QUALITY CONTROL / PHARMACEUTICAL ANALYSIS 1 and 2

UNIT 1: FIVE SECTIONS OF QUALITY CONTROL

INTRODUCTION TO QUALITY CONTROL AND QUALITY ASSURANCE 1. SPECIFICATION AND ASSAY DEVELOPMENT SECTION
QUALITY a. To coordinate with research, product development, production, sales
• Combination of attributes which when compared to a standard, serves as & management towards improvement of a product.
a basis for measuring the uniformity of a product and determines its b. To establish specification for raw and packaging materials.
degree of acceptability. c. To validate existing and tentative procedures of testing.
d. To establish specifications based on validates procedures.
• The sum of all factors which contribute directly or indirectly for the safety, e. To develop new assay methods for in-house use
effectiveness, and reliability of the product. ü Assay methods are from USP-NF but if there’s a time that the
assay methods from USP-NF is not appropriate to the drug
production, then that’s when it’s acceptable for the companies
QUALITY CONTROL particularly in their QA and QC department to make their own
• Tool which gives the assurance that a product conforms to standards and assay methods.
specification through a system of inspection, analysis and action.
o Inspect the raw materials, packaging materials, and the product f. To develop and improve specification for quality characteristics of
itself before its release. the final product being manufactured.

o For the analysis, the products are put into a variety of test.
2. CENTRAL RELEASE SECTION
o The action to be taken is whether to accept or reject the product a. To examine the records resulting from the exercise of Quality Control
and there must be a valid and reasonable basis that leads to functions through all steps of manufacturing and packaging for
that action such as using the specifications as a standard basis. completeness and accuracy and to assume responsibility for their
safekeeping and storage.
ü Examining and keeping the records of the complete production
QUALITY ASSURANCE of the product from dispensing down to distribution of these
• The activity of providing to all concerned, the evidence needed to drugs to the different outlets.
establish confidence that the activities relating to quality are being
performed adequately. b. To investigate customer complaints or inquiries on product quality.
o Quality Assurance is the name of the department not Quality c. To maintain complete and accurate records of the receipt and
Control. distribution of every lot of raw material and finished product.
d. To keep retention samples in locked areas under similar conditions
o Quality Control is solely under the scope of Quality Assurance. comparable to the market conditions.
e. To properly record and handle finished products returned by
o The roles and functions of the personnel in the QA and QC differ. pharmacies and hospitals.

QUALITY CONTROL FUNCTIONS 3. CHEMICAL CONTROL

1. Analysis Function a. To test and assay every lot of raw material, in-process products and
2. Monitor Function every lot of finished products.
3. Record Review & Release Function b. To conduct stability studies.
4. Audit Function
ü If it is a small drug company then their QC has the audit function.
However, if it’s a large drug company then the audit function is
no longer part of the functions of their QC department.

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 1

4. INSPECTION AND CHECKING SECTION DEFECTS
a. To inspect and sample every shipment of raw materials received and • Defects - is an undesirable characteristics of a product.
every lot of finished goods for distribution. • It is defined as a failure to conform to specification.
b. To examine every shipment of packaging materials
c. To examine and check all manufacturing operations, including in- CLASSIFICATION OF DEFECTS
process filling and labeling as well as periodic examination on the 1. According to measurability
quality of stocks in the warehouse. Variable defect Attribute defects
ü In actual workplace be it in community, hospital or A defect which can be measured
A defect which cannot be
manufacturing, there is someone to observe you to double directly by instruments giving
measured directly by instruments. It
check if what you’re doing is correct and appropriate. dimensions of length, weight,
shows mainly the conformance or
height, thickness, concentration,
nonconformance of the material to
d. To sample retention or reserve samples withdrawn from the volume, viscosity pH or size
specifications.
packaging line of finished products and from the warehouse for raw particles.
materials. • Balance – weight Ex.
ü Not all finished products are being released in the market for • pH meter – pH judging only by
there are reserved samples of the finished products left at the • Viscometer - viscosity color, odor, clarity.
company, in case, there’s a problem/complaint arises with the
products that need to be investigated. Another reason is for the 2. According to seriousness or gravity.
conduction of stability studies with those finished products. Critical defect Major defect Minor defect
A defect which does
A defect which may
not endanger life or
5. BIOLOGICAL AND MICROBIOLOGICAL SECTION A defect which may affect the function of
property nor will it
a. To perform and evaluate microbiological and pharmacological endanger life or property the object and
affect the function
assays. and may render the therefore may render
but remains a defect
ü It is necessary that the pharmacist knows how to perform and product non-functional. the product useless.
since it is outside the
evaluate the mentioned assays so that in case of the absence of prescribed limits.
the microbiologists and pharmacologists, then the RPh can Ex.
substitute and work on those. Ex.
Ex. Slight deviation of
The presence of a
Absence of warning in a the color of the label
b. To do sterility, pyrogenicity, bacteriological, irritation, safety, and crack in a bottle.
label for a potent drug. from the color
toxicity tests. standards.

3. According to nature.
QUALITY ASSURANCE FUNCTIONS Ocular defect Internal defect Performance defect
1. Establish systems for ensuring the quality of the product.
A defect that is A defect which is not
A defect in function.
visible. seen although present.
2. Responsible for ensuring that the quality policies adopted by a
Ex.
company are followed. Ex.
Ex. A suppository that does
A sub-potent drug
Foreign particulate not meet at body
3. It serves as the contact with regulatory agencies. product
temperature
4. Final authority for product acceptance or rejections.
SOURCES OF VARIATION
5. It helps to identify and prepare the necessary standard operation
1. Materials
procedures replated to the control of quality.
Ex. a. Variation between suppliers of same substance.
b. Variation between batches from same supplier.
c. Variation within a batch.
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 2. Machines NON-COMPLIANCE TO CGMP COULD RESULT IN:
Ex. a. Variation of equipment for the same process. • Quality Variation
b. Difference in adjustment of equipment. • Contamination
c. Aging & improper care. • Mix-ups and Errors
• Recall
3. Methods 9 Disadvantages of Recall:
Ex. a. Inexact procedures. ü It causes a lot of money lost
b. Inadequate procedures. ü Bad publicity damages the good reputation of the
c. Negligence by chance. company
ü Harmful publicity can hurt sales
4. Men ü Had adverse effect on employee
Ex. a. Improper working conditions.
b. Inadequate training and understanding. MAIN OBJECTIVE OF CGMP IS TO PRODUCE A PRODUCT THAT IS:
c. Dishonesty, fatigue & carelessness. ü SAFE: Unable to cause damage, free from danger
ü PURE: Free from contamination
ü EFFECTIVE: Producing the desired effect
TWO FACTORS RESPONSIBLE FOR ASSURING SAFETY AND THERAPEUTIC
EFFICACY OF DRUGS MATCHING TYPE
D 1. A suppository that does not melt A. Major Defect
Internal Factor External Factor at body temperature.
“General principles of Total Quality “Current Good Manufacturing A 2. The presence of a crack in a B. Ocular Defect
Control in the drug Industry” Practices” bottle.
E 3. A subpotent drug product. C. Critical Defect
Established & implemented by Established & implemented by C 4. Absence of warning in a label. D. Performance Defect
Pharmaceutical Manufacturers Food & Drug Administration. B 5. Presence of particulate matter. E. Internal Defect
Association in 1967.
Every quarter of the month FDA
All drug makers since 1967 must have visits due to Administrative Order
QA department responsible for No. 220 series of 1974.
controlling all the products they
manufactured. - Subject: which is about cGMP in
Manufacture, Processing, Packing
or Holding.

THE SCOPE OF THIS REGULATION

• Organization and Personnel
• Buildings and Facilities
• Equipment
• Components & Drug Product Containers and Closures
• Production & Process Control
• Packaging * Labelling Control
• Holding and Distribution
9 FIFO “First In First Out”
9 FEFO “First Expiry First Out”

• Laboratory Control
• Record and Reports
• Returned & Salvaged Drug Products
Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 3
 UNIT 2: PROBLEM SOLVING: RANDOM SAMPLING
STATISTICAL QUALITY CONTROL How many samples should be tested in a batch consists of 250,000
STATISTICAL QUALITY CONTROL (SQC) bottles of Antibiotic suspension?
• The monitoring of quality by application of statistical methods in all stages
of production. N = 𝟐𝟓𝟎, 𝟎𝟎𝟎 + 𝟏
= 501
STATISTICS
• Is a collection of data or numbers, and with the use of mathematics, can Using random sampling plan, determine the number of samples to be
analyze and interpret these data for the purpose of making meaningful taken from 3,000 containers of starch powder.
decisions.
N = 𝟑, 𝟎𝟎𝟎 + 𝟏
SAMPLING = 55.77 → 56
• Process of removing appropriate number of items from a population in
order to make inferences to the entire population. 3. GOVERNMENT SAMPLING OR MILITARY STANDARD – 105 D & ABC-STD-
105 D
POPULATION • Government sampling plan known as Military standard – 105 D &
• Is the totaling of all actual or conceivable items of a certain class under ABC-STD105 D
consideration.
• Originated by a committee from military agencies of the USA,
SAMPLE Great Britain and Canada.
• Is a finite number of objects selected from a population.

THREE COMMON SAMPLING PLANS

1. 100% INSPECTION
• Sampling plan of 100% inspection may be tried to minimize errors,
but normally and practically this method cannot be attained due
to personnel fatigue and other human related factors.
§ Usually applied to parenteral products.

2. RANDOM SAMPLING • It is already stated in the table how many sampling numbers can be
• Sampling plan using the square root system using the formula: used depending on the number of procured items from the supplier.

n = N +1 • The sole problem will be if the certain number of procured items from
where: the supplier cannot be seen in the given table.
n = no. of items to be taken (random)
N = total no. of the lot
FOUR BASIC QUALITY STANDARDS TO BE SPECIFIED IN THE
Example: CONSTRUCTION OF STATISTICAL SAMPLING PLAN
N = 50 items • This requires normally four basic quality standards to be specified as
follows:
n = 50 + 1 = 8 items 1. AQL – Acceptable Quality Level
ü For example, a batch of tablets is considered acceptable if it
contains 2% or less defective tablets
9 When we are testing for the weight variation, hardness test,
friability test and etc., there’s a criterion to follow in order to
determine if the product should be accepted or not.

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 9 For the weight variation, when 1 tablet doesn’t comply with BASED ON VARIABLES
the requirement, then it will be accepted & same goes if
there are 2 tablets that didn’t comply.

9 However, if there are 3-4 tablets that didn’t comply, it won’t

immediately be rejected for there’ll be a conduction of the
2nd trial that is called double sampling.

9 After the 2nd or 3rd trial, that’s when you decide whether to
accept or reject the tablets. You’ll have to accept it if the
defective tablets are 2% or less.

2. UQL – Unacceptable Quality Level BASED ON ATTRIBUTES

ü For example the same batch of tablets is said to be rejected if it
contains 4% or more defective tablets.
9 It means that 3% or less defective tablets are still accepted.

3. Producer’s risk (α)

ü Is the risk of error on the probability of rejecting a good batch.

4. Consumer’s risk (ß)

ü Is the risk of error on the probability of accepting a defective
batch.

TWO BASIC TYPES OF QUALITY CONTROL CHARTS

1. Based on variables PROBLEM SOLVING
2. Based on attributes 1. The volume of 5 vials was determined during the filling of an injectable.
Determine the UCL and LCL.
Variable Chart Attribute Chart
Variable chart is one in which Attribute chart is one in which each
several samples are tested and sample inspected is tested to
distribution of measurements can, in determine whether it conforms to
a sense, measure degrees of requirements. It is the so-called “go”
defectiveness. or “no-go” situation.

Variable chart is a chart using Attribute chart is a chart which

actual records of numerical makes use of discrete data
measurements on a full continuous classifying the number of items
scale such as: meter, grams, liter. conforming and the number of
items failing to conform to any
specified requirements.
Ø In solving the X bar, you add up all the value in the series and divide it
Examples of variable charts are the An example chart is fraction by 5.
Mean (X) and Range [R] charts. defective known as P-chart.
[P = fraction defective] Ø In solving R, you get the difference of the highest and lowest value of
each time we collected the data.

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 5

FORMULAS: PROBLEM SOLVING
DATA:

FORMULAS:

TABLE TO ESTIMATE THE 3 STANDARD DEVIATION

Ø In getting %P, just multiply the value of P to 100.

Ø This is the table where you’ll get the A2, D3, and D4 values based on
he sample size.

ANSWERS:

ANSWERS:

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 STANDARD DEVIATION ANSWERS:
• The square root of the quantity (sum of squares of deviation of individual
results from the mean, divided by one less than the number of results in
the set).
ü The one tells us how far the UCL and LCL with the target value or
average or center value.
𝚺 (𝑿 − 𝒙̄ )𝟐
𝑺= )
𝑵−𝟏

PROBLEM SOLVING
DATA:

FORMULAS:

Ø It’s up to the analysts whether he/she is going to 1 or 2 or 3 standard

deviations.

Ø In its graph, the y-axis is named X while the x-axis is named Number of
Sample. Then, we draw the LCL, UCL and the Target Mean.

Ø The UCL and LCL dictate the standard deviation (S). Both show the
Ø In the attribute the chart, the standard deviation (S) is still 2 unlike the distance with the target value or average mean.
variable chart where the S is already 3.
Ø Average Mean aka Central Line is always at the center.

RELATIVE STANDARD DEVIATION

• The standard deviation expressed as a fraction of the mean. It is
sometimes multiplied by 100 and expressed as a percentage.
𝒔
𝑹𝑫𝑺 = 𝒙 𝟏𝟎𝟎
𝒙̄

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 7

 PROBLEM SOLVING: CALCULATION OF RESULTS AND ERRORS
1. The average fill volume was 60mL with in-process specifications of Two types of Errors
57-63mL. If one standard deviation is 0.5 mL, determine the UCL & LCL. 1. Determinate error or Systematic error
Causes:
a. Personal error
o Made by the individual analyst.
o Ex. Inability to judge color changes sharply.

b. Error of method
o Caused by faulty procedures.
o Ex. Incorrect sampling and improper selection of indicator.
2. The average fill volume was 60mL with in-process specifications of
57-63mL. If two standard deviation is 0.5 mL, determine the UCL & LCL. c. Apparatus error
o Due to poor construction or calibration.
o Ex. Inaccuracy in the calibration of burets, pipets, etc.

2. Indeterminate error or Random error

Cause:
9 Manifest themselves by slight variation in a series of observations
made by the same observer under identical conditions.

3. The average fill volume was 60mL with in-process specifications of VALIDATION
57-63mL. If three standard deviation is 0.5 mL, determine the UCL & LCL. • Defined as the verification, by data and analysis that the design
objectives of a given facility, system, apparatus or procedures are
reliably fulfilled in routine operation.

STEPS INVOLED IN VALIDATION

1. Choosing the desired attributes of the products.
9 E.g.: Weight variation of capsules

2. Determining specification for those attributes

9 NLT 90% and NMT 110%

3. Selecting appropriate processes and equipment

4. The average fill volume was 250mL with in-process specifications of
9 Using analytical
245-265mL. If two standard deviation is 0.5mL, determine the UCL & LCL.
4. Monitoring and testing processes, equipment & personnel while in
operation.
9 Inspector/Checker that observes you in performing the weight
variation test.

5. Examining test procedures themselves to ensure their accuracy and

reliability.

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 8

 SCOPE OF VALIDATION
1. Process Validation
2. Assay Validation
3. Qualification of Manufacturing Equipment
4. Validation of existing products by statistical evaluation
9 E.g.: Standard Deviation

5. Cleaning Validation
9 Materials and equipment used

ANALYTICAL PARAMETERS TO BE VALIDATED (9)

ü All the 9 parameters are concerned to QC2 especially when buying Ø To determine if it is directly proportional to the concentration of the
instruments & ensuring that all these specifications, parameters, and analyte, Beer’s Law Graph is used.
attributes are present in the instruments. Ø In Beer’s Plot, absorbance is in the y-axis while the concentration is in the
x-axis.
1. PRECISION Ø After determining the reading of the absorbance for our standard, we
9 Is a measure of reproducibility of data within a series of results. plot is one-by-one.
9 It expresses a degree of agreement among individual test results Ø As we have increasing concentration of the standard solution, the
when procedure/method is applied to homogenous sample. absorbance reading is also increasing.
9 Usually expressed as SD/RSD.
5. RANGE
2. ACCURACY 9 Lowest and highest level of analyte but the method can determine
9 Is used to denotes agreement of an experimental result of the with reasonable accuracy and precision.
agreement of the main value of a series of experimental results with
the true value. 6. LIMIT OF DETECTION
9 Lowest concentration of the analyte in the sample that the method
can detect.
9 Prescribed as percentage or as parts per million.
9 After getting the true value (TV) range, we check if the actual data
results fall within the TV range. If it falls, we accept, and if not, we 7. RUGGEDNESS
reject it. 9 Degree of reproducibility of test results obtained by analyzing the
same sample under variety of normal test conditions such as different
9 Those accepted are solely the ones we add to get the average and analyst instruments, days, reagent, columns & TLC plates.
use it for its purpose such as normality concentration for 5 trials or % ü All results will pass despite that the sample will be taken at
purity of assay drugs. different analyst, reagents, instruments and etc.

9 All that fall within the true value range are not just precise but also 8. ROBUSTNESS
accurate data. 9 Is the measure of the capacity of the analytical method to remain
unaffected by small but deliberate variations in procedure.

3. SELECTIVITY (SPECIFICITY) 9. SENSITIVITY

9 Ability of the method to measure accurately the analyte of interest. 9 Capacity of the test procedure to record small variations in
concentrations.
4. LINEARITY
9 Ability of the methods to elicit test results that are directly proportional
to the concentration of the analyte.
Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 9
PROBLEM SOLVING UNIT 3:
Given the following data, calculate: QUANTITATIVE PHARMACEUTICAL CHEMISTRY
ü Average Mean (X) QUANTITATIVE PHARMACEUTICAL CHEMISTRY (QPC)
ü Range (R) • May be defined as the application of the procedures quantitative
ü Standard Deviation (SD) analytical chemistry to the analysis and determination of the purity
ü Relative Standard Deviation (RSD) and quality of drugs and chemicals used in pharmacy.
ü True Value (TV)
METHODS OF ANALYSIS USED IN QPC:
DATA: A. Volumetric Method
B. Gravimetric Method
C. Special Method
D. Instrumental Method

A. VOLUMETRIC METHOD
• Is the determination of the volume of a solution of known concentration
required to react with a given amount of a substance to be analyzed.
ANSWERS:
Example: Assay of HCl, USP XIX

HCl + NaOH " NaCl + H2O

(Analyte) (Titrant)

• The chemical reaction involved in volumetric method is NEUTRALIZATION

(others: Precipitation & Redox Reaction)

ANALYTE OR ACTIVE CONSTITUENTS

DATA: • Is the chemical substances being analyzed.

TITRANT
• Is the solution of known concentration.
• Titrant cannot be a titrant if it is only HCl, HSO4, NaOH etc. for you should
also have the concentration such as 0.1N or 0.5N.

TITRATION
• Is the act of adding and measuring the volume of titrant used in the
assay.
ANSWERS:
INDICATOR
• Is a chemical which changes color at or very near the point in the
titration where equivalent quantities of analyte and titrant have reacted.

• There’ll be no endpoint and there’s no added indicator, but it is not

always the case.

• The exemption is Potassium Permanganate because it is not only a titrant

but it is also an indicator.

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 10

 INDICATORS ARE COMPLEX ORGANIC COMPOUNDS WHICH IS USED TO MIXED INDICATORS
1. Determine the endpoint in neutralization processes. - Are used to sharpen up the color change because some indicators do no
2. Determine hydrogen-ion concentration or ph. give sharp color changes.
3. Indicate that a desired change in pH has been affected.
Example: Methyl red – methylene blue

THEORIES TO EXPLAIN THE CHANGES IN COLOR OF INDICATORS

PHYSICOCHEMICAL METHODS OF TITRATION:
ORGANIC THEORY COLLOIDAL THEORY
THEORY I. Direct Titration
Attributes the color to
Attributes the color of 𝑽 𝒙 𝑵 𝒙 𝒎𝑬𝒒𝒘𝒕
certain ions an %= 𝒙 𝟏𝟎𝟎
indicators to certain Assumes that indicators
increase in which 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
groupings of the form colloidal solutions
causes the
elements in a the change in color at
appearance of a new
compound, and the which is dependent 𝑽 𝒙 𝑵𝑭 𝒙 𝑻𝒊𝒕𝒆𝒓
color, and a decrease %= 𝒙 𝟏𝟎𝟎
change in color to a upon change in size of
in which causes the 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 (𝒎𝒈)
change in molecular the colloidal particle.
disappearance of a
structure.
color or appearance • In getting the NF in direct titration, NF = Actual Normality/Theoretical
of a different color. Normality.

• The Actual Normality can be located at the first part of the statement while
Commonly Used pH Indicators the Theoretical Normality can be located at the second part of the
INDICATOR pH RANGE COLOR CHANGE statement or the titer value, e.g., “Each mL of 0.1 N sulfuric acid..”
ACID BASE
Methyl Orange 3.2-4.4 Pink Yellow • For titer, volume will always be 1 mL.
Methyl Red 4.2-6.2 Red Yellow
Phenolphthalein 8.0-10.0 Colorless Red or Pink • In direct titration, it can be solved by two chemical factors which are
mEqwt and titer value.

RULES FOR THE USE OF INDICATORS: • In direct titration, there’s only 1 titrant used in the titration because the
1. Use 3 drops of indicator test solution for a titration unless otherwise sample easily reacts with the titrant.
directed.
PROBLEM SOLVING:
2. When a strong acid is titrated with a strong alkali, or vise versa, use methyl 1. Calculate the % of Potassium Bipthalate if 0.0758 g sample requires
orange, methyl red or phenolphthalein. 35 ml of 0.01N Sodium Hydroxide.
MW = 204
3. When a weak acid is titrated with a strong alkali, use phenolphthalein.
𝑽 𝒙 𝑵 𝒙 𝒎𝑬𝒒𝒘𝒕
%𝑲𝑯𝑪𝟖𝑯𝟒𝑶𝟒 = 𝒙 𝟏𝟎𝟎
4. When a weak alkali is titrated with a strong acid, use methyl red. 𝑾𝒔

35 𝑚𝐿 𝑥 0.01𝑁 𝑥 204/1000
5. A weak alkali should never be titrated with a weak acid or vise versa, %𝑲𝑯𝑪𝟖𝑯𝟒𝑶𝟒 = 𝑥 100
0.0758 𝑔
since no indicator will give a sharp endpoint.
%𝑲𝑯𝑪𝟖𝑯𝟒𝑶𝟒 = 94.20%
6. The appearance of a color is more easily observable than is the
disappearance.

