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1. Sample Preparation for Analysis
By: Merga D. (B. Pharm)
1 21-Jun-24

2. Introduction to Chemical Analysis
Chemical analysis includes any aspect of the
chemical characterization of a sample material.
For all chemical analyses, the analyte, which refers to
the species to be measured,
– must be in a sufficient quantity and suitable form for the
instrument used.
The majority of samples require a specific
pretreatment.
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3. Areas of chemical analysis and questions they
answer
Quantitation:
–How much of substance X is in the sample?
Detection:
–Does the sample contain substance X?
Identification:
–What is the identity of the substance in the sample?
Separation:
–How can the species of interest be separated from
the sample matrix for better quantitation and
identification?
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4. …
Sample preparation is therefore an essential
step in analysis, as important as the
measurement itself, greatly influencing its:
–Reliability
–Accuracy and
–Cost
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5. …
In designing the method we considered
sources of error and took appropriate steps,
– Such as including a reagent blank and
calibrating our instruments, to minimize their
effect.
When we collect the wrong sample or lose
analyte while preparing the sample for analysis,
–We introduce a determinate source of error.

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6. Requirements for sample preparation:
The product of the sample preparation steps must
produce a solution with the following properties:
-The analyte concentration is in the measurable
range of the instrument.
-Selectivity
-The recovery is quantitative or reproducible
from sample to sample.
-The analyte is stable until the sample is analyzed.
-The sample is compatible with the instrumental
method.
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7. Range:
The concentration of the prepared
sample must be
–Within the working concentration range of
the instrumental method.
This will determine the volume of
dilution for the sample.
–If the sample is too dilute, then the sample
must be concentrated using an appropriate
sample preparation technique.
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8. Selectivity:
The sample preparation method must not
only deliver a measurable amount of sample;
–But also the compounds accompanying the
analyte must not interfere with the analysis and
such that the sample needs to be selective.
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9. Recovery:
The recovery of a sample preparation must
be assessed,
–because the recovery determines the accuracy of the
analysis.
For drug substance and drug product analysis;
–recovery of 100% is generally required to maintain
required levels of accuracy and precision.
For biological samples;
-less than quantitative recovery is generally
acceptable if the recovery is reproducible.
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10. Stability:
After the sample is prepared and is awaiting instrumental
analysis,
–the analyte must be stable for a reasonable amount of
time.
If necessary, the sample may require one additional
step, such as pH adjustment.
-This is particularly important for trace impurity or
degradant methods.
If the method is used to report degradants, then the
sample must resist degradation.
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11. Compatibility:
The first concern in the selection of the sample preparation
solvent is to optimize recovery.
However, a secondary consideration is the sample
solvent’s effect on the analysis.
For UV analyses, the sample and the calibration standard;
must be dissolved in the same solvent to eliminate
solvent effects on the UV absorbance.
The sample and standard must also have similar pH.
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12. The need for sample pretreatment:
In order to start an analysis, a sample representative of the
batch under study is required.
This contains the species being studied called the analyte
mixed,
–in general, with a variable number of other compounds
which constitute together what is called the matrix.
When measuring traces of an analyte within other compounds
thousands of times more abundant,
–there is a fear that interferences between analyte
and other compounds may occur.
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13. …
Even the best analytical techniques cannot
remedy or compensate
–problems generated by poor sample pretreatment.
This makes a meticulous sample preparation
essential.
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14. …
There are three key issues that often
necessitate pre-treatment of a sample prior to
analysis:
1.The sample is in the wrong physical state for
the analysis method (e.g., the method requires
a liquid but you have a solid sample),
2.The sample has interfering matrix
components………..may give either a false positive
or negative reading in the measurement, and
3.The sample has too low analyte concentration
to be detected by the instrument.
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15. First, a sampling is performed, where the required
number of samples are taken.
During sampling, it is essential that samples are
taken in a representative manner.

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16. …
Often, the samples must be stored until further analysis.
The principal challenge with sample storage is to avoid
degradation and compositional changes of the sample.
If such changes occur, the final quantitation will not reflect
the original composition of the sample.
To protect samples against degradation;
They are often stored at low temperature and
protected from light, as in a refrigerator or a freezer.
After sampling and storage, samples are normally
pretreated in some way, which is termed sample
preparation.
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17. …
Sample preparation can be very simple or quite
complicated, depending on the complexity of the
sample.
Sample preparation serves the following objectives:
-The sample is compatible with the instrument for
the analytical measurement.
-The analyte is present in sufficient concentration for
the analytical measurement.
-The sample matrix that can interfere with
the analytical measurement is removed.
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18. …
In the case of tablets,
–This involves pulverization of tablets
–Dissolution of the tablet powder and
–Filtration of the solution to remove insoluble material.
At the end, the analytical measurement is performed,
where the analyte or analytes are identified and
quantified.
After the analysis, measurements are processed (data
processing),
where the results are calculated and presented in an
analytical report.
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19. Steps in Chemical Analysis:
1.Define the problem
2.Picking or selecting a method
3.Acquiring a sample or obtaining representative sample
4.Processing a sample or prepare a sample for analysis
5.Perform any necessary chemical separation or
eliminating interferences.
6.Performing the measurement
7.Calculating the results and reporting


