INSTRUMENTAL METHODS IN RESEARCH METHODOLOGY

ByDr. M. GopikrishnaReader, PG Dept. of RasashastraSJG Ayurvedic Medical College, Koppal, Karnatakaemail: rasashastra@rediffmail.com

TO IDENTIFIE NEW THINGS-

TO THROUGH MODERN LIGHT ON OLD FACTS-

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TO UNDERSTAND ALL THESE WE NEED FEW PARAMETERS TO ASSES THEM HENCEFORTH FEW OF THE INSTRUMENTS ARE MENTIONED TO KNOW THE ANALYSIS OF THE DRUGS.EG;- ROOT PRESSURE IN VARIOUS SEASONS VARY AS BILVA IN GRISHMA RITU IS RICH IN TANINS OR COLOURING AGENT IS MORE,TO IDENTIFIE WE NEED INSTRUMENTS LIKE PHYTOCHEMICAL ANALYSIS AND MICROSCOPIC ANALYSIS.

PHYTOCHEMICAL ANALYSIS:-•

-TO ASSES AND ANALYSIS.

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-WATER SOLUBLE MATERIALS WITH MANY COMPOUNDS ARE OBSERVED AND IDENTIFIED.

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-SEPARATE CHEMICAL AND ISOLATE IT AND ASSES THE DIFFERENT BONDING WITH DIFFERENT ADVANCE TECHNIQUES.

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-BIO-SYNTHETIC PATHWAY CAN BE ASSESED OF THE ISOLATED CHEMICAL.FOR THIS WE NEED TO-

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* QUALITATIVE ANALYSIS.•

*QUANTITATIVE ANALYSIS.

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SEMIQUALITATIVE AND SEMIQUANTITATIVEEG:-WATER EXTRACTS ETC

METHODS NEEDED FOR DOING ALL THESE-

1)INSTRUMENTAL–BY THIS WE CAN KNOW

PHYSICOCHEMICAL NATURE.

2)NON INSTRUMENTAL-AN ORGANOLEPTIC EVALUATION.

1)INSTRUMENTAL ANALYSIS:•

INSTRUMENTS PERFORM DIFFERENT FUNCTION EG;- REFRACTIVE INDEX OF OILS,OR SOLUBILITY OF ANY SUBSTANCE,TO KNOW ANY OF SUCH FACTORS WE NEED INSTRUMENTS TO CONFIRM THE FACTORS.

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THE CONTACT OF THE PLANT TO A INSTRUMENT , ITS REACTION TO THE SAMPLE AND COMPARED TO DESIGN THIS GRASPED SIGNAL IN A MEASURABLE UNITS I.E TO CONVERT THE ORIGINAL SIGNALS TO A CONVENTIONAL SIGNAL.CONVERT THE VARIOUS ENERGIES TO A ELECTRIC ENERGY AND CAN BE ASSESED IN A GALVANOMETERETC.(IT’S A MEASURABLE FORM).SOMETIMES TRANSFORMING SIGNALS IS DIFFICULT THERE IN SUCH PLACES WE NEED AMPLIFIERS.

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PRESENTATION OF DATA IN DIFFERENT WAYS IS TRANSFORMED,AMPLIFIED,AND GENERATED .I.E THESE OBSERVATIONS ARE DIRECTLY PRAPORTIONAL TO THE DATA.

ADVANTAGES:--TO ANALYSE A SAMPLE SMALL AMOUNT OF DRUG IS ENOUGH.-DETERMINATION OF QUALITATIVE AND QUANTITATIVE ANALYSIS IN SHORT TIME.-COMPLEX MIXTURES CAN ALSO BE ANALYSED WITH OR WITHOUT ISOLATION.-IT IS WITH SUFFICIENT RELIABILITY AND ACCURATE.

DISADVANTAGES:-

-TOO COSTLY.

-MAINTANANCE OF INSTRUMENT IS DIFFICULT.

-CHEMICAL CLEANLINESS IS ALSO COSTLY.

-SENSITIVITY DEPENDS ON ADVANCEMENT OF INSTRUMENT THEREFOR UPGRADE IT REGULARLY.

-SPECIALISED TEST FOR HANDLING OR USING IS NEEDED.

-FREQUENT NEED OF CHECKING DRUGS IS NECESSARY THEREFORE FREQUENTLY CHECK THE INSTRUMENT.

