(adapted from an article in J. Chem. Ed. by Mark Duxbury)
(Duxbury, M. J. Chem. Ed. 2003, 80, p 1180)
Introduction
Apples are relatively inexpensive and are readily obtainable year round in most developed countries from local
markets. Depending on the time of year the apples may be either fresh or have come from a cool store. The cold storage
stability of apples is limited by the tendency of some apples to develop a condition known as “Bitter Pit”. Affected
apples develop small, water soaked lesions on the surface of the fruit which gradually turn brown and become slightly
sunken after one to two months storage. The underlying apple tissue becomes spongy
Bitter Pit occurs in all the major apple producing countries in the world and affects most apple varieties to a
greater or lesser extent including the well known varieties Golden Delicious, Red Delicious and Granny Smith.
Although the condition has been intensively studied for over a century the underlying processes that lead to the
condition are still not fully understood, although it is believed that the condition is related to the physiology of the
apple rather than to attack by pathogenic organisms. However it is known that the calcium concentration in the apples
is a major factor in determining whether or not apples will develop Bitter Pit. In addition to calcium content the
potassium and magnesium content of the fruit are also a useful predictor of whether apples will develop Bitter Pit.
In general if apples have a calcium concentration greater than 30 mg/kg fresh weight they are unlikely to
develop “Bitter Pit” on storage. If the calcium concentration in the fruit is less than 19 mg/kg fresh weight the apples
are highly likely to develop Bitter Pit on storage and are usually only suitable for processing into apple juice or other
products such as cooked pie fillings. Apples with intermediate calcium concentrations between the 19 and 30 mg/kg
limits can be stored after drenching with a 3% calcium chloride solution, provided that they have a “balanced mineral
relationship”. A balanced mineral relationship is defined by the ratio of magnesium and potassium to calcium
concentration in the fruit, as follows:
[Calcium] > 0.6
[Magnesium]
[Calcium] x 10 >0.17
[Potassium]
where the concentrations are given in mg/kg fresh weight.
Typical natural elemental concentration ranges in apples are 10-50 mg/kg for calcium, 25-50 mg/kg for
magnesium and 500-1500 mg/kg for potassium. The large variations in the concentration ranges between the different
elements makes analysis by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) attractive since
the light emission output from an ICP-AES instrument is relatively linear over widely varying concentration ranges.
Experimental
In this experiment you will be utilizing ICP-AES to analyze apples you provide for Bitter Pit susceptibility. You will
also be comparing the Ca concentrations you determine with ICP to Ca concentrations determined from Flame-AAS.
Materials:
Obtain several different types of apples to test and compare across varieties.
Decide what you will use to make the elemental standards and prepare a procedure to make the required 1000 µg mL-1
stock solutions.
For Flame-AAS all solutions (standards and samples) need to be made in a buffer containing 0.218 M LaCl3, 0.01 M
CsCl, and 1.54 M HNO3. (make 500 mL)
Sample Preparation
Using a hollow laboratory Cork Hole Borer Tube (No.5) take samples approximately 4 mm in diameter from an apple
by pushing the cork hole borer through the apple and pulling out the cylindrical slice inside the Borer Tube. Use a
narrow rod to push the cylindrical slice out of the center of the hollow tube. This process quickly gives a sample with a
high surface area which speeds the subsequent digestion. Alternatively if a cork hole borer is not available slice the
apple finely with a knife. Accurately weigh approximately 1 gram of the cylindrical apple slices into a 150 mL Pyrex
beaker for your ICP sample. (Take a duplicate 1g sample from the opposite side of the apple into a second beaker).
Also collect a 2 g sample from one apple to use for Ca Flame-AAS analysis.
Sample Digestion
Nitric acid Wet Digestion Method
Warning : Concentrated nitric is a strongly corrosive acid. Wear adequate safety equipment
when handling the acid (safety glasses, shields and gloves). Carry out digestions in a fume
hood.
Add successive 15 and 10 mL aliquots of nitric acid (65% HNO3) to the 1 and 2 g apple slice samples in the Pyrex
beakers and heat on a hot plate in a fume hood; after each addition the sample is taken to near dryness. (The surface
temperature of the hotplate is held near 250 ºC throughout the digestion). During this period brown fumes (NO2) will
be given off ( and the solution will turn brown or yellow) as the easily oxidized organic material in the apple is
converted to carbon dioxide and water. Do not allow the sample to boil dry. Perform this same digestion procedure on
2 empty clean beakers to use as a method blanks for each instrument. The last addition of nitric acid is when the
boilong solution no longer produces brown vapor or turns color.
