ICH 461: SPECIAL LABORATORY METHODS

Topic 1: LABORATORY MANIPULATIVE SKILLS

Manipulative skills can be defined as psychomotor skills that relate individual cognitive function
with corresponding physical movement.
Manipulative skills play an important role for students to be able to complete science activities
effectively. In order to acquire experience in manipulating specific scientific apparatus, it is
important for students to perform various experiments using an apparatus repeatedly.
Good technique in handling and manipulating scientific apparatus is important because it can
reduce, minimize, and control misinterpretations and may minimize the error in scientific
experiments.
In science, manipulative skills emphasize the use and handling of scientific apparatus and
chemical substances during scientific investigation in the laboratory. In addition, students are
exposed to the proper technique for using, cleaning, and storing scientific equipment safely.

BASIC MANIPULATIVE SKILLS

Ability to recognize apparatus and its function
Recognition is necessary as the first step of being able to use tools or materials effectively. Once
the apparatus and its parts have been recognized, it is possible to relate it to other important
information.

Ability to identify parts and features of apparatus and its function

This category focused on the students’ ability to identify every part and feature of an apparatus
and its function. The apparatus must be distinguished in order for the students to master the
technical skills of using it.
An understanding of the basic principles of using and handling apparatus
Basic principles of using and handling apparatus in this context can be defined as fundamental
rules that the students must follow to ensure the correct result is obtained from execution of the
task. Inability to follow the rules may prevent the students from obtaining accurate results for the
experiment and can put their safety at risk. Students must take adequate precautions to ensure
reliable observations and results.
Appropriate approaches to minimize standard errors during measurement in using graduated
apparatus
This component explores the approach used by the students to minimize the standard error when
using graduated apparatus in order to obtain accurate measurements during the experiment.
Correct sequences in using the sequential apparatus
The competency in sequencing can be related to student awareness in implementing appropriate
precautionary measures during the using and handling of an apparatus. The suitable sequence of
using the apparatus should be followed by the student in order to be familiar with the apparatus,
which in turn will lead to greater efficiency in handling.
Ability to complete the task efficiently
This component described the students’ ability to use the apparatus efficiently and confidently. It
involved two criteria: the mode of action in manipulating the apparatus and the level of guidance
in performing technical skills. The skillful performance of technical skills involves complex
movement. The students’ proficiency in manipulative skills is indicated by a quick, accurate, and
highly coordinated performance. It can be recognized by their ability to use the apparatus
efficiently and confidently.
Five (5) levels hierarchy of technical skills

TOPIC 2: TECHNIQUES IN CHEMICAL ANALYSIS

There are a very large number of techniques used in chemical analysis. It
can be very useful to
classify the measurement process according to a variety of criteria:
 by the type of analytical technique – classical or instrumental
techniques;
 by the nature of the measurement data generated – single-channel
or multi-channel techniques; and
 by the quantitation method (by which the analyte concentration is
calculated) – relative or absolute techniques.

Classical vs Instrumental Techniques

In classical analysis, the signal depends on the chemical properties of the
sample: a reagent reacts completely with the analyte, and the relationship
between the measured signal and the analyte concentration is determined
by chemical stoichioimetry.
In instrumental analysis, some physical property of the sample is
measured, such as the electrical potential difference between two electrodes
immersed in a solution of the sample, or the ability of the sample to absorb
light.
 Classical methods are most useful for accurate and precise
measurements of analyte concentrations at the 0.1% level or higher.
On the other hand, some specialized instrumental techniques are
capable of detecting individual atoms or molecules in a sample!
Analysis at the ppm (μg/mL) and even ppb (ng/mL) level is routine.

Single-Channel vs Multi-Channel Techniques

Another useful distinction between analytical techniques is based on the
information content of the data generated by the analysis:
 single-channel techniques will generate but a single number for each
analysis of the sample. Examples include gravimetric and potentiometric
analysis. In the former, the signal is a single mass measurement (e.g.,
mass of the precipitate) and in the latter method the signal is a single
voltage value.
 multi-channel techniques will generate a series of numbers for a single
analysis. Multi-channel techniques are characterized by the ability to
obtain measurements while changing some independently controllable
parameter. For example, in a molecular absorption method, an absorption
spectrum may be generated, in which the absorbance of a sample is
monitored as a function of the wavelength of the light transmitted
 through the sample. Measurement of the sample thus produces a series
of absorbance values.

