University of NizwaNizwa, Sultanate of Oman

CHEM 490ADVANCE LABORATORYLABORATORY MANUAL

Academic Year 2010/2011

By

DR. FAZAL MABOOD&

FRANCISCO C. CAMACHOHANAN ALYAHYAI

HAMEEDA ALRABAANIKHALID PERVEEZ

COLLEGE OF ARTS AND SCIENCESDEPARTMENT OF BIOLOGICAL SCIENCES AND

CHEMISTRY

CHEM 490 – LABORATORY SYLLABUSSTUDY MATERIALS:Douglas A. Skoog, Donald M. West, F. James Holler, Stanley R. Crouch, “Fundamentals of Analytical Chemistry” 8th ed. 2003; Saunders College Publishing, Philadelphia

TECHNIQUES INVOLVED: This semester laboratory deals with the instrumental techniques

like spectroscopic, electroanalytical and chromatographic techniques used for the analysis and purification of unknown samples.METHOD OF INSTRUCTION:

Demonstration will be given to the students in the Laboratory prior to start each experiment. Laboratory experiments will serve to reinforce, supplement, and provide a context for the materials presented in the theory class of course CHEM 341. ASSESSMENT Total marks for lab exam 25

laboratory reports (5 marks ) Viva Voice exam (5 marks) For overall performance and practical lab exam (15 marks)

EXPECTED LEARNER OUTCOMES:

The students will be able to demonstrate a working knowledge of the proper use of instrumental techniques for the analysis of different types of analytical and environmental samples.

SAFETY: The students must agree and sign the safety sheets before they start the first laboratory session.

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LABORATORY SAFETY AND GUIDELINES

Better Safe than Sorry!!!Read the following instructions before you go into the laboratory

1. Safety glasses are required at all times. Prescription glasses are adequate, but contact lenses are not. Therefore, contact lenses are not permitted in the laboratory.

2. Always wear your laboratory coat. Do not wear clothing that hinders free movement of your hands or hangs loose outside your laboratory coat. Ladies Head scarves must be tucked into the lab coat.

3. Do not work in a laboratory if no instructor is present. Read theexperimental instructions carefully before starting the work. Note any precautionsthat must be taken.

4. Never eat, drink, or smoke in the laboratory. Never taste chemicals. Wash yourhands before you leave the laboratory.

5. Do not use your mouth to fill a pipette. There are special pipette fillers available.

6. Note the position of safety equipment such as fire extinguishers, eyewashes and first aid boxes. Report all accidents immediately to a staff member or technician.

7. Use the fume hood when doing experiments with irritatingchemicals.

8. Be careful about throwing away wastes. Always follow instructions given. Do not throw away solids in the sink. Do not leave glassware or any other solid materials, including filter papers, in the sink. Put broken glassware into the labeled buckets. Some waste liquids must be put into special bottles, not poured down the sink.

9. Do not leave a lighted burner unattended. Do not allow any part of your body or of your clothing to fall into a flame.

10. When heating anything in a test tube, do not point the open end towards yourself or towards any other person.

11. Before you leave the laboratory, turn off any water taps and burners and dispose of solid waste in the correct container. Also wash out all apparatus used and clean up the bench top.

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12. Keep your bench clean and tidy while you are working. Clean up any spills orbroken glass immediately. Keep your books and papers away from water, chemicalsand flames. Position your apparatus on the bench so that it is convenient andcomfortable to use. Put equipment you are not using out of the way, so that you donot knock it over. :

13. Finally If you are in any doubt ask. I. General Behavior1. Always read the upcoming experiment carefully and thoroughly

before entering the laboratory.

