Environmental Chemistry Laboratory: UC Berkeley, …€¦ · Environmental Chemistry Laboratory Manual, Part II 2-3 PLANNING YOUR PROJECT Because a project is less structured than

Environmental Chemistry Laboratory Manual, Part II 2-1

Environmental Chemistry Laboratory: UC Berkeley, 1993 Instructor: Dr. Susan E. Kegley

MODULE 4: INDEPENDENT PROJECTS IN ENVIRONMENTAL CHEMISTRY This module will provide you with an opportunity to find out something you've been wondering about related to environmental issues. The project will be all yours, from its inception as an idea in your mind to planning the implementation of the laboratory work, to analyzing your final results and writing them up to share as a poster presentation with the rest of the class. The project can be both frustrating and rewarding. It will be very different from doing an experiment where all the instructions are written out step by step. With each bit of experimentation, you will be discovering some new information that will take you closer to your goal. There will be times when you may get discouraged because something did not go perfectly the first time you tried it. Don't feel like you've been unsuccessful----this happens to even the best scientists and is part of the process of researching a new area. You learn from what didn't work and make modifications to learn more and perfect your methods. Some of the best projects go on to become new modules. The pesticide module had its first incarnation as a project carried out in the Spring of 1993 by Manel Silva and Julie Gerk. If you have a particularly inventive idea, you too could go down in the history of the UCB Environmental Chemistry class! On the following pages are a list of projects done by the environmental labs from the Fall of 1993.

2-2 Environmental Chemistry Laboratory Manual, Part II

Projects: Environmental Chemistry, Fall 1993 Student(s) Project Title L. Greb and

A. Ravva Uptake of Lead by Radishes Grown in Lead-Contaminated Soil

S. Tabacco, M. Benger,

and R. Cohen Are There Pesticides on Organic Fruits and Vegetables?

A. Russell, J. Freisner,

and S. Kaplan Lead in Shellfish from the Oakland Inner Harbor

J. Siem Air Quality in the Country, City, and Near the Freeway

S. Nelson, G. Robinson,

and A. Balbutin-Burnham

Testing Le Chateau Co-op

I: Lead in Water

II: Coliform in the Kitchen

J. Kim and R. Patel Chromium in Cosmetics

C. Childs, M. Robinson,

and J. Bley Make a Choice: Vitamins or Pesticides

Y. Toda and C. Bolin A Comparison of Bottled Drinking Waters

L. Briggs, S. Cowan, and

H. Huffman

Take This With a Grain of Salt

R. Pollard Does Aluminum Leach from the Container into Acidic Soft Drinks

and Juices?

D. Manning Phytoplankton Growth Limiting Factors in Lake Anza

K. Kallen and A. Mull Estrogenic Pesticides in Fruits and Vegetables

N. Kralevich and J. Lin Pesticides in Vineyard Soils, Water, Grapes, and Raisins

S. Ayers and G. Edgelow Pesticides in Grapes and the Wine Made From the Grapes

T. Aghazadeh and K. Han Nutrient Content of Fertilizers and Nutrient Soils

A. Reddi and M. Kislanka Bottled Drinking Waters

M. Atharasculis The Efficacy of Home Test Kits for Lead

J. Teel and A. Risser Mercury in Fish From the San Francisco Bay


PLANNING YOUR PROJECT Because a project is less structured than a regular experiment, it is easy to get behind if you don't spend a little time planning. Below is a suggested plan for a three week project. Not all projects will fit this model, but many do.

Time Activity 3-4 weeks before project begins • Find a partner and begin to formulate a plan.

• Talk with your instructor about the idea. Ask for some references and background information. Read it.

• Revise your plan if necessary. Decide clearly what you wish to find out by doing your project.

2 weeks before project begins • Turn in the project form, indicating any special

supplies or equipment required for your project. • Continue reading about your project topic.

1 week before project begins • Plan what you will do the first lab day. • Obtain samples if necessary, taking pictures at your

site if appropriate. • Do the calculations to determine how to make up

standards, how to make up spiking solutions, etc. • Talk again with your instructor if you have

questions. • If you have extra time in lab the week before, acid

wash any glassware you anticipate using the next week.

Week 1 of project • Using your best precision technique, make up any

necessary standards. • Work with your instructor to familiarize yourself

with the instrument you will use. • Begin sample processing. • Check your standards for accuracy, this week if

there's time.

Week 2 of project • Check your standards for accuracy on the instrument you will be using.

• Sign up for time to analyze your samples. • Continue sample processing.

Week 3 of project • Check your standards for accuracy on the instrument you will be using if you haven't done it yet.

• Finish sample processing. • Analyze your samples.


Before the poster presentation • Assemble your poster. • Brush up on your background knowledge of issues

related to your project. • Brush up on your knowledge of the instrumental

technique(s) you used and be prepared to answer questions about it.