Relative vs Absolute Techniques

Another way of classifying analytical techniques is according to the method
by which the analyte
concentration is calculated from the data:
 in absolute analytical techniques, the analyte concentration can be
calculated directly from measurement of the sample. No additional
measurements are required (other than a measurement of sample
mass or volume).
 in relative analytical techniques, the measurement of the sample must
be compared to measurements of additional samples that are
prepared with the use of analyte standards (e.g., solutions of known
analyte concentration).

Methods of Quantitation for Instrumental Analysis

Instrumental techniques are almost all relative in nature: the signal obtained
from the analysis of
the sample must be compared to other measurements in order to determine
the analyte concentration in the sample. Since these other measurements
naturally contain measurement error, relative quantitation increases the
overall error in the estimate of analyte concentration –we shall refer to this
source of error as calibration error. Calibration error can contain both
random and systematic components.
One of the advantages of classical methods over instrumental methods is the
absence of calibration error, since classical methods are absolute in nature.

The two most common methods of calibration in instrumental analysis are

(i) the use of calibration curves
(ii) the method of standard additions.
In addition, internal standards may be used in combination with either of
these methods.

Interferences in Quantitative Analysis

Ideally, the only property of a sample that affects the data collected from an
analytical procedure
is the concentration of the analyte in the analyte. Inevitably, there will be
other properties that will affect the measurements obtained in chemical
analysis; common examples of such properties include temperature, pH,
ionic strength, or solution turbidity.
Changes in these properties can thus also cause changes in the
measurements that are unrelated to analyte concentration, leading to errors;
such properties are thus called interferences, since they “interfere” with
the proper determination of analyte concentration.
Interferences may be broadly classified into two types:
1. chemical interferences, which are due to the presence of specific
chemicals in the sample, and
2. physical interferences. The effects of changing ionic strength or pH are
examples of chemical effects, while that of temperature or turbidity
are physical phenomenon.

Correction for Interferences: General Methods

Elimination
The most straightforward method of dealing with interferences is to
eliminate the source, if
possible. This may include changing procedure or equipment etc.
Control
In some cases, elimination of interferences is either not possible or too
difficult. For example, the
analyte signal is often affected by temperature of pH, and these effects
cannot be removed. In
such cases, it is possible to eliminate errors caused by the interferences by
simply controlling the
effect of the interference. For example, if pH affects the signal, we would
buffer all standards and samples to the same pH value.
Correction
There are situations where interference can neither be completely eliminated
nor controlled. For
example, in environmental analysis it is often desirable to perform rapid “on-
site” analysis, rather than transporting samples back to a laboratory.
 Topic 3: FUNCTIONAL GROUP PROTECTION AND DE-PROTECTION
Protection and deprotection is an important part of organic synthesis. During the course of
synthesis, we may want to perform reaction at only one of the two functional groups in any
single organic molecules.
The protecting groups allow the masking of a particular functional group where a specified
reaction is not to be performed. A protecting group or protective group is introduced into a
molecule by chemical modification of a functional group to obtain chemoselectivity in a
subsequent chemical reaction.
Chemoselectivity is the preferential outcome of a chemical reaction over a set of possible
alternative reactions. In another definition, chemoselectivity refers to the selective reactivity of
one functional group in the presence of others.
What is a protecting group?
A protecting group (PG) is a molecular framework that is introduced onto a specific functional
group (FG) in a poly-functional molecule to block its reactivity under reaction conditions needed
to make modifications elsewhere in the molecule.
Protecting groups are more commonly used in small-scale laboratory work and initial
development than in industrial production processes because their use adds additional steps and
material costs to the process. However, the availability of a cheap chiral building block can
overcome these additional costs.

The characteristics of protecting groups

1. It must be easy to put in

2. It must be resistant to reagents that would attack the unprotected function group.
3. It must be easily removed

In practical terms the use of protection groups adds two steps (protection-deprotection sequence)
to a synthesis, either or both of which can dramatically lower chemical yield. Crucially, added
complexity impedes the use of synthetic total synthesis in drug discovery. In contrast biomimetic
synthesis does not employ protective groups.

