University of Santo Tomas

Biochemistry
Faculty of Medicine and Surgery John Mark
Villena
Class of 2017 Section D

Laboratory Reviewer 1

Experiment 1: Identification of Sugars by Paper Chromatography

Chromatography
● technique where solutes are separated by differential migration through a porous medium
● components of a solution are separated as a result of differential affinity to;
o stationary phase- can be solid or liquid
o mobile phase- can be liquid or gas

Capillary Action
● the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and
surface tension.

Solubility
● the degree to which a material (solute) dissolves into a solvent
● if a solute dissolves into a solvent, it is soluble in it hence it has similar properties
● polar solvent can only dissolve polar solute, non-polar solvent can only dissolve non-polar solutes. (Like
dissolves like)
● this allows different solutes to have different affinities with the mobile and stationary phases which have
different polarity

*​Liquids of the mobile phase​ can travel thru a porous material by capillary action
*Solutes that are soluble in the l​ iquids of the mobile phase​ are taken along (mobile) as the liquid travels
*Solutes that are soluble in the stationary phase are adsorbed in place (stationary), hence they do not travel

Paper Chromatography
● A type of partition (separation) chromatography which uses p ​ aper (​ Whattman paper no. 1 ​(no.1 relates to
pore size)​)​ ​as the “porous medium” where solutes are separated
● components of a solution are separated as a result of differential affinity to the;
o stationary phase
▪ water trapped in the interstitial spaces of the paper (​NOT​ the paper itself)
▪ fibers of the paper has cellulose which binds with water via H-bonds
▪ polar liquid
o mobile phase
▪ solvent system used: Butanol:Ethanol:Water (52:32:10)
▪ less polar liquid (as compared to pure water in the stationary phase)

● Liquid-liquid type of Chromatography

o Liquid mobile phase, liquid stationary phase
University of Santo Tomas
Biochemistry
Faculty of Medicine and Surgery John Mark
Villena
Class of 2017 Section D

Solvent front- farthest distance covered by the solvent

Solute front- distance covered by the solute

Ratio of Fronts (Rf)

● extent of migration of the solutes, it is dependent on
o nature of the solvent (polarity)
o quality of the paper used (pore size)
o distance travelled by solvent and solute fronts
● will determine the unknown solute upon comparison with standard solutes
● if unknown solute has = Rf value with a standard solute, they have the same extent of migration, ie
they are the same solute
● not affected by temperature
Rf= solute front/ solvent front OR

STEPS
● Preparation
o prepare the standards, unknown, solvent system, materials needed
● Spotting
o apply small spots of the standards and the unknown equidistantly to avoid overlapping of lanes
● Development
o period where the chromatogram (paper with applied spots) is allowed to equilibrate with the
solvent system in an isolated environment
o “travel period” 4 hours or more in the experiment
● Visualization
o for the spots to be visible to the naked eye
o spraying with ​Aniline Acid Oxalate then heating
a. Oxalic acid, heat
Dehydration of sugars
-Furfural -> pentoses
-HMF -> hexoses

b. Aniline dye staining

Pentose= bright pink color
Hexose= negligible pink color/brown
University of Santo Tomas
Biochemistry
Faculty of Medicine and Surgery John Mark
Villena
Class of 2017 Section D

● Analysis
o computation of the Rf values and identifying the unknown.

Experiment 2: Titration of Amino Acids

Amino acids
● monomers of proteins
● amphoteric in nature
o contain an acidic moiety (COOH)
o contain a basic moiety (NH2)
o can act as an acid in the presence of a base, can act as a base in the presence of an acid.
Titration
● determination of the quantity of substance A by adding measured increments of substance B
● used to measure the total acid (or base) neutralizing capacity of a solution

Titrant- substance used to act with analyte to determine concentration; standardized solution
- use of strong acids or strong bases: react more completely with an analyte, yielding a sharper endpoint
Analyte – substance being analyzed
Indicator – used to indicate the end point, could be a dye or a pH meter
Equivalence point – theoretical, non-observable point in titration when the amount of added titrant is exactly
equivalent
to amount of analyte
End point – actual observable point in titration when physical changed occurs that is associated with reaching
the
equivalence point. Change in color or reaching a specific pH.
Titration of Amino Acids
● neutralization reaction as the added acid/base will be neutralized by the amino acid, being
amphoteric in nature
● ACID + BASE = Neutralization Reaction (salt formation)

at pH lower pH=ipH at pH higher

than the ipH than the ipH
value value
Amino Group NH3+ NH3+ NH2
Carboxyl Group COOH COO- COO-
Amino Acid net +1 0 -1
charge CATIONIC ZWITTER ANIONIC
FORM IONIC FORM FORM
University of Santo Tomas
Biochemistry
Faculty of Medicine and Surgery John Mark
Villena
Class of 2017 Section D

● pK value
o represented by plateaus in the titration curve
o areas of maximum buffering capacity, changes in pH are little to none due to the presence of
50% acid form to neutralize any base added and 50% conjugate base form to neutralize any
acid added
o the pH at which H+ begins to leave/dissociate, take for example Histidine
o it has 3 ionizable groups: 3 groups could donate H+
o it’s NH3+, its COOH, and its R-group
o the pH at which the H+ begins to leave corresponds to
its pK values
▪ pK 1​=1.82 PLATEU
● at pH 1.82, COOH begins to become
COO-
● 50% COOH, 50% COO-
● at pH 1.83, 100% COO- (inflection)
▪ pK R​= 6.00 PLATEU
● at pH 6.00, the =NH+ begins to become
=NH
● 50% =NH+, 50%=NH
● at pH 6.01, 100% =NH (inflection)
▪ pK 2= 9.17 ​PLATEU
● at pH 9.17, the NH3+ begins to become
NH2
● 50% NH3, 50% NH2
● at pH 9.18, 100% NH2 (inflection)

