Home General Cannabinoid and Terpene Info What exactly are the essential oils that we are extracting from the cannabis plant, and what are their properties?/data/pdf/2001-03-04-7.pdf as his information source: ?-9-tetrahydrocannabinol (THC) Boiling point: 157C / 315 degree Fahrenheit ( Merck 200C @0.02).Properties: Euphoriant, Analgesic, Antiinflammatory, Antioxidant, Antiemetic Cannabidiol (CBD) Boiling point: 160-180*C / 320-356 degree Fahrenheit Properties: Anxiolytic, Analgesic, Antipsychotic, Antiinflammatory, Antioxidant, Antispasmodic Cannabinol (CBN) Boiling point: 185*C / 365 degree Fahrenheit Properties: Oxidation, breakdown, product, Sedative, Antibiotic Cannabichromene (CBC) Boiling point: 220*C / 428 degree Fahrenheit Properties: Antiinflammatory, Antibiotic, Antifungal Cannabigerol (CBG) Boiling point: MP52 Properties: Antiinflammatory, Antibiotic, Antifungal ?-8-tetrahydrocannabinol (?-8-THC) Boiling point: 175-178*C / 347-352.4 degree Fahrenheit Properties: Resembles ?-9-THC, Less psychoactive, More stable Antiemetic Tetrahydrocannabivarin (THCV) Boiling point: < 220*C / <428 degree Fahrenheit Properties: Analgesic, Euphoriant Terpenoid essential oils, their boiling points, and properties ß-myrcene Boiling point: 166-168*C / 330.8-334.4 degree Fahrenheit Properties: Analgesic.

Antiinflammatory, Antibiotic, Antimutagenic ß-caryophyllene Boiling point: 119*C / 246.2 degree Fahrenheit Properties: Antiinflammatory, Cytoprotective (gastric mucosa), Antimalarial d-limonene Boiling point: 177*C / 350.6 degree Fahrenheit Properties: Cannabinoid agonist?, Immune potentiator, Antidepressant, Antimutagenic linalool Boiling point: 198*C / 388.4 degree Fahrenheit Properties: Sedative, Antidepressant, Anxiolytic, Immune potentiator pulegone Boiling point: 224*C / 435.2 degree Fahrenheit Properties: Memory booster?, AChE inhibitor, Sedative, Antipyretic 1,8-cineole (eucalyptol) Boiling point: 176*C / 348.8 degree Fahrenheit Properties: AChE inhibitor, Increases cerebral, blood flow, Stimulant, Antibiotic, Antiviral, Antiinflammatory, Antinociceptive a-pinene Boiling point: 156*C / 312.8 degree Fahrenheit Properties: Antiinflammatory, Bronchodilator, Stimulant, Antibiotic, Antineoplastic, AChE inhibitor a-terpineol Boiling point: 217-218*C / 422.6-424.4 degree Fahrenheit Properties: Sedative, Antibiotic, AChE inhibitor, Antioxidant, Antimalarial terpineol-4-ol Boiling point: 209*C / 408.2 degree Fahrenheit Properties: AChE inhibitor.

Antibiotic p-cymene Boiling point: 177*C / 350.6 degree Fahrenheit Properties: Antibiotic, Anticandidal, AChE inhibitor Flavonoid and phytosterol components, their boiling points, and properties apigenin Boiling point: 178*C / 352.4 degree Fahrenheit Properties: Anxiolytic, Antiinflammatory, Estrogenic quercetin Boiling point: 250*C / 482 degree Fahrenheit Properties: Antioxidant, Antimutagenic, Antiviral, Antineoplastic cannflavin A Boiling point: 182*C / 359.6 degree Fahrenheit Properties: COX inhibitor, LO inhibitor ß-sitosterol Boiling point: 134*C / 273.2 degree Fahrenheit Properties: Antiinflammatory, 5-a-reductase, inhibitor About The Author Related postsShare Published on Dec 29, 2015 0 Comments Statistics Notes Be the first to like this Views Actions 0 Embeds 0 1.FOOD ANALYSIS LABORATORY PRIYANKA BAL HALDIA INSTITUTE OF TECHNOLOGY 09/06/2014- 12/06/2014 2.NUTRITIONAL PARAMETERS: For any procedure to be followed we need to go through some specific norms like sequential steps- 1.

Calibration and Verification of all the instruments that are to be used.Method validation of the procedure that is to be followed.Sample preparation for the specific analyte that is to be detected.Detection of the required parameters within the sample following the SOP(Standard Operation Procedures)which are validated.FAT ESTIMATION: Fat is a nutrient.vaporizer quizIt is crucial for normal body function since it supplies maximum calorific value.1933 vaporizerChemically they are tri-esters of Glycerol and Fatty acids.top rated vapor e cigsThey are soluble in non-polar solvents but insoluble in water.atmos rx vaporizer - midnight black

Depending upon this particular property of solubility and partition co-efficient fat is estimated in a food sample.Fat can be estimated by 3 procedures depending upon the nature, chemical properties as well as its physical properties.They are:  By Soxhlets Apparatus  Acid hydrolysis  Rose gotlieb method Here we have given special emphasis upon rose gotliebs method for the estimation of fat in a milk powder sample.volcano vaporizer urban dictionaryDETERMINATION OF FAT IN MILK POWDER SAMPLE BY ROSE GOTLIEB METHOD: 1.2.e cigarette saxe gambettaProcedure: Step1: 1gm of milk powder sample was taken.To it 10ml of boiling water or luke -warm water added (so as to make it soluble).To it 2-2.5 ml of NH4OH was added.NH4OH differentiates the fat and protein layers by hydrolysing the 3. bond between them.