Protecting Groups in Organic Synthesis

The most reactive functional groups requiring protection include the following:

These commonly encountered functional groups in organic synthesis are reactive to nucleophilic
or electrophilic reagents whose selective transformation may present challenges and so regularly
require deactivation by masking with a protecting group.

Protecting Groups for Alcohols

The common protecting groups for alcohols are ether-protecting groups. Ethers are among the
least reactive of the organic functional groups. These protections replace the acidic proton on an
alcohol with an unreactive ether moiety.
Acetyl (Ac) – Removed by acid or base (see Acetoxy group).
Benzoyl (Bz) – Removed by acid or base, more stable than Ac group.
Benzyl (Bn) – Removed by hydrogenolysis.
Trityl (triphenylmethyl, Tr) – Removed by acid and hydrogenolysis.
Silyl ether (most popular ones include trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS),
tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers) – Removed by acid or
fluoride ion. (such as NaF, TBAF (tetra-n-butylammonium fluoride, HF-Py, or HF-NEt3)).
TBDMS and TOM groups are used for protection of 2'-hydroxy function in nucleosides,
particularly in oligonucleotide synthesis.
Methoxymethyl ether (MOM) – Removed by acid.
Methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT) – Removed by acid and
hydrogenolysis.
p-Methoxybenzyl ether (PMB) – Removed by acid, hydrogenolysis, or oxidation.
p-Methoxyphenyl ether (PMP) – Removed by oxidation.
Methylthiomethyl ether – Removed by acid.

Carbonyl protecting groups

Acetals and Ketals – Removed by acid. Normally, the cleavage of acyclic acetals is easier than
of cyclic acetals.
Acylals – Removed by Lewis acids.
Dithianes – Removed by metal salts or oxidizing agents.

Amine protecting groups

Carbobenzyloxy (Cbz) group – Removed by hydrogenolysis

p-Methoxybenzyl carbonyl (Moz or MeOZ) group – Removed by hydrogenolysis, more labile
than Cbz
tert-Butyloxycarbonyl (BOC) group (common in solid phase peptide synthesis) – Removed by
concentrated strong acid (such as HCl or CF3COOH), or by heating to >80 °C.
9-Fluorenylmethyloxycarbonyl (Fmoc) group (Common in solid phase peptide synthesis) –
Removed by base, such as piperidine
Benzoyl (Bz) group is common in oligonucleotide synthesis for protection of N4 in cytosine and
N6 in adenine nucleic bases and is removed by treatment with a base, most often with aqueous or
gaseous ammonia or methylamine. Bz is too stable to be readily removed from aliphatic amides.
Benzyl (Bn) group – Removed by hydrogenolysis

Carboxylic acid protecting groups

Methyl esters – Removed by acid or base.
Benzyl esters – Removed by hydrogenolysis.
tert-Butyl esters – Removed by acid, base and some reductants.
Esters of 2,6-disubstituted phenols (e.g. 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-
butylphenol) – Removed at room temperature by DBU-catalyzed methanolysis under high-
pressure conditions.
Silyl esters – Removed by acid, base and organometallic reagents.
Orthoesters – Removed by mild aqueous acid to form ester, which is removed according to ester
properties.
Oxazoline – Removed by strong hot acid (pH < 1, T > 100 °C) or alkali (pH > 12, T > 100 °C),
but not e.g. LiAlH4, organolithium reagents or Grignard (organomagnesium) reagents

Phosphate protecting groups

2-cyanoethyl – removed by mild base. The group is widely used in oligonucleotide synthesis.
Methyl (Me) – removed by strong nucleophiles thiophenole/TEA.

Orthogonal protection is a strategy allowing the specific deprotection of one protective group
in a multiple-protected structure without affecting the others. For example, the amino acid
tyrosine could be protected as a benzyl ester on the carboxyl group, a fluorenylmethylenoxy
carbamate on the amine group, and a tert-butyl ether on the phenol group. The benzyl ester can
be removed by hydrogenolysis, the fluorenylmethylenoxy group (Fmoc) by bases (such as
piperidine), and the phenolic tert-butyl ether cleaved with acids (e.g. with trifluoroacetic acid).