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20. …
20 21-Jun-24

21. Titrimetry
By: Merga D. (B. Pharm)
21 21-Jun-24

22. Titration is the process in which a concentration of
unknown solution is determined with the help of
using standard solution of known concentration.
•Titration is a quantitative technique
–provide high accuracy (99.5–100.5%) and
–precision (<0.5% relative standard deviation
(RSD)).
•Titration is an official method in the
–European Pharmacopoeia (Ph. Eur.)
–United States Pharmacopoeia (USP).
…
21-Jun-24 22

23. •In pharmaceutical analysis, titration is mainly
used for;
–quantitative analysis (assay) of APIs and
–excipients with the purpose of assessing the
purity of a given substance (analyte).
…
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24. Some terms used in titrimetry
Titrant: is a solution with known concentration which is
usually taken in a burette and used as a standard solution.
Titrand: is a solution with unknown concentration
which is being titrated and usually taken with conical
flask.
Equivalence point: the point at which exactly equivalent
amount of titrant is added to titrand.
-In theoretical concept, when equivalent amount of titrant is
added to the titrand, the indicator should change the colour
and indicate an end point.


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25. -But in actual practice, indicator does not change its color
at equivalence point.
Endpoint: defined as the point at which the operator or the
titration apparatus terminate the titration.
Indicator: A substance that changes color in response to a
chemical change.
An acid-base indicator (e.g., phenolphthalein changes
color depending on the pH.)
A drop of indicator solution is added to the titration at the
beginning; the end point has been reached when the color
changes.

…
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26. •An indicator is a weak acid or base that
changes colour between its ionised and
un-ionised forms;
– the useful range for an indicator is 1 pH
either side of its pKa value.
…
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27. Some indicators used with their pH range and colors of
each
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28. 21-Jun-24 28

29. …
Titrations are based on measurement of volumes.
Increments of a standardized solution of a reagent are
added gradually until the entire amount of analyte has
reacted.
The standardized solution is termed the titrant.
The concentration and exact volume of titrant added are
used to calculate the amount of analyte.

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30. •More generally, a titration can be described by
the following titration equation:

x(Analyte) + y(Titrant) → Products

Here x and y are the number of mol of analyte
and titrant involved to complete the titration.
The titration is completed when all the analyte
has reacted and transformed to products.
The titration has then reached the equivalence
point.
…
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31. …
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32. The titrant is added either
–manually from a burette or
–automatically from an automatic titration apparatus
termed an automatic burette or titrator.
Detection of the equivalence point can be based on
–visual inspection of colour change (Indicator
detection) or
–electrochemical measurements (Potentiometric
endpoint detection).
…
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33. •The volume of titrant consumed is read
–from the burette/titrator and
•The quantity of analyte in the sample solution can
be calculated
–on the basis of the underlying titration equation
and the exact concentration of reagent in the
titrant.
33 21-Jun-24

34. Common features of titrations
Despite the difference in chemistry, all titrations
share several common features
 Requirements for titration
–Stoichiometric ratio should be known
–No complicating side reaction
–Concentration of the titrant should be known
–Easy method of end-point detection
–The reaction should be rapid
–The reaction should be quantitative so that
sharp change will occur at the end-point
34 21-Jun-24

35. A number of requirements should be fulfilled for a
successful titration:
–The titration reaction must be well-defined and
without any side reactions.
–The reaction must be virtually complete (≈100% of
analyte must be converted to product).
–Other substances in the sample should not react with
the titrant.
–The equivalence point should be clearly detected.
–The exact concentration of titrant must be known
(standardized solution).
…
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36. Eg. Titration of a pharmaceutical ingredient and
calculation of purity (assay)
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37. 37 21-Jun-24

38. Titration curves
•Titration curve is plot of a variable related to a
relevant concentration
–as y-axis versus some measure of the amount of
titrant (e.g. volume) on the X-axis.
•A titration curve provides us with a visual picture of
how a property of the titration reaction changes
–as we add the titrant to the titrand.
•Titration curve define the required properties of an
indicator
–allow us estimate the error associated with the
titration methods.
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39. Example
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40. Standard solution:
A standard solution is reagent used to titrate
the analyte and which do have known
strength.
Should have known concentration
Two types:
–Primary standards and
–Secondary standards
40 21-Jun-24