To it Petroleum benzene (BP-60 TO 80) added at a quantity of 20-25 ml.Step 2: The total things are taken in a separating funnel (also known as mozzenors flask) and for 5 minutes shaken and simultaneously pressure released by opening the lid.The pressure inside the funnel increases due to the volatile nature of the non polar solvent and due to the variation in vapour pressure between water and petroleum benzene.if preventions not taken by releasing the pressure inside it can blast and break the flask.Step 3: Now while shaking froathing occurred and foams are formed.To avoid it 10 ml of alchohol was added which dissolves the foam hence act as anti foaming agent.Now in a separating funnel a cotton plug was kept and upon it Na2S04 (Sodium Sulphate)was added in required amount and the whole liquid in the separating funnel was allowed to pass through it.This method was followed so that if any impurities were present which are soluble in water will get absorbed in the Na2S04 bed since it’s a good water absorbent.

Now after passing through it the total organic layer was collected in a beaker which contains only organic layer containing fat within it since fat is soluble in non polar solvent.Now step 2 and 3 were followed thrice for better extraction.Now the total organic layer after 3 repeated extraction was taken in a beaker and kept in water bath and the temperature was set at the boiling point of the organic solvent used.This is because at that temperature the solvent will vapourize out and will evaporate keeping behind within the beaker only the fats which was desired to be estimated.Step 4: Now the weight of the beaker taken (W2) separately.1.2.Calculation: Weight of the empty flask = W1.Sample weight = SW Final weight of the flask = W2 Percentage (%) of fat estimated = ×100 4.PROTEIN ESTIMATION: Proteins are basically long chain compounds made up of amino acid chains connected by peptide bonds.The amino acids constitute of N-terminal which comprises the amino group and on the other hand has a C-terminal that 5. constitutes the carboxyl end.

Proteins constitute the major portion of our body since our body is made up of muscles, visceral organs, hormones, enzymes all comprises proteins.2.DETERMINATION OF PROTEIN BY AUTO KJELDAHL METHOD: The SOP that was followed in this method was IS-7219.It is accomplished basically by protein digestion method.: 2.1.Procedure: Step 1: 1gm of the sample taken.To it 0.9-1.2 gm of CuS04 added.Then 10gm of Na2SO4 added to it.To it 25 ml of H2SO4 added and it was given for digestion for 3 hrs.For first ½ hour temperature was kept at 350 degree than the temperature was risen slowly to 450 degree.It will be observed that the blank and the standard will appear green.Sample 1, 2, 3 will appear brown in colour.Now after digestion fumes that will be evolving were allowed to pass through the Neutralizer which consists of 33% NaOH and Bromothiomol as indicator.ASH CONTENT: The inorganic residue obtained by igniting a specimen of pulp, paper, other cellulosic materials in such a way that the combustible and volatile compounds are removed.

The ash content is usually expressed as the percentage of such residue based on the weight of the test sample.3.DETERMINATION OF ASH CONTENT OF ANY FOOD SAMPLE: 3.1.Procedure: Step1: The sample was taken in a crucible.Step2: the sample was kept in HOT AIR OVEN at 550 degree such that it gets ignited completely and become carbon free.3.5.CALCULATION: Weight of empty crucible = M1 Final weight of crucible after charing at 500 degree (crucible + ash) = M2.Sample weight = SW.Percentage (%) of ash = ×100 Percentage (%) of ash [on dry basis] = MOISTURE CONTENT: Moisture content or water content is the quantity of water contained in a material consisting of both monolayer water as well as capillary water.4.DETERMINATION OF MOISTURE CONTENT OF ANY FOOD SAMPLE: 4.1.Procedure: Step1: Weight of the sample and the disc taken and it was dried at a temperature Step2: After drying final weight was taken.4.5.CALCULATION: Weight before drying (dish + sample) = (M1+10).

Weight after drying (dish + sample) = M2.Percentage (%) of moisture = (M2-M1-10) × 100 CARBOHYDRATE ESTIMATION: Large group of organic compounds occurring in food and living tissues and including sugars, starch and cellulose.They contain hydrogen and water in the same ratio as that of water i.e.(2:1) and typically can be broken down to release energy in body joined by glycosidic bonds.5.DETERMINATION OF CARBOHYDRATE IN A FOOD SAMPLE: Carbohydrate cannot be determined directly by any process or experiment.It was completely calculation based.So for carbohydrate estimation we need to estimate all other nutritional parameters.CALCULATION: Percentage (%) of carbohydrate = 100-[moisture+ fat+ protein+ ash].ENERGY CALCULATION: {[9×Fat] + [4×carbohydrate] + [4×protein]} = total energy in a food sample expressed in Kcal/100gm 8.ENVIORONMENT LABORATORY INSTRUMENTS: Analytical Balance: It is used to measure the mass of any substance as per the norms of UACPA or BIS.

