Department of Medical Laboratory Science

Davao Doctors’ College

BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE

Name: Allana Marie Claire S. Rato Date Performed:

Course/Yr. /Sec.: BMLS-2C Date Submitted:

pH AND BUFFERS

PRE-LABORATORY GUIDE QUESTIONS:

1. Differentiate the following: (2 points each)

a) pH from pOH

pH (potential hydrogen) is used to easily decide if a given aqueous is acidic, basic or

neutral and it is the negative logarithm of hydrogen ion concentration in a solution while
the pOH (potential of hydroxide) is used to express the alkalinity of a solution and it is a
solution of a negative logarithm of hydroxide ion concentration.

b) K a from K b

Ka is an “acid dissociation constant” , it’s about the concentration of H+ ions and the
quantitative measure of the acid strength of the solution while the Kb is a “base
dissociation constant” , it’s about the concentration of OH- ions and the bases of
constant measure on how the base dissociates completely into its water component
ions.

c) p K a from p K b

The pKa value is a tool used to demonstrate the intensity of the acid. PKa
is the negative log of the acid dissociation constant or the Ka value. The
lower pKa value means a higher acid value. In other words, the lower
meaning means that the acid is more thoroughly dissociated in water while
the pKb is the negative base logarithm of the base dissociation constant
(Kb) of the solution and it is used to identify the strength of a base solution
or an alkaline solution.

d) K w from p K w
 Kw is called a water autoprotolysis constant or water autoionization while the pKw
is the negative logarithm of Kw

e) Molarity from normality

Molarity defines the moles of a total substance per liter of solution while
normality just defines the moles of the reactive species per liter of solution.
Normality is often a multiple molarity.

2. Why are pH and buffer important in biological systems? (3 points)

pH is an essential in the biological system because it regulates the formation or structure and
activity of macromolecules and also the buffer is important in the biological system because it is
the one who sustains the moderation changes of PH to maintain the constant pH conditions.

3. What is the Henderson-Hasselbalch equation? (3 points)

4. What are the approximate pH ranges and color changes of the following organic dyes? (2 points
each)

INDICATOR pH RANGE COLOR CHANGE

Thymol blue (acid range) 1.2 - 2.8 Red to yellow

Bromophenol blue 3.0 - 4.6 Yellow to blue

Bromocresol green 3.8 - 5.4 Yellow to blue

Bromocresol purple 5.2 - 6.8 Yellow to purple

Phenol red 6.5 - 8.4 Yellow to red

Thymol blue (alkaline 8.0 - 9.6 Yellow to blue

range)

Methyl red – methylene 4.4 - 6.3 Reddish violet to green

blue
 Methyl orange 3.2 - 4.4 Red to yellow

Phenolphthalein 8.2 – 10 Colorless to pink

5. How is the concentration of a buffer solution determined? (3 points)

The pH of the buffer is determined by two factors:

1. the steady equilibrium Ka of the weak acid and

2. the ratio of the weak base [A-] to the weak acid [HA] in the solution.

POST LABORATORY GUIDE QUESTIONS:

1. Why is there a discrepancy between the calculate pH and the experimental pH of a prepared
buffer solution? (3 points)

2. Why is it necessary to calibrate the pH meter? (3 points)

pH meter calibration is a required step to use the pH meter regardless of how the
electrode adjusts with time. Regularly calibrating your pH meter will adjust your
electrode depending on any adjustments that may have happened to ensure that your
measurements are correct and repeatable.

3. Why is the electrometric method of pH determination more accurate than the colorimetric
method? (3 points)

The method of mass loss is more precise since any unit of the substance that has
undergone a reaction is weighed. The colorimetric method of pH determination uses
acid-based indicators to have a variety of pH values for the solution and is thus less
precise than the electrometric method.

4. What is the effect of dilution on the pH of a buffer and the buffer’s capacity? (3 points)

Dilution of the buffer solution will limit its buffer capacity. You will quickly note that the pH shift
is more significant when the acid and conjugate base concentrations are diluted.