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EXTRACTION OF DRUGS:-•

PHANTA,HIMA,SATWA,ARE EXTRACTS ONLY

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-IN MODERN THE METHODS OF EXTACTIONS ARE.

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*1)MACERATION•

*2)PERCOLATION.

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*3)DECOCTION.

*1)MACERATION:-KEEPING THE POWDERED DRUG IN SUITABLE SOLVENT FOR FEW HOURS LIKE IN WATER,ETHER,METHENOL,WITHOUT APPLING HEAT TO DISSOLVE ITS SOLUBLE PORTION IN IT CALLED AS “ISOLATION MARC”

AND INSOLUBLE

PART CALLED AS “MARC”.SUB. NEEDED:-CONICAL GLASS,FILTER PAPER,EVOPARATING DISH,GLASS FUNNEL,HOT WATER BATH,ELECTRIC OVEN,ANALYTICAL WEIGHING BALANCE,PIPPETE,VOLUMETRIC FLASK,SOLVENTS,THE TEST DRUG.

PROCEDURE:TAKE 5GMS OF TEST DRUG POWDER I.E.AIR DRIED DRUG POWDER ,ADD 100ML OF WATER TO THIS AND PLACE IT IN A CONICAL FLASK SHAKE THE MIXTURE NOW AND THEN FOR 18HRS THEN FILTER IT.SEPERATE THE RESIDUE.FROM THE SOLVENT COLLECT 20ML AND PLACE IT IN A PORCELINE DISH AND DRY IT ON A HOT WATER BATH AND COLLECT THE RESIDUE AND WEIGH IT AND CALCULATE THE % OF THE EXTRACTIVE.

PERCOLATION:-IT IS THE METHOD OF EXTRACTION OF ALCOLOIDS ETC.BY PASSING A LIQUID THROUGH A COARS POWDER DRUG.THE LIQUID IS MADE TO PASS THE DRUG AND THE SOLVENT IS COLLECTED BY THE OPENING OF THE STOP CORK,ALSO THE SOLVENT IS MADE TO PASS THE COTTON PLUG AT THE NECK TO FILTER AND TO GAIN ONLY THE SOLLUBLE EXTRACT ONLY.

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USE OF PERCOLATE: COLLECT THE PERCOLATE OF 5MIN INTERVAL PF 4-5 BATCHES OF SAME SOLVENT AND ASSES FOR THE RICH ALCOLOID SAMPLE.

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EG;IF GOOD %IN FIRST SAMPLE OR 3RD

SAMPLE OR 5TH SAMPLE,THAT PARTICULAR SAMPLE CAN BE GIVEN TO THE PATIENT AND STANDARDISE THE SAMPLE.

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DECOCTION:-•

IT IS A METHOD OF EXTRACTING OF EXTRACTIVES,HERE FIRE OR HEAT TREATMENT IS USED TO EXTRACT THE SOLVENTS,HERE THE COARSE POWDER IS TAKEN IN A APPARATUS AND A ARRANGEMENT IS MADE TO PASS THE HOT LIQUID THROUGH THE DRUG AND RECYCLE IT ,AS IT PASSES THROUGH THE DRUG IT IS FILTERED THROUGH A COTTON PLUG AND IS COLLECTED AT THE CHAMBER AT THE BASE WHICH IS HEATED AND THE VAPOUR IS MADE TO PASS THROUGH A PIPE INTO THE CONDENCER AREA AND MADE TO LIQUID FORM AND AGAIN PASS THROUGH THE DRUG.REPEAT THE PROCESS AS PER REQUIREMENT AND STANDARDISE IT..

SOXLET APPARATUS IS DESIGNED FOR THE SAME PURPOSE.

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IF IT IS A COMPLEX COMPOUND SEPARATE THE EXTRACTS WITH ANY METHODS ,AS ONE METHOD IS-

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BATCH EXTRACTION:-•

IT IS A LIQUID LIQUID EXTRACTION,OR EXTRACTION OF EXTRACTIVES FROM THE LIQUIDS.

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TAKE A LIQUID SUBSTANCE IN A FUNNEL WITH CORKS ON EITHER SIDES,ALLOW IT FOR FEW HOURS AND LATER DISTINGUISH IT IN RESPECT TO ITS COLOUR OR CONSISTANCY,OF VISCOSITY,SEPARATE THEM AS PER REQUIREMENT INTO A BEAKER.

