FOOD ANALYSIS LABORATORY

PRIYANKA BAL

HALDIA INSTITUTE

OF TECHNOLOGY

09/06/2014- 12/06/2014

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.

2. Method validation of the procedure that is to be

followed.

3. Sample preparation for the specific analyte that

is to be detected.

4. Detection of the required parameters within the

sample following the SOP(Standard Operation

Procedures)which are validated.

FAT ESTIMATION:

Fat is a nutrient. It is crucial for normal body function since it supplies

maximum calorific value. Chemically they are tri-esters of Glycerol and Fatty

acids. They are soluble in non-polar solvents but insoluble in water. 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.

1. DETERMINATION OF FAT IN MILK POWDER SAMPLE BY ROSE

GOTLIEB METHOD:

1.2. Procedure:

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

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

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

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.

Observation-

1. It will be observed that the blank and the standard will appear green.

2. Sample 1, 2, 3 will appear brown in colour.

3. 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.

5.1. 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

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. They are- 1. Linearity:

If the result follows Y = MX +C. And for

linearity R2= 0.95 ;( regression co –efficient).

2. 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:

AMBIENT AIR:

1. Particulate matter size <10 µ

2. PM 2.5

3. SOX

4. NOX

5. NH3

6. CO

7. O3

8. C6H6

9. BENZO α PYRENE

10. Nickel

11. Argon

12. Lead

STACK EMISSION:

1. Particulate matter

2. SOX

3. NOX

4. O2

5. CO

6. CO2

7. Velocity of gas flow

8. Temperature

9. Acid

10. Mist

11. Hydrocarbons ( unburnt).

INDOOR AIR:

1. Particulate matter

2. Formaldehyde

3. VOC (volatile organic compound )

4. SOX

5. NOX

6. Humidity

7. Temperature

8. Co2

WORK ZONE:

1. Particulate matter.

2. SO2.

3. N02

HAZARDOUS WASTES:

1. Flash point

2. pH

3. TCLP (toxicity characteristic leaching procedure).

SOIL (EDAHIC FACTORS):

Mandatory factors for soil:

1. Sodium.

2. Potassium.

3. Calcium.

4. Phosphorous.

5. Magnesium.

Other parameters:

1. Organic carbon

2. pH.

3. Texture.

4. Porosity.

INSTRUMENTS FOR HAZARDOUS/PETROLEUM TESTING:

1. DEVELANT OPEN CUP FLASH POINT APPARATUS

1. ABEL FLASH POINT APPARATUS

2. 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.

Orsat Apparatus.

CANON FLASK VISCOMETER:

CO ANALYZER:

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. Conductivity

2. Color

3. ph

4. Fixed solid

5. Volatile solid

6. Total solid

7. Total dissolved solid

8. Total suspended solid

9. Turbidity

10. Temperature

11. Flocculation test

12. Odor

13. Salinity

14. Settle able solid

15. Sludge Volume Index (SVI)

INORGANIC PARAMETERS AND NON-

METALLIC)

1. Acidity

2. Alkalinity

3. Ammonical nitrogen

4. Chlorides (as Cl-)

5. Chloride residue (as Cl2)

6. Dissolve oxygen

7. Fluorides

8. Total hardness (as CaCO3)

9. Total kjeldahl nitrogen (TKN)

10. Nitrate nitrogen (as NO3)

11. Nitrite nitrogen (as NO2)

12. Phosphate (as PO4)

13. Sulphate (as SO4)

14. Bromides

15. Carbon dioxide

16. Chloride demand

17. Silica (as Si)

18. Cyanide (as CN)

19. Sulphides (as H2S)

INORGANIC PARAMETERS (METALS)

1. Boron (B)

2. Cadmium

3. Calcium

4. Chromium total

5. Chromium hexavalent

6. Copper

7. Iron

8. Lead

9. Magnesium

10. Mercury

11. Nickel

12. Potassium

13. Sodium

14. Zink

15. Arsenic

16. Aluminum

17. Beryllium

18. Lithium

19. Manganese

20. Selenium

21. Silver

22. Strontium

23. Tin

24. Antimony

25. Cobalt

26. 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. Pesticides: organo-chloride

7. 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. Fecal streptococci

3. Total plate count

4. Coli phage

5. Fecal coli form

6. E.coli

7. Enterococcus

LIST OF INSTRUMENTS FOR WATER ANALYSIS:

1. Ph meter

2. Conductivity meter

3. UV visible spectrophotometer

4. Turbidity meter

5. Hot air oven

6. Muffle furnace

7. AAS (Atomic Absorbance spectrophotometer )

8. ICPMS (inductive coupled plasma mass spectrometer)

9. COD digester

10. TOC analyzer

11. Ion chromatography

12. CHNS analyzer

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.

