The Perils of Drinking Water

A Teaching Module on Analytical Chemistry Techniques for Determining Drugs in our Drinking Water


Fredrick G. Hurtado1

Nicholas H. Snow, Ph. D.2

 1 Science Department, Union City High School South, Union City, NJ                      

2 Department of Chemistry and Biochemistry, Seton Hall University, South Orange, NJ


Summer Research Program for Science Teachers

August 2008



Module Topic: This teaching module was created to introduce high school students to current analytical chemistry techniques employed in the research laboratory as well as raise awareness of the current problem with pharmaceutical-tainted waters.  The analytical techniques utilized were slightly modified for the high school classroom without compromising the concepts.  These techniques highlighted in the module can be used to introduce several topics that include: Solution concentrations, solution stoichiometry, dilutions, Beers Law, equilibrium, drug delivery, absorption, adsorption and environmental concerns.


Appropriate Subject Area(s):  Advanced Placement Chemistry, Honors Chemistry and College Preparatory Chemistry.


Rationale:   Chemistry practiced in the research laboratory and the manner in which it is taught to the adolescent student is far too incongruent.  This is a serious problem for a society that is dependent upon knowledge that is current and complete.  Moreover, the problem becomes very serious when the secondary education preparation and the college expectations do not align.  Therefore, a structural change, a shift in the approach chemistry is transmitted and integrated within the k-12 curricula must take place.  An inquiry-based laboratory exercise such as the one proposed here can serve as an initial step toward such a change.  It is extremely important to expose students to this kind of rich curriculum; that allows them to be in control of their own learning.  Students must be prepared to be independent thinkers, to make informed decisions concerning human activity and the consequence that it has on the environment, and understand the interrelationship between science and technology. In a research curriculum students learn by discovering—being captivated and become engaged in doing science.


Learning Objectives: 

            Students will be able to:

1.      Use appropriate laboratory safety procedures.

2.      Utilize proper laboratory equipment

3.      Construct laboratory equipment

4.      Compose a polarity chart for chosen analgesics

5.      Create a calibration curve for a specific analgesic

6.      Calculate values from experimental measurements

7.      Interpret data from graph


Background Information:  Pharmaceuticals and personal care products, known in the water industry as PPCPs, have been detected in trace amounts in surface water, drinking water and wastewater effluent sampling conducted in both Europe and the U.S.   Environmental Health Perspectives informs in 2005, on the over 100 pharmaceuticals that have been found in rivers, lakes and coastal waters throughout Europe and the U.S.  (Hemminger, 2005).  Further research suggests that there may be some ecological harm when certain drugs are present. Chemistry and Ecology reported in 1994 on the feminization of male fish as the result of exposure to sewage effluent now known to contain ethinyl estradiol, the active ingredient in birth control pills (Hemminger, 2005).   To date, however, no evidence has been found of human health effects from pharmaceutical and personal care products in the environment; this includes drinking water sources.  The issue, however, becomes grave when one reasons the effects that the long-term exposure to these contaminants can have on people’s health.  To date, no studies on the long-term exposure-effects that trace pharmaceuticals can have on human subjects have been conducted.  It would require 3, 4 or 5 decades of data to better analyze the effects.  We just have not had the information for that long a time.


There is no debate, however, that throughout several major U.S. cities these chemicals are present in our drinking water sources.  The media first began aggressively reporting on detection of pharmaceuticals in our drinking water sources as early as March 2008, in spite of the problem having been recognized more than a decade earlier. As late as July 2008, newspapers were still reporting on antibiotics, anticonvulsants, mood stabilizers, analgesics and sex hormones that had made their way to our drinking water sources.


When pharmaceutical products are consumed, 80% or more of the drug can pass through the body unaltered (Halling-Sorensen, 1998).  As the drug is biotransformed, the metabolites can be more bioactive than the drug itself.  Wastewater-treatment plants are ill equipped to eliminate these contaminants.  As the water is recycled, it makes its way back for human consumption.  In a 2002 study, Clofibric acid, Aspirin, Clofibrate, Ibuprofen, Caffeine, Gemfibrozil, Naproxen, Carbamazepine, Ketoprofen, and Diclofenac, were found in the waters of the Seine River near Paris, France (Togola, 2007).  These and many other drugs have also been found in U.S. cities.