2. Be in the lab and ready promptly when the lab begins.

3. Absolutely no food, beverages or chewing gums will be permitted inside the lab.

4. Wash you hands frequently during the lab and of course wash them twice at the end of the lab.

5. Should any injury occur, regardless how minor it is, report it immediately to your supervisor.

6. Never pick up any broken glassware with your bare hands, regardless of the size of the pieces.

7. Never put broken glasses in a regular garbage can. A special container will be provided

8. Make sure to label and read the labels of all chemicals which your are dealing with.

9. Never use reagents from unmarked bottles.

10. In any emergency, the fastest to way to get the supervisor attention is to SCREAM.

If you are not feeling well, report it immediately to your supervisor

II. Safety Guidance and Tips

A. Do Not Pipette By Mouth - Ever

You say, "But it's only water." Even if it is, how clean do you think that 4

glassware really is? Using disposable pipettes? I know lots of people who rinse them and put them back! Learn to use the pipette bulb or automated pipetter. Don't pipette by mouth at home either. Gasoline and kerosene should be obvious, but people get hospitalized or die every year, right? I know someone who used his mouth to start the suction on a waterbed to drain it. Do you know what they put in some waterbed additives?

B. Identify the Safety Equipment

And know how to use it! Given that some people (possibly you) will need them, know the locations of the fire blanket, extinguishers, eyewash, and shower. Ask for demonstrations! If the eyewash hasn't been used in a while the discoloration of the water is usually sufficient to inspire use of safety glasses.

C. Don't Taste or Sniff Chemicals

For many chemicals, if you can smell them then you are exposing yourself to a dose that can harm you! If the safety information says that a chemical should only be used inside a fume hood, then don't use it anywhere else. This isn't cooking class - don't taste your experiments! D. Don't Casually Dispose of Chemicals Down the Drain

Some chemicals can be washed down the drain, while others require a different method of disposal. If a chemical can go in the sink, be sure to wash it away rather than risk an unexpected reaction between chemical 'leftovers' later.

E. Don't Eat or Drink in Lab

It's tempting, but oh so dangerous... just don't do it!

F. Don't Play Mad Scientist

Don't haphazardly mix chemicals! Pay attention to the order in which chemicals are to be added to each other and do not deviate from the instructions. Even chemicals that mix to produce seemingly safe products should be handled carefully. For example, hydrochloric acid and sodium hydroxide will give you salt water, but the reaction could break your glassware or splash the reactants onto you if you aren't careful!

G. Take Data During Lab

Not after lab, on the assumption that it will be neater. Put data directly in your lab book rather than transcribing from another source (e.g., notebook or lab partner). There are lots of reasons for this, but the practical one is that it is much harder for the data to get lost in your lab book. For some experiments, it may be helpful to take data before lab.

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No, I'm not telling you to dry-lab or cheat, but being able to project likely data will help you catch bad lab procedure before you are three hours or so into a project. Know what to expect. You should always read the experiment in advance. NOTE:Failure to follow these rules may automatically and without warning result in a deduction of marks. In repeated cases you may be asked to leave the laboratory.

III. Hazard Control Information

On this drawing, please mark the locations of the following equipment. There may be more that one item of each kind. Please mark them all. Walk around the lab to find each item. You may need help from your instructor or a technician. Here is the list of items to find:

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1. All exit doors2. Fume hoods3. Fire extinguishers4. Eye wash stations

5. Fife blankets6. Emergency shower7. First aid kits8. Poster with safety instructions

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The Typical Experiment has three to five separate sections as follows:

• Pre-Laboratory Assignment: Involves calculations closely connected with the laboratory. This must be completed before you come to the lab.

•Introduction: Provides general interest and background information and a summary of the experiment.

• Experimental Procedure: You must read through this before you come to your laboratory session, paying particular attention to safety information.

• Calculations: Some laboratory experiments have an example of typical calculations to help you in carrying out the ones required for your data.

• Report form: You must record your data on the report form as you acquire it. Never write your data on scrap paper with the intention of transferring it later to the report form. Although this may lead to a neater report form, there is a great danger that you will lose the data. (If your data have been lost you cannot perform the necessary calculations. At best, you will have to repeat the laboratory experiment. At worst, you will be given a failing grade for that experiment.)

NAME: ________________________________________BENCH NUMBER: ______________

Instructor: __________________________________

I, the undersigned student, have received the safety training, understood it and agree to abide by the safety guidelines. I understand the importance of eye and body protection. If I therefore fail to abide by the safety rules; I am doing so at my own risk and will not hold University of Nizwa or Dr. Fazal Mabood liable for any injury that result.