5. A solution is prepared by mixing 80 mL of 0.2 M KH 2PO4 and 120 mL of 0.2 M KH2PO4. what is its
pH. Show your solution. (5 points)

Using Henderson-Hasselbach Equation pH = pKa + log (Base/Acid)

 (Base) Na2HPO4 = (0.2 mmol/mL) x (120 mL) = 24 mmol

(Acid) NaH2PO4 = (0.2 mmol/mL) x (80 mL) = 16 mmol

pH = 7.21 + log(24/16) = 7.21 + 0.176 = 7.386 = 7.4

References:

Helmenstein, R. (2020, September 30). Chemistry Notes. Science Notes Learn Science Do Science.
https://sciencenotes.org/ph-pka-ka-pkb-and-kb-in-chemistry/

Helmenstein, R. (2020, October 07). pH Indicator Chart – Colors and Ranges. Science Notes Learn
Science Do Science. https://sciencenotes.org/ph-indicator-chart-colors-and-ranges/

Gunawardena, G. (2020, August 24). Henderson-Hasselbach Equation. Chemistry Libretexts.

https://chem.libretexts.org/Bookshelves/Ancillary_Materials/Reference/Organic_Chemistry_Glossary/
Henderson-Hasselbach_Equation
 Department of Medical Laboratory Science
Davao Doctors’ College
BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE

Name: Allana Marie Claire S. Rato Date Performed:

Course/Yr. /Sec.: BMLS-2C Date Submitted:

ISOLATION AND CHARACTERIZATION OF CARBOHYDRATES

PRE-LABORATORY GUIDE QUESTIONS:

1. Define the following terms briefly. Cite an example for each using the Fischer/Haworth
projection formula. (3 points each)
a. Aldohexose
Aldohexose is a hexose with an aldehyde group at one end.
Aldohexoses have four chiral centers for a total of 16 potential aldohexosis
stereoisomers (2). Of these, only three are popular in nature: D-glucose, D-
galactose, and D-mannose.

b. Reducing sugar
Reducing sugar is a carbohydrate that is oxidized by a weak oxidizing agent (an
oxidizing agent capable of oxidizing aldehydes but not alcohols, such as the Tollen
reagent) in a simple aqueous solution. The characteristic feature of reducing sugars
is that they create one or more aldehyde-containing compounds in an aqueous
medium.
 c. Hemiacetal

Hemiacetals are formed from aldehyde (or ketone) and one molecule of alcohol,
with the forming of a single ether bond and an OH group to the same carbon atom
(from the carbonyl group).

2. Complete the table below. (2 points each)

Test Expected Observation (Positive) Substance Indicated by a

Positive Test

I 2 solution Blue Carbohydrates and

Polysaccharide

Molisch’s Dark violet region between Carbohydrates and

junctions of two layers Polysaccharide

Benedict’s Red precipitate Reducing sugar

 Seliwanoff’s Cherry Red Ketones

Bial’s orcinol Blue-green product Pentoses

Barfoed’s White red in blue solution Monosaccharide

3. Which test can be used to differentiate the following pairs of carbohydrates? (1 point each)
a. Fructose and Galactose - both can be performed in barfoed’s test
b. Ribose and Glucose - both can be performed in Bial’s test
c. Glucose and Maltose – both can be performed in Barfoed’s test

4. Describe the reaction the takes place when starch is hydrolyzed by an enzyme. What is the
product of starch hydrolysis? (5 points)

In the presence of amylase, a sample of starch is hydrolyzed to shorter polysaccharides,

dextrins, maltose and glucose. The degree of the hydrolysis depends on how long it can
react, once the starch is fully hydrolyzed, the resulting substance is glucose.

POST LABORATORY GUIDE QUESTIONS:

1. How are carbohydrates formed in nature? Write the reaction for this process. Are animals able
to synthesize carbohydrates? (5 points)

Carbohydrates are produced by green plants from carbon dioxide and water plants
during the photosynthesis process. Carbohydrates are used as energy sources and as
important structural elements in organisms; in addition, much of the nucleic acid
structure, which includes genetic material, consists of carbohydrates. Carbohydrates are
initially synthesized in plants by a complex sequence of reactions involving
photosynthesis. -Store energy in the form of starch (photosynthesis in plants) or
glycogen (in animals and in humans).

2. What test should be performed to identify a disaccharide? Discuss its principle. (3 points)
Barfoed’s test, It is used to recognize monosaccharides from disaccharides. This test demands
that the glucose be a decrease in sugar as a reduction in the reagent happens in order to classify
the remedy. It is also capable of separating monosaccharides from disaccharides.

