Water Analysis SOP as per IP/BP/USP

Standard Operating Procedure (SOP) for Water Sample Analysis as per IP/BP/USP. Procedure for chemical testing and microbiological testing of Raw/Tap water, RO water, and purified water samples.

Procedure for Water Analysis (Chemical & Microbiological)

1.0      PURPOSE:

    • To lay down a procedure for analysis of water samples (Chemical and Microbiological Analysis)

2.0      SCOPE:

    • This Standard Operating Procedure is applicable to the Quality Control (QC) department of the pharmaceutical drug manufacturing plant.

3.0      REFERENCE:Water Analysis

4.0      RESPONSIBILITY:

    • Officer or Executive of Quality Control /Microbiology department shall be responsible for the collection of the water sample for analysis as per the sampling plan.
    • Head of the Department / Designee of respective areas & QA shall be responsible for reviewing the SOPs.
    • Head QA shall be responsible for approval of SOP.

5.0      ABBREVIATIONS USED IN SOP FOR WATER ANALYSIS:

    • CFU: Colony Forming Unit
    • EHS: Environment, Health and Safety
    • HCl: Hydrochloric acid
    • IPA : Isopropyl Alcohol
    • Ml : Millilitre
    • SCDA : Soybean Casein Digest Agar
    • TAMC: Total aerobic microbial count

6.0      PROCEDURE FOR ANALYSIS OF WATER SAMPLE:

    • Carry out the determination under conditions designed to avoid extrinsic microbial contamination of the water sample for the analysis.
    • The precautions taken to avoid contamination must be such that they do not affect any microorganisms that are to be revealed in the test (water analysis).
    • Use pre-incubated media for all tests of water analysis, where pre-incubated media are not possible to use incubate a Plate/ tube/ flask as negative control along with the test samples.
    • If the pre-incubated media is using for the Microbial testing it is not compulsory to performing the media negative control.
    • Before carrying out these tests of water analysis for enumeration of the aerobic microorganisms in the test specimens, it should be confirmed that:
      • The media used in the water analysis, promote the growth of the respective microorganisms for which they are used.
      • The test specimens of water analysis to which the tests are applied do not themselves inhibit the microorganisms and the appropriateness of the methods used for enumeration of the microorganisms in the presence of the product is determined.
    • Recommended Solutions and Culture Media:

                 [Note: This section is given for information.]

    • Preparation of R2A Agar (pH 7.2) for Water Analysis :

Yeast Extract 0.5 g
Protease Peptone 0.5 g
Casein hydrolysate 0.5 g
Glucose 0.5 g
Starch 0.5 g
Dipotassium hydrogen phosphate 0.3 g
Magnesium sulfate, anhydrous 0.024 g
Sodium pyruvate 0.3 g
Agar 15 g
Purified Water. 1000 ml
    • Note: Sterilize in an autoclave using a validated cycle.
    • Preparation of Buffered Sodium Chloride–Peptone Solution for Water Analysis :

Buffered Sodium Chloride–Peptone Solution pH 7.0
Potassium Dihydrogen Phosphate 3.6 g
Disodium Hydrogen Phosphate Dihydrate 7.2 g
Sodium Chloride. 4.3 g
Peptone (meat or casein). 1.0 g
Purified Water. 1000 ml
    • Note: Sterilize in an autoclave using a validated cycle.
    • Preparation of Casein Soybean Digest Broth for Water Analysis:

Casein Soybean Digest Broth
Pancreatic Digest of Casein 17.0 g
Papaic Digest of Soybean 3.0 g
Sodium Chloride 5.0 g
Dibasic Hydrogen Phosphate 2.5 g
Glucose Monohydrate 2.5 g
Purified Water 1000 ml

 

Check the pH if necessary adjust the pH, after sterilization the pH is to be 7.3 ± 0.2 at 25 C. Sterilize in an autoclave using a validated cycle.

