ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA

ANALYTICAL STRATEGY
 The process involving many individual steps
by which an analyte’s identity or
ANALYTICAL SCIENCE concentration in a sample is determined.
 the science that deals with the identification
and quantification of the components of FUNDAMENTAL UNITS OF
material systems. MEASUREMENT
 Analysis - the process of determining the
 SI units
level of any or all components in a material
o Standardized system of units adapted by
system.
scientists throughout the world
ANALYTICAL CHEMISTRY
 A branch of chemistry that deals with the Physical Name of Unit Abbreviation
separation, identification and quantification Quantity
of chemical compounds.
Mass Kilogram kg
 Qualitative and Quantitative analysis
Length Meter m
Qualitative Analysis - An analysis that establishes
the chemical identity of the species. Time Second s
Quantitative Analysis - An analysis that Temperature Kelvin K
determines the relative amounts of analytes in
numerical terms. Amount of Mole mol
Substance

Electric current Ampere A

Luminous Candela cd
intensity

Luminous intensity the quantity of visible light that is

emitted in unit time per unit solid angle.

 Prefixes for Units

Prefix Abbreviation Multiplier

giga- G 109

mega- M 106

kilo- K 103

deci- d 10-1

centi- c 10-2

milli- m 10-3

micro µ 10-6

nano- n 10-9

pico- p 10-12

1
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 If the exponent is negative you will have a small
number (<1) and move the decimal to the left.

-5
 A numerical system in which numbers are 5.08 x 10 0.0000508
expressed in the form A x 10n
 where A is a number with a single nonzero digit
to the left of the decimal place and n is a whole
number
 The exponent for the exponential term is equal
• Exponent = # of spaces to be moved by decimal
to the number of places the decimal point has
been moved Convert the ff:
 Positive (+) n means a large number so
the decimal moves to the right by n 93,000,000
places. 0.0000037
 Negative (-) n means a small number so
1.53x106
the decimal moves to the left by n places.
 A – coefficient 2.37x10-3
 10n – exponential term
ANSWERS:
 When converting from standard notation to
 9.3x107
scientific notation
 3.7x10-6
 If the number is one or greater you will
 1,530,000
have a positive exponent and move the
decimal to the left.  0.00237
5
801236.98 8.0123698 x 10

• In measurements there is always some amount

 If the number is less than one you will have a
of uncertainty.
negative exponent and move the decimal to the
right.

0.0000508 5.08 x 10
-5

 # of spaces moved by decimal = exponent

 When converting from scientific notation to

standard notation
o If the exponent is positive you will have
a large number (>1) and move the
decimal to the right.
5
8.0123698 x 10 801236.98

2
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
GUIDELINES FOR DETERMINING
SIGNIFICANT FIGURES
1. All nonzero digits are significant
2. Zeros may or may not be significant
 Zeros at the beginning of a number
are never significant
Ex. 0.0141 has three significant figures
 Zeros between nonzero digits are
always significant
Ex. 3.063 has four significant figures
0.001004 has four significant figures
 Zeros at the end of a number are
SIGNIFICANT if a decimal point is
PRESENT in the number
Ex. 56.00 has four significant figures
0.05050 has four significant figures
 Zeros at the end of a number are NOT
SIGNIFICANT in the number LACKS an
explicitly shown decimal point
Ex. 59,000,000 has two significant figures
6010 has three significant figures
 A substance that is dissolved in a liquid is
called a solute. The liquid in which the solute
is dissolved is the solvent. Together they
represent a solution.
 In laboratory science, biologic solute are
components in the blood known as analytes
 Solvent is a biologic fluid called plasma.
MOLARITY
 Molarity (M) is expressed as the number of
moles per 1 L of solution.
 A solution that is 1.0 M contains 1.0 mole of
solute per liter of solution
𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 ሺ𝑀ሻ
𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
=
𝑔𝑟𝑎𝑚 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
 The concentration of solution can be describe
in many ways
 Concentration is expressed as Molarity,
Normality or Percent solutions
 (included are those expression of
concentration that is routinely used in the
clinical lab)
 Gram molecular weight is obtained by adding
the atomic weight of the component elements
MOLALITY
 Molality (m) is expressed as the amount of
solute per 1 kg of solvent.
𝑀𝑜𝑙𝑎𝑙𝑖𝑡𝑦 ሺ𝑚ሻ
𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
=
𝑔𝑟𝑎𝑚 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑘𝑔 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
3
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 Sometimes confused with Molarity.
 Molarity is expressed as the number of moles
per liter of solution, while molality is expressed
as Moles/kilogram of solvent  77.4 grams of NaCl is dissolved in 250 mL of
water. What is the molar concentration of the
solution?
NORMALITY  MW of Na = 23, MW of Cl = 35
Answer: 5.34 M
 Normality (N) is the number of equivalent
weight per 1 liter of solution  How many grams of CaCl2 is needed to prepare a
𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 10 mL of 0.02M solution?
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 =  Ca (40 g/ mol) Cl (35g/mol)
𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
Answer: 0.022 grams
Where;
𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡  How many grams of HCl is needed to prepare a
𝐸𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 = 100 mL of 0.1N HCl solution?
𝑣𝑎𝑙𝑒𝑛𝑐𝑒
 (MW: 36 g/mol)
 Valence of Acids – according to the number of
hydrogen ions Answer: 0.36g HCl
 Valence of Bases- according to number of  A 10 mL of 0.5% solution composed of Barium
hydroxyl ions chloride and sulfuric acid is to be prepared. How
many mL of Sulfuric acid is needed?
 Compute first for the amount of Barium
RELATIONSHIP BETWEEN MOLARITY AND chloride
NORMALITY  Then subtract the amount of Barium
chloride to the total volume to
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 = 𝑚𝑜𝑙𝑎𝑟𝑖𝑡𝑦 𝑥 𝑣𝑎𝑙𝑒𝑛𝑐𝑒 determine the amount of Sulfuric acid
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 Answer: 0.05 mL of Barium chloride and 9.95 mL of
𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 =
𝑣𝑎𝑙𝑒𝑛𝑐𝑒 Sulfuric acid
PERCENT SOLUTIONS  5. Calculate the amount of water you need to
add to make a final concentration of 70%
 Weight/Volume (w/v) % solutions ethanol solution by using 100 mL of pure
 𝑤𝑒𝑖𝑔ℎ𝑡/𝑣𝑜𝑙𝑢𝑚𝑒% = (100%) ethanol.
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 ሺ𝑔ሻ
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 ሺ𝑚𝐿ሻ
× 100  C1V1=C2V2
Answer: 42.9 mL of water
 Most common type of solution prepared in the
clinical lab The final volume we need to make therefore is 142.9
mL. We know 100 mL of that is the 100% pure ethanol,
 Volume/Volume % solutions so the volume of water must be 42.9 mL (142.9 – 100 =

𝑣𝑜𝑙𝑢𝑚𝑒
%
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
= 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 × 100 42.9). So, adding 42.9 mL of water to 100
𝑣𝑜𝑙𝑢𝑚𝑒
mL of 100% pure ethanol will achieve a final
 Weight/weight (w/w) % solutions concentration of 70% ethanol.
 𝑤𝑒𝑖𝑔ℎ𝑡/𝑤𝑒𝑖𝑔ℎ𝑡% solution =
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
× 100

4
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
knowing that there are always 1000 grams in
one kilogram. Or we can convert lengths, say,
from kilometers to miles.

DIMENSIONAL ANALYSIS
(the study of)the relationship between  5 x 5 = 52
the quantities of substances involved in  cm x cm = cm2
a chemical reaction and the substances that 𝑐𝑚 𝑥 𝑖𝑛

are produced as a result of that chemical reaction 𝑐𝑚

EXERCISE
How many moles of water is present in 36 grams of the  : Identify the known or given quantity
same substance?
and the units of the new quantity to be
How many carbon atoms are present in 8.97 grams of determined. Write an equation with the given
ethane? quantity on the left and the units of the desired
quantity on the right.
How many moles of Na are in 5.63 grams Na2SO4?
 Multiply the given quantity by one or
more conversion factors in such a manner that
 2 moles of H2O the unwanted (original) units are canceled,
 3.59 or 4.00 x 1023 atoms of C leaving only the desired units
 0.08 moles of Na  Perform the mathematical operations
indicated by the conversion factor setup.
SAMPLE PROBLEM
A standard aspirin tablet contains 324 mg of aspirin.
How many grams of aspirin are in a standard aspirin
tablet?
Step 1:
324 mg = ? G
Step 2: Use of conversion factor
DIMENSIONAL ANALYSIS 1 𝑚𝑔
0.001 𝑔
0.001 𝑔
or 1 𝑚𝑔
0.001 𝑔
 Dimensional analysis is a general problem- 324 mg x = ?g
1 𝑚𝑔
solving method in which the units associated Step 3:
with numbers are used as a guide in setting up ቀ324
0.001
ቁ= 0.324 g
calculations 1
Or 1 g is = 1000 mg
Dimensional Analysis is a way chemists and other
scientists convert units of measurement. We can ATOMIC MASS
convert any unit to another unit of the  The mass of atoms of elements expressed in
same dimension. atomic mass units (amu)

• This means we can convert some number of MOLECULAR WEIGHT

seconds into another unit of time, such as  Sum of the atomic weights for the atoms in a
minutes, because we know that there are always molecule
60 seconds in one minute. Or we can convert  For the molecule ethane, C2H6, the molecular
some amount of mass from grams to kilograms, weight would be

5
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA

C: 2(12.0 amu)
+ H: 6(1.0 amu)
CHEMICAL FORMULAS CAN BE INTERPRETED IN TWO
30.0 amu WAYS
For example the formula N2O4

1st
 The formula N2O4 contains two atoms of
nitrogen and four atoms of oxygen in one
 The mole is the SI unit for the amount of
molecule of N2O4
substance
2nd
 It is defined as the amount of a substance that
 The formula N2O4 contains 2 moles of nitrogen
contains the same number of entities as there
atoms and 4 moles of oxygen atoms are present
are atoms in exactly 12 g of carbon-12
in one mole of N2O4
 A mole is 6.02x10 23 objects (atoms or
molecules) WHEN IT IS NECESSARY TO KNOW THE NUMBER OF
1 mole = 6.02x10 23 objects MOLES OF A PARTICULAR ELEMENT WITHIN A
6.02x10 23 objects
or
1 mole COMPOUND, THE SUBSCRIPT OF THAT ELEMENT’S
1 mole 6.02x10 23 objects SYMBOL IN THE CHEMICAL FORMULA BECOMES PART OF
THE CONVERSION FACTOR.
SAMPLE PROBLEM
For the formula N2O4
1. How many carbon dioxide molecules are there
2 moles of N atoms 1 mole of N2O4
in 1.7 moles of the same substance?  For N: or
1 mole of N2O4 2 moles of N atoms
2. How many Lithium atoms are there in 6.5 4 moles of O atoms 1 mole of N2O4
 For O: or
moles of the same substance? 1 mole of N2O4 4 moles of O atoms
 1.023 x 1024
 3.913 x 1024 SAMPLE PROBLEM
1. How many moles of Nitrogen are present in
7.36 moles of NO2?

 The molar mass is the mass, in grams, of a Nitrogen dioxide 7.36 mol of N
substance that is numerically equal to the
substance formula mass
 For example, for the compound CO2, (formula
mass of 44.01 amu)
1 mole CO2 = 44.01 g CO2
44.01 𝑔 𝐶𝑂2 1 𝑚𝑜𝑙𝑒 𝐶𝑂2
1 𝑚𝑜𝑙𝑒 𝐶𝑂2
or 44.01 𝑔 𝐶𝑂
2

SAMPLE PROBLEM
1. Calculate the mass, in grams, of a 0.9 mole
sample of glucose. (molecular formula C6H12O6)

 162 g of glucose

6
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA

 A balanced chemical equation is a chemical

equation that has the same number of atoms of
each element involved in the reaction on each
SAMPLE PROBLEMS side of the equation
1. How many Fe atoms are there in 95.8g of Fe?  An unbalanced chemical equation is brought
2. How many grams of O are there in 3.10 grams into balance by adding coefficients
of H2O?
3. How many grams Ag are there in 0.0342 mol of  A coefficient is a number placed to the left of a
AgCl2? chemical formula in a chemical equation
1.03 x 1024
2.76 g of O
3.70 g

WRITING AND BALANCING

CHEMICAL EQUATIONS GUIDELINES
 A chemical equation is a written statement that
uses chemical symbols and chemical formulas
to describe the changes that occur in a chemical
reaction
 Start with the compound that contains the
CONVENTIONS USED IN WRITING greatest number of atoms, and focus on the
CHEMICAL EQUATIONS element that has the greatest number of atoms.
 Reactants are always written on the left side of
the equation

 Pick, as the second element to balance, one

 Products are always written on the right side of whose amount is already set on one side of the
the equation equation by a previously determined coefficient

 The reactants and the products are separated

by an arrow pointing towards the products

 Pick a third element to balance.

 Plus signs are used to separate different

reactants or different products

7
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
ANSWER: 13.75 g of CO2

 The coefficients in a balanced chemical

equation can be interpreted in two ways
A separation process is a method that converts a
mixture or solution of chemical substances into two or
more distinct product mixtures.

 To remove unwanted particles

 To obtain important substances
1st  To obtain pure substances
 One molecule of N2 reacts with three molecules CENTRIFUGATION
of H2 to produce two molecules of NH3
2nd
 One mole of N2 reacts with three moles of H2 to
produce two moles of NH3
• The coefficients in a balanced chemical
equation can be used to generate mole-based
conversion factors

 Centrifugation is a process in which centrifugal

force is used to separate solid matter from a
liquid suspension

 The speed is expressed in rotations per

minute, and the centrifugal force generated is
expressed in terms of relative centrifugal
force

SAMPLE PROBLEM
1. How many grams of CO2 will be produced from
10 grams O2?

