KARL FISCHER APPARATUS

PURPOSE: To have a standard procedure to operate, standardise and calibrate Karl Fischer reagent.

KARL FISCHER TITRATION: It is a technique for the

determination of moisturecontent. The technique was developed by a chemist Karl Fischer. It is based on a reagent which reacts with water and converts the water into a non-conductive chemic There are two methods of moisture determination: Volumetric Karl Fischer Coulometric Karl Fischer The apparatus consist of a titration vessel of about 60ml capacity fitted with two platinum electrodes ,a nitrogen inlet tube, a stopper which accommodates the burette tip and a vent tube protected by a suitable desiccant. The substance being examined is introduced through an inlet tube or side arm which can be closed by a groundglass stopper, stirring is effected magnetically or by means of a stream of a dried nitrogen passed through the solution during the titration. the end point is determined by amperometry.

KARL FISCHER REAGENT:

Anhydrous methanol, sulphur di oxide iodine , primary ions.

Determination of water equivalent factor for KFR: For solids: Titre value X Factor value X 100 /Weight of sample (mg) For liquids: Titre value X Factor value / 10/weight of sample (mg) For DST: titre value X 0.1566 X 1000 / weight of DST in mg. The Water Determination Test (Karl Fischer Method) is designed to determine water content in substances, utilizing the quantitative reaction of water with iodine and sulfur dioxide in the presence of a lower alcohol such as methanol and an organic base such as pyridine,

MELTING POINT APPARATUS

Purpose : To provide a uniform procedure for the operation and calibration and maintenance of melting point apparatus. Instrument details: Name- melting point apparatus Make- polmon Model- MP-96-X Instrument specifications- OIL VOLUME: 100ml POWER REQUIRED: 20V A melting point apparatus is a scientific instrument used to determine the melting point of a substance. Some types of melting point apparatuses include the Thiele tube, Fisher-Johns apparatus. A basic melting point apparatus for the analysis of crystalline solids consists of an oil bath with a transparent window (most basic design: a Thiele tube) and a simple magnifier. The several grains of a solid are placed in a thin glass tube and partially immersed in the oil bath. The oil bath is heated (and stirred) and with the aid of the magnifier (and external light source) melting of the individual crystals at a certain temperature can be observed. In large/small devices, the sample is placed in a heating block, and optical detection is automated.The measurement can also be made continuously with an operating process.

Procedure: Properly fix the silicon oil filled glass container along with stirrer , motor are clamped to the supporting rod.Insert the thermometer of proper range in the thermometer port. Connect to the pin plug to 230 volts AC and switch on the main and switch on the stirrer .Properly fix the silicon oil filled container along with stirrer. Motor are clamped to supporting rod.Insert the thermometer of proper range in the thermometer port. Connect to the 3 pin plug to230 v and switch on the main and also switch on the stirrer and heater .Slowly increase the volts by using volts control. Fill the capillary with material and insert them through small sample ports on the Teflon cover. Adjust the viewing lens to see clearly. With volt controller raise the temperature near the MPS is very slow and not the melting point when the material melts completely . Calibration procedure for melting point apparatus: Ensure that theconnections of the instrument are proper. Operate the instrument as per the operating instruments and determine the melting point. 3. Take the reference substances from lowest range to highest range at a time for calibration.

Reference Substances Vanillin Acetanilide Urea (finely crushed) Sulphanilamide Caffeine (dried at 100C) Diclofenac Sodium

Standard Range 81.0C 83.0C 114.0C 116.0C 132.0C 135.0C 164.5C 166.5C 234.0C 237.0C 277.0C 283.0C

Acceptance criteria: The melting point of the reference substances should be within the standard range. Frequency :Once in a month Maintenance / Repairs: When the instrument does not comply with the requirement / tolerance range specified above, the instrument should be labeled OUT OF CALIBRATION and should be repaired / serviced. After repair / maintenance, calibrate the instrument. OBJECTIVE: To calibrate the equipment for reliable and accurate results SCOPE

This procedure is applicable to calibrate the Melting point apparatus, installed in quality control area. PROCEDURE for the calibration : Procedure: Switch ON the mains. Switch ON the instrument. Press menu key twice Recall will blink. Press enter. Press down key till vanillin will blink. Press enter.

Set point, gradient & maximum temperature set for vanillin will be displayed. Fill vanillin std (previously dried over silica gel for 16 hours.) into the capillary. Place the capillary in the heating chamber. Press Start. Instrument will beep when set point is reached. Press Start. Note the temperature displayed.

 Repeat the above step for Phenacetin& Caffeine. (previously dried over silica gel for 16 hour. Press Stop to start cooling.

Press menu till the submenu displaying threshold is displayed. Press menu seven times. Calibration is displayed. Enter the password. Calibration list is displayed.

Enter the difference between the rated MP & the observed MP of the 3 Standards. Rated MP is displayed on the screen in the calibration menu. Press UP to store the calibration. Press menu till original menu is displayed.

QUALITY CONTROL LABORATORY CALIBRATION REPORT OF MELTING POINT APPARATUS

CALIBRATION DATE DATE OF LAST CALIBRATION DONE Instrument details: INSTRUMENT MAKE NEXT DUE FOR CALIBRATION

INSTRUMENT NAME

INSTRUMENT IDENTIFICATION NO.

SR.NO. 1 2 3

OBSERVATION Reference Std. Observed Range Vanillin Phenacetin Caffeine

Std. Range 81C 83C 134C 136C 234C 237C

INSTRUCTIONS FOR THE MELTING POINT APPARATUS :

SAFETY PRECAUTIONS: 1. Never insert a room temperature thermometer into a hot Mel-Temp. It may shatter. 2. Do not leave a Mel-Temp on if you are not actually using it, or leave it running unattended. 3. Do not heat the Mel-Temp beyond the upper temperature of the thermometer.