Signature: ___________________________Date: _____________________

Student Name:_______________________________ID#:____________Date:_____________

Table of Contents

General Laboratory Instructions

General Laboratory Apparatuses

Laboratory Safety and Guidelines

Exp. # 1Determination of Unknown Concentration of Iron in the Soil Sample by using Atomic Absorption Spectrophotometric method

Exp. # 2Determination of Unknown Concentration of Copper in the Tea Sample by using Atomic Absorption Spectrophotometric method

Exp. # 3Determination of Unknown Concentration of Copper in the given Multivitamin Tablet Sample by using Atomic Absorption Spectrophotometric method

Exp. # 4Determination of Zn in a Multivitamin Drug Sample by Atomic Absorption Spectrophotometry

Exp. # 5 Determination of Manganese (Mn) in the given sample by atomic absorption Spectrophotometry

Exp. # 6 Separation of Benzene, Toluene, & Xylene by Gas Chromatography

Exp. # 7 Separation of Gasoline Sample Mixture by Gas Chromatography

Exp. # 8 Qualitative & Quantitative Analysis of Caffeine in Omani Coffee by using HPLC with UV-Visible Diode Array

Exp. # 9 Qualitative & Quantitative Analysis of Caffeine in Omani Coffee by using LC-MS-Ms

Exp. # 10 Determination of pKIn Value of an Indicator (Bromothymol Blue) by Using UV-VIS Spectrophotometric Method

Exp. # 11 Determination of Acetyl Salicylic Acid in an

Aspirin Tablet by Using UV-VIS Spectrophotometric Method

EXPERIMENT 1 Determination of Unknown Concentration of Iron in

the Soil Sample by using Atomic Absorption Spectrophotometric method

Preparation of Solutions

a) 100ppm Iron Solution of 100ml volume

To prepare 100ppm solution of iron of 100ml volume dissolve 0.07g of Fe(NH4)2(SO4)2.6H2O salt in distilled water and also add 2ml of concentrated H2SO4 and then dilute it upto the mark with distilled water using 100ml volumetric flask.ProcedurePrepare working standard solutions of 1, 3, 5, 7, and 9ppm from 100ppm stock solution by taking 1, 3, 5, 7, and 9ml volume from 100ppm stock solution in a100ml volumetric flasks and then dilute with distilled water using 100ml volumetric flasks.

Blank SolutionBlank solution can be prepared by adding 0.2ml concentrated H2SO4

and then dilute it upto the mark with distilled water using 100ml volumetric flask.

Sample Solution PreparationTo prepare a soil sample solution takes 2g of soil sample in a beaker. Add 5ml of concentrated HCl to it and then stirrer it with glass rod and then filter it with filter paper, then dilute with distilled water using 100ml volumetric flasks. Measure the absorbance of all solutions at using atomic absorption spectrophotometer.

EXPERIMENT 2Determination of Unknown Concentration of Copper

in the Tea sample by using Atomic Absorption Spectrophotometric Method

Preparation of Solutions

b) 100ppm Copper Solution of 100ml volume

To prepare 100ppm solution of copper of 100ml volume dissolve 0.026g of CuCl2.2H2O salt in distilled water and then dilute it upto the mark with distilled water using 100ml volumetric flask.ProcedurePrepare working standard solutions of 1, 3, 5, 7, and 9ppm from 100ppm stock solution by taking 1, 3, 5, 7, and 9ml volume from 100ppm stock solution in a100ml volumetric flasks and then dilute with distilled water using 100ml volumetric flasks.

Blank SolutionBlank solution can be prepared by adding 0.2ml concentrated HCl and then dilute it upto the mark with distilled water using 100ml volumetric flask.

Sample Solution PreparationTo prepare a tea sample solution takes 2g of tea sample in a beaker. Add 5ml of concentrated HCl to it and then stirrer it with glass rod and then filter it with filter paper, then dilute with distilled water using 100ml volumetric flasks. Measure the absorbance of all solutions at using atomic absorption spectrophotometer.