● ipH value
o isoelectric pH is the pH at which the amino acid’s net charge is 0
o this is attributed to the presence of the zwitterionic form
▪ zwitterionic form- positive and negative charges are present in the amino acid structure,
no dominating charge but they cancel each other out (pH=ipH)
▪ anionic form- negative charges predominate, deprotonated/ionized form (pH > ipH)
▪ cationic form- positive charges predominate, protonated form (pH < ipH)
o average of the 2 pK values (nearest each other)
▪ Neutral: (pK​COOH​ + pK​NH3​) / 2
▪ Acidic: (pK​COOH​ + pK​RH​) / 2
▪ Basic: (pK​NH3​ + pK​R)​ / 2
o in an a joint acid base titration curve, it is the steepest inflection

● Soren Peter Sorensen’s Discovery (role of Formaldehyde)

Analyte: Amino Acid (acting as acid: H donor)
Titrant: NaOH (acting as base: H acceptor)
o IDEAL TITRATION SCENARIO
▪ AA-COOH (acting as acid) + Na-OH (acting as base) = NEUTRALIZATION
▪ The titrant must react with the analyte to reach an endpoint
▪ The added base (titrant) must be able to react with the amino acid (analyte) to reach
the endpoint (neutralization)
o SCENARIO ​without​ COH
▪ BUT​ since the Amino Acid also has a basic moiety ie the NH2, instead of
AA-COOH + Na-OH, titrant reacting with the analyte
AA-COOH + AA-NH2, what actually happens: Analyte reacting with Analyte
▪ The analyte is reacting with itself, neutralizing itself
▪ The endpoint will not be reached as no reaction between the analyte and the titrant
occurs
o SCENARIO ​with​ COH
University of Santo Tomas
Biochemistry
Faculty of Medicine and Surgery John Mark
Villena
Class of 2017 Section D
▪ to counteract the self-neutralizing reaction of the amino acid, the basic NH2 must be
neutralized by
addition of an
aldehyde prior
to titration

▪ the formaldehyde (2 COH) will act as an acid (H donor, lowers the solution pH) to
neutralize the basic NH2 group of the amino acid forming a neutral dimethylol derivative
▪ the NH2 will now be NEUTRALIZED; ​unavailable​ for additional reactions
▪ the titration of the amino acid with a base will now be possible
▪ Na-OH (titrant) + AA-COOH (analyte) = NEUTRALIZATION END POINT

TITRATION OF A NEUTRAL AMINO

ACID

A
B
C

TITRATION OF AN
ACIDIC AMINO ACID
University of Santo Tomas
Biochemistry
Faculty of Medicine and Surgery John Mark
Villena
Class of 2017 Section D
A B C D

Aspartic Acid is an acidic Amino Acid

It has ionizable groups: COOH, NH3 and R
pK COOH = 1.88
pK R= 3.65
pK NH3 = 9.60
ipH = [(1.88+3.65)/2]= 2.77

POINTS IN THE TITRATION CURVE

● pre-pK 1
o COOH, NH3+, RCOOH
o CHARGE: +1
o 100% cationic (A)
● pK 1: COOH
o pH=1.88
o plateau: maximum buffering capacity
o 50% cationic (A), 50% zwitterionic (B)
● ipH
o pH= 2.77
o steep inflection: COO- and NH3+
o CHARGE: 0
o 100% zwitter ionic form (B)
● pK 2: R
o pH = 3.65
o plateau: maximum buffering capacity
o 50% zwitterionic (B), 50% anionic (C)
● pre-pK 3
o pH 3.66-9.59
o inflection
o 100% anionic
o CHARGE: -1
● pK 3: NH3
o pH 9.60
o plateau: maximum buffering capacity
o 50% anionic -1 (C), 50% anionic -2 (D)
● post-pK3
o pH 9.61 and up
University of Santo Tomas
Biochemistry
Faculty of Medicine and Surgery John Mark
Villena
Class of 2017 Section D
o 100% anionic D
o CHARGE: -2

TITRATION OF A BASIC AMINO ACID

A B
C D

Lysine is a basic Amino Acid

It has 2 ionizable groups: COOH, NH3 and R
pK COOH = 2.18
pK NH3 = 8.95
pK R= 10.53
ipH = [(8.95+10.53)/2]= 9.74

POINTS IN THE TITRATION CURVE

● pre-pK 1
o COOH, NH3+, RCOOH
o CHARGE: +2
o 100% cationic (A)
● pK 1: COOH
o pH=2.18
o plateau: maximum buffering capacity
o 50% cationic +2 (A), 50% cationic +1 (B)
● post pK-1
o pH 2.19-8.94
o 100% cationic +1 (B)
● pK 2: NH3
o pH = 8.95
o plateau: maximum buffering capacity
o 50% cationic +1 (B), 50% zwitterionic (C)
● ipH
o pH= 9.74
o steep inflection: COO- and NH3+
o CHARGE: 0
University of Santo Tomas
Biochemistry
Faculty of Medicine and Surgery John Mark
Villena
Class of 2017 Section D
o 100% zwitter ionic form (C)
● pK 3: R
o pH 10.53
o plateau: maximum buffering capacity
o 50% zwitterionic (C), 50% anionic -1 (D)
● post-pK3
o pH 10.54 and up
o 100% anionic (D)
o CHARGE: -1