The beakers are removed from the heat and left to cool.
For the ICP samples the contents of the beakers are diluted with deionized water and filtered through ash-less filter
paper (e.g. Whatman No540) into a 100 mL volumetric flask which is made up to the mark with deionized water.
For the 2 g Flame-AA sample dilute with the La/Cs buffer into 50 mL volumetric flasks.
(The filtration step avoids clogging of the nebulizer of the ICP by any silica residues).
Standard Preparation:
ICP
Pipette 10 mL aliquots of 1000 µg mL-1 Ca and Mg stock solutions into a 100 mL volumetric flask, dilute to the mark
with deionized water and mix to give a solution containing 100 µg mL-1 of each element respectively (Solution A).
Using a fresh pipette transfer 5 mL from Solution A into a 100 mL volumetric flask, add 10 mL of a 1000 µg mL-1 K
stock solution, dilute to the mark with deionized water and mix to give a solution containing 5 µg mL-1 Ca and Mg and
100 µg mL-1 K respectively (Solution B).
Using a fresh pipette transfer 5, 10, 15 and 20 mL aliquots of Solution B into four 100 mL volumetric flasks and dilute
to the mark with deionized water (Solutions C, D, E and F).
Solution C contains 250 ng mL-1 Ca and Mg and 5 µg mL-1 K.
Solution D contains 500 ng mL-1 Ca and Mg and 10 µg mL-1 K.
Solution E contains 750 ng mL-1 Ca and Mg and 15 µg mL-1 K.
Solution F contains 1000 ng mL-1 Ca and Mg and 20 µg mL-1 K
Flame-AAS
Use the 1000 µg mL-1 stock solutions to prepare Ca standards in a range from 1 – 6 µg mL-1
. Remember all solutions
need to diluted with the La/Cs buffer. This includes the method blank and the apple samples. Make these up in 50 mL
flasks.
Analysis
As you are running through the experiment below make sure you are collecting all the data you need to provide these
items in your lab report. This may necessitate some additional data collection
Figures of Merit Checklist:
- calibration curve
- Equation of clibration curve with standard deviations of slope and intercept
- Table of true concentrations (standards and pseudo-unknown), estimated concentrations with standard
deviations (from equations not repeat trials), and RSD’s. - Sensitivity
- Detection limit
- calibration curve, printed from Excel and in proper format for publication in the ACS Journal Analytical
Chemistry (Consult the Author Guidelines under Submission & Review (http://pubs.acs.org/journal/ancham ))
ICP
With the aid of the TA or Instructor set up the ICP-AES instrument for the analysis by selecting plasma conditions and
appropriate elemental emission lines that are free from spectral interferences.
Aspirate a deionized water blank and the working standards (Solutions C, D, E and F) into the plasma and determine
the emission intensities of each element. Use this data to prepare calibration curves of element concentration versus
intensity for each element.
Aspirate the apple samples and the method blank and determine the emission intensities for each element. Determine
the concentrations of the elements in the sample solutions from the standard calibration curves. Do not run the 2 g
sample that is only for the Flame-AA
Calculate the concentrations of the elements in the original apple samples in mg kg-1
. Use this data and the information
in the introduction to the experiment to determine whether the apples are suitable for storage (either with or without
drenching with CaCl2) or whether they are suitable only for juicing or food processing.
Flame-AAS
Set up the Flame-AA/AES instrument to perform an AA analysis for Ca. In addition to running the method blank, run
the 2 g apple sample{all diluted with the La/Cs buffer}
Calculate the Ca concentrations in both samples in mg kg-1
.
Questions
1) Are any of the apples you chose candidates for further treatment to prevent bitter pit?
2) What is and what is the purpose of a method blank?
3) How did the concentrations of Ca for the two apple samples determined by Flame-AAS compare to one another?
How did they compare to the ICP concentrations?
4) Why did we ask you to prepare a more concentrated apple sample for the Flame-AAS part of the experiment? Was there a significant difference in the error associated with the analysis of the 2 g sample and the 1 g sample for the ICP?
5) Compare and contrast the techniques of ICP and AAS for doing elemental analysis. Be sure to address the operating
parameters of the instrument and detection limit. Why did we have a La and Cs containing buffer for the AAS analysis and not for the ICP?
Sample Solution