To study the procedures and techniques utilized in the research laboratory to detect pharmaceuticals in our drinking water, a study using over-the-counter (OTC) drugs will be performed in the high school laboratory.  Qualitative and quantitative analyses will be carried out where identification, confirmation and quantitation of the drugs will take place.  Mock tainted-water samples will be analyzed for contaminants.  The contaminants will be identified from a set of standards in a thin-layer chromatography analysis.  The contaminants will then be isolated using column chromatography.  Finally, the concentration will be determined visually using a colorimetric analysis using a spectrophotometer.


Approximate Time Required:  One to two weeks, depending on the laboratory schedule.  One week is better for the standard solutions.  If the study must be scheduled to two weeks, make sure to make new standard solutions for week two.


Materials:      OTC drugs-diphenhydramine, loratadine, mezicline

Ethyl acetate

methylene chloride


ninhydrin solution

distilled water

tainted water samples (teacher should prepare these solutions ahead of time)

thin layer chromatography plates

10mL pipette


glass wool


general glassware

capillary tubes or Pasteur pipettes

solid iodine chips

watch glass

metric ruler

large test tubes

small borosilicate test tubes (cuvettes)




Scenario:  You have obtained some water samples from local bodies of water suspected of being contaminated with antihistamines, and antiemetic drugs.  Initial tests suggest that the specific drugs may be diphenhydramine, loratadine or meclizine (all available as OTC drugs).  You now have the task of determining which drug(s) is/are present in the water and to what extent.  Therefore, you have to identify and quantify the contaminants.


Part I: Making standard solutions and identifying the drugs.


  1. Crush one, two or three tablets (40 mg of active ingredient) of the drugs being careful to remove the coating without eliminating the rest of the contents.
  2. Dissolve the tablets in 20mL of distilled water.  This will result in a solution that is 2000ppm strictly by mass.
  3. Allow the solution to sit stirring overnight if possible (not required but you get a better dissolution).
  4. Filter the solutions and retain the filtrate.
  5. Prepare a thin layer chromatography (TLC) plate.
    1. Commercially available plates are 2.5cm X 7.5cm.
    2. Lightly draw a line across the width of the plate 8mm from the bottom.
    3. Lightly draw four tiny dots on the line 5mm apart from each other.
    4. Using a capillary tube or Pasteur pipette obtain a sample of each solution and spot the plate.  Use a different capillary tube or Pasteur pipette for each solution.  Each sample is to be loaded on one of the dots you drew on the line.  Make sure to keep track of which solution is on which spot.  You should write this on a laboratory journal.
  6. Prepare a solvent chamber.
    1. A 250mL beaker and a watch glass big enough to cover the mouth of the beaker can serve as a solvent chamber.
    2. Place 3mL of ethyl acetate in the beaker.
  7. Place the loaded TLC plate in the solvent chamber and cover with the watch glass.
  8. Allow the plate to sit in the solvent chamber for 10 minutes (be careful not to let the solvent front go off the plate.
  9. Remove the plate from the developing chamber and allow it to dry.
  10. Using a short-wavelength UV lamp visualize the spots.  Carefully draw a circle around each spot.
  11. Place a dot in the center of each circle you drew.
  12. Prepare an iodine chamber.
    1. Place a few chips of solid iodine in a 250mL beaker and cover with parafilm and a watch glass.
  13. Place the TLC plate in the iodine chamber and allow it to develop for 10 minutes.
  14. Quickly remove the plate and draw circles around any additional spots you see that were not seen under UV light.
  15. Again draw a dot in the center of the circle.
  16. Also draw a line across the solvent front.
  17. Obtain Rf values for all the samples.
    1. Measure the distance from the baseline to the solvent front.
    2. Measure the distance from the baseline to the center of each circle for each compound.
    3. Obtain Rf values with the following equation:


Rf =     Distance traveled by the compound

            Distance traveled by the solvent front


  1. Compare values to determine which compounds are in your tainted sample.


Part II:  Isolating the contaminants.