EXPERIMENT 3Determination of Unknown Concentration of Copper

in the given Multivitamin tablet sample by using Atomic Absorption Spectrophotometric Method

Preparation of Solutions

100ppm Copper Solution of 100ml volume

Dissolve 0.01g of copper metal in a minimum volume of 7.9 M HNO3, and then dilute to

100ml with 0.16 M HNO3 using 100ml volumetric flasks.

Procedure

Prepare working standard solutions of 1, 3, 5, 7, and 9ppm from 100ppm stock solution

by taking 1, 3, 5, 7, and 9ml volume from 100ppm stock solution in a100ml volumetric

flasks and then dilute with 0.16 M HNO3 using 100ml volumetric flasks.

Blank Solution

Blank solution can be prepared by taking 0.16 M HNO3 in a 100ml volumetric flask.

Sample Solution Preparation

Thoroughly crush a multivitamin tablet in a mortar and pestle. Add 20 mL 7.9 M HNO3,

mix thoroughly, and allow standing for five minutes.

Quantitatively transfer the solution to a funnel fitted with Whatman #1 filter paper and

filter into a 100-mL volumetric flask. Repeat the extraction two more times with 20 mL

dilute HNO3 each time. Rinse the filter with 0.16 M HNO3 and bring the volume to 100

mL with 0.16 M HNO3.

Measure the absorbance of all solutions using atomic absorption spectrophotometer.

EXPERIMENT 4

Determination of Zn in a Multivitamin Drug Sample by Atomic Absorption Spectrophotometry

Preparation of Solutions

a) 100ppm Zinc Solution of 100ml volume

To prepare 100ppm solution of zinc of 100ml volume dissolve 0.01g of zinc pillets in

concentrated HCl solution and then dilute it with 0.1 M HCl solution upto the mark using

100ml volumetric flask.

Procedure

Prepare working standard solutions of 1, 3, 5, 7, and 9ppm from 100ppm stock solution

by taking 1, 3, 5, 7, and 9ml volume from 100ppm stock solution in a100ml volumetric

flasks and then dilute with 0.1 M HCl solution upto the mark using 100ml volumetric

flask.

b) Blank Solution

Blank solution can be prepared by taking 0.1 M HCl solution in a 100ml volumetric

flask.

c) Sample Solution Preparation

Thoroughly crush any multivitamin tablet in a mortar and pestle. Add 20 mL of 6 M HCl,

mix thoroughly, and allow standing for five minutes. Quantitatively transfer the solution

to a funnel fitted with Whatman #1 filter paper and filter into a 100-mL volumetric flask.

Repeat the extraction two more times with 20 mL dilute HCl each time. Rinse the filter

with 0.1 M HCl and bring the volume to100 mL with 0.1 M HCl.

Measure the absorbance of all solutions at using atomic absorption spectrophotometer.

EXPERIMENT 5

Determination of Manganese (Mn) in the Given Sample by Atomic Absorption Spectrophotometry

EXPERIMENT 6

Separation of Benzene, Toluene, & Xylene by Gas Chromatography

Instrument

Gas chromatograph equipped with FID

Reagents

Benzene, toluene, and xylene,

Nitrogen, 99.998%

Helium, 99.995%

Acetylene gas 99.995%

Procedure

Mix all the three liquids Benzene, toluene, and xylene in a ratio of 1:1:1 in a 10ml

volumetric flask. Inject 0.2µl volume of this mixture with the help of micro syringe into

the column when the instrument gets ready to run. The operating conditions are set as

below

Injection port temperature 200 oC

FID detector temperature 250 oC

Initial temperature 60 oC and final temperature 300 oC

Rate 2 oC /min

EXPERIMENT 7

Separation of Gasoline Sample Mixture by Gas Chromatography

Instrument

Gas chromatograph equipped with FID

Reagents

Gasoline sample

Nitrogen, 99.998%

Helium, 99.995%

Acetylene gas 99.995%

Procedure

Inject 0.2µl volume of gasoline sample with the help of micro syringe into the column

when the instrument gets ready to run. The operating conditions are set as below

Injection port temperature 200 oC

FID detector temperature 250 oC

Initial temperature 60 oC and final temperature 350 oC

Rate 5 oC /min

EXPERIMENT 8

Qualitative & Quantitative Analysis of Caffeine in Omani Coffee by using HPLC with UV-Visible Diode