It can measure a weight from 0.1 – 200 gm precisely.It follows 3 basic properties.Linearity: If the result follows Y = MX +C.And for linearity R2 = 0.95 ;( regression co –efficient).Repeatability: Same instrument giving same reading for number of times.Here we need to calculate the standard deviation which is equal to 0.1%.if not then it s erroneous.RSD = % 3.Reproducibility: Result given by different analyst for same machine on calibration for which the value shouldn’t be more than 5%.4. off centre test: Checked once in 3 months.The mass is kept at four different positions within the pan and the mass and its deviation was noted.Difference shouldn’t be more than 0.002 gm.AIR: The quality of air is divided into 3 categories.They are classified as under-  Ambient air  Stack emission  Indoor air quality.PARAMETERS OF AIR: 9. AMBIENT AIR: 1.Particulate matter size <10 µ 2.BENZO α PYRENE 10.

Lead  STACK EMISSION: 1.Velocity of gas flow 8. INDOOR AIR: 1.VOC (volatile organic compound ) 4.Co2  WORK ZONE: 1.N02  HAZARDOUS WASTES: 10.Flash point 2. pH 3.TCLP (toxicity characteristic leaching procedure).SOIL (EDAHIC FACTORS):  Mandatory factors for soil: 1. Other parameters: 1.Organic carbon 2. pH.INSTRUMENTS FOR HAZARDOUS/PETROLEUM TESTING: 1.DEVELANT OPEN CUP FLASH POINT APPARATUS 1.ABEL FLASH POINT APPARATUS 11.PENSKY MORTENS FLASH POINT APPARATUS Here no water is used as a heating medium in PENSKY MORTENS flash point apparatus. Flash Point:  Fire Point: BOMB CALORIMETRE: A Bomb Calorimeter is used to detect the calorific value of any combustible product. Principle: Total Heat Gained by the Calorimeter = Total Heat Lost By The substance. Calculation: Calorific value = ORSAT APPARATUS: This apparatus is used to measure O2, CO2, CO present in the air.

First air is collected in some TEFLAR bags then connected to the apparatus.All the fluids within the vessel are marked to auto zero then as per the deviation in the height in the fluid column due to variation in the pressure the amount is calculated.CANON FLASK VISCOMETER: CO ANALYZER: 13.This device is used to measure CO present in the air.Teflar bags are connected to it and the machine is automated.A filter paper present inside that restricts the motion of all particulate matters. It works on the basis of IR technology. Calibration is done once in a year.For packed drinking water procedure followed is BIS-14543- 2004 And for unpacked drinking water is IS- 10500-2012 For packaged natural mineral water rain water river water process followed is given by IS-13428- 2005 Agencies that look for water are:  BIS  WAPCB  MoEF Water analysis Water is very important part of our life.

We cannot ignore water from our daily life.So its puerility must be analyzed.BIS give method for checking its quality.PARAMETERS FOR WATER AND WASTE WATER: 1.Color 3. ph 4.Total dissolved solid 8.Total suspended solid 9.Settle able solid 15.Sludge Volume Index (SVI) INORGANIC PARAMETERS AND NON- METALLIC) 1.Chlorides (as Cl- ) 5.Chloride residue (as Cl2) 6.Total hardness (as CaCO3) 9.Total kjeldahl nitrogen (TKN) 10.Nitrate nitrogen (as NO3) 11.Nitrite nitrogen (as NO2) 12.Phosphate (as PO4) 15.Sulphate (as SO4) 14.Silica (as Si) 18.Cyanide (as CN) 19.Sulphides (as H2S) INORGANIC PARAMETERS (METALS) 1.Vanadium ORGANIC (GENERAL) AND TRACE ORGANICS PARAMETERS: 1.Bio chemical oxygen demand (BOD) 2.Chemical oxygen demand (COD) 3.Oil and grease 4.Phenol (as C6H5OH) 5.Pesticides: oregano-nitrogen phosphorus 6.Total organic carbon 8.

Surfactants (as MBAS) 9.Tannin and lignin 10.Poly-chlorinated biphenyl (PCB’s) each 11.Polynuclear aromatic hydrocarbon (PAH) 12.Organic carbon (in solid) 13.Carbon/nitrogen ratio MICROBIAL TEST PARAMETERS: 1.Total coli form 2.Total plate count 4.Fecal coli form 6.Enterococcus LIST OF INSTRUMENTS FOR WATER ANALYSIS: 1.UV visible spectrophotometer 4.Hot air oven 6.AAS (Atomic Absorbance spectrophotometer ) 8.ICPMS (inductive coupled plasma mass spectrometer) 9.ION CHROMATOGHAPH Total solid can be detected by gravimetric method.Metal detector are AAS, ICPMS.FIVE IMPORTANT PARAMETERS TO BE DETECTED IN WATER SAMPLE….1. pH: It is a measure of the acidity or basicity of an aqueous solution.Pure water has a pH very close to 7. pH is defined as the decimal logarithm of the reciprocal of the hydrogen ion activity, aH+, in a solution.TSS (total suspended solid) : It measures the total solid particles present in sample.