Researching A Topic Once you have decided on an idea, talk to your instructor about it to see if the project is workable in the time allowed. He or she will be able to provide you with some background reading and a few leading references from which you can find other references that may be relevant to your particular topic. The library resources related to environmental issues are in a variety of locations across the campus: Chemistry (Hildebrand Hall), Engineering (Bechtel), Biosciences (Valley Life Sciences Building), Earth Sciences (McCone Hall), Public Health (Warren Hall), and the Energy and Resources Group (Building T-4, next to Evans). Some projects require talking to people in various public agencies or environmental groups to find out information about, for example, where might be the best place to sample, whom to contact to obtain permission to sample, or what other studies have been done on topics related to your project, to name a few. If you need to call people, be sure to identify yourself as a UCB student, what class you are taking, and the topic of your project. Most people are very willing to help, and you may end up meeting some interesting and influential people. Choosing a Project: Available Laboratory Resources You will work in groups of two for the projects, depending on the topic and type of sample to be analyzed. Each team of students should fill out the Project Form and turn it in. Be realistic when planning your project as regards the time necessary for the analyses. A reasonable number of samples to analyze is 3-4 per student for water or metal analysis and 2-3 for organic analysis. A good group size for water or metal analysis is 2 people, and 2-3 people can work together for organic analysis. Because you have only completed two modules by the time you have to turn in your project topic, it might be tempting to do something similar to what you have already done because it is familiar. That's OK, but be aware that there are many interesting topics out there for which you will know the techniques by the time you get to the end of the semester. Listed below are the possible analyses you can do in the Environmental Laboratory.


1) Laboratory tests requiring only minimal instrumentation pH Alkalinity Conductivity Total and fecal coliform (NOTE: Samples MUST be analyzed for bacteria content within 24 hours of taking the samples and must be refrigerated in the interim period.)

2) Ion chromatography for anions F-, Cl-, NO3

-, PO4-3, SO4

-2, SO3-2, NO2

-, Br- organic anions such as formate, lactate, acetate 3) Atomic absorption spectrophotometry for metals

Metals you can look for by Flame AA

Metals you can look for by Graphite

Furnace AA

Metals you can look for by Hydride Generation AA

Na Pb Hg Ca Cd Se Mg Cu As Ba Ni K Ag Sr Al Fe Cr Cu Zn

4) Gas Chromatography for organochlorine pesticides and PCBs in food, sediments, plants, animals. 5) Supercritical fluid extraction for organic components of environmental samples. 6) Field measurements you can do: Dissolved oxygen (DO), Conductivity/Salinity, pH 7) Special sampling devices available: Grab sampler for deep (up to 6 ft.) water samples, an inflatable raft, sampling dredge for taking underwater sediment samples. An air sampler with cassettes for trapping out particulates and chlorinated organics is available on a limited basis.


Project Form

Names of Students on the Team Give a brief description of what you intend to study: Where will you obtain samples? What substance(s) do you wish to test for? (Be realistic here) Do you need sample bottles? If so, how many? Do you need field equipment, e.g. DO meter, conductivity meter, grab sampler, sampling dredge for sediments in water, air sampler, boat? If so, please list.


What laboratory instruments will you need to use?


CARRYING OUT THE PROJECT: A FEW COMMON-SENSE RULES Notebooks When working on a project, it is most important that you keep a good notebook, writing down all that you do and your logic for doing it. All of this information, any calculations for standards, any field observations, etc., should go in your lab notebook, not on loose scraps of paper. Part of your project grade will be assessed on the completeness of your notebook, since you will be required to hand in the yellow carbon-copy pages with your project report. Labeling of Samples in the Refrigerator Anything that goes into the refrigerator must have the following information on it: Your full name (not just initials), contents of the sample container, and a date. No glass containers are allowed in the main section of the refrigerator, especially volumetric flasks (they tip over too easily!). NOTE: ANY IMPROPERLY LABELED SAMPLES MAY BE THROWN AWAY AT ANY TIME. Checking Out Extra Equipment During the course of your project, you may need to use equipment that is not in your locker. Please take responsibility for returning all borrowed equipment promptly and in good condition. If you break something, just let us know. It happens to everyone, but we will need to replace the item, so don't just sweep it under the rug! Before you check out, be sure all sample bottles are removed from the refrigerator, cleaned, and returned to the stockroom. Using the Instruments When you sign up for time on the instruments, be sure to begin preparing for your session on the instrument at least 30 minutes before you are scheduled to begin. Samples and standards must be prepared well ahead of time (it takes longer than you think!) and removed from the refrigerator so that they are at room temperature when measured. Samples should be mixed with matrix modifier if necessary (Na, Ca, Mg, K) and any extra solutions must be prepared (Hg, Se, As). Because many people need to use the instruments, your time with the instrument will be limited, so proper planning is very important. If you sign up for a time-slot to measure a particular analyte, be sure that someone from your group is there to run it or you will be "bumped" so that others who are prepared will not be thrown off schedule! Talk to your instructor if you need help planning your instrument runs.


