FACULTY OF PHARMACY

KARPAGAM ACADEMY OF HIGHER EDUCATION

(Deemed University Under Section 3 of UGC Act
1956) Eachanari Post, Pollachi Main Road,
Coimbatore-641021.

MODERN PHARMACEUTICAL ANALYTICAL

TECHNIQUES
PRACTICAL (24MPA106P)

M.PHARM DEGREE COURSE (2024-2025)

FIRST YEAR – SEMESTER I

I
 FACULTY OF PHARMACY

KARPAGAM ACADEMY OF HIGHER EDUCATION

(Deemed University Under Section 3 of UGC Act
1956) Eachanari Post, Pollachi Main Road,
Coimbatore-641021.

PROGRAM :

COURSE :

SUBJECT CODE :

NAME OF THE STUDENT:

REG. NO. :

ACADEMIC YEAR :

II
 FACULTY OF PHARMACY

KARPAGAM ACADEMY OF HIGHER EDUCATION

(Deemed University Under Section 3 of UGC
Act 1956) Eachanari Post, Pollachi Main Road,

Coimbatore-641021.

CERTIFICATE
Register No. ……………….
Certified to be the bonafide record of the work done by ..................................................... of
M.Pharm ...................... semester prescribed by the Karpagam Academy of Higher Education
in the subject of ……………………………………………... practical during the year
……………..…. at Faculty of Pharmacy, KAHE.

Signature of Staff in Charge Signature of DEAN

Signature of HOD

DATE:

Submitted for the practical examination held on ………………….

External Examiner Internal Examiner

III
 CONTENTS
DATE EXPERIEMENTS PAGE MARKS SIGNATURE
NO
1. ANALYSIS OF PHARMACOPOEIAL
COMPOUNDS AND THEIR FORMULATIONS
BY UV VIS SPECTROPHOTOMETER
2. SIMULTANEOUS ESTIMATION OF
MULTICOMPONENT CONTAINING
FORMULATIONS BY UV
SPECTROPHOTOMETRY
3. EXPERIMENTS BASED ON COLUMN
CHROMATOGRAPHY
4. EXPERIMENTS BASED ON HPLC

5. EXPERIMENTS BASED ON GAS

CHROMATOGRAPHY
6. ESTIMATION OF VARIOUS DRUGS BY
SPECTRO FLUORIMETRY
6. ESTIMATION OF SODIUM/POTASSIUM
LEVEL BY FLAME PHOTOMETRY
7. ESTIMATION BASED ON FLASH
CHROMATOGRAPHY
9. EXPERIMENTS BASED ON LC-MS

10. EXPERIMENTS BASED ON IR

SPECTROSCOPY
11. EXPERIMENTS BASED ON HPTLC

12. INTERPRETATION OF ANY TWO ORGANIC

COMPOUNDS BY HNMR

IV
1
MODERN PHARMACEUTICAL
ANALYTICAL TECHNIQUE

2
 EXPERIMENT: 01 DATE:

ANALYSIS OF PHARMACOPOEIAL COMPOUNDS AND THEIR

FORMULATIONS BY UV VIS SPECTROPHOTOMETER

AIM:
To determine the Percentage purity of the given Paracetamol Tablets by UV vis
spectrophotometry.

PRINCIPLE:
Acetaminophen (N-acetyl-p-amino-phenol) also known as paracetamol is a
widespread antipyretic and analgesic accepted as an effective treatment for the relief of
pain and fever in adults and children Paracetamol is assayed by UV Spectrophotometry
using 0.1 N NaOH as solvent and measure the absorbance of the solution at λ max 257
nm taking as the value of A1% 1cm.

PROCEDURE:
Preparation of Blank:
 Weigh 20 tablets and note the average weight of 20 tablets.
 Accurately weigh a quantity of powder equivalent to 0.15 g of paracetamol into
250 ml volumetric flask. Add 50 ml of 0.1N sodium hydroxide, dilute to 100 ml
with water, shake for 15 minutes and make up the volume with distilled water.
[SOLUTION A]
 Pipette 5 ml of Solution A into 50 ml volumetric flask and make up the volume
with distilled water. [SOLUTION B]
 Pipette 5 ml of Solution B into 50 ml volumetric flask, add 5 ml of 0.1N NaOH
and make up the volume with distilled water. [SOLUTION C] Pipette out 5 ml
of 0.1N NaOH into 50 ml volumetric flask and make up the volume with distilled
water.

Determination of Absorbance:
 The Spectrophotometer is switched on and allowed to stabilize for 15 minutes.
Baseline correction is done using blank and absorbance of the resulting solution
C was measured at 257 nm.

3
  The Percentage purity was calculated by taking 715 as A (1%, 1cm) value at
the maximum at about 257 nm.

REPORT:
The percentage purity of the given sample paracetamol was found

4
 EXPERIMENT: 02 DATE:

SIMULTANEOUS ESTIMATION OF MULTICOMPONENT CONTAINING

FORMULATIONS BY UV SPECTROPHOTOMETRY

AIM
To determine diclofenac and paracetamol by simultaneous equation method.
MATERIALS REQUIRED:
Paracetamol, Diclofenac and Methanol
PRINCIPLE:
If the solution contains more than one or several absorbing species, the
absorbance of this solution is measured by spectrophotometer with the sum of
absorbance of all the species, i.e., components/species present in the solution, do not
interact with each other. If the components x and y are present in the multi-component
drug whose molecular weight is known, the following equation can be used:
Simultaneous Equation:
Cx = A2 ay1 – A1 ay2 / ax2ay1 – ax1ay2 Cy = A1ax2 – A2ax1 / ax2ay1 –
ax2ay1
According to Beer-Lambert’s Law:
A= €ct
[t: thickness in one cm] A= €c
PROCEDURE:
Standard solution of paracetamol:
 Weigh accurately 10mg of paracetamol in a 100ml volumetric flask, add
6ml of methanol and dissolve, make up the volume to 100ml with methanol.
 Pipette out 1ml of the above solution into a 10ml volumetric flask and make up
the Volume with methanol. (Solution A)

