This is an introduction to instrumental analysis, mainly hinged on validation, error identification and minimization.

1. INTRODUCTION
By Hamu Ndwabe
B Pharm, M Pharm

2. Introduction
• Instrumental analysis is a component of analytical chemistry
that uses instruments to analyze particles and molecules.
• Classification of Analytical Methods
• Qualitative analysis gives an indication of the identity of
the chemical species in the sample and
• Quantitative analysis determines the amount of one or
more of these components

3. Sources of errors
• Incorrect weighing and transfer of analytes and standards
• Inefficient extraction of the analyte from a matrix, e.g. tablets
• Incorrect use of pipettes, burettes or volumetric flasks for volume
measurement
• Measurement carried out using improperly calibrated instrumentation
• Failure to use an analytical blank
• Selection of assay conditions that cause degradation of the analyte
• Failure to allow for or to remove interference by excipients in the
measurement of an analyte

4. Quality of analytical procedures
The International Conference on Harmonisation (ICH) has adopted the following terms for defining how
the quality of an assay is controlled.
The analytical procedure provides an exact description of how the analysis is carried out. It should
describe in detail the steps necessary to perform each analytical test. The full method should describe:
(i) the quality and source of the reference standard for the compound being analysed
(ii) the procedures used for preparing solutions of the reference standard
(iii) the quality of any reagents or solvents used in the assay and their method of preparation
(iv) the procedures and settings used for the operation of any equipment required in the assay
(v) the methodology used for calibration of the assay and methodology used for the processing of the
sample prior to analysis.

5. Selecting an analytical method
• How reproducible? - Precision
• How close to true value? - Accuracy/Bias
• How small a difference can be measured? - Sensitivity
• What range of amounts? - Dynamic Range
• How much interference? - Selectivity
• How many samples? – Efficience (time, money cost)

6. Precision
• the precision of an analytical procedure expresses the closeness of
agreement (degree of scatter) between a series of measurements obtained
from multiple sampling of the same homogeneous sample under the
prescribed conditions.
• Precision may be considered at three levels: repeatability, intermediate
precision and reproducibility.
a. Repeatability Repeatability expresses the precision under the same
operating conditions over a short interval of time. Repeatability is also
termed intra-assay precision .
b. Intermediate precision
Intermediate precision expresses within-laboratories variations:
different days, different analysts, different equipment, etc.
c. Reproducibility
Reproducibility expresses the precision between laboratories (collaborative
studies, usually applied to standardization of methodology)

7. • The Accuracy of an analytical procedure expresses the closeness of agreement
between the value which is accepted either as a conventional true value or an
accepted reference value and the value found. This is sometimes termed
trueness.
• Specificity is the ability to assess unequivocally the analyte in the presence of
components which may be expected to be present. Typically these might include
impurities, degradants, matrix
• No analytical method is completely free from interference by concomitants. Best
method is more sensitive to analyte than interfering species (interferent). The
sensitivity of method indicates how responsive it is to a small change in the
concentration of an analyte.
• LINEARITY The linearity of an analytical procedure is its ability (within a given
range) to obtain test results which are directly proportional to the concentration
(amount) of analyte in the sample.

8. • DETECTION LIMIT The detection limit of an individual analytical procedure is the lowest amount of
analyte in a sample which can be detected but not necessarily quantitated as an exact value.
• QUANTITATION LIMIT The quantitation limit of an individual analytical procedure is the lowest
amount of analyte in a sample which can be quantitatively determined with suitable precision and
accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds
in sample matrices, and is used particularly for the determination of impurities and/or
degradation products.
• ROBUSTNESS The robustness of an analytical procedure is a measure of its capacity to remain
unaffected by small, but deliberate variations in method parameters and provides an indication of
its reliability during normal usage. Robustness. The types of parameters which are assessed in
order to determine the robustness of a method include: the stability of analytical solutions; the
length of the extraction time; the effect of variations in the pH of a HPLC mobile phase; the effect
of small variations in mobile phase composition; the effect of changing chromatographic columns;
the effect of temperature and flow rate during chromatography.
• RANGE The range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including these concentrations) for which it has
been demonstrated that the analytical procedure has a suitable level of precision, accuracy and
linearity. When applied to the performance of an assay, it refers to the interval between the upper
and lower concentration of an analyte for which an acceptable level of precision and accuracy has
been established.

9. Error minimization
• Analyst has no control on random errors but systemic errors can be reduced
by following methods.
Calibration of apparatus: By calibrating all the instruments, errors can be
minimized and appropriate corrections are applied to the original
measurements.
Control determination: standard substance is used in experiment in
identical experimental condition to minimize the errors.
Blank determination: By omitting sample, a determination is carried out in
identical condition to minimize the errors occurs due to impurities present
in reagent.

10. Error minimization….
Independent method of analysis: It is carried out to maintain accuracy of the
result e. g. Iron (III) is first determined gravimetrically by precipitation method
as iron (III) hydroxide and then determined titrimetrically by reduction to the
iron (II) state.
Parallel determination: Instead of single determination, duplicate or triplicate
determination is carried out to minimize the possibilities of accidental errors.
Standard edition: This method is generally applied to physico-chemical
procedures such as polarography and spectrophotometry.
Internal standards: It is used in spectroscopic and chromatographic
determination.

11. Error analysis case study
• A batch of paracetamol tablets are stated to contain 500 mg of
paracetamol per tablet; for the purpose of this example it is
presumed that 100% of the stated content is the correct answer. Four
students carry out a spectrophotometric analysis of an extract from
the tablets and obtain the following percentages of stated content for
the repeat analysis of paracetamol in the tablets:
Student 1: 99.5%, 99.9%, 100.2%, 99.4%, 100.5%
Student 2: 95.6%, 96.1%, 95.2%, 95.1%, 96.1%
Student 3: 93.5%, 98.3%, 92.5%, 102.5%, 97.6%
Student 4: 94.4%, 100.2%, 104.5%, 97.4%, 102.1%

12. • Student 1: Precise and accurate
• Student 2: Precise and inaccurate
• Student 3: Imprecise and inaccurate
• Student 4: Imprecise and accurate