analyze a food

product for the element iron using the Beer’s Law Method. The measured iron content will be compared with the amount of iron reported by the manufacturer.

Preparation of Samples for Beer’s Law Plot:

1. Prepare 1 Liter of 8 M HCl.
2. Prepare 1 Liter of Stock Iron Solution having a concentration of 1000 μg Fe / mL.

a) Wipe several strands of Fe wire and rinse them with hexane to remove the protective oil.

b) Dissolve 1.000 g Fe wire in a beaker by adding 50 mL 1:1 HNO 3 :H 2 O ( USE HOOD )

c) Quantitatively transfer this solution to a 1 L volumetric flask and bring to volume with DI water.

3. Prepare 250 mL of Diluted Iron Solution have a concentration of 100 μg Fe / mL. Use

a 250 mL volumetric flask and DI water.

4. Beer’s Law Standard Solutions:

Sample Diluted Iron 8M HCl Final Volume

Blank 0.00 mL 125 mL 250.00 mL 1 μg Fe / mL 1.00 mL 50 mL 100.00 mL 3 μg Fe / mL 3.00 mL 50 mL 100.00 mL 5 μg Fe / mL 25.00 mL 250 mL 500.00 mL 10 μg Fe / mL 10.00 mL 50 mL 100.00 mL 15 μg Fe / mL 15.00 mL 50 mL 100.00 mL 20 μg Fe / mL 20.00 mL 50 mL 100.00 mL

Include in the Report:

1. Sketch the main components of an atomic absorption spectrophotometer.
2. Why is iron wire a good primary standard for this analysis?
3. What is the U.S. RDA of iron?
4. Outline of method used to prepare food sample including all observations
5. Prepare a Beer’s Law plot of your standards and determine the best fit line or curve

for the data and plot it. Determine the amount of Fe in your food sample from your

calibration curve.

6. Report the amount of Fe in your food sample and compare to the value reported by the manufacture.

CHL311 Instrumental Analysis Laboratory

Qualitative UV-VIS Spectrophotometry Laboratory

Introduction

UV-VIS absorption spectrophotometry can be applied both quantitatively (such as

Beer’s Law analysis) and qualitatively (compound identification and purity). This lab

will explore the use of the UV-VIS spectrophotometer to analyze various UV-VIS

absorption organic and inorganic species. Your goal should be to gain intuition into what types of chemical systems benefit most from UV-VIS spectrophotometry and those that

do not. You will also gain experience into what the various scan parameters do and why

and when you should change them.

Wavelength Accuracy

This procedure does NOT need to be performed every time you use the

spectrophotometer. However, it is important to realize that just because the readout from

the instrument indicated that 500.00-nm light is being measured, this does not absolutely

guarantee that this is the case. If wavelength accuracy is of primary concern, then you should verify the spectrophotometer’s calibration by measurement of the absorption lines

of holmium glass or emission lines of the deuterium (D 2 ) lamp. The following procedure

will allow you to check the wavelength accuracy of the spectrophotometer.

Holmium Glass Method. This method allows you to verify the wavelength calibration at three different wavelengths, 460.0 nm, 360.9 nm and 279.4 nm. The performance is

considered satisfactory if the wavelength errors of the holmium glass absorption lines are

within +/1 0.3 nm

a) With the Holmium Glass Filter in the sample compartment, measure the 460. nm absorption from between 455.0 to 465.0 nm at a 0.5 nm bandwidth, 10 nm/min scan speed, and a data interval of 0.1 nm.

b) Determine the wavelength of maximum absorbance using the Peak Pick Table.

Include in the Report:

1. Sketch the basic components (block diagram) of a UV-VIS scanning spectrometer.
2. For what types of molecules is UV-VIS spectroscopy most useful? For which is it not? As a consequence, what are the best solvents for UV-VIS spectroscopy?
3. What is meant by bandwidth? As you narrow the bandwidth of the UV-VIS

spectrometer, what might you expect to happen to the spectrum?

4. Labeled copies of all the spectra and peak data.
5. Discuss in what situations would an accurate calibration of the UV-VIS be most

important?

6. Discuss how changing the bandwidth changes the spectrum of the benzene vapor.
7. Compare the different optical material used to make cuvettes. What wavelength

ranges is each material useful for?

8. Compare the spectra of the different solvents. State which of these solvents could be

used in UV-VIS absorption spectroscopy.

9. Discuss the difference and similarities between the following spectra.

a) benzene in the vapor phase vs. benzene in cyclohexane (at 0.5 nm bandwidth)

b) benzene vs. naphthalene vs. anthracene (all in cyclohexane)

Do you find any correlation between molecular structure and the spectra?

10. How would you quantitatively describe the concentration of the organic samples you

prepared? Suggest a procedure for preparing these concentrations quantitatively.

CHL311 Instrumental Analysis Laboratory

Qualitative Fluorescence Spectrophotometry Laboratory

Introduction

Fluorescence spectroscopy is one of the most sensitive analytical techniques.

This experiment will expose you to the functioning of our Fluorescence

Spectrophotometer. You will be quantitatively examining the fluorescence of

acetylsalicylic acid and salicylic acid, compounds found in a commercial aspirin tablet.

Salicylic Acid: MW 138.12 g/mol

Acetylsalicylic Acid: MW 180.17 g.mol

Preparation of Solutions

All solutions should be prepared with 1% acetic acid in chloroform. If this stock

solvent is not available, consult your instructor. NOTE: Chloroform is toxic and

should be used only in a fume hood. Dispose of waste properly.

