II.

Qualitative Tests for the Components of Saliva

1. Carbohydrate
Carbohydrate
Place about 1/2 of the isolated mucin in a test tube. Add 1 mL of 3 M HCI and
immerse in a boiling water bath for 20 to 30 minutes. Cool and make faintly basic
with 3 M NaOH, then add 3 mL of Benedict's solution. Heat again in a boiling water
bath until a visible change occurs

Observation:
Benedict's test is the most used qualitative test for carbs and can identify reducing
sugars like glucose, fructose, and maltose. A little sample of saliva is heated while being
mixed with the Benedict's reagent. If there are reducing sugars in the saliva, they will
react with Benedict's reagent to create a colored precipitate, signifying a successful
outcome. Depending on how many reducing sugars are present, the precipitate color
can change a positive Benedict's test result.
As carbohydrates are a typical component of saliva, their presence in saliva may signal
that the salivary glands are working normally. A considerable increase in the number of
carbs, however, may be a sign of an underlying medical issue, such as diabetes or
insulin resistance. Hence, additional medical examinations and testing may be required
to ascertain the reason for the elevated quantities of carbohydrates in the saliva.

2. Protein
Dissolve the remaining portion of mucin in 1 mL of 2.5 M NaOH, then add three drops of
0.05% CuSO4 solution. Mix.

OBSERVATION:
- Protein: In our experiment for testing the carbohydrates, we first followed the
procedure, which was to dissolve the remaining portion of mucin in 1 mL of 2.5M
NaOH, then add 3 drops of a 0.05% CuSO4 solution. Then we mixed it, observed
the color change, and recorded the result. Mucin is a type of protein found in the
saliva. Its functions in the oral cavity include producing proteins against
desiccation and the environment, adding lubricants, and having antimicrobial
effects against potential pathogens. In the experiment, there was a reagent called
biuret, which is a test that is used to detect the presence of proteins in peptide
bonds, or proteins. Therefore, the biuret test can also be used to gauge the
amount of protein present in the analyte. Protein is a macromolecule and an
organic polymer that is composed of the monomer group known as amino acids.
A monomer is a smaller set of molecular units which make up polymers. Proteins
and amino acids are molecules composed of the elements carbon, hydrogen,
oxygen, and nitrogen. An amino acid has a general structure of an amino group
(CH−NH2) also called the N-terminus, a carboxyl group (-COOH) also called the
C-terminus, and a side chain or R group - which is unique to each amino acid.
The 'R' for R group is often represented as attached to the amino group
 (R-COOH). The mucin is a protein, which would result in a positive reaction
resulting in a purple color. Immunoglobulins, albumins, glycoproteins, and the
protein components of enzymes make up the majority of the proteins found in
saliva.

3. Nitrate
Mix 1 ml of 0.05 N H2 SO4 two drops of 1% KI and two drops of starch solution. Add 2 ml of
saliva and mix

Water pollution is a major global problem, and regular monitoring of pollutant ions is important to
keep groundwater and drinking water safe. Nitrate ions have a significant impact on human
health and the environment, and excessive use adversely affects ecosystems and the natural
environment. Nitrate can be detected by laboratory-based methods or sensor-based in situ
methods to develop monitoring systems. Some of these systems have high sensitivity and high
limits of detection (LOD), but the instruments are expensive. If the proposed detection method
uses a sensor-based handheld detection system, others have reasonable sensitivity at low cost.
This review describes various detection and measurement methods for nitrate in water. Various
properties of detection methods. B. Includes LOD and system limits. The thinner the reactant
solution, the slower the reaction. The most important thing to understand here is that chemical
reactions depend on collisions of reactant particles (collision theory). The more concentrated a
reactant is, the more likely it is to collide with other reactants and cause a chemical change.
In the test, various densities of Na2S2O3 React with HCl. This experiment includes a timer so
you can see how changing the concentration changes the overall rate of reaction.
In our experiment for Nitrate, we followed first the procedure which is Mix 1 ml of 0.05 N H2
SO4 two drops of 1% KI and two drops of starch solution. Add 2 ml of saliva and mix. As we
observed, after a minute of mixing, the solution's color turned into yellowish color or pale yellow.
When KI ( potassium iodide) is added together with the starch solution, the solution's color starts
to change into light purple. We also observed a black strand in the solution and after a few
seconds, it slowly disappeared.