5
Standard solution of Aceclofenac:
 Weigh accurately about 10mg of aceclofenac in 100ml volumetric flask, add
6ml of methanol and dissolve, and make up the volume to 100ml with
methanol.
 Pipette out 1ml of the above solution into a 10ml volumetric flask and makeup
the volume with methanol (solution B)
Preparation of sample solution:
Pipette out 1ml of the given sample solution in 10ml volumetric flask and
make up the volume with methanol (solution C)
Determination of absorbance:
 The spectrophotometer is switched on and allowed to stabilize for 15 minutes
Base line correction is done using blank.
 Scan solution A and solution B in wave length range of 400- 200nm.
 Determine the λmax of solution A and Solution B.
 Measure the absorbance of solution A at λ1 and λ2 and calculate the
absorptivity. [paracetamol – ax1, ax2]

 Measure the absorbance of solution B at λ1 and λ2 and calculate the

absorptivity [Aceclofenac- ay1, ay2]
 Measure the absorbance of solution C [sample solution] at λ1 and λ2 (A1 and
A2)
 Calculate the Concentration of paracetamol and Aceclofenac present in the
given. sample solution C.

REPORT:

6
 EXPERIMENT: 03 DATE:

EXPERIMENTS BASED ON COLUMN CHROMATOGRAPHY

AIM:
To demonstrate the principles of column chromatography and the separation
of a
mixture of compounds using a chromatographic column.
MATERIALS REQUIRED:
 Chromatography column (glass or plastic)
 Silica gel or other suitable stationary phase
 Mixture of compounds to be separated
 Solvents (e.g., hexane, ethyl acetate, methanol)
 Glass wool or fritted disc
 Sample applicator (e.g., pipettes, syringes)
 Collection tubes or flasks
 UV lamp or other visualization techniques (optional)
 Safety goggles and gloves
 Funnel and filter paper (for solvent filtration
SAFETY PRECAUTIONS:
 Safety goggles and gloves are essential to protect against potential chemical
splashes and skin contact with solvents, which can cause irritation or harm.
 Conducting the experiment in a well-ventilated area or under a fume hood helps
minimize exposure to solvent vapors, which can be harmful if inhaled in high
concentrations.
 Familiarize yourself with the Safety Data Sheets (SDS) for all chemicals used in
the experiment to understand their potential hazards and proper handling.
COLUMN PACKING:

7
 The selection of an appropriate stationary phase, such as silica gel, is crucial for
effective separation. Silica gel is commonly used due to its polarity and ability
to interact with a wide range of compounds.
 Packing the column evenly and without air bubbles ensures consistent flow of
the solvent and optimal separation efficiency.
 The addition of glass wool or a fritted disc at the bottom of the column helps
support the stationary phase and prevent it from flowing out of the column during
elution.
SAMPLE LOADING:
 Dissolving the mixture of compounds in a suitable solvent is necessary to create
a homogeneous sample solution for application onto the column.
 Carefully applying the sample onto the stationary phase ensures that it is evenly
distributed and does not spread out too much, which could affect separation
efficiency.
 Proper sample loading technique is important to prevent sample breakthrough or
overloading, which can result in poor separation and loss of resolution.
ELUTION:
 The choice of solvent system depends on the polarity of the compounds in the
mixture. A gradient of solvents or a single solvent of appropriate polarity can be
used to elute the compounds from the column.
 Elution is typically performed by gravity flow, allowing the solvent to pass
through the column at a controlled rate. This ensures adequate interaction
between the compounds and the stationary phase for separation to occur.

 Monitoring the elution process visually allows the operator to observe the
movement of the solvent front down the column and make adjustments as needed
to optimize separation.
FRACTION COLLECTION:
 Collecting fractions in separate tubes or flasks enables the isolation of individual
compounds or groups of compounds for further analysis.
 Fraction collection can be based on either volume or time intervals, depending
on the elution profile of the compounds and the desired level of resolution.

8
 Visualization techniques such as UV lamps can be used to detect fluorescent
compounds, aiding in fraction collection and analysis.
ANALYSIS:
 Analyzing each fraction using complementary analytical techniques such as thin-
layer chromatography (TLC), gas chromatography (GC), or high-performance
liquid chromatography (HPLC) allows for the identification and quantification of
separated compounds.
 Comparing the retention factors (Rf values) of the compounds in the fractions
with those of reference standards helps confirm their identities and assess the
success of the separation.
FRACTION COMBINING:
 Combining fractions containing similar compounds helps concentrate the
separated compounds and reduce the number of fractions for further analysis.
 Careful consideration should be given to the selection of fractions to combine
based on preliminary analysis results to avoid contamination or loss of purity.
CLEANUP:
 Proper disposal of used solvents and chromatography materials according to
laboratory waste disposal guidelines is essential to minimize environmental
impact and ensure safety.