Here is steps to calculate TSS…  Take 50 ml sample and pass to preweighted filter paper (w1)(paper dried at 105˚C for 1 hr)  Again dry the filter paper at 105˚C till constant weight is achieved. Take final weight W2 TSS (mg/l) 3.COD (Chemical Oxygen Demand : It is the no.of ml of oxygen required to chemically oxidize 1l of water.BOD (Biochemical Oxygen Demand): BOD is number of ml of oxygen required to biologically oxidize 1l of water.Oil and grease: Steps to find oil and grease in sample:  Take 1000 ml oil mixed sample in a separating funnel  Add (1:1) 5ml HCl to maintain pH less than 2. Add 30ml n-hexane shake well add another 2 times n-hexane 30ml each. Wait for layer separation  On o separating funnel put a filter paper and sodium sulfate and wet it with n-hexane. Now drain the water layer. Pass the organic layer through sodium sulfate into weighted beaker(W1)  Evaporate n-hexane in water bath.

 Take final weight(W2) O&G (mg/l) PARAMETERS FOR PACKED DRINKING WATER: 1.Total Dissolved Solid 6.11.Sulfate 12.Alkalinity 13.Calcium 14.Magnesium 15.Residual Free 16.E Chloride 17.Phenolic Compounds 18.Anionic Surface Active Agent 19.Sulfides 20.Cyanide.PACKAGED DRINKING WATER BOTTLES.PRIYANKA 13/06/2014 – 17/O6/2014 RESIDUE ANALYSIS LABORATORY 21. to vials for detecti 1.ROTA EVAPORATOR: This instrument is used for evaporating the liquid that is obtained after extraction.It works on the principle of BOILING POINT.The boiling point of any liquid is that particular temperature at which its vapour pressure becomes equal to the atmospheric pressure and the liquid starts boiling and under goes phase change.In rota evaporator at time one sample is being used.There is a water bath which provides the heating medium.The temperature is set as per the boiling point of the solvent that is to be vaporized.

The flask is attached to the condenser and kept rotating in the water bath clockwise.As soon as the temperature rises, the liquid starts passing through the condenser where it gets concentrated and free from any impurity.And thereby what was left after completion of the process was collected by taking out the round bottom flask.Precaution:  Pressure should be gently released while opening otherwise it could crack the flask. The instrument should be always calibrated and verified.SYNCHORE EVAPORATOR: Principle similar as that of rota evaporator.Mainly used if the amount of sample that is to be vaporised is more than 100-200 ml.Advantage  At time more than 1 sample can be worked together because it is having capacity of 6 samples. Saves time and labour saving service.3. pH METER: Principle: Used for measuring pH of any solution.pH is actually negative log[H+ ] ions.It ranges from 0 – 14. pH = 7 (neutral)  pH > 7 (acidic)  pH < 7 (basic) 22.

Calibration: pH meter is calibrated by using several buffers like acidic buffer, basic buffer.CENTRIFUGATION CHAMBER: In centrifugation chamber we use to centrifuge any analyte or commodity.There is a pot inside fitted within the machine inside.Within it remain centrifugation tubes where we need to put our samples.Always they are balanced with equal amount of weight diametrically opposite.Then by means of software the speed was adjusted in terms of RPM (revolution per minute) with the time which indicates how long it would centrifuge.Then after the revolution completes machine automatically indicates and it is stopped and opened.Then the tubes were taken out slowly.The analyte within it was separated into two part supernatant and 5.MULTI REAX VORTEX: It is basically a shaking agent used for thorough mixing of the components in solution.ULTRA SONIC BATH SONICATOR: Used to de-gas the mobile phase in any process.SILENT CRUSHER: It allows taking out or extracting trace elements from inside the cell matrix which enters into the cell components by means of endosmosis.

TURBO VAP EVAPORATOR: It too works the same as that of rota evaporator or synch-ore evaporator.But the difference lies in the volume of the sample evaporated.Generally ria –vials are vaporized using turbo evaporator.FUME HOOD: Generally when we deal with acids it’s better to use fume hood.Because it absorbs all the toxic, harmful, poisonous fumes that generates after addition of corrosive acids in the analyte.As a result of which volatile matters also oozes out with the fumes so for precaution they should be performed within the fume hood.10.MICRO BALANCE AND ANALYTICAL BALANCE: 23.Micro balance is mainly used to weigh the samples ranging from 1mg - 6mg.Whereas analytical balance is used to measure weight from 5mg – 220 mg.11.REFRIGERATOR: The refrigerator is used to store standards for preservation at low temperature for long time.Temperature maintained inside it is (2-8) ᵒC.Mother Stock are kept within the refrigerator.12.GRINDER: Basically in EFRAC we used 2 grinders.

