How does the length of aging of various commercially-available cow cheeses (0-5 weeks old) affect sodium chloride content determined by Volhard’s Method?

Content of sodium chloride in food is crucial because it directly affects the action of the sodium-potassium pump, which controls cellular function and homeostasis. The sodium-potassium pump is in charge of actively moving potassium ions \(\left(\mathrm{K}^{+}\right)\) into the cell and sodium ions \(\left(\mathrm{Na}^{+}\right)\) out of the cell. The pump has a role in controlling the resting membrane potential of the cell, by preserving the concentration gradients of sodium and potassium ions across the cell membrane. For my own personal interest, determination of sodium chloride concentration in a common food type such as cheese is very important for my overall health. While sodium is a crucial nutrient, higher consumption of salt than needed can lead to serious problems such as high blood pressure, heart disease, and stroke (Salt and Sodium/ Nutrition source, 2019). Monitoring sodium intake is important for maintaining overall health. This is because I practice basketball at a very high level. In addition, high-intensity and highendurance sports such as basketball can put strain and pressure on the heart and blood vessels of the body (O'Keefe et al., 2012). Given the effects that salt can have on blood pressure and general cardiovascular health, knowing how much salt is in different types of cheese helps me regulate my sodium intake. This research project supports my personal goal of eating a balanced diet and lowering the risks related to consuming too much salt.

Cheese is a dairy product obtained through the coagulation of milk. It is crafted in various forms, colors, flavors, and through diverse methods. Main components of cheese are milk and dairy fats, water, lactose, proteins, minerals and salt. These components together make different types of cheese unique in terms of taste, texture, and nutritional composition. One of the most important features of cheese is its age. The age of cheese refers to the period of maturation or aging that occurs after the cheese is produced. The time that the cheese spends in the maturation process significantly influences its taste, texture, and aroma. The age of cheese can vary from a few weeks to several years, depending on the type of cheese and the traditional or specific aging conditions. Chemical content of cheese consists of different percentages of carbohydrates, fat, water, minerals, proteins and other compounds inside the cheese (Chemistry of cheese, 2021).

In this research, 6 types of cow cheese will be analysed. Sodium chloride, known informally as table salt, is perhaps one of, if not the, most commonly used spice in the world. Finding itself in nearly every food item, sodium chloride is the source from which we obtain most of our body's sodium (CDC, 2023). Sodium is an extremely important mineral within the human body, where it is responsible for the rising phase of action potentials. Action potentials are electrical discharges responsible for many essential functions within our body; for example, within the nervous system, action potentials are the signals that spread from one neuron to another allowing our brain and spinal cord to control the rest of our body (Byrne, 2021). In pancreatic cells, the completion of action potentials is what releases insulin into the bloodstream. Perhaps most interestingly for my own personal interest, action potentials are responsible for the initiation of muscle contraction by muscle cells (Byrne, 2021). However, excess levels of sodium in the body can result in various cardiovascular issues, such as heart disease and high blood pressure leading to potential strokes (Chan, 2023). Since sodium chloride is our largest source of sodium, maintaining a balanced level of sodium chloride in the body is essential for health and survival - this is why many multicellular organisms store sodium chloride in their extracellular fluid (Pubchem, 2023).

Salting extracts the whey and regulates the acidity of the cheese and ripening, and increases the shelf life of cheese. Salting, also, affects the formation of the crust and the consistency of the cheese, and prevents the growth of undesirable bacteria. The salt content in mature cheese ranges from 1 (or less) to \(5\%\) (or more), depending on the type of cheese. However, the actual concentration of salt is much higher, because salt dissolves only in the aqueous phase of the cheese (Sirevi, 2017).

Regarding the experimental methods for the investigation of sodium chloride content, Volhard's titration method was chosen. Titration was determined as the best method to determine sodium chloride content, as determining the direct sodium content would require ICP-OES, a method which was not possible given the current laboratory equipment at my disposal. Thus, the most appropriate method to determine sodium chloride content was through determination of chloride content by titration then calculating sodium chloride by the stoichiometric ratio of 1:1 (WHO, 2020).