    • Preparation of MacConkey Broth for Water Analysis:

MacConkey Broth
Pancreatic Digest of Gelatin 20.0 g
Lactose monohydrate 10.0 g
Dehydrated Ox Bile 5.0 g
Bromocresol Purple 10 mg
Purified Water 1000 ml

 

Check the pH if necessary adjust the pH, after sterilization the pH is to be 7.3 ± 0.2 at 25C. Sterilize in an autoclave using a validated cycle.

  • Preparation of “MacConkey Agar” for Water Analysis:

MacConkey Agar
Pancreatic Digest of Gelatin 17.0 g
Peptones (meat and casein) 3.0 g
Lactose monohydrate 10.0 g
Sodium Chloride 5.0 g
Bile Salts 1.5 g
Agar 13.5 g
Neutral Red 30.0 mg
Crystal Violet 1 mg
Purified Water 1000 ml

 

Check the pH If necessary adjust the pH, after sterilization the  pH is to  be 7.1 ± 0.2 at 25C. Boil for 1 minute with constant shaking, then sterilize in an autoclave using a validated cycle.

    • Preparation of “Rappaport Vassiliadis Salmonella Enrichment Broth” for Water Analysis:

Rappaport Vassiliadis Salmonella Enrichment Broth
Soya Peptone 4.5 g
Magnesium Chloride Hexahydrate 29.0 g
Sodium Chloride 8.0 g
Dipotassium Phosphate 0.4 g
Potassium Dihydrogen Phosphate 0.6 g
Malachite Green 0.036 g
Purified Water 1000 ml

 

Dissolve, warming slightly. Sterilize in an autoclave using a validated cycle, at a temperature not exceeding 115C. The pH is to be 5.2 ± 0.2 at 25C after heating and autoclaving.

    • Preparation of Agar – Xylose Lysine Deoxycholate for Water Analysis:

Xylose Lysine Deoxycholate Agar
Xylose 3.5 g
l-Lysine 5.0 g
Lactose Monohydrate 7.5 g
Sucrose 7.5 g
Sodium Chloride 5.0 g
Yeast Extract 3.0 g
Phenol Red 80 mg
Agar 13.5 g
Sodium Deoxycholate 2.5 g
Sodium Thiosulfate 6.8 g
Ferric Ammonium Citrate 0.8 g
Purified Water 1000 ml

 

Check the pH if necessary adjust the pH after heating it is 7.4 ± 0.2 at 25  C. Heat to boiling, cool to 50C, and pour into Petri dishes. Do not heat in an autoclave.

    • Preparation of Cetrimide Agar for Water Analysis:

Cetrimide Agar
Pancreatic Digest of Gelatin 20.0 g
Magnesium Chloride 1.4 g
Dipotassium Sulfate 10.0 g
Cetrimide 0.3 g
Agar 13.6 g
Purified Water 1000 ml
Glycerol 10.0 ml

 

Heat to boiling for 1 minute with shaking. Adjust the pH, so that after sterilization it is 7.2 ± 0.2 at 25C. Sterilize in an autoclave using a validated cycle.

    • Preparation of Mannitol Salt Agar for Water Analysis:

Mannitol Salt Agar
Pancreatic Digest of Casein 5.0 g
Peptic Digest of Animal Tissue 5.0 g
Beef Extract 1.0 g
d-Mannitol 10.0 g
Sodium Chloride 75.0 g
Agar 15.0 g
Phenol Red 0.025 g
Purified Water 1000 ml

 

Heat to boiling for 1 minute with shaking. Check the pH if necessary adjust the pH, after sterilization the pH is to be 7.4 ± 0.2 at 25C. Sterilize in an autoclave using a validated cycle.

    • Microbial enumeration tests: Test for Total Aerobic Microbial Count

    • Membrane Filtration: Use a filtration apparatus designed to allow the transfer of the filter to the medium.
      • Use membrane filters having a nominal pore size not greater than 0.45 µm.
      • Use the sterile filtration unit (previously sterilized as per the validated autoclave cycle).
      • Assemble the filtration unit under Laminar airflow by keeping the sterile 0.45 µm membrane filter on the holder of the filtration unit, moisten the membrane with selected diluent/Sterile water.
      • Transfer a suitable quantity (1 ml for purified water) of the sample on membrane filter and filter immediately and wash the filter through 100 ml sterile water.
      • For the determination of total aerobic microbial count (TAMC), transfer the membrane filter to the surface of the R2A Agar and incubate the plates at 30–35°C for the 5 days in the inverted position.
      • After incubation period, count the number of colonies and report the results in cfu/ml.
    • For Test Negative Control:
    • Transfer a suitable quantity of diluent on the membrane filters and filter immediately.
    • Transfer the membrane on to the R2A Agar and incubate the plate at 30–35 °C for 5 days.
    • After incubation period, negative control plates should not show any growth.
    • If growth occurs in the negative control plates test is invalid. Failed negative control needs investigation.
    • Plate-Count Methods:

    • Perform plate-count methods at least in duplicate for each medium and use the mean count of the result.
    • Pour-Plate Method
    • For TAMC: Transfer aseptically 1 ml of the sample to each of two Petri dishes about 9 cm in diameter, add 15 to 20 ml of sterile R2A agar previously maintained at not more than 45°C (If larger Petri dishes are used, the amount of agar medium is increased accordingly) and gently mix the plates for the equally distribution of the sample and allow for solidification.
    • After solidification incubates the plate at 30–35°C for 5 days.
    • After incubation period check the plates for the growth and count the number of CFU and take the arithmetic mean of the counts per medium, calculate the total number of cfu/ ml.
    • Media Negative Control

    • For TAMC: Perform the negative control by adding the 15 to 20 ml of R2A agar previously maintained at not more than 45°C to a sterile Petri dish and allow for solidification.
    • After solidification incubates plates in an inverted position at 30–35°C for 5 days.
    • After incubation period, negative control plates should not show the growth. If growth occurs in the negative control plates test is invalid. Failed negative control needs the investigation.
    • Test Negative control: Perform a negative control using the chosen diluent in place of the test preparation. There must be no growth of microorganisms. Failed negative control needs investigation.
    • Testing of Products for Specified Pathogens:

    • Sample Enrichment:
    • Add 100 ml of water sample in 100 ml of double strength Soyabean casein digest medium.

or

    • Filter 100 ml of water sample and immerse the membrane filter in 100 ml Soyabean casein digest medium
    • Mix well, and incubate the sample at 30° – 35 ° C for 18 to 24 hrs.
    • Tests for Escherichia coli (water Analysis):

    • After incubation period, shake the container; transfer 1 ml of Soybean–Casein Digest Broth to 100 ml of MacConkey Broth.
    • Incubate at 42-44°C for 24 to 48 hours.
    • After incubation period Subculture on a plate of MacConkey Agar and incubate at 30-35°C for 18 to 72 hours.
    • Interpretation: Growth of colonies indicates the possible presence of Escherichia coli. This is confirmed by identification tests.
    • The product complies with the test if no colonies are present or if the identification tests are negative.
    • Test Negative control: To verify testing conditions, perform a negative control using the chosen diluent or media in place of the test preparation.
    • There must be no growth of microorganisms. Failed negative control needs investigation.
    • Identification Test 1 (water analysis):

Note: Biochemical test or identification by automated methods can be used for confirmatory identification.

    • Transfer well-isolated suspected colonies from MacConkey agar plate to 5 ml of 5% Peptone water or MacConkey Broth or a suitable medium contained in a test tube.
    • Incubate the tube at 42- 44°C for 24 hours.
    • Add 0.5 ml of Kovac’s reagent to the tube, shake well and allow to stand for one minute. If a red colour is observed in the reagent layer, indole is present.
    • The preparation being examined passes the test if such colonies are not seen or if the confirmatory biochemical tests are negative.
    • Test Negative control: To verify testing conditions perform a negative control using the chosen diluent in place of the test preparation. There must be no growth of microorganisms.
    • Test for Salmonella:

    • After incubation period transfer 0.1 ml of Soybean–Casein Digest Broth to 10 ml of Rappaport Vassiliadis Salmonella Enrichment Broth.
    • Incubate at 30-35°C for 18 to 24 hours. Sub culture on a plate of Xylose Lysine Deoxycholate Agar, and incubate at 30-35°C for 18 to 48 hours.
    • Interpretation
    • The possible presence of Salmonella is indicated by the growth of well- developed, red colour with black center clear to slightly opalescent gel forms on Xylose Lysine Deoxycholate Agar: This is confirmed by identification tests.
    • The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.
    • Test Negative control: To verify testing conditions perform a negative control using the chosen diluent or media in place of the test preparation. There must be no growth of microorganisms. Failed negative control needs investigation.
    • Identification Test 2 (water analysis):

Note: Biochemical test or identification by automated methods can be used for confirmatory identification.

    • Transfer separately a few of the suspect colonies to Triple sugar iron agar in tubes, using surface and deep inoculation.
    • This can be done by first inoculating the surface of the slope and then making a stab culture with the same inoculating needle and incubating at 30 – 35°C temperature for 24 hours.
    • The presence of Salmonella is provisionally confirmed if, in the deep inoculation but not in the surface culture, there is a change of color from red to yellow and usually a formation of gas, with or without production of Hydrogen sulphide in the agar.
    • Precise confirmation may be carried out by appropriate biochemical test such as Urea broth.
    • Transfer separately a few of the suspect colonies to 10 ml of Urea broth contained in the test tube and incubate at 30 – 35°C temperature for 24 hours.
    • Upon incubation, the absence of red color in the Urea broth tube indicates the presence of Salmonella.
    • The preparation being examined passes the test if, colonies of the type described do not appear or if the confirmatory biochemical tests are negative.
    • Test Negative control: To verify testing conditions, perform a negative control using the chosen diluent or media in place of the test preparation. There must be no growth of microorganisms. Failed negative control needs investigation.
    • Test for Pseudomonas aeruginosa:

    • After incubation period, subculture on a plate of Cetrimide Agar, and incubate at 30-35°C for 18 to 72 hours.
    • Interpretation: Growth of Greenish colony indicates the possible presence of aeruginosa. This is confirmed by identification tests.
    • The product complies with the test if colonies are not present or if the confirmatory identification tests are negative.
    • Test Negative control: To verify testing conditions perform a negative control using the chosen diluent or media in place of the test preparation.
    • There must be no growth of microorganisms. Failed negative control needs investigation.
    • Identification test: Oxidase test (water analysis)

Note: Biochemical test or identification by automated methods can be used for confirmatory identification.

    • Place 2 or 3 drops of a freshly prepared 1 % w/v solution of N, N, N’N’-Tetramethyl-4-phenylene diamine dihydrochloride on filter paper and smear with the suspect colony.
    • If a purple color is produced, the test is positive. Alternatively, readymade impregnated discs can also be used.
    • Test for Staphylococcus aureus (water Analysis):

    • After incubation period, subculture on a plate of Mannitol Salt Agar, and incubate at 30-35°C for 18 to 72 hours.
    • Interpretation: The possible presence of aureus is indicated by the growth of yellow or white colonies surrounded by a yellow zone. This is confirmed by identification tests.
    • The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.
    • Test Negative control: To verify testing conditions perform a negative control using the chosen diluent or media in place of the test preparation.
    • There must be no growth of microorganisms. Failed negative control needs investigation.
    • Identification Test: Coagulase Test:

Note: Biochemical test or identification by automated methods can be used for confirmatory identification.

    • With the aid of an inoculation loop, transfer representative suspected colonies from surfaces of Mannitol Salt Agar to individual tubes, each containing 0.5 ml of mammalian, preferably rabbit or horse plasma with or without suitable additives.
    • Incubate in a water bath at 37°C temperature, examining the tubes after 3 hours and subsequently at suitable intervals up to 24 hours.
    • If coagulation in any degree is observed, the test is positive.
    • Carry out a positive control by adding a volume of broth (24 hours old) containing 10 to 100 cells of Staphylococcus aureus Culture in soyabean casein digest medium of Staphylococcus aureus (ATCC 6538 / NCTC 10788) to a tube containing 0.5 ml of plasma.
    • Carry out a negative control by incubating an un-inoculated tube containing 0.5 ml of plasma.
    • Perform positive and negative control test.
    • For the test to be valid the positive control tube should show coagulation within 3 hours of incubation and there should be no coagulation in negative control tube even after 24 hours of incubation.
    • Test for Coliforms:
      • Filtered 100 ml samples and transfer the membrane filter to pre incubated plate of M-Endo agar and incubate at 30-35°C for 24 hrs.
      • Interpretation of results: Golden Green colonies with metallic surface sheen.
      • Perform the negative control.
    • Tests for Candida albicans:
      • Filter 100 ml of water sample and immerse the membrane filter in 100 ml Sabouraud Dextrose Broth.
      • Incubate at 30 to 35°C for 3 to 5 days.
      • After incubation period Subculture on a plate of Sabouraud Dextrose Agar and incubate at 30 to 35°C for 24 to 48 hours.
      • Interpretation: Growth of white colonies may indicate the presence of Candida albicans. This is confirmed by identification tests.
      • The product complies with the test if such colonies are not present or if the confirmatory identification tests are negative.
    • Test for Shigella:
      • Filter 100 ml of water sample and immerse the membrane filter in 100 ml Soyabean casein digest medium.
      • Incubate the sample at 30° – 35 °C for 18 to 24 hrs.
      • After incubation period transfer 1.0 ml of Soybean–Casein Digest (Enrichment) Broth to 100 ml of GN Broth and incubate at 30 to 35 °C for 24 to 48 hours.
      • Sub culture on a plate of Xylose Lysine Deoxycholate Agar, and incubate at 30 to 35 °C for 18 to 48 hours.
      • Interpretation
        • The possible presence of Shigella is indicated by the growth of red colored translucent colonies, without black centre on Xylose Lysine Deoxycholate Agar: This is confirmed by identification tests.
        • The sample complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.
        • Test Negative control: To verify testing conditions perform a negative control using the chosen diluent or media in place of the test preparation. There must be no growth of microorganisms. Failed negative control needs investigation.
        • Identification test: Biochemical test or identification by automated methods can be used for confirmatory identification.

Procedures for Chemical Analysis of Water:

    • Description: Check by physical examination like clear, colorless and odorless liquid.
    • Total Dissolved Solids: Take a 100 ml sample in 200 ml pre-weighed Evaporating Disk or dry beaker. Evaporate it on hot plate completely and after that dry on 105ºC in the oven for 2 hours, then cool on desiccator and weigh the beaker and calculate the result.

Calculation:                              

(W2 – W1) x 100 x 10000   =   PPM

100

W1 = Weight of empty beaker

W2 = Weight of beaker + residue (after drying)

    • Total Hardness (water analysis):

    • Take 100 ml sample to a 250 ml conical flask, add 2 ml ammonia-ammonium chloride buffer and 5 mg mordant black indicator and shake well.
    • Titrate with 0.01M EDTA to a permanent blue color persists.

Calculation:       

T.V x Strength of EDTA x 0.100 x 100 x 10000 =   PPM

Volume of sample

T.V = Volume of EDTA consumed

    • Chlorides Test (water analysis):

    • Take a 50 ml sample in 250 ml conical flask and titrate with 0.1 N Silver nitrate solution using potassium chromate as an indicator.
    • The color change yellow to brick red is endpoint.
    • Each ml of 0.01N Silver nitrate corresponds to 0.003546g of chloride.

Calculation: 

T.V x Strength of 0.1 N AgNo3 x 0.03546 x 100 x 10000 =   PPM

Volume of sample

T.V = Volume of 0.1 N AgNo3 consumed

(Or)

    • Chlorides : To 10 ml of sample, add 1 ml of 2M Nitric Acid and 0.2 mL of 0.1 M Silver Nitrate, the appearance of the   solution does not change for at least 15 minutes.
    • Sulphates Test (water analysis):

    • Place 100ml of 0.0908% w/v solution of potassium sulphate to form 500-ppm sulphate.
    • Pipette out 1 ml of dilute solution in a nessler cylinder, add 2 ml of dilute HCL and dilute to 45 ml water and 5ml of barium sulphate reagent, stir and allow to stand for 5 minutes.
    • Test: Place 1ml of sample in a nessler cylinder, add 2ml of dilute HCL sol. and dilute to 45 ml with water, add 5ml barium chloride reagent, stir immediately and allow to stand for 5 minutes.
    • The turbidity produced in test solution should not be greater than standard solution.

(Or)

    • Sulphates : To 10 ml of sample, add 0.1 ml of 2M Hydrochloric Acid and 0.1 ml of Barium Chloride Solution. The appearance of the solution does not change for at least 1 hour.
    • Nitrate Test (water analysis)

    • To 5 ml of sample in a test tube immersed in ice, add 0.4 ml of a 10% w/v solution of Potassium Chloride, 0.1 ml of Diphenylamine solution and add drop wise with shaking 5 ml of Sulphuric Acid.
    • Transfer the tube to a water bath at 50°C and allow to stand for 15 minutes.
    • Any blue colour in the solution is not more intense than that in a solution prepared at the same time and in the same manner using a mixture of 4.5 ml of nitrate free water and 0.5 ml of nitrate standard solution (2 ppm NO3).
    • Total Alkalinity (water analysis)
    • Take 100 ml Sample in 250 ml conical flask, and titrate with 0.1 N HCl solution using 0.1 ml methyl orange solution as indicator.
    • The colour change yellow to red is end point.

Calculation: 

T.V x Strength of 0.1N HCl x 0.040 x 100 x 10000 =    PPM

Volume of sample

T.V. = Volume of 0.1N HCl

    • Ammonium Test (water analysis) :

    • To 20 ml of sample add 1 ml of alkaline potassium tetraiodomercurate solution and allow to stand for 5 minutes,
    • when viewed vertically the solution is not more intensely coloured than a solution prepared at the same time by adding 1 mL of alkaline potassium tetraiodomercurate solution to a solution containing 4 ml of Ammonium Standard Solution (1 ppm NH4) and 16 ml of Ammonia free water (0.2 ppm).
    • Calcium & Magnesium Test (water analysis) :

    • To 100 mL of sample, add 2 ml of ammonium chloride buffer pH 10.0, 50 mg of mordant black 11 triturate and 0.5 ml of 0.01 M Disodium Edetate, a pure blue colour is produced.
    • Residue on Evaporation Test (water analysis) :

    • Evaporate 100 ml to dryness on a water bath and dry to constant weight at 105°C.
    • The residue weighs not more than 1 mg (0.001%).
    • Heavy Metals Test (water analysis) :

    • Determined by Method D on 12 ml of a solution prepared in the following manner.
    • In a glass evaporating dish evaporate 150 ml to 15 ml on a water bath.
    • Use Lead Standard Solution (0.1 ppm) to prepare the solution.
    • Method D:
    • Take two cylinders, one with 12 ml of evaporated water and second with 10 mL of standard solution & 2 ml of the evaporated water and mix.
    • To each of the cylinders add 2 ml of Acetate buffer pH 3.5, mix, add, 1.2 ml of Thioacetamide reagent, allow to stand for 2 minutes.
    • Then view downwards over a white surface; the color produced with the test solution is not more intense than that produced with the standard solution.
    • Acidity or Alkalinity Test (water analysis) :

    • To 10 ml of sample, freshly boiled and cooled in a borosilicate glass flask, add 0.05 mL of methyl red solution, the resulting solution is not red.
    • To 10 mL, add 0.1 ml of Bromothymol Blue solution, the resulting solution is not blue.
    • Silica Test (water analysis) :

    • To 50 ml sample add in rapid succession 1.0 ml 1+1 HCl and 2ml ammonium molybdate reagent.
    • Mix by inverting at least 6 times and let stand for 5 to 10 minutes .
    • Add 2.0 ml oxalic acid solution and mix thoroughly.
    • Read colour after 2 minutes but before 15 minutes, measuring time from addition of oxalic acid.
    • Because the yellow colour obeys beer’s law, visually compare with permanent artificial color standard using K2CrO4 and borax solution as given in the table:
Value in Silica (µg) Potassium Chromate Solution (ml) Borax Solution (ml) Water (ml)
0 0.0 25 30
100 1.0 25 29
200 2.0 25 28
400 4.0 25 26
500 5.0 25 25
750 7.5 25 22
1000 10.0 25 20
    • To 25 ml sample add 25 ml of Calcium hydroxide TS: The mixture remains same.
    • Conductivity Test (water analysis) :

    • As per respective SOP for Calibration and Operation of Conductivity meter, measure the conductivity of water at 25°C. 
    • Oxidisable Substances Test (water analysis) :

    • To 100 ml of sample, add 10 ml of dilute Sulphuric Acid and 0.1 ml of 0.02 M Potassium Permanganate and boil for 5 minutes, the solution remains faintly pink.
    • Sulphide Test (water analysis) :

    • Measure from a burette in to a 500 ml flask an amount of iodine solution estimated to be an excess over the amount of sulfide present. Add distilled water, if necessary, to bring volume to about 20 ml. pipette 200 ml sample into flask, discharging sample under solution surface.
    • If iodine colour disappears, add more iodine until colour remains.
    • Back- titrate with 0.01 N Na2S2O3 solution adding a few drops of starch solution as end point is approached, and counting until blue colour disappears.

Calculation:

Sulfide (in ppm): (Blank T.V – T.V) x Strength of 0.01N Na2S2O3 x 0.016 x 100 x 10000                                                                      Volume of Sample

    • Total Suspended Solids (water analysis):

    • Set up the filtration assembly. Wet the filter with a small volume of water to place the filter properly.
    • Mix the sample by using a magnetic stirrer at the optimum speed to shear larger particles & for obtaining uniform (preferably homogenous) particle size.
    • Use Centrifuge force to separate particles on the basis of size and density, which results in poor precision when point of sample withdrawal is varied.
    • After stirring, pour out the required volume to the seated glass fiber filter, for homogeneous sample, pour out from the approximately mid point of container but not in vortex. (Choose a point, both in mid depth and midway between wall and vortex).
    • Wash filter through three successive 10 ml volumes of reagent grade water, allowing complete drainage between washings.
    • After complete filtration, continue suctions for about three minutes.
    • Sample with high dissolved solids may require additional washings.
    • Carefully remove filter from filtration apparatus, add aluminum weighing dish as a support (if required).
    • Alternatively remove the crucible and filter combination from the crucible adapter, if a Gooch crucible is used.
    • Dry for at least 1 hour at 103-105°C in an oven, cool in a desiccator (to balance the temperature) and then weigh it.
    • Repeat the cycle of drying, cooling, desiccating and weighing until a constant weight is obtained or until a weight change is less than 4 % of the previous weight or 0.5 mg (whichever is less).

Calculation:          

Total suspended Solids mg/L =       (A – B) X 10000 

                                                                           Volume of sample mL

 

Where:  A= Weight of filter + dried residue (mg)

B= Weight of filter (mg)

    • Total Organic Carbon (TOC) 

      • Refer SOP for Cleaning and Operation of TOC Analyser.
    • Apparatus Requirements.