3. An unknown carbohydrate solution tested (+) with Molisch’s reagent formed a red ppt. with
Barfoed’s reagent within 3 minutes and turned blue-green when mixed with Bial’s orcinol
reagent. What is this carbohydrate? Insert its Haworth projection structure. (5 points)
 4. Erythrose is an aldostetrose. Describe the result when it is subjected to each of the following
test: (3 points each)

a. Molisch’s Test
In this procedure, erythrose or tetrose does not give a positive reaction.

b. Barfoed’s Test

A small volume of brick red precipitate is observed in a positive reaction.

c. Benedict’s Test
Red cuprous oxide

d. Seliwanoff’s Test
A cherry red color is being used in a positive reaction

5. What product is formed when the aldehyde end of glucose is oxidized? (2 points)

If the aldehyde end of the molecule is oxidized, the name of the compound is – onic acid. When
the aldehyde end of glucose is oxidized, the compound is called gluconic acid. If the alcohol is
oxidized at the end opposite of aldehyde, the product is called uronic acid.

6. How do hexoses affect alkaline Cu2+ complex ions, and what use is made of this reaction? (3
points)

Sugars containing aldo or keto can reduce copper in alkaline solutions (Fehling solution) to
create a brick-red coloring of cuprous ions. These sugars are considered reducing sugars and the
reaction, while not unique to reducing sugars, is used for both qualitative and quantitative
purposes.

7. Do all three disaccharides act as reducing agents? Why or why not? (3 points)

No because sucrose is not sugar-reducing, but both maltose and lactose are. Reducing agents
are giving electrons up to other molecules. Aldehyde (double-bonded oxygen) groups do this.

References:
 Department of Medical Laboratory Science
Davao Doctors’ College
BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE

Name: Allana Marie Claire S. Rato Date Performed:

Course/Yr. /Sec.: BMLS-2C Date Submitted:

PHYSICAL AND CHEMICAL PROPERTIES OF LIPIDS

PRE-LABORATORY GUIDE QUESTIONS:

1. Based of polarity, what are the types of lipids present in egg yolk? Provide examples for each
type. (5 points)

The yolk lipid fraction consists of 66 percent triglycerides, 28 percent phospholipids, 5 percent
cholesterol such as and minimal concentrations of other lipids.

Triglycerides - stearic acid, oleic acid, palmitic acid

Phospholipids – phosphatidylcholine and fatty acid

Cholesterol – fatty acids

2. Discuss the principle involved in the Liebermann-Burchard test. Explain the importance of each
reagent used in this test. (5 points)

The Liebermann – Burchard or acetic anhydride test is used to detect cholesterol. The creation
of a green or green-blue color after a few minutes is positive. Lieberman – Burchard is a reagent
used in a colorimetric test to measure cholesterol, which gives a vivid green color. This color
starts with a purplish, pink color, going through a light green color and then a very dark green
color. The color is due to the hydroxyl group (-OH) of cholesterol reacting with the reagents and
increasing the conjugation of the non-saturation of the adjacent fused ring because this test
uses acetic anhydride and sulfuric acid as reagents, vigilance must be exercised so as not to
inflict serious burns.

3. What is a cholesterol? Explain its good and bad effects. (5 points)

Cholesterol is a fat-like material in the body. It is created by the body which is also present in
food. While it is required for good health, too much cholesterol will weaken your arteries and
increase your risk for heart disease. It’s not inherently “bad.” Your body needs it to build cells
and make vitamins and other hormones. But too much cholesterol can pose a problem.
 Cholesterol comes from two sources your liver makes all the cholesterol you need. The
remainder of the cholesterol in your body comes from foods from animals. For example, meat,
poultry, and dairy products all contain dietary cholesterol these same diets are rich in saturated
and trans fats, these fats make the liver more cholesterol than you might otherwise have. For
certain individuals, this increased production means that they go from a normal amount of
cholesterol to a level that is not safe. Few tropical oils – such as palm oil , palm kernel oil and
coconut oil – contain saturated fats that can raise the amount of bad cholesterol. These oils are
commonly used in baked goods.