8
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
CLASSIFIED BASED ON SEVERAL CRITERIA  Decantation is the process of pouring a liquid
gently so as to not disturb a solid in the bottom
 Benchtop or floor model of the container.
 Refrigeration
 Rotor head (fixed, hematocrit, swinging bucket, Applications in the clinical laboratory
or angled)  Clinical microscopy URINALYSIS – AFTER
CENTRI – DECANT
APPLICATIONS IN THE CLINICAL LABORATORY  Blood banking – red blood cell suspension
 Clinical chemistry – to separate blood (RCS) preparation
components, since serum is mostly intented for
chemical analytes of blood
 Clinical microscopy – to perform microscopic
FILTRATION
examination, to obtain the microscopic
sediments of urine (Urinalysis)
 Blood banking – to separate blood
components in the blood bag (packed RBC,
plasma, platelets)
 Hematology – HEMATOCRIT
 Filtration can be used instead of centrifugation
for the separation of solids from liquids
 When the filter paper is placed inside the funnel,
the solution slowly drains through the filter
paper within the funnel and into a receiving
vessel. The liquid that passes through the filter
paper is called the filtrate.

DECANTATION  Modern-day chemical analysis can involve very

complicated material samples—complicated in
the sense that there can be many substances
present in the sample

INTERFERENCE
 Species other than the analyte that affect the
final measurement are called interferences, or
interferents

 substances other than the analyte generate an

instrumental readout similar to the analyte,
such that the interference adds to the readout
of the analyte

9
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 an interference can also suppress the readout  Fractional distillation involves repeated
for the analyte (e.g., by reacting with the evaporation–condensation steps before the
analyte) distillate is actually collected

INTERFERENCES CAUSE ERRONEOUS FRACTIONAL DISTILLATION

ANALYTICAL RESULTS – ERROR IN THE RESULTS
 These repeated steps occur in a fractionating
RECRYSTALLIZATION column (tube) above the original heated
container—a column that contains a high
 Recrystallization is a purification technique for surface area of inert material for condensing the
a solid, usually organic. The separation is based vapors
on the solid’s solubility in a liquid solvent

 The key to the procedure is to use a minimum

amount of solvent, such that the solid will just
dissolve at the elevated temperature (usually
the boiling point, if the solid is stable at that
temperature). While maintaining this elevated
temperature, any impurity that has not
dissolved can be filtered out. The insoluble
impurities are thus removed.

 Soluble impurities, however, are still present

in the hot filtrate. These are removed by
cooling the filtrate. Cooling the filtrate causes
the solid being purified to crystallize

 If the solubilities of the soluble impurities have
not been exceeded during the cooling step, they
will stay dissolved and be separated during the
second filtering
DISTILLATION
 Distillation is a method of purification of
liquids contaminated with either dissolved
solids or miscible liquids
 This method consist of
 boiling and evaporating the mixture
 recondensation of the vapors in a
condenser
 The separation is based on the fact that the
contaminants have different boiling points
than the liquid to be purified
 The substances with lower boiling points are
therefore separated from substances that have
high boiling points.
 Boiling point – basis of the procedure  solid 
liquid
Used to separate a mixture of liquids – fractional
distillation – but it should have different boiling points
LIQUID–LIQUID EXTRACTION
 In this method, the sample containing the
analyte is a liquid solution, typically a water
solution, that also contains other solutes.
 The need for the separation usually arises
from the fact that the other solutes, or perhaps
the original solvent, interfere in some way with
the analysis technique chosen.
 a method in which the analyte is removed from
the original solvent and subsequently dissolved
in a different solvent (extracted) while most of
the remainder of the sample remains
unextracted, i.e., remains behind in the original
solution.
10
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
Involves Two Liquid Phases  shaking–venting step is then repeated several
times such that the two liquids have plenty of
 The original solution opportunity for the intimate contact required
 Extracting solvent for the analyte to pass into the extracting solvent
to the maximum possible extent
 Criteria for successful separation of analyte
 these two liquids be immiscible
 the analyte be more soluble in the
extracting solvent than the original
solvent
Immiscible – (of liquids) not forming a
homogeneous mixture when added together, so
separation is possible
Soluble – able to dissolve  Following this
procedure, the funnel
SEPARATORY FUNNEL is positioned in a
padded ring in a ring
 The separatory funnel is manufactured stand and left
especially for solvent extraction undisturbed for a
period of time to allow
the two immiscible
layers to once again
separate

 After mixing – allow it

to stand for some time
– it is positioned in a
ring

 The sample and solvent are placed together in
the funnel, and the funnel is tightly stoppered
and, while holding the stopper in with the index
finger, shaken vigorously for a moment
SOLID–LIQUID EXTRACTION
 This method is used when the analyte needs to
be extracted from a solid material sample
rather than a liquid
 Analyte – extracted from a solid rather than a
liquid
 The weighed solid sample, preferably finely
divided, is brought into contact with the
extracting liquid
 The mixture is shaken or stirred for a period of
time, sometimes at an elevated temperature
 Following the extraction, the undissolved solid
material is then filtered out and the filtrate
analyzed
 FILTRATE – is analyzed

11
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
peanut butter from the jar is analyzed for
sodium
Example:
DEFINITION OF TERMS
 1. Amount of iron in a blood – iron –analyte;
REAGENT blood- sample

 is a compound or mixture added to a system to

cause a chemical reaction
 It may be used to tell whether or not a
specific chemical substance is present by
causing a reaction o occur with it.

ANALYTE
 a substance or sample being analysed

BULK SYSTEM
 a term for the material under investigation
(group)

SAMPLE
 representative portion of the bulk system
 The analytical laboratory technician analyzes
these samples by subjecting them to certain
rigorous laboratory operations that ultimately
 Suitable for use in most analytic laboratory
result in the identity or quantity of the analyte
procedures
in question
 the American Chemical Society established
 The key is that the sample must possess all the
specifications for AR grade chemicals
characteristics of the entire bulk system with
 Labels on reagents state the actual impurities for
respect to the analyte and the analyte
each chemical lot or list the maximum allowable
concentration in the system
impurities
 In other words, it must be a representative
 a chemical compound of a known high standard
sample – it must truly represent the bulk
of purity.
system
 NEXT  GRADE FOR CHEMICALS AND
REAGENTS

 The bulk system in the case of analyzing

toothpaste for fluoride is the toothpaste in  Ultrapure chemicals have been put through
the tube. The bulk system in the case of additional purification steps for use in specific
determining the ammonia level in the water in procedures such as chromatography, atomic
an aquarium is all the water in the aquarium. absorption, immunoassays, molecular
 We therefore collect a representative portion of diagnostics, standardization, or other
the bulk system and bring this portion into the techniques that require extremely pure
laboratory for analysis chemicals.

 Hence, a portion of the water in a lake is

analyzed for mercury and a portion of the
12
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA

 DISTILLED WATER
 DEIONIZED WATER
 RO WATER
 ULTRAFILTERED WATER
 REAGENT GRADE WATER – CLRW/SRW

RO – REVERSE OSMOSIS WATER

CLRW – CLINICAL LABORATORY REAGENT WATER OR
SRW – SPECIAL REAGENT WATER

 United States Pharmacopeia (USP) and the

National Formulary (NF).  Distilled water has
 FOR DRUG MANUFACTURING been purified to
 Chemicals that are used to manufacture drugs, remove almost all
may be pure enough for use in most chemical organic materials,
procedures using distillation
process in which
water is boiled and
vaporized
 These designations indicate that the impurity
 Distilled water has
limitations are not stated and that preparation
been purified to
of these chemicals is not uniform
remove almost all organic materials, using
distillation process in which water is boiled and
vaporized

 Deionized water has

some or all ions
removed, usually
done from previously
treated water, such as
prefiltered or
distilled water
 AKA: TRIPLE
DISTILLED WATER
 Clinical laboratory –
bacteriology: water used in media preparation

 is used for commercial and industrial purposes;

however, like many others,
 it is not pure enough to be offered for food, drug,
or medicinal use of any kind.
 Reverse osmosis is a process that uses pressure
to force water through a semipermeable
membrane, producing water that reflects a
filtered product of the original water

13
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 is a special type of filtration that uses a semi-
permeable, thin membrane with pores small
enough to pass pure water through while
rejecting larger molecules such as dissolved
salts (ions) and other impurities such as bacteria
 Produced from the process of ultrafiltration,
which removes particulate matter,
microorganisms, and any pyrogens or
endotoxins
 Reagent grade water can be obtained by initially
filtering it to remove particulate matter,
followed by reverse osmosis, deionization, and a
0.2 mm filter or more restrictive filtration
process.
 Water used for reagents
 Many process undergone – in order to become
extremely pure
I II III
 Type I Water - Used for test methods
requiring minimum interference, such as trace
metal, iron and enzyme analyses
 Type II Water - Type II water is used for
general laboratory applications and is
acceptable for most analytic requirements,
including reagent, quality control and standard
preparation
 Type III Water - Type III water is acceptable
for glassware washing and filling autoclaves, but
not for analysis or reagent preparation
 CONCENTRATION
 COLLIGATIVE PROPERTIES
 Concentration – is a general term that
expresses the quantity of solute contained in a
given amount of solution. (HOW MUCH SOLUTE
THERE IS IN A SOLVENT?)
 Colligative properties – properties
of solutions that depend on the ratio of the
number of solute particles to the number
of solvent molecules in a solution, and not on the
nature of the chemical species present 
SOLUTE:SOLVENT
 A solution is a homogenous mixture of two or
more substances with each substance retaining
its own chemical identity
SOLVENT
 A solvent is the component of solution that is
present in the greatest amount, the medium in
which the other substances present are
dissolved
SOLUTE
 A solute is a component of a solution that is
present in a lesser amount relative to that of the
solvent.
DILUTION
 Dilution is the process in which more solvent is
added to a solution in order to lower its
concentration
DILUTE SOLUTION
 A dilute solution is one in which there is
relatively little solute or one which has been
made to a lower solute concentration per
volume of solvent as when making a dilution.
CONCENTRATED SOLUTION
 A concentrated solution has a large quantity of
solute in solution
SATURATED SOLUTION
 A saturated solution is a solution in which there
is an excess of undissolved solute particles
SUPERSATURATED SOLUTION
 A supersaturated solution has an even greater
concentration of undissolved solute particles
than a saturated solution of the same substance.
14
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
INTERNAL INDICATORS (USED IN
PRECIPITATION METHOD)

 – VOLHARD METHOD
 – MOHR METHOD
 – FAJAN
METHOD

 Is also known as ARGENTOMETRIC ANALYSIS COLORED ION (VOLHARD

 PRECIPITATE
 DERIVED FROM ARGENTUM = silver
 In titration, analyte and titrant react to reach the
endpoint (which is the change in color)
 While, in precipitation method, analyte and the
volumetric solution react to yield compounds
with limited solubility (PRECIPITATE)
 Analyte is titrated with a standard solution of a
precipitating agent in the presence of an
appropriate indicator.
METHOD)
JACOB VOLHARD
 Titrant/VS/Standard Solution = Ammonium
thiocyanate (NH4SCN)
 Indicator = Ferric Alum or FAS (Ferric
Ammonium Sulfate)
 Secondary precipitate = Ferric thiocyanate
Fe(SCN)3
 Endpoint = Reddish brown color
 Analyte + Titrant ➖> Primary precipitate
 Excess titrant + Indicator ➖ > Secondary ppt.

1. AMMONIUM THIOCYANATE
2. SILVER NITRATE
POTASSIUM THIOCYANATE CAN ALSO BE USED IN
REPLACEMENT OF AMMONIUM THIOCYANATE
KARL FRIEDRICH MOHR
 It applies the solubility product principle,
which states that in a saturated solution of an
ionic compound, the product of the molar
activities has a constant value at any particular
temperature and pressure.
 Ionic product exceeds the Ksp value of the
compound – precipitation will proceed
 Which means that if Ksp should be exceeded, if
not, there would be back reaction. Thus,
precipitation will not proceed.
ENDPOINT
 The endpoint is determined by KAZIMIERS FAJAN
 Formation of precipitate
 Appearance of turbidity
 Instrumental methods
 Internal indicators
COLORED SECONDARY
PRECIPITATE
(MOHR METHOD)
 Titrant/VS/Standard Solution = Silver Nitrate
 Indicator = Potassium chromate (K2CrO4)
 Secondary precipitate = Silver chromate
 Endpoint = White to Reddish colored mixture
Colored secondary precipitate – compound
COLORED ADSORPTION
PRODUCT
(FAJAN METHOD)
 Titrant/VS/Standard Solution = Silver Nitrate
 Indicator = DCF, TEE, Eosin Y
 Endpoint = Yellowish green to reddish pink
15
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
SOLVE HERE !
 DCF – Dichlorofluorescein
 TEE – Tetrabromophenolphthalein ethyl ester
 Eosin Y – Tetrabromofluorescein

 The gram eq wt. of a substance in the

precipitation method is obtained by dividing the
molecular weight by the total valence of the
precipitating or precipitated ion.

MW/val of cation
ADDITIONAL NOTES FOR WEEK 3
 AgBr; eq = 1
 KBr, eq = 1
 BaCl2 = 2
 Hg = 2
 SrCl = 2

COMPOUNDS ASSAYED CLASSIFICATION OF CHEMICALS

 Determination of Halogens
 Thiocyanate
 Cyanide
STANDARD SOLUTIONS
 SILVER NITRATE - a colorless crystalline solid
becoming black on exposure to light. A silver salt
with powerful germicidal activity. It can
potentially be used as cauterizing or sclerosing
agent.
 AMMONIUM THIOCYANATE – a colorless
crystalline solid. It is soluble in water. It is used
in chemical analysis, in photography, as a
fertilize, and other uses.
 POTASSIUM THIOCYANATE – an important
salt of the thiocyanate anion. The compound has
a low melting point relative to most inorganic
salts.
In standardizing silver nitrate, the primary standard is
Sodium Chloride and the indicator is DCF.
• Analytical Reagents or Reagent Grade chemicals
are commonly used inside the analytical and
•
clinical laboratories.
About 96-98% purity.
• Used in analytical procedures and in
manufacture of food and purer chemicals
• Good quality chemicals but do not meet an
official standard.
• Usually used in educational purposes and basic
qualitative analyses.
• Stands for United States Pharmacopeia or
National Formulary
• These chemicals pass the standard for use in
medicines, food ingredients, and dietary
supplements.
• “Safe for human consumption”

16
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
• Used commercially and industrially but is not OTHER TOOLS FOR MEASUREMENT
pure enough for any kind of analysis or food • Mohr pipette
manufacturing use. • Serological pipette
• Graduated Cylinder
• May also be referred to American Chemical • Erlenmeyer Flask
Society Grade (ACS) • Beaker
• Exceeds most chemicals in purity. • Reagent Bottles
• 98-99% purity.