 Thoroughly cleaning the chromatography column with an appropriate solvent

removes any residual stationary phase or sample material and helps maintain its
performance for future use.
 By incorporating these detailed explanations and considerations, students can
gain a comprehensive understanding of column chromatography principles and
procedures, enhancing their knowledge and skills in analytical chemistry
technique.

REPORT:

9
EXPERIMENT: 04 DATE:

EXPERIMENTS BASED ON HPLC

AIM:
To analyze the given sample (paracetamol) using high performance
liquid chromatography (HPLC).
PRINCIPLE:
High-Performance Liquid Chromatography (HPLC) is a powerful analytical
technique used to separate, identify, and quantify components in a mixture based on
their interactions with a stationary phase and a mobile phase. In HPLC, the
stationary phase is typically packed into a column, while the mobile phase flows
through the column, carrying the sample components with it.
1. Stationary Phase: The stationary phase is a solid or liquid material that is
immobilized inside the chromatography column. It interacts with the sample
components based on various mechanisms such as adsorption, partitioning, size
exclusion, or ion exchange. In the case of paracetamol analysis, a commonly used
stationary phase is a C18 column, where the packing material consists of
hydrophobic alkyl chains bonded to the silica support.
2. Mobile Phase: The mobile phase is a solvent or solvent mixture that flows
through the column, carrying the sample components along. The choice of mobile
phase composition, pH, and flow rate is crucial for achieving efficient separation of
analytes. Common mobile phase solvents include water, acetonitrile, and methanol.
In paracetamol analysis, a suitable mobile phase might consist of a mixture of water
and an organic solvent like acetonitrile.
3. Separation Mechanism: The separation of components in HPLC occurs based
on their differential interactions with the stationary phase and mobile phase.
Components with stronger interactions with the stationary phase spend more time in
the column, resulting in longer retention times, while components with weaker
interactions elute more quickly. In the case of paracetamol, separation is achieved
based on differences in polarity, hydrophobicity, and other molecular properties.
4. Detection: After separation, the components eluting from the column are detected
by a detector, commonly a UV-Vis detector, which measures absorbance at a
specific

10
wavelength. Paracetamol absorbs UV light at a wavelength of around 243 nm,
making UV detection suitable for its analysis.
5. Quantification: The concentration of paracetamol in the sample is determined
by measuring the peak area or height in the chromatogram. The peak area or height
is proportional to the concentration of the analyte in the sample. Quantification is
typically performed by comparing the peak area or height of the analyte to that of a
known concentration standard.
Materials and Equipment:
 Waters Alliance HPLC instrument
 HPLC-grade solvents (e.g., acetonitrile, water)
 HPLC column (e.g., C18 column)
 Sample vials
 Analytical balance
 Pipettes and pipette tips
 Sample containing paracetamol
PROCEDURE:
1. Column Equilibration:
Before sample analysis, it's crucial to ensure that the HPLC column is
properly equilibrated with the mobile phase under the desired operating conditions.
This step involves flushing the column with the mobile phase for a sufficient period
to stabilize the chromatographic system and achieve reproducible retention times.
Equilibration conditions such as flow rate, temperature, and solvent composition
should be optimized for the specific column and sample being analyzed.
2. Sample Preparation:
Prepare the sample solution containing paracetamol by accurately weighing a
suitable amount of the sample and dissolving it in a solvent compatible with the
mobile phase. If necessary, the sample may need to be filtered to remove particulate
matter or other impurities that could interfere with the analysis. The concentration
of the sample solution should be adjusted to fall within the linear range of the
calibration curve.

11
3. Instrument Setup:
Configure the Waters Alliance HPLC instrument according to the established
method parameters. This includes setting the injection volume, column temperature,
mobile phase flow rate, and detection wavelength. Ensure that the instrument is
properly calibrated and that all components are functioning correctly before
proceeding with the analysis.
4. Sample Injection and Analysis:
Inject the prepared sample solution into the HPLC instrument using an
autosampler or manual injection system. The injection volume should be optimized
to ensure accurate and reproducible results. The sample is then carried by the mobile
phase into the column, where separation occurs based on the interactions with the
stationary phase. Paracetamol and other components in the sample are eluted from
the column and detected by the UV-Vis detector.
5. Data Acquisition and Analysis:
Acquire chromatographic data using the HPLC software, which displays
chromatograms showing the signal intensity (peak area or height) as a function of
retention time. Identify and quantify the peak corresponding to paracetamol by
comparing its retention time and peak characteristics with those of the standard
solutions. Calculate the concentration of paracetamol in the sample using the
calibration curve or external standard method.
6. Data Interpretation and Reporting:
Analyze the chromatographic data to ensure the accuracy and reliability of
the results. Evaluate the chromatogram for peak purity, symmetry, and resolution.
Prepare a report summarizing the experimental procedure, including details of sample
preparation, chromatographic conditions, and data analysis. Present the results of
the paracetamol analysis, including the calculated concentration and any relevant
observations or conclusions.

REPORT:

12
 EXPERIMENT: 05 DATE:

EXPERIMENTS BASED ON GAS CHROMATOGRAPHY

AIM:
To demonstrate the principles of gas chromatography (GC) and the
separation of
a mixture of compounds using a gas chromatograph.
MATERIALS REQUIRED:
 Gas chromatograph instrument
 Injection syringe
 Gas chromatography columns (packed or capillary)
 Sample vials containing mixture of compounds (e.g., a mixture of alcohols,
alkanes, or fragrances)
 Carrier gas (e.g., helium, nitrogen)
 Detector (e.g., flame ionization detector, mass spectrometer)
 Computer with chromatography software (if applicable)
 Safety goggles and gloves
SAFETY PRECAUTIONS:
 Safety goggles and gloves should be worn to protect eyes and skin from
potential chemical splashes or contact.
 Ensure the experiment is conducted in a well-ventilated area to prevent the
buildup of volatile fumes.

 Familiarize yourself with the Material Safety Data Sheets (MSDS) for all
chemicals used in the experiment to understand their potential hazards and
proper handling procedures.
INSTRUMENT SETUP:
Gas chromatography instruments consist of various components, including an injection
port, column, detector, and data acquisition system

13
  Allow sufficient time for the instrument to reach its operating
temperature, typically around 200-300°C for the injection port and column.
 Check that the carrier gas flow rate is within the recommended range to
ensure efficient separation of compounds.
 Verify that the detector is properly calibrated to ensure accurate detection
and quantification of analytes.
SAMPLE PREPARATION:
 Carefully select and prepare the sample to be analyzed, ensuring it
is representative of the mixture under investigation.
 Sample preparation techniques such as dilution or derivatization may
be necessary to improve analyte detection and separation.
 Label sample vials with relevant information such as the composition of
the mixture, concentration, and any pre-treatment steps undertaken.
INJECTION:
 The injection of the sample into the gas chromatograph must be done swiftly
to minimize sample degradation and ensure reproducible results.
 Proper injection technique is essential to prevent sample carryover
and contamination of subsequent analyses.
 Avoid overloading the column with sample, as this can lead to peak
broadening and reduced resolution.
SEPARATION:
 Gas chromatography separates compounds based on differences in their
volatility and affinity for the stationary phase.
 The stationary phase, typically a high-boiling-point liquid or solid coated
onto the column, interacts with analytes as they pass through the column.
 Compounds with higher volatility elute from the column faster than those
with lower volatility, resulting in separation based on retention time.

14
DETECTION:
 Gas chromatography detectors measure the concentration of analytes as they
elute from the column.
 Common detectors include flame ionization detectors (FID), thermal
conductivity detectors (TCD), electron capture detectors (ECD), and mass
spectrometers (MS).
 Each detector has its own detection mechanism and sensitivity towards
different types of compounds.
DATA ANALYSIS:
 Chromatography software or integrated data systems are used to
analyze chromatograms and extract relevant information.
 Peaks in the chromatogram represent individual compounds, and their
retention times are used to identify and quantify analytes.
 Peak area or height is proportional to the concentration of the compound in
the sample, allowing for quantitative analysis.
INTERPRETATION:
 Interpretation of chromatographic data involves comparing retention times
and peak shapes with known standards or reference materials.
 The resolution, efficiency, and selectivity of the separation can be
evaluated based on the chromatogram.
 Peak identification may be confirmed using retention time standards
or complementary detection techniques.
CLEANUP:
 Dispose of all waste materials, including used sample vials, syringes, and
any contaminated consumables, according to laboratory waste disposal
guidelines.
 Properly shut down the gas chromatograph and return any borrowed
equipment to its designated storage location.
 Clean the injection port and column as per manufacturer recommendations
to maintain instrument performance.
15
REPORT:

16
 EXPERIMENT: 06 DATE:

ESTIMATION OF VARIOUS DRUGS BY SPECTROFLUORIMETRY

AIM:
To estimate the concentration of Quinine sulphate present in the given sample
by
fluorimetry.
PRINCIPLE:
When any molecule absorbs UV/visible radiation, its electrons transmit from a
singlet ground state to a singlet excited state and as this excited state is not stable, it
emits the radiation and returns to a stable singlet ground state. This phenomenon of
emission of radiation is known as fluorescence. The fluorimetry is the measurement of
this emitted radiation. The emitted radiation (fluorescence intensity) is directly
proportional to the concentration of the substance present which can be measured by
fluorimeter. Molecules such as quinine sulphate having conjugated double bonds,
especially pi bonds are particularly suitable for fluorescence. Quinine sulphate in 0.1
N sulphuric acid gives blue fluorescence and the fluorescence intensity can be
measured by fluorimeter, with an excitation wavelength of 360nm using a primary
filter and with an emission wavelength of 485nm using a secondary filter.
PROCEDURE:
 A stock solution of Quinine sulphate (1mg/ml) was prepared.

 Pipette out 10 ml of 1mg/ml Stock solution into 100 ml volumetric flask and
make up the volume with 0.1N Sulphuric acid. [SOLUTION A]
 Pipette out 10 ml of solution A into 100 ml volumetric flask and make up the
volume with 0.1N Sulphuric acid. [SOLUTION B]
 A series of standard solution ranging from 0.5 to 2.5 µg/ml was prepared
from solution B.
 Pipette 1 ml from the given unknown sample solution into 10 ml volumetric
flask and make up the volume with 0.1N Sulphuric acid.
 The Fluorescence intensity was set to 0% using 0.1N Sulphuric acid and 100%
using highest concentration of standard solution.

17
 The % Fluorescence intensity of standard solutions and unknown sample
solution was measured.
 A graph of Concentration Vs Fluorescence intensity was plotted and the
unknown sample concentration was determined from the graph.

REPORT:

The amount of quinine sulphate present in the given sample was found to be

18
 EXPERIMENT: 07 DATE:

ESTIMATION OF SODIUM/POTASSIUM LEVEL BY FLAME

PHOTOMETRY

AIM:
To estimate the concentration of Sodium and Potassium ion present in the
given
sample by flame photometry.
PRINCIPLE:
A liquid sample containing a metallic salt solution is introduced into a flame,
the process involved in flame photometry are complex, but the following is a
simplified version of the events. The salt is vaporized are converted into gaseous state.
A part (or) all of the gaseous molecule is progressively dissociated to give free neutral
atoms (or) radicals. These neutral atoms are excited by the thermal energy of the
flame. the excited atom which are unstable, quickly emit photons, return to low
energy state, eventually reaching the unexcited state. The measurement of the emitted
photon(i.e.) radiation, forms the basis of flame photometry.
PROCEDURE:
 From 1 mg/ml stock solution of sodium and potassium, pipette out 10 ml of
potassium and 10 ml of sodium solution in to a 100 ml volumetric flask and make
up the volume with distilled water. [SOLUTION A]
 A series of standard solution ranging from 10 to 50 µg/ml was prepared from
solution A.
 Pipette 1 ml from the given unknown sample solution into 10 ml volumetric
flask and make up the volume with distilled water.
 A sodium and potassium filter were selected.
 The air pressure of 0.4 to 0.5 kg/cm2 and flame intensity in the burner was set.
 The flame intensity was set to 0% using distilled water and 100% using
highest concentration of standard solution.
 The % flame intensity of standard solutions and unknown sample solution
was measured.

19
  A graph of Concentration Vs Flame intensity was plotted and the unknown
sample concentration was determined from the graph.

REPORT:
The amount of sodium and potassium ions present in the given sample was
found
to be

20
EXPERIEMENT:08 DATE:

EXPERIMENTS BASED ON FLASH CHROMATOGRAPHY.

AIM:
To demonstrate the working of flash chromatography.
MATERIALS REQUIRED:
 silica gel (typically "silica 60") as the stationary phase,
 a suitable solvent system (usually a mixture of non-polar and polar solvents),
 a flash chromatography column,
 a sample to be purified,
 a solvent reservoir,
 a fraction collector, and
 a UV detector (for monitoring elution).

PRINCIPLE:
The principle is that the eluent which is a liquid, under gas pressure (normally nitrogen
or compressed air) rapidly pushed through a short glass column. The glass column is
packed with an adsorbent of defined particle size with large inner diameter. The most
used stationary phase is silica gel 40 –63μ m, but obviously packing with other particle
sizes can be used as well. Particles smaller than 25μm should only be used with very low
viscosity mobile phases, because otherwise the flow rate would be very low. Normally
gel beds are about 15 cm high with working pressures of 1.5 –2.0 bars. Originally only
unmodified silica was used as the stationary phase, so that only normal phase
chromatography was possible.
SOLVENT SYSTEMS
This chromatography is usually carried out with a mixture of two solvents, with a polar
and a non-polar component.
•Hydrocarbon: pentane, petroleum ether, hexanes.
•Ether and dichloromethane
21
•Ethyl acetate
•Ether/petroleum ether; Ether/Hexane; Ether/Pentane; Ethyl Acetate/Hexane;
Methanol/Di-chloromethane.

COLUMN SELECTION:
Typical Volume of eluant Required for packing and elution

22
PROCEDURE
Pump systems:
Pump Controller: A pressure ranges up to either 10 bar or 50 bar gives optimum
separation results fora broad range of applications. The pump modules can be controlled
by three different units.
 The Pump Controller C610 (for isocratic separation up to 10 bar).
 The Pump Manager C615 (for isocratic and gradient separation up to 50 bar).
 The Control Unit C620.

Type of pumps: Pump Module C-601:10 bars is used for fast isocratic Flash separations.
Pump Manager C-615: It’s with two Pump Modules C-601/C-605 for binary solvent
gradients. Pump Module C-605,50 bars: It’s with a Pump Module C-601/C-605 is used
for isocratic Flash separations.12/24
Vacuum Pump/peristaltic Pump: Transfer Solvent from Mobile phase Reservoir to Flash
Pump.
Sample Injection Systems: Injection systems are designed to facilitate column loading
with liquids and low solubility oils and solids. Regardless of the nature or quantity of the
material.
Injection Valve: For the sample injection of 0–5ml.
Glass Columns, Filling Sets & Column Valves
Glass Columns: A wide range of columns offer maximum flexibility for every situation.
Depending on the nature and the quantity of the sample offers a series of column types
which vary in form, size and performance.
Plastic + Glass Column: Plastic + Glass-coated Glass Columns are available for larger
sample amounts and higher-pressure applications on a high safety level. The columns are
designed for sample amounts from 1–100 g and pressures up to 50 bar during preparative
separations. Easy fixation on a support rod by using the corresponding pivoting clamp.
Plunger Column C-695: Robust, chemically resistant and biocompatible plunger
columns are designed for optimum operational performance and safety. Volume changes

23
in soft gel can be equalized & column length 460 mm. An integrated cooling jacket allows
separations under constant conditions at a high-quality level.14/24
Pre-columns: column is minimizing dead volumes and enhance the life time of the main
column by trapping contaminants. The small Pre-column, fits to Glass Columns of inner
diameter of ID 15, 26, 36 and 49 mm. The large Pre-column, fits to Glass Columns of ID
70- and 100-mm inner diameter.
Filling Sets for Glass Columns
Dry Filling Set: The Dry Filling Set is employed for filling glass columns with silica gel
using compressed gas. Silica gel in the size range of 25 – 200 μm can be packed with this
method.
Slurry Filling Set: The Slurry Filling Set is used for wet filling and conditioning of glass
columns with silica gel particles smaller than 25 μm.
Fraction Collector: For simple separations a column, pump and pump controller may be
enough. For a greater level of automation with precision, performance and ease of use the
Fraction Collector can be incorporated into most setups.
Fraction Collector C-660: The intelligent, height-adjustable Fraction Collector with
dialogue language options for preparative chromatography. The C-660 collects the
separated substances according to time, volume or peak. During each run, up to 12 litres
can be collected in a maximum of 240 glass tubes.
Detectors:
In the absence of adequate UV/Vis absorption, likely for sugars or polymers, a
Differential Refractometer (RI Detector) in combination with a UV/Vis detector is the
preferred setup.
UV Monitor C-630: Filter Photometer with four standards built in filters at 200 nm, 220
nm, 254 nm and280 nm. Delivered with built in Deuterium Lamp and a preparative flow
cell.
UV Photometer C-635: Spectral Photometer with a wavelength range between 190 nm
and 740 nm. Delivered with built in Deuterium Lamp and a preparative flow cell.

24
Differential Refractometer: RI detector mostly used in combination with a UV/Vis
detector for the analysis of low/Vis absorbing substances. Delivered with a preparative
cell. Flow rate of 100ml/min
REPORT:

25
EXPERIMENT:09 DATE:

EXPERIMENTS BASED ON LC-MS

AIM:
To demonstrate the principles of LC-MS chromatography and the separation of a mixture
of compounds using LC-MS chromatography.
MATERIAL REQUIREMENT:
Solvents: Water, Acetonitrile, Methanol, Isopropanol, Additives like formic acid, acetic
acid, or ammonium acetate.
Columns: C18 columns (most common). Other specialized columns depending on the
analyte polarity and characteristics.
Sample preparation reagents:
Depending on the sample matrix, may include extraction solvents, precipitation agents,
solid phase extraction (SPE) cartridges.
Gases:
Nitrogen (for nebulization and curtain gas), Air (for ionization source)
PRINCIPLE:
The Liquid Chromatography-Mass Spectrometry (LC-MS) is hyphenated analytical
technique which is combination of Liquid Chromatography (LC) and Mass Spectrometry
(MS). HPLC (LC) separates the components of mixtures by passing through
chromatographic column. Generally, the separated components cannot be positively
identified LC alone. Mass Spectrometry is also used for identification of unknown
compounds, known compounds and to elucidate their structure. Mass spectrometry is
alone not good for identifying mixtures because mass spectrum mixture is actually
complex of overlapping spectra from separated individual components. It is difficult to
connect Liquid chromatography (LC) with Mass spectrometry (MS). An interface is used
to transfer the liquid eluents from LC to MS.LC-MS is more significantly used in invite
dissolution, bioavailability, bioequivalence and pharmacodynamics studies.

26
PROCEDURE:
The Liquid Chromatography (LC) is a high-performance liquid chromatography in which
separation of components of mixture can be carried out by using liquid mobile and solid
stationary phase, There are different types of chromatography like normal phase liquid
chromatography, Reversed phase chromatography, Ion-exchange liquid chromatography,
Chiral separation and affinity liquid chromatography.
Pump: It consists of material which is inert towards solvents or any mixed composition
of aqueous buffer and organic solvents. It delivers high volume of mobile phase up to
10mL/min. There are three major types of pumps are used i.e. reciprocating pump,
Syringe pumps and constant pressure pumps.
Sample injector: It is used to introduce sample volume into the chromatographic system.
Generally sample volume from 1µL to 100µL can be injected. The injection volume can
be increase by injector loop up to 2mL volume. There are two major types of injectors
used i.e. Automatic injectors and Manual injectors. Automatic injectors are more
comfortable and user friendly and are more accurate and precise as compare to manual
injectors.
Columns: It is stationary phase which consists of silica material in combination with
carbon chain. Generally the column length used is about 50mm to 300mm. The columns
used in HPLC are consists of Octadecyl (C18), Octyl (C8), Cyano, Amino, Phenyl
packing’s. The columns are used on the basis of nature of compounds to be separated.
Detectors and recorder: The detectors is most important part of HPLC .There are
different types of detectors used are UV-Visible detectors, PDA detectors, Refractive
index (RI) detectors, Electrochemical detector, Fluorescence detectors and conductivity
detectors. The signal received from detector can be recorded as peak and respective data
can be stored in a software
Mass spectrometry: Mass Spectrometry is analytical technique based on the
measurement of the mass to charge ratio of ionic species related to the analyte under the
investigation.MS can be used to determine the molecular mass and elemental composition
27
of an analyte as well as in depth structural elucidation of the analyte.

Ionization/Ion Source and Interfaces: The Liquid chromatography separates mixture

of components which are in liquid form, usually contains methanol, acetonitrile and
water. This liquid containing mixture of components is transferred into the ion source of
mass spectrometer. As ion source is under high vacuum.
Direct liquid Introduction (DLI): The ionization in Direct Liquid Introduction (DLI) is
generally accomplished by vaporizing solvent as a chemical ionization and reagent gas.
Both the normal and reverse phase solvent system have been used. Reverse phase solvents
used are methanol/water, acetonitrile/ water mixture up to 60% water. In general buffer
with salts are not allowed as there is chance of capillaries to plug when heated.
Atmospheric-Pressure Ionization (API): In Atmospheric-pressure ionization (API)
contains three major steps i.e. Nebulisation, Evaporation and Ionization. There are two
main modes of API are Electrospray Ionization (ESI) and Atmospheric-pressure
ionization (APCI). In Atmosphericpressure ionization (API), when stream of liquid
(solvent) containing a sample is passed through narrow capillary tube and nebulized at
large chamber, mist of small droplets is produced.
REPORT:

28
EXPERIMENT: 10 DATE:

EXPERIMENTS BASED ON IR SPECTROSCOPY

AIM:
To perform the IR spectroscopy of the given sample.
PRINCIPLES
A covalent bond between two atoms can be thought of as two objects with
masses m₁ and m₂ that are connected with a spring. Naturally, this bond stretches and
compresses with a certain vibrational frequency. This frequency is given by Equation
I, where k is the force constant of the spring, c is the speed of light, and u is the
reduced mass. The frequency is typically measured in wavenumbers, which are
expressed in inverse centimetres (cm-1). The frequency is proportional to the strength
of the spring and inversely proportional to the masses of the objects. Thus, C-H, N-H,
and O-H bonds have higher stretching frequencies than C-C and C-O bonds, as
hydrogen is a light atom. Double and triple bonds can be considered as stronger
springs, so a C-O double bond has a higher stretching frequency than a C-O single
bond.
Infrared light is electromagnetic radiation with wavelengths ranging from 700
nm to 1 mm, which is consistent with the relative bond strengths. When a molecule
absorbs infrared light with a frequency that equals the natural vibrational frequency
of a covalent bond, the energy from the radiation produces an increase in the
amplitude of the bond vibration. If the electronegativities (the tendency to attract
electrons) of the two atoms in a covalent bond are very different, a charge separation
occurs that results in a dipole moment. For example, in a C-O double bond (a carbonyl
group), the electrons spend more time around the oxygen atom than the carbon atom
because oxygen is more electronegative than carbon. Hence, there is a net dipole
moment resulting in a partial negative charge on oxygen and a partial positive charge
on carbon.
On the other hand, a symmetrical alkyne does not have a net dipole moment
because the two individual dipole moments on each side cancel each other. The
intensity of the infrared absorption is proportional to the change in the dipole moment
when the bond stretches or compresses. Hence, a carbonyl group stretch will show an
intense band in the IR, and a symmetrical internal alkyne will show a small, if not
invisible, band for stretching of the C-C triple bond.

29
PROCEDURE:

 Turn on the IR spectrometer and allow it to warm up.

 Collect a background spectrum.
 Using a metal spatula, place a small amount of sample under the probe
 Twist the probe until it locks into place.
 Record the IR spectrum of the unknown sample.
 Repeat if necessary to obtain a good quality spectrum.
 Record the absorption frequencies indicative of the functional groups present.
 Clean the probe with acetone.
 Turn off the spectrometer.
 Analyze the obtained spectrum.

REPORT:

30
EXPERIMENT:11 DATE:

EXPERIMENTS BASED ON HPTLC

AIM:
To demonstrate the principles of HPTLC and the separation of a mixture of compounds
using HPTLC chromatography.
MATERIALS REQUIREMENT:
HPTLC plates: These are glass plates pre-coated with a thin layer of high-performance
silica gel, often with a fluorescent indicator (like F254) for visualization under UV light.
Developing solvents: A mixture of solvents carefully chosen based on the sample
components to achieve optimal separation on the plate.
Sample application device: A micro-syringe or capillary tube used to precisely apply the
sample solution onto the plate.
Developing chamber: A sealed chamber where the HPTLC plate is placed for solvent
development.
Densitometer: An instrument that scans the developed plate at a specific wavelength to
quantify the separated components by measuring their absorbance.
UV lamp: Used for visualizing the separated components on the plate, especially when
using fluorescent indicators.
Compressed air/nitrogen: Required for sample application and sometimes for plate drying.
PRINCIPLE:
It is a powerful analytical method equally suitable for qualitative and quantitative analytical
tasks. Separation may result due to adsorption or partition or by both phenomenon,
depending upon the nature of adsorbent used on plates and solvents system used for
development, The mobile phase solvent flows through because of capillary action. The
components move according to their affinities towards the adsorbent. The components with
more affinity towards the stationary phase travels slower and the components with lesser
affinity towards the stationary phase travel faster.