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Separatory Funnel Extraction Procedure•

1. Inspect your separatory funnel.

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The organic chem teaching labs have acquired several different types of sep funnels over the past years, with different types of stopcocks and stoppers, as illustrated in the photo below. The Teflon stopcocks work better than the ground glass stopcock; if you have a sep funnel with a ground glass stopcock, you may exchange for a Teflon style.

The sep funnel on the left is a 60 mL size, the others are 125 mL. The organic chem teaching labs are downscaling slowly to this smaller size.

There are two different styles of stopper, too. Some students swear by the plastic style, some by the ground glass style. The disadvantage of the ground glass style is that it can lodge permanently in the sep funnel if it is not removed and stored separately after use. Whichever style you have, make sure that the stopper fits snugly in the top of the flask.

2. Support the separatory funnel in a ring on a ringstand.The rings

are located on

the back shelves and they come in many sizes. Test to make sure that you haven’t chosen too large a ring before setting the funnel in it. You can add pieces of cut tygon

tubing

to the ring to cushion the funnel.

Make sure the stopcock of the separatory

funnel is closed!

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3. Add the liquid to the separatory funnel.•

Place a stemmed funnel in the neck of the separatory funnel. Add the liquid to be extracted, then add the extraction solvent. The total volume in the separatory funnel should not be greater than three- quarters of the funnel volume. Insert the stopper in the neck of the separatory funnel.

pour in liquid to be extracted . . .

pour in the solvent . . .

add a stopper

4. Shake the separatory funnel.Pick up the separatory

funnel with the stopper in

place and the stopcock closed, and rock it once gently. Then, point the stem up and slowly open the stopcock to release excess pressure. Close the stopcock. Repeat this procedure until only a small amount of pressure is released when it is vented.

Now, shake the funnel vigorously for a few seconds. Release the pressure, then again shake vigorously. About 30 sec total vigorous shaking is usually sufficient to allow solutes to come to equilibrium between the two solvents.

Vent frequently to prevent pressure buildup, which can cause the stopcock and perhaps hazardous chemicals from blowing out. Take special care when washing acidic solutions with bicarbonate or carbonate since this produces a large volume of CO2

gas.

Let the funnel rest undisturbed until the layers are clearly separated.

While waiting, remove the stopper and place a beaker or flask under the sep funnel.

5. Separating the layers.

Carefully open the stopcock and allow the lower layer to drain into the flask. Drain just to the point that the upper liquid barely reaches the stopcock.

If the upper layer is to be removed from the funnel, remove it by pouring it out of the top of the funnel.

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6. Perform multiple extractions as necessary.•

Often you will need to do repeat extractions with fresh solvent. You can leave the upper layer in the separatory funnel if this layer contains the compound of interest. If the compound of interest is in the lower layer, the upper layer must be removed from the separatory funnel and replaced with the drained-off lower layer, to which fresh solvent is then added.

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Yes, it can be confusing! Plus, the beginning student often does not know in which layer resides the compound of interest. The best advice: Always save all layers until the experiment is completely finished!

7. Store your separatory funnel with the cap (stopper) separate from the funnel!

Please, remove the stopper before storage!!

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DECANTATATION:-•

ALLOW THE SOLID PART TO SETTAL AND ONLY THE LIQUID PART TO BE REMOVED OR SEPERATED ITS DECANTATION,THIS CAN BE DONE EVEN WITH A CENTRIFUGAL MACHINE AND SEPERATED.

DISTILLATION:-

SEPERATION OF VOLATILE SUBSTANCESFROM A DRUG IN A DISTILATION APPARATUS.

CHROMATOGRAPHY:-IT IS A TECHNIQUE TO SEPARATEINDIVIDUAL COMPONENTS INA MIXTURE,IN A SPECIFIC MOBILE ANDSTATIONARY PHASE.

CHROMA= COLOUR.

GRAPHY= WRITING .

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1ST INVENTED BY M.TSWETT IN 1906 BY A BOTONIST,HE USED CACO3 CRYSTALS IN A COLOUM AND POURED EXTRACTIVES IN COLOUM,ALSO POURED TEST SOUTION IN IT AND DEVELOPED COLOURED BANDS IN CACO3 .THEREFORE THIS SYSTEM WAS NAMED AS “SYSTEM OF COLOURED BANDS”.LATER NAMED AS CHROMATOGRAPHY WHICH TOOK TREMENDAROUS CHANGES TODAY TO SEPARATE ALMOST ANY SUBSTANCE IN A COMPLEX SOLVENT.