Working as Partners Ideally, the project should be a joint endeavor between two or three interested and motivated people, all of whom are working together to gather data and find relevant information. In reality, the level of commitment of each partner to the project can vary. Keep in mind that anyone who works hard and plans their project well, will get a good grade. The person who works less by not showing up for all group consultations and activities or by not fully participating in the planning and implementation of the project will be graded accordingly. All members of a group do not typically get the same grade. To get a good grade on this module, it is necessary to be a full participant in the whole process of the project. The Poster Presentation Scientists from many disciplines present their data in poster format at professional meetings. This type of presentation of your project results will introduce you to this way of sharing results with your colleagues. Each team of students will produce one poster. Please use at most two pieces of poster board, available from local art stores. Important: You must make copies of all information on the poster on 8.5" x 11" paper and hand this in as your report. This will allow future students to use your data. A good poster should make it easy for people to understand what you did, why you did it, and the significance of your results. Please include the following on your poster: • the names of the students and the title of the project • an introduction with background information on why you are studying the

topic you chose • pictures of your site or other relevant pictures • a map with sample sites marked, if appropriate • an outline of all of the procedures used • a description of how any instrument used in the project works • data, in the form of tables, graphs, etc. Be sure to label all axes and

use units. • interpretation of data (compare to other known values, list the standards

that you used, state the detection limits of the instrument, etc.) Are your data significant or in the noise of the instrument?

• conclusions---overall, what did you learn? Remember: People looking at your poster will not be familiar with your abbreviations. Have a friend who is not in the class take a look at your poster to help you be sure that all labeling schemes make sense.


At the poster session: Be ready to answer questions about the project, the techniques used, and the calculations you did. Have one person stand with the poster while other walks around to look at others' posters, then switch. The Report • Prepare one report per group. • Include all the information you have on the poster, printed on 8.5” x 11”

paper. • The report must be handed in AT THE POSTER SESSION.


SAMPLING Some (but not all) projects may require you to collect samples. This could be water, clams, fish, sediments, soils, plants, or air. Deciding Where to Sample Some factors to consider include, but are not limited to, the following: • The topography of the area. A map of the area is always useful. • What do you want to know about the site? A representative analysis?

The extent of the pollution? The maximum concentrations present? • What are the potential sources of the contaminant(s)? Knowing the

history of the site is useful information to have. Do your homework! • What are the potential exposure routes for humans, animals, plants, etc.

in the sampling area? Eating, drinking, breathing, skin exposure? • Might the pollutant have migrated due to natural forces? • What are the effects of normal weather patterns, e.g. currents, prevailing

winds, stream flows, etc.? • What are the effects of recent weather, e.g. rain, snow, high stream flows,

seasonal temperature changes, etc.? • Do you want multiple samples from the same site? • Do you want composite samples or individual samples? • How many samples should you take? Sample Blanks In order to be sure your samples are truly representative of the site, and that contamination is not causing artificially high or low readings, it is normal to include some blank samples in the analysis. Blank samples help to determine if there are extraneous sources of pollutants being tested for. For example, if the sample bottles are not completely clean, some contamination might be introduced into your sample. If any of the reagents or glassware being used in the procedure contain trace amounts of the pollutant you are testing for, you need to know this and correct for it. There are several different types of blanks, described in detail below. Field BlankA field blank is taken when there might be the possibility of air contamination (including rain or fog) of the sample during the sampling process. For water sampling, the field blank is a bottle of pure de-ionized water that is taken to the sample site and left open and exposed to the air for the same amount of time that the real samples are exposed to the air. The bottle is then taken back to the lab and analyzed in the same manner as the real samples. Trip Blank


The trip blank is used to measure possible contamination from the container or any preserving reagents added to the samples during sample handling and storage. For water sampling, the trip blank is a bottle of pure de-ionized water that is taken to the sample site but never opened, returned to the lab, and analyzed with the other samples. The trip blank would enable you to detect possible leaching of contaminants from the sample container. Equipment Blank The equipment blank tests for possible contamination from sampling equipment and is taken by rinsing the equipment after it has been cleaned and analyzing the rinsate for possible contamination. Background SamplesWhen you are taking samples in an area suspected to be contaminated by human activities, it is important to know what the natural background concentration of the pollutant is in that area. For example, there is concern over mercury pollution in the South San Francisco Bay. Certain industries are suspect as the polluters, but it is also true that mercury occurs naturally in the south bay hills and was mined there in the early part of the 20th century. Determination of background mercury levels in this case is important to assess the amount of damage caused by industry. A background sample is taken near the site of interest to determine if the site is truly contaminated by human activities or is just normal for the area. Taking the Sample: A Few Tips • Label the sample bottle BEFORE putting the sample in. Use colored tape

and be sure to include your name, a sample ID number, the location, and the identity of the sample. Use only indelible pens to label your containers.

• Remember that fingers are dirty. Don't touch the inside of the sample container.

• Be sure you are using the proper sampling utensils, as discussed at the beginning of this section.

• If you are taking a water sample, position the bottle so the mouth of the bottle is pointing upstream and your fingers (holding the bottle) are downstream of the bottle opening. Fill the bottle to the top.

• If you are taking a water sample, first rinse the sample bottle with a bit of the sample. This is a good habit to get into for both sampling and carrying out the analytical procedures.

• If you are taking a soil or sediment sample, avoid possible contamination of the samples by cleaning the sampling equipment well in between samples.