• Uses centrifugal force to separate suspended

BALANCES elements in a solution.
•
 A weighing instrument with the • Filters are used to remove suspended particles
platform enclosed by glass doors in all in a solution by the use of gravity (sometimes
directions. Sensitive enough that a slight assisted by a vacuum)
breeze could alter the reading.
INSTRUMENT CARE
 A lesser precision instrument than the 1. Like all other materials, it is important to read
analytical balance. The platform has no and understand about the different
enclosures as the readings usually only requirements to care for laboratory
includes 1/10th of a gram. instruments.
2. For machines, annual calibration is
 A crude weighing tool that uses recommended. This should be done by the
counterweights to measure the weight manufacturer/distributor’s authorized
of an analyte. technicians.
3. Pipettes should be washed regularly with mild
CALIPER detergent and water. Take note that some
• A tool for precisely measuring length or chemicals can etch or damage glasswares, and
diameter. thus alter the volume that it can
• 2 different sides can be used to measure inner contain/transfer.
and outer diameter. 4. Instruments should not be used irresponsibly.
There is no “one-size-fits-all” for these
VOLUMETRIC GLASSWARE instruments, and so, it is important for you to
• Volumetric glasswares are used for precise and find out if the instrument/glassware can handle
accurate measurements as they are typically the the experiment that you will be doing.
instruments with the lowest margin of error.
CONTINUATION OF PRECIPITATION METHOD
AUTOMATIC PIPETTES
• Pipettes with a built-in vacuum system that
allows for consistent aspiration of a variable or
fixed volume.
• Precision is high.
• Accuracy is only high if regularly maintained and PRECIPITATION
calibrated.  Most precipitation gravimetric methods were
developed in the nineteenth century
17
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 In precipitation gravimetry an insoluble
compound forms when we add a precipitating
reagent, or precipitant, to a solution that
contains our analyte. In most cases the
precipitate is the product of a simple metathesis
reaction between the analyte and the
precipitant; however, any reaction that
generates a precipitate potentially can serve as
a gravimetric method.
 after the precipitate has been separated from
the solution, it has in some cases to be dried and
then weighed; in others, it has to be dried and
ignited before weighing.
 the operations of drying and ignition and the
necessary apparatus will now be described.
 Precipitates that are dried (below 250°C) or
ignited (above 250°C) should be collected on
filter paper, porcelain filtering crucibles or silica
filtering crucibles. The temperature at which
precipitates are dried or ignited can be
determined by a study of thermogravimetry.
THERMOGRAVIMETRY
 is a technique in which a change in the weight of
a substance is recorded as a function of
temperature or time. It is used in conjunction
with other techniques like differential thermal
analysis (DTA), gas chromatography and mass
spectrometry.
 Crystalline precipitates absorb water or
solvent can be easily removed by heating the
precipitates e. g. cuprous thiocyanate.

 After separating the precipitate from its

supernatant solution, we dry the precipitate to
remove residual traces of rinse solution and to
WHAT IS PRECIPITATION GRAVIMETRY?
remove any volatile impurities.
 The temperature and method of drying depend
 an analytical technique that uses a precipitation
on the method of filtration and the precipitate’s
reaction to separate ions from a solution
desired chemical form.
 The chemical that is added to cause the
 Placing the precipitate in a laboratory oven and
precipitation is called
heating to a temperature of 110oC is sufficient the precipitant or precipitating agent.
to remove water and other easily volatilized
 Sometimes you might hear people referring to
impurities.
precipitation gravimetry simply as gravimetric
 Higher temperatures require a muffle furnace, a analysis, which is a broader class of analytical
Bunsen burner, or a Meker burner, and are techniques that includes precipitation
necessary if we need to decompose the gravimetry and volatilization gravimetry.
precipitate before its weight is determined.
 A absorbs moisture, we must WHAT IS A PRECIPITATION REACTION?
remove it before we weigh the precipitate, this  In this topic, we will go through an example of
is accomplished by folding the filter paper over finding the amount of an aqueous ionic
the precipitate and transferring both the filter compound using precipitation gravimetry.
paper and the precipitate to a porcelain or
platinum crucible.
 can not
withstand high temperatures and are dried in an
oven at a temperature below 200oC.
 The used in Gooch
crucibles can be heated to a maximum
temperature of approximately 500oC.

18
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
Soluble silver salts such as silver(I) nitrate can be used as
precipitating agents to determine the amount of halide
ions present in a sample. Not only does the mass of the
precipitate tell you about the concentration of the halide  Since we are assuming that the mass of the
ions in solution, the color is also distinctive for different precipitate is all AgCl(s), we can use the
silver salts. This picture shows test tubes containing molecular weight of AgCl to convert the mass of
yellowish AgI (left), cream-colored AgBr (middle), and precipitate to moles.
white AgCl (right)
 Mol of AgCl(s) = 1.032 g AgCl x 1 mol AgCl
143.32 g AgCl
Example: Determining the purity of a mixture
= 0.007201 mol AgCl
containing MgCl2 and NaNO3

 As a result of the mishap, we have 0.7209 g of a

mysterious mixture containing MgCl2 and NaNO3.  We can convert the moles of AgCl(s) , the
We would like to know the relative amount of each precipitate, to moles of MgCl2(aq) using the
compound in our mixture, which is fully dissolved molar ratio from the balanced equation.
in water. We add an excess of our precipitating  Mol of MgCl2(aq) = 0.007201 mol AgCl x 1 mol
agent silver(I) nitrate, AgNO3(aq), and observe the MgCl2 /2 mol AgCl
formation of a precipitate, AgCl(s). Once the = 0.0036 mol MgCl2
precipitate is filtered and dried, we find that the
mass of the solid is 1.032 g.
What is the mass percent of MgCl2 in the  Since we are interested in calculating
original mixture? the mass percent of MgCl2 in the original
 Since this is a stoichiometry problem, we will want mixture, we will need to convert moles of MgCl2
to start with a balanced chemical equation. Here we into grams using the molecular weight.
are interested in the precipitation reaction  Mass of MgCl2 = 0.0036 mol MgCl2 x 95.2 g
between MgCl2(aq) and AgNO3(aq) to make MgCl2 / 1 mol MgCl2
AgCl(s), when AgNO3(aq) is in excess. = 0.3427 g MgCl2
 You might remember that precipitation reactions
are a type of double replacement reaction, which
means we can predict the products by swapping
 The mass percent of MgCl2 in the original
the anions (or cations) of the reactants. We might
mixture can be calculated using the ratio of the
check our solubility rules if necessary, and then
mass of MgCl2 from Step 3 and the mass of the
balance the reaction. In this problem we are
mixture.
already given the identity of the precipitate, AgCl
(s). That means we just have to identify the other  Mass% MgCl2 = 0.3427 g MgCl2/ 0.7209 g
mixture x 100%
product,Mg(NO3)2 (aq), and make sure the overall
reaction is balanced. The resulting balanced = 47.54% MgCl2 in mixture,,
chemical equation is:
MgCl2(aq) + 2AgNO3(aq)  2AgCl(s) + Shortcut: We could also combine Steps 1 through
Mg(NO3)2(aq) 3 into a single calculation which will involve careful
checking of units to make sure everything cancels out
 The balanced equation tells us that for every 1 mol
properly:
MgCl2 (aq), which is the compound we are
interested in quantifying, we expect to make 2 mol
AgCl(s), our precipitate. We will use this molar
ratio to convert moles of AgCl(s) to moles of
MgCl(2). We are also going to make the following
assumptions:
END OF PRELIM 19
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
IMPORTANCE
 “No analytical method, no matter how simple or
sophisticated, no matter how specialized or
routine, no matter how easy or difficult, and no
matter how costly, will produce the correct
result if the sample is not correctly obtained and
prepared.”
REPRESENTATIVE SAMPLE
 The critical part of any sampling task is to obtain
a sample that represents the bulk system as well
as possible
 The sample must possess all the characteristics
of the entire bulk system with respect to the
analyte and the analyte concentration in the
system
It must truly represent the bulk system
If there are suspected variations, small
samples must be taken from all suspect
locations
Composite samples – the small samples
taken are mixed and made homogenous to
give the final sample to be tested
Selective sample – analysis on the
individual samples
 If the bulk system is homogenous for a particular
component, then one sample is taken from one
location at random in the bulk system
 The sample integrity must be strictly maintained
and preserved
 Preservative, refrigeration, storage container
 The procedures to account for the integrity of
each specimen by tracking its handling and
storage from point of specimen collection to
final disposition of the specimen.
 Documentation of sample handling
 It is very important to document who has
handled the sample, what responsibility each
handler has at various junctures between the
sampling site and the laboratory, and what
actions the sample handler has taken relating
to sample integrity while the sample was in
his or her custody.
MAINTAINING SAMPLE INTEGRITY
 All sample custodians must maintain the sample
in its original physical and chemical condition so
that it remains representative of the bulk system
20
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
in terms of the analyte identity and
concentration.
TYPE OF URINE SAMPLES
 Sample stability should also be considered in Random specimen (Routine Testing)
handling specimens

 Give examples such as exposure to light First morning sample (8 hr sample/used for
routine screening, pregnancy, orthostatic )
(bilirubin det), delayed transpo (decrease
in glucose), bacterial contamination
Midstream clean catch (Routine and
 Unstable analytes in the sample must be bacterial)
protected from decomposition

24 hour sample (Quantitative Chemical Test)

BLOOD SAMPLES FECAL SAMPLE

 Used primarily for the study of cellular elements  Used for the detection of gastrointestinal
of the peripheral blood and those components of disorders such as diarrhea, and identification of
the plasma pathogenic bacteria or parasites

PHLEBOTOMY
 Collection of blood samples for laboratory
analysis to diagnose and monitor medical
conditions
URINE SAMPLE
 Frequently collected urine samples include
random, first morning, midstream clean-catch, CEREBROSPINAL FLUID
24-hour samples, catheterized and suprapubic  CSF sample is collected to diagnose meningitis,
aspirations subdural hemorrhage and other neurological
 Analyzing urine aids in the diagnosis of disease, disorders
screening asymptomatic populations for  Tube 1 – for chemistry
undetected disorders, and monitoring the  Tube 2 – for microbiology
progress of disease and the effectiveness of  Tube 3 – hematology
therapy Lumbar puncture between the 3rd, 4th, or 5th lumbar
vertebrae
21
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
SEMEN SPUTUM
 Semen samples are collected and tested to  Sputum is mucus or phlegm collected from the
evaluate fertility and postvasectomy procedures trachea, bronchi and lungs
Abstain for 3 days and not longer than 5 days  Used to test for active tuberculosis and
pneumonia
Deep coughing and then expelled into a sterile
container

 Biopsy is a medical procedure that involves

taking a sample of a tissue and examined under
a microscope

SYNOVIAL FLUID
 Synovial fluid, or “joint fluid”, is a viscous fluid
found in the cavities of the movable joints that
lubricates and reduces friction between bones
during joint movement
 It can be used to determine the pathologic origin
of arthritis

22
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
ACID HEMATIN FOR HEMOGLOBIN
DETERMINATION

IMPORTANCE
 Manual methods have long been the foundation
that built the advanced technology we have
today.
 Most of our automated procedures came from
manual methods of analysis.
 Laboratory scientists, when in doubt of a certain
result, should always manually check the sample
before releasing the result. Why?
HEMATOCRIT
 Because nothing is more certain that a result  Upon centrifugation of blood, the volume of the
that you have seen and verified yourself. red blood cells represents the packed red cell
 (ex. Hematology machines – produce volume or hematocrit
erroneous result = verify with a blood smear) MICROHEMATOCRIT

 Hematology
 Hemoglobin determination
 Hematocrit determination
 RBC count
 WBC count
 Platelet count
 Differential WBC count RED BLOOD CELL
HEMOGLOBIN  Also known as erythrocytes, they are small cells
 Identified by Felix Seyler and proved that this shaped like biconcave discs
was the true coloring matter of the blood  Primary function is to ferry oxygen in blood to
 An iron-bearing protein, hemoglobin’s main all cells of the body
function is to transport oxygen from the lungs to WHITE BLOOD CELL
tissues and transport carbon dioxide from the  Also known as leukocytes, the white blood cells
tissues to the lungs for exhalation. help defend the body against damage by
HEMOGLOBIN DETERMINATION bacteria, viruses or parasites
 Manual: Drabkin’s Cyanmethemoglobin Method CELL COUNTING: WBC/RBC

23
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
WHITE BLOOD CELLS

Granulocytes
• Neutrophils
• Eosinophils
• Basophils
Agranulocytes
• Lymphocyte
• Monocyte
STAINING OF BLOOD SMEAR
 Wright stain CHEMICAL EXAMINATION
DIFFERENTIAL COUNTING  Reagent strips currently provide a simple, rapid
means for performing medically significant
chemical analysis of urine, including pH, protein,
glucose, ketones, blood, bilirubin, urobilinogen,
nitrite, leukocytes, and specific gravity
Reflectance photometry - uses the principle that light
reflection from the test pads decreases in proportion to
the intensity of color produced by the concentration of
the test substance.