  1. Prepare column chromatography set-up.
    1. Obtain a 10mL disposable glass pipette and measure the diameter of the width.
    2. Place a small wad of glass wool or cotton and push it down to the conical end of the tube.
    3. Above that place a small amount of sand.
    4. Fill the pipette with silica to a height equal to 8 times the diameter you measured.  Gently tap the side of the pipette to pack the silica.
    5. Fit the bottom end of the pipette with 1 to 2 inches of rubber tubing and clamp it to keep liquids from escaping.
    6. Slowly fill the column with ethyl acetate to about 3mL above the silica. Make sure to drip the solvent on the walls of the pipette to slow the filling process.
  2. Obtain 10mL each of the tainted water sample(s) and evaporate the water by gentle heating.  Save the remaining 10mL for Part III.
  3. Re-suspend the crystals in 10mL of 1:1 methylene chloride—ethanol mixture (an organic solvent).
  4. Run a TLC plate as in Part I, to spot check for the drugs.
  5. Unclamp the column and let the solvent run out until the silica is almost exposed.  Do not allow the solvent to run below the silica.
  6. Quickly place a 1mL aliquot of the dissolved crystals in the column and allow the sample to run.  Just before the silica is exposed add ethyl acetate to the column.  Never allow the column to run dry.  You must continue to refill the column with this solvent.
  7. Place a small test tube at the bottom to collect the fractions of solvent and solution.  The test tube has to be replaced after each fraction collected.
  8. Collect 3, 3mL fractions, and then start collecting 20, 0.5mL fractions.
  9. Re-clamp the rubber tubing and cover the top of the column with parafilm.  Remember the column should always have solvent above the silica.
  10. Run TLC plates for each of the fractions collected keeping track of the fractions on the plate.  Each plate must also include a standard for each identified in part I.
  11. Pull together all the fractions with the same spots (same Rf values as the standard) into one large test tube.  You should have a large test tube for each different Rf value on your TLC plates.
  12. Identify the drugs in the tainted water sample by comparing the Rf values.
  13. Evaporate the solvent in each test tube by gentle heating.  You can place the test tube in a warm water bath.
  14. Re-suspend the crystals in 10mL of distilled water and save for part III.


Part III:  Colorimetric Assay


Part A:  Optical Activation of drugs.


  1. Obtain 5mL of each standard solution and place them in a test tube.
  2. Obtain 5mL of distilled water and place them in a test tube.  This will serve as a blank to zero a spectrophotometer.
  3. Obtain 5mL of the isolated drug solutions and place each in a test tube.
  4. Add 1.5mL of 1.25% (w/v) aqueous solution of ninhydrin to each of your standards, the blank and your samples.
  5. Place ~150mL of water in a 250mL beaker.  Heat the water to 95°C.
  6. Place all the test tubes in the hot water bath for 15 minutes.  Make sure to keep the temperature at 95°C.  Make sure that the water does not splash.
  7. Remove the test tubes from the water bath and allow them to cool to room temperature.
  8. Set your samples to the side and prepare your standards for a calibration curve.


Part B:  Serial Dilution, Calibration Curve and Quantitation of Drug Sample.