Array INTRODUCTIONHigh performance liquid chromatography (HPLC) is a widely used method of choice for the separation and quantification of mixtures of soluble compounds in liquids.Separation of the constituents in a mixture is controlled by the chemistry of the mobile phase and stationary-phase materials in the column as well as the detector type used.Many commercial brands of cola drinks and coffee are caffeinated. Caffeine is a polar compound containing UV-active chromophores and hence can be detected with a UV detector attached to the HPLC system. In this experiment you will utilize a HPLC system with a UV detector (measures UV absorbance at 254 nm) as an analytical tool for the separation, identification and quantification of caffeine in soda or brewed Omani coffee.The separation is carried out using a polar mobile phase (or eluent) and a non-polar stationary phase stationary phase. The non-polar stationary phase consist of long chain octadecyl hydrocarbon groups bonded to a silica support , often referred to as C-18 column.EXPERIMENTAL PROCEDUREMobile phase (Eluent)Freshly prepared 80:20 vol mix acetic acid (1M, pH 4.5) and methanolGlasswareThe glassware used should be cleaned, rinsed well with distilled water and finally with small quantities of methanol to remove the water.

Preparation of Caffeine stock and standardsWeigh accurately 100.0 mg of caffeine (to the nearest 0.1 mg) and transfer into a 50 mL volumetric flask. Dissolve in the mobile phase solution, dilute to volume with the mobile phase solution, and mix well. Prepare serial dilution of this stock solution to obtain standards of about 0.50, 0.25, 0.050 and 0.025 mg/ml. Use a 0.22μm HPLC disk filter to remove particulate material from all standards and samples before any injection. Inject duplicate of 15-μL aliquots of each of the serial caffeine standards and record the chromatograms. Record the retention time and the area under each peak in the standards. Make a plot of peak area against concentration of caffeine. Beverages. Analyze either soda or coffee. Obtain caffeinated and caffeine–free soda or coffee product. Place 10.0 mL of beverage in a 50 mL volumetric flask and use the mobile phase to take it to the mark. Mix the content of the flask by shaking and pour into a clean 100 mL beaker; and sonicate for 10 min. Filter the degassed sample through a 0.22μm HPLC disk filter. Inject a 15-μL aliquot into the HPLC system at least three times. AFTER AN INJECTION, DO NOT INJECT THE NEXT UNTIL YOU’RE SURE THE ENTIRE SAMPLE HAS ELUTED. From a comparison of the retention times of the constituents in the beverages with the retention time recorded for the standard caffeine solution, identify the caffeine peak in the chromatograms of the beverages. Record the area of the caffeine peak in the beverages and calculate the concentration of caffeine in your samples.Instrumental parameterColumn = Reversed phase C-18 column (preconditioned by flowing the mobile phase)Mobile phase: Freshly prepared and degassed 80: 20 vol mix 1M acetic acid (pH 4.5) and methanol.

Flow rate: 1.5 mL/minWavelength of Detection: 254 nm.

EXPERIMENT 9

Qualitative & Quantitative Analysis of Caffeine in Omani Coffee

by using LC-MS-Ms

EXPERIMENT 10

Determination of pKIn Value of an Indicator (Bromothymol Blue) by Using UV-VIS

Spectrophotometric Method

Preparation of Solutions

a) Bromothymol Blue

To prepare bromothymol blue solution dissolve 0.1g of bromothymol blue in 20ml of

ethanol and dilute upto the mark with distilled water using 100ml volumetric flask.

b) O.1M Dipotssium hydrogen phosphate (K2HPO4) Solutions of 250ml volume

Dissolve 4.35g of dipotssium hydrogen phosphate salt in distilled water and dilute it

upto the mark with distilled water using 250ml volumetric flask.

c) O.1M Dihydrogen potassium phosphate (K H2PO4) Solutions of 250ml volume

Dissolve 3.4g of dihydrogen potassium phosphate salt in distilled water and dilute it

upto the mark with distilled water using 250ml volumetric flask.

d) 4M Sodium Hydroxide Solution

Dissolve 16g of Sodium Hydroxide in distilled water and dilute it upto the mark with

distilled water using 100ml volumetric flask.

