SOP on Good Laboratory Practices (GLP)

 Objective:To lay down a procedure for operation of “Hot Plate”.

 Scope: This Standard Operating Procedure is applicable to Quality Control Department followed
at pharmaceutical company name with location.

 Responsibility:QC personnel shall be responsible to follow the procedure as per SOP.

 Accountability:Department Head & QA Head shall be accountable for implementation of this SOP.
 Abbreviations and Definitions:
 SOP: Standard Operating Procedure; a document where step by step instructions are cited to serve as
support for methods or manners of fulfilling a function or functions reliably and consistently.
 QC: Quality Control
 GLP:Good Laboratory Practice
 procedure

 Weighing:
o Make sure that the balance pan and surrounding area are cleaned and dry.
o Check ‘0’ of the balance.
o Place a weighing paper and tare the balance.
o Carefully transfer the sample to be weighed onto the weighing paper.
o Record the weight (gross weight) according to the balance least count and transfer the sample
quantitatively to the flask.
o Again weigh the same paper and record the weight (tare weight) according to the balance least
count.
 Points to be kept in mind while using an analytical balance:
o Always use balances, which are calibrated periodically against absolute standard weights.
o Always choose a balance such that the value of three times the standard deviation of the
reproducibility of the balance, divided by the amount to be weighed, does not exceed 0.001.

 While weighing, the quantity actually used must not deviate by more than 10 % from that stated in the
test procedure of the individual.
 Always use a correct size of butter paper or tare vessel.
 Always check the balance cleanliness.
 Always use weighing bottles instead of butter paper for weighing liquid samples or solids that readily
lose or gain weight on standing.
 Do not weigh hot or warm sample.
 Do not use filter or ordinary paper for weighing purpose.
 Do not weigh off center.
 Do not touch weighing vessels with bare fingers.
 Do not weigh magnetic stir bars.
 Do not use contaminated spatula.
 Never weigh lumps.
 Never leave balance doors open.

 Pipetting :
o Take a clean dry pipette.
o Place the tip of the pipette below the surface of the liquid and suck the sample up into pipette
with the help of a pipetting bulb about 1 inch above the mark.
o Allow the pipette to drain completely to a waste container.
 o Once again, suck up the sample to just above the mark.
o Clean the outer surface of the pipette which was dipped in the sample with a tissue paper.
o If the sample is colourless /slightly coloured and clear, slowly drop the lower meniscus of the
sample to the mark of the pipette and if it is highly coloured then slowly drop the upper
meniscus of the sample to the mark of the pipette.
o Keep the pipette in a vertical position and then touch against the wall of the receiving vessel to
drain the tip.
o For non viscous samples, allow the pipette to drain for about 15 sec after the liquid has been
dispensed.
o For viscous samples, allow the pipette to drain for about 45 sec after the liquid has been
dispensed.
o If the sample is viscous like syrup/suspension, wash the pipette with the diluents and add the
washings to the measured portion.
 Points to be kept in mind while pipetting :
o Always use such pipettes whose tips are not broken.
o While observing the meniscus always keep the pipette in vertical position at the level of eyes.
o Never blow out the tip of the pipette while draining.
 Dilutions :
o Select clean and dry apparatus of correct size for dilutions.
o Place the tip of the pipette below the surface of the liquid and suck the sample up into pipette
with the help of a pipetting bulb about 1 inch above the mark.
o Allow the pipette to drain completely to a waste container.
o Once again, suck up the sample to just above the mark.
o Clean the outer surface of the pipette which was dipped in the sample with a filter paper or a
tissue paper.
o Slowly drop the meniscus of the sample to the mark of the pipette.
o Keep the pipette in a vertical position and then touch against the wall of the receiving
volumetric flask to drain the tip.
o Rinse the pipette if necessary with the diluents and add the washings to the measured portion.
o Add more of the diluents until the volume is just below the mark of the volumetric flask.
o Make up the final volume with the diluents drop by drop with the help of a pipette and mix.
 Points to be kept in mind while doing dilutions:
o Be careful not to lose sample during transfer, such as liquid dripping out of a pipette tip before
the tip is in the receiving vessel.
o Be careful not to overshoot or undershoot the mark on either pipettes or volumetric flasks.
o During dilution, mix the contents frequently in order to maintain a homogeneous blend.
o When the combination of two substances is either exothermic or endothermic, allow the
mixture or solution to come to room temperature prior to achieving final volume.
o When using microlitre pipettes, be sure that they are properly calibrated.
 Filtration :
o Gravity Filtration :
 Choose an appropriate size of filter paper (as per requirement) from an approved Fold it
in cone shape and place it into a suitable clean funnel.
 Place the funnel stem into the receiving vessel such that the bottom tip of the stem
touches the wall of the receiving vessel.
 Pour few ml of sample to be filtered on the filter paper and discard the filtrate.
 Continue the filtration by pouring sample in portions, allowing each portion to filter
before adding the next.
 Do not fill the funnel (with filter paper) to more than about 90 percent of its capacity.
 o Vacuum Filtration :
 Take a clean vacuum filtration cup.
 Place the filter media (membrane filter) in the filtration cup.
 Wet the membrane filter with appropriate solvent.
 Place the filter assembly on a clean vacuum filter flask and connect the sidearm of the
flask to a source of vacuum.
 The size of the vacuum filter flask selected should be at least 20 % larger than the
volume to be filtered so as not to suck liquid into the sidearm of the flask.
 Filter the sample in portions, adding each subsequent portion after the previous one has
been approximately 80 % filtered. Do not allow the filter media to go dry between
portions.
 On completion of filtration, break the vacuum by releasing the vacuum from the flask
sidearm slowly and gradually.
 Remove and disassemble the filter funnel assembly, discard the membrane filter, and
clean the filter assembly for subsequent use.
o Centrifuging :
 Examine the centrifuge to make sure that it is leveled.
 Place the sample to be centrifuged into the centrifuge tube.
 Place the tube in the centrifuge head’s tube holders .Always use an even number of
tubes, such that they are placed across each other so that the weight of material in the
centrifuge is evenly balanced. If only one sample is to be centrifuged, then
counterbalance it with a tube containing water.
 Make sure that the centrifuge cover is closed and any guards or locks are properly
engaged.
 Set the speed knob and time knob at the required parameters.
 Switch on the centrifuge.
 Switch off the centrifuge after the specified time and allow it to come to a full stop.
Never remove samples from a centrifuge that has not come to a full stop.
 Remove the tube from the centrifuge.
o Manual Titration :
 Prepare the sample as directed in the Standard Test Procedure of the sample.
 Prepare the blank (if mentioned ), consisting of all reagents but not the sample.
 Select a clean and dry burette, class A or B grade (after calibration) with 0.01 ml or 0.1
ml subdivisions and size accurate to need. Rinse it with titrant two times, making sure
that no liquid clings to the inner walls of the burette after the rinses are drained. If
liquid clings to the walls, clean that burette before use or use a different one.
 Fill the burette with the titrant up to the 0 ml mark.
 Make sure that the stopcock fittings are snug and there are no leaks.
 Remove the bubbles, if any in the burette tip.
 Slowly drop the titrant level until the lower meniscus, if the titrant is colourless/slightly
coloured like Sodium Hydroxide; or the upper meniscus, if the titrant is highly coloured
like Potassium Permanganate; is level with the 0 ml mark.
 Titrate the blank and record the titration values in the analyst worksheet.
 Titrate the sample, going to the same color as that of the titrated blank endpoint, and
record the volume of titrant consumed in the analyst worksheet.
 The volume of titrant consumed by the sample (V) is calculated as,
V= A–B

where, A = Volume of titrant consumed in the sample titration.

B = Volume of titrant consumed in the blank titration.

 Points to be kept in mind while doing manual titration:

o Always use standardised titrant for titration.
o Perform the titration slowly, drop by drop towards the end point.
o Continuously swirl the sample/blank containing flask while performing titration.
o While using the indicator solution, use approximately 0.1 ml of indicator solution unless
otherwise specified in the Standard Test Procedure.
o Select a burette of such size that the titrant volume represents not less than 30 % of the nominal
volume. Where less than 10 ml of titrant is to be measured, a 10 ml burette or a micro burette
generally is required.
o The tips of the burettes should restrict the outflow rate to not more than 500 μl per second.
 Distillation:
o Take a clean and dry distillation flask.
o Take a measured quantity of sample in it and add other reagents required as specified in the
Standard Test Procedure. Also add glass beads to avoid bumping and for constant heating.
o Place the distillation flask on the heating mantle and connect it to the condenser through a
connector tightly.
o Place the receiving vessel near the tip of the condenser.
o Check the water connections and start it.
o Set the temperature of the heating mantle and start the heating.
o Collect the required amount of distillate and then switch off the heating mantle.
o Let the apparatus to cool down and take away the receiving vessel.
 Kjeldahl’s method for Nitrogen determination:
o Sample digestion:
 Accurately weigh the sample and transfer it into a clean and dry Kjeldahl flask.
 Add 2g mixture of Copper sulphate and Potassium sulphate (1: 10).
 Add 20 ml of Concentrated Sulphuric Acid, allowing it to rinse down the walls of the
flask.
 Mix the contents and the flask, place in a 45º angle position, over an open flame until
the solution is clear green for 30 min.
 Allow to cool and add 150 ml of laboratory water. Mix the contents of the flask and
again cool.
o Sample Distillation and Titration.
 Pour the contents of the kjeldahl flask to the distillation flask and rinse it with few
portions of water adding the washings to the distillation flask. Add more of the water so
as to have a volume of approximately 1000 ml .Add 80 ml of 50 % NaOH and
immediately close the distillation flask with the connecting tube. Start the flow of water
through the condenser. Place the tip of the condenser in collecting vessel containing
acid and 2-3 drops of methyl red or as specified in the Standard Test Procedure. Start
the burner and distill until about 300 ml of distillate is collected. Titrate
the resulting solution with alkali or as specified in the Standard Test Procedure.
o Basic Techniques for performing TLC :
 Prepare the mobile phase as directed in the Standard Test Procedure.
 Take a clean and dry TLC chamber and rinse it with the mobile phase. If mentioned in
the Standard Test Procedure, saturate the chamber with the mobile phase prior to use.
 Take a precoated TLC plate of appropriate size. As specified in the Standard Test
Procedure treat the plate prior to use like heating or developing the unspotted plate in
the mobile phase etc.
  Prepare the sample and standard solutions required for spotting as per the Standard Test
Procedure.
 Spot the plate using a micro liter syringe or fixed volume capillary pipette. If multiple
applications are to be made to a spot, allow each to dry before applying the next.
Drying can be facilitated by using a gentle stream of dry air to the spot.
 After spotting, and when all of the spots are dry, place the plate in the TLC chamber
containing the mobile phase. Allow the solvent to migrate the distance prescribed in the
Standard Test Procedure being performed.
 Remove the plate from the TLC Chamber, let it to dry, and visualize as called for in the
Standard Test Procedure.
o Points to be kept in mind while performing TLC :
 Always use a TLC plate 1.5-2.0 inch more in length than the distance to be developed.
 Always prepare fresh mobile phase sample and standard solutions and spraying
reagents.
 Always spot at a distance of 1.0- 1.5 cm above the baseline of the plate.
 The mobile phase level in the chamber should not be more than the application line of
the spots on the plate.
 Place the plate straight or with the least angle, in the TLC chamber for faster and better
development.
 For better resolution, make sure that the spots are small, tight and round.
o Basic techniques for the safe use of vacuum in the analytical laboratory.
 Connect the source of vacuum, either a vacuum pump or a vacuum line, to the
apparatus (oven or filter flask) using heavy-wall vacuum tubing. Do not use regular
tubing, as the walls of such tubing might collapse under reduced pressure.
 When using vacuum in conjunction with an oven, be sure that there is a needle valve or
stopcock between the vacuum source and the apparatus to allow for proper control of
vacuum.
 Filtration with the vacuum filter is normally done using full vacuum, connecting the
vacuum tubing directly to the filter flask.
 Turn on the vacuum pump or the vacuum line.
 For ovens, using the needle valve or stopcock, slowly apply vacuum until the desired
mm of vacuum are achieved.
 When vacuum is no longer needed, break the vacuum as follows:
o Filter flasks:
 Slowly and gradually remove the vacuum tubing from the sidearm of the filter flask,
allowing the vacuum to dissipate gradually. Be careful not to cause bumping of liquid
by removing the vacuum too rapidly.
 Turn off the vacuum pump or line.
o Vacuum Ovens:
 Close the vacuum inlet valve, and turn off the vacuum pump. Then bleed off the
vacuum gradually over a period of a minute or two so as not to disturb samples
contained within the oven.
 The air being sucked back into the oven during release of vacuum should be passed
through a drying tube to avoid reintroducing moisture into the oven.
 After the pressure in the oven has achieved equilibrium, close the bleed valve. The oven
can now be opened for removal or introduction of samples.
o Special Precautions while using vacuum oven :
o When pulling vacuum on vessels containing volatile or corrosive solvents, place a cold trap or
scrubber between the vacuum source and the vessel to which vacuum is being applied.
 Whenever vacuum is being applied to glassware, be sure that the glassware used is rated for
use with vacuum. Failure to do so may result in serious injury due to glassware implosion.

 Forms and Records (Annexures)

Not Applicable

 Distribution
o Master copy – Quality Assurance
o Controlled copies – Quality Assurance, Quality Control
 History
Date Revision Number Reason for Revision

– 00 New SOP