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 2. In titration of 1.2163g sample of Sodium carbonate with 23.45 of 2. Calculate the Magnesium Hydroxide of Milk of Magnesia, 12.32g of
0.9066 N Sulfuric Acid, calculate the % of Sodium Carbonate. Each mL which was dissolved in 50mL of 1.0340N Sulfuric Acid, producing a
of 1N H2o4 is equivalent to 53 mg of Na2CO3. mixture that the required 24.50mL of 1.1255N sodium Hydroxide. Each
mL of 1N Sulfuric Acid is equivalent to 29mg of Mg(OH)2.
𝑽 𝒙 𝑵𝑭 𝒙 𝑻𝒊𝒕𝒆𝒓
% 𝑵𝒂𝟐𝑪𝑶𝟑 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 (𝒎𝒈) (𝑽 𝒙 𝑵𝑭) − (𝑽 𝒙 𝑵𝑭) 𝒙 𝑻𝒊𝒕𝒆𝒓
%= 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 (𝒎𝒈)
0.9055 𝑁
23.45 𝑚𝐿 𝑥 𝑥 53 𝑚𝑔
%𝑵𝒂𝟐𝑪𝑶𝟑 = 1𝑁 𝑥 100 1.0340 𝑁 1.1255 𝑁
`50𝑚𝐿 𝑥
1216.30 (𝑚𝑔)
%= 1𝑁 a − `24.50𝑚𝐿 𝑥 1𝑁 a 𝑥 29 𝑚𝑔 𝑥 100
12320
%𝑵𝒂𝟐𝑪𝑶𝟑 = 92.64%
% = 5.68%

II. Residual Titration

III. Direct Titration using Blank
(𝑽 𝒙 𝑵) − (𝑽 𝒙 𝑵) 𝒙 𝒎𝑬𝒒𝒘𝒕
%= 𝒙 𝟏𝟎𝟎 (𝑽𝒃𝒍 𝒙 𝑽𝒔𝒂) − 𝑵 𝒙 𝒎𝑬𝒒𝒘𝒕
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 %= 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
(𝑽 𝒙 𝑵𝑭) − (𝑽 𝒙 𝑵𝑭) 𝒙 𝑻𝒊𝒕𝒆𝒓
%= 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 (𝒎𝒈)
(𝑽𝒃𝒍 𝒙 𝑽𝒔𝒂) − 𝑵𝑭 𝒙 𝑻𝒊𝒕𝒆𝒓
%= 𝒙 𝟏𝟎𝟎
• In residual titration, we are using two titrants in the assay because the 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 (𝒎𝒈)
sample do not readily react with the 1st titrant, hence, there’s a need for
the 2nd titrant to finish the reaction. • The 1st titration is with the sample, and in the 2nd titration, the Erlenmeyer
flask contains the same reagent but it doesn’t contain the analyte.
• If the titer is in milligram (mg), then the weight of the sample should also
be in mg. the same rule applies if the titer is in grams (g). • Direct Titration using Blank is done if we do not want to standardized the
solution and for correction purposes.
• It is not always that the % purity is the unknown for it can also be the
volume, normality, NF, mEqwt, titer and weight of the sample which will PROBLEM SOLVING:
be the unknown. 1. If 0.1125g of Manganese Dioxide consumed 16.35mL and 13.58mL of
0.1095N Potassium Permanganate during the Blank and Sample
PROBLEM SOLVING: titration respectively, calculate the % MnO2.
1. If 1.2500 g sample of Zinc Oxide was treated with 50.00 mL of 1.1230N MW = 86
Sulfuric acid and 24.50 mL of 0.9765 Sodium Hydroxide, calculate the % (𝑽𝒃𝒍 𝒙 𝑽𝒔𝒂) − 𝑵 𝒙 𝒎𝑬𝒒𝒘𝒕
%= 𝒙 𝟏𝟎𝟎
ZnO. 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
MW = 81
(𝑽 𝒙 𝑵) − (𝑽 𝒙 𝑵) 𝒙 𝒎𝑬𝒒𝒘𝒕 (16.25𝑚𝐿 𝑥 13.68𝑚𝐿) − 0.1095 𝑥 85/2000
%= 𝒙 𝟏𝟎𝟎 %= 𝑥 100
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 0.1125 𝑔

(50𝑚𝐿 𝑥 1.123𝑁) − (24.50𝑚𝐿 𝑥 0.9765𝑁) 𝑥 81/1000 % = 10.76%

%= 𝑥 100
1.2500 𝑔

% = 104.41%

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 12

IV. Determination of content per tablet
3. About 0.2465g of Calcium Carbonate was used in the assay and
𝑽 𝒙 𝑵 𝒙 𝒎𝑬𝒒𝒘𝒕 𝑨𝒗𝒆. 𝑾𝒆𝒊𝒈𝒉𝒕 consumed 24.65 mL of 0.0940M EDTA solution. Each mL of 0.1M EDTA is
𝑪𝒐𝒏𝒕𝒆𝒏𝒕/𝒕𝒂𝒃 = 𝒙
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 𝑻𝒂𝒃 equivalent to 10mg of CaCO3. Calculate the % Calcium Carbonate.

𝑽 𝒙 𝑵𝑭 𝒙 𝑻𝒊𝒕𝒆𝒓 𝑨𝒗𝒆. 𝑾𝒆𝒊𝒈𝒉𝒕 𝑽 𝒙 𝑵𝑭 𝒙 𝑻𝒊𝒕𝒆𝒓

𝑪𝒐𝒏𝒕𝒆𝒏𝒕/𝒕𝒂𝒃 = 𝒙 %= 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 𝑻𝒂𝒃 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆

𝑨𝒄𝒕𝒖𝒂𝒍 𝒂𝒎𝒐𝒖𝒏𝒕 𝒐𝒃𝒕𝒂𝒊𝒏𝒆𝒅 𝟐𝟒. 𝟔𝟓 𝒎𝑳 𝒙 𝟎. 𝟗𝟒𝟎𝑴 𝒙 𝟏𝟎𝒎𝒈

%= 𝒙 𝟏𝟎𝟎 %= 𝒙 𝟏𝟎𝟎
𝑳𝒂𝒃𝒆𝒍𝒆𝒅 𝑨𝒎𝒐𝒖𝒏𝒕 𝟐𝟒𝟔. 𝟓 𝒎𝒈

• e.g., Content Uniformity of Sodium Bicarbonate tablets % = 94 %

• You cannot compute for the % purity/labeled claim (LC) without first
getting the amount of content per tablet.
STANDARDIZATION
PROBLEM SOLVING: • The determination of the normality or molarity of a solution.
1. If 0.1085g sample of Sodium Bicarbonate (325mg) was used in the
assay and utilized 10.35mL of 0.1135N Sulfuric Acid, what is the content STANDARD SOLUTION
per tablet and % of NaHCO3. The weight of the 10 tablets is 3.6500g. • Is a solution of known normality or molarity.
• Also known as Titrant
𝑽 𝒙 𝑵 𝒙 𝒎𝑬𝒒𝒘𝒕 𝑨𝒗𝒆. 𝑾𝒆𝒊𝒈𝒉𝒕
𝑪𝒐𝒏𝒕𝒆𝒏𝒕/𝒕𝒂𝒃 = 𝒙
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 𝑻𝒂𝒃 NORMALITY
• Is defined as the number of equivalents of solute per liter (equiv/L) or
10.35𝑚𝐿 𝑥 0.1135𝑁 𝑥 84/1000 3.6500𝑔 milliequivalents per milliliter (meq/mL) of solution.
𝑪𝒐𝒏𝒕𝒆𝒏𝒕/𝒕𝒂𝒃 = 𝑥
0.1085 𝑔 10
MOLARITY
= 𝟎. 𝟑𝟑𝟏𝟗𝟓𝒈 𝒐𝒓 𝟑𝟑𝟏. 𝟗𝟓𝒎𝒈 • Is an expression of the concentration of solution in terms of moles per liter.

𝑨𝒄𝒕𝒖𝒂𝒍 𝒂𝒎𝒐𝒖𝒏𝒕 𝒐𝒃𝒕𝒂𝒊𝒏𝒆𝒅

%= 𝒙 𝟏𝟎𝟎
𝑳𝒂𝒃𝒆𝒍𝒆𝒅 𝑨𝒎𝒐𝒖𝒏𝒕 TWO WAYS TO ACCOMPLISH PREPARATION OF STANDARD SOLUTIONS
I. Primary Standard
331.95 𝑚𝑔 • By the use of a carefully weighed sample of a substance of known
%= 𝑥 100
325 𝑚𝑔 purity.
• The sample is solid.
% = 102.14 %

2. A 1.000 g sample containing Sodium Carbonate (MW=106) is FORMULAS:

dissolved in water and titrated with 0.1000M HCl. The buret reading at
the phenolphthalein end point is 17.5 mL. Calculate the % of Na2CO3 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑷𝒓𝒊𝒎𝒂𝒓𝒚 𝑺𝒕𝒅.
𝑽𝒙𝑵=
𝒎𝑬𝒒𝒘𝒕
𝑽 𝒙 𝑴 𝒙 𝑴𝑾/𝟏𝟎𝟎𝟎
%= 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑷𝒓𝒊𝒎𝒂𝒓𝒚 𝑺𝒕𝒅.
𝑽𝒙𝑵=
𝑴𝑾
17.5 𝑚𝐿 𝑥 0.1000 𝑀 𝑥 106/1000 ( )
𝟏𝟎𝟎𝟎
%= 𝑥 100
1.000 𝑔

% = 18.55 %
Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 13
FORMULAS: NORMALITY TO CALCULATE mEqwt OF A SAMPLE

𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
𝑽𝒙𝑵=
𝒎𝑬𝒒𝒘𝒕

𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
𝑵=
𝑽 𝒙 𝒎𝑬𝒒𝒘𝒕

𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
𝑽=
𝑵 𝒙 𝒎𝑬𝒒𝒘𝒕

Weight of Sample (Ws) = V x N x mEqwt EXAMPLE

FORMULAS: MOLARITY

𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
𝑽𝒙𝑴=
𝑴𝑾/𝟏𝟎𝟎𝟎

𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
𝑴=
𝑽 𝒙 𝑴𝑾/𝟏𝟎𝟎𝟎

𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
𝑽= GRAM EQUIVALENT WEIGHT (GEW)
𝑴 𝒙 𝑴𝑾/𝟏𝟎𝟎𝟎
• Is defined as that weight in grams which is chemically equivalent to 1
Ws = V x M x (MW/1000) gram-atom of Hydrogen (1.0079 g).

GRAM MILLIEQUIVALENT WEIGHT (GmEw)

II. Secondary Standard • Is defined as GEW/1000.
• By the use of another standard solution of known concentration (N
or M). Determine the Molecular Weight, Gram Equivalent Weight and
• The sample is Liquid. Equivalent Weight

FORMULAS: NORMALITY

𝑵𝟏 𝑽𝟏 = 𝑵𝟐 𝑽𝟐

𝑵𝟐 𝑽𝟐
𝑵𝟏 =
𝑽𝟏

𝑵𝟐 𝑽𝟐 Give the MW, GEW, and mEqwt:

𝑽𝟏 =
𝑵𝟏

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 14

 PROBLEM SOLVING:
1. If a sample of Sodium bicarbonate weighing 1.2466 g required 29.23 𝑵𝟏 𝑽𝟏
𝑽𝟐 =
mL of the acid in the titration, what is the Normality of the solution? 𝑵𝟐
MW = 84
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 0.0987𝑁 𝑥 18.4𝑚𝐿
𝑵= 𝑽𝟐 =
𝑽 𝒙 𝒎𝑬𝒒𝒘𝒕 0.1188𝑁

1.2466 𝑔 𝑽𝟐 = 15.29 𝑚𝐿
𝑵=
84
29.23𝑚𝐿 𝑥 1000 6. About 0.3986 g sample of Potassium biphthalate requires 28.45 mL of
NaOH solution. What is the Molarity of NaOH?
𝑵 = 0.5077 𝑁 MW = 204
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
2. If a 0.5438 g of Sodium carbonate is titrated with 1.1039 N Sulfuric 𝑴=
𝑽 𝒙 𝑴𝑾/𝟏𝟎𝟎𝟎
acid, what volume of the acid should be required to produce an
endpoint? 0.3986 𝑔
MW = 106. 𝑴=
28.45𝑚𝐿 𝑥 204/1000
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
𝑽=
𝑵 𝒙 𝒎𝑬𝒒𝒘𝒕 𝑴 = 0.687 𝑀

0.5438 𝑔 7. Determine the weight of Calcium carbonate used in the

𝑽=
106 Standardization of 23.56 mL solution with a Molarity of 0.1295.
1.1039 𝑁 𝑥 2 𝑥 1000
MW = 100
Ws = V x M x (MW/1000)
𝑽 = 9.29 𝑚𝐿
Ws = 23.56mL x 0.1295M x (100/1000)
Ws = 0.30510 g or 305.10 mg
3. About 27.47 mL of 1.1165 N NaOH Solution was required in the
titration of Potassium biphthalate (KHC8H4O4), calculate the weight of
8. In the titration of 0.1187M Sulfuric acid, about 0.4063 g Potassium
the sample.
carbonate was utilized. What volume of the acid would be the
MW = 204
necessary to produce an end point?
Ws = V x N x mEqwt
#$% MW = 138
Ws = 27.47mL x 1.1165N x &$$$ 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
𝑽=
Ws = 6.2567 g 𝑴 𝒙 𝑴𝑾/𝟏𝟎𝟎𝟎

4. Determine the Normality of the 25.8 mL solution if 32.5 mL of 0.1149 N 0.4063 𝑔

𝑽=
Standard solution was used to reach the end point of titration. 0.1187 𝑀 𝑥 138/1000

𝑵𝟐 𝑽𝟐 𝑽 = 24.80 𝑚𝐿
𝑵𝟏 =
𝑽𝟏
9. Calculate the Molarity of the solution (25.66 mL) if 33.18 mL of
0.1149𝑁 𝑥 35𝑚𝐿 0.1245M solution was consumed to produce an endpoint.
𝑵𝟏 =
25.8 𝑚𝐿 𝑴𝟐 𝑽𝟐
𝑴𝟏 =
𝑽𝟏
𝑵𝟏 = 0.1447 𝑁
0.300𝑀 𝑥 400 𝑚𝐿
5. About 18.4 mL of 0.0987 N Acid solution was utilized in the 𝑴𝟏 =
10.0 𝑀
Standardization of 0.1188 N solution to reach the end point. Calculate
the volume consumed in the titration. 𝑴𝟏 = 0.16099 𝑜𝑟 1610 𝑀
Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 15
 10. What volume of concentrated (10.0M) HCl should be used to TITER
prepare 400 mL of 0.300 M HCl? • Is defined as the weight of a substance chemically equivalent to 1 mL
𝑴𝟐 𝑽𝟐 of standard solution.
𝑽𝟏 =
𝑵𝟏 • Formulas:
Titer = V x N x mEqwt
0.300 𝑀 𝑥 400 𝑚𝐿 Titer = V x M x (MW/1000)
𝑽𝟏 =
10.0 𝑀
PROBLEM SOLVING:
𝑽𝟏 = 12.00 𝑚𝐿 1. Calculate the titer value for 0.08 N H2SO4. Sodium oxalate MW = 134
Titer = V x N x mEqwt
&'%
Titer = 1 mL x 0.08N x # ) &$$$
11. If 25.00 mL of HCl required 26.25 mL of 0.0952 N NaOH in a titration,
what volume of the solution would produce 0.2867 g of AgCl? Titer = 0.00536 g or 5.36 mg
MW = 143
𝑵𝟐 𝑽𝟐 Titer statement:
𝑵𝟏 = Each mL of 0.08 N Sulfuric acid is chemically equivalent to 0.00536 g or
𝑽𝟏
5.36 mg of Sodium oxalate.
0.0952𝑁 𝑥 26.25 𝑚𝐿
𝑵𝟏 = 2. Calculate the titer value for 0.5 M HCl. Calcium carbonate MW = 100
25.00 𝑚𝐿
Titer = V x M x (MW/1000)
𝑵𝟏 = 0.09996 𝑜𝑟 0.10𝑁 = 1 mL x 0.5 M x (100/1000)
= 0.05g or 50 mg
0.2867 𝑔
𝑽= Titer statement:
143
0.10 𝑁 𝑥 1000
Each mL of 0.5 M HCl is chemically equivalent to 0.05 g or 50 mg of
Calcium carbonate.
𝑽 = 20.05 𝑚𝐿
3. Calculate the titer values for 5N H2SO4:
1. Potassium Bicarbonate (MW = 100)
MOLALITY 2. Calcium Carbonate (MW = 100)
• Is defined as the number of moles of solute per kilogram of solvent. 3. Magnesium Oxide (MW = 40)

• This method of expressing concentration is based on the mass of solute

(expressed as moles) per unit mass (1.00 kg) of solvent.

4. Calculate the titer values for 0.02M HCl:

1. Sodium Oxalate (MW = 134)
2. Zinc Oxide (MW = 81)
3. Potassium Carbonate (MW 138)

EQUIVALENT (equiv)
• Is the number of gram equivalents involved in a quantitative procedure.

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 16

 COMMONLY USED APPARATUS IN QPC A. Acidimetric Analysis
Two (2) types of Volumetric Apparatus:- Acidimetry in Aqueous Solvent
1. That made to deliver a definite volume of liquid, such as; burets
and pipets. ACIDIMETRY
- The direct or residual titrimetric analysis of bases using an accurately
2. That made to contain a definite volume of liquid, such as; measured volume of acid.
volumetric flasks and graduated cylinder.
Ø Analyte: Base
BURETS Ø Titrant: Acid
• Graduated glass tubes of uniform bore throughout the whole length,
used in the measurement of variable quantities of liquid. Acidimetry Direct Titration Methods
• Two types of Burets: Title of Exercise Sample Titrant Indicator Endpoint
1. Acid Burette: Assay of 1N
ü Use if the titrant is Acid Sodium NaHCO3 H2SO4 Methyl orange T.S. Pink
ü Made out of Glass Bicarbonate
1N
2. Base Burette or Mohr Burette Assay of NaOH NaOH Phenolphthalein T.S. Pink
H2SO4
ü Use if the titrant is Basic
ü There is a portion that is made out of rubber. Acidimetry Residual Titration Method / Back Titration
Title of Sample Titrant Indicator Endpoint
PIPETS Exercise
• Transfer or delivery pipet 1N H2SO4
• Measuring pipet Zinc Oxide Methyl orange
Assay of ZnO Yellow
1N T.S.
VOLUMETRIC FLASK NaOH
• Are used to make up standard solutions to a given volume. 1N H2SO4
Assay of Milk Milk of Methyl red T.S.
GRADUATED CYLINDER Yellow
of Magnesia Magnesia 1N
• They are graduated to contain a given volume of liquid at standard NaOH
temperature.
ACIDIMETRY RESIDUAL TITRATION METHOD / BACK TITRATION
Reasons:
CHEMICAL REACTIONS USED IN TITRIMETRY ü When the basic sample is insoluble in water.
1. Neutralization (acid-base) in aqueous and non-aqueous solvents
2. Precipitation ü When the rate of its reaction with the standard acid is relatively slow.
3. Complexation
4. Oxidation – Reduction ü When the substance to be assayed does not give a distinct, sharp
endpoint with an indicator by direct titration.
1. Neutralization (In Aqueous Solvent)
• Are chemical processes in which an acid (proton donor) reacts
with a base (proton acceptor). NITROGEN DETERMINATION BY THE KJELDAHL METHOD
• Method I is a macro method in which the ammonia is distilled into
• The products of a neutralization reaction in aqueous solution are excess boric acid solution.
water and a salt. Ex. NaCl
o Aqueous Solvent since the sample is easily dissolved in Water. • Method II is a semimicro method in which smaller samples are
employed using semimicro Kjeldahl Apparatus.