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IN 1930-FEW VARITIES OF TLC AND ION EXCHANGE CROMATOGRAPHY WAS INTRODUCED.

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IN 1941 PARTITION AND PAPER CHROMATOGRAPHY WAS DEVELOPED.

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IN 1952 GAS CHROMATOGRAPHY WAS INTRODUCED.

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TLC IS A TECHNIQUE PURELY BASED ON RATE OF MOUNT OF COMPONENT THROUGH A MEDIUM AND A STATIONARY PHASE,BINDING CAPACITY OF A MIXTURE IS SEPERATED .

2 PHASES IN CROMATOGAPHY.*STATIONARY PHASE -SOLID FORM OR A LIQUID IN SOLID FORM.*MOBILE PHASE- EITHER LIQUID OR GAS TECHNIQUEIN MOBILE PHASE PASSES THE STATIONARY PHASETRANSPORTS THE SEPARATE COMPONENT AT DIFFERENTSPEED AT DIRECTION OF FLOW OF MOBILE PHASE.

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PRINCIPLE:•

SEPERATION OF SINGLE COMPONENT FROM A MIXTURE IN STABLE AND MOBILE PHASE.

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2 PHASES NEEDED-

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1)STATIONARY PHASE -USING SILICA GEL ALSO CELLULOS POWDER ETC.

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-2)MOBILE PHASE- ETHANOL,BENZINE,CARBONTETRACHLORIDE,ETC . CAN BE USED.

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APPLICATIONS OF TLC:-•

*IT IS MUCH BENIFICIAL WITH INDIVIDUAL COMPONENTS IN A MIXTURE.

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*FOR CHECKING PURITY OF THE SAMPLE,ALSO FOR THE PURIFICATION PROCESS.

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*HELPFUL IN CHEMISTRY LAB TO IDENTIFIE THE REACTION.

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*FOR IDENTIFICATION OF INDIVIDUAL COMPONENTS.•

*STANDERD PARAMETER USEFUL FOR STANDERDISATION OF PHARMACEUTICAL PROCEDURE IN INDUSTRIES.

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*ISOLATION OF MANY ORGANIC COMPOUNDS LIKE ALCOLOIDS,AMIDES,ACIDS ARE POSSIBLE.

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*ALSO IN BIOCHEMICAL ANALYSIS IN METABOLITES LIKE PLASMA,SERUM ANALYSIS,URINE ANALYSIS.

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ADVANTAGES:•

*SIMPLE AND EASY TEST IN STANDERDISATION.

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*TIME IS 20-40MIN ,ITS VERY FAST.•

*SEPARATE INDIVIDUAL COMPONENTS FROM SMALL AMOUNT OF SAMPLE.

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*ITS HIGHLY SEPERATION METHOD IN INDIVIDUAL COMPONENTS.

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*LESS EXPENSIVE.•

*VERY EASY FOR DETECTION OF SAMPLE.

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DIS ADVANTAGES:•

NOT POSSIBLE TO SEPARATE THE COMPONENTS IN LARGE SCALE.

Procedure for TLC1. Prepare the developing container.The developing container for TLC can be a specially designed chamber, a jar with a lid, or a beaker with a watch glass on the top:

In the teaching labs, we use a beaker with a watch glass on top.

Pour solvent into the beaker to a depth of just less than 0.5 cm.

To aid in the saturation of the TLC chamber with solvent vapors, line part of the inside of the beaker with filter paper.

Cover the beaker with a watch glass, swirl it gently, and allow it to stand while you prepare your TLC plate.

2. Prepare the TLC plate.TLC plates used in the organic chem. teaching labs are purchased as 5 cm x 20 cm sheets. Each large sheet is cut horizontally into plates which are 5 cm tall by various widths; the more samples you plan to run on a plate, the wider it needs to be.

Plates will usually be cut and ready for you when you come to lab.Handle the plates carefully so that you do not disturb the coating of adsorbent or get them dirty.

Measure 0.5 cm from the bottom of the plate. Take care not to press so hard with the pencil that you disturb the adsorbent.

Using a pencil, draw a line across the plate at the 0.5 cm mark. This is the origin: the line on which you will "spot" the plate.

Under the line, mark lightly the name of the samples you will spot on the plate, or mark numbers for time points. Leave enough space between the samples so that they do not run together, about 4 samples on a 5 cm wide plate is advised. Use a pencil and do not press down so hard that you disturb the surface of the plate. A close-up of a plate labeled "1 2 3" is shown to the right.