41. …
•A primary standard is a highly purified compound
(99.99%) material that serves as reference.
Requirements:
–Must be easy to obtain, to purify, to dry and
preserve in pure state
–It should be 100.00 % pure although 0.01 to
0.02 % impurities is tolerable if accurately
known.
–It should be stable at atmospheric conditions
–It should be soluble in the conditions applied
41 21-Jun-24

42.  In general, primary standard is a chemical reagent with the
following characteristics:
–High chemical purity (99.99%)
–Well-defined chemical composition (including the amount of
crystal water)
–High stability during storage or in contact with air and light
–High solubility in the titration solvent (water or organic solvent)
–Non-volatile
–Strong electrolyte
–Non-hygroscopic
…
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43. …
Examples of primary standards in Ph. Eur
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44. …
•Standards that do not meet all these criteria are
called secondary standards
•A secondary standard is a chemical that has
been standardized against a primary standard
for use in a specific analysis.
–Exact concentration is determined by
standardization
–In many cases the titrant is not available in a
stable form of well-defined composition
44 21-Jun-24

45. …
•To achieve high accuracy in titration, it is
essential to know the exact concentration of
titrant.
•Thus, the titrant has to be standardized prior to
use and for standardization, a primary standard is
needed
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46. …
The titrant must be standardized (usually by
volumetric analysis) against a compound that
is available in a stable, highly pure form (i.e., a
primary standard).
Standard solutions are prepared in two ways:
1.Direct method: weigh and dissolve -for a
primary standard
2.Indirect method: standardization- for
secondary standard
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47. Eg. Standardization of 0.1 M hydrochloric acid with sodium
carbonate as primary standard
7/14/2022

48. …
Equivalence and End point
Equivalence point: the point where the reaction
b/n the titrant and analyte is completed
-Can not be determined experimentally
End point: the point at which completion of
reaction is usually shown by a change of color
caused by a substance called an indicator.
–This point is referred as end point
48 21-Jun-24

49. •The titration is stopped when the endpoint of
titration is reached.
•The endpoint is defined as the point at which the
operator or the titration apparatus terminate the
titration.
•Ideally, the endpoint coincides with the
equivalence point.
•If not, the result is prone to titration error
…
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50. Applications of titrimetry

51. Advantages and limitations of titrimetry

Advantages:

52. Limitations:
Non-selective (particularly for acid-base
titration)
Time-consuming if not automated and require
greater level of operator skill
Require large amount of sample
Reaction of standard solution should be rapid
and complete

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53. Type of Titration
•Based on method of titration
–Direct titration and Back titration
•Further based on the nature of chemical reaction
–Acid-base titration (aqueous and non-aqueous
types)
–Precipitation titration
–Complexometric titration and
–Redox titration
53 21-Jun-24

54. Aqueous Acid–Base Titrations
•Most titrations of pharmaceutical ingredients are
acid–base titrations.
•Acid–base titrations are used to assay basic or
acidic pharmaceutical ingredients.
•In aqueous solution, strong acids and bases are
entirely dissociated.
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55. Direct titration
•A known amount of titrant is added from a burrete to a
titrand sample taken in a flask.
•Here one substance is analyzed for its quantity
– by another substance of known volume and concentration.
•Eg. Standardization of 0.1 M hydrochloric acid with
sodium carbonate as primary standard
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56. Back titration
A back titration is a titration method where
the concentration of an analyte is determined
–by reacting it with a known amount of excess reagent.
The remaining excess reagent is then titrated with
another second reagent.
The second titration's results show how much of the
excess reagent was used in the first titration
–the original analyte's concentration can then be calculated.
This methods is also suitable for weakly reactive or non-
reactive substance estimation.
56 21-Jun-24

57. …
•In a regular titration, the original sample is
titrated.
•In a back titration, a known amount of
reagent is added to a solution and allowed
to react, and the excess is titrated.
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58. When do we use back titration?
If the end point of the reverse titration is easier to
identify than the end point of the normal titration.
If the reaction between the analyte and the titrant is
very slow.
Volatile substance: E.g; Ammonia, some of w/c
would be lost during the titration.
Insoluble substance. e.g- require excess volumetric
reagent to effect a quantitative reaction.
Substance w/c requires heating with a volumetric
reagent during
– the determinations in which decomposition or loss of
reactant or product would occur in the process.
E.g: formaldehyde solution
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59. Example:
•What was the percent by mass of aspirin
in the tablets when analyst dissolved four
aspirin tablets with a total mass of 1.427 g
in water and added 50.00 mL of 0.500
mol/L sodium hydroxide solution.
•Then, titrated the excess sodium hydroxide to
a phenolphthalein end-point with 31.92 mL of
0.289 mol/L hydrochloric acid.
Back titration…
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60. …
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61. …
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62. …
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63. 63 21-Jun-24