Preserving and Storing the Sample


You may not always be able to analyze a sample as soon as you arrive back in the laboratory. It is important to store the sample correctly, so no changes occur in the sample. The presence of oxygen (if the sample bottle is not completely full) and bacteria in a sample are the biggest culprits for changing the concentrations of different analytes in water. Soils and sediments are more forgiving. Storage procedures are given below for different types of samples.


Water Samples • Storage before filtration should be at 4°C for no more than 2 days. • Storage after filtration should be at 4°C for no more than 30 days. • If analysis for trace metals is being carried out, the sample should be

acidified to pH 2 or lower. At neutral pH, metal ions will adsorb onto the surface of the container or precipitate out as hydroxide or carbonate salts.

Sediment or Soil Samples • Samples should be stored at 4°C for no more than 30 days. Tissue Samples • Refrigerate if analysis will take place within a week. • Freeze if samples need to be stored for longer periods of time, up to 90

days. Labware, Sampling Utensils, and the Sample Container It is possible to introduce contamination into your sample from the sampling utensils, sample container, or the lab glassware used to process the sample. All efforts should be made to eliminate these types of interferences. The optimum containers for samples will vary, depending on what contaminants you will be analyzing for. The cleaning procedures for different types of analyses are listed on pp. 14-16 of the lab manual for the first part of the course. Water samples, testing for general water quality parameters: Clean plastic bottles. Water samples, testing for trace metals: Clean glass bottles with Teflon-lined caps or clean plastic bottles. Water samples, testing for trace organics: Clean glass bottles with Teflon-lined caps. Water samples, testing for bacterial contaminants: Sterile plastic bottles. NOTE: The acid cleaning procedure will serve to sterilize your containers. To avoid contamination from airborne bacteria, dry the container and its cap upside down and cap it while it is still upside down. Snow and ice samples, testing for general water quality parameters or trace metals: Un-opened, unused-used Ziplock™ bags. Snow and ice samples, testing for trace organics: Clean glass bottles with Teflon-lined caps.


Sediment samples, testing for trace metals: Clean glass bottles with Teflon-lined caps. Dry or damp soil samples, testing for trace metals: Either Zip-lock™ bags or clean glass bottles with teflon-lined caps. Sediment samples, testing for trace organics: Clean glass bottles with teflon-lined caps. PREPARATION OF STANDARDS Standards must be prepared for the components you are measuring. For Ca, Mg, Na, and K, standards are prepared using de-ionized water as the diluent. For all other metals, 0.2% HNO3 should be used to dilute the concentrated stock standard to the desired concentration. If de-ionized water is used by accident, the high pH of the water will cause the metals to precipitate out of solution and the standard will be useless. Remember the handy formula:

V1C1 = V2C2

where V1 is the volume of the concentrated standard of concentration C1 that must be measured out to obtain the desired volume V2 of the diluted standard that has concentration C2. The quantity C2 is simply the target concentration. The volume V2 is the volume of the volumetric flask in which you will make up the standard. Important conversion factors are: 103 µg/mg, 103 mL/L, 103 g/kg. Remember that in dilute aqueous solution, ppm = mg/L and ppb = µg/L. In soil or tissue, ppm = mg/kg = µg/g and ppb = µg/kg = ng/g. Calculate the volume of concentrated standard necessary to prepare 100 mL of each standard. There are pipettes available that measure from 0.1 to 1.0 mL in 0.1 mL increments, so amounts as small as 0.1 mL can be very accurately measured. Have your instructor check your calculations, then prepare the standard, using clean pipette tips and acid-washed volumetric flasks for all liquid transfers. Be as precise as possible and remember: There never seems to be enough time to do a task exactly as you should, but on the other hand, there's always time to do it over again! Save yourself from having to re-do it by doing it very carefully the first time!


VERY IMPORTANT: It is critical that you label your standards correctly. The necessary information to have on the label of the bottle is: your full name, date, identity of standard, concentration of standard, and solvent used (water, 0.2 % HNO3, organic solvent, etc.)


Concentrations of Standards for Flame AA

Metal Concentration of Stock Solution

(mg/L)

Standards to Prepare (mg/L)

Other Necessary Solutions

Wavelength of Analysis (nm)

Na 1,000 0.25,0.5,1.0 CsCl 6.32 g/L 589.0 Ca 1,000 1.0,2.5,5.0 KCl 9.52 g/L 422.7 Mg 1,000 1.0,5.0,10.0 LaCl3 8.86 g/L 285.2 K 1,000 0.5,1.0,2.0 CsCl 6.32 g/L 766.5 Fe 1,000 1.0,2.5,5.0 none 248.3 Cu 1,000 1.0,2.5,5.0 none 324.8 Zn 1,000 1.0,2.5,5.0 213.9

Concentrations of Standards for Graphite Furnace AA

Metal Concentration of

Stock Solution (mg/L)

Standards to Prepare

(µg/L in 0.2% HNO3)



Pb 1,000 10, 50 0.2 mg (NH4)2HPO4

283.3

Cd 1,000 1, 2 0.2 mg (NH4)2HPO4

228.8

Cr 1,000 2, 5 0.05 mg Mg(NO3)2 357.9 Cu 1,000 1, 2 none 324.8 Ni 1,000 1, 2 none 232.0 Al 1,000 20, 100 none 309.3

*NOTE: If you are analyzing water samples with concentrations of metal ions expected to be low, graphite furnace AA is the best technique.