A titration is a technique where a solution of known concentration is used to determine the concentration of an unknown solution (Purdue University, 2019). On the other hand, there is a titration method where the concentration of an unknown compound is determined by reacting with a known amount of excess reagent, which is called back-titration. Equivalence Point is the point in a titration when the amount of titrant added is equivalent to the amount of analyte present. At this stage, the reaction is complete, and the solution is neutralized. End point refers to the point at which colour change in a system or solution occurs. (Vedantu, 2022)

Volhard's method is a back-titration method commonly used for determining halide concentration. This method involves treating the sample with excess silver nitrate \(\left(\mathrm{AgNO}_{3}\right)\) to precipitate the halide, then titrating the excess silver nitrate with ammonium thiocyanate \(\left(\mathrm{NH}_{4} \mathrm{SCN}\right)\) and any ferric indicator to produce a red color at the endpoint (Ricca, 2022). The balanced chemical reactions for this process are as follows:

\[\begin{aligned}& \mathrm{Cl}^{-}{ }_{(\mathrm{aq})}+\text { excess } \mathrm{AgNO}_{3(\mathrm{aq})} \rightarrow \mathrm{AgCl}_{(\mathrm{ppt})}+\mathrm{AgNO}_{3 \text { (aq) }} \\& \mathrm{NH}_{4} \mathrm{SCN}_{(\mathrm{aq})}+\mathrm{AgNO}_{3(\mathrm{aq)}} \rightarrow \mathrm{AgSCN}_{(\mathrm{ppt})}+\mathrm{NH}_{4} \mathrm{NO}_{3} \text { (aq) } \\& \text { Excess } \mathrm{SCN}^{-}{ }_{(\mathrm{aq})}+\mathrm{Fe}^{3+} \rightarrow \mathrm{FeSCN}^{2+}{ }_{(\text {aq })}(\text { Ricca, 2022) }\end{aligned}\]

\(\mathrm{FeSCN}^{2+}\) complex is red and indicates the endpoint of the back-titration as there is no more silver for the \(\mathrm{SCN}^{-}\) to react with.

NaCl content in cheeses was analyzed by many chemists. The results of the analyses conducted with Volhard's method showed that the cheeses contain \(0.97 \%-4.72 \%\) in 100 g of cheese (Rajković, Sredović, & Miloradović, 2010).

Null hypothesis \(\left(\mathrm{H}_{0}\right)\) is that the sodium chloride content of the cow cheeses will not be affected by their age. Alternative hypothesis \(\left(\mathrm{H}_{1}\right)\) posits that the sodium chloride content found within the cow cheeses will be higher within cheeses that have been aged for longer (cheeses whose aging process is between 3-5 weeks old).

Independent variable: The age ( \(0,1,2,3,4,5\) weeks) of the 6 cow cheeses that have been tested. Tested cheeses are Low-fat curd cheese ( 0 weeks), full-fat curd cheese (1 week), Pljevaljski cheese ( 2 weeks), Jastrebački cheese (3 weeks), Sjenički cheese ( 4 weeks), Zlatar cheese ( 5 weeks).

Dependent variable: The sodium chloride content of each variety of cheese.

In order to use chemicals during the experiment, it was needed to understand safety and environmental issues before using it. During the procedure, I use chemical safety equipment, which includes coat, googles, mask and gloves. I followed safety data sheet for the proper disposal procedures, in order to reduce environmental impact. Chemicals were not poured down the drain, they were rightly disposed of in designated containers.

1. Potassium permanganate \(\left(\mathrm{KMnO}_{4}\right)\)

• It can cause eye and skin burns.
• Acute or chronic health hazards result from the substance.
• The substance is hazardous to the aquatic environment.

2. Nitric Acid \(\left(\mathrm{HNO}_{3}\right)\)

• It is sensitive to air
• The substance itself does not burn, but in contact with combustible substances it increases the risk of fire and can fuel any existing fire substantially

3. Silver nitrate \(\left(\mathrm{AgNO}_{3}\right)\) (New Jersey Department of Health, 2009)

• It can affect you when inhaled (irritation of nose, throat, lungs) and passing through the skin
• As it is corrosive, contact with it can lead to a serious burning of skin and eyes with possible eyes damage.
• Exposure to silver nitrate can also cause nausea, vomiting, dizziness.