    • This test method is performed using a calibrated instrument.
    • The suitability of the apparatus must be periodically demonstrated as described below.
    • In addition, it must have a manufacturer’s specified limit of detection of 0.05mg of carbon per liter (0.05 ppm of carbon) or lower.
    • Reference Standards:

    • 1,4-Benzoquinone RS, Sucrose RS.
    • Reagent water: Use water having a TOC level of not more than 0.10mg per liter.
    • Glassware preparation:

    • Organic contamination of glassware results in higher TOC values.
    • Therefore, use glassware and sample containers that have been scrupulously cleaned of organic residues. Any method that is effective in removing organic matter can be used.
    • Use reagent water for the final rinse.
    • Standard Solution:
    • Dissolve in the reagent water an accurately weighed quantity of USP Sucrose RA, previously dried at 105°C for 3 hours obtain a solution having a concentration of 1.19mg of Sucrose per liter (0.50mg of carbon per liter)
    • Test solution:

Note: Use extreme caution when obtaining samples for TOC analysis. Then it can be contaminated during the process of sampling and transportation.

    • Collect the test solution in a tight container with minimal headspace, and test in a timely manner to minimize the impact of organic contamination from the closure and container.
    • System suitability Solution:
    • Dissolve in Reagent water an accurately weighed quantity of USP 1, 4 -Benzoquinone RS to obtain a solution having a concentration of 0.75mg per liter
    • Reagent water control:
    • Use a Suitable quantity of reagent water obtained at the same time as that used in the preparation of the standard solution and the system suitability solution.
    • Other control solutions:
    • Prepare appropriate reagent blank solutions or other specified solutions needed for establishing the apparatus baseline or for calibration adjustments following the manufacturer’s instructions, and run the appropriate blanks to zero the instrument.
    • System Suitability:

    • Test the reagent water control in the apparatus and record the response r­­w Repeat the test using the standard solution, and record the response, rs.
    • Calculate the corrected standard solution response, which is also the limit response, by subtracting the reagent water control response from the response of the standard solution.
    • The theoretical limit of 0.50 mg of carbon per liter is equal to the corrected standard solution response, rss.
    • Calculate the corrected system suitability solution response of the system suitability solution, rss – rw calculate the response efficiency for the system suitability solution by the formula.

100 {(rss – rw) / (rs – rw)}.

    • The system is suitable if the response efficiency is not less than 85% and not more than 115% of the theoretical response.
    • Procedure to perform TOC test in water analysis:

    • Perform the test on the test solution, and record the response, ru.
    • The test solution meets the requirements if ru is not more than the limit response, rs – rw.
    • This method also can be performed alternatively using on-line instrumentation that has been appropriately calibrated, standardized, and has demonstrated acceptable system suitability.
    • The acceptability of such online instrumentation for quality attribute testing is dependent on its location in the water system.
    • These instrument locations and responses must reflect the quality of the water used.
    • Iron Test (water analysis) :

    • Standard iron solution:
    • In a clean and dried Nessler cylinder, transfer 2ml iron solution (20 ppm Fe) and dilute with 30ml of water.
    • Add 2ml of 20% w/v solution of iron-free citric acid and 0.1ml thioglycolic acid. Mix, make alkaline with iron-free ammonia solution, dilute to 50ml with water and allow to stand for 50 min.
    • Sample solution: Transfer accurately about 1.0ml of the sample to a Nessler’s cylinder. Add 30ml of water, 2ml of 20% iron-free citric acid and 50ml with water and allow to stand for 5 min.
    • Interpretation: Not more than 0.3 ppm and any color produced by the sample should not be greater than that produced by the standard preparation.

 

 

pharmabeginers

Niranjan Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an updated pharmaceutical blogging platform. Email: [email protected]
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