Concentrations of Standards For Hydride Generation AA

Metal Concentration of Stock Solution

(mg/L)

Standards to Prepare

(µg/L in 0.2% HNO3)



Hg 1,000 mg/L 1,000 µg/L • 1% NaOH • 3% NaBH4

made up in 1% NaOH

253.7


As 1,000 mg/L 1,000 µg/L • 1% NaOH • 3% NaBH4

made up in 1% NaOH

193.7

Se 1,000 mg/L 1,000 µg/L • 1% NaOH • 3% NaBH4

made up in 1% NaOH

196.0


Preparing Organic Standards: When you are preparing organic standards, keep the following points in mind: • Volumetric flasks should be acid washed, then rinsed with acetone several

times, then rinsed with hexane several times, then rinsed again with the solvent used as the diluent. Be sure to dispose of all waste in the appropriate container.

• Be sure to always wear gloves when weighing out or handling concentrated standards.

• You will be using small volumetric flasks when diluting organic standards. There are only a limited number of these flasks available, so be sure to clean them immediately after you use them. Do not leave them in the oven to dry, as they will warp and will no longer measure volumes accurately.

• Do not leave volumetric flasks containing organic solvents open on the countertop, as the liquid will quickly evaporate and the volume will no longer be accurate.

• Place all diluted standards in properly labeled (full name, date, identity of sample, concentration of sample, and solvent used) teflon-capped vials and store them in the freezer until you are ready to use them.

See your instructor for help with preparing organic standards. If other groups will be using the same set of standards, it may be useful to get together and make enough of each for everyone, since only one set of standards is required for each GC run.


SOME SPECIAL TIPS THAT MIGHT BE USEFUL The remainder of this section of the manual describes special tips which are useful for various types of analyses. Since there are many groups performing many different kinds of projects, it is impossible to include specific details of every task in a reasonable sized manual! Your instructor will be happy to guide you in the right direction if you have a need which is not addressed here. The folks working on the Wild Dolphin Project need to read Section 3 of the manual as well for general details about the samples which they will be studying. Even if you are not involved in the Wild Dolphin Project analyses, you may find the information in Section 3 useful, especially if you need to dissect a fish! Everyone should refer to Appendix A: Polychlorinated Biphenyls In Bay Sediments, Water & Fish, which contains a lot of general lab technique as well as a good overview of a particular problem, including background information. Coliform Tests It is important to carry out coliform tests as soon as possible after sampling. If the test cannot be performed right away, store the samples in a refrigerator at 4°C. Tests performed after 3 days are not reliable. If you suspect high coliform counts, filter less sample (diluted to 100 mL before filtering) for the test. The table below provides guidelines for sample volumes for the total coliform test for a variety of water sources. For the fecal coliform test, use ten times the suggested volume in the table.

Suggested Sample Volumes for Membrane Filter Total Coliform Test

Volume (X) To Be Filtered

mL Water Source 100 50 10 1 0.1 0.01 0.001 Drinking water X Swimming pools X Wells, spring X X X Lakes, reservoirs X X X Water supply intake X X X Bathing beaches X X X River water X X X X Chlorinated sewage X X X Raw sewage X X X Source: Standard Methods for the Examination of Water and Wastewater, 17th edition, American Public Health Association (Washington, DC, 1989),


p. 9-91.


Analyzing Tissue Samples (Animal or Plant Material) for Heavy Metals Digest tissues like soil in 30% HNO3, but weigh out more sample (~5-7 g, instead of 1g). Because the water content of tissues is so high, you will need to process more sample for the same level of sensitivity. If all tissue doesn't dissolve by the end of the 30 minute digestion period, add another 10 mL of acid and heat for another 15 minutes. Concentrations may be low; you may not need to dilute the sample. Using a Spiking Solution to Determine the Effectiveness of a Procedure In order be sure you are not losing the analyte which you will be measuring, spiked samples are also prepared. To spike a solution, prepare a sample which is exactly like another, but before any preparation in laboratory such as digestion, etc., add a known amount of the analyte you are looking for, in the concentration range you expect for the samples or slightly higher. This sample will be analyzed along with its unspiked control, and you should see the full amount of analyte you added in addition to that which was originally in the sample. If you don’t, did you lose some in the preparation process? Is there some interference in the sample which is altering the detection of your analyte? Analyzing for Hg, Se, or As Using Hydride Generation: You may already be familiar with mercury analysis using the Cold Vapor Method on the AA. The procedure is almost identical for selenium and arsenic, except that the quartz cell is heated. When sodium borohydride reacts with Se or As in solution, the gaseous reaction products are not free atoms, as is the case with mercury, but are volatile hydrides which travel up the tube to the cell. The hot quartz cell dissociates the hydride gas into free atoms which can then be measured by the instrument. Caution: Since the formation of the hydride molecule releases hydrogen, which is also carried to the heated cell, you must be sure to pause at least ten seconds after attaching the reaction vessel to the instrument before pressing the plunger to be absolutely sure that ALL air has been purged from the system. This is due to the following series of simplified reactions:

As3+ + 2BH4- ---> AsH3 (gas) + H2 (gas) + 3/2 B2H6

AsH3 + Heat ---> As


H2 + O2 + Heat ---> BO2M! Of course, this is not a problem with mercury analysis since the cell isn't heated. In fact, if you wait more than a couple of seconds before pressing the plunger for mercury analysis you run the risk of purging your sample of mercury as well as air!