Using 25 mL volumetric flasks, prepare the following:

a) 25 mL of 2x10-5^ M Acetylsalicylic Acid solution

b) 25 mL of 2x

• M Salicylic Acid Solution

Spectrofluorometer Setup

1. Consult instructor about the operation of the spectrometer.
2. Set the parameters to these initial settings.

Excitation Slit 10 nm Emission Slit 5 nm Scan Speed 120 nm/min

Excitation and Emission Spectra for acetylsalicylic acid

(Print a copy of all spectra)

1. Fill a quartz cuvette at least ¾ full with your acetylsalicylic acid sample.
2. Set the Emission Wavelength to 0 nm (All wavelengths pass).
3. Scan the excitation range from 200-500 nm.

Include in the Report:

1. Sketch the basic components (block diagram) of a spectrofluorometer.
2. What are the differences between this instrument and the UV-VIS spectrometer?
3. Instrumentally, what is the difference between an excitation spectrum and an emission

spectrum?

4. Which types of molecules will have the highest fluorescence intensity?
5. Hand in printouts of all spectra, with appropriate lablels, including instrumental

settings.

6. Based on your spectra, discuss which excitation and emission wavelengths are most suitable for the analysis of acetylsalicylic acid and salicylic acid in an aspirin tablet.
7. Discuss the effect of changing the excitation and the emission slit width. What

changes and why?

CHL311 Instrumental Analysis Laboratory

Fourier Transform Infrared Absorption Laboratory

Qualitative Analysis of Aspirin Tablet

Introduction

Infrared (IR) radiation with wavelengths of 700 nm to 50,000 nm is found in the

electromagnetic spectrum between the visible and microwave regions. It can be applied

to the analysis of organic molecules by causing molecular rotation and/or molecular vibrations (stretching or bending of bonds) in the molecules. This experiment will

measure the absorption of infrared light by salicylic acid and acetylsalicylic acid. A

commercial aspirin tablet containing both of these molecules will be analyzed and the

spectra will be compared.

Materials

Polystyrene Calibration Film KBr (Potassium Bromide) Salicylic Acid Acetylsalicylic Acid Commercial Aspirin Tablet

Procedure

1. Preparation of KBr Pellets

The sample container material must be transparent to infrared radiation in order to

measure the absorption of the sample. Halide salts (including KBr) have excellent transparency in the IR. Liquid samples are typically placed between two large, polished

crystals of KBr (known as salt plates) that are placed in the spectrometer. Solid samples

are measured by grinding the solid sample into a fine powder and mixing a small amount of sample powder with powdered KBr. Halide salts have the property of “cold flow” in

which they have glass-like transparent or translucent properties when sufficient pressure is applied to the finely powered materials.

a) Blank KBr Pellet

i. Grind 200 to 300 mg of KBr into a fine powder using a mortar and pestle.

ii. Following the “Operating Instructions for Evacuable 13 mm KBr Die”, create a KBr pellet. Do not apply vacuum. After making each KBr pellet, rinse the metal pellets (D) with acetone into a waste beaker IN THE HOOD.

2. Obtain IR Spectra

Consult your instructor as to the operation of the Perkin-Elmer 1600 Series FTIR.

Obtain all spectra using the following parameters unless otherwise noted. Save

all scans to a 3.5” floppy disk.

Resolution: 2.0 cm- Apodization: Strong Range: 4400 cm

• to 450 cm -

Mode: Ratio Number of Scans: 4

a) IR Background Spectrum

Obtain a background IR spectrum without a sample in the instrument.

b) Polystyrene Calibration Film Spectrum

i. Place the polystyrene calibration film in the sample holder and obtain the

spectrum at a resolution of 16.0 cm-1.

ii. Change the resolution back to 2.0 cm-1^ and obtain another spectrum of the

polystyrene calibration film.

c) Blank KBr pellet Spectrum

Place the blank KBr pellet in the sample holder and obtain the spectrum.

d) Salicylic Acid Spectrum

Place the salicylic acid/KBr pellet in the sample holder and obtain the spectrum.

e) Acetylsalicylic Acid Spectrum

Place the acetylsalicylic acid/KBr pellet in the sample holder and obtain the

spectrum.

f) Commercial Aspirin Spectrum

Place the aspirin /KBr pellet in the sample holder and obtain the spectrum.

g) Dispose of rinse acetone properly.

Include in the Report:

1. Sketch the main components of an FTIR infrared absorption spectrophotometer.
2. Hand in copies of all spectra acquired during this experiment.
3. Explain why the IR Background spectrum has peaks even though the sample chamber

was empty. (See Figure 16-9, pg. 397 of text)

4. Compare the polystyrene calibration film spectra obtained at a resolution of 16.0 cm- and 2.0 cm-1. What are the differences?
5. Compare the polystyrene calibration film spectrum obtained at a resolution of 2.0 cm-

with the spectrum appearing in the text in Figure 16-1, page 382.

6. Examine the salicylic acid spectrum. Using the structure of salicylic acid below and

Table 17-2, pg. 410 in the text, identify the major peaks in the spectrum.

7. Examine the acetylsalicylic acid spectrum. Using the structure of acetylsalicylic acid below and Table 17-2, pg. 410 in the text, identify the major peaks in the spectrum.
8. Examine the commercial aspirin spectrum and determine spectral contributions from

salicylic acid and acetylsalicylic acid.