One of 20 kg and the other of 50kg.When it was asked to measure the amount of pesticide present in a trench or a block of farming plot the plot was divided into 4 quadrants and from each corner samples are taken and one from centre and grinded in the respective grinders as per weight.Then after crushing they were taken for sample preparation and further detection.SAMPLE PREPARATION Basically three steps are followed while sample preparation.They are as follows- 1.Clean up’ and 3.For extraction basically we follow QUECHERS METHOD, which means Quick Easy Cheap Effective Rugger and Safe.Extraction is mainly of 3 types-  SOLID LIQUID EXTRACTION. LIQUID LIQUID EXTRACTION. SOLID PHASE EXTRACTION.Now brief ideas of the above topics are given below. PRACTICAL PROCEDURES OF SAMPLE PREPARATION: 24. Solid Liquid Extraction: Step1- Grind the sample in a mixer grinder.To detect the compounds some standards will be provided.

From them working standards will be prepared of known concentration by means of serial dilution.Step2- We will prepare two samples.One control sample and the other spike sample.To the spike sample we will add 1 ml of the spike. What Is Spike?Ans- Spike is a known concentration of a mother stock bearing similar compounds as that of the elements that are going to be detected within the commodity so as to check whether the process followed is correct or not and to see the percentage of recovery.Spike calculation- Step3- Now 10 ml of ACN added to the sample (Control + spike).Step4- We vortex the sample to homogenize the solution properly.Step5- After that 1gm of NaCl and 4gm of Na2SO4 was added to it.Step6- It was further centrifuged at 8000 RPM for 10 minutes.Step 7- Then from it 4 ml of the solution after centrifugation was taken in Ria- Vials.Step8 – To it now 50mg PSA (Primary Secondary Amine), 100 mg C18, 2-5mg of GCB (Graphite Carbon Black) was added.

Step9- Again the vials were centrifuged at 5000 RPM for 5 minutes.Step10- Then from them 1ml of the sample was taken in a fresh Ria-Vial and allowed to vaporize via Turbo vaporizer.Step11- Now if the sample is to be detected by GC (Gas Chromatography), then to it Ethyl Acetate added and if by UPLC then DCM (Di Chloro Methane) was to be added. Liquid Liquid Separation: Liquid liquid extraction was mainly performed on the basis of immiscibility nature of the solvent separated on the basis of-  Partition Co-efficient.The solvent which is mainly used here for extraction is DCM (Di Chloro Methane).Now the sample preparation for this process as per in-house validation is described below- Step1- 1lt of the sample taken in a measuring cylinder and poured into separating funnel or Mozzeners Flask.Step2 – pH of the sample was tested using pH probe (calibrated) or by pH strip.Step3- To it 80- 120 gm NaCl was added.Step3- Now DCM was added to it for 3 times 60 ml each and the process was followed so that for better extractions.

Step 4- Now the separating funnel is allowed to shake for 5 minutes for complete mixing of the polar and non polar solvents for fine extraction.Step5- After some time we will notice that the two layers got departed.The organic layer goes down due less specific gravity where as the aqueous layer remains up due much higher density.Step6- Now the solution was allowed to pass through a funnel containing Na2SO4, which is guarded by a cotton plug at the bottom.Na2SO4 is good water absorbent.It absorbs moisture and the impurity that are water soluble.Step 7- Now the extracted sample was allowed to vaporize by means of rota evaporator.Step8- After evaporation the concentrated form that will stick to the wall of the round bottom flask will be extracted out with hexane (4- 5 ml).Step9- Then from it 4- 4.5ml of the sample taken in Ria- vials and vaporized out through turbo evaporator and finally the remaining extract was treated with either Ethyl Acetate for running through GC or ACN for running through UPLC.

 Solid Phase Extraction: In case of solid phase extraction the analyte is extracted in solid belt (C18, silica) cartridges for getting higher recovery.The analyte was collected in belt and negative pressure applied to it.Basically the phenomenon depends upon the principle of ionisation i.e.hydrophilic lyophilic balanced reverse phase.The procedures that are followed for solid phase extraction is as follows- Step1- Conditioning [CH3OH or Methanol is mainly used to activate the functional groups].Step2- Equilibrate [generally water is used].Step3- Loading [passing the sample].Step4- Washing [to remove the impurities].Step5- Illusion [solvent used to remove the analyte]. No evaporation is required for the above process. This process is mainly used for extraction of antibiotics. Types Of Cartridges used- I. Weak Cation Exchange [WCE].Strong Cation Exchange [SCE].Mixed Cation Exchange [MCE].Weak Anion Exchange [WAE].V. Strong Anion Exchange [SAE].

INSTRUMENTS USED FOR DETECTION: 1.UPLC (MS/MS)[ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING MASS SPECTROMETRY DETECTOR]:  Principle: The basic principle upon which UPLC (MS/MS) works is the variation in there stationary and mobile phases.Generally in the early decades chromatography was performed normally using NON POLAR MOBILE PHASE (hexane, DCM) and POLAR STATIONARY PHASE that is Water.But nowadays Reverse phases are used.STATIONARY PHASE is considered to be NON POLAR and MOBILE PHASE to be POLAR.Why do we need to change the phase?a) Since all the drugs are soluble in water.b) In human body 98% is water and it is mobile in nature since it takes part in basic circulatory system mixed with blood. Detector used: In MS we analyze by mass: charge ratio of any element, so we need to ionize the compounds.Generally we use FORMIC ACID, BUFFER contents to ionise the compounds.GC- MS/FID/ECD [GAS CHROMATOGRAPHY using MASS SPECTROMETRY,FLAME IONISATION DETECTOR OR ELECTRON CAPTURE DETECTOR]:  Principle : The basic principle is same since here also reverse phase chromatography is used.