4. Thiocyanate

To two drops of saliva, add three drops of freshly prepared 0.1 M FeCl3 solution and a few
drops of 1 N HCI.p

Observation:
 In our experiments, adding 2 drops of saliva to 3 drops of 0.1M FeCl3 solution and a few
drops of 1N HCl resulted in a yellowish color, indicating the presence of thiocyanate ions in
saliva. Salivary thiocyanate levels as a potential biomarker: Salivary thiocyanate levels can be a
useful biomarker for identifying smokers and tobacco smoke exposure. Saliva samples are
readily available and can be collected non-invasively, making them a convenient tool for routine
health screenings. In addition, saliva samples are easy to store and more stable than other
bodily fluids such as blood, urine, and serum. Effect of e-cigarette use on thiocyanate levels:
E-cigarette use is marketed as a safer alternative to smoking traditional cigarettes. However,
recent studies have shown that e-cigarettes also release harmful chemicals such as
thiocyanate. Several studies have reported elevated salivary thiocyanate levels in e-cigarette
users compared to nonsmokers, suggesting e-cigarettes may not be as safe as previously
thought. It has been.

Health Effects of Elevated Thiocyanate Levels:

Thiocyanic acid is a toxic substance and can have adverse effects on human health.
Elevated thiocyanate levels are associated with an increased risk of developing a variety of
health conditions, including cardiovascular disease, cancer, and respiratory problems.
Monitoring helps identify individuals at risk of developing these diseases. Importance of quitting
smoking: Quitting smoking is the most effective way to reduce exposure to harmful chemicals
like thiocyanate. Smoking cessation programs that combine behavioral support and medication
have proven to be highly effective in helping smokers quit. In addition, salivary thiocyanate
levels can be used by healthcare professionals to help smokers quit and monitor their progress.

The present study has several limitations that should be considered when interpreting
the results. The sample size was small, and the study did not consider confounding factors such
as age, gender, or other lifestyle factors. Additionally, the study did not consider the impact of
environmental factors such as air pollution. This also affects thiocyanate levels in the body.
Therefore, future studies using larger sample sizes and more rigorous methods are needed to
confirm the results of this study.

In this study, salivary thiocyanate levels in smokers were estimated and compared with
e-cigarette users, nonsmokers and smokers. To better understand how smoking affects the oral
dose of thiocyanate, we studied human saliva. 1N HCL.p was added to prepare the thiocyanate.
In our experiments, two drops of saliva He added to three drops of 0.1M FeCl3 solution and a
few drops of 1N HCl gave a yellowish color, indicating the presence of thiocyanate ions in
saliva. Saliva is a biological substance that can be used as a simple, non-invasive diagnostic
tool to detect disease. Taking a saliva sample is quick, easy and above all painless. Saliva has
historically been used to identify a variety of autoimmune diseases, including diabetes, heart
disease, tooth decay, and other oral conditions.

Saliva sample results show a dangerous correlation between a person's relative

exposure to cigarette smoke and the amount of thiocyanate in the body. Thiocyanate as an
additive in compositionally engineered perovskite films has been validated and its impact on
perovskite solar cell performance has been studied. A perovskite precursor spiked with a small
amount of formamidinium thiocyanate is deposited by a one-step solution process to produce
perovskite films. We find that the domain size increases significantly when thiocyanate ions are
incorporated into perovskite films. In addition, trace amounts of thiocyanate groups on
perovskite films, measured by time-of-flight secondary ion mass spectrometry, identify
thiocyanates present within the perovskite mass, especially near the bottom of the film.

5. Glucose
Using a saliva sample collected 15 minutes after eating, add 1 mL of Benedict’s solution to 2 mL
of saliva, then heat in a boiling water bath.

Observation:

In this experiment where we used a saliva sample collected 15 minutes after eating, add 1 mL
of Benedict’s solution to 2 mL of saliva, then heat in a boiling water bath we observed that
reducing sugar results in a favorable outcome since the solution's color changes from blue to
brick-red and a brick-red precipitate forms. As the color of the solution changes from yellow to
dark blue during the iodine test, starch yields a good result. The simplest type of carbohydrate,
known as a monosaccharide or simple sugar, is glucose. A polysaccharide, or polymer of sugar,
is a starch made up of many glucose units bound together by glycosidic linkages. The salivary
amylase enzyme, which hydrolyzes starch into glucose and breaks down the glycosidic linkage
between glucose that is found in starch, is best when it is heated to a temperature of 37°C,
which is close to that of the human body. This saliva passes Benedict's test with flying colors.
The salivary amylase enzyme is denatured at 95°C because the enzyme loses its structural
integrity. Small carbohydrates called reducing sugars can operate as reducing agents for metal
ions like Ag+ or Cu2+. They typically include one or two sugar units. Because they oxidize
aldehydes and change color when reduced, these metal salts have long been utilized for testing
purposes. Sugars can interact with these salts because an acyclic aldehyde and their cyclic
hemiacetal functional group are in equilibrium. Acetals are inert. Decide whether or not a sugar
has a hemiacetal to quickly determine if it is a reducing sugar.

