Punjab University College of Pharmacy,

University of the Punjab, Lahore, Pakistan.

M.Phil. Pharmacognosy (Semester – I)
PCOG – 713 – Pharmacognosy Lab
Final Term Syllabus:

• Analysis of Chemical Structure

• Laboratory Safety
• Laboratory Safety Protocol
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Topic 1. Analysis of Chemical Structure

Overview:
• Spectroscopy Introduction & Types
o Infrared (IR) spectroscopy: What functional groups does the molecule contain?
o Mass spectrometry (MS): What is the atomic weight of the molecule and its common
fragments?
o Ultraviolet-visible (UV-vis) spectroscopy: To what extent does the molecule contain a system
of conjugated pi bonds?

Spectroscopy:
• Spectroscopy is the measurement and interpretation of electromagnetic radiation absorbed or emitted
when the molecules or atoms or ions of a sample move from one energy state to another energy state.
• It is used in qualitative and quantitative analysis for the identification of chemical structures of
compounds through the spectrum emitted from or absorbed by them.

Basis of Spectroscopy:
• It is the study of how electromagnetic radiation, across a spectrum of different wavelengths, interacts
with molecules - and how these interactions can be quantified, analyzed, and ultimately interpreted to
gain information about molecular structure.
• Principle is based on the measurement of spectrum of a sample containing atoms / molecules.
• Spectrum is a graph of intensity of absorbed or emitted radiation by sample verses frequency (ν) or
wavelength (λ).
• Spectrometer is an instrument design to measure the spectrum of a compound.

Types – Based on Principle:

• Absorption Spectroscopy:
o An analytical technique which concerns with the measurement of absorption of
electromagnetic radiation.
o e.g., UV (185 - 400 nm) / Visible (400 - 800 nm) Spectroscopy, IR Spectroscopy (0.76 - 15
μm)
• Emission Spectroscopy:
o An analytical technique in which emission (of a particle or radiation) is dispersed according to
some property of the emission & the amount of dispersion is measured.

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 o e.g., Mass Spectrometry

UV Spectroscopy:
UV spectroscopy is a type of absorption spectroscopy in which light of the ultra-violet region (200-
400 nm) is absorbed by the molecule which results in the excitation of the electrons from the ground state to
a higher energy state.
• It uses electronic transitions to determine the bonding patterns.
• A technique in which light of a shorter wavelength is employed to provide information about organic
molecules containing conjugated p-bonding systems.
• The UV radiation region extends from 10 nm to 400 nm and the visible radiation region extends from
400 nm to 800 nm.
o Near UV Region: 200 nm to 400 nm
o Far UV Region: below 200 nm
o Far UV spectroscopy is studied under vacuum condition.
• The common solvent used for preparing sample to be analyzed is either ethyl alcohol or hexane.
Principle:
• Basically, spectroscopy is related to the interaction of light with matter.
• As light is absorbed by matter, the result is an increase in the energy content of the atoms or molecules.
• When ultraviolet radiations are absorbed, this results in the excitation of the electrons from the ground
state towards a higher energy state.
• Molecules containing π-electrons or nonbonding electrons (n-electrons) can absorb energy in the form
of ultraviolet light to excite these electrons to higher anti-bonding molecular orbitals.
• The more easily excited the electrons, the longer the wavelength of light they can absorb. There are
four possible types of transitions (π–π*, n–π*, σ–σ*, and n–σ*), and they can be ordered as follows:
σ–σ* > n–σ* > π–π* > n–π*
• The absorption of ultraviolet light by a chemical compound will produce a distinct spectrum that aids
in the identification of the compound.
Electronic Transitions:

The possible electronic transitions can graphically shown as:

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Applications:
• Qualitative & Quantitative Analysis:
o It is used for characterizing aromatic compounds and conjugated olefins.
o It can be used to find out molar concentration of the solute under study.
• Detection of impurities:
o It is one of the important method to detect impurities in organic solvents.
• Detection of isomers are possible.
• Determination of molecular weight using Beer’s law.
• It is useful in the structure elucidation of organic molecules, such as in detecting the presence or
absence of unsaturation, the presence of heteroatoms.
• UV absorption spectroscopy can be used for the quantitative determination of compounds that
absorb UV radiation.
• UV absorption spectroscopy can characterize those types of compounds that absorb UV radiation thus
used in the qualitative determination of compounds. Identification is done by comparing the absorption
spectrum with the spectra of known compounds.
• This technique is used to detect the presence or absence of a functional group in the compound. The
absence of a band at a particular wavelength is regarded as evidence for the absence of particular group.
• Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through
the reaction cell and the absorbance changes can be observed.
• Many drugs are either in the form of raw material or in the form of the formulation. They can be
assayed by making a suitable solution of the drug in a solvent and measuring the absorbance at a
specific wavelength.
• Molecular weights of compounds can be measured spectrophotometrically by preparing the suitable
derivatives of these compounds.
• UV spectrophotometer may be used as a detector for HPLC.