Generally thermally stable compounds are detected by GC.Working temperature of this instrument is 300ᵒC.Three types of injectors are used here- a. Split mode- When the sample quantity is less and we have to assure whether our sample is ok or not.It splits the injecting mechanism in 2 or 3 subdivisions.For e.g.- suppose 5ml of sample to be taken, it can be taken in twice as 3ml + 2ml.b. Split less mode- When the sample quantity is less we can directly inject 5 ml in the vials for detection.c. Head space mode- When we are working will alcohols, generally we need to check the purity of the sample we use this mode.The samples are taken within the vials and kept in auto injector where high temperature is provided.Alcohols are volatile by nature so they quickly get converted into vapour 29. phase and then this vapour is directly taken by the injector as mobile phase. Other parameters used: Nearly 250- 350ev high voltage is applied at high temperature to vaporize as well as ionise the samples and pass through the columns Columns are long cylindrical rods of varying diameter and length made up of packed C18 or polysiloxane performs the function of stationary phase Methanol/ACN/Acetone can’t be used directly because they can choke the columns.

GC MS/MS [GAS CHROMATOGRAPHY USING MASS SPECTROPHOTOMETRY(Quantitative)/MASS SPECTROPHOTOMETRY(Qualitative)]: 30. Principle- The process works step by step.Firstly we use to scan and tune any analyte so as to make it acquainted with the machine and see whether the particular analyte is present in the sample or not.For this we take 50- 550mg of sample and provide it oven temperature.Now we will detect its RETENTION TIME (time for which the analyte will remain bound to the stationary phase) and the parent ions to see which gives the maximum response.This gives the qualitative response. Calibration- GC MS/MS is calibrated by PFTBA. Generally pesticides residues are detected by it which is not thermally stable.HRGC/HRMS [HIGH RESOLUTION GAS CHROMATOGRAPHY/HIGH RESOLUTION MASS SPECTROSCOPY]:  Principle- Electromagnets are there with columns from which the compounds after passing through the electromagnetic field in vacuum splits up into spectral bands where they are detected.

The whole instrument is kept in air not in tough with the ground so as 31. to avoid minimum vibration that may cause fluctuation in the flow of electromagnetic radiations. Can give result up to 5 points after decimal so as to verify accuracy. Used to detect DIOXIMES, FURANS. For verification reference standards are used like PFK (per fluoro kerosene) which is injected in between the mid of the run at anytime for double verification.DETERMINATION OF OC COMPOUNDS IN EGG SAMPLE AND SAMPLE ANALYSIS: The commodity that was provided to us was an egg sample.The method of extraction that was followed is solid liquid extraction procedure by means of Quenchers dye. Preparation Of Calibration Curve- 10 ppm of known standard was provided.From it 5 working standards formed namely that of concentration 200ppb, 100ppb, 50ppb, 10ppb so as to plot the calibration curve. Methods Followed- A.2.0351 gm of the control sample and 2.0344 gm of spike sample taken.

To it 1ml of spike standard inoculated.C. 10 ml of ethyl acetate added to it.2gm NaCl and 3 gm Na2SO4 added.E. Allowed to vortex it.F. Then it was centrifuged for 10000 RPM for 10 minutes.G. Then the debris was discarded and the supernatant was taken by my micro pipette of 4 ml and put into Ria- vials.H. Then to it 70 mg of PSA, 120 mg of C18 and 200 mg of MgSO4 was added.I. Kept for some time and then again centrifuged at 5000 RPM for 5mins.J. Then from it 1 ml of the sample was taken in fresh Ria – vials and allowed to evaporate by the turbo evaporator.Finally to it EA was added and transferred on in GC- MS/ECD/FID 33.HALDIA INSTITUTE OF TECHNOLOGY.RESIDUE ANALYSIS LABORATORY 35.REAL TIME PCR A real-time PCR, also called as quantitative polymerase chain reaction (qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR) which is used to amplify and simultaneously quantify a targeted DNA molecule.

Basically it is used to detect strands of particular DNAs so as to judge whether the substance is genetically modified organism or not.The procedure followed was the general principle of polymerase chain reaction; its key feature is that the amplification of DNA was detected at the instantaneous reaction progress so called as Real Time.Gene expression analysis can be done using 1.SYBR® Green I dye chemistry.Basically here we use to detect 2 particular strands-  35S(Cauliflower Mosaic Virus)  N0S (Agrobacterium tumificans) The polymerase chain reaction proceeds in 3 subsequent ways- A. Denaturation of template (95ᵒC) B. Annealing of primer to template (60ᵒC) C. Extension of primer (60ᵒC) BIOLOGICAL SIGNIFICANCE: It involves TaqMan chemistry which uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during the PCR cycle.Initially, intercalator dyes were used to measure a real time PCR product.

