01 - INSTRUMENTATION
● Sample compartment - holds transparent tube or
pH METER
● Measures H+ ion concentration and pH of an
unknown solution
● Designates the intensity or degree of acidity
● pH formula
● Equipped with: glass electrode, reference electrode
○ Modern pH meters has temperature probes
which allows temperature corrections
Glass Electrode - thin, pH sensitive glass membrane
Reference Electrode - salt bridge with saturated KCl solutions
● Most common: Calomel Electrode
SPECTROPHOTOMETRY
● Method to measure how much the chemical
substance absorbs light by measuring the intensity of
light as a beam of light passes through sample
solutions
● Basic principle: “Each compound absorbs or
transmits lights over a certain range of wavelength.”
● One of the most useful quantitative analysis
● Uses spectrophotometers
ULTRAVIOLET-VISIBLE SPECTROPHOTOMETER (UV-VIS)
● Makes the sample absorb light in the visible and near
UV region of the electromagnetic spectrum
COLORIMETRIC ASSAYS
● Measures the amount of light transmitted through a
sample at a given wavelength
WAVELENGTH OF LIGHT SOURCE
UV-VIS SPECTROPHOTOMETER
Ultraviolet Range - 185-400 nm
Visible Range - 400-700 nm
INFRARED SPECTROPHOTOMETER
Infrared Range - 700-15,000 nm
UV-VIS ESSENTIAL PARTS
● White light source
● Monochromator - disperse light into its components’
wavelength
cell containing sample
● Detector - measure amount of light transmitted
● Read out device - displays the reading
Tungsten Lamp - light source in the visible region
Deuterium Lamp - light source in the ultraviolet region
Source > entrance slit > dispersing device > exit slit > cuvette
(sample holder) > detector (as shown in the diagram in the
discussion)
TRANSMITTANCE
● Amount of monochromatic light absorbed by a
sample is determined by comparing the intensities of
incident light (Io) and the transmitted light (I)
● Also the ratio of the intensity of the transmitted light
to the incident light
● Transmittance formula
ABSORBANCE
● Amount of light absorbed by a sample
● Absorbance formula
BEER-LAMBERT’S LAW
● Determination of the concentration of the absorbing
species in the analyte solution
● States that: “The intensity of the transmitted light
decreases exponentially as anyone of these factors
increase.”
● Beer-Lambert’s Law formula
● According to this law, absorbance is directly related
to the concentration if the measurements are made
at a fixed wavelength in a cell of constant path which
is usually 1 cm
Molar extinction coefficient - measure of how strongly a
solution absorbs light in a particular wavelength
● For two solutions of the same compounds,
one with a known concentration (standard
solution) and the other with an unknown
concentration, the concentration of the
unknown solution can be calculated from
the measured absorbance of the unknown
solution and the absorbance of the
standard solution.
● Insert formulas here
 GENESYS 5 SPECTROPHOTOMETER 02 - pH MEASUREMENT
● Offers 5 nm spectral slit width and has a wavelength AND BUFFER PREPARATION
range of 200-1100 nm
● Has a split beam optical system ● pH
○ Negative log of the [H+]
MULTISKAN GO MICROPLATE SPECTROPHOTOMETER
○ For determination of acidity and alkalinity
● Used to measure UV/VIS absorbance from 200 to ○ pH formula
1000 nm on 96 to/or 384 - well microplates ● pOH
● Incubate up to 45 ℃ and shake the microplate ○ Negative log of [OH-]
○ pOH formula
Microplate - allows smaller sample volume (microliter)
FORMULAS
IMPORTANT POINTS TO CONSIDER IN UV/VIS ANALYSIS
● Write formulas in another paper
1. Errors, due to inadequate or missing reagents,
POTENTIOMETER AND COLORIMETRIC pH DETERMINATION
solutions should be well mixed prior to absorbance
reading Potentiometric pH Determination
2. Enough time should be given for the complete color ● Involves the use of a pH meter
development of the solution before the absorbance ● Measures the voltage between two electrodes and
is measured display the results
3. Assays should be performed in duplicate or triplicate
4. Beer-Lambert’s Law is accurate = absorbance of Colorimetric pH Determination
0.05-0.70 ● Use of indicator dyes which produce color depending
> 0.70 - underestimates real absorbance on the sample
<0.05 - not accurate
5. Calibration curve should never be extrapolated COLORIMETRIC INDICATORS
beyond the highest absorbance value measured. If
the sample is not within the calibration curve, INDICATOR ACID BASE pH RANGE
sample solution should be diluted so that its
absorbance falls within the calibration curve. Thymol blue (1st) R Y 1.2 - 2.8

APPLICATIONS OF THE UV-VIS SPECTROPHOTOMETER Thymol blue (2nd) Y B 8.0 - 9.6

● Reaction Kinetics Bromophenol blue Y V 3.0 - 4.6
● Quantitation
● pKa Determination Bromocresol green Y B 3.8 - 5.4
● DNA Structural Information
Bromocresol purple Y P 5.2 - 6.8
CENTRIFUGATION
Methyl orange O Y 3.2 - 4.4
● Process used to separate or concentrate materials
suspended in a liquid medium using the effect of Methyl red R Y 4.2 - 6.3
gravity on the particles in suspension
Centrifugal Force - fictitious force, peculiar to a particle Chlorophenol blue Y B 4.8 - 6.4
moving on a circular path
Centrifuge - device used in centrifugation …….. according to Bromothymol blue Y B 6.0 - 7.6
size, shape, density/viscosity of the medium and the rotor
speed Cresol red Y R 7.2 - 8.8
Phenol red Y R 6.8 - 8.4 ● Can be either polar, nonpolar, acid, or base