4. Ammonium Thiocyanate ( \(\mathrm{NH}_{4} \mathrm{SCN}\) ) (New Jersey Department of Health and Senior Services, 1996)

• Ammonium Thiocyanate can affect you when breathed in and may be absorbed through the skin. Contact can irritate and burn the skin and eyes.
• Breathing Ammonium Thiocyanate can irritate the nose and throat.
• Repeated exposure can cause headache, nausea, vomiting, loss of appetite and weight loss.

Very small quantity of each cheese type was used for the experiment, while the rest was utilized for food consumption.

For the experiment, 6 different varieties of cow cheese were acquired at the local supermarket, aged for varying periods of time before being put on the market. Cheeses were selected with different published aging times (0-5 weeks) in order to have a range of ages to test from. For each cheese, 5 repetitions of the Volhard titration were completed, for a total of 30 tests. 5 titrations were completed for each cheese in order to have enough data to eliminate potential outlier data. Before the trial, experiment was tried with Mohr's method, however obtained results were inaccurate as it can only be carried out between pH 7 and 10. In acidic conditions, chromate \(\mathrm{CrO}_{4}{ }^{2-}\) is converted to chromic acid which does not react with excess silver ions. ("Mohr's and Volhard's Method of Precipitation Titration - HSC Chemistry," n.d.). Volhard's method was chosen as very useful for analysis of a halide. This is because the acidic pH , at which Volhard's method must be completed, halide analysis is more effective since other positively charged ions such as carbonate do not interfere with the analysis (Collegedunia, 2023). Other advantages of Volhard's method are the fact that it has a very distinct color change, and also it provides accurate results due to back-titration.

Glassware and equipment:

• Standard titration stand with \(10 \mathrm{~cm}^{3}\) burette (uncertainty \(= \pm 0.025 \mathrm{~cm}^{3}\) ) and burette holder
• \(250 \mathrm{~cm}^{3}\) Erlenmeyer flask
• \(10 \mathrm{~cm}^{3}\left( \pm 0.1 \mathrm{~cm}^{3}\right)\) measuring cylinder
• \(25 \mathrm{~cm}^{3}\left( \pm 0.5 \mathrm{~cm}^{3}\right)\) measuring cylinder
• \(50 \mathrm{~cm}^{3}\left( \pm 0.5 \mathrm{~cm}^{3}\right)\) measuring cylinder
• \(100 \mathrm{~cm}^{3}\left( \pm 0.5 \mathrm{~cm}^{3}\right)\) measuring cylinder
• \(1 \mathrm{~cm}^{3}\) pipette
• Hot plate for boiling

Cheese samples from the local area (standard aging length in parentheses):

• Low-fat curd cheese ( 0 weeks), full-fat curd cheese (1 week), Pljevaljski cheese ( 2 weeks), Jastrebački cheese ( 3 weeks), Sjenički cheese ( 4 weeks), Zlatar cheese ( 5 weeks)

Solutions:

• 1.5 moldm \(^{-3}\) nitric acid solution was prepared (to maintain an acidic solution)
• 0.1 moldm \(^{-3}\) silver nitrate standard solution
• \(\quad 0.1000 \pm 0.0005 \mathrm{moldm}^{-3}\) ammonium thiocyanate standard solution (for the back-titration)
• \(5\%\) potassium permanganate solution (for proper digestion/dissolution of cheese)
• Saturated ferric ammonium sulfate solution (as an indicator)
• Distilled water

Preparation of \(\mathrm{HNO}_{3}\) acid: \(104.53 \mathrm{~cm}^{3}\) of \(65\%\) nitric acid, density \(1.391 \mathrm{~g}/\mathrm{cm}^{3}\) was diluted to \(1 \mathrm{dm}^{3}\) with distilled water.

Preparation of \(5\% \mathrm{KMnO}_{4}\) solution: To prepare \(150 \mathrm{~cm}^{3}\) of a \(5\%\) solution of \(\mathrm{KMnO}_{4}\) it's needed to dissolve 7.7415 g of \(\mathrm{KMnO}_{4}\) in distilled water. After the solid is completely dissolved, the solution was diluted to a final volume with distilled water.

Preparation of saturated ferric ammonium sulfate solution: 10 g of \(\mathrm{FeNH}_{4}\left(\mathrm{SO}_{4}\right)_{2} * 12 \mathrm{H}_{2} \mathrm{O}\) is dissolved in the mixture of \(80 \mathrm{~cm}^{3}\) of water and \(10 \mathrm{~cm}^{3}\) of \(\mathrm{HNO}_{3}(1:1)\).