The primary disadvantage of these dyes is that they detect accumulation of both specific and non specific dyes. How TaqMan sequence detection chemistry works: Step1: An oligonucleotide probe is constructed containing a reporter fluorescent dye on the 5’end and a quencher dye on the 3’end.While the probe is intact the proximity of the quencher dye greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance transfer (FRET) through space.Step2: If the target sequence is present, the probe anneals downstream from 1 of the primer sites and is cleaved by the 5’nuclease activity of Taq DNA polymerase as this primer is extended.Step 3: Cleavage of the probe separates the reporter dye from the quencher dye, increasing the reporter dye signal On the other hand removal of the probe from the target strand, allowing primer extension to continue to the end of the template strand.Thus inclusion of the probe does not inhibit the overall PCR process.

Step 4: Additional reporter dye molecules are cleaved from their respective probes with each cycle resulting in an increase in the fluorescence intensity proportional to the amount of amplification. ADVANTAGES OF TaqMan CHEMISTRY: 1.Specific hybridization between probe and target is required to generate fluorescent signal.Probes may be labelled with different, distinguishable reporter dyes, which allow amplification of two distinct sequences in one reaction tube.Post PCR process was eliminated which reduces assay labour and material cost.Primary disadvantage was the synthesis of different probes was required for different sequences.MACHINE HANDELING: In a real time PCR everything is automated.Just what was required was that there are some wells which need to be filled with sample that to be detected.Firstly DNA s was isolated using kit needed to add as per SOP.Secondly PRIME and PROBE were added.Within the kit there were master mix (DNTP with Taq polymerase, negative control (RNS free water), ROx dye that gives fluorescence’s.

Prime probe mix contains positive control with it in some different coloured vials.Taking 130µl of master mix + 5.2µl of dye + 10µl of prime probe mix it was fed to the wells.Then they were covered with a plastic polymeric strip so as to avoid contamination from 1 well to another consecutive well 38.Within the PCR there are Thermal Cyclic Block at the base which provides heat to the inoculum , a Halogen detector which catches the fluorescence and a camera at the top that detects and captures and detects the images.MEDIA PREPARATION: Medias are mainly prepared to provide enrichment to the bacteria’s for their growth.Basically specific Medias are prepared for specific organisms.PCA – Total bacterial count.YEDCA – Total yeast and mould count.BPA - Staphylococcus sp.MB (DS/SS) – E.coli 5.PLATING TECHNIQUES: Plating is mainly of three types- 1.Pour plate Here the organisms are inoculated before and then the medium is poured and rotated clockwise and anti clockwise.

Spread plate Here the medium is already made and the organisms are spread upon the plate by means of a bent glass rod.Streak plate Here the culture is taken in a nichrome loop and taken in solid media and parallel lines are drawn to isolate pure culture.STAINING TECHNIQUES:  THE GRAM STAIN This is a DIFFERENTIAL STAIN.It requires a PRIMARY STAIN and a COUNTERSTAIN.It divides most of the EUBACTERIA into two large groups: GRAM POSITIVE bacteria and GRAM NEGATIVE bacteria.The basic procedure goes like this: 1.Take a heat fixed bacterial smear.Flood the smear with CRYSTAL VIOLET, 1 minute, then wash with water.Flood the smear with IODINE, 1 minute, and then wash with water.Flood the smear with ETHANOL-ACETONE, quickly, and then wash with water.Flood the smear with SAFRANIN, 1 minute, and then wash with water.Blot the smear, air dry and observe. EXPERIMENT: o 1.)Perform the gram stain on smears made form the following organisms:  Staphylococcus aureus  Escherichia coli 2.

) Use a sterile applicator stick to obtain a sample of the "crud" on your teeth at the gum line.Mix the sample with a loopful of water on a microscope slide and allow the specimen to air dry.Heat fixes and performs the gram stain on this sample.Observe all of your specimens under oil immersion and record cell morphology and arrangement and gram reaction.GRAM (+) organisms holds onto the crystal violet-iodine complex more tightly than the GRAM (-) bacteria do.However this is not an absolute phenomenon!Sometimes gram (+) cultures will appear gram (-) or GRAM VARIABLE (a mixture of gram (+) and gram (-) cells).What are some of the reasons for this?Too harsh heat fixation 3.Too thick a smear 4.Improper washing between steps 5.Too old a culture 6.Impure or mixed culture 41.It is believed that the gram staining characteristics of an organism is a function of its cell wall.Therefore any time the cell wall is damaged the gram stain characteristics of gram positive cells will change.

On the other hand, gram (-) cells never become gram (+) if the stain procedure is done correctly.Gram (+) cells have thicker cell walls - more peptidoglycan and teichoic acid.Gram (-) cells have Lip polysaccharide (Endotoxin) in the outer membrane of their cell walls.Gram (+) cells is generally more sensitive to those antibiotics which interfere with cell wall.Gram (+) cells is more sensitive to lysozyme - a peptidoglycan digesting enzyme.The gram morphology of some common bacteria: Gram (+) = Staphylococcus, Streptococcus, Bacillus, Clostridium Gram (-) = the coliforms: (Escherichia, Klebsiella, Serratia), the enteric pathogens: Salmonella, Shigella, Campylobacter  THE CAPSULE STAIN - Most bacteria have some kind of CAPSULE.This viscous surface layer is also known as the SLIME LAYER, the GLYCOCALYX or the EXTRACELLULAR POLYMERIC SUBSTANCE (EPS).Most bacterial capsules are composed of polysaccharide however some genera produce polypeptide capsules.