  1. The following procedure is performed for each drug found in your tainted water sample.
  2. Set up a spectrophotometer before you work with the samples.  Turn on the spectrophotometer and leave it on for 5 minutes before you place any samples in the machine.
    1. In a cuvette place 2.0mL of the blank standard.
    2. Wipe the cuvette and place it in the spectrophotometer
    3. Set the wavelength to 400nm.
    4. Turn the absorbance button until the needle reads: Transmittance 100% / 0 Absorbance.
  3. Remove the cuvette form the spectrophotometer and place it to one side.  You may need this cuvette later.
  4. Prepare a serial dilution for each standard.
    1. Split the 10mL of the standard solution into two 5mL portions and place them in 2 separate test tubes.  Label one test tube 1A and the other 1B.
    2. Add 5mL of distilled water to test tube 1B, and transfer 5mL to a third test tube.  Label this new test tube 1C.
    3. Add 5mL of distilled water to test tube 1C, and transfer 5mL to a fourth test tube.  Label this new test tube 1D.
    4. Add 5mL of distilled water to test tube 1D, and transfer 5mL to a fifth test tube.  Label this new test tube 1E.
    5. Add 5mL of distilled water to test tube 1E and save.
  5. Obtain a 2mL aliquot of a standard 1A solution and place it in a cuvette.
  6. Wipe the cuvette and place it in the spectrophotometer.
  7. Read the absorbance and record this value in your laboratory journal.
  8. Repeat steps 5 – 7 for standards 1B – 1E.
  9. Plot a graph of Absorbance against sample number (concentration).
  10. Obtain reading for your drug sample as well.  Make sure that the sample matches the standard.
  11. Determine the concentration of the drug by drawing a line from the Y-axis to the sketched line of the graph and then extending it down to the X-axis.




All elements of the K-12 science program must be consistent with the other National Science Education Standards
and with one another and developed within and across grade levels to meet a clearly stated set of goals.

The program of study in science for all students should be developmentally appropriate, interesting, and relevant to students' lives; emphasize student understanding through inquiry; and be connected with other school subjects.

The science program should be coordinated with the mathematics program to enhance student use and understanding of mathematics in the study of science and to improve student understanding of mathematics.

The K-12 science program must give students access to appropriate and sufficient resources, including quality teachers, time, materials and equipment, adequate and safe space, and the community.

All students in the K-12 science program must have equitable access to opportunities to achieve the National Science Education Standards

Schools must work as communities that encourage, support, and sustain teachers as they implement an effective science program.





STANDARD 5.1 (Scientific Processes) All students will develop problem-solving, decision-making and inquiry skills, reflected by formulating usable questions and hypotheses, planning experiments, conducting systematic observations, interpreting and analyzing data, drawing conclusions, and communicating results.


STANDARD 5.3 (Mathematical Applications) All students will integrate mathematics as a tool for problem solving in science, and as a means of expressing and/or modeling scientific theories.


STANDARD 5.4 (Nature and Process of Technology) All students will understand the interrelationships between science and technology and develop a conceptual understanding of the nature and process of technology.


STANDARD 5.6 (Chemistry) All students will gain an understanding of the structure and behavior of matter.


STANDARD 5.10 (Environmental Studies) All students will develop an understanding of the environment as a system of interdependent components affected by human activity and natural phenomena.




1)      Crimi, Christina M. and Snow, Nicholas H. “Analysis of Pharmaceutical Residual Solvents Using Comprehensive Two-Dimensional Gas Chromatography.” (2008) Chromatography The Website of LCGC North America.

2)      Frutos, P. Torrado, Susana. Perez-Lorenzo, M.E. and Frutos, G. “A Validated Quantitaive Colorimetric Assay for Gentamicin.” (2000) Journal of Pharmaceutical and Biomedical Analysis 21:1149-1159.

3)      Halling-Sorensen B, Nielsen SN, Lanzky PF, Ingerslev F, Holtem Lutzhoft HC, Jorgensen SE (1998) Chemosphere 36:357-393.

4)      Hemminger, Pat. “Damming the Flow of Drugs into Drinking Water.”  Environmental Health Perspectives. Volume 113. No. 10. October 2005.

5)      Librera, William L. Ed. D. “New Jersey Core Curriculum Content Standards for Science.” (2004) New Jersey Core Curriculum Content Standards. PTM# 1505.24:E1-E27.

6)      Snow, Nicholas H. “Determination of Free-Energy Relationships Using Gas Chromatography.” (1996) Journal of Chemical Education 73:592-597.

7)      Togola, Anne and Budzinski, Helene. “Analytical Development for Analysis of Pharmaceuticals in Water Samples by SPE and GC-MS.” (2007) Analytical and Bioanalytical Chemistry 388:627-635.

8)      The United States Pharmacopeia–National Formulary (USP–NF).  The Official Compendia of Standards, USP 29-NF 24 2005.  Pharmacopeial Convention Inc., November 2005.  USP <467>.