6. Inorganic Phosphate

Mix 1 ml of saliva and 1 ml of ammonium molybdate solution. Heat and observe.

Observation:

Mixing 1 ml of saliva and 1 ml of ammonium molybdate solution and heating the mixture
can be used to test for the presence of phosphates in the saliva.

The ammonium molybdate solution reacts with phosphate ions to form a yellow
precipitate of ammonium phosphomolybdate. Heating the mixture can help to accelerate
the reaction and make the results more visible.
 To perform the test, first, add 1 ml of ammonium molybdate solution to 1 ml of saliva in a
test tube. Mix the solution well, and then heat the mixture gently over a flame. You can
use a Bunsen burner or a hot plate for this step.

Observe the solution carefully for any changes in color or appearance. If phosphates are
present in the saliva, a yellow precipitate should form. The intensity of the yellow color
can be used to estimate the concentration of phosphates in the saliva.

It is important to note that other substances in the saliva, such as proteins and amino
acids, can interfere with the test and give false-positive results. Therefore, this test
should be used as a screening tool and confirmed with other methods if necessary.

In summary, mixing 1 ml of saliva and 1 ml of ammonium molybdate solution and heating

the mixture can be used to test for the presence of phosphates in the saliva. The
formation of a yellow precipitate indicates the presence of phosphates in the saliva.
However, the test should be used as a screening tool and confirmed with other methods
if necessary.

7. Chloride

Mix 1mL of saliva with three to five drops nitric acid followed by five drops of AgNO³

Observation:

As we followed the procedure by mixing 1 mL of saliva, then 5 drops of nitric acid followed
by 5 drops of AgNO³ solution which results in a transparent cloudy (vague) substance. The
purpose of this experiment is to measure the amount of components in saliva–chloride.

The chloride ion concentration in the sweat test, which is considered to be the gold standard
indicator for the diagnosis of cystic fibrosis, is a requirement for making the diagnosis of cystic
fibrosis ( 60 mEq/mL). Furthermore, salivary glands express the CFTR protein similarly to sweat
glands.A positive sweat chloride test shows that the child or person being tested most likely has
cystic fibrosis. (CF)

When compared to healthy control subjects, patients with cystic fibrosis had higher sweat and
saliva chloride concentrations (p-value 0.001).
When sweat chloride concentration and saliva chloride concentration were correlated,
Spearman's Rho (correlation coefficient) showed a positive result of 0.475 (95% CI=0.346 to
0.587).
What ion is present in saliva by the above result?

- Based on the result upon the observation, the ion presents in saliva in chloride testing is
chloride ions. Chloride ions are used to activate the salivary amylase and also catalyzes
the body's chemical reactions and helps with digestion.
The opening of Cl- channels in the apical membrane of salivary gland acinar cells
initiates the fluid secretion process, whereas the activation of Cl- channels in both the
apical and the basolateral membranes of ductal cells is thought to be necessary for NaCl
reabsorption.

In order to travel into the luminal compartment, chloride secretion creates an electrical
driving force (lumen-negative) for trans-epithelial sodium secretion via the paracellular
pathways. Together, their movements produce the osmotic driving force for water flow,
which results in an isotonic secretory product.

Substances Tested Tested reagents Results Conclusion

for

Carbohydrate NaOH, Benedict’s Positive - A little quantity of

sol’n, heat the Benedict's test
substance was
sample (in this case,
saliva) is combined
with Benedict's
reagent which is a
solution of
sodium carbonate,
copper sulfate, and
sodium citrate The
mixture is then
heated,
and if there are any
decreasing sugars,
 they will
react with the
reagent's copper
ions.
turning a blue tint to
turn green
depending on the
environment, red,
orange, or yellow
amount of sugar that
reduces.
Thus, if a qualitative
Benedict's test is
performed,
performed on a color
sample and a saliva
sample.
As change happens,
it reveals the
sugars and carbs are
reduced in
the sample.