Phenolphthalein CLESS R 8.4 - 10.0 Glucogenic acid - glucose/sugar

Ketogenic acid - ketone

DENATURATION
BUFFERS AND THEIR IMPORTANCE
● Process of changing or deviating from the native
BUFFER SOLUTION conformation
Denaturing Agents: Heat, extreme pH, Mercaptoethanol
● Contain either a weak acid or weak base and its salt.
Both of these must be present. They can resist TYPES OF PROTEINS
changes in pH when small amounts of either acid or
base are added. ● Based on shape:
● Must contain relatively large concentration of acid to ○ Globular - (Globule) Albumin, Hemoglobin,
react with any hydroxide ions that may be added / Insulin, Catalase
Must contain relatively large concentration of base ○ Fibrous - (Large) Collagen, Keratin
to react with any hydrogen ions that may be added ● Functions
● The buffer components must not consume each ○ Collagen - structural protein; has 2 amino
other in neutralization reactions. The remedies by acids
using an acid-base conjugate pair. ○ Elastin - blood vessels
Example: ○ Myoglobin - storage proteins, muscles
CH₃COOH / CH₃COONa Buffer ○ Hemoglobin - transport proteins, carries O2
Adding acid, the reaction would be: blood
CH₃COO⁻ + H⁺ ↔ CH₃COOH ○ Insulin & Glucagon - hormones, control
Adding a base would result: blood sugar
CH₃COOH + OH⁻ ↔ CH₃COO⁻ + H₂O ○ Immunoglobulins - antibodies
HENDERSON-HASSELBALCH EQUATION ISOLATION/SEQUENCING
● Used to estimate the pH of a solution ● By polarity; salting out, precipitation
● H-H Equation formula ● By charge
● By size
BUFFER CAPACITY
Chromatography - separating components or solutes of a
● The number of moles of an acid or base necessary to mixture on the basis of relative amounts of each solute
change the pH of a buffer by 1 distributed between a moving fluid stream called the mobile
phase (liquid/gas) and a contiguous stationary phase
03 - ISOLATION AND (solid/liquid)
GENERAL QUALITATIVE TESTS FOR AMINO ACIDS
CHARACTERIZATION OF PROTEINS
TEST REAGENT POSITIV PRESENCE
PROTEINS E
● Constitute 90% of the organic matter of cell
Xanthoproteic Test HNO₃ Yellow/o Phenylalanine
● Polymers of amino acids linked by a peptide bond
range
ppt
AMINO ACIDS

● Building blocks of proteins Fohl’s Test PbCH₃COO Black ppt Methionine

● Contains carboxyl acid and amino functional groups (PbS)
Hopkin-Cole Test Glyoxylic Violet Tryptophan b. REAGENT: NaOH, CuSo₄
Acid Ring c. POSITIVE RESULT: Blue > Pink (Peptide
bonds present, 2 or more AA present)
Ninhydrin Test Ninhydrin 1: Violet General test d. NEGATIVE RESULT: Blue (1 or none)
Reagent 2: Yellow for free amino 2. Ninhydrin Test
acids a. For α-amino acid
b. REAGENT: Ninhydrin’s solution
Millon-Nasse Hg Pink/Old Phenolic c. POSITIVE: 1 - Violet; 2 - Yellow
Rose rings/Tyrosine 3. Xanthoproteic Test
a. Detects aromatic side chain
Sakaguchi Test α-naphthol Red/Win Arginine b. REAGENT: HNO₃
e Color c. POSITIVE: Yellow ppt
4. Millon’s Test
a. Detects AAs containing phenol group
b. POSITIVE: Brick Red/Old Rose, Presence of
ISOLATION OF ALBUMIN Tyrosine
5. Hopkins-Cole Test
● Protein made by liver
a. Detects indole rings
● Keeps fluid from leaking out of blood vessels,
b. POSITIVE: Violet ring
nourishes tissues, and transports hormones,
6. Sakaguchi Test
vitamins, drug, and other substances throughout the
a. For Arginine
body
b. REAGENT: α-naphthol
Albumin Deficiency
c. POSITIVE: Red Wine Color
● Severe liver disease
7. Nitroprusside Test
● Kidney disease
a. For Sulfur containing AA
b. Ex. Cysteine
ISOLATION OF GLUTEN FROM WHEAT FLOUR
c. POSITIVE: Red
Gluten 8. Pauly Test
● Family of proteins found in grains, wheat, spelt, a. Tyrosine/Histidine
barley b. POSITIVE: Red Color
● Glutenin & Gliadin - main proteins in Gluten c. NEGATIVE: Glycine
● Celiac Disease - gluten intolerance
THIN LAYER CHROMATOGRAPHY
● When wheat flour is washed, this process removes
starch that when tested with I2 solution, positive
● Common technique used to identify compounds,
results give a violet/purple solution.
determine purities
● The insoluble material left is gluten
● Used to separate substances according to polarity
Semi-Quantitative Method
ISOLATION OF MYOGLOBIN FROM MUSCLE
● Stationary Phase - special finely ground matrix (silica
Myoglobin & Hemoglobin gel alumina) is located on a glass plate/metal/plastic
● Vital to oxygen transport and storage in vertebrates film as a thin layer
● Myoglobin - muscles; Hemoglobin - blood
● Myoglobin can be isolated using Ammonium Sulfate
precipitation

QUALITATIVE TESTS

1. Biuret Test
a. Detect peptide bonds