 Benedict’s test
PLATELETS  Heat Acetic Acid
 Platelets are needed for the clotting process that  Benedict’s test - When reducing sugars are
occurs when blood vessels are ruptured or heated in the presence of an alkali, they get
broken converted to powerful reducing compounds
PLATELET COUNT known as enediols. Enediols reduce the cupric
 Direct Platelet count ions (Cu2+)present in the Benedict's reagent to
 Indirect Platelet count cuprous ions (Cu+) which get precipitated as
insoluble red copper(I) oxide(Cu2O)
 In hot alkaline solution glucose reduce copper
salts to cuprous oxide
 Biuret method, by which copper salts in
alkaline solution form a purplecomplex with
substances containing two or more peptide
Microscopic Physical Chemical bonds
Examination examination examination

PHYSICAL EXAMINATION
 The physical examination of urine includes the
determination of the urine color, clarity, and
specific gravity

24
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
MICROSCOPIC EXAM

Gram Stain /
Sputum
Culture and AFB staining
coiling
Sensitivity

GRAM STAIN
 The most commonly used differential stain in
clinical microbiology laboratory

URINE ANTIMICROBIAL SUSCEPTIBILITY TESTING

 Used to identify the susceptibility patterns of the
bacterial isolate to the antimicrobial agents
CULTURE AND SENSITIVITY

The iQ 200 Automated Urine Microscopy Analyzer

(Iris Diagnostics, Chatsworth, Calif.) automatically
analyzes and classifies urine particles into 12
categories. The sample is mixed and aspirated to a
SPUTUM SMEAR
planar flowcell where 500 digital photomicroscopic
images are taken per sample. The system uses Auto
Particle Recognition (APR) software that classifies
urine particles in the photographs based on size,
shape, texture, and contrast into 12 categories—
RBCs, WBCs, WBC clumps, hyaline casts, unclassified
casts, squamous epithelial cells, nonsquamous
epithelial cells, bacteria, yeast, crystals, mucus, and
sperm

25
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
AFB STAINING
IMPORTANT NOTES!
TWO TEST FOR HEMOGLOBIN DETERMINATION
MANUAL: DRABKIN’S CYANMETHEMOGLOBIN
METHOD
 here we used Drabkin’s Reagent. It is used for
quantitative choloremetric determination of
hemoglobin concentration in whole blood.
 PRINCIPLES: This method lies in conversion of
hemoglobin. Icoconvert yung hemoglobin na
nakita sa whole blood to cyanmethehemoglobin
by the addition of postassium cyanide and ferri
cyanide. With the absorabance value of 540
nanometers. So we will use photoelectric
calorimeter or spectrophometer.
ACID HEMATIN
PRINCIPLES:
 The blood is mix with normal hydrochloric acid.
In the conversion of hemoglobin to acid hematin
which is a brown color.
 In the container our solution is same color.
 Then we will now see the values of how much
the hemoglobin.
HEMATOCRIT
 Is done to measure the RBCs in blood.
 Also known as packed cell volume.
 Measure of the ratio of the volume occupied by
RBC.
 If mababa hematocrit indicates anemia, a
condition that lack of RBCs to deliver oxygen.
MICROHEMATOCRIT
 Determination of packed cell volume or
hematocrit using a small quantity of whole
blood.
 After that - Submit into centrifugation.
WHITE BLOOD CELL
 Granulocytes – means have granules.
 Agranulocytes –doesn’t have granules.
How to Read:
 Need to do Blood Smear.
 Usage of Wright Stain
DIFFERENTIAL COUNTING
 Gives relative percentage of each type of WBC
and help reveals abnormal WBC population.
PLATELETS COUNT
 To avoid reading it repeatedly, in zigzag motion
starting from the bottom to up. (left to right
first).
GRAM STAIN
 It is a complex and differential staining
procedure use to differentiate the gram positive
and gram negative bacteria.
 Gram positive contains thick layers of
peptidoglycan. Stain purple.
 Gram Negative thin layer of peptidoglycan and
stain pink.
26
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 Crystal Violet
 Iodine / Gram’s Iodine
 Acid Alcohol/ alcohol
 Safranin
 Smear of bacteria.
 Stain the smear with crystal Violet. This is the
primary stain. Stay for 1 minute.
 Rinse to running water or ilulubog lang.
 Iodine then stay 1 minute. Rinse again.
 Acid Alcohol. Decolorizer.
 Then wash again.
 Add the safranin.
ANTIMICROBIAL SUSCEPTIBILITY
TESTING
 Determine what particular antibiotic or anti-
fungal drugs to stop growth of bacteria or fungi.
CULTURE AND SENSITIVITY
 Pag maliit yung hallow zone is the bacteria is
resistance doon sa antibiotic.
 Pag nakakapatay, susceptible ang bacteria.
SPUTUM SMEAR
 It allows rapid and reliable identification of
patient with tuberculosis or pneumonia.
 Gumagawa ng Sputum Coiling.
 Choose a new and clean slides
 Label the slides.
 Use wood applicator stick.
 Collect the thick or greenish part.
 Evenly distribute the specimen.
 Allow to dry.
 Then Heat Fix – para di mawashout ang
specimen.
- Bunsen Burner
- Alcohol Burner
- Electrical Heating Block.
 If using burner pass the slide 2 to 3 times.
 If using Electrical Heating Block (-2 Hours, 65C –
70C)
 Main aim in staining: Differentiate bacteria into
acid fast group and non-acid fast group by the
use of stain.
 By the use of this stain: Ziehl- Neelsen Staining
- Carbol-Fuschin (Primary Dye)
- 20% Sulphuric Acid or Acid Alcohol
(Decolorizer)
- Methylene Blue Dye (Counterstain) or
Malachite Green
 Carbol Fuschin for about 3 mins. (5-10 mins)
 Rinse and Shake
 Destain with Acid Alcohol. Drop and drop until
the color remove.
 Rinse and Shake Again.
 Methylene Blue Dye for 2 minute.
 Rinse Again and Shake.
 Blot Gently with Kimwipe.
 Acid Fast bacilli will be red.
 Heating.
CLINICAL CHEMISTRY
 Usage of Spectrophotometry.
 Measure how much a chemical substance
absorbs light by measuring the intensity of the
light as a beam of light passes through the
solution.
27
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 Instrumental analysis is a field of analytical
chemistry that investigates analytes using
scientific instruments. Block diagram of an
analytical instrument showing the stimulus and
measurement of response.
 The use of an instrument to measure a compon
ent, to detect the completion of a quantitative r
eaction, or to detect change in the properties o
f a system.
 Instrumental analysis is a field of analytical
chemistry thatinvestigates analytes using scie
ntific instruments.
 And Electrophoresis (Which is a separation
techniques)
 In this topic, the general concepts of
chromatography and discuss the different
classifications of it
 Also: We will be discussing the electrophoresis –
and the different types of it.
 Chromatography is the separation of the
components of a mixture based on the different
degrees to which they interact with two
separate material phases
 Technique for separating the components,
or solutes
 The result of this analysis is written in Color
Chroma- color Graphein- write
MOBILE PHASE – moving phase
STATIONARY PHASE – does not move
 Mixture is dissolved in the mobile phase and it
will move going to the stationary phase
 All types relies with these 2 phases
 Just like the ink of the pen you accidentally
write to a paper then smudged it with water–
WHAT DID YOU SEE? Because black dye is a
bunch of different other dyes and components .
This is the basic example of paper
chromatography
 Because it is composed of different components
– different colors
 The mixture (sample) is usually introduced
into the mobile phase, which is then made to
move through the stationary phase.
 The components are attracted to and slowed
by the stationary phase to varying degrees, and
as a result, they move along with the mobile
phase at varying rates, and are thus separated
 The general concept of chromatography – how
will the procedure of chromatography takes
place PAANO NANGYAYARI
 The mobile phase can be either a gas or a liquid,
while the stationary phase can be either a liquid
or solid.
 One classification scheme is based on the
nature of the two phases
 To fully understand the classification we
should know the ff: Is the mobile and
stationary phase liquid, solid or gas?
 The stationary phase is the phase that
doesn't move and the mobile phase is the
phase that does move. The mobile phase
28
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
moves through the stationary phase picking
up the compounds to be tested.
 And what is the separation method used,
what is the basis ex.) Polarity

 All techniques which utilize a gas for the

mobile phase come under the heading of gas
chromatography. All techniques that utilize a
liquid mobile phase come under the heading of
liquid chromatography

 It is more useful, however, to classify the

techniques according to the nature of the  Stationary phase is the solid medium but Liquid
interaction of the mixture components with in nature. The Mobile phase is also liquid
the two phases
 The stationary phase actually consists of a thin
liquid film chemically bonded to the surface of
finely divided solid particles
 Such a stationary phase cannot be removed from
the solid substrate by heat, reaction, or
dissolving in the mobile phase.
 The mobile phase is a liquid that moves through
a liquid stationary phase as the mixture
components partition or distribute themselves
between the two phases and become separated.
 The separation mechanism is thus one of the
dissolving of the mixture components to
different degrees in the two phases according to
their individual solubilities in each.

 1ST CLASSIFICATION – BOTH PHASE (LIQUID)

separation of similar substances by repeated
extraction by two immiscible liquids.

 If the stationary phase is somewhat polar, it

will retain polar components more than it will
nonpolar components, and thus the nonpolar
components will move more quickly through
the stationary phase than the polar
components.
 The reverse would be true if the stationary
phase were nonpolar.

29
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 The mobile phase for partition
chromatography can also be a gas.
Stationary phase -liquid
IN SUMMARY
 Partition chromatography is a type of
chromatography in which the stationary phase
is a liquid chemically bonded to the surface of a
solid substrate, while the mobile phase is either
a liquid or gas.
 The mixture components dissolve in and out of
the mobile and stationary phases as the mobile
phase moves through the stationary phase, and
separation occurs as a result
 Dissolve in and out and then the separation
happens when the mobile phase moves
through the stationary phase
 Stationary phase -liquid
 The separation mechanism is adsorption
 Adsorption – it is defined as the adhesion of
a chemical species onto the surface of particles
ADSORPTION CHROMA- STATIONARY SOLID PHASE
 The stationary phase consists of finely divided
solid particles packed inside a tube, and the
mixture components adsorb or stick to the
surface of the solid
CONCEPT
 The difference with partition – partition – liquid
stationary phase
 for this solid – the components will adhere into
the stationary phase
 STATIONARY PHASE SOLID
 The nature of the adsorption involves the
interaction of polar molecules, or molecules
with polar groups, with a very polar solid
stationary phase.
 The mobile phase can be either a liquid or a gas
 Nature of the adsorption – polarity
 Interaction of polar molecules with a polar
stationary phase
 It is a method for separating mixtures of ions,
both inorganic and organic.
 Method for ions
 The stationary phase consists of very small
polymer resin beads that have many ionic
bonding sites on their surfaces. These sites
selectively exchange ions with certain mobile
phase compositions as the mobile phase moves.
 Ions that bond to the charged site on the resin
beads are thus separated from ions that do not
bond
30
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA

CONCEPT
 Separation – bonding (affinity with the
stationary phase – ability to bond)
 the beads are charged, compounds having the
same charged maybe repelled by the column ,
meaning they will travel quickly.,

Cation echanger.

ION EXCHANGE FOR DEIONIZATION

OF WATER
 Wide columns packed with a mixture of an
anion exchange resin that exchanges
dissolved anions for hydroxide ions, and a
cation exchange resin that exchanges
dissolved cations for hydrogen ions are used
because water that is passed through such a
column becomes free of ions (deionized) since
the hydrogen and hydroxide ions combine to
form more water.
 Cation exchange chromatography, more
specifically, uses a negatively charged ion
exchange resin with an affinity for molecules
having net positive surface charges.
SPECIAL APPLICATION: deionization of water
 Hydroxide – negative charge
 Hydrogen – positive charge
 Combined to form water

 The stationary phase material can be either an

anion exchange resin, which possesses
positively charged sites to exchange negative
ions, or a cation exchange resin, which
possesses negatively charged sites to exchange
positive ions.
 The mobile phase can only be a liquid.

Anion – exchange negative ions (since negative

charge) then it possess positive charged sites where
the positive ion will be attracted

31
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 The two resins: anion and cation resins will
exchange cations and anions to produce water
 The Salt is used for the ion
 It is a technique for separating dissolved species
on the basis of their size.
Basis: SIZE
Aka: Gel filtration chromatography
 The stationary phase consists of porous
polymer resin particles. The components to be
separated can enter the pores of these particles
and be slowed from progressing through this
stationary phase as a result.
 The mobile phase for this type can only be a
liquid.
 Thus, the separation depends on the sizes of
the pores relative to the sizes of the
molecules to be separated.
 Porous – with pores (holes)
  The advantages of this method include good
separation of large molecules from the small
molecules
 Configurations can be broadly classified into
two categories: Planar methods and the
column methods
Arrangement of chromatography
 —physical state (gas or liquid) and
positioning, and how and in what direction
the mobile phase travels in terms of gravity,
capillary action, or other forces.
A. PLANAR METHOD
 The planar methods utilize a thin sheet of
stationary phase material and the mobile phase
moves across this sheet, either upward
(ascending chromatography), downward
(descending chromatography), or
horizontally (radial chromatography).
32
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
2 types of chromatography under the planar
method:
 Paper chromatography
 Thin Layer chromatography
PAPER CHROMATOGRAPHY
 Paper chromatography makes use of a sheet of
paper having the consistency of filter paper
(cellulose) for the stationary phase.
 Since such paper is hydrophilic, the stationary
phase is actually a thin film of water
 Capillary action is the ability of a liquid to
unintentionally adsorbed on the surface of the
flow in narrow spaces without the assistance
paper.
of, and in opposition to, external forces such as
 Thus, paper chromatography represents a form
gravity
of partition chromatography only.
 The most common stationary phase used –
 The mobile phase is always a liquid.
silica gel – highly polar stationary phase for
Why partition? Because the filter paper is
adsorption chromatography
hydrophilic – meaning it is just like a water
 Another common – pure cellulose – same
uninterntionally absorbed on the surface of the
material with paper chromatography thus
water
under the Partition chromatography .

 These Retardation factors, which are fractions

less than or equal to 1, are compared to those
of standards to reveal the identities of the
components.

WATER IS HELD IN THE PORES OF A FILTER PAPER

 It travels up in the paper.
 How attracted it is to the paper or the solvent
depending on its chemical properties.
THIN LAYER
CHROMATOGRAPHY
 The stationary phase is a thin layer of material
spread across a plastic sheet or glass or metal
plate.
 TLC can be any of the four types, including
adsorption, partition, ion exchange, and size Rf factors  ratio of the distance traveled by
exclusion component to the distance traveled by the solvent
 The mobile phase for TLC is always a liquid. or the mobile phase

33
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 Quantitative analysis is also possible. The spot
representing the component of interest can be
cut (in the case of paper chromatography) or
scraped from the surface (TLC), dissolved, and
quantitated by some other technique, such as
spectrophotometry.
B. COLUMN METHOD
• Column methods utilize a cylindrical tube to
contain the stationary phase, and the mobile
phase moves through this tube either by
gravity, with the use of a high pressure pump,
or by gas pressure
It is a solid-liquid technique in which the stationary
phase is a solid & the mobile phase is a liquid or gas.
Stationary phase is held in a narrow tube through
which the mobile phase is forced under pressure or
under the effect of gravity
Types:
1. Open column chromatography
2. Instrumental chromatography
OPEN COLUMN
CHROMATOGRAPHY
 A type of chromatography that consists of a
vertically positioned glass tube in which the
stationary phase is placed
 It is typical for this tube to be open at the top
(hence the name open-column
chromatography), to have an inner diameter
on the order of a centimeter or more, and to
have a stopcock at the bottom
 All four types (adsorption, partition, ion
exchange, and size exclusion) can be used with
this technique.
 Stopcock – just like faucet to regulate the flow
(stop and not to stop)
 raditional column chromatography is
characterized by addition of mobile phase
under atmospheric pressure and the
stationary phase is packed in a glass column.
34
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 The mixture to be separated is placed at the
top of the column and allowed to pass onto
the stationary phase by opening the stopcock.
 The mobile phase is then added and
continuously fed into the top of the column
and flushed through by gravity flow. The
mixture components separate on the
stationary phase as they travel downward and
are then collected as they elute from the
column.
The components present into the mixture will travel
downward and will be separated with the stationary
phase
 A typical fraction collector consists of a
rotating carousel of test tubes positioned
under the column such that fractions of eluate
are collected over a period of time
 A substance obtained from elution –
ELUATE
 Elute - remove (an adsorbed substance) by
washing with a solvent, especially in
chromatography.
INSTRUMENTAL
CHROMATOGRAPHY
 A column along with a continuous mobile phase
flow system (that does not use gravity for the
flow), a device for introducing the mixture to
the flowing mobile phase, and an electronic
sensor at the end of the column incorporated
into a single unit
 These are Gas chromatography and high-
performance liquid chromatography.
 Add a degree of efficiency and speed to the
chromatography concept.
AUTOMATED – with machines and instruments –
more efficient and faster than non-instrumental
chromatography
 A separation technique that utilizes an electric
field
 It is a general term that describes the migration
and separation of charged particles (ions)
under the influence of an electric field.
35
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 An electric field is an electrically charged region TYPES OF ELECTROPHORESIS:
of space, such as between a pair of electrodes 1. Paper ( glass plate + cellulose + glass plate)
connected to a power supply. 2. Gel
 The technique utilizes the varied rates and 3. Capillary
direction with which different organic ions (or
large molecules with charged sites) migrate
while under the influence of the electric field.

 Rates and direction variation  The soaked cellulose sheet is sandwiched

 Electrophoresis is an electrokinetic process
which separates charged particles in a fluid
using a field of electrical charge.
 A power supply is needed for connection to a
pair of electrodes to create the electric field. The
medium (cellulose or gel) and sample to be
separated are positioned between the
electrodes
between two horizontal glass plates with the
ends dipped into vessels containing more
electrolyte solution.
 The electrodes are also dipped into these
vessels.
 The sample is spotted in the center of the sheet,
and the oppositely charged ions then have room
to migrate in opposite directions on the sheet

 Electrophoresis is for separating ions, since only

ions will migrate under the influence of an
electric field, negative ions to the positive
electrode and positive ions to the negative
electrode
 Useful in biochemistry experiments

1. Ions of opposite charge will migrate in different

directions and become separated on that basis
2. Ions of like charge, while migrating in the same
direction, become separated due to different
migration rates
 Factors influencing migration rate are charge
values (e.g., –1 as opposed to –2) and different
mobilities. The mobility of an ion is dependent
on the size and shape of the ion as well as the
nature of the medium through which it must
migrate
The samples are spotted in the center of the paper,
high voltage is applied, and the spost migrate
according to their charges.
 Qualitative analysis is performed much as with
paper chromatography, by comparing the
distances the individual components have
migrated to those for standards spotted on the
same sheet.

36
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA

 The thin gel slab is contained between two

glass plates. The slab is held in a vertical
position and has notches at the top where the
samples to be separated are spotted or
streaked.
 Only downward movement takes place, and
thus only one type of ion, cation or anion, can
be separated, since there is only one direction
to go from the notch.

For this type of electrophoresis – only downward

movement takes place meaning only one type of ion
is separated either cation or anion

 A tracking dye can be added to the sample so
that the analyst can know when the experiment
is completed
 The slab can be removed from the glass plates
and a staining dye can be applied that binds to
the components, rendering them visible
 Components with different mobilities
through the gel show up as different bands or
streaks on the gel.
 Qualitative analysis is performed as with paper
electrophoresis—standards are applied
alongside the samples and the components
identified by their positions relative to the
standards
the leading edge of the sample solvent is visible via
the tracking dye).
 a useful extension of basic gel electrophoresis in
protein analysis
 In this technique, a series of ampholytes is
placed on the slab via electrophoresis
 An ampholyte is a substance whose molecule
contains both acidic and basic functional groups
(solution of different ampholytes have different
pH values)
 Different ampholyte molecules differ in size and
therefore will have varying mobilities in the
electrophoresis experiment
 Thus, these molecules migrate into the slab, take
up different positions along the height of the
slab, and create a pH gradient through the height
of the slab.
 Amino acid molecules have different mobilities
in different pH environments and also have their
charges neutralized at particular pH values,

37
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
rendering them immobile at some position in
the gel
 Mixture components migrate to a particular
location in the gel.
 The pH at which the sample component is
neutralized is called the isoelectric point, and
this technique is called isoelectric focusing, since
samples are separated according to their
components’ isoelectric points.
 It is electrophoresis that is made to occur inside
a piece of small-diameter capillary tubing
 The tubing contains the electrolyte medium, and
the ends of the tube are dipped into solvent
reservoirs
 An electronic detector is on-line and allows
detection and quantitative analysis of mixture
components.
 ANODE – positive electrode (negative ions will
migrate) ; CATHODE – negative electrode
(positive ions will migrate)
 The very small volume of sample (5 to 50
nL/nanoliter) is injected on one end of the tube,
typically the end dipped into the reservoir
containing the positive electrode. When the
experiment begins, the positive ions migrate
quickly through the capillary tube toward the
negative electrode in the opposite reservoir and
separate on the basis of their mobilities, as in the
other electrophoresis techniques.
 Ultimately, they pass individually through the
detector and generate peaks on a recorder trace
in a way very similar to that of HPLC
 For the last topic in midterm period: We will be
discussing the quality control (which is a very
important procedure done in all sections of the
clinical laboratory)
 Quality control (QC) is one of the most
important impacts on laboratory testing—it
ensures both precision and accuracy of patient
sample results.
1. Objectives and definition of quality control
2. Parameters of quality control
3. Kinds of quality control
OBJECTIVES OF QUALITY CONTROL
 To check the stability of the machine
 To check the quality of reagents
 To check technical (operator) errors
QUALITY CONTROL
 Quality control is a system of ensuring accuracy
and precision in the laboratory by including
quality control reagents in every series of
measurements
 It is a process of ensuring analytical results are
correct by testing known samples that
resemble patient samples
 Involves the process of monitoring the
characteristics of the analytical processes and
detects analytical errors during testing, and
ultimately prevent the reporting of inaccurate
patient test results
PARAMETERS OF QUALITY CONTROL
 Sensitivity
 Specificity
 Accuracy
 Precision or Reproducibility
Parameters – numerical or other measurable factor
that defines a system
PARAMETERS OF QUALITY CONTROL
 PRACTICABILITY – degree by which a method is
easily repeated
 RELIABILITY – ability of an analytical method to
maintain accuracy and precision over an
extended period of time during which
equipment, reagents and personnel may change
 DIAGNOSTIC SENSITIVITY
 DIAGNOSTIC SPECIFICITY
38
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
DIAGNOSTIC SENSITIVITY
 It is the ability of the analytical method to detect
the proportion of individuals with the disease
 It indicates the ability of the test to generate
more true-positive results and few false-
negative
 Screening tests require high sensitivity so that
no case is missed
Sensitivity (%)
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑖𝑠𝑒𝑎𝑠𝑒𝑑 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑤𝑖𝑡ℎ 𝑎 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑡𝑒𝑠𝑡
x 100
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑖𝑠𝑒𝑎𝑠𝑒𝑑 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑡𝑒𝑠𝑡𝑒𝑑
DIAGNOSTIC SPECIFICITY
 It is the ability of the analytical method to detect
proportion of individuals without the disease
 Reflects the ability of the method to detect true
negatives with very few false positive
 Confirmatory tests require high specificity to
be certain of the diagnosis
Specificity (%)
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑡ℎ𝑒 𝑑𝑖𝑠𝑒𝑎𝑠𝑒 𝑤𝑖𝑡ℎ 𝑎 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑡𝑒𝑠𝑡
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑡𝑒𝑠𝑡𝑒𝑑 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑡ℎ𝑒 𝑑𝑖𝑠𝑒𝑎𝑠𝑒
100
 100% sensitivity and specificity indicate that the
test or method DETECTS every patient WITH
the disease and that the test is NEGATIVE for
every patient WITHOUT the disease
KINDS OF QUALITY CONTROL
 Intralab Quality Control (Internal QC)
 Interlab Quality Control (External QC)
INTRALAB QUALITY CONTROL
 Involves the analyses of control samples
together with patient specimens
 Detects changes on the performance between
the present operation and the “stable” operation
 It is important for the daily monitoring of
accuracy and precision of analytical methods
CHARACTERISTICS OF AN IDEAL QC MATERIAL
 Resembles human sample
 Inexpensive and stable for long periods
 No communicable diseases
=  No matrix effects
 With known analyte concentration
 Convenient packaging for easy dispensing and
storage
Matrix effect – alteration to the results
INTERLAB QUALITY CONTROL
 It involves proficiency testing programs that
periodically provide samples of unknown
concentrations to participating clinical
laboratories
 It is important in maintaining long-term
accuracy of the analytical methods
 Next kind
 ANNUALLY – once a year
 Specimens are despatched in pairs to
= participating laboratories. Perfromed by many
x lab at the same time and monitored by one,
periodic monitoring for the performance of
the lab
CONDUCT OF EXTERNAL QC TESTING
 A series of unknown samples are sent to the
laboratory from the reference laboratory or
authorized program provider
 Unknown samples must be tested by the
laboratorians who regularly perform analysis of
patient specimens using the same reagents and
equipment for actual patient specimens, and the
results are submitted to the program provider
RATIONALE OF THE EXTERNAL
QC/PROFICIENCY TESTING
 To ensure clinicians that patient results are
accurate
 Allows each laboratory to compare and evaluate
test results or outcomes with those laboratories
that used the same methods (reagents and
equipment)
39
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
reaction between the two is judged complete.
The volume of reagent needed to complete the
titration is determined from the difference
between the initial and final volume readings.
INTRODUCTION
 The EQUIVALENCE POINT in a titration is
 A technique for determining the concentration
reached when the amount of added titrant is
of a solution by measuring the volume of one
chemically equivalent to the amount of analyte
solution needed to completely react with
in the sample.
another solution. Titration process involves
 the point in a titration
addition of solution of known concentration
when the amount of added
from a burette to the measured volume of
standard reagentis exactly
analyte.
equivalent to the amount of analyte.
THE PRINCIPLE:  END POINT: the point in a titration when a
physical change occurs that is associated
 It is based on the complete chemical reaction
with the condition of the chemical
between the analyte and the titrant of known
equivalence.
concentration.
 INDICATORS are often added to the analyte
ANALYTE + TITRANT  Product solution to produced an observable physical
change at or near the equivalence point.
 Volumetric titrimetry involves measuring the
volume of a solution of known concentration
that is needed to react essentially completely
with the analyte.
 Gravimetric titrimetry differs only in that the
mass of the reagent is measured instead of its
volume.
 Coulometric titrimetry, the reagent is a constant
direct electrical current of known magnitude
that consumes the analyte.

 A standard solution (or a standard titrant) is

a reagent of known concentration that is used
to carry out a titrimetric analysis.
 A titration is performed by adding a standard
solution from a buret or other liquid-dispensing
device to a solution of the analyte until the
40
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 During titration.
The titrant is added to the
PHYSICAL CHANGE flask with swirling until
the color of the indicator
 the appearance or disappearance of a color,
persists. In the initial
 the change in color,
region of the titration,
 the appearance or disappearance of turbidity.
titrant may be added
rather rapidly, but as the
Typical setup for
end point is approached,
carrying out a titration.
increasingly smaller
The apparatus consists
portions are added; at
of a buret, a buret stand
the end point, less than
and clamp with a white
half a drop of titrant
porcelain base to
should cause the
provide an appropriate
indicator to change color.
background for viewing
indicator changes, and a
wide-mouth
Erlenmeyer flask
containing a precisely
known volume of the
solution to be titrated.
The solution is normally
delivered into the flask
using a pipet, as shown
in.
Figure 1: The titration process

 Detail of the buret graduations. Normally, the

buret is filled with titrant solution to within 1 or
2 mL of the zero position at the top. The initial
volume of the buret is read to the nearest ± 0.01
mL. The reference point on the meniscus and the
proper position of the eye for reading are
depicted in figure.

 Before the
titration begins. The
solution to be titrated,
an acid in this example,
is placed in the flask
and the indicator is
added as shown in the
photo. The indicator in
this case is  Titration end point. The end point is achieved
phenolphthalein which when the barely perceptible pink color of
turns pink in basic phenolphthalein persists.
solution.  The flask on the left shows the titration less than
half a drop prior to the end point; the middle
flask shows the end point.
41
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 The final reading of the buret is made at this
point, and the volume of base delivered in the
titration is calculated from the difference
between the initial and final buret readings. The
flask on the right shows what happens when a
slight excess of base is added to the titration
mixture. The solution turns a deep pink color,
and the end point has been exceeded. In color
plate 9, the color change at the end point is much
easier to see than in this black-and-white
version.
TITRATION ERROR (ET)
 the difference in volume or mass
between the equivalence point and the end point
Et = Vep – Veq
 Vep : the actual volume of reagent required to
reach the end point
 Veq: the theoretical volume to reach the
equivalence point
• A primary standard is a highly purified
compound that serves as a reference material in
all volumetric and mass titrimetric methods. The
accuracy of a method is critically dependent on
the properties of this compound. Important
requirements for a primary standard are
 High purity
 Atmospheric stability
 Absence of hydrate water so that the
composition of the solid does not change
with variation in relative humidity
 Ready availability at modest cost
 Reasonable solubility in the titration
medium
 Reasonably large molar mass so that the
relative error associated with weighing the
standard is minimized.
SECONDARY STANDARD
 a compound whose purity has been established
by chemically analysis and that serves as the
reference material for a titrimetric method
 The accuracy of a titrimetric method can be no
better than the accuracy of the concentration of
the standard solution used in the titration. Two
basic methods are used to establish the
concentration of such solutions. The first is the
direct method in which a carefully weighed
quantity of a primary standard is dissolved in a
suitable solvent and diluted to a known volume
in a volumetric flask.
 The second is by standardization in which the
titrant to be standardized is use to titrate
 (1) a weighed quantity of a primary
standard,
 (2) a weighed quantity of a secondary
standard, or
 (3) a measured volume of another
standard solution
TWO BASIC METHODS ARE USED
CONCENTRATION OF SUCH SOLUTIONS:
(1) Direct method ～ ～ careful weighed quantity
of primary standard is dissolved in a suitable
solvent and dilute to exactly know volume.
(2) Standardization: the titrant to be
standardized is used to titrate weighed
quantity of a primary standard weighed
quantity of a secondary standard measured
volume of another standard solution
 The concentrations of standard solutions are
generally expressed in units of either molarity c
or normality cN.
 Molarity gives the number of moles of reagent
contained in one liter of solution,
 Normality gives the number of equivalents of
reagent in the same volume.
M= moles/L
N= equivalents/L
SOME USEFUL ALGEBRAIC
RELATIONSHIPS (VOL)
 Most volumetric calculations are based on two
pairs of fundamental equations that are derived
from definitions of millimole, mole, and molar
concentration. For the chemical, we may write
42
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
Amount (mol) = mass (grams)
molar mass (gm/mole)
Amount (mmol) = mass (grams)
mmolar mass (gm/mmole)
 We may derive a second pair from the definition  Calibration of glassware and tedious cleaning to
of molar concentration. That is, amount (mol) = ensure proper drainage are completely
volume (L) x concentration (mole/L) amount eliminated.
(mmol) = volume (mL) x concentration  Temperature corrections are unnecessary
(mmole/mL) because weight molarity does not change with
temperature, in contrast to volume molarity.
SOME USEFUL ALGEBRAIC  Weight measurements can be made with
RELATIONSHIPS (MASS) considerably greater precision and accuracy
 Weight titrations are more easily automated
 WEIGHT OR GRAVIMETRIC TITRIMETRY – Mass of than are volumetric titrations.
titrant is measured TITRATION CURVESIN
 Weight molarity (MW) : the number of moles of
reagent in 1 Kg Solution
TITRIMETRIC METHODS
 End point ～～ physical change that near
 Weight Molarity= Mole
equivalent point
solution (Kg)
Ex. TWO MOST WIDELY USED END POINT
0.1 MW NaCL  changes in color due to the reagent, the analyte,
= 0.1 mol of the NaCl in 1 Kg of solution or an indicator
= 0.1 mmol of the NaCl in 1 g of solution  change in potential of an electrode that responds
to the concentration of the reagent or the
analyte
TYPES OF TITRATION CURVES
 Titration curve: plots of a concentration-
related variable as a function of reagent volume.
TWO GENERAL TYPES OF TITRATION CURVES
 sigmoidal curve
 linear segment curve
THE P-FUNCTION OF ANALYTE IS PLOTTED AS A
FUNCTION OF REAGENT VOLUME
 Measurements are made on both sides the
equivalent point

43
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
CONCENTRATION CHANGES DURING TITRATIONS
 The equivalent point in a titration is
characterized by major changes in the relative
concentrations of reagent and analyte.
 ACID BASE TITRATION
 COMPLEXOMETRIC TITRATIONS
 REDOX TITRATION
 PRECIPITATION TITRATION
 ACID –BASE TITRATION – a sample of known
concentration of acid is estimated with known
concentration base or vice versa.
 COMPLEXOMETRIC TITRATION – as the name
indicates, the end point is achieved by the
formation of a complex molecule.
 Once complex is formed, the complex is stable
and no further reaction takes place.
Titrant + Titrand  complex
Ca+2 + EDTA-4 à  CaEDTA-2
 REDOXTITRATION – is based on the redox
reaction (oxidation-reduction) between the
analyte and titrant.
 PRECIPITATION TITRATION – the titrations which
are based on the formation of insoluble
precipitates, when the solutions of two reacting
substances are brought in contact with each
other are called Precipitation titration
 Titration is widely applied to analytical
technique. Some areas where titration is used
are given below
 Agriculture, oil industry, chemical industry,
pharmaceuticals, food industry
LABORATORIES
 Reasons why titration is widely used in
laboratories
 Titration is an established analytical
techniques
 It is fast
 It is a very accurate and precise technique
 Titration offers a good price/performance
ratio as compared to more sophisticated
techniques
 It can be used by low skilled and low
trained operators
 No need for highly specialized chemical
SOLUTION PREPARATION
 In many chemical reactions, solution
concentrations are expressed and prepared
considering the amount of substance present in
the actual solution (mole).
Karamihan sa reagents ay iiexpressed natin sa
molarity, molality or normality, in titrations mas
maganda I use ang moles for reference point.
CONCENTRATION
 The concentration of a solution is the expression
of how much of a substance is present and
dissolved within a solvent.
 SOLUENT – substance being dissolved.
 SOLVENT – substance that dissolves.
44
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 Concentration can be expressed in many MOLARITY
different units.
 Molarity (moles relative to volume of
whole solution)
 Molality (moles relative to mass of
solvent)
 Percent concentration (mass relative to
volume, volume relative to volume, mass
relative to mass)
Pag sinabi pong percent concentration , where
looking for how much of the soluent are present for
relative to the amount of the solvent. PERCENT CONCENTRATIONS
 (Mass / Volume) x 100%
MOLARITY  (Volume / Volume) x 100%
 (Mass / Mass) x 100%

DILUTIONS
 Dilutions can be done to adjust the volume
of a ready-made solution.
 To do so, follow the formula: C1V1=C2V2
STANDARD SOLUTIONS
 Standard solutions are solutions of known
concentration. They are used as a
reference point for unknown analytes.
N= mole (given weight/ MW)  However, if a commercially produced
/ volume= Liters only. standard solution is scarce, there is a
process called “standardization”
MOLALITY  Standardization can be done in two ways:
by comparing to a known substance or by
comparing with a primary standard.
PRIMARY STANDARD
 A primary standard is a substance that can
be weighed accurately and thus would no
longer need standardization.
 Karamihan sa reagents ay iiexpressed
natin sa molarity, molality or normality, in
titrations mas maganda I use ang moles for
reference point.
 Molality is expressed in mol/ kg of TITRATION
solvent.
 Instead of volume , were getting mass.  Is a laboratory experiment used to
 Mass of solvent.. determine the concentration of an
 Hindi po siya yung buong mixture unknown analyte using a substance of
known concentration.

45
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
GLASSWARES
 Acid and Base Burettes
 Burette Clamp
 A titrant
 Indicator
 Analytical Balance

BURETTES

BURETTE CLAMP

END POINT TITRATION

INDICATOR

END OF MIDTERM 46
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
AUTOMATION
 The process whereby an analytical instrument
performs many tests with only minimal
involvement of an analyst
 Controlled operation of an apparatus, process,
or system by mechanical or electronic devices
without human intervention
 To increase the number of tests performed by
one laboratorian in a given period
 To minimize the variation in results from one
laboratorian to another
 Automation eliminates the potential errors of
manual analyses such as volumetric pipetting
steps, calculation of results, transcription of
results etc.
 Instruments can use very small amounts of
samples and reagents
DEFINITION OF TERMS
BATCH TESTING
 All samples are loaded at the same time, and a
single test is conducted on each sample
PARALLEL TESTING
 More than one test is analyzed concurrently on
a given clinical specimen
SEQUENTIAL TESTING
 Multiple tests analyzed one after another on a
given specimen
RANDOM ACCESS TESTING
 Any test can be performed on any sample in any
sequence
 A type of testing which any specimen is analyzed
by any available process in or out of sequence
with other specimens and without regard to
their initial order
OPEN REAGENT SYSTEM
 A system other than manufacturer’s reagents
can be utilized for measurement
CLOSED REAGENT SYSTEM
 A system where the operator can only use the
manufacturer’s reagents
CARRY OVER
 The transport of a quantity of analyte or reagent
from one specimen reaction into and
contaminating a subsequent one
 These are the continuous flow, centrifugal
analyzer, and discrete analyzer
 All three can use batch analysis (large numbers
of specimen in one run), but only discreet
analyzers offer random-access, or STAT,
capabilities
CONTINUOUS FLOW ANALYZER
 Liquids (reagents, diluents, and samples) are
pumped through a system of continuous tubing.
 Samples are introduced in a sequential manner,
following each other through the same network.
 A series of air bubbles at regular intervals serve
as separating and cleaning media.
 Continuous flow, therefore, resolves the major
consideration of uniformity in the performance
of tests because each sample follows the same
reaction path.
 Continuous flow also assists the laboratory that
needs to run many samples requiring the same
procedure.
 The major drawbacks that contributed to the
eventual demise of traditional continuous-flow
analyzers in the marketplace were significant
carryover problems and wasteful use of
continuously flowing reagents.
47
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA

 It is uniformed
 No 1 problem- carry over

CENTRIFUGAL ANALYZER
 Centrifugal analysis uses the force generated by
centrifugation to transfer and then contain
liquids in separate cuvettes for measurement at
the perimeter of a spinning rotor
 Centrifugal analyzers are most capable of
running multiple samples, one test at a time, in a
batch.
 Batch analysis is their major advantage because
reactions in all cuvettes are read virtually
simultaneously
 Multiple samples one test at a time
DISCRETE ANALYZER
 Discrete analysis is the separation of each
sample and accompanying reagents in a
separate container.
 Discrete analyzers have the capability of
running multiple tests one sample at a time or
multiple samples one test at a time.
 They are the most popular and versatile
analyzers and have almost completely replaced
continuous-flow and centrifugal analyzers.
 Multiple test or multiple samples
 However, because each sample is in a separate
reaction container, uniformity of quality must be
maintained in each cuvette so that a particular
sample’s quality is not affected by the particular
space that it occupies.
 Major advantage: Random access capability
which allows STAT samples to be easily tested
 A common medical abbreviation for urgent or
rush. From the Latin word statim, meaning
"immediately."
 More than half of all instrumental methods of
analysis involve the absorption or emission of
light. Such instrumental methods can be
referred to as spectrochemical methods
 The science that deals with light and its
absorption and emission by solutions and other
material substances is called spectroscopy or
spectrometry
 The broad term for the instrument used is
spectrometer, while a slightly more specific
term (when a light sensor known as a phototube
is used) is spectrophotometer
 In spectrochemical analysis procedures, the
degree to which light is absorbed, or the
intensity of light that is emitted, is related to
the amount of an analyte present in the
sample tested
 IMPORTANT A spectrophotometer is an
instrument that measures the amount of
light that can pass through a solution. It is
apparent that less light is allowed to pass
through a highly turbid or colored solution
than through a clear solution.
 Standards are materials containing a
precisely known concentration of a substance
for use in quantitative analysis.
A standard provides a reference that can be
used to determine unknown concentrations or
to calibrate analytical instruments.
 To standardize an analytical method we
use standards containing known amounts of
analyte.

48
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA

CHARACTERIZING ENERGY

LIGHT  Light is a form of energy, and each wavelength

or frequency has a certain amount of energy
 The modern characterization of light is that it associated with it. This energy is considered to
has a dual nature be the energy associated with a single photon of
 Some qualities of light are best explained if we the light.
describe it as consisting of moving particles,
often called photons CONCLUSIONS
 Other qualities are best explained if we describe
 Frequency and energy are directly proportional
light as consisting of moving electromagnetic
 Wavelength, on the other hand, is inversely
disturbances, referred to as electromagnetic
proportional to frequency and energy
waves

WAVELENGTH
 The length of an electromagnetic wave is called
its wavelength, and its symbol is the lowercase
Greek letter lambda, λ
 In a set of repeating waves, wavelength is the  Wavelengths can vary in distance from as little
physical distance from a point on one wave, as fractions of atomic diameters to as long as
such as the crest of the wave, to the same point several miles.
(the crest) on the next wave.  This suggests the existence of an extremely
 They can be expressed in meters, centimeters, broad spectrum of wavelengths
nanometers  This electromagnetic spectrum of light is so
 Wavelengths of electromagnetic waves vary broad that we break it down into regions
from as short as atomic diameters to as long as  The region of wavelengths that we see with our
several miles. eyes is called the visible region. It is located
approximately in the middle of the
electromagnetic spectrum and has
wavelengths that vary from approximately 350
nm to approximately 750 nm—a very narrow
region compared to the entire spectrum and to
 Relationship
the other regions
 Due to electromagnetic wave – example: radio
waves
 A term to describe the entire range of light
that exists
 The region of light – different range of
FREQUENCY wavelength
 The number of the moving electromagnetic
waves that pass a fixed point in 1 sec is called the
frequency of the light. Its symbol is the
lowercase Greek letter nu, ν
 It is expressed in waves (most often called
cycles) per second, or hertz (Hz)

 Symbol – nu, v
 Unit - hertz
49
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
OTHERS INCLUDE THE ULTRAVIOLET REGION, THE
INFRARED REGION, THE X-RAY REGION, AND THE RADIO
AND TELEVISION WAVE REGION
RADIO AND TELEVISION WAVES
 are on the long end of the electromagnetic
spectrum, on the order of kilometers long. These
are very low energy waves that do no harm as
far as our health and safety are concerned
MICROWAVES
 have wavelengths on the order of a centimeter
and are also of low energy, but they can be
dangerous because their absorption causes the
generation of much internal heat.
INFRARED
 In another familiar region, the infrared region,
the wavelengths are extremely short by
comparison. While the wavelengths are shorter
and have higher energy than radio and
television waves or microwaves, they also cause
us no harm.
ULTRAVIOLET
 When we consider the ultraviolet region, the
wavelengths are shorter still, meaning more
energy. They are known to cause harm, such as
sunburn
 the remote controls we use to control
 our stereos and VCRs utilize infrared light
X-RAYS
 are extremely short wavelengths of extremely
high energy, penetrating our skin and tissue and
causing harm
GAMMA RAYS
 have wavelengths on the order of atomic
diameters and cause extreme damage to the
human body due to their extremely high energy.
 radioactive substance (nuclear explosion)
 The wavelengths within the visible region of the
electromagnetic spectrum are associated with
the colors we see
 The visible region of the spectrum provides a
good starting point to understanding the
process of light absorption
 Objects display a color because some visible
light wavelengths are absorbed.
 Why does a red sheet of paper appear red? All
the wavelengths of visible light from the sun and
the light bulbs on in the room are absorbed
except for the red wavelengths, and these are
reflected to our eye.
 Those wavelengths of light that are not absorbed
by a sample of matter are the wavelengths that
are reflected, or transmitted, to our eye and
give the sample its color.
 Many instruments that are used to measure the
absorption of light by atoms, molecules, and
complex ions are constructed to measure the
ultraviolet and visible regions of the
electromagnetic spectrum.
 Others are constructed to measure the infrared
region of the spectrum.
 If molecules are being measured in the
ultraviolet and visible regions, the technique is
referred to as ULTRAVIOLET-VISIBLE (UV-VIS)
SPECTROPHOTOMETRY
 If molecules are being measured in the infrared
region, the technique is referred to as INFRARED
(IR) SPECTROMETRY
 If atoms are being measured, the technique is
referred to as ATOMIC SPECTROSCOPY
50
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA

 An absorption spectrum is a plot of the amount

of light absorbed by a sample vs. the wavelength
of the light
 The amount of light absorbed is called the
ABSORBANCE
 An absorption spectrum is obtained by using
a spectrometer to scan a particular wavelength
region and to observe the amount of light
absorbed by the sample along the way

ABSORPTION AND
TRANSMISSION SPECTRA
 The absorption and transmission spectrum is
unique for each compound or ion, and thus the
absorption spectrum is a molecular
fingerprint
 This means that absorption and transmission
spectra are useful for identification and for
detecting impurities or other sample
components

 Consider a solution of copper sulfate. It displays

a blue color, which means that the blue portion
of the visible region is not absorbed, but
transmitted to our eyes, while the red portion is
absorbed.
 The spectrum clearly shows that wavelengths in
the blue and violet regions (350 to 500 nm) are
not absorbed, while wavelengths in the red
region (650 to 750 nm) are absorbed.

 The absorption vs. wavelength information for

molecules and complex ions may also be
displayed as a transmission spectrum, rather
than an absorption spectrum, by plotting the
amount of light transmitted by a sample rather
than the light absorbed.

51
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
transmittance, he or she must convert it to
TRANSMITTANCE
absorbance
 The intensity of light striking the light sensor A = 2 - log %T
(more often called a detector) when a blank
solution (no analyte species) is held in the path
of the light is given the symbol I0
 The blank is a solution that contains all chemical
 It states that the concentration of the unknown
species that are present in the standards and
substance is directly proportional to the
samples to be measured (at equal concentration
absorbed light (absorbance) and inversely
levels) except for the analyte species. Such a
proportional to the amount of transmitted light
solution does not display any absorption due
(% transmittance)
to the analyte, and thus I0 represents the
 A statement of Beer’s law is:
maximum intensity that can strike the detector
at any time. A = abc
 When the blank is replaced with a solution of the  in which A is the absorbance; a is the
analyte, a less intense light beam will be absorptivity, or extinction coefficient; b is the
detected because it is absorbed by the analyte. pathlength; and c is the concentration.
The intensity of the light for this solution is given
the symbol I PATHLENGTH
 The fraction of light transmitted is thus I/I0.  is the distance the light travels through the
This fraction is defined as the transmittance (T): measured solution. It is the inside diameter of
the sample container placed in the light path.

ABSORPTIVITY
 is the inherent ability of a chemical species to
absorb light and is constant at a given
wavelength, pathlength, and concentration. It is
a characteristic of the absorbing species itself.

 It was stated that the absorptivity is constant

at a given wavelength, implying that it
changes as the wavelength changes
 The GREATEST ANALYTICAL SENSITIVITY
Io = incident light (input)
I = transmitted light (output) (the ability to detect and measure small
concentrations accurately) occurs at the
WAVELENGTH at which the absorptivity is a
ABSORBANCE MAXIMUM.
 This is the same wavelength at which the
 Transmittance is that it is not linear with
concentration absorbance is a maximum in the molecular
absorption spectrum for that species. This
 A plot of transmittance or percent transmittance
wavelength is often referred to as the
vs. concentration would not be an acceptable
standard curve because it is not a straight line WAVELENGTH OF MAXIMUM
 Absorbance then is a parameter that increases ABSORBANCE.
linearly with concentration and is important
for quantitative analysis. If the analyst measures

52
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 For iron-o-phenanthroline, the wavelength of  Principle of UV-VIS instrumentation
maximum absorbance is approximately 510 nm.  Beam coming from the light
 For toluene in heptane, it is approximately 263
nm
 For benzene in heptane, it is approximately 255
SOURCES
nm  Special light sources are used in order to provide
 For potassium permanganate, it is an optimum-quality light beam for the region of
approximately 520 nm. the spectrum utilized.
 An ideal light source is one that emits an
intense continuous spectrum of light across
an entire region of the spectrum, such as the
visible region, while also exhibiting a long life.
 A light source used frequently for visible light
absorption studies is the tungsten filament
source
 If an instrument is meant strictly for visible light
studies, then this lamp is the only one present in
the instrument. Such an instrument is often
referred to as a colorimeter

 The light used in the spectro – is not an

ordinary light but a special one
 Colorimeter – may also be known as visible
spectrophotometer

 A light source used frequently for ultraviolet

 Ultraviolet–visible (UV-VIS) molecular
spectrochemical methods utilize light in the
ultraviolet and visible regions of the
electromagnetic spectrum to analyze laboratory
samples for molecular compounds and complex
ions.
absorption studies is the deuterium lamp.
 If an instrument is meant strictly for ultraviolet
work, then the deuterium lamp is the only light
source present and the instrument is called a
UV spectrophotometer
 Often, both a tungsten filament lamp and a
deuterium lamp are present and are
individually selectable.
 A light source that can be used for both
ultraviolet and visible studies, the xenon arc
lamp, may be used.
 The instrument used is called UV-VIS
spectrophotometer

TUNGSTEN FILAMENT LAMP

 Such a source is very bright and emits light over
the entire visible region and into the near
infrared region.

53
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
DEUTERIUM LAMP
 Deuterium is the name given to the isotope of
hydrogen that has one neutron in the atomic
nucleus
 The lamp contains deuterium at a low
pressure. Electricity applied to electrodes in the
lamp results in a continuous UV emission due to
the presence of the deuterium.
 Its wavelength output ranges from 185 nm to
about 375 nm, satisfactory for most UV
analyses
Isotope of H  greater mass compared with H
XENON ARC LAMP
 This lamp contains xenon at a fairly high
pressure and the light is formed via a discharge
across a pair of electrodes.
 A continuous ultraviolet and visible emission
is emitted due to the presence of the xenon.
 In some instruments, the electronic circuitry
creates regular pulses of light that are very
intense and therefore more useful. This results
in a longer life for the lamp.
VERY INTENSE, LONGER LIFE
 In order to plot the absorption spectrum of a
compound or complex ion, we must be able to
carefully control the wavelengths from the
broad spectrum of wavelengths emitted by the
source so that we can measure the absorbance
at each wavelength.
 In order to perform quantitative analysis by
Beer’s law, we need to be able to carefully
select the wavelength of maximum
absorption, also from this broad spectrum of
wavelengths
 These facts dictate that we must be able to filter
out the unwanted wavelengths and allow
only the wavelength of interest to pass.
 In reality, there is no such thing as a single
wavelength. In the visible region, the spectrum
of colors is continuous, meaning that there is no
sharp delineation between green light and blue
light, for example, or where one wavelength
ends and the adjacent wavelength begins.
 The electromagnetic spectrum is a continuous
wavelength band. Thus wavelength selection
in a spectrochemical instrument actually
consists of the selection of a narrow
wavelength band from the larger band
 The width of the band that is allowed to pass is
called the bandwidth. The narrowness of the
band that is allowed to pass varies from one
design to another and is called the resolution.
 Thus a high resolution (narrow bandwidth) is
ideal, although many applications do not
require the best available resolution and money
is often saved by purchasing instruments with a
low resolution (wide bandwidth).
 The width of the band that is allowed to pass is
called the bandwidth. The narrowness of the
band that is allowed to pass varies from one
design to another and is called the resolution.
 Thus a high resolution (narrow bandwidth) is
ideal, although many applications do not
require the best available resolution and money
is often saved by purchasing instruments with a
low resolution (wide bandwidth).
 The most inexpensive way to isolate a
wavelength band is with the use of
absorption filters
 For visible light, such a filter would consist of
colored glass, the color of the glass indicating
what region of the visible spectrum is passed.
 Thus, if the wavelength called for in a given
method is in the red region of the visible
spectrum, a red colored glass filter is chosen.
 Absorption filters have the lowest resolution
54
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
Kung nasaan na wavelength siya – yun yung
magiging color nya
different narrow wavelength range emerges
from the exit slit at each position of rotation.
 The light emerging from this exit slit is
therefore monochromatic and is passed on to
the sample compartment to pass through the
sample
Once it disperses – the narrow band will be
selected by exit slit (the one who will exit into the
exit slit, filtered, is the narrow band only)

 The word “monochromator” is derived from the

Latin language, “mono” meaning “one” and
“chromo” meaning “color.”
 It is a device more sophisticated than an
absorption filter that isolates the narrow band
of wavelengths from visible and ultraviolet
sources
 A monochromator is made up of three parts:
- An entrance slit, a dispersing
element, and an exit slit
 A SLIT is a small circular or rectangular hole cut
in an otherwise opaque plate, such as a black
metal plate.
 The ENTRANCE SLIT is where light enters the
monochromator from the source. Its purpose is
to create a unidirectional beam of light of
appropriate intensity from the multidirectional
light emanating from the source
 After passing through the entrance slit, the beam
encounters a DISPERSING ELEMENT.  The rotation of the dispersing element is
accomplished by manually turning a knob on
 The dispersing element disperses the light into
the face of the instrument or by internal
its component wavelengths
programmed scanning controls.
 For visible light, for example, this would mean
 The position of the knob is coordinated with the
that a beam of white light is dispersed into a
wavelength emerging from the exit slit such that
spray of rainbow colors, the violet–blue
this wavelength is read from a scale of
wavelengths on one end to the red wavelengths
wavelengths on the face of the instrument, on a
on the other, with the green and yellow in
readout meter, or on a computer screen
between
 Some instruments improve (narrow) the
 The narrow band of the spectrum is then
bandwidth by using filters ahead of the
selected by the EXIT SLIT dispersing element.
 As the dispersing element is rotated, the spray  The dispersing element is either a diffraction
of colors moves across the exit slit such that a grating or a prism.

55
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
PRISM
 is a three-dimensional triangularly shaped
glass or quartz block. When the light beam
strikes one of the three faces of the prism, the
light emerging through another face is
dispersed.

DIFFRACTION GRATING
 is like a highly polished mirror that has a large
number of precisely parallel lines or grooves
scribed onto its surface. Light striking this
Breaking down of the regions
surface is reflected, diffracted, and dispersed
into the component wavelengths
Diffraction grating is used more often than prism

 Following the wavelength selection by the

monochromator, the beam passes on to the
sample compartment where the sample
solution, held in the cuvette, is positioned in its
path
 The sample compartment is an enclosure with a
lid that can be opened and closed in order to
insert and remove the cuvette.
 When the lid is closed, the compartment should
be relatively free of stray light

 After wavelength selection by the

monochromator it will pass into the sample
compartment
 Stray light – detected light of any
wavelength that is outside the bandwidth
of the wavelength selected  cause errors

56
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA

 Photomultiplier tubes or photodiodes (light

sensors) are used as detectors in UV-VIS
spectrophotometers

PHOTOMULTIPLIER TUBE
 A PHOTOMULTIPLIER TUBE is a light sensor
combined with a signal amplifier. The light
emerging from the sample compartment strikes
the photosensitive surface and the resulting
electrical signal is amplified.
 The photomultiplier tube consists of a PHOTODIODES
photocathode, an anode, and a series of
dynodes for multiplying the signal  Utilizes materials such as silicon
 A DYNODE is an electrode that, when struck  Silicon can be doped with impurities to make it
with electrons, emits other secondary either electron rich (an n-type semiconductor)
electrons. The dynodes are situated between or electron poor (a p-type semiconductor).
the photocathode and the anode. A high voltage  When an n-type semiconductor is in contact
is applied between the photocathode (an with a p-type semiconductor, electronic changes
electrode that emits electrons when light occur at the boundary, or junction
strikes it) and the anode.  A photodiode is a p–n junction constructed with
 When the light beam from the sample the top p layer so thin that it is transparent to
compartment strikes the photocathode, light. Light shining through the p layer creates
electrons are emitted and accelerated, because additional free electrons in the n layer that can
of the high voltage, to the first dynode, where diffuse to the p layer, thus creating an electrical
more electrons are emitted current that depends on the intensity of the
 These electrons pass on to the second dynode, light.
where even more electrons are emitted  This small current is easily amplified and
 When the electrons finally reach the anode, the measured.
signal has been sufficiently multiplied
 This amplified signal is then sent on to the
readout
 The accumulation of electrons striking the
anode produces a current signal, measured in
amperes, that is proportional to the initial
intensity of the light.

57
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 Cuvettes used for UV-VIS spectrophotometry
must be transparent to all wavelengths of light
for which it is used
 If visible light is used, the material must ideally
be completely clear and colorless, which
means that inexpensive materials, such as
colorless plastic and ordinary colorless glass,
are perfectly suitable
 Ordinary colorless glass and plastic are not
transparent to light in the ultraviolet region.
 For ultraviolet spectrophotometry, the cuvettes
must be made of quartz
 If two or more different cuvettes are used in an
analysis, one should be sure that they are
matched
 Cuvettes that have scratches or cleaning
procedures that may cause scratching should be
avoided.
 Cotton swabs can be used for scrubbing, rather
than metal-handled brushes.
 Any liquid or fingerprints adhering to the
outside wall must be removed with a soft, lint-
free cloth or towel.
 In addition to scratches, fingerprints, water
spots, etc., the analyst should be aware that any
foreign particulate matter suspended in the
sample or standards would also be a problem.
 Particulates in the path of the light would reflect
and scatter the light, lessening the intensity of
the transmitted light, and would result in an
error in the reading.
 The problem is solved by making sure the
samples are well filtered
 Some spectrometric experiments, however, are
designed to actually measure such particles. The
analysis of samples for suspended particulate
matter by spectrophotometry is an analysis for
the turbidity of those samples
 An interference is a contaminating substance
that gives an absorbance signal at the same
wavelength or wavelength range selected for the
analyte.
 For qualitative analysis this would show up as
an incorrect absorption spectrum
 For quantitative analysis, this would result in
a higher absorbance than one would measure
otherwise
With more analyte bec with interference so
Higher abs-higher conc
MAINTENANCE
 The basic considerations for spectrophotometer
maintenance are safety and cleanliness
 Solutions spilled on sensitive electronic circuits
can render them inoperative. Any spills should
be cleaned up immediately
 Instrument operators may also want to conduct
periodic wavelength calibrations. It is important
to know that the wavelength displayed or
dialed in really is the wavelength passing
through the sample compartment
One way to check for proper wavelength
calibration is to prepare a solution of an
analyte for which the wavelength of maximum
absorbance is well known. If the maximum
absorbance does not occur at this wavelength, it
may be that the wavelength control is out of
calibration
58
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 The living matter is composed of mainly six
elements: carbon, hydrogen, oxygen, nitrogen,
phosphorus and sulfur. These elements together
constitute about 90% of the dry weight of the
human body
 BIOANALYSIS may be defined as laboratory
analysis of biomolecules.
 BIOMOLECULES are organic compounds with
biological activity, generally important only in
biological systems, or cells
 BIOCHEMISTRY is the study of structure and
function of biomolecules.
MONOSACCHARIDES
 BIOTECHNOLOGY a related concept,
concerns the industrial applications of
biochemical techniques.
 The four groups of biomolecules are
carbohydrates, lipids, proteins, and nucleic
acids. All four are essential to life and are
therefore found in living cells.
 All are organic compounds, based on the
element carbon. In addition to carbon, all four
also contain hydrogen and oxygen.
 Proteins, nucleic acids, and a few lipids contain
nitrogen. Of the four, proteins alone contain
sulfur, while nucleic acids and some lipids also
contain phosphorus.
 The two most important biomolecules in
modern bioanalysis are proteins and nucleic
acids.
 Organic compounds accounts for 25-30% of the
cell weight
 They are nucleic acids, proteins, polysaccharides
(carbohydrates) and lipids.
 Proteins accounts 10-20% of the weight of the
cell.
 Nucleic acids account 7-10% of the cell weight.
 Polysaccharides usually account for 2-5% of the
cell weight
 About 3% of cell weight is due to lipids. Lipids
content may be higher in adipocytes or fat cells.
 Proteins may account more of cell weight in cells
like erythrocytes.
CARBOHYDRATES
 Carbohydrates, which include sugars and
starches, contain carbon, hydrogen and oxygen
 Carbohydrates are classified according to size as
monosaccharides, disaccharides or
polysaccharides
 Carbohydrates provide a ready, easily used
source of food energy for cells
 Monosaccharides are the smallest
carbohydrates.
 Common examples of aldoses are glucose,
galactose, and ribose. Ribulose and fructose
are ketoses.
 Monosaccharides are commonly classified
according to the number of carbon atoms in
their chemical backbones.
 A three-carbon monosaccharide is called a
triose, five-carbon a pentose, and six-carbon a
hexose. Glucose, galactose, and fructose are
hexoses, while ribose and ribulose are
pentoses.
 In nature, monosaccharides exist in two forms,
rings and open-chain carbon skeletons
59
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
DISACCHARIDE
 A disaccharide consists of any two
monosaccharides covalently linked together.
 Sucrose = glucose + fructose
 Lactose = glucose + galactose
 Maltose = glucose + glucose
 In nature, they tend to be exclusively either plant
or animal products. Sucrose (table sugar) and
maltose (malt or grain sugar) are produced only
by plants, while lactose (milk sugar) is
exclusively an animal product.
POLYSACCHARIDES
 The largest and most complex carbohydrates
are the polysaccharides. They are polymers,
long chains of repeating chemical units.
 Examples of common polysaccharides are
starches, plant products that are major
macronutrients in the human diet, and cellulose,
found in plant cell walls.
 In the human diet, cellulose is referred to as
fiber, indigestible but beneficial for normal
intestinal motility
LIPIDS
 All totally hydrophobic (water fearing)
 Most lipids are insoluble in water but readily
dissolve in other lipids or organic solvents
 The most abundant lipids in the body are
triglycerides (triacylglycerol), phospholipids
and steroids.
 Triacylglycerols, commonly referred to as fats
and oils, consist of three fatty acids linked to a
molecule of glycerol, a three-carbon alcohol.
Fatty acids are long-carbon-chain molecules,
each with a single carboxyl functional group.
 In nature, triacylglycerols are widespread and
are crucial energy storage molecules (and
function as insulation in mammals).
 Phospholipids have a hydrophilic and
hydrophobic region. It is present in cell
membranes and allow cells to be selective about
what may enter or leave
 Steroids are the only common lipids without
fatty acids. Chemically, they consist of a fused
carbon ring structure consisting of four rings,
three rings of which are six membered and one
ring of which is five membered.
 Some steroids, such as cortisone, function as
hormones. Cholesterol is an important
component of membranes in animal cells.
60
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
PROTEINS
 Proteins account for over 50% of organic matter
in the body, and they have the most varied
functions of the organic molecules
 The building blocks of proteins are small
molecules called amino acids
 Amino acids are joined together in chains to
form large, complex protein molecules
 They are large polymers (many have molecular
weights in excess of 100,000) with intricate
three-dimensional structures and tremendous
diversity.
 Amino acids are the monomer units.
 Historically, amino acid sequences have been
used to trace possible evolutionary
relationships among organisms, since they are
coded by DNA base sequences.
 Mutations therefore are manifested structurally
by different amino acid sequences of proteins.
1-Proteins are almost certainly the most structurally
complex molecules found in nature.
are short chains of amino acids linked by
peptide bonds. Most biologically active peptides contain
two to ten amino acids.
 ASPARTAME, a dipeptide (aspartic acid +
phenylalanine) artificial sweetener marketed
under the trade name Nutrasweet
 GLUTATHIONE, a tripeptide (glutamic acid +
cysteine + glycine) antioxidant believed to be an
anticancer agent
 ENKEPHALINS, pentapeptides found in the
central nervous system that are natural
analgesics
 OXYTOCIN, a hormone composed of nine
amino acids, which induces labor by stimulating
contraction of uterine muscles
strandlike proteins that appear most often in body
structures. They are very important in binding
structures together and for providing strength in certain
body tissues
– they are
mobile, generally spherical molecules that play crucial
roles in virtually all biological processes. Some help
provide immunity, others help growth and
development, and some are biological catalysts
(enzymes)
 Enzymes are functional proteins that act as
biological catalysts
 A catalyst is a substance that increases the rate
of a chemical reaction without becoming part of
the product or being changed itself
 However, they do not only increase the speed of
chemical reactions, they also determine just
which reactions are possible at a particular time
 Without enzymes, biochemical reactions would
occur far too slowly to sustain life
 Enzymes are very specific in their activities, each
controlling only one (or a small group of)
chemical reaction(s) and acting only on specific
molecules
61
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
NUCLEIC ACIDS
 Only two nucleic acids exist. They are DNA
(deoxyribonucleic acid) and RNA
(ribonucleic acid).
 Like proteins, nucleic acids are polymers, with
nucleotides being the monomer units.
 The role of DNA is fundamental: they make up
the genes, which provide the basic blueprint
of life
 Not only do they determine what type of
organism you will be, but they also direct ones
growth and development–and they do this
largely by dictating protein structure
DNA
 its is the genetic material found within the cell
nucleus. It has two roles.
 One is to replicate itself before a cell divides
 Second, it provides instructions for building
every protein in the body.
RNA
 is considered as a “molecular slave” of DNA. It is
because RNA carries out orders for protein
synthesis issued by DNA
ELECTROPHORESIS
 Electrophoresis refers to a group of techniques
used to separate and study molecules with
electrical charges.
 Based upon these charges, both sign and
magnitude, biomolecules such as proteins,
peptides, amino acids, nucleic acids, and
fragmented nucleic acids migrate in an electrical
field.
 The direction of migration is determined by the
sign of the charge. The speed and distance of
migration are determined by the magnitude of
the charge.
 For example, molecules with charges of ++++
and + will both move toward the cathode
(negative electrode), but the ++++ sample will
move faster and therefore farther.
 Matrices for separation include paper, various
gels, and liquids.
 In paper electrophoresis, a piece of absorbent
filter paper soaked in a current-carrying buffer
is the matrix for separation. This type of
electrophoresis is most commonly applied to
amino acids and peptides.
 The two most common types of gels are
polyacrylamide (polyacrylamide gel
electrophoresis (PAGE)) and agarose.
 The essential component in all PAGE gels is
acrylamide, a potential neurotoxin. Agarose is a
polysaccharide that is much safer to handle.
 PAGE is usually run vertically, while agarose gels
are horizontal.
 PAGE is most commonly used with proteins,
while agarose gel electrophoresis is the favored
method for nucleic acids and their fragments,
but is also effective for proteins.
 In capillary electrophoresis, separation
occurs in a glass capillary tube containing buffer
only
CHROMATOGRAPHY
 Chromatographic techniques are also powerful
tools for the analysis of biomolecules. Various
types of chromatography are useful, especially
for analyzing proteins and nucleic acids.
PAPER CHROMATOGRAPHY
 This method is very useful for separating
amino acids found in food samples.
 The most effective matrix for separation is an
absorbent cellulose-based filter paper. A very
effective mobile phase is 70% isopropyl
alcohol in water.
62
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
 Although the 20 amino acids are chemically very
similar, they may be successfully separated by
this method. Amino acids interact with the
stationary phase to different extents, thus
moving at different speeds.
 Chemical differences among amino acids that
determine migration speed include molecular
weight, charge, and polarity.
THIN-LAYER CHROMATOGRAPHY
 TLC has similar applications to paper
chromatography. The stationary phase is a
coating, such as silica gel, on a glass or plastic
plate.
 Depending on the TLC plate used, components
may be separated based on differences in
molecular weight, charge, or polarity.
 TLC with a 70% isopropyl alcohol mobile
phase and a silica gel plate is an effective
substitute for paper chromatography separation
of amino acids.
 Nucleotides may be separated on a special silica
gel plate and a 20% ethanol (in water) mobile
phase.
SPECTROSCOPY
 Various spectroscopic methods are also useful in
the analysis of biomolecules. The most notable is
UV–visible (UV-VIS) spectroscopy
 Proteins and nucleic acids absorb UV light, with
absorption maxima between 200 and 300 nm.
Nucleic acid solutions are colorless, with no

absorption of visible light.
Some proteins, called conjugated proteins,
have a chromophore that absorbs visible light as
well as UV. Examples of such proteins include
hemoglobin and cytochrome C, both red in color,
with absorption maxima in the 520 to 580 nm
region.
 All proteins have some UV absorption at 280
nm, and all nucleic acids and nucleotides absorb
at 260 nm.
 These wavelengths are therefore useful in
monitoring the presence of these molecules in
various solutions.
IMMUNOASSAY
 Antibodies are complex proteins directly
involved in immune responses against foreign
agents such as viruses and bacteria. They are
produced by the immune system in response to
the entry of anything foreign into our bodies.
 The laboratory measurement of antibodies in
biological samples is frequently used to
diagnose various bacterial and viral diseases.
 The general laboratory method, called
immunoassay , usually utilizes a UV-VIS
absorption technique.
 Since proteins, including antibodies, and the
products of the reaction of antibodies with drugs
absorb UV and visible light, their presence in
blood, urine, semen, and other body fluids may
be detected by this method.
 It is widely accepted that the majority of medical
decisions are made using laboratory data. It is
therefore critical that results generated by the
laboratory be accurate.
 The most important aspect of the job of the
analyst is to assure that the data and results that
are reported are of the maximum possible
quality. This means that the analyst must be able
to recognize when the test instrument is
breaking down and when a human error is
suspected.
ERRORS
DETERMINATE ERRORS
 are errors that were known to have occurred, or
at least were determined later to have occurred,
in the course of the lab work.
INDETERMINATE ERRORS
 on the other hand, are errors that are not
specifically identified and are therefore
impossible to avoid.
63
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA

Since the errors cannot be specifically identified, a MEASURES OF

statistical analysis must be performed to determine
whether the results are far enough “off-track” to
merit rejection
SPREAD
 Spread is the measure of how the data are
MEASURES OF distributed. It represents the relationship of
all the data points to the mean

CENTER
 The three most commonly used descriptions
of the center of a dataset are the mean,
median and the mode

RANGE
 The range is simply the largest value in the
data minus the smallest value, which
represents the extremes of data one might
MEAN encounter
 Mean is often called the average. DEVIATION
 It is calculated by summing the observations  How much each measurement differs
and dividing by the number of observations from the mean is an important number and
Σ𝑥 is called the deviation
Mean (x̅ ) =  A deviation is associated with each
𝑛
measurement, and if a given deviation is large
MEDIAN compared to others in a series of identical
measurements, the proverbial red flag is
 The median is the middle of the data after the
raised. Such a measurement is called an
data have been rank ordered
outlier
 It is the value that divides that data in half STANDARD DEVIATION
MODE  The standard deviation represents the
 The mode is the most frequently occurring “average” distance from the center of the
value in a dataset. data (mean) and every value in the data set
 The significance of SD is that the smaller it is
numerically, the more precise the data (the
64
ANCH 111_1ST YEAR_2ND SEM © ABEGAIL G MAGBANUA
more the measurements are “bunched” points there are bunched around the mean and
around the mean). the sharper the drop-off away from the mean,
the smaller the standard deviation and the
(d12 + d22 + d32 + …ሻ more precise the data
√
𝑛−1

Σሺ𝑥 − 𝑚𝑒𝑎𝑛ሻ2
√
𝑛−1
EXAMPLE.
 Determine the SD of the ff values: 5, 4, 6, 5, 3, 7,
5

 Analytical laboratories, especially quality

assurance laboratories, will often maintain

COEFFICIENT OF graphical records of statistical control so that

scientists and technicians can note the history of
the device, procedure, process, or method at a
VARIATION glance. The graphical record is called a control
chart and is maintained on a regular basis, such
 It is another way of expressing the SD as daily.
 Calculated by dividing the SD by the mean and  It is a graph of the numerical value on the y-
multiplying by 100 to express it as a percentage axis vs. the date on the x-axis.
𝑆𝐷
CV = x 100
𝑚𝑒𝑎𝑛

 For an infinite data set, plot of frequency of

occurrence vs. the measurement value yields a
smooth bell-shaped curve.
 This curve is called the normal distribution
curve because it represents a normal
distribution of values for any infinitely repeated
measurement.
 The normal distribution curve is a picture of LEVEY-JENNINGS CONTROL CHART
the precision of a given data set. The more
END OF SEMESTER 65