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3. Spot the TLC plate•

The sample to be analyzed is added to the plate in a process called "spotting".

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If the sample is not already in solution, dissolve about 1 mg in a few drops of a volatile solvent such as hexanes, ethyl acetate, or methylene chloride. As a rule of thumb, a concentration of "1%" or "1 gram in 100 mL" usually works well for TLC analysis. If the sample is too concentrated, it will run as a smear or streak; if it is not concentrated enough, you will see nothing on the plate. The "rule of thumb" above is usually a good estimate, however, sometimes only a process trial and error (as in, do it over) will result in well-sized, easy to read spots.

add a few drops of solvent . . .

. . . swirl until dissolved

The solution is applied to the TLC plate with a 1µL microcap.

Microcaps come in plastic vials inside red-and-white boxes. If you are opening a new vial, you will need to take off the silver cap, remove the white styrofoam plug, and put the silver cap back on. A small hole in the silver cap allows you to shake out one microcap at a time. Microcaps are very tiny; the arrow points to one, and it is hard to see in the photo.

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Take a microcap and dip it into the solution of the sample to be spotted. Then, touch the end of the microcap gently to the adsorbent on the origin in the place which you have marked for the sample. Let all of the contents of the microcap run onto the plate. Be careful not to disturb the coating of adsorbent. dip the microcap into solution - the arrow points to the microcap, it is tiny and hard to see

make sure it is filled - hold it up to the light if necessary

touch the filled microcap to TLC plate to spot it - make sure you watch to see that all the liquid has drained from the microcap

rinse the microcap with clean solvent by first filling it . . .

. . . and then draining it by touching it to a paper towel

do this rinse process 3 times!

here's the TLC plate, spotted and ready to be developed

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4. Develop the plate.•

Place the prepared TLC plate in the developing beaker, cover the beaker with the watch glass, and leave it undisturbed on your bench top. Run until the solvent is about half a centimeter below the top of the plate (see photos below).

place the TLC plate in the developing container - make sure the solvent is not too deep

The solvent will rise up the TLC plate by capillary action. In this photo, it is not quite halfway up the plate.

In this photo, it is about 3/4 of the way up the

plate.

The solvent front is about half a cm below the top of the plate - it is now ready to be removed

Remove the plate from the beaker.

quickly mark a line across the plate at the solvent front with a pencil

Allow the solvent to evaporate completely from the plate. If the spots are colored, simply mark them with a pencil.

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5. Visualize the spots•

If your samples are colored, mark them before they fade by circling them lightly with a pencil.

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Most samples are not colored and need to be visualized with a UV lamp. Hold a UV lamp over the plate and mark any spots which you see lightly with a pencil.

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Beware! UV light is damaging both to your eyes and to your skin! Make sure you are wearing your goggles and do not look directly into the lamp. Protect your skin by wearing gloves.

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If the TLC plate runs samples which are too concentrated, the spots will be streaked and/or run together. If this happens, you will have to start over with a more dilute sample to spot and run on a TLC plate.

this is a UV lamp

here are two proper sized spots, viewed under a UV lamp (you would circle these while viewing them)

The plate to the left shows three compounds run at three different concentrations. The middle and right plate show reasonable spots; the left plate is run too concentrated and the spots are running together, making it difficult to get a good and accurate Rf reading.

Here's what overloaded plates look like compared to well-spotted plates. The plate on the left has a large yellow smear; this smear

contains the same two compounds which are nicely resolved on the plate next to it. The plate to the far right is a UV visualization of

the same overloaded plate.

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DETECTION OF THE ISOLATED COMPONENT:-

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1)non specific method:-•

By colours-florensent phase

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Iodine chamber.•

H2 so4 spray.

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u.v chambers.•

Color spray reagents can be used.

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2)specific method:-•

For different types of components-

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i)phenolic compounds and tannins-ferric chloride spray is advised.

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ii)for alcoloids-dragandroffs reagent.•

iii)for amino acids-ninhydrin in acetone.

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iv)for cardiac glycosides-spray with 3,5dinitro benzoic acid.

FURTHER EVALUATION OF SEPERATED COMPONENTS:-

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QUALITATIVE AND QUANTITATIVE-•

QUALITATIVE ANALYSIS:-

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-By visual assessment by observing size,density,number of spots with different reagents of components can be identified.