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 17

 Title of Exercise Sample Titrant Indicator Endpoint ACIDIMETRY IN NON-AQUEOUS SOLVENTS
Determine the Sample substances:
Nitrogen content of Beef 1N Methyl red – Gray 1. Amine
Alcohol soluble extract H2SO4 Methyl blue color 2. Amine salts
substance in Beef 3. Heterocyclic nitrogen compounds
Extract 4. Alkali salts of organic acids
5. Alkali salts of weak inorganic acid
ALKALIMETRY 6. Amino acids
- The direct or residual titrimetric analysis of acids using an accurately
measured volume of alkali. Solvents: The choice of solvent is determined by the basic character of the
substance to be assayed.
• Inorganic acids
9 Methyl red or phenolphthalein can be used as indicator. Neutral solvents Acid solvents
- Acetonitrite - Formic acid
• Organic acids - Alcohols - Glacial acetic acid
9 Phenolphthalein is used. - Chloroform benzene - Propionic acids
- Dioxane - Acetic anhydride
Ø Analyte: Acid - Ethyl acetate - Sulfonyl chloride
Ø Titrant: Base
Titrant: Perchloric acid in glacial acetic acid.
Alkalimetry, Direct Titration
Title of Sample Titrant Indicator Endpoint Indicators:
Exercise 1. Crystal violet
1N 2. Methylrosaniline chloride
Assay of HCl HCl Methyl red T.S. Pink 3. Quinaldine red
NaOH
Assay of Boric acid 1N 4. a–naphtholbenzein
Phenolphthalein T.S. Pink 5. Malachite green
Boric acid HBO3 NaOH
Tartaric
Assay of 1N Title of Exercise Sample Titrant Indicator Endpoint
acid Phenolphthalein T.S. Pink
Tartaric acid NaOH
C4H6O6 Assay of Methacholine 0.1N
Crystal
Methacholine chloride Perchloric Blue green
violet
Alkalimetry, Residual Titration chloride C8H18ClNO2 acid
Title of Exercise Sample Titrant Indicator Endpoint
1N H2SO4
Assay of Aspirin Aspirin Phenolphthalein ALKALIMETRY IN NON-AQUEOUS SOLVENTS
C9H8O4 1N NaOH T.S. Sample substances:
Note: Aspirin hydrolysis products: Acetic acid and Salicylic acid 1. Acid halides
2. Anhydrides
NON-AQUEOUS TITRIMETRIC ANALYSIS 3. Acids
Reasons: 4. Amino acids
• Weakly reactive of sample in water 5. Enols such as barbiturates and xanthene
• Poor solubility 6. Ionides
7. Phenols pyrroles
8. Sulfonamides
9. Organic salts of inorganic acids

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Solvents: 3. Adsorption indicators
Solvents for the titration of weak Solvents for medium-strength a. Dichlorofluorescein (DCF)
acids such as enols acidic substances. b. Eosin y T.S. or Tetrabromofluorescein
Strong bases: - Dimethylformamide c. Tetrabromophenolphthalein ethyl ester (TEE)
- Ethylenediamine - Pyridine
- N-butylamine Standard Solutions
- Morpholine • 0.1N Silver Nitrate
• 0.1N Ammonium thiocyanate
Titrants:
1. Sodium Direct Titration Method
2. Methoxide Title of Exercise Sample Titrant Indicator Endpoint
3. Lithium methoxide Assay of
4. Potassium methoxide (is not used since it may produce a gelatinous Sodium Lauryl Sodium 0.1N Potassium
product) Sulfate for NaCl Lauryl Sulfate AgNO3 chromate T.S. Red
5. Sodium aminomethoxide content
6. Sodium triphenylmethane (phenols and pyrroles)
Residual Titration Method (Volhard Method)
Indicators: Title of Exercise Sample Titrant Indicator Endpoint
1. Azol violet – (weak acids) 0.1N Ferrium
2. Thymol blue – (intermediate to strong acids) Assay of NaCl NaCl AgNO3 Ammonium Red
3. Thymolphthalein Sulfate (T.S.)
4. p-hydroxyazobenzene 0.1N
NH4SCN
Title of Exercise Sample Titrant Indicator Endpoint
Assay of 0.1N ARGENTOMETRIC METHODS
Phenytoin Phenytoin Sodium Azo violet Blue • Argentum is the Latin name of Silver (Ag)
methoxide • The interaction between Silver Nitrate and Sodium Chloride
NaCl + AgNO3 à AgCl + NaNO3
2. Precipitation
— Volumetric precipitimetry PARAMETERS CONSIDERED
§ The reaction is requiring the formation of relatively insoluble A. Precipitate formed must be insoluble
substances or precipitates to cause the reaction to go to sufficient B. Precipitation process should be fast and rapid
completion. C. Co-precipitation effects must be minimal
D. Detection of Equivalence point must be apparently visible
DETERMINATION OF THE ENDPOINT
1. Cessation of precipitation or the appearance of a turbidity. TWO CATEGORIES OF ARGENTOMETRIC METHODS
2. Use of internal indicators. 1. Direct titration with Silver Nitrate
3. Instrumental methods i.e, potentiometric or amperometric. 2. Ammonium Thiocyanate Silver Nitrate Titration (Volhard Method)

Indicators: 3. Complexation Methods

1. Ferric ammonium sulfate T.S. — Quantitative analysis of inorganic pharmaceutical products containing
Endpoint: Red color of Ferric thiocyanate metal ions, such as; Al, Bi, Ca, Mg, and Zn.

2. Potassium chromate T.S. TYPES OF METALS

Endpoint: Red color of Silver chromate against a white of 1. Monovalent (Na +, K +) – form unstable and weak complexes with EDTA,
silver chloride thus not a candidate sample for complexation method.

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2. Divalent ( Ca+2, Mg +2, Zn +2) MASKING
— The term used to indicate the determination of a metal in the
3. Polyvalent (Al +3, Bi+3) presence of another metal.
Ø both Divalent & Polyvalent are good candidate for complexation
because they form stable and strong complexes. MASKING CAN BE ACCOMPLISHED BY
1. Adjusting the pH of the titration medium.
Titrant: 2. Auxiliary complexing agent.
Disodium Ethylenediaminethetraacetate (EDTA)
Example:
Indicator: 1. Triethanolamine
1. Azo dyes 2. Thioglycols
2. Phthleins 3. Potassium cyanide
3. Triphenylmethane dyes 4. Ammonium fluoride
4. Eriochrome black T 5. Ascorbic acid
5. Hydroxynaphthol blue (HNB) 6. Citrates
7. Tartrates
COMPLEXATION REACTION
• When a metal ion combine with a molecule which can donate electrons, DIRECT TITRATION METHODS
the resulting compound is called a COMPLEX. — The metal ions which are determined by direct titration with EDTA are
Ca2+, Mg2+, and Zn2+.
• If the combining molecule contains two or more groups that donate
electrons, this complex is called a CHELATE. Residual Titration
— The indirect or residual method is applied to the analysis of Aluminum
• EDTA will react with metal ions to form a water soluble, stable complex or and Bismuth compounds.
chelate compound.
" The reaction is with polyvalent metal ions such as Al3+, Bi3+, and 4. Oxidation-Reduction Methods
divalent metals such as Ca2+, Cu2+, Hg2+, Mg2+, and Zn2+. — Involve in a change in valence of the reacting substances.
VILEORA VDGEROA
" Monovalent metals such as Na+, K+ yield weak or unstable Valence Valence
complexes. Increase Decrease
Loss Gain
• With a divalent metal such as calcium or magnesium there are six bonds Electron Electron
or point of attachment between the metal and the EDTA: 2 are ionic; 2 Oxidation Reduction
are coordinate; 2 are ordinate bonds. With the trivalent metal ion such as Reducing Oxidizing
Al3+ one of the ordinate bonds would become ionic in character. Agent Agent

Standard Solution
Oxidizing Agents Reducing Agents
• Ferric ammonium sulfate • Ferrous ammonium sulfate
• Potassium permanganate • Oxalic acid
• Potassium dichromate • Potassium arsenate
• EDTA molecule which provides groups for attachment to metal ion is • Potassium bromate • Titanium chloride
called a LIGAND. • Potassium iodate • Sodium thiosulfate
• Potassium ferricyanide
• The four oxygen and the two nitrogen atoms of the EDTA molecule • Ceric sulfate
capable of entering a complexation reaction with metal ion to from a • Iodine
hexadentate molecule. • Bromine

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I. Permanganate Methods III. Iodimetric and Iodometric Methods
• Potassium Permanganate Solution (Titrant) IODIMETRY
• is a direct analysis of reducing agent such as thiosulfate and arsenates by
Characteristics: the use of Iodine.
- Standardized easily
- Retains its concentration over long period of time IODOMETRY
- The reactions in solution are rapid • is an indirect analysis of oxidizing agent such as Ferric and Cupric salts is
- It also serves as indicator in titration where it is used reduced with excess potassium iodide and the iodine thus formed is
titrated with Sodium thiosulfate.
Direct Titration Methods
Title of Exercise Sample Titrant Indicator Endpoint Standard Solutions:
Assay of H2O2 H2O2 0.1N KMnO4 0.1N KMnO4 Pink — 0.1N Iodine
— 0.1N Sodium thiosulfate
Indirect Titration Methods — 0.1N Potassium arsenite
Title of Exercise Sample Titrant Indicator Endpoint
Assay of Cherry Indicator: Starch T.S.
Cherry
juice for Malic 0.1N KMnO4 0.1N KMnO4 Pink
juice Note: In hot water, starch burst and form a colloidal dispersion of ß–
acid
amylase, known as soluble starch, and a–amylase and amylopectin
Residual Titration Methods as the insoluble starch. The interaction of Iodine with starch results to
Title of Exercise Sample Titrant Indicator Endpoint an intensely blue-colored solution.
0.1N KMnO4
Assay of Sodium Sodium Direct Titration Methods
0.1N KMnO4 Pink Title of Exercise Sample Titrant Indicator Endpoint
Nitrite Nitrite 0.1N Oxalic
acid Assay of Strong 0.1N Starch T.S.
Iodine Iodine Potassium Blue
II. Ceric Sulfate Titration Method arsenite
Advantages:
1. The solutions are stable even on boiling. Indirect Titration Methods
Title of Sample Titrant Indicator Endpoint
2. Sodium oxalate or arsenic trioxide may be used as primary standard. Exercise
Assay of 0.1N
3. The cerous ion is colorless & does not obscure the indicator endpoint. Sodium Disappearance
Sodium Sodium Starch T.S.
Hypochlorite of blue color
Hypochlorite thiosulfate
4. The intermediate products are formed in the reduction of ceric
cerium.
Residual Titration Methods
5. Rather high concentrations of chloride ion are not oxidized by ceric Title of Exercise Sample Titrant Indicator Endpoint
salts, so that ferrous iron can be determined in the presence of 0.1N KMnO4
chlorides. Assay of Sodium Sodium
0.1N KMnO4 Pink
Nitrite Nitrite 0.1N Oxalic
6. The Ferrous phenanthroline ion (Ferroin) is an indicator of choice in acid
the titration with ceric salts.

Examples:
ü Assay of Ferrous sulfate tablets
ü Assay of Ascorbic acid

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IV. Oxidation-Reduction with 0.1N Bromine 4. Preparation &
• Bromine is employed as an oxidizing agent in the assay of aniline, phenol, Standardization
Benzoic acid Thymol blue Blue
and resorcinol. of 0.1N Sodium
Methoxide
• The standard solution employed does not contain bromine as such but 5. Preparation &
rather an equivalent amount of potassium bromate and excess KBr. Standardization Ferric
Bromine is liberated when the solution is acidified. of 0.1N 0.1N AgNO3 Ammonium Red-brown
Ammonium Sulfate
Title of Exercise Sample Titrant Indicator Endpoint Thiocyanate
Reaction: AgNO3 + NH4SCN ’ AgSCN” + NH4NO3
0.1N Disappearance FeNH4(SO4)2 + 3NH4SCN ’ Fe(SCN)3 + 2(NH4)2SO4
Assay of Phenol Phenol Starch T.S.
Bromine of blue color 6. Preparation &
Sodium
Standardization 0.1N KMnO4 Pink color
oxalate
of 0.1N KMnO4
V. Oxidation-Reduction with Potassium Iodate
• Potassium Iodate may be used as the oxidizing agent in the assay of 7. Preparation &
iodides, arsenite and other reducing agents. Standardization 0.1N Oxalic
0.1N KMnO4 Pink color
of 0.1N Oxalic acid
• The method depends upon the formation of iodine monochloride in acid
strong HCl solution. 8. Preparation &
Standardization Orthophenan
Arsenic trioxide Blue
Title of Exercise Sample Titrant Indicator Endpoint of 0.1N Ceric throline
sulfate
Assay of 0.05M 9. Preparation &
Potassium Starch Disappearance Standardization
Potassium Potassium Arsenic trioxide Starch T.S. Pink
Iodide T.S. of color of 0.1N Iodine
Iodide Iodate
solution
10. Preparation &
VI. Diazotization Assays with Sodium Nitrite Standardization Potassium Disappearance of
Starch T.S.
• The assays of sulfa drugs and other compounds containing an acrylamino of 0.1N Sodium dichromate blue color
group. thiosulfate
11. Preparation &
Potassium
Standardization
bromate and Disappearance of
Preparation And Standardization Of Solutions of 0.1N Bromine, Starch T.S.
Potassium color
Title of Exercise Sample Indicator Endpoint Koppeschaar’s
bromide
1. Preparation & solution
Sodium Methyl red
Standardization Faintly pink 12. Preparation &
Carbonate T.S.
of 1N HCl Standardization
Sulfanilamide Starch T.S. Blue ring
Reaction: Na2CO3 + 2HCl ’ 2NaCl + H2O + CO2“ of 0.1M Sodium
2. Preparation & Potassium Nitrite
Phenolphthal
Standardization biphthalate Pink
ein T.S.
of 1N NaOH (KHC8H4O4)
Reaction: KHC8H4O4 + NaOH ’ KNaC8H4O4 + H2O
3. Preparation &
Potassium
Standardization Crystal violet
biphthalate Emerald green
of 0.1N Perchloric T.S.
(KHC8H4O4)
acid

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 B. GRAVIMETRIC METHOD 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑻𝒂𝒃𝒍𝒆𝒕
= 𝒙
• Is the measurement of the weight of a substance in a sample or 𝑻𝒂𝒃 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 𝑺𝒂𝒎𝒑𝒍𝒆 𝑺𝒊𝒛𝒆
calculation of the weight of a substance in a sample from the weight of
a chemically equivalent amount of some other substance. PROBLEM SOLVING:
Given the data below, how much Progesterone was present in 5
• Is the separation by extraction, precipitation or other means of the capsules?
constituent to be determined either in the natural state or in the form of Wt. of 10 capsules = 6.8975 g
a definite compound the composition of which is known to the analyst, Wt. of sample = 0.3620 g
and the weighing of the resulting product. Wt. of residue = 0.0507 g

GRAVIMETRIC APPARATUS 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑻𝒂𝒃𝒍𝒆𝒕

= 𝒙
• PLAIN CRUCIBLE 𝑻𝒂𝒃 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 𝑺𝒂𝒎𝒑𝒍𝒆 𝑺𝒊𝒛𝒆
- Used in ignition of drugs and precipitants
𝑃𝑟𝑜𝑔𝑒𝑠𝑡𝑒𝑟𝑜𝑛𝑒 0.0507 𝑔 6.8975 𝑔
• GOOCH FILTRATION CRUCIBLE = 𝑥
5 𝐶𝑎𝑝𝑠𝑢𝑙𝑒𝑠 0.3620 𝑔 10 𝑐𝑎𝑝𝑠
- Designed for the separation of precipitate by suction filtration.
= 0.096603 g x 5
• DESICCATOR = 0.4830 g or 483 mg
- Used to maintain a dry atmosphere for objects that might be
affected by moisture or CO2.
II. Chemical Method - is the separation by precipitation, ignition of
• CONSTANT WEIGHT a compound, electrolysis of the constituents in the form of a
- Two consecutive weighings do not differ by more than 0.5 mg/g of definite compound the composition of which is known to the
substance taken for the determination, the second weighing analyst, and the weighing of the final product.
following an additional hour of drying.
𝑾𝒙𝑬
- When two consecutive weighings after heating and cooling do not % = 𝒙 𝟏𝟎𝟎
𝑺
differ by more than 0.2 mg. Where:
W = Weight of Precipitate or Product
LAWS AND THEORIES OF CHEMISTRY E = Chemical Factor or Gravimetric Factor
Ø Law of Mass Action
Ø Reversible Reaction 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑈𝑛𝑘𝑛𝑜𝑤𝑛
= 𝑥 100
Ø Solubility Product Principle 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑃𝑟𝑜𝑑𝑢𝑐𝑡
Ø Common Ion Effect S = Weight of Sample

PROBLEM SOLVING:
GRAVIMETRIC ANALYSIS If a 0.4600 g sample of Sodium Iodide yielded 0.7200 g of AgI, what
• The precipitation is guided by the following: was the iodine content of the sample in % w/w?
1. Concentration of the solute and the precipitating agent MW of Iodine = 127
2. Reaction Time MW of Silver Iodide (AgI) = 235
3. Reaction Temperature 𝑾𝒙𝑬
4. Amount of other substances present in solution % = 𝒙 𝟏𝟎𝟎
𝑺

Two (2) Methods: 127

0.7200 𝑔 𝑥 235
I. Physical Method - is the separation by extraction of the % = 𝑥 100
constituents in the natural state, and the weighing of the final 0.46000 𝑔
product.
% = 𝟖𝟒. 𝟓𝟗 %
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 C. SPECIAL METHODS RESIDUE ON IGNITION
I. ASH DETERMINATIONS — The ash content of chemical is determined by ignition to dull redness.
• Apparatus: Electric Furnace — Negligible amount (quantity not exceeding 500 mcg or 0.5 mg)
Ø Temperature Equivalent for Ignition
ü Very Dull heat = 500-550°C 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆
% 𝑹𝒆𝒔𝒊𝒅𝒖𝒆 𝒐𝒏 𝑰𝒈𝒏𝒊𝒕𝒊𝒐𝒏 = 𝒙 𝟏𝟎𝟎
ü Dull Red heat = 550-700°C 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
ü Bright Red heat = 800-1000°C
ü Yellow Red heat = 1000-1200°C LOSS ON IGNITION
ü White Heat = 1200-1600°C — Provides a means of determining the percentage of test material which
is volatilized and driven off under the condition specified.
ASH CONTENT
• Ash content of a crude drug is taken to be the residue remaining after 𝑾𝒔 − 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑫𝒓𝒊𝒆𝒅 𝑺𝒂𝒎𝒑𝒍𝒆
% 𝑳𝒐𝒔𝒔 𝒐𝒏 𝑰𝒈𝒏𝒊𝒕𝒊𝒐𝒏 = 𝒙 𝟏𝟎𝟎
incineration. 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆
% 𝑨𝒔𝒉 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 Loss on Drying = Ws – Wt. of Dried Sample

PROBLEM SOLVING: SULFATED ASH

About 0.9935g of the incinerated material was obtained from 4g sample of — Is the residue after the carbonized mass is treated with 1 mL of
acacia. Calculate the Ash Content of the sample. concentrated H2SO4 and further incinerated after evaporating off the
H2SO4 under a hood.
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆
% 𝑨𝒔𝒉 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = 𝒙 𝟏𝟎𝟎 % 𝑺𝒖𝒍𝒇𝒂𝒕𝒆𝒅 𝑨𝒔𝒉 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆

0.9935 𝑔 II. WATER CONTENT DETERMINATION

% 𝐴𝑠ℎ 𝐶𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑥 100
4𝑔 — Used to ensure uniformity of water content limits in the drug. Drugs official
in the USP and N.F. contain varying quantities of water either as water of
% 𝐴𝑠ℎ 𝐶𝑜𝑛𝑡𝑒𝑛𝑡 = 𝟐𝟒. 𝟖𝟒 % crystallization (hydrates) or as water in the adsorbed form.

METHODS OF WATER CONTENT DETERMINATION

ACID INSOLUBLE ASH • We determine the water content because we do not want the drug
— Is the part of the total ash which is insoluble in diluted hydrochloric acid. product to be destroyed by e.g., presence of molds.
— This is also referred to the silica so you can either use % Silica or % Acid-
Insoluble Ash. 1. Gravimetric, for drugs containing no constituents other than water,
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆 volatile at 105 0C.
% 𝑨𝒄𝒊𝒅 − 𝑰𝒏𝒔𝒐𝒍𝒖𝒃𝒍𝒆 𝑨𝒔𝒉 = 𝒙 𝟏𝟎𝟎 𝑾𝒔 − 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑫𝒓𝒊𝒆𝒅 𝑺𝒂𝒎𝒑𝒍𝒆
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 % 𝑴𝒐𝒊𝒔𝒕𝒖𝒓𝒆 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
PROBLEM SOLVING:
What is the % Silica adhering to the drug if the residue obtained after the PROBLEM SOLVING:
incineration and treatment of diluted HCl was 0.6795g from a 2g acacia Determine the % Water Content of a 10g sample of guava leaves. The
leaves. weight after drying was 7.8953g.
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆 𝑾𝒔 − 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑫𝒓𝒊𝒆𝒅 𝑺𝒂𝒎𝒑𝒍𝒆
% 𝑺𝒊𝒍𝒊𝒄𝒂 = 𝒙 𝟏𝟎𝟎 % 𝑴𝒐𝒊𝒔𝒕𝒖𝒓𝒆 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆

0.6795 𝑔 𝟏𝟎 𝒈 − 𝟕. 𝟖𝟗𝟓𝟑 𝒈
% 𝑺𝒊𝒍𝒊𝒄𝒂 = 𝑥 100 % 𝑴𝒐𝒊𝒔𝒕𝒖𝒓𝒆 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = 𝒙 𝟏𝟎𝟎
2𝑔 𝟏𝟎 𝒈

% 𝑺𝒊𝒍𝒊𝒄𝒂 = 𝟑𝟑. 𝟗𝟖 % % 𝑴𝒐𝒊𝒔𝒕𝒖𝒓𝒆 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = 𝟐𝟏. 𝟎𝟓 %

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 2. Gravimetric, for drugs containing ether-soluble constituents volatile at III. EXTRACTIVE AND CRUDE FIBER CONTENTS
105 0C. Extractive
— Is the withdrawal of desired constituents from crude drugs through the
3. Azeotropic or Toluene distillation in the N.F. and Xylene distillation in the use of selected solvents in which the desired constituents are soluble.
USP.
Extractives
4. Karl Fischer Electronic Titration Method. — The products of extraction.
Reagent: Karl Fischer reagent is consists of:
Iodine, Sulfur dioxide, Pyridine, Methanol SOLVENT OR MENSTRUUM
1. Ether
TO STANDARDIZE THE SOLUTION 9 Dissolve almost all constituents of the plant sample except for H2O
a. Primary standard: Sodium tartrate (Na2C4H4O6–2H2O) soluble.
b. Secondary standard: a water-methanol solution of known water
concentration. 2. Alcohol
𝑺𝒙𝑭 9 Is a good solvent for resinous matter
% 𝑴𝒐𝒊𝒔𝒕𝒖𝒓𝒆 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 (𝒎𝒈)
Where: 3. Diluted Alcohol
S = Volume of Karl Fischer reagent 9 Product of the combined solvent of water and alcohol and its
F = Water equivalence Factor concentration may vary such as 50%, 60%, 70% or 80%.
W = Weight of the Sample
4. Water
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑷𝒓𝒊𝒎𝒂𝒓𝒚 𝑺𝒕𝒂𝒏𝒅𝒂𝒏𝒓𝒅 9 Used for tannins
𝑭 = 𝒙 𝟎. 𝟏𝟓𝟔𝟔
𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝑲𝒂𝒓𝒍 𝑭𝒊𝒔𝒄𝒉𝒆𝒓 𝑹𝒆𝒂𝒈𝒆𝒏𝒕
5. Hexane
PROBLEM SOLVING: 9 Is a good solvent for fats and fatty oils
1. Calculate the % moisture in Starch if 12.00mL of Karl Fischer reagent,
having a water equivalence factor of 6.30 was consumed by a 5.500g 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆
% 𝑬𝒙𝒕𝒓𝒂𝒄𝒕𝒊𝒗𝒆 = 𝒙 𝟏𝟎𝟎
sample. 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
𝑺𝒙𝑭
% 𝑴𝒐𝒊𝒔𝒕𝒖𝒓𝒆 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = 𝒙 𝟏𝟎𝟎
𝑾𝒔 (𝒎𝒈)
CRUDE FIBER
12 𝑚𝐿 𝑥 6.30 — Is the residue, consisting chiefly of cellulose, that remains undissolved
% 𝑴𝒐𝒊𝒔𝒕𝒖𝒓𝒆 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = 𝑥 100
5500 𝑚𝑔 after successive treatment with boiling acids and alkali.