4. What are the etiologies of the following disease and provide their pathophysiology: (5 points
each)

a) Gaucher’s disease

Gaucher disease is a rare genetic disorder caused by a lack of enzyme called

glucocerebrosidase. It results to excessive lipid buildup in the spleen, liver, lungs, bones
and, in other cases, the brain. An individual with Gaucher 's disease inherits a mutated
copy of the GBAgen from either of his or her parents.

It Classified into three forms, Types 1, 2 and 3. Type 1 being the most common category.
It causes enlargement of the liver and spleen, bone pain, and fractures. Sometimes it
involves complications with the lungs and kidneys. Type 2 typically occurs in children
and induces significant brain damage. Affected babies do not grow up to the age of 2. In
Type 3, the enlargement of the liver and spleen manifests, the brain is progressively
affected.

b) Neimann-Pick disease

It refers to a group of genetic metabolic disorders that cause excessive accumulation of

lipids in the brain, spleen, liver, lungs, and bone marrow. Caused mutations in various
genes related to the body's metabolism of fat (cholesterol and lipids). The defects in the
Niemann-Pick gene are transmitted from parents to children in a pattern called
autosomal recessive inheritance.

Classified into three types, the symptoms of type A, B and C can include: lack of muscle
tone, brain degeneration, heightened sensitivity to touch, spasticity, and slurred
expression.
Type A, the most extreme form, happens during adolescence in most Jewish
communities. Children afflicted by this condition typically survive up to 18 months of
age.
Type B typically happens in pre-teen years. An enzyme called sphingomyelinase is
responsible for the build-up of toxic quantities of sphingomyelin, which happens in type
A and type B.
 Type C occurs in late adolescents and adults, a deficiency of NPC1 or NPC2 protein
causes significant brain injury and failure to look up and down, trouble walking and
chewing, and gradual loss of vision and hearing.

c) Respiratory distress syndrome

A type of neonatal respiratory disease that is most commonly cause by a lack of a

surfactant in the lungs. The fetus' lungs tend to make surfactants during the third
trimester of pregnancy, or at weeks 26 through labor and childbirth. Surfactant coats
the insides of air bags, or alveoli, in the lungs. Inadequate pulmonary surfactants are the
main source of RDS. The structurally immature and surfactant-deficient lungs have
decreased compliance and a susceptibility to atelectasis; other causes in preterm infants
that raise the risk of atelectasis include shortened alveolar radius and poor chest walls.

d) Taysach’s disease

Defective chromosome 15 gene (HEX-A) cause Tay-Sachs disease. This faulty gene
causes the body not to produce a protein called hexosaminidase A. Without this
enzyme, the molecules called gangliosides build up in the brain's nerve cells, killing brain
cells.

Tay-Sach 's disease is inherited in an autosomal recessive way. The disease arises from
variations (mutations) in the gene known as the HEXA gene, which controls the
development of the enzyme hexosaminidase A.

POST LABORATORY GUIDE QUESTIONS:

1. Differentiate thin-layer chromatography from column chromatography based on (1) the

separation of their components in the stationary and mobile phases, (2) solvent
migration, and (3) type of support used. (2 points each)

Thin layer chromatography (TLC) is a chromatographic method used to distinguish the

components of a mixture using a thin stationary phase supported by an inert surface. It
can be carried out on an analytical scale as a way of tracking the success of a reaction or
on a preparatory scale for the purification of small quantities of a substance. In all
chromatography there is a mobile phase and a stationary phase. The stationary phase is
the phase that doesn't move and the mobile phase is the phase that does move. The
mobile phase moves through the stationary phase picking up the compounds to be
tested. Thin layer chromatography is achieved exactly as it says-using a thin, uniform
layer of silica gel or alumina coated on a sheet of glass, metal or rigid plastic.

The silica gel (or aluminum) is a stationary process. The stationary phase of thin-layer
chromatography often frequently involves a material that is fluorescent in UV light. The
mobile phase is an effective liquid solvent or solvent mixture.
 2. What is the purpose of using ninhydrin and iodine in the thin-layer chromatography? (3
points)

Identification of amino acid is important in the assessment of protein structure. Thin-

layer chromatography (TLC) is an effective and affordable technique used to classify
amino acids. Amino acids are colorless molecules that can be detected on a
chromatogram using the Ninhydrin reagent. Ninhydrin reacts with amino acids to give
coloured compounds, mainly brown or purple.

References: