LIST OF EXPERIMENTS

1. Determination of absorption maxima and effect of solvents on absorption maxima

of organiccompounds
2. Estimation of dextrose by colorimetry
3. Estimation of sulfanilamide by colorimetry
4. Simultaneous estimation of ibuprofen and paracetamol by UV spectroscopy
5. Assay of paracetamol by UV- Spectrophotometry
6. Estimation of quinine sulfate by fluorimetry
7. Study of quenching of fluorescence
8. Determination of sodium by flame photometry
9. Determination of potassium by flame photometry
10. Determination of chlorides and sulphates by nephelo turbidometry
11. Separation of amino acids by paper chromatography
12. Separation of sugars by thin layer chromatography
13. Separation of plant pigments by column chromatography
14. Demonstration experiment on HPLC
15. Demonstration experiment on Gas Chromatography

Faculty Incharge Principal

Program Name: Pharm.D. Course Name: Pharmaceutical Analysis Practical
Year: III Year Course Code: 3.2

LIST OF EXPERIMENTS
1.Separation and identification of Amino Acids by Paper Chromatography.
2. Separation and identification of Sulpha drugs by TLC technique.
3. Effect of pH and solvent on the UV spectrum of given compound.
4. Comparison of the UV spectrum of a compound with that of its derivatives.
5. Determination of dissociation constant of indicators using UV-Visible spectroscopy.
6. Conductometric titration of mixture of acids with a strong base.
7. Potentiometric titration of a acid with a strong base.
8. Estimation of drugs by Fluorimetric technique.
9. Study of quenching effect in fluorimetry.
10. Colourimetric estimation of Supha drugs using BMR reagent.
11. Simultaneous estimation of two drugs present in given formulation.
12. Assay of Salicylic Acid by colourimetry.
13. Determination of Chlorides and Sulphates in Calcium gluconate by Nepheloturbidimetric
Method. 14. Determination of Na/K by Flame Photometry.
15. Determination of pKa using pH meter.
16. Determination of specific rotation.
17. Comparison of the IR spectrum of a compound with that of its derivatives.
18. Demonstration of HPLC.
19. Demonstration of HPTLC.
20. Demonstration of GC-MS.
21. Demonstration of DSC.
22. Interpretation of NMR spectra of any one compound.

Faculty Incharge Principa

STANDARD OPERATING PROCEDURE

TITLE: INFRA RED RAYS
Objective

To lay down the operation for Infra Red rays in PharmaceuticalAnalysis Department.

Purpose

The purpose of this equipment is to determine the molecular structure, identification of chemical
species.

Procedure

1. Acquiring FTIR Spectra

1.1. Before Commencing work

 Obtain a bottle of alcohol and several paper towels. Kimwipes should also be
available near the FTIR.
 Clean the keyboard, mouse, GLOVES, and any area you will be exposed to
(monitor excluded) with the solvent-soaked paper towels.
 DO NOT spray directly onto surfaces.
 Discard ALL the paper towels (whether they were used or not) into the
designated waste container.
 Wait 5 minutes before commencing work.

1.2. Before Commencing Analysis

 DO NOT PLACE SAMPLE onto CRYSTAL(A); keep anvil (C) in the up position.
 ENSURE THAT THE ATR CRYSTAL (A) IS CLEAN OF ANY MATERIAL.
 Clean the crystal with the alcohol and let it dry.

1.3. Starting the Software (if not already done)

 Click the OPUS icon (select OPUS 7.0 or 7.5).
 Username & Password (please ask TA/TRACES Staff).
 Allow the Alpha-P to run diagnostic test.
 Anvil (C) remain in the up position during background runs.
 When finished you will be prompted to ‘CONTINUE’ or ‘RUN Background’.
 Run ‘Background’ once completed, press ‘CONTINUE’.
1.4. Setting up FTIR
 Analysis flow follows the ICONS from LEFT to RIGHT

 Click on the Measurement icon

 Start Background Measurement; keep anvil (C) in the up position.
 The green bar indicates the progress of the measurement (sample and background).
 Enter Sample Name once the buttons are no longer grayed out.
 1.5. Sample Loading
 Liquid Samples
Introduce the liquid sample (1 drop) on to the ATR crystal (A) keep anvil
(C) in the up position

 Solid Samples
Place the solid sample so that it covers the ATR crystal (A). Gently press
down on the anvil (C) till it make contact the sample. If necessary, rotate the
anvil arm (B) to ensure full contact is made with the crystal. In most cases,
solid samples that are ground to a finer powder, result in sharper FTIR

3 sample holder
1 Ellen key
Sample holder window
Die (with small pin hole) and punch
First place the die with a pin point facing down
Fill the sample in the sample holder
Place on the die
Place the punch over the sample Check the
pressure is zero Tighten the vertical screw
Then tighten the horizontal screw so that the
pressure is about 1.7 ton.
Leave for about 2 minutes Ratio of sample to KBr
1:300
The pellet formed should be as transparent as
possible The sample pellet should be washed Use gloves while handing as the moisture from
with acetone/chloroform. the hands may give wrong results.
Always keep KBr, sample holder other To open the block, remove the bottom holder
attachment in Dessicator. first by rotating lightly, then the sample pellet
from the punch by slight rotation.
spectra.

Pellet Press Sampling (for Powdered samples)

Place the pellet holder window in the Instrument such that the 2 knobs on
holder are facing away from the IR lamp path. Start the Labsolution software,
as discussed previously
Place the KBr pellet, Start scan, black areas represent the moisture peaks.
Go to Instrument, initialization for the preliminary scan. Initialization
succeeded. 3 Green blocks on right side. BKG scan. (for KBr
background)
 Go to Measurement. Window opens. Go to measurement mode. Select
Absorbance/transmittance. Usually 45 scans (can be 22-45 scans also), Resolution of 2-4
depends on Pharmacopoeia. Range of 400-4000/cm. BKG scan. Save the datas always in
drive D not in C.
Peaks at 2400 or 600 indicates that of CO2.
Then place the sample pellet. Take sample scan. Peak table below the graph.
To differentiate the CO2 peaks and sample peaks at same wave frequency, Go to
manipulation, Go to atmospheric correction, click Ok. Peak pick table, go to manipulation,
peak pick, window opens, select threshold can be 45 or more. Noise level 0.100, if 0.00
peaks will be very near.
Peak ratio: add peak, delete peak etc.
Purity index: Click Purity, window opens, Intensity can be changed. Take standard peak,
and sample peak, drag the standard peak over the other one. Send to source to reference.
Calculate. Purity is shown in purity index.
1.6. FTIR Sample Analysis
 Select Start Sample Measurement
 The green bar indicates the progress of the measurement

2. Evaluating FTIR Spectra

2.1. If the sample requires Baseline Correction click the icon

2.2. Click on the icon to run a standard peak pick of the spectra.
 Right-click mouse and to choose Single Peak Pick for manual selection.

2.3. Click on the icon to print your spectra.

2.4. Click on the icon to remove your spectra from the workspace.
2.5. Remove your sample and clean the surrounding area and the ATR CRYSTAL (A)
BEFORE departing with an alcohol. Place all waste into the appropriate waste bottle.

3. Post FTIR Analysis

 Obtain a bottle of alcohol and several paper towels. Kimwipes should also be
available near the FTIR.
 Clean the keyboard, mouse, GLOVES, and any area you will be exposed to
(monitor excluded) with the solvent-soaked paper towels.
 DO NOT spray directly onto surfaces.
 Discard ALL the paper towels (whether they were used or not) into the
designated waste container.
 Wait 5 minutes before next users can use the area and instrument.
 Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: UV-VISIBLE SPECTROPHOTOMETER

Objective

To perform the operation for in uv -visible spectrophotometer in Pharmaceutical Analysis

Department.
Purpose

The purpose of this equipment is to measuring the Transmittance or Absorbance of a sample .

Procedure

1. Plug in to ensure the power supply.

2. Switch ON the main power supply and instrument mains.

3. The instrument will auto ensure that the program is functioning properly by
displaying OK on the screen. This will take approximately 3-4 min.
4. From the Configure drop-down menu, select
Parameters.
i. You may use the default parameters
ii. OR Adjust Wavelength Range before starting the test. Wavelength range is
between 200-800 nm.
iii. Recording Range can be changed at any time. It is recommended to set
Scan Speed to fast and Sample Interval to Auto.
5. For determining maximum wavelength for the given sample, go to SPECTRUM
mode.
6. Select the wavelength scanning range between 800-200nm.
7. Fill the two cuvettes with blank solution and place both of them in two separate
cuvette holder.
8. Click on BASE Correction to detect and nullify any background interference.
9. After the base correction is done the instrument will beep.
10. Replace one of the cuvettes with the sample under study.
11. Press the START button and wait till the scanning is completed.
12. A graph will be displayed on the screen with the highest peak as the maximum
absorption for the given sample.
13. For obtaining linear curve/ calibration curve, go to PHOPTOMETRIC mode.
14. Clink on GO TO WAVELENGTH and set the maximum absorbed wavelength.
15. Fill the two cuvettes with blank solution and place both of them in two separate
cuvette holder.
16. If some amount of the light will be absorbed by the blank, the reading will be
displayed on the screen, then click on Auto Zero and wait until it reads 0.000A
17. Replace one of the cuvettes with the different serially diluted samples under study.
 18. Note the absorbance values for each diluted sample and plot a graph of
ABSORBANCE
v/s CONCENTRATION.

Precautions:

 Before start of the instrument, ensure to remove silica bag from the cuvette
holder chamber.
 Ensure that cuvettes are cleaned thoroughly after every using.
 The concentrations of the solutions under study by dilute so as to obtain
accurate results.

Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: FLUORIMETER
Objective
To perform the operation for Fluorimeter in Pharmaceutical Analysis Department.
Purpose
The purpose of this equipment is to measuring the Fluorescence or light emitted by different
fluorescing objects.
Procedure

1. Connect A.C. power pack to the main unit by inserting the cable socket into the panel
behind the main unit. Hook the power pack to 230V AC mains. Switch on the stabilizer.
 2. Switch ON the power pack unit.
3. Switch ON the mercury lamp
4. Allow 15 min for warming the mercury lamp.
5. Insert the primary filter (366nm peak) which looks dark violet in the slot between big
lens & sample hole. i.e. in the path of incident light.
6. Switch ON detector for activating the main unit (detector & amplifier) & observe digital
display.
7. Select the range say 2ppm max. concentration of quinine sulphate range with sensitivity
switch.
8. Take 0.1 N H2SO4 as blank solution in a tube provided & insert in the sample holder.
9. Adjust “Zero” by using the controls coarse & fine.
10. Replace blank tube with 1ppm quinine sulphate solution & set 100.0 by means of GAIN
control & light iris on digital display. OR take 2ppm solution & set 199.0 on digital
display in same manner.
11. Take unknown solution & read its concentration in the range selected. If the concentration
is more than 1.999 ppm change to higher range & repeat steps 7,8 9 & 10. Whenever the
range is changed set ZERO with BLANK solution & set 100.0 with the solution of
appropriate concentration is need to follow.
12. For short periods of non-use, the instrument need not be switched off the mercury lamp,
since the lamp takes time for cooling & restart. You may switch off the detector part of
the unit.

Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: FLAME PHOTOMETER
Objective
To perform the operation for Flame Photometer in Pharmaceutical Analysis Department.
Purpose
The purpose of this equipment is to determine the electrolytes in body fluids.
Procedure
1. Prepare the standard solutions well in advance. The substance should be dried at
180°C for 1 hour. (635mg/250ml=1000ppm for NaCl, then diluting 1ml to 10 ml to get
100ppm) (477mg/250ml-1000ppm (KCl); 624mg/250ml CaCO3, dissolve in 1:1 HCl
minimum qty, 1000ppm).
2. Prepare different dilutions from the 100ppm stock solution. For better readings
concentration of less than 40ppm should be taken.
3. Connect the Unit 1 with the air compressor unit provided with the pipe. Switch on the
air compressor unit.
4. Maintain the air pressure reading between 0.4-0.6 kg/cm2 in unit 2.
5. Connect the LPG Gas cylinder with the pipe provided to Unit 1 at the back of the unit
and maintain the gas flow slowly rotating the gas knob anticlockwise and keep on pressing the
auto ignitor knob provided at the back of main unit (glitters brick red) to lit the flame.
6. Put the Chimney at the position provided.
7. Select the desired filter.
8. Take the double distilled water in the sample holder. Keep on the mixing chamber
platform.
9. Connect the plastic capillary tubing with the fine metal capillary. Dip the capillary tube in
the sample.
10. Sample will be aspirated and color of flame may change depending upon the sample.
11. Allow the water to be aspirated for cleaning of the capillary tube.
12. Adjust the blank reading to Zero with Zero knob.
13. Remove the blank replace with the standard solution of higher concentration and
adjust the reading with calibration knob to the ppm of solution.
14. Clean the capillary tube with double distilled water and take the reading of another
concentrations to get a calibration curve.
15. After each reading of the sample, the capillary should be washed with double distilled
water.
16. After every 20-30 minutes of operation, the instrument should be re-calibrated
by repeating the operation of calibration.

PRECAUTIONS:
1. Air compressor should always be switched on first, then only the LPG gas should be
ignited.
2. Flame should not be too high, should form a cone of flame, can be viewed from the
window provided. If too high and not maintained, the flame may burn the unit and other parts.
3. Never try to look from the top of the igniting chamber while the flame is burning
(harmful for eyes). Always view from the window provided.
4. Always use clean glass apparatus and double distilled water.
 Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: UV CABINET
Objective
To describe the procedure for Operation UV Cabinet in Pharmaceutical Analysis Department.
Purpose
The purpose of this equipment is used for viewing longwave and short wave Ultraviolet lights and
white light of chromatograms.
Procedure

1. Connect the plug to main switch and switch on the mains.

2. Switch ON the ON/OFF switch.

3. Red coloured light will glow, indicating power supply is connected.

 4. Switch ON DAY LIGHT marked switch.

5. Red indicator light will be ON.

6. Open the front door of cabinet and place the pre-coated (coated with silica gel G) TLC plate
inside UV cabinet and close the door.

7. Three switches are provided on the UV cabinet.

8. Switch 1 – For General DAY LIGHT.

9. Switch 2 – For UV Light at short wavelength (254 nm).

10. Switch 3 – For UV light at long wavelength (365 nm).

11. Switch off DAY LIGHT.

12. Switch on the required UV light, i.e., short wave (254 nm), long wave (365 nm).

13. Visualize the sample from outside the chamber through glass provided to visualize the
sample inside the chamber.

14. After visualizing the sample note down the observation, switch off the UV light and remove
plate from chamber.

15. Switch ‘OFF’ the instrument when not required.

Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: NEPHELOTURBIDIMETER
Objective
To describe the procedure for Operation Nepheloturbidity meter in Pharmaceutical Analysis
Department.

Purpose
The purpose of this equipment is used for measure the how much of light transmission and scattered
from sample .
Procedure

1. Fill turbidity vial has white line around top of glass with downward arrow) to the
line (about 15ml) with unfiltered water. Take care to handle the sample cell by
 the top. Cap the cell.

2. Wipe the cell with a soft,lint free cloth to remove water spots and fingerprints.

3. Press I/O the instrument will turn on. Place the instrument on a flat surface. Do
not hold the instrument while making readings.

a. Put the sample vial in the instrument cell compartment so its diamond
mark aligns with the raised orientation mark in front of the cell
compartment. Close the cover.

b. Set automatic range by pressing the RANGE key. The display will
show “AUTO RNG”.

c. Select signal averaging by pressing ‘signal average’ key,display should

show”SIG AVG”

d. PRESS READ- the display will show”—NTU” and the light bulb icon
will flash 10 times. The final aveage will be displayed as numbers in
NTU after the lamp symbol turns off.

Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE
TITLE: HOT AIR OVEN
Objective
To provide a procedure for Operation of Hot Air Oven in Pharmaceutical Analysis Department.

Purpose
The purpose of this equipment is used to sterilise materials like glassware, chemicals and sealed
containers.
Procedure
1. Load the properly packed material to be sterilized within the oven,
2. Close the door.
3. Connect the plug to the power source and switch on the power.
4. Switch on the mains of oven.
5. LED lamp for the power indicator will glow
 6. Set required temperature (160oC for 2 hours for sterilization of glassware) using
temperature adjustment knobs.
7. Heaters will start, the LED for heating will start glowing and the temperature rises
steadily to the Set temperature.
8. Note the time at which the set temperature is reached.
9. After completion of sterilization, switch off the MAINS and the power source.
Cleaning:
 Wipe the surface, walls, top, bottom and trays of the oven with dry lint free cloth on the
daily basis so that there will be no dust particles in the oven
 Wipe all the parts and the outer surface of the Oven with wet lint free cloth soaked in
purified water, on weekly basis and fill the cleaning record.

Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: COLORIMETER
Objective
To provide a procedure for Operation of Colorimeter in Pharmaceutical Analysis Department.

Purpose
The purpose of this equipment is used to measure the absorbency of light.

Procedure

1. Switch on the Colorimeter.

2. Wait until the fluctuation will stop.
3. Set the percentage transmission at zero.
4. Take the Cuvette and clean it very properly. There should not be any trace on the surface of
 the Cuvette.
5. Fill the sample in the Cuvette which we are going to measure.
6. Place the Cuvette very slowly in the Cuvette chamber.
7. Note down Optical Density of the given sample.
8. Switch off the instrument.
Precautions

1. This instrument is a precision measuring instrument.

2. This instrument is not waterproof and should not be used in high humidity environment
or water mist.
3. Keep the instrument clean and tidy.
4. If the instrument will not be used for a long time, remove the battery.
5. The power adapter other than our company's special one cannot be used, otherwise the
instrument may be burnt down.
6. After the instrument is used, the colorimeter and the whiteboard cover should be placed
in the instrument box and stored properly.
7. The instrument should be stored in a dry, cool environment to avoid damage to the
instrument.

Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: DIGITAL BALANCE
Objective
It covers the procedure for operation of balance in Pharmaceutical Analysis Department.

Purpose
To ensure correct method of operation of balance.

Procedure

Before weighing:
1. One minute is required after turning the power on in order that the scale functions well.
2. Calibration may be required before weighing
3. Read “CALIBRATION” first and if necessary, calibrate your scale for accurate weighing.
Weighing procedure
 Press (on/off) to turn on the screen.
 When power is turned on, all display segments appear for a few seconds and “0” will appear on
the display.
 Select the weighing unit with “SET”
 Press SET to select a weighing unit “g”, “kg”, “lb”, “oz”
 Once the unit has been selected, the selected unit will be displayed to weight value
 Start weighing.
If you do not use a container before weighing
 Verify the reading is”0”.if not ,press(ZERO)to display “0”
 Place objects on weighing platform to weigh.
 When the reading becomes stable, the stable indicator is displayed.
If you use a container before weighing
 Place an empty container on the platform
 Wait for the stability indicator to be displayed and press “ZERO”
 Place the objects to be weighed in the container

Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: DIGITAL PH METER
Objective
To describe the procedure for operation of Digital P h Meter in Pharmaceutical Analysis
Department.

Purpose
The purpose of this equipment is used to obtain more accurate PH Measurements.

Procedure

1. Connect the power cable to 230V A/c 3 pin power socket (Earth to pH meter must be perfect).
2. Fix the electrode in to the stand clip.
3. Insert the combined Electrode in socket written “COM”.
4. Keep the “SELECTOR” in pH position and check/read switch to check position and temp comp
at 300c
5. Switch ‘ON’ the instrument by switch which is at rear of the instrument.
6. Adjust ‘Calibrate’/SET-7.00 control to make the reading 7:00pH.
7. Wash the Electrode with distilled water and wipe of the moisture.
8. Dip electrode in standard 4.00 pH buffer stir the beaker for a while.
9. Bring the switch to read position; the reading shall be 4.01 at 300c.
10. If it is not happening adjust ‘Imp.Adj/SET-BUFFER:to do so.
11. Keep the switch in check position switch to pressed (or) check position. Remove the electrode
from buffer, wash wipe of moisture and dip in unknown solution.
12. After a minute put the switch to read position and read pH of the solution.
Maintenance Of Eletrode
1. Shake the electrode gently to ensure that the internal buffer solution fills the whole bulb and no
air bubbles are entrapped.
2. Electrolyte used is saturated KCL filled the electrode through the filing hole.
3.The level of electrolyte should never be less than 3cm from bottom.
i. To ensure pressure equalization the cap of filling hole should be removed or perforated with a
pin.
ii. Soak the electrode in distilled water always.
iii. Electrodes which have a slow response due to driving out of the membrane (due to keeping
them in store for a long time or not kept in distilled water or used in extreme conditions may
be reactivated by soaking in 0.1N HCL for overnight period and washed and kept in distilled
water again for 24 hours.

Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE
TITLE: POLARIMETER
Objective
To describe the procedure for operation of Polarimeter in Pharmaceutical Analysis Department.

Purpose
The purpose of this equipment is used for determining the polarization properties of light beams
and samples.

Procedure

1.Polarimeter are used to measure the polarization of transverse waves as they exit an object .This
almost always refers to the way light refracts as it enters and leaves a transparent object, such as
type of glass, crystal or film.
2.A Polarimeter is a laboratory instrument used to determine the angle of optical rotation of plane –
polarized light passing through a sample of material.
3.Select a light source .This may be a direct light source such as a light bulb, neon sign or TV
screen, or an indirect light source such as a mirror or cloud.
4.Set the polarimeter to zero degrees and view the selected object through the polrimeter. Rotate
the lens of the polarimeter filter slowly until you have rotated it in a complete circle. Check for any
changes in brightness. The light is un polarized if its brightness does not change as you rotate the
lens.
5.Locate the polarimeter setting at which the light is brightest. Settings of zero and 180 degrees
indicate a completely vertical polarization, and settings of 90 and 270 degrees indicate complete
horizontal polarization.
6.Determine the degree of polarization .Locate the setting where the light is faintest and estimate
the brightness of the faintest setting as a percentage of the brightest light. If no light at all can be
seen on this setting, the light is completely polarized. If the brightness doesn’t change, the light is
completely un polarized

Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: POTENTIOMETER
Objective
To lay down the procedure for operation of Potentiometer in Pharmaceutical Analysis Department.

Purpose
The purpose of this equipment is used to determine the (H +) ion concentration in logarithmic
variation in linear relation to voltage generated at glass membrane according to Nernst Equation.

Procedure

1. Connect A.C power pack to the main unit by inserting the cable socket in to the panel plug
behind the main unit

2. Hook the power pack to 230V A.C mains

3. Switch ON the instrument by turning the control

4. Put the side switch to 100% slop position

 5. Set the function switch to mv mode-By position

6. Rinse the electrode pair with distilled water and wipe with tissue paper

7. Immerse the electrode in distilled water

8. Set the temperature to the solution on the TEMP control

9. Set the function key to the mv position

10. Press the gray button for taking the readings

11. After use wash the electrode with the distilled water and immerse then in a beaker
containing distilled water

12. Switch OFF the instrument

Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: CONDUCTOMETER
Objective
To lay down the procedure for operation of Conductometer in Pharmaceutical Analysis Department.

Purpose
The purpose of this equipment is used to analyse ionic species and to monitor a chemical reaction
by studying the electrolytic conductivity of reacting species.

Procedure
1.First connect the power supply to the conductometer after inserting the cable socket in to the
panel plug behind the main unit having voltage supply of 230V.
2. Next switch on power switch and wait for 10 minutes for stabilization of instrument.
3. Immerse the conductivity electrode (platinum electrode) into the container having distilled water
and change the right knob to cell constant which should display approx 0.860.
4. Now change the temperature regulating knob to fix the temperature between 25-30oC or any
suitable temperature mentioned in the procedure for specific sample.
5. Next adjust the sensitivity knob in accordingly by the type and concentration of electrolytes
present in the sample solution to be analyzed.
6. Now immerse the conductivity electrode (platinum electrode) into the container having distilled
water and change the right knob in that way to get the reading in the digital display to zero.
7. Next immerse the conductivity electrode (platinum electrode) into the container having sample
solution and wait for few minutes until a stable reading is observed in digital display which is the
conductivity value for the sample solution.
8. By changing different sample dilutions in electrode container different conductivity

Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: REFRACTOMETER
Objective
To lay down the procedure for operation of Refactrometer in Pharmaceutical Analysis Department.

Purpose
The purpose of this equipment is used to measure the refractive index of samples.

Procedure
 Open the wooden box and carefully remove the instrument from box.
 Place the instrument on table where adequate light can be reflected through mirror.
 Open the prism lock and place 2-3 drops of sample onto the lower prism using a clean
pipette, making sure that you do not touch the prism with the pipette.
 Allow the liquid to smear on lower prism surface quickly, close the prism lock.
 Adjust the eye piece cross wire with the knob by rotating it in a clockwise or
anticlockwise direction so that the sharp demarcation line passes through center.
 In case of any difficulties for making these adjustments or uneven interfaces or blobs of
black colour is seen, that is probably because enough sample on the PRISM is not
available.
 If a rainbow of colours is observed, turn the Fine Adjust knob until a sharp interface is
obtained.
 If observation is close to the index of refraction of sample, the view in the eyepiece
shows a dark region on the bottom and a lighter region on the top.
 Once a crisp demarcation between the light and dark regions is reached, turn the
handwheel on side to place the borderline exactly on the center of the crosshairs as
 shown below,

 Sharp demarcation is produced in the form of circle having half light and half dark
portion.
 A knob provided just above the prism can do fine adjustment.
 Note down the reading from the scale with the help of magnified lens of the instrument
as below,
 The left side of scale indicate Refractive Index Range of scale is 1.300 to
1.700.
 The right side scale (sugar at 20° 1996) is not used here as it indicate the
concentration of sugar in water.
 View the scale and read your R.I. where the horizontal line crosses the
right scale.
 The example shown here has a refractive index of 1.342.
 The refractive index is unit less number.

 Open the prism and wipe the upper and lower plates with a tissue moistened with
acetone. Leave open to dry.
 Prepared by Verified by Principal

STANDARD OPERATING PROCEDURE

TITLE: HIGH PEFORMANCE LIQUID CHROMATOGRAPHY
Objective
To lay down the procedure for operation of High Peformance Liquid Chromatography in
Pharmaceutical Analysis Department.

Purpose
The purpose of this equipment is used to confirm the identity of drug and provide quantitative and
qualitative results.

Procedure
 1. Start UP:
 Switch on the main button.
 After that start the on/off button on high performance liquid chromatography Instrument.
 Display will show on screen ;
 SHIMADZU Conditioning ….. , Seeking Home ….. , LAMP preheating ….. , Checking
Wave ….. , © 2017 Shimadzu Corporation. All rights reserved.
 Above all conditions are ok, then put ID Admin & Password then press OK.
 After that “Ready” display will be shown on screen.
 Start the computer (Software), Select user and Password, appear on the Monitor.
 Double click on Lab Solution icon. Login Window will appear.
 Enter User Name and Password. Click on login tab.
 Lab Solution Software will open.
 Run Method:
 Select the project and click “Ok”. Realtime analysis window” will appear.
 To select method, click on “File” then select “Open Method File” and choose method click
“Download and Close”.
 Run Sequence:
  To Create new Sequence, click on Window tab then click on “show window” and select
“Realtime Batch.”,
 Right Click on Sequence and select table style, table style window will appear. Select
following options.
 Vial
 Enter the number where vial is kept.
 Tray Name
 Enter the number where sample is kept.
 Sample Name
 Name of Product.
 Sample ID
 Identification of sample eg. Blank , Standard solution Etc.
 Method File
 Select required method.
 Data File
 Give Suitable Data file name.
 Inj. Volume
 Put the value which is indicated in specifications.
 To save the sequence file, click on “File” and select “Save Batch File”. Give suitable batch
sequence name.
 To start a batch sequence , select sequence and Click on “Batch” and click on “Start.”
 Integration:
 Click on Window tab then click on “show window” and select “Data Analysis Tools”,
 Select the project and click Ok. Double click on Post Run Icon.
 Select the data to integrate and click on “Wizard” icon. Compound wizard table window will
appear. Window shows options; width, Slope, Drift, Min. Area / Height. Put value as
required.
 Click Next, Select retention time and click next. Give compound name and click on “Finish”
tab.
 Select “Apply to Method” icon and Save.
 To integrate the data create on 3D plus HPLC system, Select “Method View -Multi
Chromatogram Table”.
 To integrate the data create on 3D plus HPLC system, Select “Method View -Multi
Chromatogram Table”.
 Click on “Integration” and set the value of width, Slope, Drift, Min. Area / Height etc. as
required.
 Click on “Compound” and give the compound name and select retention time.
 After integration select “Apply to Method” icon and Save.
 To integrate data with multiple wavelength repeat the above steps.
 For calculating Peak Purity, Click on “Purity” under “Method View” and set the purity
parameters.
 Set the Noise Spectrum, Click in the check box and put the Retention time Range as
required.
 Click on check box to compute noise spectrum from current data for peak purity or Click on
“Open Data File” to select alternate data to compute noise spectrum.
 Results of Peak Purity Such as Impurity, Peak purity index, Single point Threshold and
Minimum peak purity Index will appear under “Purity View”.
 Create Project:
 To Create new project, Click on “Window” tab then click on “show window” and select
 “administrator tools”.
 Administration tools will appear then Click on “project administration”.
 Project Administrator window will appear. Click on “Add” to add new project.
 Give project name and click “Next”.
 Select User and then Click “Next”.
 Select Instrument and then Click “Next”.
 Verify the details such as project name, Selected User, Selected instrument and click on
“FINISH”.
 Create Method File:
 Select the project and click “Ok”. Realtime analysis window will appear.
 To Create new Method File, click on “File” then click on “New Method File”, Click “New”.
 Set the instrument parameters such as LC Time Program, Wavelength, and Temperature etc.
as per Standard Test Procedure.
 For PDA detector set the instrument parameters such as LC Time Program, Temperature etc.
as per Standard Test Procedure. Wavelength range is 190 nm to 800 nm. For single
wavelength , set the wavelength near to the wavelength given in Standard Test Procedure.
For example for 254 nm set the wavelength range 200 nm to 300 nm.
 Set the Auto purge time to 3 min., Purge Time (Rinse Port) 20 min., Rinse Deep Time to 5
Sec. Set the maximum Pressure not more than 449 kgf/cm2.
 Select “Download and Close” to save method file. Give File name and Click “Save”.
 To save the file in template , click on “File” and select the ” Save Method File As Template”.
Saved templates are used for making new method files.
Create Report Format:
 Click on Window tab then click on “show window” and select “Data Analysis Tools”.
 Select the project and click Ok. Double click on Post Run Icon. Post Run Analysis window
will appear.
 Click on Window tab then click on “show window” and select “Report Generator”.
 Click on “Item” and Select Sample Information, Chromatogram, peak table, Summery
(compound) etc. from “common”, “Analysis Common” options.
 To set the chromatogram parameters such as Peak Top comment, Setting Scale etc., right
click on chromatogram then click on Properties and set the parameters as required.
 To set the Peak table parameters such as Name, Area, Area %, Retention Time, Relative
Retention Time etc. right click on peak table then click on Properties and set the parameters
as required.
 Save the report format and give the name as Assay, RSD etc
 Prepared by Verified by Principal

Course outcome for Instrumental Methods of Analysis Practical (BP705P)

CO-I The students will learn about the absorption maxima and effect of solvents.
CO-II The students will estimate the percentage of few bulk drugs by Colorimetric.
CO-III The students will learn about Assay of any two bulk drugs by UV-spectro
photometry.
CO-IV The students will learn about quenching effect in fluorescence drugs and
determine the electrolytes by using Flame Photometry and Nepheloturbidity
meter.
CO-V The students will learn about the different chromatographic development
techniques like TLC, Column and paper Chromatography .
CO-VI The students will appreciate and demonstrate the various chromatographic
techniques like Gas Chromatography an HPLC.
 LIST OF EQUIPMENTS
B. Pharm. Instrumental Analysis Lab (BP705P)

SI.NO EQUIPMENT NAME QUANTITY

1 UV- Visible Spectrophotometer 2

2 HPLC 1
3 FTIR 1
4 Flame Photometer 1
5 PH Meter 2
6 Conductometer 1
7 Potentiometer 2
8 Colorimeter 2
9 Nephelo Turbidity meter 2
10 Fluorimeter 1
11 Polarimeter 1
12 Refractometer 1
13 UV-Cabinet 2
14 Digital Balance 1mg 1
15 Digital Balance 10mg 1
16 Hot Air Oven 1
17 Vaccum Distillation Unit 1
18 Vaccum Pump 1
19 TLC Chambers 5
20 TLC Sprayers 3

Faculty Incharge HOD Principal