% 𝑴𝒐𝒊𝒔𝒕𝒖𝒓𝒆 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = 𝟏. 𝟑𝟕 % 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆 − 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑨𝒔𝒉

% 𝑪𝒓𝒖𝒅𝒆 𝑭𝒊𝒃𝒆𝒓 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
2. Calculate the Water Equivalence factor (F) of Karl Fischer reagent if a
250mg sample of Sodium Dihydrate required 18.5mL of Karl Fischer
reagent.
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑷𝒓𝒊𝒎𝒂𝒓𝒚 𝑺𝒕𝒂𝒏𝒅𝒂𝒏𝒓𝒅
𝑭 = 𝒙 𝟎. 𝟏𝟓𝟔𝟔
𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝑲𝒂𝒓𝒍 𝑭𝒊𝒔𝒄𝒉𝒆𝒓 𝑹𝒆𝒂𝒈𝒆𝒏𝒕 IV. CONSTANT OF FATS, FATTY OILS, WAXES, BALSAM, RESINS, ETC
1. Acid Value also known as Acid number, Acidity index, defined as the
𝟐𝟓𝟎 𝒎𝒈 number of milligrams of KOH necessary to neutralize the free acids in 1
𝑭 = 𝒙 𝟎. 𝟏𝟓𝟔𝟔
𝟏𝟖. 𝟓 𝒎𝑳 g of oil, fat, wax, resin, balsam, and similar substances.

𝑭 = 𝟐. 𝟏𝟐 𝒎𝒈/𝒎𝑳 𝒎𝒈𝑲𝑶𝑯
𝑽 𝒙 𝑵 𝒙 𝒎𝑬𝒒𝑲𝑶𝑯 (𝟓𝟔. 𝟏𝟏)
𝑨𝒄𝒊𝒅 𝑽𝒂𝒍𝒖𝒆 =
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 25
 PROBLEM SOLVING: 4. Iodine Value or Iodine Number
If a 1.1g sample of rosin required 28.00mL of 0.1100N NaOH in the titration of • Is the number of grams of iodine absorbed under specified
the free fatty acids, determine the Acid Value of Rosin. conditions by 100 g of oil, fat, wax, or other substances.

28 𝑚𝐿 𝑥 0.1100𝑁 𝑥 (56.11) • This value is a quantitative measure of the proportion of

𝑨𝒄𝒊𝒅 𝑽𝒂𝒍𝒖𝒆 =
1.1 𝑚𝑔 unsaturated fatty acids present, both free and uncombined as
esters, that have the property of absorbing iodine.
𝑨𝒄𝒊𝒅 𝑽𝒂𝒍𝒖𝒆 = 𝟏𝟓𝟕. 𝟏𝟎𝟖

2. Saponification Value or Saponification Number or Koettsdorfer Number, REASONS OF DETERMINATION

defined as the number of milligrams of KOH required to neutralize the 1. It serves to characterize them and to indicate whether they are pure or
free acids and saponify the esters contained in 1 g of fat, fatty or admixtures.
volatile oil, wax, resin, balsam, and similar substances. Drying oil Non-drying oil Semi-drying oil
Have a very high
(𝑽𝒃𝒍 − 𝑽𝒔𝒂) 𝒙 𝑵 𝒙 𝟓𝟔. 𝟏𝟏 iodine number, above
𝑺𝒂𝒑𝒐𝒏𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏 𝑽𝒂𝒍𝒖𝒆 = Have relatively low Have intermediate
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 120, since they contain
iodine numbers below iodine values, that is
a large proportion of
100. between 100 and 120.
PROBLEM SOLVING: unsaturated fatty
Find the Saponification Value of cottonseed oil if a 1.588g sample refluxed acids.
with 25.00mL of about 0.5N Alcoholic KOH, required 14.80mL of 0.5125N HCl Ex.: Ex.: Ex.:
for the residual titration. The blank requires 26.50mL of 0.5125N HCl to bring Linseed oil and Cod Olive oil and almond Cottonseed oil and
the endpoint of titration. liver oil oil sesame oil

(𝑽𝒃𝒍 − 𝑽𝒔𝒂) 𝒙 𝑵 𝒙 𝟓𝟔. 𝟏𝟏 2. It does not only serves to indicate in a definite manner the class to which
𝑺𝒂𝒑𝒐𝒏𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏 𝑽𝒂𝒍𝒖𝒆 = an unknown fat or oil belongs.
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆

(26.50 𝑚𝐿 − 14.80 𝑚𝐿) 𝑥 0.5125𝑁 𝑥 56.11 3. Serves as a means of detecting adulteration.

𝑺𝒂𝒑𝒐𝒏𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏 𝑽𝒂𝒍𝒖𝒆 =
1.588 𝑔

𝑺𝒂𝒑𝒐𝒏𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏 𝑽𝒂𝒍𝒖𝒆 = 𝟐𝟏𝟏. 𝟖𝟕 METHODS OF IODINE VALUE DETERMINATION:

1. Hübl
3. Ester Value or Ester Number 2. Hanus
• Is defined as the number of milligrams of KOH required to 3. Wijs
𝒈
saponify, the esters in 1 g of a fatty or volatile oil, fats, wax, (𝑽𝒃𝒍 − 𝑽𝒔𝒂)𝒙 𝑵 𝒙 (𝟎. 𝟏𝟐𝟔𝟗) 𝒙 𝟏𝟎𝟎
𝒎𝑬𝒒𝑰𝒐𝒅𝒊𝒏𝒆
balsam, resin. 𝑰𝒐𝒅𝒊𝒏𝒆 𝑽𝒂𝒍𝒖𝒆 =
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
• The Ester number may also be calculated in the same manner as
the Saponification value.
PROBLEM SOLVING:
• Tn substances that do not contain free acids ester number is
About 0.2246g of Olive Oil utilized 25.17mL and 12.85mL of 0.1147N Sodium
equal the Saponification value.
Thiosulfate during the blank and sample titration respectively. Calculate
the Iodine Value of the oil.
Saponification Value = Ester Value + Acid Value
(25.17 𝑚𝐿 − 12.85 𝑚𝐿)𝑥 0.1147𝑁 𝑥 (0.1269) 𝑥 100
• When free acids are present, the ester value is the difference 𝑰𝒐𝒅𝒊𝒏𝒆 𝑽𝒂𝒍𝒖𝒆 =
0.2246 𝑔
between the acid and Saponification value.
𝑰𝒐𝒅𝒊𝒏𝒆 𝑽𝒂𝒍𝒖𝒆 = 𝟕𝟗. 𝟖𝟒
Ester Value = Saponification Value – Acid Value

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 26

 5. Unsaponifiable Matter Assay for Phenol Content
— Is defined as the substances present in oils and fats that are not Apparatus: Cassia Flask
saponified by alkali hydroxides but are soluble in ordinary fat 𝑽𝒐𝒍. 𝒐𝒇 𝑶𝒊𝒍 − 𝑽𝒐𝒍. 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒂𝒍 𝑶𝒊𝒍𝒚 𝑳𝒂𝒚𝒆𝒓
% 𝑷𝒉𝒆𝒏𝒐𝒍 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = 𝒙 𝟏𝟎𝟎
solvents. 𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝑶𝒊𝒍

— When oil and fats are saponified, there remains a small amount PROBLEM SOLVING:
of residue that may consist of phytosterol in vegetable oils and In Phenol content determination of a volatile oil, the layer in the graduated
cholesterol in animals. neck of the Cassia flask read 1.3mL obtained from a 10mL sample of the oil
after treatment with KOH solution. Determine the % Ketone content of the
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆 oil.
% 𝑼𝒏𝒔𝒂𝒑𝒐𝒏𝒊𝒇𝒊𝒂𝒃𝒍𝒆 𝑴𝒂𝒕𝒕𝒆𝒓 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 𝑽𝒐𝒍. 𝒐𝒇 𝑶𝒊𝒍 − 𝑽𝒐𝒍. 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒂𝒍 𝑶𝒊𝒍𝒚 𝑳𝒂𝒚𝒆𝒓
% = 𝒙 𝟏𝟎𝟎
𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝑶𝒊𝒍
V. ASSAY OF VOLATILE OILS
> Volatile oils also known as ethereal oils or essential oils. 10 𝑚𝐿 − 1.3 𝑚𝐿
% = 𝑥 100
10 𝑚𝐿
Chemical components of Volatile Oil
1. Hydrocarbons % 𝑲𝒆𝒕𝒐𝒏𝒆 = 𝟖𝟕%
2. Aldehydes
3. Alcohols
4. Ketones Assay for Volatile Oil in Spirits
5. Acids Apparatus: Babcock Bottle
6. Sulfur compounds
𝑽𝒐𝒍 𝒐𝒇 𝑶𝒊𝒍 + 𝑲𝒆𝒓𝒐𝒔𝒆𝒏𝒆 − 𝑽𝒐𝒍 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒂𝒍 𝑶𝒊𝒍𝒚 𝑳𝒂𝒚𝒆𝒓
% 𝑽𝒐𝒍. 𝑶𝒊𝒍 𝒊𝒏 𝑺𝒑𝒊𝒓𝒊𝒕 = 𝒙 𝟏𝟎𝟎
𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝑶𝒊𝒍
Assay for Ester Content

(𝑽𝒃𝒍 − 𝑽𝒔𝒂) 𝒙 𝑵 𝒙 𝒎𝑬𝒒𝒘𝒕

% 𝒘/𝒘 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
VI. ASSAY OF ALKALOIDS AND AMINE DRUGS
Alkaloids may be defined as chemical substances:
Assay for Alcohol Content
Apparatus: Acetylization Flask (1) Which are obtained from plant, animal or synthetic sources.
𝑨 𝒙 𝟕. 𝟖𝟏𝟑
% 𝑻𝒐𝒕𝒂𝒍 𝑴𝒆𝒏𝒕𝒉𝒐𝒍 = (2) Contain organic nitrogen(s) within their chemical structure.
𝑩 − (𝑨 𝒙 𝟎. 𝟎𝟎𝟐𝟏)
(3) Posses physiological activity (e.g., Morphine – anesthesia used in
Assay for Aldehyde Content
major operations).
Methods:
1. Hydroxylamine method
ALKALOIDAL TEST SOLUTION
(𝑽𝒃𝒍 − 𝑽𝒔𝒂) 𝒙 𝑵 𝒙 𝒎𝑬𝒒𝒘𝒕 • Mercuric Iodide T.S. (Valser’s reagent)
% 𝑨𝒍𝒅𝒆𝒉𝒚𝒅𝒆 = 𝒙 𝟏𝟎𝟎 • Iodide T.S. (Wagner’s reagent)
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
• Mercuric Potassium Iodide T.S. (Mayer’s reagent)
Ø Dragendorff’s reagent
2. Bisulfite method
§ Another alkaloidal test solution that is not used in QC is
𝑽𝒐𝒍. 𝒐𝒇 𝑶𝒊𝒍 − 𝑽𝒐𝒍. 𝑹𝒆𝒔𝒊𝒅𝒖𝒂𝒍 𝑶𝒊𝒍𝒚 𝑳𝒂𝒚𝒆𝒓 the Dragendorff’s reagent.
% 𝑨𝒍𝒅𝒆𝒉𝒚𝒅𝒆 = 𝒙 𝟏𝟎𝟎
𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝑶𝒊𝒍
Choice of Indicator for Alkaloidal Titration
ü Methyl Red
Ø If you have a small amount of oil, use the Hydroxylamine method.
Ø If you have a bigger amount of oil, use the Bisulfite method.

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 27

Methods: VII. CLASSIFICATION OF ASSAYS OF CRUDE DRUGS
1. Gravimetric Proximate Assay
2. Volumetric — If the percent of extractive represents the total of a class of plant
3. Spectrometric principles, such as; alkaloids, glycosides, etc.
4. Electrometric
5. Physiological Ultimate Assay
— If the percent of extractive from a crude drug represents a single
Gravimetric chemical species, such as morphine.

𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆
% 𝑨𝒍𝒌𝒂𝒍𝒐𝒊𝒅𝒂𝒍 𝑬𝒙𝒕𝒓𝒂𝒄𝒕𝒊𝒗𝒆 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 D. INSTRUMENTAL METHOD
Spectrometry
PROBLEM SOLVING: — Method of analysis which deals with the measurement of the spectra.
What is the % Alkaloid content present from 50g Acacia leaves if the
residue obtained was 1.267g after the extraction? — A “spectra (spectrum)” is a continuum of color formed when a bean
of white light is dispersed (passage through a prism) so that its
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑹𝒆𝒔𝒊𝒅𝒖𝒆 component wavelengths are arranged in order based on the
% 𝑨𝒍𝒌𝒂𝒍𝒐𝒊𝒅𝒂𝒍 𝑬𝒙𝒕𝒓𝒂𝒄𝒕𝒊𝒗𝒆 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆 different refraction according to wavelength.

1.267 𝑔
% = 𝑥 100
50 𝑔

% 𝑨𝒍𝒌𝒂𝒍𝒐𝒊𝒅𝒂𝒍 𝑬𝒙𝒕𝒓𝒂𝒄𝒕𝒊𝒗𝒆 = 𝟐. 𝟓𝟑𝟒 %

Volumetric by Residual

(𝑽 𝒙 𝑵) − (𝑽 𝒙 𝑵) 𝒙 𝒎𝑬𝒒𝒘𝒕
% 𝑨𝒍𝒌𝒂𝒍𝒐𝒊𝒅 = 𝒙 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
Ø The commonly used colors in the analysis are blue, green, and
PROBLEM SOLVING:
red.\
Calculate the % Caffeine if 1.0885g sample of Coffee powder utilized
Ø In colorimeter, the old model, we change the filter to these 3
21.5mL of a 0.0264N H2SO4 and 18.7mL of 0.0249N NaOH. Each mL of 0.02N
commonly used colors.
H2SO4 is equivalent to 3.8858mg of Caffeine (C8H10N4O2).
SPECTROSCOPY
(𝑽 𝒙 𝑵𝑭) − (𝑽 𝒙 𝑵𝑭) 𝒙 𝑻𝒊𝒕𝒆𝒓
% 𝑪𝒂𝒇𝒇𝒆𝒊𝒏𝒆 = 𝒙 𝟏𝟎𝟎 — Study of radiated energy and matter to determine their interaction.
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝒂𝒎𝒑𝒍𝒆
— When we allow the light to pass through the sample, there’s only one kind
0.0264𝑁 0.0249𝑁
`21.5 𝑚𝐿 𝑥 0.02𝑁 a − `18.7 𝑚𝐿 𝑥 0.02𝑁 a 𝑥 3.8858 𝑚𝑔 of light or monochromatic radiation that must enter the solution so that
% = 𝑥 100 this will be detected by the detector and give the numerical data that
1088.5 𝑔
we want to know from the sample such as absorbance, transmittance to
% 𝑪𝒂𝒇𝒇𝒆𝒊𝒏𝒆 = 𝟏. 𝟖𝟐 % determine the concentration.

— e.g., In the Assay of Biogesic tablets containing Paracetamol, at what

particular wavelength are we going to dtermine the absorbance reading
of the sample?

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 DEFINITION OF TERMS SPECTROPHOTOMETRY
BASIC SPECTROPHOTOMETRY — Branch of spectrometry which embraces the measurement of the
absorption of radiant energy of definite and narrow wavelength
approximating monochromatic radiation (single wavelength).
— Monochromator is positioned between the light and the sample so that
only one light radiation will pass through the solution.
— If there’s no monochromator like prism and diffraction grating in front of
the light, the movement of the light is in different directions.

SPECTROPHOTOMETER
— Is an instrument used to measure the intensity of light at different
Ø The 3 readings in spectrophotometry are the concentration, wavelength.
absorbance and transmittance.
WAVELENGTH (Λ)
Electromagnetic Spectrum — Is the length of a complete wave or cycle.
— Termed used to define the complete system of energy propagated in
wave form. Energy of this nature is termed radiant energy.
— The movement of wavelength from radio waves to gamma waves are
getting smaller and thinner

Units of measurement of wavelength

1 µm = 10-4 cm
1 nm = 10-7cm
1Å = 10-8cm

Radiant energy waves that are of importance to spectrophotometry:

(a) Ultra Violet (UV) – 200 to 380 nm
(b) Visible (Vis) – 380 to 780 nm
COMPLETE ELECTROMAGNETIC SPECTRUM (c) Infrared (IR) – Near IR - 780 to 3,000 nm
Medium IR - 3.0 to 15 µm
Far IR - 15 to 300 µm

WAVE NUMBER (ν)

— Number of waves per centimeter and is equal to 1/wavelength(cm)
9 Unit of measurement = cm-1

FREQUENCY (v)
— Number of complete cycles which pass a given point per second (cps).
9 1 Hertz = 1 cps

CHROMOPHORE
— Defined as a functional group which absorbs radiant energy in the UV or
Visible region of the spectrum.
— Examples:
RADIANT ENERGY ü Ethylene
— Refers to the energy in the UV, Vis and IR regions of the electromagnetic ü Ketone
spectrum. ü Acetylene
ü Aldehyde
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 THEORIES OF RADIANT ENERGY (2) 2. IR Spectroscopy
1. Energy in the electromagnetic spectrum occurs in bundles called photons (Vibrational Energy Transitions)
and quanta. — Electromagnetic radiation is passed through a sample and is
• Equation which relates the energy of a photon to the frequency of absorbed by the bonds of the molecules in the sample
radiation causing them to stretch or bend.
E=hv — There is no transition from higher to lower energy level.
where: E = energy (ergs) — Electrons undergo vibration.
h = universal constant (Planck’s constant)
= 6.62 X 10-27 ergs
v = frequency (Hz) FUNDAMENTAL LAWS OF SPECTROPHOTOMETRY
BEER’S LAW
2. Energy in the electromagnetic spectrum is propagated in wave form. — States that the power of a transmitted radiant beam decreases
• Expressed mathematically as: exponentially as the concentration of the solution containing the
c=λ v absorbing chemical species increases arithmetically.
where: c = velocity of light (3 X 1010 cm/s)
λ = wavelength (cm)
v = frequency (Hz)

PROBLEM SOLVING:
Calculate the energy in ergs associated with the radiant energy at 200 nm. 𝒍𝒊𝒈𝒉𝒕 𝒊𝒏𝒄𝒊𝒅𝒆𝒏𝒄𝒆
𝑨 = 𝐥𝐨𝐠
𝒕𝒓𝒂𝒏𝒔𝒎𝒊𝒕𝒕𝒆𝒅 𝒍𝒊𝒈𝒉𝒕

BEER’S PLOT
— A plot the absorbance values against a series of known solute
concentrations.
— Should yield a straight line (linear)
— This plot is used for the determination of unknown solute concentration
ü In the y-axis, it is the absorbance and the x-axis, it is the
Ø h and c values are constant. concentration of the solution.

Ø The delta before E means that in UV Visible region, if we use to read

absorbance, concentration and transmittance, there’s an excitation of
electrons from lower to higher energy level.

Ø Electron in higher energy level is not stable, hence, there’s a need for
an immediate return for electrons to be in the lower energy level.

Ø In Delta E, we measure for the difference of the two energy levels.

RADIANT ENERGY TRANSITIONS

1. UV and Vis Spectroscopy LAMBERT’S OR BOUGUER’S LAW
(Electronic Energy Transitions) — States that the power of a transmitted radiant beam decreases
— Radiation is passed through a solution of a compound. The exponentially as the thickness of the solution containing the
electrons become excited and will occupy a higher absorbing chemical species increase arithmetically.
quantum state and in the process absorbs some of the
energy passing through the solution.

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 BEER-LAMBERT OR BEER-BOUGUER LAW I. UV SPECTROSCOPY
— Relates the power of the incident and transmitted radiant beams to — The measurement of UV absorption spectra is primarily used to detect
the thickness and concentration of the solution containing the the presence of conjugated hydrocarbons.
absorbing chemical species.
— The region we refer to as UV consists of the region from 1000 - 4000 or
MATHEMATICAL RELATIONSHIP OF THE COMBINED LAW IS: 100 - 400 nm.

— UV is subdivided into the near UV and the far UV (vacuum).

Far = 100 - 190 nm
Where: Near = 190 - 400 nm
A =Absorbance
a =Absorptivity COLORIMETRY
b =Pathlength — Spectrophotometric measurement in the visible region was referred to
c =Molarity(moles/L) colorimetry.
TRANSMITTANCE — A colorimeter is a device used to test the concentration of a solution by
— Ratio of the radiant power transmitted by the solution to the radiant measuring its absorbance of a specific wavelength of light.
power transmitted by the blank (solvent)
— The amount of light which passes through a solution

𝑷 (𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏)
%𝑻=
𝑷 (𝑩𝒍𝒂𝒏𝒌)

ABSORBANCE
— Also called optical density, absorbency or extinction coefficient

𝐥𝐨𝐠 𝟏
𝑨=
𝑻
— log [P(blk)/ P(solution)] — The device is most commonly used to determine the concentration of a
known solute in a given solution by the application of the Beer-Lambert
law, which states that the concentration of a solute is proportional to
CONCENTRATION the absorbance. Example: 0.4M Copper Sulfate
— Usually expressed in g/L, mg/mL , ug/mL, M

THREE METHODS OF COLORIMETRY CALCULATIONS

ABSORPTION SPECTRUM (BEER’S PLOT) I. Proportionality
— It is the graph obtained when absorbance is plotted against a II. Graphing
wavelength. III. Beer’s Law

DIFFERENT KINDS OF SPECTROPHOTOMETRIC TECHNIQUES I. Proportionality

A. Absorption Spectrophotometry — Focuses on the idea that the absorbance of a solution is directly
— It is the measurement of the interaction between electromagnetic proportional to its concentration.
radiation and the molecules of chemical substances.
— When using this approach it is necessary to be sure that the values
— Techniques employed are UV, Visible, IR and Atomic Absorption given are for different concentration of the same chemical
Spectrophotometry. measured under the same conditions (wavelength and path length).