Procedure (Mitrovic, 1954):

1. Precisely measure out 2.00 g of the cheese sample and place into a \(250 \mathrm{~cm}^{3}\) Erlenmeyer flask.
2. In the same flask, \(10 \mathrm{~cm}^{3}\) of \(0.1 \mathrm{moldm}^{-3}\) silver nitrate solution as well as \(25 \mathrm{~cm}^{3}\) of \(1.5 \mathrm{moldm}^{-3}\) nitric acid solution. Put the flask on the hot plate and begin boiling.
3. As the solution boils, slowly add \(5 \mathrm{~cm}^{3}\) of \(5\% \mathrm{KMnO}_{4}\) solution. Adding these drops will cause color to form, do not add more until color has disappeared. If color stops forming after drops have been added, take the solution off the hot plate.
4. Once the solution stops boiling, check for undigested cheese particles on the surface. If some remain, repeat step 3 until all cheese is digested in the solution. There will be white silver chloride precipitate on the bottom of the flask. This means that all excess silver nitrate will be in the aqueous portion of the solution, and is ready to be titrated.
5. Filter the solution to remove the silver chloride precipitate, and put the filtrate into a conical flask. Add \(150 \mathrm{~cm}^{3}\) of distilled water and \(1 \mathrm{~cm}^{3}\) of the ferric ammonium sulfate solution for an indicator. Place the flask under the burette in the titration stand.
6. Titrate the solution using 0.1000 moldm \(^{-3}\) ammonium thiocyanate in the burette until an endpoint is reached. The endpoint of this reaction is reached when the solution turns dark red due to the \(\mathrm{FeSCN}^{2+}\) complex being formed indicating there is no more silver nitrate in solution.

Qualitative observations

Younger cheeses such as low-fat curd, full-fat curd and Pljevaljski were clearly softer and moister as they haven't undergone an extended aging process. In contrast to that, remaining cheeses were firmer due to the processes of maturation and loss of moisture contributing to the formation of a denser structure. Especially in Zlatar cheese, holes in cheese were obvious.

When mixing in the potassium permanganate, there was a foul odor occurring in the vicinity. This indicates the cheese was properly being digested in the solution. In addition, at the endpoint of titration, the red color remained in the solution for some time, indicating that the titrations were performed in the right environment- a higher temperature in the laboratory would have been indicated by the color fading quickly due to the decomposition of the \([\mathrm{FeSCN}]^{2+}\) complex. In the tables below, there are raw data about the initial and final volumes of NH4SCN, after which comes the processed data for volumes of NH4SCN used for each type of cheese. This put a good surface for the calculation of mass of NaCl in 100 g in each cheese type which is visible in table 4, after which I calculated uncertainties and errors in the tables 5,6 and 7.

In order to calculate the average volume of ammonium thiocyanate used to reach the equivalence point in each sample, the following sample calculation was used (Cheese 1 used for sample calculation):

\[\Delta \text { Vaverage }=(0.85+0.85+0.85) / 3=\mathbf{0.85} \mathbf{cm}^{\mathbf{3}}\]

To calculate the chloride content and thus the sodium chloride content, stoichiometric calculations must be done (Cheese 1):

Volume of \(\mathrm{AgNO}_{3}\) that reacted with chloride: \(10.0 \mathrm{~cm}^{3}-0.85 \mathrm{~cm}^{3}=9.15 \mathrm{~cm}^{3}=0.00915 \mathrm{dm}^{3}\)

Moles of silver nitrate \(=0.1 \mathrm{~mol} \mathrm{dm}^{-3} \times 0.00915 \mathrm{dm}^{3}=9.15 \times 10^{-4} \mathrm{~mol} \mathrm{Ag}\)

Because of the \(1:1\) stoichiometric ratio of \(\mathrm{Ag}:\mathrm{Cl}\) and Cl to NaCl, this means \(9.15 \times 10^{-4} \mathrm{~mol}\) chloride ions were present in solution, or \(9.15 \times 10^{-4} \mathrm{~mol}\) of sodium chloride in the original cheese sample.