Instructions for Using the Gas Chromatograph *****NEED TO ADD THIS***** Increasing the Sensitivity of an Analysis: If you suspect low concentrations, you can increase the sensitivity of the analysis. There are several ways in which you can do this: (1) take a larger sample size, (2) reduce to a smaller final volume of extract; that is, concentrate the sample, or (3) analyze a greater portion of the sample. See your instructor for help in determining which method is the best for your application.

Analysis of Water Samples for Lead Content If you wish to determine lead in drinking water, there are several procedures that you MUST follow if your data is to be useful. First-draw samples are those that are taken from a tap that has not been used in at least 12 hours. The lead in these samples is a good measure of whether or not lead is leaching out of the pipes within the house. If you let the water run for two minutes, and then take a sample, you will obtain a measure of whether or not the pipes that deliver the water contain lead that is leaching out into the water. All samples must be preserved as soon as possible after taking them by adjusting the pH to <1.5. This is done by addition of a few drops of Optima Grade or Trace Metal Grade concentrated HNO3. The purpose of acidifying the solution is to prevent the lead ions from sticking to the sides of the container. Up to 95% of the lead ions will be unavailable for analysis if you skip this step!!! NOTE: The concentration of lead in drinking water is usually quite low (<50 µg/L). With concentrations this low, graphite furnace AA is the ONLY technique that is sensitive enough to reliably measure concentrations. Preparation of Water Sample for Lead Analysis In order to analyze your sample for lead content, you must first digest the sample by heating it in the presence of a strong acid. Digestion serves to solubilize any lead that might be present in forms that are not normally water soluble, including PbO, PbSO4, Pb(OH)2, PbS, and PbCO3. The sample is then filtered and diluted to a specified volume of 100 mL. 1) All glassware used in this procedure must be acid washed and rinsed with de-ionized water before use. You will need:

100 mL graduated cylinder 400 mL beaker watch glass


100 mL volumetric flask short stem funnel plastic sample bottle

2) Shake your sample bottle well and measure out 100 mL of sample into a graduated cylinder. Pour the water into a 400 mL beaker. 3) Add 5 mL of concentrated (100%) HNO3 (nitric acid) to the sample and cover the beaker with a watch glass to keep particles from the air out of your sample. CAUTION!! Nitric acid is very corrosive. Be sure you have your safety glasses on, Wear gloves, and be careful. If you spill acid on yourself, wash it off with lots of water. If you spill acid on the bench top, clean it up. 4) Place the beaker on one of the hot plates in the hood and heat the sample so that it boils gently for 30-45 minutes or until the volume is reduced to 60-75 mL. Vigorous boiling should be avoided. At the end of the heating period, remove the sample from the hot plate and allow it to cool. 5) Filter the sample directly into a (CLEAN) 100 mL volumetric flask. To prepare the filter, fold a piece of Whatman 41 filter paper into quarters and place it in a short stem funnel. Clamp the volumetric flask and place the funnel with filter paper in the top. BEFORE you pour any sample through the filter, rinse it with ~5 mL of 5 % HNO3 and discard the rinsings. This avoids contamination of your sample with any lead that may be in the filter. 6) Pour the sample into the funnel. Rinse the watchglass with a few mL of 5 % HNO3 and pour the rinsate into the funnel. Rinse the beaker twice with 5 mL of 5 % HNO3 by adding the acid and swirling the liquid around in the beaker. Finally, rinse the funnel and its contents with two 5 mL portions of deionized water. 7) Remove the funnel and dilute the sample to 100 mL with deionized water. Pour the sample into one of your (CLEAN) plastic sample bottles and label it carefully. Your sample is now ready for analysis by graphite furnace AAS. 8) Discard the used filter paper into the trash can. 9) Analyze your sample by graphite furnace AA.



Supercritical Fluid Extraction Techniques This part of the manual contains a summary of the operational procedures for the Hewlett Packard Supercritical Fluid Extraction System. For more details, refer to the grey Hewlett Packard binder that contains the manuals on the shelf above the SFE instrument or consult with your instructor. For a description of the technique, refer to the lab manual from the first half of the course, pp. 170-171. For an example of preparing a SFE sample, see pp. 152-154. The following topics are discussed: • Creating an extraction method • Preparing a Sequence • Running an Extraction • Printing Reports Creating an extraction method: In essence, a method is a set of extraction steps outlining how extraction and rinsing is to take place. In turn, the extraction steps themselves, contain setpoints which define the conditions of each step. 1) At the main screen, select "Method" in the menu bar and then "New Method". 2) In the box, type the name of the method and click OK to save the new method. If a method of the same name exists, a warning message will appear asking "METHOD has been changed; save the changes?" Note: the program automatically appends the extension "EXT" to the method name. 3) Next, the extraction fluids must be selected. Click on "Extraction Fluids" representation (the picture of the gas cylinder) at the main screen. The default Extraction fluid is always CO2 and if your method requires subsequent extraction fluids, select the relevant ones and enter their parameters. Click OK when done. 4) Click on "Rinse Solvents" representation at the main screen to choose the final solvent(s) in which the analyte will be dissolved. These are the solvents (such as hexane, acetone, toluene, etc.) that will rinse the analyte off the trap and into the GC vials after the supercritical carbon dioxide extracts them from the matrix and into the trap. Click the scroll bar next to the Rinse Solvent 1 to access a drop-down list of solvent selections. The melting and