IR Spectroscopy:
A technique which is used in organic chemistry to detect the presence or absence of common functional
groups. It measures the bond vibration frequencies in a molecule and is used to determine the functional group.
Infrared (IR) spectroscopy or vibrational spectroscopy is an analytical technique that takes advantage
of the vibrational transitions of a molecule. It is one of the most common and widely used spectroscopic
techniques employed mainly by inorganic and organic chemists due to its usefulness in determining the
structures of compounds and identifying them. The method or technique of infrared spectroscopy is conducted
with an instrument called an infrared spectrometer (or spectrophotometer) to produce an infrared spectrum.
Principle:
• Infrared Spectroscopy is the analysis of infrared light interacting with a molecule.
• The portion of the infrared region most useful for analysis of organic compounds have a wavelength
range from 2,500 to 16,000 nm, with a corresponding frequency range from 1.9*1013 to 1.2*1014 Hz.
• Photon energies associated with this part of the infrared (from 1 to 15 kcal/mole) are not large enough
to excite electrons, but may induce vibrational excitation of covalently bonded atoms and groups.
• It is known that in addition to the facile rotation of groups about single bonds, molecules experience a
wide variety of vibrational motions, characteristic of their component atoms.
• Consequently, virtually all organic compounds will absorb infrared radiation that corresponds in
energy to these vibrations.
• Infrared spectrometers, similar in principle to other spectrometer, permit chemists to obtain absorption
spectra of compounds that are a unique reflection of their molecular structure.

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 • The fundamental measurement obtained in infrared spectroscopy is an infrared spectrum, which is a
plot of measured infrared intensity versus wavelength (or frequency) of light.
• IR Spectroscopy measures the vibrations of atoms, and based on this it is possible to determine the
functional groups.
• Generally, stronger bonds and light atoms will vibrate at a high stretching frequency (wavenumber).
Applications:
• It has been of great significance to scientific researchers in many fields such as:
o Protein characterization
o Nanoscale semiconductor analysis & Space exploration
o Analysis of gaseous, liquid or solid samples
o Identification of compounds
o Quantitative analysis
o Information regarding functional groups of molecules and constitution of molecules can be
deduced from IR spectrum
o To know about interaction among molecules

Mass Spectroscopy:
• Mass Spectrometry (MS) is an analytical chemistry technique that helps identify the amount and type
of chemicals present in a sample by measuring the mass-to-charge ratio and abundance of gas-phase
ions.
• In this instrumental technique, the sample is converted to rapidly moving positive ions by electron
bombardment and charged particles are separated according to their masses.
• A mass spectrum is a plot of relative abundance against the ratio of mass/charge (m/e).
• These spectra are used to determine the elemental or isotopic signature of a sample, the masses of
particles and of molecules, and to elucidate the chemical structures of molecules and other chemical
compounds.
Principle:
• In this technique, molecules are bombarded with a beam of energetic electrons.
• The molecules are ionized and broken up into many fragments, some of which are positive ions. Each
kind of ion has a particular ratio of mass to charge, i.e. m/e ratio (value).
• For most ions, the charge is one, and thus, the m/e ratio is simply the molecular mass of the ion.
• The ions pass through magnetic and electric fields to reach the detector where they are detected and
signals are recorded to give mass spectra
Applications:
• Environmental monitoring and analysis (soil, water, and air pollutants, water quality, etc.)
• Geochemistry – age determination, soil, and rock composition, oil and gas surveying
• Chemical and Petrochemical industry – Quality control
• Identify structures of biomolecules, such as carbohydrates, nucleic acids
• Sequence biopolymers such as proteins and oligosaccharides
• Determination of the molecular mass of peptides, proteins, and oligonucleotides.
• Monitoring gases in patients’ breath during surgery.
• Identification of drug abuse and metabolites of drugs of abuse in blood, urine, and saliva.
• Analyses of aerosol particles.
• Determination of pesticides residues in food.
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 Topic 2. Laboratory Safety
Introduction:
• Laboratory safety rules are a major aspect of every pharmacy lab.
• Each student in pharmacy laboratory must follow specific safety rules and procedures.
Why is Lab Safety Important?
Lab safety rules and symbols are needed so that students do not injure themselves or their classmates.

Lab Safety Rules:

• Wear protective clothing.
o Protective clothes
o Gloves are essential
o Lab coats are required
o Safety glasses (goggles) may be required to avoid splashes
• Laboratory personnel should not wear sandals.
o Do NOT Wear: Jewelry, Loose or Baggy clothing
• Avoid touching objects (e.g., pencils, cell phones, door handles) while wearing gloves.
• Pencils, labels, or any other materials should never be placed in your mouth.
• Caution must be taken when using gas burners. Be sure gas burners are turned off when finished.
• Long hair must be tied back or covered to minimize fire hazard or contamination of experiments.
• Do not eat food or drink water in the lab.
o Do not use lab glassware as food or water containers.
• Protect your hands safety:
o Wash hands after every lab
o Handle glassware, sharp tools and heated containers carefully.
• Electrical safety:
o Unplug electrical equipment after use
o Keep all electrical cords and wires away from water
• Chemical safety:
o Never touch, taste or smell a chemical unless instructed to do so.
o Never mix chemicals unless instructed to do so
o Keep lids on chemical containers when not in use
• Do not take any cultures out of the lab for any reason.
o All cultures should be handled as potentially pathogenic
o Liquid cultures must always be kept in a test tube rack
• Do not engage in practical jokes or horseplay in the lab.
• Keep nonessential books and clothing far away from your work area.
• Wipe the bench tops down with disinfectant both before you begin your work and after you have
completed your work.
• Dispose of waste products according to instructions.
• Report all accidents, no matter how minor, to your supervisor!!