Mass of sodium chloride in cheese 1 (full-fat curd cheese):

\(\mathrm{m}=\mathrm{n} \times \mathrm{M}=9.15 \times 10^{-4} \mathrm{~mol} \times 58.44 \mathrm{~g} \mathrm{~mol}^{-1}=0.0535 \mathrm{~g}\) in the 2.00 g sample

(Molar mass of sodium chloride \(\left.=58.44 \mathrm{~g} \mathrm{~mol}^{-1}\right)\)

\(0.0535 / 2.00 \times 100 \%=2.67 \%\) of sodium chloride by mass, e.g. \(2.67 \mathrm{~g} \mathrm{NaCl} / 100.0 \mathrm{~g}\) of cheese.

First analysis comes from the Table 4. Highest mass of NaCl in 100 g is seen in the youngest cheese, with the value of 2.67, after which the mass of NaCl in 100 g in cheese 2 heavily decreases to 2.23. Then, inward trend from \(2^{\text{nd}}\) to \(5^{\text{th}}\) cheese time comes up. Again, the \(6^{\text{th}}\) type of cheese doesn't correlate to the inward trend, as the mass of NaCl in 100 g of cheese 6 is lower than in \(5^{\text{th}}\) cheese. Finally, in a graph above, it's the same, as it's obvious that upward trend between the \(1^{\text{st}}\) and \(4^{\text{th}}\) week (2-5 cheese types) can be observed, with the first and last cheese type deviating from it. This leads to the conclusion that aging process influence the sodium content in cheese. Reasons for deviation of first and last type of cheese will be discussed in Evaluation section.

Total % uncertainty was calculated on the cheese 1 as followed:

%uncertainty for \(\mathrm{V}_{\text{AgNO3}}-\mathrm{V}_{\mathrm{NH}4\mathrm{SCN}}=\frac{0.15 * 100\%}{9.15}=1.639 \approx \mathbf{1.6\%}\)

%uncertainty for mass of cheese which is a constant ( \(2.00 \pm 0.01 \mathrm{~g}\) ), so %uncertainty for mass of all cheeses will be the same.

%uncertainty for mass of cheese \(=\frac{0.01 * 100\%}{2.00}=\mathbf{0.50\%}\)

Total uncertainty for mass of NaCl in 100 g of cheese is: \(1.6+0.50=\mathbf{2.10\%}\)

In the table below are present total % uncertainties for each cheese type.

In order to calculate the percentage error for mass of NaCl in each type of cheese, it's needed to use formula:

\[\text { Percentage error }=\frac{(\text { Experimental value-Theoreticalvalue })}{\text { Theoretical value }} * 100\%\]

Experimental value is obtained by conducting the experiment, so for the cheese 1 it's 2.67 g in 100 g of cheese. I found theoretical value for \(\% \mathrm{NaCl}\) in different types of cow cheeses from \(0-5\) weeks of aging process (Ostojic & Jovic, Dairy Institute, 1981) translated from (Ostojić & Jović, PROUČAVANJE TEHNOLOGIJE KRIVOVIRSKOG SIRA, 1981)

In the table below, theoretical values for \(\%\) of NaCl are shown.

So, for the first cheese, percentage error would be:

\[\% \text { error }=\frac{2.67-2.27}{2.27} * 100\%=17.6\%\]

In the table below, all values of % error are shown.

Upon examination of the graph of data points, mass of NaCl in 100 g of cheeses clearly increases from 1-4 weeks of aging process, so it is clear that the length of the cheese aging process does affect the mass of sodium chloride in the cheese. Interestingly, the cheese with the highest sodium chloride content was the semi-fat curd cheese, which was also the cheese aged for the shortest period of time, which should naturally lead to the shortest shelf life, since aged cheeses are more acidic leading to slightly more bacterial resistance (Fulton, 2021). It is possible that the manufacturers of this cheese have added the most salt in order to preserve the cheese and artificially increase its shelf life, something otherwise not possible due to the lack of aging. Another possibility is that the youngest cheese contained a lot of water, which would lead to the increase of NaCl content in it.

Although percentage errors of NaCl for \(1^{\text{st}}\) and \(3^{\text{rd}}\) cheese are high, this doesn't necessarily mean that the results of the experiment aren't accurate. As mentioned in the background, values for \(\% \mathrm{NaCl}\) in cheese vary between 0.96 and \(4.42\%\). Both theoretical and my experimental values for all cheese calculated belong to this category.