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Formula: — Formula:
𝑪𝒖 𝑨𝒖 𝑨 = 𝒂𝒃𝒄 𝑨=
𝑨
= (𝒂)(𝒃)
𝑪𝒔 𝑨𝒔
Where:
Where:
A = stands for the absorbance which is measured with an
C = Concentration of the sample and reference standard
instrument
A = Absorbance
𝑨𝒖 a = molar absorptivity
𝑪𝒖 = 𝒙 𝑪𝒔 b = pathlength measured in centimeters
𝑨𝒔
— The symbol “A” stands for the absorbance which is measured with an
PROBLEM SOLVING:
instrument. The symbol “a” is a proportionality factor called the molar
A standard solution with a concentration of 0.14 M is measured to have an
absorptivity which is how much light will be absorbed by 1 cm of a 1
absorbance of 0.43. A sample solution of the same chemical is measured
M solution of this chemical. Its value depends on what the chemical is
under the same conditions and has an absorbance of 0.37. What is the
and also on what wavelength (or color) of light is being used.
concentration?
𝑨𝒖
𝑪𝒖 = 𝒙 𝑪𝒔 — The symbol “b” stands for the pathlength measured in centimeters.
𝑨𝒔

0.37 — The symbol “c” stands for concentration measured in molarity.

𝑪𝒖 = 𝒙 0.14
𝐴0.43
PROBLEM SOLVING:
= 𝟎. 𝟏𝟐𝟎𝟓 𝑴 The absorptivity of a particular chemical is 1.5 M/cm. What is the
concentration of a solution make form this chemical if a 2.0 cm sample has
an absorbance of 1.20?
𝑨
II. Graphing 𝑨=
— The graphing method is called for when several sets of data are (𝒂)(𝒃)
available for concentration and absorbance.
1.20
𝑨=
— Graphing the data allows you to check the assumption that Beer’s (1.5𝑀/𝑐𝑚)(2.0 𝑐𝑚)
Law is valid for a straight–line relationship for the data.
𝑨 = 𝟎. 𝟒𝟎 𝑴

II. UV/VIS SPECTROPHOTOMETER

— Can measure the absorbance, concentration and % Transmittance of
a sample.
— Sample container: Cuvette
— Fused silica cuvette: UV
— Glass/plastic cuvette: Vis

THE COMPONENTS INCLUDE

Light sources
- Quartz halogen/ Tungsten Lamp – Vis
- Deuterium Lamp – UV
III. Beer’s Law
— Is often written in the form of this equation: A = abc as a way of Monochromator
summarizing and quantifying the relationship between the - Used to dispersed light into the constituent wavelengths, which is
absorbance, the nature of the absorbing chemical, the pathlength further selected by a slit.
of the solution and the concentration of the solution.

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Optics APPLICATIONS OF UV/VIS SPECTROPHOTOMETRY
- It is designed to split the light beam so that the beam passes to 2 — Standard method for determining the physical-chemical properties of
sample compartments. drug molecules prior to formulation and for measuring the release from
formulations.
ü e.g. partition coefficient, solubility and release of a drug from
a formulation.

III. INFRARED (IR) SPECTROSCOPY

— Involves measuring how much infrared radiation is absorbed or

transmitted by a sample.
ü Identification of the functional group (Qualitative Analysis)
TYPES OF DETECTORS ü MEASUREMENT of the height of the result (Quantitative Analysis)
Detectors Wavelength Region
Photo tubes 150-1000 nm THREE REGIONS OF INFRARED
Photomultiplier tubes 150-1000 nm Ø Near Infrared
Silicon diodes 190-1100 nm Ø Middle infrared
Photodiode array 190-1100 nm - Most widely used region as it is involved with molecular vibrations
typically involved in organic molecules.
Ø Detectors are the most sensitive part of the instrument. Ø Far Infrared
Ø Need a detector that can read 200-780 nm.

WHAT IS THE IMPORTANCE OF STUDYING SPECTROMETRY?

The significance of spectrometric analysis includes?
1. It is used in the determination of the identity of a compound (Qualitative
Analysis)
ü Each functional group is capable of absorbing/transmitting light at a
specific wavelength, knowing the functional group present in a given
sample can help in identifying a certain compound (just like a
puzzle).

2. It is used in the determination of the quantity of the active ingredient

(Quantitative Analysis) ü The y-axis of the IR Spectrum is transmittance and the x-axis is the
ü A quantitative analysis can be done by using the spectrometric wave number.
equation that can help in determining the amount of an active
ingredient Infrared Wavelength Ranges (Old IR)
Ø In UV Vis, we’re after the quantity of the active ingredient. Near IR Middle IR Fair IR
Ø In IR, we’re after the quantity of the active compound. 780 – 3000 nm 3 – 15 µm 15 – 300 µm

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 NEW MODEL OF IR COMPONENT OF FOURIER TRANSFORMED INFRARED SPECTROPHOTOMETER
Ø Near IR (NIR) = 12,900 to 4,000 cm-1
• Interest in the NIR has grown in recent years, particularly in the food
and foodstuffs industries, where it is routinely used for quantitative
analysis.

Ø Mid IR (MIR) = 4,000 cm to 200 cm-1

• MIR is the most widely used region as it is involved with molecular Ø INFRARED SOURCES
vibrations typically involved in organic molecules. It provides a wealth NEW OLD
of structural information, as well as quantitative data. Nerst Glower
Various Ceramic (CLAY) Material
Silicon Carbide “Globar”
Ø Far IR (FIR) = 200 cm to 10 cm-1
• FIR is principally concerned with rotational spectra, and crystal lattice
vibrations. Ø MONOCHROMATORS
OLD NEW
Metal Halide Prism
Gratings
NaCl Prism

ü When we are using IR Spectroscopy, it gives us IR Spectrum. DIFFERENT TYPES OF MATERIALS THAT ARE USED IN PREPARING PELLETS ARE
ü IR Spectrum, come from molecular motion/ vibration of the electrons For Transmission Range
of the sample. KBr 4000 – 400 cm -1
ü Due to the molecular motion, it gives the functional groups of the KCl 4000 – 500 cm -1
given sample. NaCl 4000 – 600 cm -1
Csl 4000 – 200 cm -1
MOLECULAR MOTIONS
TWO (2) FUNDAMENTAL VIBRATIONS OF ATOMS EXISTING — KBr is the most commonly used matrix in pellet making.
1. Stretching (distance increases or decreases along the bond axis). — Spectroquality KBr is recommended.
ü Symmetrical
ü Antisymmetrical Ø DETECTORS
- Thermocouples
2. Bending also known as deformation. - GOLAY Detector
(change in the bond angles occur) - Bolometer
ü Scissoring EXAMPLE OF AN IR SPECTRUM
ü Rocking
ü Wagging
ü Twisting

PARTS OF IR SPECTROPHOTOMETER
FOURIER TRANSFORMED INFRARED SPECTROPHOTOMETER

Ø Move the mouse near the peak and if there’s a wave number, it means
that the peak is significant.
Ø If the peak is not significant, there’s no wave number that will appear.
Ø Y-axis à % Transmittance & X-axis à Wave Number.
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 GOOD QUALITY IR SPECTRUM • Water is NOT USED as solvent
1. Strongest Band: 3-15% T a. It strongly absorbs most of the IR radiation
2. Baseline: 80-95% T b. It would destroy the sodium chloride cells which hold the sample
3. Well defined band shapes
4. Little noise in the spectrum
5. Resolution should be adequate

FUNDAMENTAL VIBRATIONS IN IR SPECTROPHOTOMETRY

1. Skeletal vibrations (Fingerprint region)
— Mainly due to carbon chain
— Used to identify sample
— 1300 – 400 cm-1 SAMPLING TECHNIQUES USED IN IR SPECTROSCOPY
— 8– 15 um Solid Sampling Techniques
1. KBr Pellet Technique - (pressing a sample into a salt disk)
2. Characteristics Group Vibrations (Functional Group) 2. Nujol Mull Technique - (suspensions in mineral oil)
— Characteristics group frequencies use for identification of sample 3. Cast Film Technique - (evaporated films on salt plates)
— Functional group determination 4. Free Standing Films – (casting or pressing the film)
— 4000 to 1300 cm-1
— 3 – 8 um KBr Pellet Technique
GROUP FREQUENCY REGION — Most commonly used matrix in pellet making
— Improper grinding causes:
a. Sloping baselines
b. Christiansen effect- scattering of light

— After we dissolve the sample, e.g., alkaloid with a chloroform (organic

solvent), just drop it, let it dry and put in the sample compartment of the
IR.

Nujol Mull Technique

CRITERIA FOR A GOOD SOLVENT — Useful with samples that undergo ion exchange.
9 The sample must be soluble to a desired extent in the solvent. — Mulling agents include: Nujol, Fluorolube
9 The solvent must be chemically inert toward the sample.
9 Solvent transparency Liquid Sampling Technique
9 There are regions where the solvent transmits at least 25 % in the 1. Sealed Cells (used for volatile liquids)
incident radiation. 2. Demountable cells
3. Capillary Film - Suspending thin film of sample between two windows.
COMMON INFRARED SOLVENTS 4. Smear Technique - Sample is spread over the surface or an IR window.
a. Carbon disulfide
b. Carbon Tetrachloride Cast Film Technique
c. Tetrachloroenthylene — Used in demountable cells.
d. Chloroform
e. Dioxane LIQUID SAMPLING TECHNIQUE
f. Cyclohexane 1. Sealed Cells (used for volatile liquids)
g. Benzene 2. Demountable cells
3. Capillary Film - Suspending thin film of sample between two windows.
Usual concentration of samples in solution = 10% solution. 4. Smear Technique – Sample is spread over the surface or an IR
window.
Usual cell thickness = 0.05 mm

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 ADVANCE SAMPLING TECHNIQUE CALCULATIONS
1. Attenuated Total Reflectance (ATR) Convert Transmittance value 79.5 and 46.0% to Absorbance
— Uses a transmitting crystal 1 100
𝐴 = = log
— Analysis of bulk properties of solid samples and liquid samples 𝑇 %𝑇

2. Diffuse Reflectance FTIR (DRIFTS) 100

𝐴= = log 100 − log 79
— IR energy is directed towards a sampling cup containing powdered 79.5
or rough samples. = 2.000 – 1.9004
— Analysis for powder samples and matte surfaces. A1 = 0.0996

3. Specular Reflectance
— Allows thin coatings layers on reflective surfaces.
— Analysis for samples of lubricating or polymeric coatings.

APPLICATIONS OF IR SPECTROSCOPY
— Qualitative fingerprint check for the identity of raw material and for
identification of drugs.
PROBLEM SOLVING: (Absorbance vs. Transmittance)
1. Convert the following values of %T to absorbance values:
— Used for preliminary checking of the presence or absence of a carbonyl
A. 90% B.70% C. 95%
group which is difficult to check by other method.
A. A = log100 – log90
— For structure elucidation.
= 2 – 1.954242609
= 0.046
QUALITY ANALYSIS
• Identify the structure and function group
B. A = log100 – log70
present in the sample.
= 2 – 1.84509804
= 0.155
ü Each peak has a wave number.
ü To know the equivalent of the wave
C. A = log100 – log95
number, there’s a library of all
= 2 -1.977723605
organic compounds where we are
= 0.022
given to interpret the functional group present in the sample.
2. Convert the following values of Absorbance to %T:
QUANTITATVE ANALYSIS
A. 0.045 B. 0.530 C. 0.634
• Purity of the sample
A. %T = 2 – 0.045
ü Measure the peak to have the
= antilog 1.955
reading for Au and As.
= 90.16
ü Make a tangent line, then draw the
height (base and peak of the
B. %T = 2 – 0.530
spectrum), then subtract
= antilog 1.47
ü Don’t use Transmittance since it uses in the reading, must compare to
= 29.51
absorbance
ü Getting the measurement of A1 and A2 then proceed to the
C. %T = 2 – 0.634
computation of the absorbance reading of the sample and the
= antilog 1.366
reference standard.
= 23.23

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B. FLAME SPECTROSCROPY • e.g. Sodium – flame color is yellow
FLAME SPECTROSCOPY (Atomic Spectrophotometry) Potassium – violet
- Is used in the assay of metallic elements in the blood (Na+, K+, Li+, Calcium – brick red
Ca++) Strontium – crimson red
- Is used in clinical chemistry
— Atoms are thermally excited so that they emit light and the radiation
Types: emitted is measured.
1) Atomic Emission Spectrophotometry ü There’s an excitation of electrons in the outmost orbital.
2) Atomic Absorption Spectrophotometry ü E.g., Sodium has an atomic number of 11 and it is in 3S1, once it’s
heated and it’s in the outermost part of the electron from higher o
ATOMIC EMISSION SPECTROPHOTOMETRY lower energy level, the excited electrons of sodium will go to 3p, 4s.
• “flame photometry”, deals with the measurement of emitted light. however, it needs to go back to have measurement.
• The instrument has no hollow cathode lamp.
— The intensity of the emission energy is a function of the concentration of
ATOMIC ABSORPTION SPECTROPHOTOMETRY the elements being assayed.
• Measurement of the absorption of light by free metallic atoms. — The emission lines of each elements are unique.
• The instrument has hollow cathode lamp. — It corresponds to transitions from ground state to a higher energy state.
— Transitions are referred to as resonance lines.
FLAME SPECTROPHOTOMETER
- A spectrophotometer and a flame source are the essential COMPONENTS OF AN ATOMIC EMISSION SPECTROPHOTOMETER
instruments for analytical use. Flame
• The sample is volatilized in natural gas/compressed air flame or
sometimes using air/acetylene flame.

Monochromator/Filter
• Narrow band of emitted radiation is selected.

Detector
• The intensity of the selected radiation is then measured using a
photosensitive cell.

ATOMIC SPECTROPHOTOMETRY
APPLICATIONS OF AES IN PHARMACEUTICAL ANALYSIS
(Flame Spectroscopy)
— Quantification of alkali metals and its salts and some alkaline earth
Flame Spectroscopic Methods Include:
metals.
— Flame Emission Spectrophotometry
— Determination of metallic impurities in some of inorganic salts used in
— Atomic Absorption Spectrophotometry (AAS)
preparing solutions.
ATOMIC EMISSION SPECTROPHOTOMETRY (AES)
— It plays an important role in the control of sodium, potassium and
Atomic Absorption Spectroscopy
lithium in a number of raw materials and formulations.
— It involves the measurement of light absorbed by metal ions.
— The light source is the hollow cathode lamp and it is specific for each
Flame Emission Spectrophotometry
element and it contains an inert gas, Neon or Argon.
— Use in the assay of Lithium carbonate USP.
— Major advantage of AAS over flame emission is increase sensitivity.
— Use in the assay of other elements like sodium, potassium, calcium in
— Atoms of a metal are volatilized in a flame & their absorption of a
blood or other biological fluids.
narrow band of radiation produced by a hollow cathode lamp,
— Identification is used by the color of the flame that may produced for
a specific element because of their atoms. coated with the particular metal being determined, is measured.

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 ATOMIC ABSORPTION MAXIMUM FLAME TEMPERATURE WITH VARIOUS MIXTURE OF FUELS
FLAME TEMPERATURE
FUEL
In Air In Oxygen
Illuming Gas 1700 2700
Propane 1925 2800
Butane 1900 2900
Hydrogen 2100 2780
Acetylene 2200 3050
Cyanogen 2330 4550
> The sample has no container so its holder is a tray.
Ø Acetylene is used of the time.
COMPONENTS OF AN ATOMIC ABSORPTION SPECTROPHOTOMETER
SAMPLE: ONLINE ACTIVY

Light source
• Hollow cathode lamp coated with the element being analyzed.

Flame
• The flame is usually air/acetylene or nitrous oxide/ acetylene which
are required to volatilized salts of elements.

Monochromator/Filter
• It is used to narrow band the width of the band of radiation being
examined.

Detector
• The intensity of the selected radiation is then measured using a
photosensitive cell.

FLAME & FLAME TEMPERATURE

A FLAME IS USED:
1. For transforming the sample to be analyzed from the liquid or solid
state into the gaseous state.

ü From all the collected data, make a Beer’s Plot or Absorbance

2. For decomposing the molecular compounds of the investigated Spectrum.
element into simpler molecules or atoms. ü From where it falls, that’s the concentration of the unknown sample
which is the Nickel.
3. For exciting the latter particles to light emission.
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 APPLICATIONS OF AAS IN PHARMACEUTICAL ANALYSIS NEPHELOMETRY
— Determination of metal residues remaining from the manufacturing - Is based on the measurement of the brightness of the light reflected
process in drugs. by a cloud of finely divided particles suspended in a liquid.
— Limitations: o Measurement of the amount of Light Scattered in a
- Only applicable to metallic elements. Suspension.
- Each element requires a different hollow cathode lamp.
o Due to having specific hollow cathode lamp for each
element, AAs becomes expensive to use.

C. MOLECULAR EMISSION SPECTROSCOPY

Fluorescence Spectrophotometry
- It is the measurement of the emission of light from the chemical
substance while it is being exposed to UV, Visible and other
electromagnetic radiation usually 20 to 30 nm. NEPHELOMETER
ü Used in the clinical laboratory to quantitate the rate of insoluble
Fluorometry antigen-antibody complex formation during the assay of specific
— When certain chemical substances are excited electronically by the serum proteins.
absorption of UV or visible radiation, they emit light at a longer
wavelength called luminescence (radiative process). APPLICATIONS OF NEPHELOMETRY
— Non–radiative process: collision deactivation. — Can be used in chemical analysis. e.g. Laboratory Test
— Use in the analysis of vitamins (Thiamine and Riboflavin), determination
of ethinylestradiol tablets. Determination of Rheumatoid Factor
- Indicates rheumatoid arthritis and/or systemic lupus erythematosus
Raman Spectroscopy (Inelastic light scattering)
- It is a light scattering process in which the specimen under
examination is irradiated with intense monochromatic light (usually a TURBIDIMETRY
laser light) and the light scattered from the specimen is analysed for - In which the transmitted light is measured after radiant energy passes
frequency shifts. through a turbid solution or suspension.

TURBIDIMETER
APPLICATION OF RAMAN SPECTROSCOPY
— For identification of complex samples.
— Provides additional fingerprint identity information complementary to
mid-IR spectroscopy.

ü Filter acts like the monochromator

D. LGHT SCATTERING SPECTROSCOPY
LIGHT SCATTERING APPLICATIONS OF TURBIDIMETRY
— It is the measurement of light scattered because of the substance — Use in the analysis of antibiotics, Calcium pantothenate and Vitamin B12
microscopic optical density in homogeneities of solutions. — Term used by the USP to designate solution of turbidity is turbidance.
Two techniques are used:
A. Nephelometry TURBIDANCE
B. Turbidimetry - As a measure of the light scattering effect of suspended particles.
- The term used by the USP to designate solution turbidity.
— Light scattering phenomenon o May be determined by the turbidimetric or nephelometric
o Tyndall effect method.

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 PROBLEM SOLVING:
1. Using the spectrophotometer to measure the concentration of a sample, — The unit of mass is Dalton (Da)
the following data were obtained:
— Dalton (Da) is defined as 1Da = (1/12) of the mass of a single atom of
As = 0.26 the isotope carbon-12 (12C).
Au = 0.38
Cs = 15 mg/ml — Mass spectrometers can be divided into three fundamental parts,
namely the ionization source, the analyzer , and the detector.
Calculate the concentration of the sample
𝑨𝒖 — Determines the molecular weight of an organic compound.
𝑪𝒖 = 𝒙 𝑪𝒔
𝑨𝒔
— Analytical spectroscopic tool primarily concerned with the separation
0.38 of molecular species according to their mass.
𝐶𝑢 = 𝑥 15
0.26
— Mass spectrometer
= 𝟐𝟏. 𝟗𝟐 𝒎𝒈/𝒎𝒍 • An instrument that measures the masses of individual molecules
that have been converted in ions (molecules that have been
electrically charge).
MATCHING TYPE
A. Measurement of the amount of PRINCIPLE
C 1. SPECTROPHOTOMETRY
light scattered by a suspension. — Charge molecules are generated in a high vacuum region using a
B. Measurement of the amount of variety of methods for ion production.
D 2. COLORIMETRY
light absorbed by a suspension.
C. Measurement of the amount of — The ions are generated in their gas phase so that they can then be
A 3. NEPHELOMETRY
light absorbed by a solution. manipulated by the application of electric or magnetic fields to enable
D. Measurement of the light in the the determination of their molecular weight.
E 4. FLAME PHOTOMETRY
visible region of the spectrum.
E. Measurement of the intensity of
B 5. TURBIDIMETRY light emitted by an element when
subjected to high temperature.

E. MASS SPECTROSCOPY
— It is an analytical technique used to measure the mass-to-charge ratio
of ions.

— An analytical technique for the determination of the elemental APPLICATION OF MASS SPECTROSCOPY
composition of a sample or a molecule. — Provides a highly specific method for determining or confirming the
identity or structure of drugs and raw materials.
— It is most generally used to find the composition of a physical sample by
generating a mass spectrum representing the masses of sample — Usually in conjunction with either gas chromatography (GC-MS) or
components. liquid chromatography (LC-MS) provides a method for characterizing
impurities in drugs and formulations.
— It is used for the elucidation of the chemical structures of molecules
(peptides) or other chemical compounds.

— Mass spectrometry is an analytical tool used for measuring the

molecular mass of a sample.