3. Do not touch the heating block.

FRIABILITY TEST APPARATUS PURPOSE : To provide uniform procedure for the operation and check of friability apparatus. Instrument details : Name- friability apparatus Make- DBK instrument Model DBK502016 Instrument id no.- CL-01 Power requirements - 230 volts The strength of a tablet plays a very important role in its marketing and dissolution. The mechanical strength of tablet or granules can be determined by its hardness and through friability test.

Measuring the hardness of a tablet is not a reliable indicator for tablet strength as some formulations when compressed into very hard tablets tend to 'cap' or lose their crown portions on attrition. Such tablets tend to powder, chip and fragment.

They not only lack elegance and consumer acceptance but also spoil the areas of manufacturing such as coating and packaging. In friability test the tablets are prone to abrasion hence enabling us to check for the tablet strength under application of force in different methods. The friability test is carried out in an instrument called a friabilator. A friability testing apparatus should stimulate the conditions that the product will be exposed to during the process of production. This test is a method to determine physical strength of uncoated tablets upon exposure to mechanical shock and attrition. The commonly used friabilator in laboratories is the Roche Friabilator. Friability is affected by various external and internal factors like: 1) Punches that are in poor condition or worn at their surface edges, resulting in 'whiskering' at the tablet edge and show higher than normal friability values. 2) Friability test is influenced by internal factors like the moisture content of tablet granules and finished tablets. Moisture at low and acceptable level acts as a binder Specifications of Roche Friabilater: Speed : 25 RPM Accuracy : 1 RPM Counter : 1 to 9999 revolutions No. of Drums One or Two as per model : Display : 4 Digits LED Dimensions : 26 cm x 20 cm x 25 cm

Supply :

230 V / 50 Hz 1 Ph. (Optional 110 V 60 hz).

Operation procedure of friabilater: Connect the power cord to an appropriate electrical outlet. Unscrew the locking nut to release the drum. Brush any loose dust from the tablets to be tested. Accurately weigh the tablets. Load the tablets into the brum. Place the plastic cover over the drum. Hold the cover firmly in place and slide thedrum body onto the shaft. Place the locking nut ontothe end of the shaft. Tighten the locking nut into position. Turn the knobto the desirednumber of rotations. Wait until the drum returns to a stationary position. Remove locking nut.Carefully remove the drum from the shaft. Remove the tablets and brush away any loose powder from themCheck for any obviously cracked,

cleaved or broken tablets since this will indicate that the sample tablets have failed the friability test. Reweigh the tablets.

Calculate the percentage of weight loss using the following formula: % weight loss = initial weight final weight / Initial weight X 100%.\

Operation And Calibration Of Friability Test Apparatus

1.0

OBJECTIVE: To lay down the procedure for operation and calibration of friability test apparatus

2.0

RESPONSIBILITY: Quality Control Chemist / QA Chemist

3.0

ACCOUNTABILITY: Quality Control Manager / QA Manager.

4.0 4.1

PROCEDURE : GENERAL CLEANING

Clean the apparatus free of dust with dry cloth from out side every day. Clean the rotating disc and unloading pan from inside with dry cloth at the start and end of every operation. Wet cloth may be used occasionally if cleaning is not proper with dry cloth but this should follow drying of the inner surface by first wiping with dry cloth and exposure to atmosphere air.

4.2

OPERATING INSTRUCTIONS

Ensure that apparatus is properly connected with power supply. Weigh tablets to be tested and record the weight {WI}. Switch on the main switch of power supply. Press MODE key and select count mode. Press SET button and set the RPM to 25 with the help of numerical keys (0 to 9) & press ENTER Key located on the front side of the instrument. Load the weighed tablets in the drum, close the lid and secure it by tightening he screw and press ENTER and then press START to run test. After the completion of set rotations, the apparatus will automatically unload the tablets into the tray. Observe the tablets. De-dust the tablets, weigh and record the weight {W2}. Calculate the percentage Friability by the following formula; =

Initial Wt. of Tablets (W1) Wt. of tablets After 100 rotation (W2) X 100 Initial Wt. of Tablets (W1) 4.3 CALIBRATION PROCEDUR

Switch on the power supply. Set the RPM to 25 and start the machine simultaneously with the stop watch. Count the actual rotations and not the time required for the same. Similarly set the RPM to 100 and note the time required and actual rotations. Apparatus is in proper working condition if, Time required for 25 rotations is 1 min 05 sec. Time required for 100 rotations is 4 min 20 sec. Record the observation in the calibration record as per Annexure 1. Affix a Calibration Status label on the instrument. In case of any discrepancy, report the observations to QC manager / QA Manager and notify the defect to Engg. Department. Affix an UNDER MAINTENANCE label on the

Friability test apparatus Instrument name Instrument Ser. No. Make & Model Calibration date Calibration due on : : : Electrolab. : : TIME ACCEPTANCE CRITERIA 1 min 05 sec 4 min 2 min

Sr. SET ROTATIONS ROTATION No. ROTATION DISPLAYED OBSERVED MA NUALLY 1 2 25 100

Dissolution test apparatus In the pharmaceutical industry, drug dissolution testing is routinely used to provide critical in vitro drug release information for both quality control purposes, i.e., to assess batch-to-batch consistency of solid oral dosage forms such as tablets, and drug development, i.e., to predict in vivo drug release profiles. In vitro drug dissolution data generated from dissolution testing experiments can be related to in vivopharmacokinetic data by means of in vitro-in vivo correlations (IVIVC). A well established predictive IVIVC model can be very helpful for drug formulation design and post-approval manufacturing changes.

The main objective of developing and evaluating an IVIVC is to establish the dissolution test as a surrogate for human bioequivalence studies, as stated by the Food and Drug Administration (FDA). Analytical data from drug dissolution testing are sufficient in many cases to establish safety and efficacy of a drug product without in vivo tests, following minor formulation and manufacturing changes (Qureshi and Shabnam, 2001). Thus, the dissolution testing which is conducted in dissolution apparatus must be able to provide accurate and reproducible results. Several dissolution apparatuses exist. In United States Pharmacopeia (USP) General Chapter <711> Dissolution, there are four dissolution apparatuses standardized and specified.[3] They are: USP Dissolution Apparatus 1 - Basket (37C) Dissolution Apparatus 2 - Paddle (37C) USP Dissolution Apparatus 3 - Reciprocating Cylinder (37C) USP Dissolution Apparatus 4 - Flow-Through Cell (37C) USP Dissolution Apparatus 2 is the most widely used apparatus among these four. The performances of dissolution apparatuses are highly dependent on hydrodynamics due to the nature of dissolution testing. The designs of the dissolution apparatuses and the ways of operating dissolution apparatuses have huge impacts on the hydrodynamic.

Dissolution test apparatus:

FEATURES OF DISSOLUTION APPARATUS: The 8/14 station microcontroller based instrument is designed to conduct the test simultaneously in all vessels in similar conditions as per IP/BP/USP Standards Equipped with a large graphic LCD, to display Speed of stirrer can be controlled and maintained by advanced microcontroller based system 150 Test Methods with 18 different time intervals can be programmed Water bath contains a non-corrosive tank pate Incorporated with circulation pump to maintain the constant temperature in water-bath Individual vessels centring is possible Password protection at different level Off line printing facility features selective printing as per date and product selection Facility to customize test reports with users company name and instrument number Memory to store 48 test records Power failure indication program is incorporated Supports USP apparatus 1,2,5,6 and intrinsic test Wobble and vibration free instrument.

Specification:

Number of vessels: VDA-8 D: 8 /VDA-14 D: 14 Stirrer RPM: 25 200 Speed accuracy: Better than 1 RPM Stirrer depth adjustment: 25 mm Test time interval: 1 minute to 23.59 Hours

Temperature range: 3C above room temperature up to 40C Temperature resolution: 0.1C Tank capacity: VDA-8D: 30 Litres (approx) /VDA-14D: 50 Litres (approx) Vessel capacity: 1 Litre Heating device: Encapsulated heating chamber with water inlet and outlet, having built in auto-cut off (over temperature) device Display: Graphic LCD Display Input: 4 x 4 tactile keyboard + PS -2 keyboard Printer: Parallel Interface for 80 Columns Dot Matrix Printer Dimension: 710x 620 x 640 mm and 850 mm: Total height when platform is positioned at topmost level -1140 x 570x 640mm Weight: VDA-8D: 50 kg. /VDA-14D: 100 kg Power: VDA 8D: 230 V, 50 Hz, 2.5 KW (max)/ VDA 14D: 230 V, 50 Hz, 3.25 KW (max).

Disintegration apparatus
Disintegration test is widely used in the pharmaceutical industry for evaluation of disintegration capability of formulations (ex:tablets) and quality control of different dosage forms. Purpose Disintegration tests are performed as per the pharmacopoieal standards. Disintegration is a measure of the quality of the oral dosage form like tablets and capsules. Each of the pharmacopoeia like the USP, BP, IP etc each have their own set of standards and specify disintegration tests of their own. USP, European pharmacopoeia and Japanese pharmacopoeia have been

harmonised by the International conference on Harmonisation (ICH) and are interchangeable. The disintegration test is performed to find out the time it takes for a solid oral dosage form like a tablet or capsule to completely disintegrate. The time of disintegration is a measure of the quality. This is because, for example, if the disintegration time is too high; it means that the tablet is too highly compressed or the capsule shell gelatin is not of pharmacopoeial quality or it may imply several other reasons. And also if the disintegration time is not uniform in a set of samples being analysed, it indicates batch inconsistency and lack of batch uniformity.

Disintegration Test Apparatus Coming to the test, the disintegration test is conducted using the disintegration apparatus. Although there are slight variations in the different pharmacopoeias, the basic construction and the working of the apparatus remains the same. The apparatus consists of a basket made of transparent polyvinyl or other plastic material. It has 6 tubes set into the same basket with equal diameter and a wire mesh made of stainless steel with uniform mesh size is fixed to each of these six tubes. Small metal discs may be used to enable immersion of the dosage form completely. The entire basket-rack assembly is movable by reciprocating motor which is fixed to the apex of the basket-rack assembly. The entire assembly is immersed in a vessel containing the medium in which the disintegration test is to be carried out. The vessel is provided with a thermostat to regulate the temperature of the fluid medium to the desired temperature.

Disintegration Test Method The disintegration test for each dosage form is given in the pharmacopoeia. There are some general tests for typical types of dosage forms. However, the disintegration test prescribed in the individual monograph of a product is to be followed. If the monograph does not specify any specific test, the general test for the specific dosage form may be employed. Some of the types of dosage forms and their disintegration tests are: 1.Uncoated tablets- Tested using distilled water as medium at 37+/-2 C at 29-32 cycles per minute; test is completed after 15 minutes. It is acceptable when there is no palpable core at the end of the cycle (for at least 5 tablets or capsules) and if the mass does not stick to the immersion disc. 2.Coated tablets- the same test procedure is adapted but the time of operation is 30 minutes. 3.Enteric coated/ Gastric resistant tablets- the test is carried out first in distilled water (at room temperature for 5 min.; USP and no distilled water per BP and IP), then it is tested in 0.1 M HCL (upto 2 hours; BP) or Stimulated gastric fluid (1 hour; USP) followed by Phosphate buffer, pH 6.8 (1 hour; BP) or Stimulated intestinal fluid without enzymes (1 hour; USP). 4.Chewable tablets- exempted from disintegration test (BP and IP), 4 hours (USP). These are a few examples for illustration. The disintegration tests for capsules, both hard and soft Gelatin capsules are also performed in a similar manner. Also, the USP also provides disintegration tests for suppositories, pessariesetc Applications of Disintegration test : 1.Disintegration test is a simple test which helps in the preformulation stage to the formulator.

2.It helps in the optimisation of manufacturing variables, such as compressional force and dwell time. 3.This test is also a simple in-process control tool to ensure uniformity from batch to batch and among different tablets 4.It is also an important test in the quality control of tablets and hard gelatine capsules. Advantages of Disintegration tests: This test is simple in concept and in practice. It is very useful in preformulation, optimisation and in quality control. Disadvantages: Disintegration test cannot be relied upon for the assurance of bioavailability.

Calibration of Disintegration Test Apparatus

A) Number of Cycles (With a constant frequency of 29 to 32) per minute: 1. Record the frequency of moving up and down of the Basket rack assembly, in a given time as shown below.

Start Time (Mins.)

End Time (Mins.)

Side A (Left hand side of the operator). No. Of cycles (A) No. of cycles per min. (A / 5)

Side B (Right hand side of the operator). No. Of cycles (B) No. Cycles per min. (B / 5)

0 15 30 45 60

5 20 35 50 65

2. Record the frequency (of moving up and down) manually with respect to time. 3. Do not stop the instrument in between the operation. 4. Measure the frequency initially and after each fifteen minutes interval from the start, for 5 minutes each. 5. Record five readings.

6. Divide the observed reading by five & note as frequency of moving up & down per minute. 7. Perform the test for both the basket rack assembly positions (side A & side B) individually. Acceptance criteria - The No. of cycles per minute should be within 29 to 32 throughout the period of operation. B) Calibration for Temperature Maintenance: 1. Set the temperature to 37C. 2. Fill the beaker with water. 3. Attach the basket rack assembly & start the constant frequency of moving up & down. 4. Insert the calibrated thermometer in one of the tubes of the basket rack assembly. 5. Wait till temperature reaches between 36oC to 38oC. 6. Start recording the readings. 7. Record the temperature readings initially & after each 15 minutes interval up to 120 minutes from the start. 8. Perform the test for both the basket rack assembly positions (side A & side B) individually. 9. While performing the test, do not keep thermometer inside the basket rack assembly constantly, but insert 2 to 3 minutes prior to the measurement to give stable reading.

Temperature (In C) Time (In Mins.) Side A (Left hand side of the operator). Side B (Right hand side of the operator).

Initial After 15 After 30 After 45 After 60 After 75 After 90 After 105 After 120

Acceptance criteria - The temperature should remain within 37 1C, throughout the operation. C) Calibration of the Distance Traveled: 1. Attach the basket rack assembly firmly. 2. Attach white paper firmly without kinks on the instrument, parallel to the path of the arm of basket rack assembly.

3. Select one point on the horizontal arm of the assembly & mark the same on the paper (pointed marker or pen can be used) when assembly is not moving & at its highest position. 4. Start the instrument by pressing START / STOP key & followed by pressing the respective timer key. 5. As soon as assembly reaches the lowest position, mark the same point again on the paper (while doing this activity take the time to decide the exact lowest position & then mark). 6. Measure the distance between two marks using calibrated ruler. 7. Remove the paper. 8. Perform the test initially, after 60 minutes from the start & after 120 minutes from the start. 9. Perform the test for both the basket rack assembly positions (side A & side B) individually. Acceptance criteria - Distance travelled is between 53 to 57mm. Time in mins. Distance traveled by the basket Side A (Left hand Side B (Right hand side of the side of the operator) operator)

Initial After 60 After 120

Frequency : Once in a Month. Maintenance / Repair : When the instrument does not comply with the requirements specified above; the instrument should be labelled as Out of Calibration and should be repaired / serviced. After repairing / servicing, calibrate the instrument before use. PRECAUTIONS Ensure that the tank is filled with Purified / Distilled water without vessel up to the mark of the instrument, before putting on the heater switch. Do not over tight the paddle / basket, and also not to hold the paddle / basket when it is in operation. Note - Sinkers shall be used if the dosage form unit floats. Close the vessels with the cover during Operation.

High pressure liquid chromatography [HPLC]

High-performance liquid chromatography (formerly referred to as high-pressure liquid chromatography), HPLC, is a chromatographictechnique used to separate the components in a mixture, to identify each component, and to quantify each component. HPLC is considered an instrumental technique of analytical chemistry (as opposed to a gravimetric technique). In general, the method involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent. Each analyite in the sample interacts slightly differently with the adsorbent material, thus retarding the flow of the analytes. If the interaction is weak, the anallyteflow off the column in a short amount of time, and if the interaction is strong, then the elution time is long. HPLC has been used in medical (e.g.

detecting vitamin D levels in blood serum), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and manufacturing (e.g. during the production process of pharmaceutical and biological products). Chromatography can be described as a mass transformation process involving adsorption. HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with a sorbent, leading to the separation of the sample components. The active component of the column, the sorbent, is typically a granular material made of solid particles (e.g. silica,, polymers, etc.), 2-50 micro-meters in size. The components of the sample mixture are separated from each other due to their different degrees of interaction with the sorbent particles. The pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile and/or methanol) and is referred to as a "mobile phase". Its composition and temperature plays a major role in the separation process by influencing the interactions taking place between sample components and sorbent. These interactions are physical in nature, such as hydrophobic (dispersive), dipole-dipole and ionic, most often a combination thereof

Types of chromatography: Partition chromatography Normalphase chromatography Reversed-phase chromatography (RPC) Bio-affinity chromatography Aqueous normal-phase chromatography

A chromatogram of complex mixture (perfume water) obtained by reversed phase HPLC;

CALIBRATION OF KARL FISCHER TITRATOR Calibration procedure: Remove the Karl Fischer reagent from the reservoir and clean it

with water. Then empty the burette and clean it with the water and flush the water 3 to 4 times in pipe connected with burette. Then fill the reservoir with water and operate the instrument as per SOP. 1. Collect the water in tarred 25 ml beaker and note down the weight. 2. Similarly measure the Burette volume for 2 ml, 5 ml, and 10 ml as above. 3. Divide the observed weight by 0.99602 (Weight/ml of water at 25 0C) and find out the obtained volume.

Acceptance Criteria : The obsereved volume should not deviate by 1 % of set volume Precision : Precision can be determined by analysing the 5 replicate samples of the disodium tartrate di-hydrate (C4H4Na2O6.2H2O). 1. Operate the instrument as per the SOP and perform the factor determination using sodium tartrate di-hydrate and record the result as per the format. 2. Repeat the factor determination for another 4 times using the same sample and record the results as per format. 3. Calculate the Mean, SD and RSD. Acceptance Criteria :RSD should not be more than 1.00 %. Accuracy : Operate the instrument as per the SOP and perform the factor determination using 10 mg water in triplicate and record the result as per the format. 1. Calculate the % Moisture, SD and RSD.

After calibration over, affix the calibration tag on it. Frequency : once in every three month

Calibration procedure: Instrument Code No Make Model : ____________ : ___________ : ____________ : ___________

Determination of Kf reagent factor.

Sr. No.

Weight of Water (In gm)

Factor of KF reagent (In mg H2O/5 ml)

Mean factor of KF reagent (In mg H2O/5 ml)

Remark

1. 2. 3.

Determination of water content of Di-Sodium Tartrate (15.66% 0.3 %)

Sr. No. Wt. of DiSod. Tartrate (In gm) Water of DiSod. Tartrate (In % ) Mean Water of DiSod. Tartrate (In % ) Remark

1. Accuracy : Sr. No 1 2 3 5 % RSD NMT 2.0 % Weight taken Burette reading % Moisture in mg in ml Limit

Calibration Status

: Satisfactory / Not Satisfactory

OPERATION & CALIBRATION OF HPLC :

1.0.

OBJECTIVE: The lay down a procedure for operation and calibration procedure for HPLC system.

2.0. 2.1 2.1.1. 2.1.2.

RESPONSIBILITY: The Officer - Quality Control shall be: Responsible for operating the HPLC system as per the SOP. Responsible for timely calibration of HPLC system as per the SOP. The Executive - Quality Control shall be: Responsible for ensuring the adherence of SOP. Responsible for maintenance operation. ACCOUNTABILITY: Head - Quality Control PROCEDURE: PROCEDURE FOR GENERAL CLEANING: Ensure that the power supply to the instrument is switched off and main cord is removed from supply. Clean the instrument with a clean dry cloth everyday. A wet cloth dipped in dilute soap solution may be used occasionally. Precaution must be taken to clean the instrument immediately with dry cloth. OPERATING INSTRUCTIONS: Ensure that the instrument is properly connected to the power supply. Fix the column and prepare the mobile phase.

2.2 2.2.1 2.2.2 3.0. 4.0. 4.1 4.1.1 4.1.2

4.1.3

4 4.2.1 4.2.2

4.3 4.3.1 4.3.2

PREPARATION OF MOBILE PHASE: Use HPLC grade solvent/ HPLC grade water / HPLC grade reagent only. Filter the mobile phase through 0.45 membrane filter after mix it in required proportion and degas on ultrasonic bath for about 5 minutes. Affix the label on the container of Mobile Phase as shown below.

4.3.3

MOBILE PHASE Material Prepared on Valid up to Prepared By : : : :

4.4 4.4.1 4.4.2

Operating Procedure: Turn On the mains of LC-2010AHT unit. Turn on the computer and double click on the icon class VP from the menu window. In the main menu window, double click on the instrument icon.

4.4.3

4.4.4

After the LC2010 AHT beeps indicating connection to the PC has been established the instrument window opens. Place the tube leading to the purge/rinse degasser in 50:50 volume of (Methanol or Acetonitrile: water) Click on the purge icon on the direct control toolbar. Select the tubing to be purged and set purging time for each. Click on the purge button; display a window showing the progress of purge and auto purging shell start. Select the new method from the list opened by clicking on the new method button. untitled method shell display with Default method parameter on the LC setup window. Configure the LC parameters by clicking on the pump, oven and detector icons in the LC setup. Placing the mouse pointer on them highlights LC component icons. Clicking the icon, opens the window to set and confirm that components parameters.

4.4.5

4.4.6

4.4.7

4.4.8

4.4.9

4.4.10

4.4.11 4.4.12

Select file>Method>Save as and give a name for the method. After creating method, down load it to the LC-2010AHT main unit by clicking on down load button. When the down load is complete, a message window appears and then Click OK The LC-2010AHT controller shell be activated, and check that the correct parameter has been downloaded successfully. To create a new sequence: Click on the sequence wizard button in LC setup Select Create new file and click on the next button. For making single run, Click on the single run button in the LC setup window or the single run button on the toolbar, specify a method file to be used in a respective dialog box and specify the path to save the data files. And click on the start command. To make a series of a sequence, Click on the sequence run button in the LC setup or the sequence run button on the toolbar, specify the start vial number, injection volume, method file, data path, sample IDs, data file names and after the setting is complete Click on the finish button to save the sequence file created in a respective folder by giving a desired name. After creating a sequence, view Sequence Wizard in the LC setup, select the sequence and click on the start button to start acquisition. Before injecting system suitability solution ensure that proper base line is obtained .For system suitability test, inject six continuous injections of same standard and RSD should not be

4.4.13

4.4.14 4.4.15 4.4.16 4.4.17

4.4.18

4.4.19

4.4.20

more than 2.0 %, otherwise it is specified in standard test procedure. 4.4.21 After every ten injection of sample (5 samples in duplicate), the standard solution shall be injected in triplicate and the RSD shall be calculated and ensure that it is within the limit. For terminating acquisition, select control>Extend run and enter a value for time added to the end of the run. To shorten the acquisition time. Specify a negative value. Setting integration event, Click file> open to open the data file. 4.4.24 The default values can be used for integration, however it is recommended to optimize slope in accordance with the base line in advance to prevent undesired peaks such as noise from being integrated. The default values: Width 5, Slope 200, Drift 0 T. DBL 1000, MINIMUM AREA 0. For Creating report templates Select file> print setup, and setup the printer connected. Click on the edit custom report to open the custom report window. SHUT DOWN PROCEDURE: Switch-off the computer. Switch-off the LC-2010AHT main unit power. CALIBRATION PROCEDURE:

4.4.22

4.4.23

4.4.25

4.4.26

4.4.27

4.5 4.5.1 4.5.2 4.6

4.6.1 4.6.1.1

AUTO-VALIDATION: Check the various components meet the performance and functionality criteria by carrying Auto validation and the system checks for the following parameters during auto validation Wavelength accuracy (nm) Lamp energy Pulsation (Mpa) Temperature (C) Absorbance accuracy (%) Drift (Mau) Noise (mAU) Gradient A% B% Gradient A% C%

4.6.1.2

Press VP to display the VP/TOP screen, press F3 (VALID) to open the validation menu, and then 3WXYZ (Enter) to display Auto validation screen. Connect the resistor tubing to the pump outlet, and till the mobile phase reservoirs. When performing in the gradient mode, use 0.05% aqueous acetone in the Port A and HPLC grade water in ports B, C & D.

4.6.1.3

4.6.1.4

4.6.1.5

In the auto validation screen select Gradient (GE) mode and select solvent combinations A/B, A/C and A/D. Press F1 to start auto validation In GE mode it starts purging port B, C AND D. Wait for the system to finish purging. After the system finishes purging, the next screen is displayed and the wavelength accuracy test beings. Tests follow one after the other and it takes about 3hrs to complete the Auto-validation. After the gradient concentration accuracy, the results are displayed from all tests in Gradient (GE) mode. To save the results press F4 (SAVE) Down load all the results to the class VP software by pressing the Control> Validation run on the Menu bar, Select complete and enter a name for the file and save the file. Click print to print the report.

4.6.1.6 4.6.1.7

4.6.1.8

4.6.1.9

4.6.1.10 4.6.1.11

4.6.1.12 ACCEPTANCE CRITERIA

PARAMETERS Wavelength accuracy (nm) Lamp energy Pulsation (Mpa) Temperature (c) Absorbance accuracy (%) Drift (mAU/h) Noise (mAU) Gradient A% B% Gradient A% C% Gradient A% D%

CRITERIA 1

>800 <0.50 <0.20 3.0

<0.50 <0.02 2.0 2.0 2.0

Note: Flow rate calibration is part of Auto validation in which system checks for gradient and flow accuracy 4.6.2 4.6.2.1 FOR PUMP: Disconnect the column and connect the inlet and outlet tubings with a union.

4.6.2.2

Prime all the lines at 5 ml/min flow rate with water and ensure that flow line is free from air bubbles. Set the flow rate at 1ml / min and collect the mobile phase (water) in a dry pre-weighed beaker and collect the mobile phase for 10 min. Weigh the beaker to get the weight of mobile phase. Calculate the flow rate by dividing the weight obtained with 10 (run time) and calculate the volume by dividing with weight per ml. Calculate the corresponding flow rate. Carry out the experiment in duplicate. Record the observations in Annexure 1. Acceptance criteria: Flow rate should be in between 0.99 to 1.01 ml / min. FOR GRADIENT VALVE: 4.6.3.1 Install union in place of column and flush solvent lines (A, B, C and D) with respective solvents at flow rate of 2ml/min. Prepare 56 mg / litre of Propyl Paraben in Methanol. Fill reservoir A and B with methanol and reservoir C and D with 56 mg / lit of Propyl Paraben in Methanol as mobile phase. 4.6.3.4 4.6.3.5 INSTRUMENT SET UP: Enter the following time program: Time Initial 2 Flow (ml) 2.0 2.0 % A 50 90 % B 50 0 % C 0 10 % D 0 0

4.6.2.3

4.6.2.4

4.6.2.5

4.6.3

4 6 8 10 12 14 16 18

2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

50 90 50 0 50 0 50 50

50 0 50 90 50 90 50 50

0 0 0 10 0 0 0 0

00 10 0 0 0 10 0 0

4.6.3.6 4.6.3.7 2. 4.6.3.8

Use detector at wavelength of 254nm. Record the printout of gradient valve test as per Annexure

The gradient valve test shall be accepted if percentage RSD of peak heights is not more than 5.0%. CALIBRATION OF INJECTOR: CHECK FOR PRECISION: Purge the injector system with 100% water to ensure the complete washing of injector. STANDARDPREPARATION: Transfer about 50 mg of Uracil to a 250ml volumetric flask. Add 100ml of Methanol, sonicate to dissolve and make up the

4.6.4 4.6.4.1 4.6.4.1.1

4.6.5 4.6.5.1

volume with Methanol to obtain a solution containing about 0.02 mg/ml of Uracil. 4.6.5.2 4.6.5.3 4.6.5.4 Filter the solution through 0.45m membrance filter. Use HPLC grade Methanol as the mobile phase. Instrumental Set Up Column mm, 5.0m Flow rate : Hypersil ODS or equivalent 250 x 4.6

: 1.0 ml/min

Injector Volume : 20m litre Detector : 254 nm

4.6.5.5

Inject the standard preparation six times in the system. The peak areas observed shall be consistent. The relative standard deviation for area counts calculated shall not be more than 1.0%. Record the observation in the format as mentioned in Annexure 3. CHECK FOR LINEARITY: Inject 10, 20, 30, 40 and 50m litre of standard preparation in duplicate. Calculate the average area counts corresponding to each set of injection. Tabulate the average area against each injection. Plot a graph for area counts vs. m liter the resulting graph shall be

4.6.5.6

4.6.5.7

4.6.6 4.6.6.1

linear. The correlation co- efficient calculated shall be not less than 0.99. 4.6.6.2 4.6.7 4.6.7.1 Record the observations in Annexure - 3. CALIBRATION OF DETECTOR: Standard preparation. Mobile phase, Instrument set- up same as mentioned in calibration of Injector. Run the chromatograph at different wavelength (252 nm 264 nm with 2 nm increment) The largest peak response shall be at 258 nm 2nm. Record the results in the detection calibration record as per the Annexure - 4. Affix a calibration status label on the instrument containing Calibrated On, Due On and Signature. Report to Head QC, if any discrepancy observed during calibration or operating the instrument and affix Under Maintenance label on the instrument.

4.6.7.2

4.6.7.3 4.6.7.4

4.6.7.5

4.6.7.6

MICROBIOLOGY (fromgreek, mkros, "small"; , bios, "life"; and , -logia) is the study of microscopicorganisms, either unicellular (single cell), multicellular(cell colony), or a-cellular (lacking cells).Microbiology includes the disciplines virologymycology parasitology, bacteriology and so on.

Eukaryotic microorganisms exhibit cell organelles and include fungiand protists, whereas prokaryotic organismswhich all are microorganismsare conventionally classified as lacking organelles and include eubacteria and archaebacteria. Microbiologists traditionally relied on culture, staining, and microscopy. Apparently, however, only some 1% of the microorganisms present in some environments are culturable.Microbiologists often rely on extraction or detection of nucleic acid either DNA or RNA sequences. Viruses are not always classified as organisms,[3] as they have been identified either as very simple microorganisms or very complex molecules . Pironsnever considered microorganisms, have been investigated by virologists, however, as the clinical effects traced to them were originally presumed due to chronic viral infections, and virologists took searchdiscovering "infectious proteins".

The Laboratory of Microbiology is engaged in research and education in biotransformations and interactions of microorganisms as well as their control. Moreover, it contributes to the exploitation of the generated knowledge in the application areas of Health & Food, Bioproducts& Energy and Environment & Sustainability. Microbiology Laboratory Equipment Introduction Proper use of laboratory equipment is necessary for successful experiment outcomes. Before using laboratory equipment you should understand how the equipment works, how to operate it, safety considerations in using the equipment, and how to clean and put away the equipment.

Bunsen Burner Bunsen Burner Quiz Microscope Operating Guide Oil Immersion Phase Contrast Instrument Diagram Microscope Parts Self Quiz Microscope Quiz Light Path Quiz Pipettor Calibration and Care Parts Spectrophotometry

FUMIGATION OF MICROBIOLOGY LAB 1.0 OBJECTIVE To lay down a procedure for Fumigation of Microbiology Lab. 2.0 SCOPE This SOP is applicable to Quality Control Department (Microbiology Lab). 3.0 RESPONSIBILITY Microbiologist 4.0 ACCOUNTABILITY Department Head. 5.0 PROCEDURE 5.1 Use safety goggles and hand gloves during fumigation. 5.2 Put OFF the A/C unit . 5.3 Take Potassium Permanganate (about 15 gm.) in a petri dish and keep in the area where fumigation is to be carried out. 5.4 Place the petri plate on a polyethene bag. 5.5 Add about 25 ml of 35 % Formaldehyde solution to Potassium Permanganate . 5.6 Immediately close the area. 5.7 Label the area as Area Under Fumigation so that nobody enters the room. 5.8 Keep the room under fumigation for a minimum of 8 - 12 hrs. 5.9 Defumigate the room by putting on the A/C unit . 6.0 FREQUENCY : 6.1 once a week and after completion of any civil work. Hazards of Formaldehyde : 1. It is toxic to inhalation and ingestion and causes irritation to eyes and respiratory tract. 2. Skin contact can cause dermatitis. Also cause skin burns. 3. Exposure to a concentration of 10 to 20 PPM can cause difficulty in breathing.

4. Slightly corrosive in nature, suspected carcinogen. Fire Hazards : 1. Vapors of Formaldehyde are flammable and may form an explosive mixture with air. 2. It should be stored in a cool and well ventilated area. 3. Avoid storing below 16 Degree C because it forms a polymer Paraformaldehyde which is

Aseptic Technique
Introduction Attitude: Number one rule: always think of the patient. A well thought out training program for aseptic technique should focus on safety and accuracy. Safety: a.) The finished product should be free of contamination (particles, bacteria, extraneous material). b.) The solution should be clear -- all medications should be completely dissolved. c.) All compounding materials should be checked for expiration date, outer integrity, etc. Accuracy: Guidelines must be set up to ensure the right drug, right dose, and right concentration. This includes using the appropriate syringe size to measure out fluid volumes in order to minimize errors. Another example would be to require that all syringes be drawn back to the original amount of each individual dose and placed next to the admixture to facilitate checking by the pharmacist. If a filter needle was required, it should also be present.

Dangers of poor aseptic technique: Patients who are receiving intravenous therapy tend to be the most critical. Every precaution must be taken to avoid contamination. The IV route is the most dangerous route of administration because it bypasses all of the body's natural barriers. An improperly prepared solution when administered can have very serious consequences: infections, emboli, occlusions and even.........

Laminar Flow Hoods: 1. Provide clean air to the working area. 2. Provide a constant flow of air out of the work area to prevent room air from entering. 3. The air flowing out from the hood suspends and removes contaminants introduced into the work area by personnel. The most important part of a laminar flow hood is a high efficiency bacteria-retentive filter. Room air is taken into the unit and passed through a pre-filter to remove gross contaminants (lint, dust etc). The air is then compressed and channeled up behind and through the HEPA filter (High Efficiency Particulate Air filter) in a laminar flow fashion--that is the purified air flows out over the entire work surface in parallel lines at a uniform velocity. The HEPA filter removes nearly all of the bacteria from the air.

Why control room air? The environmental control of air is of concern because room air may be highly contaminated. Example: Sneezing produces 100,000 - 200,000 aerosol droplets which can then attach to dust particles. These contaminated particles may be present in the air for weeks. (Have you ever viewed the air around you when you open the curtains on a sunny day?)... Limitations: With poor technique it is easy to overcome the established airflow velocity and introduce reverse currents that can re-introduce contaminants into the work area. Laminar hoods should remain on 24 hours a day. If turned off for any reason, it should be on for at least 30 minutes and thoroughly cleaned before reusing.

Examples of vertical and horizontal laminar flow hoods:

Aseptic technique: guidelines & key points A direct path must be maintained between the filter and the area inside the hood where the manipulations are being performed. Air downstream from non-sterile objects (such as solution containers, hands etc.) becomes contaminated from particles blown off these objects. To best illustrate this very important point click on the two examples of proper aseptic technique: (manipulation of a vial and an ampoule).

The hands should never obstruct airflow around the area where the needle enters the vial or ampoule. Also, when pulling back the plunger of a syringe, the fingers should not come in contact with any part of the plunger--except the flat part at the end. Poor manipulation of the syringe is the most frequent cause of contamination.

Always minimize clutter. Waste and other items should never enter the hood. All calculations should be done before entering the hood. Wash hands and arms before compounding or re-entering the hood. Also, remove any jewelry from the hands and wrists. It is important that you keep your hands within the cleaned area of the hood as much as possible. Do not touch your hair, face or clothing. Excess dust should be removed from items before introducing them into the hood. Arrange objects in a manner to get full benefit of the laminar flow of air. Critical items should be placed as close to the air source as possible. In a horizontal hood, items should be placed no closer than 3 inches from the very back of the hood (nothing should touch the filter). Occasionally, you may stack a few items (eg IVPB's), however they must be stacked from lower to higher starting from the back of the hood. Also, it should be limited to a maximum of 3 to 4 items. When working in a horizontal laminar flow hood, all work must be performed at a distance of no less than 6 inches from the front edge of the work surface. At a distance of less than 6 inches, laminar

flow air begins to mix with the outside air and contamination is possible. Never become so engrossed in your work that you forget this basic rule. Avoid spraying or squirting solutions onto the HEPA filter. Always aim away from the filter when opening ampoules or adjusting syringes. Outer pouches and wraps should be removed at the edge of the work area as the sterile contents are pulled into the work area. Never bring these items into the main work area. Large objects should never be placed near the back of the hood. Not only do these objects contaminate everything downstream, but they also disrupt the laminar flow pattern of air which normally suspends the contaminants and removes them from the area. Remember that hand cleanliness is further reduced each time more bottles and other non-sterile items are handled. Before and after preparing a series of IV admixtures, or anytime something is spilled, the work surface of the laminar flow hood should be thoroughly cleaned with alcohol. A long side to side motion should be used starting at the back of the hood and then working forward. The acrylic plastic sides should also be cleaned periodically. It is possible to overcome the established airflow velocity by a strong reverse current produced by coughing, quick movements, talking etc. Keep all of these to a minimum in order to maintain a sterile environment. Do not talk, cough or sneeze into the hood

The contents of glass ampoules should always be filtered before adding to an IV admixture. Needles should be used for a maximum of 8 to 10 punctures. It may be necessary to change the needle more frequently if it becomes increasingly difficult to enter a vial. Always disinfect all rubber stoppers and ampoule necks with alcohol before entering with a needle.