Lab Safety Equipment:

• Safety shower
• Eye wash
• Fire Blanket
• Fire Extinguisher
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Lab Safety Symbols:

Biological Safety Levels:

• BSL – 1:
o Not known to cause disease in healthy adult humans
o Safety equipment:
▪ Minimal requirements
o Facilities:
▪ Open bench top
• BSL – 2:
o Associated with mild to moderate disease in humans
o Safety equipment:
▪ Biological Safety Cabinet and personal protective equipment as needed
o Facilities:
▪ BSL-1 plus the availability of a mechanism for decontamination
• BSL – 3:
o Associated with serious or potentially lethal disease in humans
o Safety equipment:
▪ Biological Safety Cabinet and personal protective equipment required
o Facilities:
▪ BSL-2 with self-closing double door access and single-pass negative directional airflow
• BSL – 4:
o Associated with high risk of life-threatening disease in humans and/or animals
o Safety equipment:
▪ Biological Safety Cabinet
▪ Full-body air-supplied, positive pressure personnel suit
o Facilities:
▪ BSL-3 plus dedicated air and exhaust, decontamination procedures for exit, separate
building, etc.
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Topic 3. Lab Safety Protocol

Muhammad Muneeb (01-MCOG-M22)

Introduction:
• Laboratory safety rules are a major aspect of every pharmacognosy lab.
• Each student in pharmacognosy laboratory must follow specific safety rules and procedures.

Hazards of Clinical Laboratory Work:

Clinical laboratory personnel and employees are subjected to the risk of the followings:
• Biological hazards (infection-infectious agents)
• Chemical hazards
• Physical hazards

Biosafety Levels
Based on the degree of hazard posed by the infectious agents, labs are divided into four biosafety
levels.
Biosafety Level 1:
• Agents are not known to cause disease in healthy adults; however, some organisms may cause disease
in immunocompromised individuals
• Agents include Bacillus subtilis, infectious canine hepatitis virus, non-pathogenic E. coli species
• Standard practices are required for laboratory work at this level and work may be done on an open
bench top
Biosafety Level 2:
• Agents associated with human disease, generally required for any human-derived blood, bodily fluids,
tissues in which infectious agent may be unknown
• Agents include measles virus, Salmonella species, pathogenic Toxoplasma, Clostridium botulinum,
hepatitis B virus
• Biosafety cabinets or other approved containment devices autoclave for glassware proper disposal of
needles and sharp objects
Biosafety level 3:
• Agents with potential for respiratory (aerosol) transmission and may cause serious and potentially
lethal infection
• Agents include Mycobacterium tuberculosis, brucellae, and a wide variety of viruses including
immunodeficiency viruses
• Standard practice required along with a strict controlled access to the lab, special clothing and
decontaminating all waste
Biosafety level 4:
• Dangerous and novel agents with high risk of life-threatening disease, aerosol-transmitted
• Other related agents with unknown risk of transmission are also studied
• All agents are viruses, include Marburg virus, Ebola virus, and Lassa fever
• Maximum containment and decontamination procedures are used in this level laboratories, which is
found in only a few reference and research laboratories

Basic Safety Requirements in Laboratory:

• The laboratory must be kept neat, orderly, clean and the bench tops should be free of non-essential
material
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 • Protective laboratory clothing (Uniforms coats, gowns) must be available and worn properly and
fastened by all personnel including students, visitors, trainees
• Suitable footwear with closed heels and toes and with non-slip soles should be worn in the laboratory
• Long hair must be tied back or restrained
• Do not bring food, beverages or tobacco products into the laboratory
• Do not apply cosmetics in the laboratory
• Do not eat or drink in the laboratory
• Oral pipetting is laboratory prohibited. Use appropriate pipetting devices that bypass use of the mouth
• Clean your workbench with disinfectant at the beginning and at the end of the laboratory exercise.
Report any spilled reagent or culture however minor, to the instructor
• Wash your hands with soap and water before and after the lab. Don’t use towels
• Never remove cultures, reagents, or other materials from the laboratory unless you have been granted
specific permission
• Inoculated media placed in the incubator must be properly labeled with your name, date, and nature of
the specimen
• All reagents and equipment must be returned to their proper place at the end of the lab
• Pencils, labels, or any other materials should never be placed in your mouth
• Caution must be taken when using gas burners. Be sure gas burners are turned off when finished
• All used cultures and contaminated glassware should be put into a designated container to be
autoclaved
• Contaminated plastic and other disposables are to be discarded into a separate container also to be
autoclaved

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