Protein NaOH, CuSo4 Biuret Reaction - It is a

- The violet characteristic
color is a reaction of
positive test compounds
for the containing a
presence of peptide bond
protein, and (including
the intensity proteins). In
of the color is alkaline
proportional to medium
the number of peptide bonds
peptide bonds form a
in the complex of
solution. violet color
with Cu2+
ions.
- Proteins
present in
saliva are
mainly
immunoglobul
ins, albumins,
glycoproteins
and the
protein
components
 of enzymes.

Nitrate H2SO4, KI, starch Positive - Iodine KI

reagent to the
Carboxylic acids solution or
directly to
and sulfonic acids ingredients
can react with such as
sodium potatoes,
bread,
bicarbonate crackers, and
(NaHCO3) flour. In the
(NaHCO3) to presence of
starch, a
produce carbon blue-black
dioxide and water color is
(Figure 6.51). formed. In the
Other mainstream absence of
functional groups starch
amylose, the
(most phenols color remains
and alcohols) are orange or
not acidic enough yellow. The
to produce a gas amylopectin in
with bicarbonate. starch does
not impart
color, nor
does cellulose
or
disaccharides
such as
sucrose in
sugar.

- Amylose in
starch is
responsible
for forming a
deep blue
color in the
presence of
iodine. The
iodine
molecule
slides into the
amylose coil.
Iodine - KI
Reagent:
- Since iodine
is poorly
 soluble in
water, an
iodine reagent
is prepared by
dissolving
iodine in
water in the
presence of
potassium
iodide. This
creates a
linear triiodide
ion complex
that slides into
the starch
tangles,
resulting in an
intense
blue-black
color.
-

CNS FeCl3 HCI Positive FeCl3 is used to

indicate the presence
of a phenol group by
producing a purple
color. In this
experiment, the
crystalline product
turned colored as a
result of still having
salicylic acid present
because it contains a
phenol group.

Both phenol and

salicylic acid give
violet color with
neutral ferric chloride
solution because it
has a phenolic
compound.

Compounds such as
enols, hydroxamic
acids, sulfinic acids,
and oximes give
positive results.

FeCl3 (iron(III)
 chloride) and HCl
(hydrochloric acid)
can be used together
in various chemical
reactions. For
example, they can be
used to convert
benzene to
chlorobenzene.

Glucose Benedict's reagent, Positive Both simple and

heat complex carbs are
broken down by the
digestive process into
glucose. All of the
cells in the body use
this as fuel. If there is
too much glucose,
the body will either
store it as fat or
consume it right
away. Glucose is
converted to fat by
the liver.
In this experiment,
we'll examine some
common meals and
beverages to see if
we can identify any
that, if any, have a
significant
concentration of
simple carbs.

Benedict's solution is
a specialized reagent
that can be used to
check for simple
carbohydrates like
glucose. Benedict's
solution is blue, but if
there are any simple
carbs, it will turn
another color. Green
or yellow indicates a
low level, while red
indicates a high
amount. Benedict's
solution contains
copper (II) sulphate,
and the Cu2+ ions
are what give the
solution its blue color.
If sugars are present,
a precipitate will also
form, and the amount
of this gives an
indicator as to the
proportion of sugars
in the test sample.
When the mixture is
heated and there are
any simple sugars
present, the copper
will be reduced and a
red copper (I) oxide
precipitate will
appear. These sugars
are referred to as
lowering sugars as a
result.

Benedict’s Test is
used to test for
simple
carbohydrates.
Benedict's test
identifies reducing
sugars
(monosaccharides
and some
disaccharides), which
have free ketone or
aldehyde functional
groups. Benedict’s
solution can be used
to test for the
presence of glucose
in urine. When
glucose is mixed with
Benedict's reagent
and heated, a
reduction reaction
causes the
Benedict's reagent to
change color. The
color varies from
green to dark red
(brick) or
rusty-brown,
 depending on the
amount of and type of
sugar. Glucose never
gives violet colour
precipitate with
Benedict's solution.

Inorganic phosphate Ammonium molbdate Positive Ammonium

molybdate is a salt
that is commonly
used as a reagent in
analytical chemistry,
particularly in the
detection and
quantification of
phosphorus
compounds. When
ammonium
molybdate is mixed
with inorganic
phosphate ions in the
presence of an acid,
it forms a
yellow-colored
complex known as
ammonium
phosphomolybdate.
This complex is
insoluble in water and
can be easily
precipitated out of
solution.