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F. NUCLEAR MAGNETIC RESONANCE (NMR) Ø The sample must be pure and free from particulate matter and
— Most powerful tool used for elucidating the structure of organic certainly any magnetic contaminates. Most samples are
molecules. analyzed as solutions in the 2 to 15% wt/wt concentration range.

— Determination of structural formula of an organic compound. Ø The best solvent for PMR do not contain Hydrogen or free of
protons (CCl4, CS2, CDCl3).
— Diagnosis of diseases.
- Magnetic Resonance Imaging (MRI) TMS – (Tetramethyl Silane, (CH3)4Si) the most common reference material in
PMR.
— Transition between energy levels can be generated by radiant energy 1. All 12 protons in this compound are identical and have a very high
only if the molecules are placed in magnetic field. degree of shielding from the electropositive central silicon atom.

— The radio frequency radiation ranges from 0.1 – 100 MHz wavelength. 2. It exhibits a single sharp resonance line at a high magnetic applied field,
well beyond where most organic protons will resonate.
— Unlike UV, visible and/or infrared spectroscopy NMR does not involve
electrons or bonds but the nuclei of compound. 3. It is chemically inert and is soluble in most nonpolar solvent.

— Only nuclei with an odd sum of protons and neutrons have magnetic 4. It is fairly volatile (bp = 26.5 0C) and is not soluble in water.
moments: 1H and 13C are the most often studied.

— If you put 1H in magnetic field, the separation is not equal. Hence, if it is

exposed in a magnetic field, there’ll be some that will go to the low
energy level and some that will go to the high energy level. However,
there’ll always be an excess of protons in the low energy level.

— There’s always an excess in the low energy level so that when the radio
frequency pass through, this excess will be lifted up by the radio
frequency and when it fall then that’s where the measurement of NMR
spectrum will take place.

— Unlike if you put 2H in magnetic field, the separation is totally equal that
there’s no occurrence for the rising of radio frequency if it enters the
sample.

— If there’s no rise and fall of protons and neutrons, then there’ll be no

NMR spectrum.

— Same goes to 12C and 13C.

*Assigned a chemical shift of zero.

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 PHYSICAL PROPERTIES OF NUCLEI PROCESS BASIC INSTRUMENTATION OF NMR
a. Spin 1. Magnet
b. Nuclear magnetic moment 2. Stable radiofrequency transmitter
3. Radio receiver and detector
• Since all nuclei possess charge, a spinning nucleus such as the proton 4. A coil of wire surrounding the sample (sensor)
generates a magnetic field. The strength of the magnetic field is 5. Cell containing the sample
measured in terms of magnetic moment (µ). 6. Method of sweeping through the spectrum
7. Recorder to display absorption peaks
2 ORIENTATION OF PROTONS IN A MAGNETIC FIELD

ü If there’s no magnetic field, then there’s no orientation that’ll happen to

the sample; and if they do not have spin & nuclear magnetic moment,
there’ll be no protons that’ll go up to the higher energy level. ü There should be a magnet that surrounds the sample compartment
because without it, there’s no separation of the protons.
ü The picture on the left means there’s no magnetic field exposure of the
protons.

ü The picture on the right has protons moving up and there are also protons
moving down when we exposed it to the magnetic field.

SATURATION
• Is observed when both energy levels become equally populated with
a slight excess of protons in the lower energy level.

RELAXATION
— Refers to any process that removes nuclei from an excited state.
— Two (2) Basic relaxation mechanisms: CHEMICAL SHIFT
1. Spin-spin Relaxation (Transverse) – involves exchange of energy
— The applied magnetic – field strength at which a given nucleus in a
between two proximal processing nuclei.
molecule absorbs energy relative to an arbitrary reference.
— Values are usually expressed as “delta” values in parts per million
2. Spin-Lattice Relaxation (Longitudinal) - transfer of energy to lattice
(ppm).
components as nuclei returns from higher to lower spin state.

PROTON-NMR

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 SPIN-SPIN COUPLING CHROMATOGRAPHY
• Formation of multiplets in the delta scale (NMR spectrum). — Greek words: chroma – color
• Measure, use to determine the percent purity of the sample. graphein – to write

— Mikhail Semyonovich Tsvet “Tswett” – Russian botanist separate plant

pigments by passing solutions through glass columns packed with
calcium carbonate.

— It is a collective term for a family of laboratory techniques for the

separation of mixtures.

— It involves passing a mixture dissolved in a “mobile phase” through a

“stationary phase” which separates the analyte to be measured from
other molecules in the mixture and allows to be isolated.
THE NMR SPECTRUM PROVIDES 4 DIFFERENT KINDS OF INFORMATION
1. The number of signal grouping in a spectrum provides information — It may be preparative or analytical.
about the no. of different proton environments in a compound.
— According to USP, it is defined as a procedure by which solutes are
2. The chemical shift indicates the electron density around the various separated by a differential migration process in a system consisting of
hydrogen nuclei and therefore helps to identify the proton type. two phases, one of which moves continuously in a given direction & in
which the individual substances exhibit different mobilities by reason of
3. The integrator allows the relative no. of equivalent protons of each type differences in adsorption, partition, solubility, vapor pressure, molecular
to be determined. size or ionic charge density.

4. The splitting pattern for a given signal indicates the no. of protons on
the adjacent carbons. COLUMN CHROMATOGRAPHY
— Use for separation, identification and determination of the chemical
TYPE OF PROTON (ppm) APPROXIMATE CHEMICAL SHIFT components in complex mixture.
Amino (RNH2) 1-5
Carboxylic (RCOOH) 10.5-12 Two Phases In Chromatography
Phenolic (ArOH) 4-11.5 — Stationary Phase
Aldehyde (RCHO) 9-10 • Fixed phase (may be porous or finely divided solid or a liquid
that has been coated in a thin layer on an inert supporting
Aromatic (ArH) 6-8.5
material.
Hydroxylic (ROH) 3.5-4
Primary Proton (RCH3) 0.9
— Mobile Phase
Secondary (RCH2) 1.3
• Pure liquid or gas or mixture of solutions that moves through or
Tertiary Proton (R3CH) 1.5 over the fixed phase.

MAGNETIC RESONANCE IMAGING (MRI)

- Is a useful non-invasive and non-destructive diagnostic tool for CLASSIFICATION OF CHROMATOGRAPHIC METHODS
imaging soft tissues such as the brain, heart and muscles and for 1. Adsorption
discovering tumors in many organs. 2. Partition
3. Ion exchange
- Together, NMR and MRI revolutionized the practice of chemistry and 4. Size exclusion
medicine by providing fast, non- destructive and non-invasive means 5. Electrophoresis
for the observation of matter from the atomic to the macroscopic 6. Affinity
scale.
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1. Adsorption Chromatography - Polymeric substance containing numerous pores of molecular
— Stationary phase dimensions.
- Solid on which a sample components are adsorbed
— Mobile phase
— Mobile phase - Containing analytes are solvated molecules are separated
- May be a liquid (liquid-solid) or a gas (gas-solid) according to their size by their ability to penetrate a sieve like
Example: structure.
- Column Chromatography (CC) — e.g.
- Thin Layer Chromatography (TLC) - Soft gels – dextran (Sephadex), agarose (Sepharose) or
polyacrilamide (Bio-gel)-gel filtration.
2. Partition Chromatography - Semi-rigid or rigid gels – polystyrene, glass beads or alkylated
— Stationary phase dextran –gel permeation.
- Liquid supported on inert solid.
— Mobile phase
— Mobile phase - Liquid or gas.
- Is a liquid (liquid-liquid partition) or a gas (gas-liquid).
Example: — Use for the separation of solutes with different molecular size.
Paper Chromatography — Use extensively for separation of macromolecules of biological origin
- Type of partition chromatography in which the stationary and for purification of synthetic –organic polymers.
phase is a layer of water adsorbed on a sheet of paper.

Liquid-Liquid Partition LIQUID CHROMATOGRAPHIC MODES (SUMMARY)

— Normal-Phase Chromatography Ø NORMAL-PHASE or LIQUID-SOLID (NP, LSC)
- Polar stationary phase (e.g. Water or methanol) ü Separation based on adsorption/desorption of the analyte onto a
- Non-polar mobile phase (e.g. hexane) polar surface (silica).
*This favors retention of polar compounds and elution of non-polar
compounds Ø REVERSED-PHASE (RPC)
ü Separation based on analytes’ partition coefficients between the
— Reversed-Phase Chromatography mobile phase and the bonded stationary phase.
- Non-polar stationary phase
- Polar mobile phase Ø ION-EXCHANGE (IEC)
ü Separation based ion ion-exchanging with the counter-ions and ionic
3. Ion-Exchange Chromatography interaction with the bonded ionic group.
— Stationary Phase
- Ion exchange resin -polymeric matrix with the surface of which Ø SIZE-EXCLUSION (SEC or GPC)
ionic functional groups (e.g. carboxylic acids and amines) ü Separation based in analytes’ molecular size and sieving action of
have been chemically bonded. the column.

— Mobile Phase
- Liquid 4. Affinity Chromatography
— Utilizes high specific interactions between one kind of solute molecule
— Method of choice for inorganic ions and attempts of reversed phase and a second molecule covalently attached (immobilized) to the
method unsuccessful. stationary phase.

4. Size Exclusion Chromatography (SEC) — Immobilized molecule can be an antibody to a particular protein
— Also called Gel filtration or Molecular–sieve chromatography. o Used in protein determination.
— Stationary phase

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5. Electrophoresis TWO TECHNIQUES PERFORMED IN ELUTION ANALYSIS
• It is defined as the migration of electrically charged molecules 1. If the composition of the solvent is not change during the course of
suspended and dissolved in an electrolyte under the influence of an development (a single solvent), the technique is termed isocratic
external electrical field (electric current). elution analysis.

2. Is the composition of the solvent uses different mobile phase which is

• It was introduced by Tiselius for the purification of proteins. It has been
capable of overcoming long elution times is termed gradient elution
used widely, especially for the separation of complex mixtures of
analysis.
biological substances such as proteins, nucleic acids and
ü The maximum number of mobile phases to be used is 4.
polysaccharides.
THEORY OF CHROMATOGRAPHY
— Plate Theory
- The chromatographic system is a series of discrete layers of
theoretical plates. (Martin and Synge).

— Rate Theory
- The dynamics of the solute particle passes through the void
spaces between the stationary phase particles in a system as well
as its kinetics. (Giddings).

A CHROMATOGRAM MAY BE EVALUATED QUALITATIVELY

— By determining the Rf value or Retardation Factor or Ratio Front or
Retention Factor.

— Rf is a measure of the fraction of its total elution time that any

compound spends in the mobile phase.

— Use in complete chromatography like paper and thin layer

chromatography (mobile phase allowed to develop in a predetermine
DIFFERENT TECHNIQUES IN CHROMATOGRAPHY
endpoint and then stopped).
— Paper Chromatography
— Thin Layer Chromatography (TLC)
IN CONTINUOUS CHROMATOGRAPHY
— Column Chromatography (CC)
— Mobile phase is allowed to continue indefinitely until the solutes elute
— Gas Chromatography (GC)
from the end of the stationary phase.
— High Performance Liquid Chromatography
— The capacity factor is being measured.
I. Column Chromatography
— Retention time tR – the time that elapses from the start of the
chromatogram to the elution maximum of the solute.

ANOTHER PARAMETER USED TO DESCRIBE THE RETARDATION OF A SOLUTE

CAPACITY FACTOR (k’)
- k’ is measure of peak retention or how many times the peak is
retained vs an unretained peak (to)
- k’=(tR –tO )/tR‘/tO
- Relationship to the void and the first peak
o The capacity factor is being measured.

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 II. Paper Chromatography
— Principle: differences of partition coefficients of substances between
two immiscible liquids.
ü The paper becomes liquid due to the water content. However, there
are paper who doesn’t have any water content which makes that a
solid type of chromatography or adsorption.

ü A type of planar chromatography as well as of that thin layer

RETENTION VOLUME (VR)
chromatography.
- tR = retention time
- tO = retention time of an unretained solute
— Solid adsorbent: filter paper (cellulose); (whatman, schleicher, schwell)
- Wb = Based width of peak (4 𝜎 width)
— Chromatogram is determined by the Rf value, use for identification.

𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒔𝒐𝒍𝒖𝒕𝒆 𝒎𝒐𝒗𝒆𝒔

𝑹𝒇 =
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒔𝒐𝒍𝒗𝒆𝒏𝒕 𝒎𝒐𝒗𝒆𝒔

• Use in the identification of Digoxin, USP.

ü Use in the separation of Trisulfapyrimidine drugs.

Ø Retention Time – a time to take for the solute to elute. Three Methods or procedures for the preparation of
Ø Retention time tR – the time that elapses from the start of the Paper Partition Chromatogram
chromatogram to the elution maximum of the solute. 1. Descending chromatography
- Which is accomplished by allowing the mobile phase to flow
RETENTION VOLUME (VR) downward on the paper slip.
- It is equal to the volume of the mobile phase required to elute a
compound from the system. 2. Ascending chromatography
o It is equal to the product of the retention time and the - In which the mobile phase is allowed to rise upward on the paper
flow rate of the mobile phase. by capillary attraction.

ADSORBENTS USED IN CC: weakest (1)→strongest(11) 3. Radial chromatography

1. Sucrose - In which the mobile phase moves out in concentric circles from
2. Starch the center of a circular piece of paper.
3. Insulin
4. Talc
5. Calcium Carbonate III. Thin Layer Chromatography
6. Calcium Phosphate — Involves the spotting of a sample of a mixture of components at one
7. Magnesia end of an adsorbent-coated glass plate followed by passage of a
8. Silica Gel solvent through the adsorbent separating the components of a sample.
9. Magnesium silicate ü Allowing the molecules to separate just like what you did in the
10. Alumina dye mixture
11. Charcoal
— Common adsorbents used:
ü To have a very good stationary phase depends on the material ü Silica gel G (SiO2 x H2O)
that you’ll use. ü Alumina (Al2O3 x H2O)

ü Silica gel is the most used. — Usual dimension of the plate: 20 X 20 cm

— Layer thickness: 250 µm
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 TLC DETECTION COMMONLY USED DERIVATIZING AGENTS
— Chromatogram is examined under UV radiation Compound Class Reagent Color Produced
— termed Fluorescence quenching General Iodine vapor Brown
- UV short wave - 254 nm General Sulfuric acid (50%) Black
- UV long wave - 360 nm Acids Bromocresol green Yellow
Aldehydes and
STATIONARY PHASE Dinitrophenylhydrazine Yellow-red
ketones
> Silica gel Amines and Amino
9 Most frequently used stationary phase in adsorption TLC Ninhydrin Fluorescent
acids
9 If it is adsorption, there’s no water incorporated to the silica gel. Alkaloids Mercuric nitrate Yellow to brown
9 Partition chromatography is used in the silica gel of the stationary Barbiturates Diphenylcarbazone Purple
phase if it has a water content. Carbohydrates Aniline phthalate Gray-black
Lipids Bromthymol blue Light-green
REVERSE PHASE SEPARATION:
Steroids Antimony trichloride Various
• Silica gel coated with silicone oil
• ODS (octadecylsilyl) PLANAR CHROMATOGRAPHY
9 Surface of silica gel is acidic due to the presence of silanol • A separation technique in which the stationary phase is present as or
hydroxyl group. on a plane.
9 Use in the analysis of acidic compounds and polar compounds
such as amino acids and sugars. Ø PAPER CHROMATOGRAPY
• A method invented by the British biochemists, Archer John Porter
> Alumina (Aluminum oxide) Martin and Richard Laurence Millington Synge.
9 Has a basic surface.
9 Use for the separation of basic and weakly polar compounds. • An analytical technique for separating and identifying mixtures that
or can be colored, especially pigments.
> Polyamide (nylon)
9 A long chain polymer. Ø THIN LAYER CHROMATOGRAPHY
9 Has many free amide and carboxyl groups on its surface. • A widely employed technique similar to paper chromatography.
9 An adsorbent with strong hydrogen bonding capabilities.
9 Readily bonds phenol, carboxylic acids, quinones and nitro • It involves spotting of sample or a mixture of samples at one end of
compounds of which requires polar solvents like methanol and an adsorbent- coated glass plate followed by passage of a solvent
dimethylformamide to displace them. through the adsorbent for the purpose of separating the components
of a mixture.
> Cellulose-polysaccharide
9 Contains numerous neutral hydroxyl groups and can adsorb PROBLEM SOLVING:
water or polar solvents by hydrogen bonding (useful in partition
Using TLC, calculate the Rf values of the three samples found in the
TLC).
chromatogram. Given the following data:
Ø Less active and less frequently used adsorbents: Distance travelled by the solvent = 50 mm
ü Calcium phosphate
Distance travelled by the 1st spot = 42 mm
ü Calcium carbonate
ü Diatomaceous earth Distance travelled by the 2nd spot = 38 mm
Distance travelled by the 3rd spot = 25 mm
> To ensure the stationary adheres firmly on the backing plate and does
not flake off during development, binders are added to the adsorbent. 𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒔𝒐𝒍𝒖𝒕𝒆 𝒎𝒐𝒗𝒆𝒔
𝑹𝒇 =
— Calcium sulfate (gypsum) 𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒔𝒐𝒍𝒗𝒆𝒏𝒕 𝒎𝒐𝒗𝒆𝒔
— Starch
— Carboxymethylcellulose 1st spot Rf = 0.84 // 2nd spot Rf = 0.76 // 3rd spot Rf = 0.50

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 IV. Gas Chromatography Coefficient B
• Also known as “gas-liquid chromatography” or “gas-liquid partition - Coefficient of longitudinal diffusion – Van Deemter equation – the
chromatography”. concentration of solute is lower at the edges of the band than in the
center and continuously diffusing through the mobile phase.
• A separation technique in which the mobile phase is a gas.

• Specifically gas-liquid chromatography Coefficient C

9 Involves a sample being vaporized and injected onto the head - Coefficient of mass transfer – is concerned with the transfer of the
of the chromatographic column. The sample is transported solute between the two phases.
through the column by the flow of inert, gaseous mobile phase.

9 The column itself contains a liquid stationary phase which is INSTRUMENTAL COMPONENTS OF GAS CHROMATOGRAPHY
adsorbed onto the surface of an inert solid. BASIC INSTRUMENTATION OF GC:

9 Results are shown on a chart paper as the peak position on the

chromatogram measured in terms of the retention column.

ADVANTAGES OF GAS CHROMATOGRAPHY

ü Fast analysis
ü Efficient, providing high resolution
ü Sensitive
ü Non-destructive
ü Highly accurate quantitative analysis
CARRIER GAS
ü Requires small samples
ü Reliable, relatively simple & inexpensive • The carrier gas must be chemically inert.
• Commonly used gases include nitrogen, helium, argon, and carbon
dioxide.
DISADVANTAGES OF GAS CHROMATOGRAPHY • The choice of carrier gas is often dependant upon the type of detector
ü It is limited to volatile samples. which is used.
ü Not suitable for thermally labile samples.
ü Fairly difficult for large, preparative samples.
• The carrier gas system also contains a molecular sieve to remove water
and other impurities.
ü Requires elaborate instrument such as mass spectroscopy, for
confirmation of peak identity.
SAMPLE INJECTION PORT
• The most common injection method is where a microsyringe is used to
APPLICATIONS OF GAS CHROMATOGRAPHY inject sample through a rubber septum into a flash vaporizer port at the
9 Limit tests for solvent residues and other volatile impurities in drug head of the column.
substances. • The temperature of the sample port is usually about 50°C higher than
the boiling point of the least volatile component of the sample. For
9 Characterization of volatile oils. packed columns, sample size ranges from tenths of a microliter up to 20
microliters.
THEORY OF GAS CHROMATOGRAPHY
Coefficient A COLUMN
- Eddy diffusion or multiple-path coefficient – is concerned with the • There are two general types of column, packed and capillary (also
different paths travelled by the molecules of a particular solute during known as open tubular).
their passage through the column.

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 • Packed columns are usually made of glass contain a finely divided, LIQUID CHROMATOGRAPHY
inert, solid support material (commonly based on diatomaceous earth) 9 Is a separation technique in which the mobile phase is a liquid.
coated with liquid stationary phase.
9 Can be carried out either in a column or a plane.

9 Present day LC that generally utilizes very small packing particles and
a relatively high pressure is referred to as high performance liquid
chromatography (HPLC).

• Capillary columns are made from fused silica, usually coated on the
outside with polyamide to give the column flexibility.

V. High Pressure Liquid Chromatography (HPLC)

• Most advance chromatographic method.
DETECTOR • Most commonly used analytical method in drug testing.
FUNCTIONS OF DETECTOR
ü Measures the concentration of the sample injected in the column. • Results are obtained in the recorder chart showing the peak in the
ü It generates a signal proportional to the sample concentration. chromatogram.

METHOD OF CHOID FOR

• Non-Volatile Substance
• Substance with high polarity or ionic samples
• Substance with high molecular weight
• Thermally instable and decomposable substance

BASIC INSTRUMENTATION: HPLC

• The most common are the:
9 FLAME IONIZATION DETECTOR (FID),
9 ELECTRON CAPTURE DETECTOR (ECD)
9 THE THERMAL CONDUCTIVITY DETECTOR (TCD)

• TCDs are essentially universal and can be used to detect any

component other than the carrier gas.

• FIDs are sensitive primarily to hydrocarbons, and are more sensitive to

than TCD.

RECORDER
• To graphically re-produce the output of the detector and record
result called Chromatogram.

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 COMPONENT PARTS OF HPLC

DETECTORS FOR HPLC

HPLC Solvent
ü Are glass or stainless-steel containers capable of holding up to a liter
of mobile phase which may consists of pure organic solvents or
aqueous solutions of salts or buffers.

HPLC Pump
ü Pumps are needed to force the mobile phase through the column
ü 2 Types of Pumps: Ø Data Acquisition
1. Mechanical – which deliver at a constant flow rate 9 Integrator – the main function is to graphically reproduce the output
2. Pneumatic – which produce a constant pressure of the detector and record the result called chromatogram.
9 CHROMATOGRAM
ü It is the output signal from the detector of the instrument
Injector
ü The sample solution is injected through a self- sealing rubber or Teflon
disc using a microliter syringe.

HPLC Column
ü Guard Column – this contains solid support coated with a higher % of
liquid phase than the analytical column in order to saturate the
mobile phase and retard dissolution.

ü Analytical Column – in which the actual separation take place, is a HPLC CHROMATOGRAM
stainless-steel tube, usually 25 cm in length with an internal diameter
of 2 to 4.6 mm

Detector
ü Measure the concentration of the sample injected
ü on the column.
ü It generates a signal proportional to the sample concentration.

o Run first the blank then the reference std., and then the sample.

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 COMMONLY USED HPLC STATIONARY PHASES 2. Zone Electrophoresis (moving boundary)
• ODS silica gel (octadecylsilane) - The sample is introduced as a narrow zone or spot in a column
slab or film of buffer.
• Octyl silane and Butyl silane silica gels
• Silica gel e.g. Gel electrophoresis
ü The gel (agar, starch or polyacrylamide) is used as a stabilizing
APPLICATIONS OF HPLC medium.
• HPLC with UV/Vis detector provides accurate and precise method for
quantitative analysis of pharmaceutical products. Polarimetry (Rotatory Power)
• Used for monitoring the stability of pure drug substance in formulations • Deals with the study of rotary power of substances.
• Rotary Power – also known as “optical activity”.
FORMULA:
𝑨𝒓𝒆𝒂 𝑷𝒆𝒂𝒌 𝒔 𝑪𝒔
• It is the measurement of optical rotation or optical activity of a
𝒙 substance.
𝑨𝒓𝒆𝒂 𝑷𝒆𝒂𝒌 𝒖 𝑪𝒖
• Substances that may show optical rotatory power are CHIRAL.
𝑪𝒖 =
𝑨𝒓𝒆𝒂 𝑷𝒆𝒂𝒌 𝒔
𝒙 𝑪𝒔
- When the direction of the rotation is toward the right [+];
𝑨𝒓𝒆𝒂 𝑷𝒆𝒂𝒌 𝒖 Clockwise rotation (+ isomer)> Dextrorotatory

Where: - When the direction of the rotation is toward the left [-]; Counter
C = concentration of the sample and standard clockwise rotation (- isomer)> Levorotatory
Cm = height of chromatograph of the sample standard

PROBLEM SOLVING: • Importance; establish identity of substances, Establish purity of samples,

Indication of its therapeutic value.
Given the date, calculate the concentration of the unknown sample:
Data:
INSTRUMENTS: TWO TYPES OF POLARIMETER
Area Peaks = 15472118
Area Peaku = 11158642 1. Polariscope or Polarimeter – used for liquid substances
Cs = 6.5 mg/mL 2. Polarizing Microscope – used for solid substances
𝟏𝟏𝟏𝟓𝟖𝟔𝟒𝟐
𝑪𝒖 = 𝒙 𝟔. 𝟓 9 It is the technique of measuring the “polarization of light.”
𝟏𝟓𝟒𝟕𝟐𝟏𝟏𝟖
9 It is the measurement of optical rotation or optical activity of a
= 4.688 mg/ml or 4.69 mg/ml substance.

Polarimetry
VI. ELECTROPHORESIS 9 Measures the extent to which a substance interacts with plane
polarized light (light which consists of waves that vibrate only in one
plane).

9 Substances that may sow optical rotary power are CHIRAL.

9 If the substance rotates plane polarized light to the left or to the right,
it is called optically active.
2 ELECTROMETRIC METHODS
1. Free Solution 9 To be optically active, a compound must have a chiral center.
- A buffered solution of proteins in a U-shaped cell is subjected in
electric current which causes the proteins to form a series of 9 A chiral center is a carbon that has 4 different groups attached to it.
layer in order of decreasing mobility.

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 ENANTIOMERS AND CHIRALITY ANGLE OF ROTATION
Chiral center • The concentration of the solution
- Asymmetric carbon -4 different groups are attached to it. • The pathlength of the solution through which the light travels
- You must have at least one asymmetric carbon to have enantiomers. • The wavelength of light
- The terms Chiral carbon, asymmetric carbon and carbon • The chemical configuration of the substance
stereocenter are essentially synonymous.
FACTORS AFFECTING ANGLE OF ROTATION
ü The concentration of the optically active substance
ü Solvent
ü Temperature (25oC)
ü Wavelength of the polarized light
EXAMPLES OF ORGANIC SUBSTANCES THAT ARE OPTICALLY ACTIVE
ü Nature of the substance
— Volatile oils
ü Length of sample tube
— Alkaloids
— Sugars
SPECIFIC ROTATION OF A LIQUID
9 e.g. Specific rotation of official sucrose NLT +65.9°
• Is defined as the angular rotation in degrees through which the plane
APPLICATION OF POLARIMETRY polarization of polarized monochromatic light is rotated by passage
• Establish both its identity and purity through 1 dm (100mm) of the liquid, calculated on the basis of a
• Indication of its therapeutic value specific gravity of 1.

POLARIMETER • Temperature specified in official standards for the measurement of

optical activity is 25 ºC.

REFRACTOMETRY
• Is a technique that measures how light is refracted when it passes
through a given substance (an unknown compound) and the amount
by which the light is refracted determined the refractive index.

• The light source of polarimeter is solely sodium. • It deals with the study and measurement of index of refraction of
• It has 2 prisms which are the polarizer and analyzer prism. substance in order to assess their composition of purity.

LIGHTSOURCE • IMPORTANCE
9 Sodium lamp ü To determine the identity of an unknown substance based on its
refractive index
PRISM ü To assess the purity of a particular substance
9 Polarizer: polarizes the original light source ü To determine the concentration of one substance dissolves in
9 Analyzer: used to examine the polarized light after passing through a another
solution 9 Determine of the identity and purity of drug products.
9 It may use to determine the strength and purity of solution or
POLARIMETERTUBE the proportions in which liquids are mixed.
9 Contains the substance being examined
REFRACTIVE INDEX/INDEX OF REFRACTION
TELESCOPE • Is defined as the ratio of velocity of light in air to the velocity of light in
9 Where the field of view is observed the medium being measured or the ratio of the sine of angle of
incidence to the sine of angle of refraction.

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 𝑽𝒆𝒍𝒐𝒄𝒊𝒕𝒚 𝒂𝒊𝒓 II. An Internal Measurement Scale
𝒏𝑫 =
𝑽𝒆𝒍𝒐𝒄𝒊𝒕𝒚 𝒍𝒊𝒒𝒖𝒊𝒅 1. Eyepiece is used to observe both the total reflection borderline and
the instrument’s internal measurement.
• Refractive index of a substance is the ratio of the velocity of light in air
to the velocity of light in a substance. 2. Measurement Scale.
9 Upper Scale – nD – 1.30 to 1.71
9 Lower Scale – % Total Dissolved Solid – 0 to 85%
• It is valuable in the identification of a substance and the detection of
impurities.
3. Dual Reticle
• Refractive index is temperature dependent. 9 Provides precise settings on the reflection borderline and
accurate readout of the measurement scale.
• Is defined as the ratio of velocity of light in air to the velocity of light in 9 Referring to the vertical line.
the medium being measured.
4. Total reflection borderline.
REFRACTOMETER 9 Horizontal line that separates the dark field from light field.
• An optical instrument used to measure the refractive index
• They are best known for measuring refractive indices of liquids but they
also measure RI of gases and solids (such as glass and gemstones).
• Calibrated using distilled water (1.3325 at 25 °C)
• Temperature selected for measurement is at 25 °C
> Dark field – Lower part
• D line of Sodium is used as the wavelength > Light Field – Upper part

IMPORTANCE OF REFRACTOMETRIC MEASUREMENTS III. A Compensating Prism System

1. Determine of the identity and purity of drug products. 1. Fine adjustment knob / Fine compensator dial
9 Removes rainbow color seen in the viewing film.
2. It may used to determine the strength and purity of solution or the 9 It is because one color should one be seen.
proportions in which liquids are mixed.
2. Coarse adjustment knob / Handwheel
PARTS OF REFRACTOMETER
9 Sets the total reflection borderline exactly at cross hair
intersection.

IV. Other Parts

1. Water Jacket
9 Maintain the desired temperature which is 20 0C or 25 0C
except for rose oil which at 30 0C.
9 Made of water inlet – water outlet.
I. Refracting Prism Assembly with Illuminator 9 Carries the liquid to the upper prism.
Upper prism Lower prism Hinged light
Prism field lamp 2. Thermometer Compartment
case case shield
Contains the Prevents stray 9 Holds the thermometer.
Provide the required 9 Provides an accurate indication of the temperature.
measuring light from
Contains the illumination (white
prism and entering the
illuminating light) located at the — Ernst Abbe designed it in 1689.
acts as a front of the
prism. end of the — Can be used with ordinary white light.
sample measuring
adjustable arm. — It must calibrated using water at 1.3325 at 250C or 1-
compartment. prism.
bromonaphthalein.
Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 53
 — Since refractive index values vary with temperature and Ø Potentiometric is a technique for detecting the end point of titration
wavelength, these variables must be held constant. where there is a change in the concentration of the reactants and thus
— The absolute refractive index of air is 1.000294. a big shift in the electrode.

4 TYPES OF ELECTROCHEMICAL METHODS Ø It involves the measurement of changes in the EMF of the cell brought
1. POTENTIOMETRY about adding a titrant, i.e. the monitoring of the potential serves only to
• A branch of electrochemistry which deals with the study and locate the equivalence point of titration.
measurement of electrode potential ( Eº ).

• Potentiometric method gives the pH values using commercially

available pH meter.

• Requires a reference and indicator electrodes.

• The measured potential may be used to determine the analytical

quantity of interest, generally the concentration of some component of
TITRATION CURVE
the analyte solution.

• The potential that develops in the electrochemical cell is the result of

the free energy change that would occur if the chemical phenomena
were to proceed until the equilibrium condition has been satisfied.

• This concept is introduced in quantitative analysis in relation to

electrochemical cells that contain an anode and a cathode.

• In the electrochemical cells, the potential difference between the

cathode electrode potential and the anode electrode potential is the
potential of the electrochemical cell. TYPES OF POTENTIOMETRIC TITRATION
1. Acid-base
POTENTIOMETRIC TITRATIONS 2. Precipitation
• A technique similar to direct titration of a redox reaction 3. Complexation
• No indicator is use, instead the voltage across the analyte, typically an 4. Redox
electrolyte solution is measured. 5. Non-Aqueous
• The monograph in the USP and NF usually designate the electrode pair 6. Measurement of pH
which is to be used for a particular potentiometric titration ü The number of electrodes to be used depends on the number of
• For the purpose of measuring pH of pharmaceutical solutions and techniques available for potentiometric titration.
products, a properly standardized pH meter equipped with glass and
calomel electrodes may be used DIFFERENT ION SELECTIVE ELECTRODES
— Fluoride
FACTORS THAT INFLUENCE THE ACCURACY OF INDICATOR TITRATION — Chloride
ü Lighting conditions — Nitrate
ü Color or turbidity of the solution — Nitrite
ü Colorblindness — Ammonium
ü Fatigue of the operator — Lead
ü Indicator or end point techniques may not available — Copper
— Mercury
— Barium
— Cadmium
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 pH Determination: • Titration of the sample in methanol with Karl Fischer reagent
Glass Electrode 9 Components: Pyridine, Iodine, Methanol, Sulfur dioxide
• Is the most popular of all indicator for pH determinations. 9 Karl Fischer reagent should be freshly prepared and standardized
• The electronic measuring device is pH meter. with sodium tartrate.

2. VOLTAMMETRY
• It is a method of analysis based on the measurement of current
• Include all the electrochemical techniques which involve the resulting from electrolysis of an electroactive species at a given
application of an external potential in the system and utilize the electrode potential.
current potential relatively arising at a polarizable microelectrode to
calculate the concentration of electroactive species
• APPLICATION
2 TYPES OF ELECTROCHEMICAL ANALYSIS BASED ON VOLTAMMETRIC
9 is for the electrometric titration of water called the Karl Fischer
method for water determination
PRINCIPLES
1. Polarography
PARTS OF THE POLAROGRAPH
2. Amperometry
1. ELECTROLYTIC CELL
POLAROGRAPHY
9 An electronic chemical vessel where experiments are performed
and it contains an electrolyte
• A method of analysis based on the measurement of current resulting
from electrolysis of an electroactive species at a given electrode 2. ELECTRODE
potential. 9 Indicator Electrode
• Application is for the electrometric titration of water called the Karl - It is a means of converting the current to a measurable signal
Fischer method for water determination.
9 Reference Electrode
• It is a method for analyzing the composition of a dilute electrolytic - Electrode measuring electric potential of working electrode,
solution. usually a saturated calomel electrode

• Two electrodes are placed in the solution: One has a fixed potential 3. SUPORTING ELECTRODE
(voltage) called the reference electrode, and the other has a variable 9 It is added to the solution to suppress the migration of
potential and is called the polarizable electrode. electroactive species towards the electrodes by electrostatic
9 As voltage is applied to the polarizable electrode, the resulting attraction
change in the current through the solution is monitored. 9 Example: KCl

9 By plotting the pairs of values for voltage and current, a series 4. RECORDER
of current-voltage curves (polarograms) can be generated. 9 Record the result called Polarogram
9 POLAROGRAM
9 The general name for this method is voltametry; the term - is a plot of current flowing in the cell as a function of the
polarography is used when the polarizable electrode is a applied potential.
dropping mercury electrode (DME).

9 Current-voltage curves, which look like a series of steps called

polarographic waves, can be used to determine the reduction
potentials of any reducible species present in the solution.

• Application is for the electrometric titration of water called the Karl

Fischer method for water content determination.

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The shape of polarogram depends on the: • Either DME or platinum electrode can be used with a saturated
a. Method of analysis selected calomel electrode as the reference electrode.
b. Type of indicator electrode • The greatest use is for titration where a slightly soluble precipitate is
9 Dropping Mercury electrode (DME) – which is used for reduction formed.
reactions
9 Platinum electrode(stationary or rotating) – is used for oxidation 3. COLUOROMETRY
reactions. pH meter
• Is a unit of measure which describes the degree of acidity or alkalinity
c. Potential ramp that is applied. of a solution
• Is derived from
METHODS OF POLAROGRAPHY 9 “p”- the mathematical symbol of the negative logarithm
• Classical or Linear sweep voltammetry 9 “H”- the chemical symbol of Hydrogen
• Cyclic voltammetry or triangular wave
• Differential pulse polarography • pH–is the negative logarithm of Hydrogen
• DC polarography or pulse • It was introduced by Soren Peter Lauritz Sorensen (Danish biochemist)
in 1909.
APPLICATIONS • Can be measured either electrometrically or colorimetrically.
• Water analysis of natural, waste and fresh water
• Determination of nitrate, nitrite, chloride, iodide,cyanide, oxygen PARTS OF pH METER
• Determination of organic and toxic materials such as herbicides,
pesticides, insecticides, etc.

FORMULA
𝑪𝒎𝒔 𝑪𝒔
𝒙
𝑪𝒎𝒖 𝑪𝒖
Where:
C = concentration of the sample and standard
Cm = height of chromatograph of the sample standard 1. pH Scale – Indicate the pH reading of the sample
2. Selector Switch – adjust the instrument to pH solution
PROBLEM SOLVING: 3. Zero Adjustment knob – for calibration of pH meter
A polarogram of a reference standard solution containing 0.06mg/mL 4. Temperature knob – controls the temperature
measured 12.00 cm and the polarogram obtained from the sample solution 5. Electrode – used to measure hydrogen ion activity in the solution
measured 12.45 cm. Calculate the concentration of the unknown sample. 9 Indicator electrode – develops a potential that varies according to the
concentration of hydrogen ion
12.45 ü Example: Glass Electrode
𝐶𝑢 = 𝑥 0.06
12.00
9 Reference Electrode – maintains a constant potential
Cu= 0.0623 mg/ml

AMPEROMETRY
• This method is based in the principle of polarography, with the
exception that the voltage is maintain constant during a titration
procedure.

• Involves determination of a single substance only.

*Glass electrode is the commonly used type of electrode in pH meter.

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 BUFFER SOLUTIONS USE RECOMMENDED TESTS AND SPECIFICATIONS FOR QC AND QA OF TABLETS
• These solutions of known pH value allow the user to adjust the system to AND CAPSULES
read accurate measurement. DISSOLUTION TEST
DISSOLUTION
USE OF BUFFER SOLUTIONS • It is define as the process by which a solid substance enters in the
ü To calibrate the pH measurement system solvent to yield a solution.
ü Serve as a reference standard
• It is controlled by the affinity between the solid substances and the
solvent.
STANDARD BUFFER SOLUTION
ü Hydrochloric acid buffer
Tablet/ Capsules deliver drug through a series of physical and phase changes
ü Acid Phthalate buffer
as described below:
ü Neutralized Phthalate buffer
• Tablet or Capsule DISINTEGRATION → Aggregates
ü Phosphate buffer
• Aggregates DE-AGGREGATION → particles
ü Alkaline borate buffer
• Particles DISSOLUTION → Drug in Solution
ü StandardBuffersolutionsrequiredapH7.0, pH4.0 or pH 10.0

PROBLEM SOLVING:
FACTORS THAT INFLUENCE THE DISSOLUTION CHARACTERISTICS OF DRUG:
1. Gatorade, a popular anti-thirst drink has a hydrogen ion concentration of
— Wettability of the dosage unit.
8.0 x 10 -4 mole per liter. Calculate its pH and pOH.
— Penetration ability of the dissolution medium.
— The swelling process.
— Disintegration and de-aggregation of dosage forms.

WHY DISSOLUTION TESTING?

• Bio-availability
• Meet Legal Requirements for Compendial Drugs
• Quality Control tool for Drug Product Manufacturing
• Stability

2. Calculate the pH of Pepsi Cola if the concertation of the H3O+ in this DISSOLUTION RATE
solution is 0.00347 M - Defined as the amount of active ingredient in a solid-dosage form
dissolved in unit time under standardized conditions of liquid/solid
interface, temperature and media composition.
9 In liquid/solid interface, it is the joining of the solvent with the
capsule/tablet.
9 Temperature: 37C
9 Media composition: Water is most frequently used but if there’s a
need to use stimulated intestinal or gastric fluid, then it can also
be used.

Noyes-Whitney Equation
4. CONDUCTIMETRY • Mathematical expression for dissolution rate
𝒅𝒘
= 𝑲 𝑺 (𝑪 𝒔𝒂𝒕 − 𝑪 𝒔𝒐𝒍)
𝒅𝒕

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 Where: • Calibrator Tablets
𝒅𝒘
= dissolution rate - Salicylic acid tab (USP non-disintegrating tab)
𝒅𝒕
K = dissolution constant - Prednisone tab (USP disintegration tab)
S = surface area of the solid
C sat = conc. of a saturated solution • Preferred medium is Distilled water
C sol = conc. at any given time
• Experiment conducted should be at 37°C
• Usual volume is 500 mL to 1000 mL
THEORETICAL CONCEPTS OF DISSOLUTION TESTING
INTRINSIC DISSOLUTION RATE • Other dissolution medium used:
- The rate of dissolution of a pure pharmaceutical active ingredient - Buffered aqueous solution (pH 4 to 8)
when conditions such as surface area, temp., agitation, stirring - Diluted acid ( 0.001 N to 0.1 N HCl)
speed, pH, ionic strength of the dissolution is kept constant.
• Common operating speeds:
APPARENT DISSOLUTION - 100 rpm for basket
• Total mass of a solid dissolved in unit time. - 50 rpm for paddle
• More significant than intrinsic dissolution.

RATE LIMITING FACTOR

• Complex because the transfer of solid active ingredient from its
delivery system involves sequential processes.
• Disintegration> De-aggregation> Dissolution

FACTORS AFFECTING THE RATE OF DISSOLUTION:

• Physicochemical properties of the drug.
• Drug product formulation.
• Effect of processing factors on dissolution rate.
• Factors related to dissolution test parameters.
DISINTEGRATION TEST
COMPLETE DISINTEGRATION
FACTORS RELATED TO DISSOLUTION TEST PARAMETERS
- The state in which any residue of the unit, except fragments of the
• Aqueous medium insoluble coating or capsule shell, remaining on the screen of the test
• The temperature of the medium apparatus is a soft mass having no palpably firm core
• The agitation rates

DISSOLUTION STANDARDS
• USP Policy (General)
- All tab and cap NLT 75% of the labeled content is dissolved in
NMT 45 minutes in 900 mL.

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 APPARATUS COMPOSITION PROBLEM SOLVING:
• 1L Low-form beaker for immersion of fluids. 1. Given the data obtained after disintegration test
• Thermostatic arrangement for heating.
35-39 °C
• Device for raising and lowering the basket at a constant frequency of
29 to 32 cycles per minute.
• Basket-rack assembly moves vertically along its axis.

Basket-Rack Assembly: ANSWER

• Consist of six open ended transparent tubes 7.75 cm long, diameter
21.5 and thickness 2mm.
• Tubes are held in a vertical position by two plastic plates each about 9
cm in diameter and 6mm in thickness.
• Attached under the lower plate is a 10 mesh screen No.23.
• Accessory: Disks – sp.gr. 1.18-1.20
five 2mm holes
v-shaped planes perpendicular 2. Situation I: Following data were obtained after disintegration on
to the end of the cylinder Ethambutol HCl + Isoniazid + Pyroxidine HCl tablet.

DISINTEGRATION TEST PROCEDURE

Uncoated Tablets
- 1 tablet is placed in each of the six tubes of the basket and operate 1. Determine the average time when tablet disintegrate
the apparatus using water maintained at 37+/- 2 °C. At the end of
the time specified, lift the basket and observe tablets.
2. time the last table disintegrates
Requirement:
- All tab should disintegrate completely, if 1 or 2 fails to disintegrate
repeat additional 12 tablets. NLT 16 of the total 18 tablets tested
should completely disintegrates. 3. Disposition on the batch tested

DISINTEGRATION TIME REQUIREMENT

• Uncoated Tablets - 30 minutes 4. Time when the first tablet completely disintegrate
• Coated Tablets - up to 2 hours
• Sublingual Tablets - 3 minutes 5. If the disintegration test specification is reduced to NMT 10 minute. How
• Buccal Tablets - 4 hours many tablet will have failed results

FACTORS AFFECTING DISINTEGRATION OF TABLETS

1. Physical and Chemical Properties of granulation
2. Hardness and porosity of tablets
3. Disintegrating agent used

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 FRIABILITY TESTING TABLET HARDNESS
• Drum is made of transparent synthetic polymer with polished internal — The resistance of the tablet to chipping, abrasion or breakage under
surface and subject to static build up - 285 mm (i.d.). conditions of storage, transportation and handling.
• Drum rotates at a speed of 25 rpm.
— Hardness determination are made throughout the tablet runs to
• Each turn the tablet falls about 130 mm onto the drum. determine the need for pressure adjustment on the tableting machine.
ü If the tablet is too hard, it may not disintegrate in the required
Friability Test: period of time or meet the dissolution specification.
ü Tablets weighing < 650 mg- 20 tablets ü If the tablet is too soft, it will not withstand the handling during
ü Tablets weighing > 650 mg- 10 tablets subsequent processing such as coating or packaging and
ü Tablets are to be rotated 100 times shipping operations.
ü Loose dust are removed by air pressure or soft brush using no. 10 sieve
ü % weight loss is calculated — A commonly used rule of thumb describes a tablet to be of proper
ü Requirement: Weight loss should not be > 1 % hardness if it is firm enough to break with a sharp snap when it is held
between the second and third fingers and using the thumb as the
FACTORS AFFECTING TABLET FRIABILITY fulcrum, yet, doesn’t break when it falls on the floor.
1. Moisture content of granules
2. Particle size of granules — The Stokes-Monsanto is an instrument that measures the force required to
3. Compression pressure break the tablet when the force generated by a coil spring is applied
diametrically to the tablet. The force is measured in kilograms and when
FORMULA: used in production, hardness of 4 kg is considered to be minimum for a
ü % Weight Loss is equal to the difference of Weight of tablets before satisfactory tablet.
testing and Weight of tablets after testing divided by the Weight of
tablets before testing multiplied by 100. — The Strong-Cobb hardness tester also measures the diametrically applied
force required to break the tablet. In this instrument the force is produced
𝑾𝒕. 𝒐𝒇 𝑻𝒂𝒃𝒔 𝒃𝒆𝒇𝒐𝒓𝒆 𝒕𝒆𝒔𝒕𝒊𝒏𝒈 − 𝑾𝒕. 𝒐𝒇 𝑻𝒂𝒃𝒔 𝒂𝒇𝒕𝒆𝒓 𝒕𝒆𝒔𝒕𝒊𝒏𝒈 by a manually operated air pump. As the pressure is increased a plunger
% 𝒘𝒕. 𝒍𝒐𝒔𝒔 = 𝒙 𝟏𝟎𝟎
𝑾𝒕. 𝒐𝒇 𝑻𝒂𝒃𝒔 𝒃𝒆𝒇𝒐𝒓𝒆 𝒕𝒆𝒔𝒕𝒊𝒏𝒈 is forced against the tablet placed on anvil. The final breaking point is
indicated on a dial calibrated into 30 arbitrary units.
PROBLEM SOLVING:
1. Calculate the % Weight Loss of the tablet if the initial weight and final — Another instrument is the Pfizer hardness tester which operates on the
weight obtained were 6.2948 and 6.2066 g respectively. same mechanical principle as ordinary pliers. The force required to break
the tablet is recorded on a dial and may be expressed as either kilograms
𝟔. 𝟐𝟗𝟒𝟖 − 𝟔. 𝟐𝟎𝟔𝟔 or pounds of forces.
% 𝒘𝒕. 𝒍𝒐𝒔𝒔 = 𝒙 𝟏𝟎𝟎
𝟔. 𝟐𝟗𝟒𝟖
— Schleuniger apparatus also known as the Heberlein and electrically
operated test equipment and most widely used apparatus to measure
% 𝒘𝒕. 𝒍𝒐𝒔𝒔 = 1.40 % tablet hardness.

CONTENT UNIFORMITY TEST USP TABLET THICKNESS

• Is intended to established within limits that a product has uniform MICROMETER
amounts of an active ingredient in batch preparations of — Is used to measure very small thicknesses and diameters of wires and
pharmaceutical dosage form. spheres.
• A sample of 30 tablets is selected and ten are individually assayed.
— this instrument, invented by William Gascoigne in the 17th century, is often
• NLT 85% and NMT 115% used to measure diameters of wires and spheres.

Edit: Loraine Angelie C. Bermas // Content: Jaica Chinnie S. Pintor 60

— Tablet thickness is determined with a caliper or thickness gauge which PROBLEM SOLVING:
measures the thickness in millimeters. 1. What is the Final reading of the tablet thickness, if the reading from the
sleeve was 3.7 and the reading taken from the thimble was found in line
— A plus or minus 5% may be followed, depending on the size of tablet. with thirteenth (13th) division on the sleeve?

Final Reading = 3.7 + 13 (0.01)

THREE TYPES OF MICROMETERS = 3.7 + 0.13
1. An External micrometer is typically used to measure wires, spheres, = 3.83 mm
shafts and block.
2. An Internal micrometer is used to measure the opening of holes. 2. Given the data, calculate:
3. A Depth micrometer is used to measure depths of clots and steps. a. The True Value based on ±5%
b. Average Mean
PARTS OF A MICROMETER c. Standard Deviation (S)
d. UCL and LCL

DATA

FORMULA TO DETERMINE THE THICKNESS

Final Reading = Main Scale Rdg. + Circular Scale Rdg. x Least count (LC)

• Least Count (LC) = 1/n (pitch of screw)

= 1/50th of 0.5 mm
= 0.02 x 0.5
LC = 0.01 mm
It’s because when you close the anvil and the spindle, it is not very
closer to each other, hence, there’s still a small distance between the
two.

Data:
Main Reading = 6.5
Circular Scale Reading = 7

• Final Reading (FR)

= 6.5 + 7 (0.01)
= 6.5 + 0.07
= 6.57 mm

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 UNIFORMITY OF DOSAGE FORM CRITERIA:
a. Weight variation Test Stages Condition Specifications Remarks
b. Content Uniformity Test None of the samples
1 10 samples 85%-115% exceed the
WEIGHT VARIATION TEST specification
• Weight variation is used to ensure that each tablet contains the If more than 1
proper amount of drug Repeat the test with
exceeds the 85%-115%
• It involves the determination of individual weights of the samples additional 20 samples
specification
tested and computation for the average weight. 1 sample exceeds Repeat the test with
• The test was carried out by weighing 20 tablets individually using 75%-1125%
the specification additional 20 samples
analytical balance, then calculating the average weight and None of the samples
comparing the individual tablet weight to the average Additional 20
2 85%-115% exceed out the
• The weight of the tablet is the quantity of the granulation which samples
specification
contains the labeled amount of the therapeutic ingredient
• Twenty tablets are weighed individually and the average weight is COMPENDIAL REQUIREMENTS FOR PARENTERALS AND SEMISOLIDS
calculated • In parenteral dosage forms, its quality control have 4 basic area there
are: Sterility, Freedom form Pyrogens, Freedom from particulate matter
𝑨𝒄𝒕𝒖𝒂𝒍 𝑾𝒕. −𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝑾𝒕. and leakers.
% 𝑾𝒕. 𝒗𝒂𝒓𝒊𝒂𝒕𝒊𝒐𝒏 = 𝒙 𝟏𝟎𝟎
𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝑾𝒕. • The achievement of sterile, non-pyrogenic and particulate free
parenteral product provides a quality product for the patients and
Average Weight (mg) % Weight Variation consumers
<150 ± 10
150 to 324 ± 7.5 QUALITY CONTROL TESTS FOR PARENTERALS
> 324 ±5 STERILITY TEST
• Sterility test is a microbiological test suitable for revealing the
CRITERIA: presence of viable forms of bacteria, fungi and yeast in or on
ü NMT 2 Tablets s should differ by more than the % weight variation pharmaceutical articles
ü No tablet differ by more that double the % weight variation • Test Organism and Culture Media
9 Soybean Casein Digest Medium for Streptococcus species
Tablet Weight (mg) % Difference 9 Fluid Thioglycollate Medium for Clostridium species.
130 mg or less 10 9 Subtilis
130 -324 mg 7.5
> 324 mg 5 METHODS OF STERILITY TEST
1. Direct Inoculation Method - involves the direct transfe.ir of the
CONTENT UNIFORMITY TEST USP pharmacopeial article to test media.
• Is intended to established within limits that a product has uniform
amounts of an active ingredient in batch preparations of 2. Membrane Filtration Method - method of choice for sterility testing; it
pharmaceutical dosage form. uses a suitable membrane filter consisting of an assembly that
facilitates handling of articles and that allows the processed
• A sample of 30 tablets is selected and ten are individually assayed. membrane to be removed aseptically for inoculation of appropriate
media.
• However, the 20 tablets in 30 tablets will be used only, if the first 10
tablets is found not complying with the requirement / specification. 3. Biological Indicators - the most preferred method which makes use of
the different methods of sterilization.
• NLT 85% and NMT 115%. 9 Moist heat and Ethylene oxide - Bacillus stearothermophilus; Dry
heat - Bacillus subtilis.

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 LEAKER TEST The most potent pyrogenic substances are produced by:
• Done using negative pressure within incompletely sealed ampules while 1. Gram-negative bacteria (endotoxin)
submerging them in a dye solution 2. Gram-positive bacteria
3. Fungi
Leaker Test for Ophthalmic Ointments
ü By Classical Blotting Paper Method-the sealed and filled ophthalmic Properties:
ointment collapsible tubes are place in an absorbent paper, heated 1. Lipid in nature
in a 60C ±3 oven for 8 hours 2. Phosphorus attached to a polysaccharide or a protein or both

SAFETY TEST Signs and Symptoms of Pyrogen contamination:

• A test done using white mice or guinea pigs. It is used for determining the 1. Febrile reaction in human beings
safety of the plastic devices used in parenteral products 2. Chills
3. Pains in back and legs
PARTICULATE MATTER TESTS 4. Malaise
• A test for the presence of particles or particulates.
• Equipment Sources of Pyrogens:
ü Electronic Particulate Counter 1. Water
ü Membrane Filtration Technique 2. Equipment
3. Solute
CLARITY TEST
• A test done to prevent the distribution and use of parenteral products Pyrogen Test
that contain any particulate matter. — Rabbit Test
— LAL Test
MINIMUM FILL TEST FOR SEMISOLIDS
• Done for the assessment of content uniformity in semisolid products like RABBIT TEST (IN VIVO)
creams, ointments, lotions, aerosols, jellies and pastes Test animals:
- Use healthy mature rabbits
- Weight of the Rabbit: same weight if possible
PYROGEN TESTING
Bacterial Endotoxin [LAL] Test Procedures:
• Limulus Amoebocyte Lysate test 1. Use only those rabbits whose control temperature do not vary
• A test for estimating the concentration of bacterial endotoxins that (between) by more than one degrees centigrade from each other and
maybe present in a sample. do not use any rabbit having a temperature exceeding 39.8 0C.

Pyrogen Test 2. Unless otherwise specified in the individual monograph inject to an ear
• Also known as “rabbit test" vein of three rabbits 10mL of the test solution per kg of body weight,
• It is designed to limit to an unacceptable level the risks of febrile completing each injection within 10 minutes after start of
reactions in the patient to the administration or injection of a administration.
product.
• It involves measuring the rise in temperature of rabbits following 3. Record the temperature at 1, 2 and 3 hours subsequent to the injection.
administration of a test solution

PYROGEN Test Interpretation:

— Any substance that produces a fever. 1. If no rabbit shows an individual rise in temperature of 0.6 0C or more
— Are products of the metabolism of microorganisms or portions of the above its respective control temperature and if the sum of three (3)
protein coat of bacteria. individual maximum temperature rises does not exceed 1.4 0C, the
product meets the requirements for the absence of pyrogens.

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 2. If any rabbit shows an individual rise of 0.6 0C or more, or if the sum of PROBLEM SOLVING:
the three individual temperature rises exceeds 1.4 0C, continue the test
using other rabbits.

3. If not more than three of the eight (8) rabbits show individual rises in
temperature of 0.6 0C or more and if the sum of the eight individual
maximum temperature rises does not exceed 3.7 0C, the material under
examination meets the requirements for the absence of pyrogens.

After injection rabbit temperature are recorded at 30 mins intervals between

1 & 3 hour

If rabbit shows an individual temperature rise of 0.5oC or more the test is

LIMULUS AMEBOCYTE LYSATE (LAL) TEST (IN VITRO)
continued with additional 5 rabbits
— Also known as Bacterial Endotoxin Test (BET)
THE RESULT OT PYROGEN TEST:
— Test to detect minute quantities of bacterial endotoxins and other
pyrogens using an extract from the circulating amebocytes from the
horse shoe crab (Limulus polyphemus).

— Positive result (Gel formation).

LAL is an aqueous extract of blood cells (amoebocytes) from the blue-

blooded horseshoe crab, Limulus Polyphemus, LAL is being used for endotoxin
detection

METHODS OF ENDOTOXIN TESTING

• Since1940s: Rabbit Pyrogen Test
• Since1970s:
ü Limulus Amoebocyte
ü Lysate (LAL) Test
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 • Almost all products interfere to a certain extent with the LAL test STABILITY OF PHARMACEUTICAL PRODUCT
• A summarizing study by Guilfoyle & Munson: ü Studies are undertaken by Q.C. to confirm the shelf life stability of the
ü 587 products were test product as indicated by the expiry date appear on the label of the
ü 78% of them were interfering with LAL product.

LIMULUS AMOEBOCYTE LYSATE (LAL) TEST ü Stability of a drug can also be defined as the time from the date of
• Based on clotting reaction of horseshoe crab (Limulus Polyphemus) manufacture and packaging of the formulation until its chemical or
blood cell (Amoebocyte) lysate to endotoxin biological activity is not less than a predetermined level of labeled
• Developed in 1960’s by Drs. Bang and Levin potency and its physical characteristics have not changed appreciably
• Faster or deleteriously.
ü More economical
ü More sensitive than rabbit Pyrogen Test (all methods accepted TWO METHODS OF STABILITY TESTING
by the FDA, USP, EP & JP 1. Accelerated Testing (Stress Testing)
2. Long term method using Arrhenius Equation
TYPES OF LAL TEST
5 Types Of Stability
1. Physical
2. Chemical
3. Therapeutic
4. Microbiological
5. Toxicological
1) Gel Clot Technique
- Based on gel formation EXPIRATION DATING
— Is defined as the time in which the preparation will remain stable
2) Turbidimetric Technique when stored under recommended conditions.
- Based on turbidity — Is expressed traditionally in terms of month and year.

3) Chromogenic Technique EXPIRATION DATE

- Based on development of color — Shelf life plus date of manufacture.

SHELF LIFE
• The period of stability of a preparation from date of manufacture until
its chemical or biological activity is not less than 90% of the labeled
potency.

A simple means of estimating shelf life:

1. Select the best prototype formulation based on short-term stability
PROCEDURE OF LIMULUS AMOEBOCYTE LYSATE (LAL) TEST
data, and
1. To u se the commercial product, a laboratory analyst reconstitutes the
2. Predict both estimated and minimum shelf life values for the
vial of freeze-dried LAL with endotoxin-free water.
formulation.
2. An equal amount of reconstituted LAL, usually 0.1 mL, is then added to
This estimate is subject to
the sample of solution in a small, glass, endotoxin-free test tube.
1. Type 1 or alpha error (setting the date too early so that the product will
be destroyed or recalled from the market at an earlier time than is
3. The mixture is then incubated at 370C for one hour.
actually necessary).
4. At the end of this time, the mixture is examined for gel formation by gently
2. Type 2 or beta error (setting the date too late so that harmful even
inverting the tube.
occurs in an unacceptably large proportion of cases).
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 PHARMACEUTICAL PRODUCT STABILITY EVALUATION OINTMENTS
1. Physical — A stable ointment is one in which retains homogeneity throughout the
Physical Factors: heat, light and moisture shelf life period.

2. Chemical — The main stability problems seen in ointments are “bleeding” and
Chemical causes of drug deterioration have classified into: changes in consistency due to aging or changes in temperature.
- Incompatibility
- Oxidation – Reduction — When fluid components such as mineral oil separate at the top of an
- Hydrolysis ointment, the phenomenon is known as bleeding and can observed
- Racemization visually.
- Decarboxylation
- Formation of ppt. — Among other tests performed are visual appearance, color, odor,
viscosity, softening range, consistency, homogeneity, particle size
3. Biological distribution and sterility.

PRODUCT SABILITY AND THEIR STABILITY PROFILE PROBLEM SOLVING: SRABILITY COMPUTATION
EMULSIONS FORMULATION
Appearance Viscosity
Color Phase Separation
Odor Strength
pH

ORAL SOLUTIONS AND SUSPENSIONS

Appearance Crystallization
Color Redispersibility
Odor Suspensibility
Taste Pourability QUESTION
Clarity Viscosity
Precipitation pH
Strength

TABLETS
Appearance Moisture
Color Humidity Effect
Odor Dissolution
(except chewable tablet)
Taste Strength
Hardness
Friability

CAPSULES
Appearance Dissolution
(except liquid filled gelatin capsules)
Color Strength
Shapes Brittleness
Moisture

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 ADDITION OF OVERAGE penetrate matter. Gamma radiation can damage the human body.
— Overage are added to pharmaceutical formulation to keep the content Concrete or lead is typically used to shield people from gamma
of the active ingredient within the limits compatible with therapeutic radiation.
requirements for a predetermined period of time.
— The International Pharmaceutical Federation has recommended that Radiation Measurements
overages be limited to a maximum of 30% over the labeled potency of — Roentgen: The roentgen measures the energy produced by gamma
an ingredient. radiation in a cubic centimeter of air. It is usually abbreviated with the
capital letter "R". A milliroentgen, or "mR", is equal to one one-thousandth
2 types of overage of a roentgen. An exposure of 50 roentgens would be written "50 R".
Manufacturing overage
- Overage added to a preparation to compensate loss during — Rad: Or, Radiation Absorbed Dose recognizes that different materials that
manufacturing of the preparation. receive the same exposure may not absorb the same amount of energy.
A rad measures the amount of radiation energy transferred to some mass
Stability overage of material, typically humans. One roentgen of gamma radiation
- Is the excess added to a preparation to extend its shelf life. exposure results in about one rad of absorbed dose.

— Rem: Or, Roentgen Equivalent Man is a unit that relates the dose of any
RADIOACTIVITY radiation to the biological effect of that dose. To relate the absorbed
— Radioactive substances are used in medical practice as diagnostic aids dose of specific types of radiation to their biological effect, a "quality
and the therapeutic agents. factor" must be multiplied by the dose in rad, which then shows the dose
in rems. For gamma rays and beta particles, 1 rad of exposure results in 1
— Radioactivity is the property of certain atoms (which are not stable) to rem of dose.
spontaneously disintegrate by emitting either energetic particles or rays
of pure energy (or both) from the nucleus or center of the atom. OTHER MEASUREMENTS TERMS
— Standard International (SI) units which may be used in place of the rem
— The daughters of a radioactive substance are the other substances which and the rad are the sievert (Sv) and the gray (Gy).
are created as byproducts in the process of radioactive disintegration; in
many cases, the daughters of a radioactive substance are also RADIOPHARMACEUTICALS
radioactive. Are used in the field of nuclear medicine as tracers in the diagnosis and
treatment of many diseases.
— Ionizing Radiation is the term used to describe the various energy forms Ø Iodine-125 (60 d): used in cancer brachytherapy (prostate and
which can be emitted by the disintegration of radioactive atoms; these brain), also diagnostically to evaluate the filtration rate of kidneys
include energetic particles -- alpha, beta, and neutrons; rays of pure and to diagnose deep vein thrombosis in the leg.
energy -- gamma rays and x-rays.
Ø Rhenium-188 (17 h): used to beta irradiate coronary arteries from an
THREE KINDS OF RADIATION EMITTED BY RADIOACTIVE ELEMENTS angioplasty balloon.
1. Alpha radiation is made up of particles that include two neutrons and
two protons each. Alpha radiation travels only a few inches in air. A Ø Indium Chlorides In 133m Inj: used to blood- pool studies including
sheet of paper or skin will block alpha radiation, but it is harmful if it is visualization of aneurysms and for liver, lung and bone imaging.
taken into the body through eating, drinking or breathing.
RADIOISOTOPES USED IN MEDICINE
2. Beta radiation particles are smaller, though they have more energy ü Iodine-131(8d): Widely used in treating thyroid cancer.
than alpha particles. Beta radiation can travel up to 12 to 15 feet in
air and can penetrate skin. About an inch of shielding - glass, wood, ü Iridium-192: Used as an internal radiotherapy source for cancer
plastic or metal - will stop most beta particles. treatment.

3. Gamma radiation is made up of energy waves similar to light or radio ü Palladium-103(17d): Used to make brachytherapy permanent
waves but with more energy. It can travel great distances and implant seeds for early-stage prostate cancer.
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 ü Phosphorus-32(14d): Used in the treatment of polycythemia vera APPLICATION
(excess red blood cells). • To achieve accurate diagnosis and to prevent false diagnosis or
analytical interference.
ü Rhenium-186(3.8d): Used for pain relief in bone cancer.
Note:
ü Strontium-89(50d): Very effective in reducing the pain of prostate and ü Biochemical interferences occur when the drug enters some
bone cancer biochemical or physiological process, resulting in a change in the
level of some important biochemical.
IMAGING USING NUCLEAR MEDICINE
-End

BIOMEDICAL ANALYTICAL CHEMISTRY (BAC)

• The discipline which uses the principles and techniques of analytical
chemistry and biochemistry was often called Clinical Chemistry or BAC

BIOCHEMICAL SUBSTANCES
ü Serum Albumin
ü Bilirubin
ü Blood Urea Nitrogen(BUN)
ü Cholesterol
ü Glucose
ü Total Protein
ü Uric Acid
ü Inorganic ion such as: Calcium, Chloride, Potassium and Sodium

INSTRUMENTS USED
ü AAS
ü HPLC&GC
ü UV/Vis SPECTROMETER
ü FLUROMETER
ü GLUCOSE ANALYZER

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