2. 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.

4. BOD (Biochemical Oxygen Demand):

BOD is number of ml of oxygen required to biologically oxidize 1l of water.

5. 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. Shake well.

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. Color

2. Odor

3. Taste

4. Turbidity

5. Total Dissolved Solid

6. Ph

7. Nitrate

8. Nitrite

9. Fluoride

10. Chloride

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

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.

2. 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)

Calibration: pH meter is calibrated by using several buffers like acidic buffer,

basic buffer.

4. 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.

6. ULTRA SONIC BATH SONICATOR:

Used to de-gas the mobile phase in any process.

7. 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.

8. 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.

9. 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:

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. Extraction ,

2. Clean up’ and

3. Concentrate the sample.

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:

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.

Distribution 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].

II. Strong Cation Exchange [SCE].

III. Mixed Cation Exchange [MCE].

IV. 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.

2. 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

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.

3. GC MS/MS [GAS CHROMATOGRAPHY USING MASS

SPECTROPHOTOMETRY(Quantitative)/MASS

SPECTROPHOTOMETRY(Qualitative)]:

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.

4. 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

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.

B. To it 1ml of spike standard inoculated.

C. 10 ml of ethyl acetate added to it.

D. 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

HALDIA INSTITUTE OF TECHNOLOGY. 18/06/2014 - 21/06/2014.

RESIDUE ANALYSIS LABORATORY

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. TaqMan® chemistry.

2. 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.

2. Probes may be labelled with different, distinguishable reporter dyes, which allow

amplification of two distinct sequences in one reaction tube.

3. Post PCR process was eliminated which reduces assay labour and material cost.

DISADVANTAGES:

1. 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

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.

1. PCA – Total bacterial count.

2. YEDCA – Total yeast and mould count.

3. BPA - Staphylococcus sp.

4. MB (DS/SS) – E.coli

5. PDA – Fungus.

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.

2. Spread plate

Here the medium is already made and the organisms are spread upon the plate by

means of a bent glass rod.

3. 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.

2. Flood the smear with CRYSTAL VIOLET, 1 minute, then wash with water. [PRIMARY

STAIN]

3. Flood the smear with IODINE, 1 minute, and then wash with water. [MORDANT]

4. Flood the smear with ETHANOL-ACETONE, quickly, and then wash with water.

[DECOLORIZE]

5. Flood the smear with SAFRANIN, 1 minute, and then wash with water.

[COUNTERSTAIN]

6. 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.

3.) 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?

1. Overdecolorization

2. Too harsh heat fixation

3. Too thick a smear

4. Improper washing between steps

5. Too old a culture

6. Impure or mixed culture

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.

1. Gram (+) cells have thicker cell walls - more peptidoglycan and teichoic acid.

2. Gram (-) cells have Lip polysaccharide (Endotoxin) in the outer membrane of their cell

walls.

3. Gram (+) cells is generally more sensitive to those antibiotics which interfere with cell

wall.

4. 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"

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. DON'T HEAT FIX!

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.

FUNGAL STAINING:

1. 1st lactophenol cotton blue added

2. Then culture added

3. Then separated

4. Observed under microscope.

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

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.

350 3500

3500 350000

350000 3500000

3500000 nd

At rest At op.

0 0

0 2000

2000 20000

20000 Nd

CULTURE MAINTANCE:

ELISA READER( enzyme-linked immunosorbent assay)

ELISA is a test that uses antibodies and colour change to identify a substance.

Use to determine

1. Food allergens

2. Hormone

3. Toxins

Types of elisa

DIRECT ELISA:

INDIRECT ELISA

SANDWICH ELISA TECHNIQUE: