(E5) ZINC IN PET FOOD
Zinc is an essential nutrient for both people and animals. It is used by mammalian enzymes,
structural proteins and hormones. It is needed for many aspects of the immune system, including
iodine metabolism. Zinc is also needed in cell replication and so is necessary for healing wounds,
maintaining the intestinal mucous, and for healthy nail and hair growth. There is also some
evidence that zinc helps shorten the duration of, or lessen the severity of, colds (however there is
debate on this issue). Zinc is found in high levels in shell fish, beans, dairy products, and cereals
that are fortified with zinc and nuts.
Because pets tend to eat a very non-diverse diet, ensuring that pet foods have the appropriate
levels of zinc is very important. The Association of Feed Control Officials (AAFCO), the
organization which regulates the sale and distribution of animal food, recommends a minimum
of 120 mg zinc/kg dog food and a maximum of 1000 mg zinc/kg dog food. The AAFCO also
recommends that dog food has a minimum of 150 mg zinc/kg dog food if the dog food will be
used to feed northern breeds of dogs such as Huskies or Alaskan Malamutes. This is because
these northern breeds tend to have genetic disorders which prevent them from being able to
uptake zinc. Zinc deficiencies have been observed in these northern breeds, in dogs that have
diets high in calcium (which hinders the uptake of zinc) and in dogs that are fed generic dog
food. Symptoms of zinc deficiency include scaling of the skin (particularly on the legs and
face), hair loss, lesions on the face, and a dull and dry coat. Zinc supplements have also been
shown to benefit a dog’s skin and coat even when zinc deficiencies are not present. Zinc
toxicity, though rare, is also seen in small dogs which ingest non-food items such as pennies
minted after 1982 (when the amount of zinc in pennies was increased), nuts and bolts, and some
zinc containing sun screens or lotions. Symptoms of zinc toxicity in dogs include lack of
appetite, vomiting, lethargy and pale gums. To treat zinc toxicity the zinc items are removed
from the gastrointestinal system and a chelation agent such as EDTA may be used to bind to the
excess zinc.
In this experiment you are going to test two types of pet foods for their zinc content. One sample
you test will be a brand provided by your teaching assistant, the other will be a sample of food
you provide from home. These pet foods will be tested to ensure their zinc content is within the
range recommended by the AAFCO.
Experimental Summary:
The zinc content in a pet food is determined by first digesting the sample with acid to
extract the zinc into the aqueous phase. The resulting solution is analyzed by flame atomic
absorbance spectroscopy. This procedure was taken from Butala et al., J. Chem. Ed. 72, 1995,
441-444 and Stemporzewski et al., J. Chem. Ed. 51, 1974, 332-333, J. Chem. Ed. 53, 1976, 165.
Information on how to use the instrument and the instrument parameters can be found in
Appendix A. Read this over carefully before coming to class.
Note: This experiment uses a standard addition technique to account for matrix effects and
extraction efficiency. You will be told the concentrations of spikes to use to create the standard
addition curve. In reality, if you were going to use a standard addition you would first analyze
your sample and then determine the appropriate amount of standard to spike in. Instructions for
the amount to spike are included here to reduce the amount of time needed for the experiment.
Additional References:
1. J. W. Hill and C. S. Hill, Chemistry for Changing Times, Macmillan: New York, 5th Ed.,
1988, pp. 415-416.
2. D. G. Crosby, Environmental Toxicology and Chemistry, Oxford University Press: New
York, 1998, pp. 213-218.
3. C. Baird, Environmental Chemistry, Freeman: New York, 1995, pp. 354-363.
4. C. Baird, Environmental Chemistry, Freeman: New York, 1995, pp. 376-380.
5. http://veterinarynews.dvm360.com/dvm/Medicine/Canine-zinc-responsivedermatosis/ArticleStandard/Article/detail/162405
6. http://www.peteducation.com/article.cfm?c=2+1659+1662&aid=718
7. White, S.D., Bourdeau, P., Rosychuk, R.A.W., Cohen, B., Bonenberger, T., Fieseler,
K.V., Ihrke, P., Chapman, P.L., Schultheiss, P., Zur, G., Cannon, A. and Outerbridge, C.
(2001), Zinc-responsive dermatosis in dogs: 41 cases and literature review. Veterinary
Dermatology, 12: 101–109. doi: 10.1046/j.1365-3164.2001.00233.x
8. http://www.petplace.com/dogs/zinc-toxicity-in-dogs/page1.aspx
9. Science, M., Johnstone, J., Roth, D.E., Guyatt, G., and Loeb, M., (2012). Zinc for the
treatment of the common cold: a systematic review and meta-analysis of randomized
controlled trials, Canadian Medical Association Journal, 184, E551.
Analytical Focus:
Use of standard addition matrix spikes to determine extraction efficiency
Prelab Questions:
1. Why are you using nitric acid for your dilutions? (Hint: Look up how zinc solubility is
affected by pH.)
2. Do the necessary calculations for your dilutions of the stock zinc solution. For these
calculations, assume the concentration of the stock solution is exactly the value given in the lab
manual. (Note: for your lab report and actual calibration curve you will need to calculate
your concentrations from the actual stock solution provided to you in lab.) Using this
assumed stock concentration, calculate the volume of stock solution you will need for each
dilution, and the final volume and concentration of each dilution. Do not make more than 2
serial dilutions. Your final volume should be either 10 mL or 25 mL. (These stock solutions
should be as exact as possible so keep that in mind when choosing what glassware you should
use).
3. You make up a standard addition curve with an equation of y=0.1913x+0.059 (y is
absorbance x is mg zinc spiked in per L of sample solution). Assume that the sample you took
your AA measurement was diluted to a final volume 100 mL and each dog food sample was
exactly 0.1000 g. What would your amount of zinc in dog food be in units of mg zinc/kg dog
food?
4. Visit the NIST Atomic database (http://www.nist.gov/pml/data/asd.cfm) and explore the
emission spectrum of Zn in the spectral range from 200 to 600nm. What is the wavelength of the
strongest Zn atomic emission line?
Samples:
Your TA will provide you with one dog food samples. In addition, your group will be
required to provide one pet food (dog or cat) sample for analysis. Bring about a cup of
your own pet food in a Ziploc bag. If no one in your group has a pet ask another member of
the class if they can bring you a sample. If the pet food bag states a Zn concentration, be
sure to record that value.
Safety:
This procedure requires the use of concentrated acid. Use gloves and goggles throughout
the procedure and dispose of all waste as instructed. Be sure to have sodium bicarbonate readily
available for acid spills. DO NOT PUT SODIUM BICARBONATE ON AN ACID SPILL
ON YOUR SKIN!!!! IT WILL ONLY MAKE IT WORSE!!! If any acid does get on your
skin wash with copious amounts of water. Take care not to put your arms on the desk you are
working on as this is a very easy way to get acid on your arms if there is any spilled acid on the
desk. Also remember that while filtering your filter paper has acid on it and should not be
touched.
Reagents Provided by the TA:
Read the labels on the reagent bottles for exact masses and volumes used. The general
preparation procedures for the provided reagents are as follows:
Concentrated nitric acid
Zinc stock standard (50 ppm Zn): Approximately 0.4550 g of zinc nitrate hexahydrate
(Zn(NO3)2.6H2O) were added to a 2 L volumetric flask and diluting to the mark with 1%
HNO3 (v/v). Check container for actual amounts.
Nitric acid, 1% (v/v): Provided in a squirt bottle to bring your dilutions to the mark. Do not use
for cleaning.
Nitric acid, 10% (v/v): (For cleaning) 50 mL concentrated nitric acid was SLOWLY added to
~300 mL of distilled water then enough distilled water to bring the total volume to ~500 mL was
SLOWLY added.
Reagents to be Prepared by the Student(s):
Zinc standard addition standards: All zinc standards should be made by diluting the zinc stock
standard with the 1% HNO3. Prepare samples 10 ppm Zn solution, 15 ppm Zn solution,
25 ppm Zn solution, and 40 ppm zinc solutions.
Instrumentation:
A flame-based atomization and ionization procedure will be used in concert with an
atomic absorption spectrometer. Instructions for how to use this instrument are presented in
Appendix A. Please read these instructions before coming to class.
Procedure Outline:
1. Clean all glassware.
2. Prepare samples.
a. Homogenize pet food and weigh out 6 0.1000 g samples of each type into a 100
mL beaker.
b. Spike each of the 6 samples of the student brought pet food with 1 mL of the 50
ppm, 40 ppm, 25 ppm, 15 ppm, 10 ppm, and 0 ppm Zn solutions respectively.
c. Spike each of 6 samples of the TA provided pet food with 1 mL of the 50 ppm, 40
ppm, 25 ppm, 15 ppm, 10 ppm, and 0 ppm Zn solutions respectively.
3. Begin digestion of samples.
4. Finish digestion.
a. Cool samples.
b. Filter samples to remove particulates.
c. Dilute samples to the appropriate volume for analysis
5. Analyze samples
Procedure Details:
1. Thoroughly clean and rinse the glassware with 10% HNO3.
2. Prepare each of your samples for digestion.
a. Homogenize ~1-2 g of the TA-provided sample with a mortar and pestle. Weigh
six 0.1000 g samples (note this mass should be exact, make sure that it is to the
last digits place) and transfer them to six 100 mL beakers. Do the same with the
student provided sample.
b. Spike 1.0 mL of each of the stock solutions into the student provided samples (1
stock solution per sample). Do the same for the TA provided samples. Place the
samples in the oven at 188 ºC and allow them to partially dry (~7 minutes).
c. Take the samples out of the oven (using hot glove) and allow them to cool for ~ 5
minutes.
3. Add ~20 mL of concentrated nitric acid to each sample, mix the slurry, and cover with a
watch glass. Place the beakers on a hot plate (in the hood!!) and turn up the heat until the
samples are barely boiling or very close to boiling (keep an eye on them) the hot plate
should be set at ~3 or less. Digest the samples for 45 minutes. Never let the solutions go
to dryness. If they start to go dry, add more concentrated nitric acid. One person in the
group must be watching the digesting samples at all times! Some nitrogen dioxide will
come off your samples so keep them in the fume hood while they are digesting.
4. Remove the digested samples from the hot plate. Uncover them in the fume hood and
allow all the colored gas to come off them before you take them out.
a. Allow the samples to cool in the air then allow them to further cool in an ice bath
(do not put the hot samples directly into the ice bath as the glass beakers may
shatter).
b. WHEN THE SAMPLES ARE COOL, filter each sample through Whatman-41
filter paper and collect the filtrate in a 100 mL volumetric flask. Rinse the
remaining solid with 1% nitric acid several times, adding the rinsate to the filtrate.
Rinse the beaker with 1% nitric acid and add that to the volumetric flask. If the
samples appear cloudy or large particles are observed, the sample must be filtered
again. Large particles will clog the aspirator on the AA.
c. Dilute to the mark with 1% nitric acid.
5. Analyze each of your samples using the technique on the instrument WITH the smith
hieftje correction. Be sure that each of your samples falls within the linear dynamic
range of your instrument. If necessary, dilute the samples with 1% nitric acid to bring
them into range.
6. Analyze a blank sample to get the LOD and LOQ for this instrument.
Waste Disposal and Clean-up:
All samples and standards should be combined into one waste container. All solid waste
(gloves, filter paper, etc) should be placed in the solid waste container.
Calculations:
Determine the zinc concentrations by making a standard addition plot of signal vs.
concentration. Remember, the values you determine with your standard addition curve are the
concentrations of zinc in the aqueous solutions that you analyzed with the instrument, NOT the
concentrations of zinc in each of the samples. To obtain these concentrations you must convert
the aqueous concentration to a pet food concentration by considering the extraction and dilution
procedures.
Report Considerations:
1. Discuss the significance of the standard addition method with respect to your results.
2. Calculate the limit of detection and limit of quantification for Zn on the AA instrument.
3. Using the limit of detection for Zn on the instrument and considering the extraction and
dilution procedures, calculate the method detection limit for Zn in dog food.
4. Construct a standard addition curve and use it to find the mg zinc per kg pet food for each
of your samples. Don’t forget to include error bars on your numbers.
5. Comment on the reproducibility of your analysis.
6. Comment on the smith hieftje method. How is it used to account for contamination in
your sample?
7. Comment on the values and error bars you obtain in your unknowns. Do they match
values given on the bag (if known)? Are they in the limits recommended by the
AAFCO?
8. Why did your original masses of pet food need to be exactly 0.1000 g (or as close as you
could get as possible)? Why couldn’t you just divide your absorbance by the mass of the
sample?