boiling points will be entered automatically. Up to 2 different rinse solvents can be selected at a time. Click OK when done. 5) Next, the setpoints of an extraction step of the method need to be entered. The setpoints of each step can be defined in the subareas: -Extraction Conditions -Analyte Trap -Fraction Output These can be accessed by clicking the respective areas in the picture of the instrument at the main screen. 6) Click on the "Extraction Conditions" picture on the main screen. In the box of "Extraction Conditions", the values for the following extraction setpoints can be entered: -Extraction Fluid -Flow Rate -Carbon Dioxide Density -Chamber temperature -Equilibrium time -Extraction time 7) Click on the "Analyte Trap" picture on the main screen. In the "Analyte Trap" box, the values for the following trap setpoints can be entered: -Nozzle temp. -Trap temp. -Trap packing -Compensation 8) Finally, click on the "Fraction output" picture on the main screen. In the "Fraction Output" box, the values for the following final rinse and collection setpoints can be entered: -Rinse Solvent -Volume -Rate -Nozzle temp. -Trap temp. -Vial Number *Note: When entering values and editing this substep, click "Modify Substep" to place the correct values in the table. They will be lost if only OK is clicked. To add another substep (such as another vial), enter the values above and click on "Add Substep" to add a new line to the table. When done, click "OK" to return to the main screen.


9) To get a summary of the setpoints entered, click on "Extract" and select "Step Setpoints" in the menu bar. The screen will show all the values for the setpoints. To edit the setpoints in this box, click on the desired box. Note: clicking on the desired box will go to the same box as if you were to click on the picture representation of the same setpoint on the main screen. When done, click "OK" to return to the main screen. 10) If the method requires more than one extraction step, click "Extract" and then "Steps" at the menu bar. Click "Add Step" to create a copy of step one. The message will appear "Vial capacity exceeded; free vials used." Disregard this (because you will edit the fraction output substep). Repeat if more steps are needed. Click OK and proceed to modify the setpoints for the additonal steps by the procedure above. 11) To save the method, select "Method" and "Save Method" in the menu bar. Preparing a Sequence A sequence is a list of thimble positions and the preparation methods to be used with each one. 1) In the menu bar, select "Sequence" and then "New Sequence". Enter the name of the sequence and when done click "Create". 2) Then click on "Sequence" again, and choose "Build Sequence". Then click on the down arrow in the method selection box to select the method for the first carousel position number 1. To add the same method to the next thimble position, click on "Add Copy". To add a new method, go to the method selection and select the method as you did before. Up to eight methods may be chosen (as there are only eight thimble positions.) To delete the method from the last position, click "Delete Thimble". Note: You can only delete from the bottom up of the sequence. When done, click OK. 3) Logging in the sample: In the main screen, click on the "Carousel Login" notebook picture. This is where the thimbles are identified, any comments are typed in, and the name of the operator. Enter the relevant info and click OK when done. 4) Go to "Sequence" on the menu bar, and choose "Save Sequence" to save your work.


Running the Extraction Note: Have your instructor help with this part. 1) Make sure the instrument is turned on. Turn on the two carbon dioxide gas cylinders to the left of the machine. 2) The system now needs to be primed to remove any air out of the solvent lines. Remove the prime port plug on top of the extractor behind the output tray. Find the priming syringe that is to the left of the port, and makes sure the plunger is fully depressed. Screw the 10mL priming syringe into the port. 3) To prime the rinse solvents, return to the computer and select "Run" in the menu bar, and then "Maintenance and Test." The machine is now in the test mode. Select the "Prime Rinse Solvent to Waste" test on the computer. Select the solvent to be primed (either 1 or 2). Click on "Send" and you will hear the pump running and making noises. 4) With the pump running, return to the machine and slowly pull the syringe plunger out until the solvent line is filled with solvent and no bubbles are visible. Note: do not pull too fast as this could cause cavitation (boiling) of the solvent in the pump. When the syringe is full of solvent, remove it from the port and deposit the solvent into the flask labeled "Waste" (it is to the right of the instrument). Draw up more solvent from the port with the syringe if there are still air bubbles present. If bubbling persists, there may be a leak somewhere. Tell the instructor about the situation. When finished priming, return to the computer and click "Stop" to stop the pump. Remove the syringe and replace the prime port plug. *Note: if your method extraction requires more than one solvent, all solvents must be primed by selecting the appropriate solvent line in the above Maintence and Test window and repeating the above procedure. 5) Go to "Sequence" in the menu bar, and select "Load Sequence". Select the appropiate one to run. Click OK when done. 6) Load the thimble samples in the correct carousel positions as listed in the "Carousel Login." It is CRITICAL that the thimbles have NO TAPE whatsoever on them as tape will cause the thimble to get stuck in the instrument. Label the thimbles using a permanent marker on the metal part of the thimble. Finally, make sure the thimbles are properly loaded in the slots. 7) Place pre-weighed, empty, capped and crimped vials in the appropriate positions of the output vial tray.