Heating a solution
containing
ammonium
molybdate and
inorganic phosphate
can cause the
formation of
ammonium
phosphomolybdate to
accelerate, resulting
in an increase in the
rate of precipitation.
This can cause the
 solution to appear to
be "boiling" as the
precipitate forms and
settles back down,
creating a turbulent
motion in the
solution.

Chloride HNO3, AgNO3

REFERENCES:
Kalburgi, V.C., Naik. L.K., Kokatnur, V.M., and Warad, S. (2014, April-Jun). Estimation and
correlation of salivary thiocyanate levels in healthy and different forms of tobacco users having
chronic periodontitis: A cross-sectional biochemical study. National Library of Medine. Retrieved
from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067780/#:~:text=Salivary%20thiocyanate%20(S
CN)%20has%20been,to%20blood%20and%20urine%20testing.

Flieger, J., Kawka, J. and Tatarczak-Michalewska, M. (2019, October 21). Levels of the
Thiocyanate in the Saliva of Tobacco Smokers in Comparison to e-Cigarette Smokers and
Nonsmokers Measured by HPLC on a Phosphatidylcholine Column. National Library of
Medicine. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832790/#:~:text=Salivary%20thiocyanate%20co
ncentration%20in%20nonsmokers,3%2C4%2C5%5D.

G. (n.d.). LAB REPORT 12 SALIVARY AMYLASE - PDFCOFFEE.COM. pdfcoffee.com.

https://pdfcoffee.com/lab-report-12-salivary-amylase-pdf-free.html

Digestion and Absorption of Carbohydrates (2022) Libre Text Medicine.

https://med.libretexts.org/Under_Construction/Purgatory/Book%3A_Human_Nutrition_1e_(
University_of_Hawaii)/04%3A_Carbohydrates/4.02%3A_Digestion_and_Absorption_of_Car
bohydrates#:~:text=Saliva%20contains%20the%20enzyme%2C%20salivary,glucose%2C%
20called%20dextrins%20and%20maltose.

L. (2022, July 4). Starch and Iodine. Chemistry LibreTexts.

https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_Modules_
Biological_Chemistry)/Carbohydrates/Case_Studies/Starch_and_Iodine

L. (2022a, April 7). 6.4D: Individual Tests. Chemistry

LibreTexts.https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemi
try_Lab_Techniques_(Nichols)/06%3A_Miscellaneous_Techniques/6.04%3A_Chemical
Tests/6.4D%3A_Individual_Tests
Original article Chloride and sodium ion concentrations in saliva.(4, July–August 2019).
Volume 95, Issue 4, Pages 443-450. Jornal de Pediatria;
https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.sciencedirect.com/sci
ence/article/pii/S0021755717310537&ved=2ahUKEwjnm_f6qo_-AhXPDN4KHSmSD48QFn
oECAwQBQ&usg=AOvVaw1b6vM3tEJxgKQsUzlPC4yr
Jornal de Pediatria

Qualitative Tests for Carbohydrates: Benedict - 511 Words | Studymode. (n.d.). Qualitative
Tests for Carbohydrates: Benedict - 511 Words | Studymode.
https://www.studymode.com/essays/Qualitative-Tests-For-Carbohydrates-Benedict-s-Test-3
66930.html

Ashenhurst, J. (2017, September 12). Reducing Sugars. Master Organic Chemistry.

https://www.masterorganicchemistry.com/2017/09/12/reducing-sugars/

Glucose on reacting with Benedict’s solution may give the following precipitates except.
(n.d.). Toppr Ask.
https://www.toppr.com/ask/question/glucose-on-reacting-with-benedicts-solution-
may-give-the-following/

Salivary amylase is activated by which ion? - Byju's

https://www.google.com/url?sa=t&source=web&rct=j&url=https://byjus.com/question-ans
wer/salivary-amylase-is-activated-by-which-ion/&ved=2ahUKEwjtrrr6lZD-AhVVQPUHHZ
3OApIQFnoECAwQBQ&usg=AOvVaw2seClxcznoP-ZRJ1x9r2PD

Melvin, J.E. (1999). Chloride channels and salivary gland function.

Crit Rev Oral Biol Med.
https://www.google.com/url?sa=t&source=web&rct=j&url=https://pubmed.ncbi.nlm.nih.gov/1075
9422/&ved=2ahUKEwjtrrr6lZD-AhVVQPUHHZ3OApIQFnoECAcQBQ&usg=AOvVaw1eKPdRv1
h3pTcrRWp5IFHr