31
PROCEDURE:
Carrier gas The carrier gas must be chemically inert. Commonly used gases include
nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependant
upon the type of detector which is used. The carrier gas system also contains a molecular
sieve to remove water and other impurities.
Sample injection port For optimum column efficiency, the sample should not be too large,
and should be introduced onto the column as a "plug" of vapour - slow injection of large
samples causes band broadening and loss of resolution. The most common injection
method is where a microsyringe is used to inject sample through a rubber septum into a
flash vapouriser port at the head of the column. 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 packed columns, sample size ranges from tenths of a microliter up to 20
microliters.
Capillary columns, on the other hand, need much less sample, typically around 10 -3 mL.
For capillary GC, split/splitless injection is used. Have a look at this diagram of a
split/splitless injector; The injector can be used in one of two modes; split or splitless. The
injector contains a heated chamber containing a glass liner into which the sample is injected
through the septum. The carrier gas enters the chamber and can leave by three routes (when
the injector is in split mode). The sample vapourises to form a mixture of carrier gas,
vapourised solvent and vapourised solutes. A proportion of this mixture passes onto the
column, but most exits through the split outlet. The septum purge outlet prevents septum
bleed components from entering the column.
Columns There are two general types of column, packed and capillary (also known as
open tubular). Packed columns contain a finely divided, inert, solid support
material(commonly based on diatomaceous earth) coated with liquid stationary phase.
Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.
Capillary columns have an internal diameter of a few tenths of a millimeter. They can be
one of two types; wallcoated open tubular (WCOT) or support-coated open tubular
(SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with
32
liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined
with a thin layer ofsupport materialsuch as diatomaceous earth, onto which the stationary
phase has been adsorbed. SCOT columns are generally less efficient than WCOT
columns. Both types of capillary column are more efficient than packed columns, These
have much thinner walls than the glass capillary columns, and are given strength by the
polyimide coating. These columns are flexible and can be wound into coils. They have
the advantages of physicalstrength, flexibility and low reactivity.
Column temperature For precise work, column temperature must be controlled to
within tenths of a degree. The optimum column temperature is dependant upon the
boiling point of the sample. As a rule of thumb, a temperature slightly above the average
boiling point of the sample results in an elution time of 2 - 30 minutes. Minimal
temperatures give good resolution, but increase elution times. If a sample has a wide
boiling range, then temperature programming can be useful. The column temperature is
increased (either continuously or in steps) as separation proceeds.
Detectors There are many detectors which can be used in gas chromatography. Different
detectors will give different types of selectivity. A non-selective detector responds to all
compounds except the carrier gas, a selective detector responds to a range of compounds
with a common physical or chemical property and a specific detector responds to a single
chemical compound.
Detectors can also be grouped into concentration dependant detectors and mass flow
dependant detectors. The signalfrom a concentration dependant detector is related to the
concentration of solute in the detector, and does not usually destroy the sample Dilution
of with make-up gas will lower the detectors response. Mass flow dependant detectors
usually destroy the sample, and the signal is related to the rate at which solute molecules
enter the detector. The response of a mass flow dependant detector is unaffected by make-
up gas. Have a look at this tabular summary of common GC detector.
The effluent from the column is mixed with hydrogen and air, and ignited. Organic
compounds burning in the flame produce ions and electrons which can conduct electricity
through the flame. A large electrical potential is applied at the burner tip, and a collector
33
electrode is located above the flame. The current resulting from the pyrolysis of any
organic compounds is measured. FIDs are mass sensitive rather than concentration
sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the
detector's response. The FID is a useful general detector for the analysis of organic
compounds; it has high sensitivity, a large linear response range, and low noise. It is also
robust and easy to use, but unfortunately, it destroys the sample.

REPORT:

34
EXPERIEMENT:12 DATE:

INTERPRETATION OF ANY TWO ORGANIC COMPOUNDS BY HNMR

AIM:
To interpretate two organic compounds by HNMR
MATERIALS REQUIRED:
Magnet: A large magnet that creates a magnetic field that splits energy levels
Nuclei: The atomic nuclei that are being studied
Spin: The intrinsic spin properties of the nuclei
Chemical shift: The difference between the resonant frequency of the nuclei and the
reference molecule
Diffusion rate: The rate at which the nuclei diffuse
THEORY:
Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a
magnetic field absorb and re-emit electromagnetic radiation. This energy is at a specific
resonance frequency which depends on the strength of the magnetic field and the
magnetic properties of the isotope of the atoms. Proton nuclear magnetic resonance
(proton NMR, hydrogen-1 NMR, or 1H NMR) is the application of nuclear magnetic
resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules
of a substance, in order to determine the structure of its molecules. Carbon-13 nuclear
magnetic resonance (most commonly known as carbon-13 NMR or 13C NMR or
sometimes simply referred to as carbon NMR) is the application of nuclear magnetic
resonance (NMR) spectroscopy to carbon. It is analogous to proton NMR (1H NMR)
and allows the identification of carbon atoms in an organic molecule just as proton
NMR identifies hydrogen atoms. As such 13C NMR is an important tool in chemical
structure elucidation in organic chemistry. 13C NMR detects only the 13C isotope of
carbon, whose natural abundance is only 1.1%, because the main carbon isotope, 12C, is
not detectable by NMR since it has zero net spin.

35
PROCEDURE:
a) Compute the "number of unsaturated units or rings" from the composition CxHyNz:
UR = (x + 1) - y/2 + z/2

b) In this formula, halogens are equivalent to hydrogens. Oxygen atoms do not enter into
the calculation.
A. Use the following examples for interpretation:
a) Note the general groupings of signals and assume that signals which possess similar
chemical shifts correspond to similar carbon atoms in the molecule.
b) Count the number of distinct peaks in the spectrum. This is equal to the minimum
c) number of chemically different carbon atoms in the molecule. The more peaks, the
higher the complexity. There may be more carbon atoms than this number only if two
carbon atoms happen, by accident, to have the same chemical shift.
d) Assign each of the carbon atoms to a functional group type based on information from
a standard table of chemical shifts. Remember that the values in the Table are approximate
and an exact match is unlikely. However, from experience, you will become accustomed
to the maximum deviation which signals a poor correlation.
e) The areas under the peaks in 13C NMR do not correspond to the actual number of
carbon atoms in the molecule (although the opposite is true for 1H NMR).
In general, however, there is a clue to the kinds of hydrogens attached to a carbon atom
that can be taken from the size of the peaks. If there is one or more hydrogens directly
attached to the carbon atom, the size of the peak tends to be strong. If there are no
hydrogens attached to the carbon atom, the size of the peak tends to be weak.
Try to piece together a structure which is consistent with the number of peaks, their
chemical shifts and the relative sizes of the peaks.
REPORT:

36
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