8) Click on the "Keyboard" representation on the main screen. Then click "Start" to run the extraction. 9) To view the online status of the instrument, select "Run" at the menu bar, and click on "Instrument Status." When the status is "Idle", the extraction is complete. Printing At the end of each thimble extraction, a sample fraction report is printed. Other listings can be printed on request, going to the Print menu on the main screen and selecting the various options. They are: • Method Listing: A full description of a sample preparation method • Sequence Listing: A full description of a sequence • Logfile Listing: A list of activities (commands or events) performed during

either the current or the previous SFE session. The file ARCHIVE.LOG contains the logfile information from earlier runs.


Determining Anions in Salt Water by Ion Chromatography (IC) This part of the manual gives the procedure you would use if you determine that it is necessary to filter out chloride ions from salt water which would normally block out all of your data peaks of interest due to its high concentration in seawater samples. For details on the theory of IC, refer to the manual from the first half of the semester, pp. 18-22. The presence of specific anions in solution can be identified using an ion chromatograph (IC), which serves to separate and quantify different anions. Use the filtered sample for this analysis. Follow the detailed instructions below to use the IC to obtain concentrations of the above anions in mg/L or ppm. If necessary, dilute sample using pipets and a volumetric flask. Be sure to note the dilution factor in your notebook. NOTE: The dilution factor can be calculated by dividing the total volume of the diluted sample by the volume of concentrated sample that was used to make up the dilute sample, e.g. if you diluted 5 mL of sample to 100 mL total volume, the dilution factor is 100 ÷ 5 = 20. Loading The IC 1) Rinse the IC syringe three times with deionized water, collected from a clean beaker. Use about 5 mL for each rinse. The rinsings can be squirted into the sink. Remember not to dip your fingers into the deionized water or your sample! 2) Pull up 3-4 mL of deionized water into syringe. 3) Air is bad for the IC, so you should remove any bubbles from the syringe by holding the syringe tip up while tapping the side of the syringe with your finger. When the bubbles have floated to the top, push them out of the tip of the syringe with the plunger. 4) While holding the syringe tip up, screw the Dionex Onguard Filter to the top. Be careful not to touch the tip of the filter! 5) Keeping the syringe tip pointed up, push 1-2 mL of de-ionized water through the syringe. This is just to wet the filter, but try not to leave any air bubbles inside the filter. 6) Remove the filter and set it aside on top of a clean KimwipeTM. 7) Empty any remaining deionized water in the syringe into the sink.


8) Rinse the syringe with 5 mL of the sample you intend to run. Empty the rinsate into the sink. Remember not to dip your fingers into the sample! 9) Pull up 4-5 mL of sample into syringe. While holding the syringe tip up, push out any bubbles by tapping the side of the syringe. 10) Keeping the syringe tip up, reattach the filter making sure it is secure. Remember to keep the syringe tip up, in order to keep air from re-entering the filter. 11) Push 1-2 mL of sample through the filter. Making sure no air bubbles remain. Do not worry about little air bubbles near the plunger; just remember not to push the plunger in all the way in when you inject the sample. 12) If there is any sample on the outside of the syringe tip or on the injection port, remove it with a KimwipeTM. 13) With the filter attached, screw the syringe into the injection port. Push 3 mL of sample into the IC. This will take a surprising amount of force, so inject slowly but firmly. If the plunger will not move at all, ask for assistance. Do not force it. The sample loop contains 50 mL (that’s 0.050 mL), so 3 mL of sample will rinse this loop out well. 14) Dispose of the used filter in the beaker labeled “Used Filters.” Running the IC with the Computer 1) Go to the computer. If the Run program is open and the method has been selected go to the third item below. If you are not sure, start at the first item. • Double click on the Run icon. • Pull down Load, and select Method…. When the window pops up, double click on manan5.met. • Pull down Run, and select Start…. When the window pops up, input a filename and the dilution factor if the sample was diluted. Leave the rest as it is. • Clicking OK will inject the sample onto the column and start the run. 2) The computer screen will show a plot of the conductivity versus time as the run proceeds. The run takes 7 minutes. Printing Out a Run


It is possible to print out a previous run while a new sample is running on the IC. To print out a chromatogram, close the windows by clicking on the square in the upper-left corner and pulling down to Close. It is OK to close all windows. Do the following: • Double click on the Optimize icon. • Pull down File , and select Open Data File…. When the window pops up, click the box marked “Include sample information” and double click on the file you want. • Pull down File and select Print…. When the window pops up, click the boxes marked Report and Chromatogram so that they have x's in them. Click the OK button.

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