Hydrochloric acid Solution

Use concentrated HCl.

Procedure

Record the absorbance of bromothymol blue solution at low pH and then at high pH

Absorbance at low pH

To measure the absorbance of bromothymol blue solution at low pH take 1ml of

bromothymol blue solution in 100ml volumetric flask, add it a few drops of

concentrated HCl and then dilute it with distilled water. After addition of

concentrated HCl to this solution the color of indicator solution will turn yellow in

acidic medium. Take a few ml of this solution in a covet and then record or scan the

wavelength of this solution in the range from180nm to 600nm in order to find the

optimum wavelength for acidic color (λ1max).

Absorbance at High pH

To measure the absorbance of bromothymol blue solution at high pH take 1ml of

bromothymol blue solution in 100ml volumetric flask, add it a few ml of 6M sodium

hydroxide solution and then dilute it with distilled water. After addition of sodium

hydroxide solution to it the color of indicator solution will turn blue in basic medium.

Take a few ml of this solution in a covet and then record or scan the wavelength of

this solution in the range from180nm to 800nm in order to find the optimum

wavelength for basic color (λ2max).

Measurement of Absorbance of indicator solutions at different pH using λ1max

and λ2max

Take seven different volumetric flask of 100ml volume capacity. To each flask add

the prepared solutions in the ratio as given below in the able.

Vml of Indicator

soultion

1 1 1 1 1 1 1

Vml of (K2HPO4)

Solutions

0 5 10 5 10 5 1

Vml of(K H2PO4)

Solutions

5 10 5 1 1 0 5

pH range of

solution

4.6 5.9 6.4 7.0 8.12 9.0 7.5

Absorbance at

λ1max

Absorbance at

λ1max

Dilute all the solutions upto 100ml volume capacity with distilled water and then

measure the absorbance of above solution both on λ1max and λ2max wavelengths.

EXPERIMENT 11

Determination of Acetyl Salicylic Acid in an Aspirin Tablet by Using UV-VIS Spectrophotometric Method

Introduction

The most common quantitative method in UV-visible spectroscopy is single component

analysis. Here we analyze an analgesics tablet containing acetyl salicylic acid as the only

active ingredient.

Reagents

Acetyl salicylic acid, an analgesic tablet containing acetyl salicylic acid as the only active

ingredient (for example, aspirin)

Preparation of Solutions

a) Prepare a standard solution of acetyl salicylic acid

To prepare standard solution of acetyl salicylic acid dissolve 0.013g of acetyl

salicylic acid in distilled water and dilute upto the mark with distilled water using

100ml volumetric flask. (Make sure that the acetyl salicyclic acid is completely

dissolved. Use a magnetic stirrer if necessary.)

Procedure

Determine the tablet weight.

Break the tablet into small pieces and prepare a sample solution:

tablet material (about 50 mg in 500 ml distilled water)

Stir this solution for at least 30 min.

Use distilled water for the reference measurement in the wavelength range from

200 to 400 nm.

Measure the acetyl salicylic acid standard absorbance spectrum in a wavelength

range from 200 to 400 nm.

Use the filtered sample solution to measure the sample spectrum in the range from

200to 400 nm

Evaluation

1. Determine the wavelength of the absorbance maximum of your standard solution.

2. Get the absorbance value Astd λmax of your standard solution at the absorbance

maximum.

3. Calculate the molar concentration cstd of your acetyl salicylic acid standard

solution. The molecular weight of acetyl salicylic acid MW is 180.15 g / mol.

4. Determine the sample concentration using Beer’s law:

According to Beer’s law we determine the molar extinction coefficient ελ using the

tandard absorbance value Astd max first.

Get the absorbance value Asmp max of your sample solution at the absorbance

maximum.

Based on the molar extinction coefficient ελmax and the measured sample absorbance

Asmpλmax we can calculate the sample concentration.

Determine the total amount of acetyl salicylic acid contained in your tablet.

With the molecular weight of acetyl salicylic acid MW we can calculate the weight