Capsular material is very moist (slimy) and any heating will cause it to shrink - it is for this reason that we will not heat fix the slide before staining.Also, heating may cause the bacterial cell to shrink resulting in a clear zone around the cell - which may cause cells which don't have capsules to appear as if they do.The polymers which make up the capsule tend to be uncharged and as such they are not easily stained.For this reason we use a NEGATIVE STAIN to visualize them.That is, we use a stain which stains the background against which the uncolored capsule can be seen.Our procedure, the Gin's Method, uses india ink to color the background and crystal violet to stain the bacterial cell "body" 42.This structure helps the bacterial cell to ATTACH TO SURFACES and to AVOID BEING PHAGOCYTOSED.For instance, the oral streptococci produce a glucan based EPS which helps them to attach to the teeth.When this material begins to accumulate on the teeth it is referred to as dental plaque.

As a general phenomenon, organisms with capsules tend to be more virulent presumably because of their resistance to phagocytosis and killing.Streptococcus pneumoniae exists in a smooth form (encapsulated) and a rough form (non- encapsulated).Only the smooth form is lethal for mice. EXPERIMENT o a. Use a loop to mix a drop of water, a drop of india ink and a small amount of Klebsiella pneumoniae together at the end of a slide.b. Use another slide to spread the smear like a blood smear.(As the instructor will demonstrate.)Allow the smear to air dry.c. Flood the smear with crystal violet, 1 minute.Wash with water, blot, dry, observe.Compare your observations with the illustrations on page 30 of Leboffe and Pierce. ENDOSPORES - The Schaeffer - Fulton Stain These are very resistant structures made by only a few genera of bacteria.The two genera which we will study are:  Clostridium is an anaerobic organism that forms spores.Tetanus, botulism, gas gangrene and pseudomembranous colitis are diseases caused by different species in this genus.

Bacillus is a common aerobic genus whose species can form endospores.Anthrax and Bacillus cereus food poisoning are two diseases caused by members of this genus.Spores are extremely resistant structures, difficult to destroy with heat or other physical and chemical disinfecting agents.Endospore destruction is the standard for testing the operation of an autoclave. EXPERIMENT o Prepare a smear of Bacillus megaterium, allow the smear to dry and then heat- fix.o Place the slide on the staining rack in the sink and flood the smear with malachite green stain.o Heat the stain to steaming by passing a lit bunsen burner over the smear.Don't overheat the stain!Once the steaming stops, pass the bunsen burner over the slide again.As the stain evaporates add more stain.Continue this procedure for 5-10 minutes. (Safety Note: Please remember that the acetone-alcohol decolorizer from the gram stain experiment is extremely flammable.Do not perform this flaming step while people are gram staining!)

o Wash the smear gently and thoroughly with running water.o Counterstain with aqueous safranin for 1 minute.o Wash the slide with water, blot gently and allow the smear to air dry. SIMPLE STAINING  Purpose: To recognize the three basic shapes of bacterial cells. Principle: In order to observe most bacterial cells using bright field microscopy the cells must be dark enough to see, that is they must have contrast to the light.To create contrast a simple stain can be used.Simple stains use basic dyes which are positively charged.These positive dyes interact with the slightly negatively charged bacterial cell wall thus lending the colour of the dye to the cell wall.1st lactophenol cotton blue added 2.Then culture added 3.BIOCHEMICAL TEST: This test is done to check whether bacteria is present or not.We can say them as conformity test too.They are as follows:  Oxidase test The oxidase test is a test used in microbiology to determine if a bacterium produces certain cytochrome c oxidases.

It uses reagent such as N,N,N′,N′- tetramethyl-p-phenylenediamine (TMPD) or N,N-dim ethyl-p- phenylenediamine (DMPD), which is also a redox indicator.The reagent is a dark-blue to maroon colour when oxidized, and colourless when reduced.e.g: Pseudomonaus aeruginosa  Catalytic test Some bacteria have catalyzed enzyme.Which decompose hydrogen peroxide to water and oxygen.Catalytic enzyme protect the bacteria from oxidative damage.Eg- E.coli  Coagulase test Certain bacteria can coagulate fibrin plasma into fibrinogen. Indol test 45.It is biochemical test performed on a bacterial species to determine the ability of organism to convert tryptophan into the indol.Positive indol test give red ring on the surface.E.g -E.coli CLASSIFICATION OF AREA: Viable(cfu/2hr) Non viable * Gloves Per hand Air limit Per m3 Contact plate limit Class A < 1 <1 <1 <1 Class B <5 <5 <10 <5 Class C <50 ND <100 <25 Class D <100 ND <200 <50 *non viable (particle count) .5µl diameter 5 l diameter Class A Class B Class C Class D At rest At op.