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-separately measure the spot in mm is directly proportional to substance present in that spot.

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RETARDATION OR RETENTION FACTOR:‐Rf

• Measuring Rf

values

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measurements are often taken from the plate in  order to help identify the compounds present. 

These measurements are the distance travelled

by  the solvent, and the distance travelled

by individual 

spots.

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When the solvent front gets close to the top of the  plate, the plate is removed from the beaker and the  position of the solvent is marked with another line 

before it has a chance to evaporate.

The Rf

value for each dye is then worked out using the formula:

These measurements are then taken:

For example, if the red component travelled

1.7 cm from the base line while the solvent had travelled

5.0 cm, then the Rf

value for the red dye is:

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If you could repeat this experiment under exactly the same conditions, then the Rf values for each dye would always be the same. For example, the Rf value for the red dye would always be 0.34. However, if anything changes (the temperature, the exact composition of the solvent, and so on), that is no longer true. You have to bear this in mind if you want to use this technique to identify a particular dye

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QUANTITATIVE ANALYSIS:-•

Carried out in 2 ways-

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Direct method:-•

a) on the plate i.e., after spray of different reagents.

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b)by assessing the density of elute.•

c)measurement of spot area in mm is proportion to amount of quantity more in the sample.

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d)densitometer:- method where intensity of color of substance is measured in chromatogram –in situ method.

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E)densitometer-method where optical density of separated spots is measured.

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f)spectrophotometer-instrument which gives qualitative and quantitative analysis. by wave length of maximum absorption of different spots and compared with standard spots.

densitometer

spectrophotometer-instrument

Indirect method:-•

Components scraped is assessed further with different test or different chromatograms etc. in different titrations.

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Microanalysis can be performed by colorimeter ,electroporosis.etc.,

HPTLC:-HIGH PERFORMANCE THIN LAYER CROMATOGRAPHY-

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In this a pre coated stationary phase is used and the chemicals used are extremely small sized particles so that adsorbent capacity is highly active.

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Instead of maneuver samples the standard samples are available here for spotting and a new type of development chamber which requires less amount of solvent for development, more efficacy in separation and shorter analysis time because of advance type of densitometer scanner and improved data possessing capacity by which you will know the readings in computer.

PAPER CROMATOGRAPHY:-•

Technique in which analysis of unknown substance with the help of the mobile phase and a stationary phase is specially designed filter paper also known as whattman chromatography paper, all principals of chromatography holds good.

Adsorption ChromatographyAdsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibriation

between

the mobile and stationary phase accounts for the separation of different solutes.

Partition ChromatographyThis form of 

chromatography is based  on a thin film formed on the 

surface of a solid support by  a liquid stationary phase. 

Solute equilibriates between the mobile phase 

and the stationary liquid.

Types of Chromatography -

Radial chromatography

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Ascending chromatography

-

Descending chromatography

Radial Chromatography In this type of chromatography, as the pigment separates, the different colours

move

outwards.

Ascending Chromatography The solvent moves upwards on the separating media

Descending Chromatography The solvent moves downwards on the separating media.

Ion Exchange  Chromatography

In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations

onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.

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Column Chromatography•

Procedure for Microscale Flash Column Chromatography

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In microscale flash chromatography, the column does not need either a pinchclamp or a stopcock at the bottom of the column to control the flow, nor does it need air-pressure connections at the top of the column. Instead, the solvent flows very slowly through the column by gravity until you apply air pressure at the top of the column with an ordinary Pasteur pipet bulb.

(1) Prepare the column.The column is packed using a simple dry‐pack method. 

Plug a Pasteur pipet

with a small amount of cotton; use a wood applicator stick to tamp it down lightly. Take care that you do not use either too much cotton or pack it too tightly. You just need enough to prevent the adsorbent from leaking out.

Add dry silica gel adsorbent, 230-400 mesh --

usually the jar

is labeled "for flash chromatography." One way to fill the column is to invert it into the jar of silica gel and scoop it out . . .

. . . then tamp it down before scooping more out.

Another way to fill the column is to pour the gel into the column using a 10 mL beaker.

Whichever method you use to fill the column, you must tamp it down on the bench top to pack the silica gel. You can also use a pipet

bulb to force air into the

column and pack the silica gel.

When properly packed, the silica gel fills the column to just below the indent on the pipet. This leaves a space of 4–5 cm on top of the adsorbent for the addition of solvent. Clamp the filled column securely to a ring stand using a small 3-pronged clamp.

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(2) Pre-elute the column.•

The procedure for the experiment that you are doing will probably specify which solvent to use to pre- elute the column. A non-polar solvent such as hexanes is a common choice.

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Add hexanes (or other specified solvent) to the top  of the silica gel. The solvent flows 

slowly down the column; on the                           column above,it

has flowed down 

to the point marked by the arrow.

Monitor the solvent level, both as it flows through the silica gel and the level at the top. If you are not in a hurry (or busy doing something else), you can let the top level drop by gravity, but make sure it does not go below the top of the silica. Again, the arrow marks how far the solvent has flowed down the column.

Speed up the process by using a pipet

bulb to force the solvent

through the silica gel -

this puts the flash in microscale

flash

chromatography. Place the pipet

bulb on top of the column,

squeeze the bulb, and then remove the bulb while it is still squeezed. You must be careful not to allow the pipet

bulb to

expand before you remove it from the column, or you will draw solvent and silica gel into the bulb.

When the bottom solvent level is at the bottom of the column, the pre-elution process is completed and the column is ready to load.

If you are not ready to load your sample onto the column, it is okay to leave the column at this point. Just make sure that it does not go dry --

keep the top

solvent level above the top of the silica (as shown in the picture to the left) by adding solvent as necessary.

(3) Load the sample onto the silica gel column.Two different methods are used to load the column: the wet method and the dry method: wet and dry. Below are illustrations of both methods of loading a crude sample of ferrocene

onto

a column. In the wet method, the sample to be purified (or separated into components) is dissolved in a small amount of solvent, such as hexanes, acetone, or other solvent. This solution is loaded onto the column.

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Wet loading method

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The column is being loaded by the wet method.  Follow the thumbnails below to see close‐up details 

of the sample as it is allowed to sink into the  column. Once it's in the column, fresh eluting  solvent is added to the top and you are ready to 

begin the elution process (see step 4).

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Sometimes the solvent of choice to load the sample onto the column is more polar than the eluting solvents. In this case, if you use the wet method of column loading, it is critical that you only use a few drops of solvent to load the sample. If you use too much solvent, the loading solvent will interfere with the elution and hence the purification or separation of the mixture. In such cases, the dry method of column loading is recommended.

Dry loading method

First dissolve the sample to be 

analyzed in the minimum 

amount of solvent and add 

about 100 mg of silica gel. Swirl 

the mixture until the solvent 

evaporates and only a dry 

powder remains. Place the dry 

powder on a folded piece of 

weighing paper and transfer it to 

the top of the prepared column. 

Add fresh eluting solvent to the 

top ‐‐

now you are ready to 

begin the elution process (see 

step 4). 

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(4) Elute the column.•

Force the solvent through the column by pressing on the top of the Pasteur pipet

with a

pipet

bulb. Only force the solvent to the very top of the silica: do not let the silica go dry. Add fresh solvent as necessary.

The photo at the left shows the solvent being forced through the column with a pipet

bulb.

The series of 5 photos below show the colored compound as it moves through the column after successive applications of the pipet

bulb process.

The last two photos illustrate collection of the colored sample. Note that the collection beaker is changed as soon as the colored compound begins to elute.The process is complicated if the compound is not colored. In such experiments, equal sized fractions are collected sequentially and carefully labeled for later analysis.

•

(5) Elute the column with the second elution solvent.

•

If you are separating a mixture of one or more compounds, at this point you would change the eluting solvent to a more polar system, as previously determined by TLC. Elution would proceed as in step (4).

•

(6) Analyze the fractions.•

If the fractions are colored, you can simply combine like-colored fractions, although TLC before combination is usually advisable. If the fractions are not colored, they are analyzed by TLC (usually). Once the composition of each fraction is known, the fractions containing the desired compound(s) are combined.

•

Gas Chromatography: Procedure•

(1) Add the sample to be injected to the syringe.

•

A 25µL glass Hamilton syringe is used to inject the GC samples. Only 2-4 µL of sample is injected onto the column, which means that you fill only a small part of the barrel with sample. Examine the syringe carefully before you fill it. The divisions are marked "5 -

10 -

15 -

20 -

25".

This is a 25 µL glass Hamilton syringe. You only inject 2.5 µL, so it will NOT be filled to the top.

Place the tip of the needle in the liquid. Slowly draw up a small amount of liquid by raising the plunger, then press on the plunger to expel the liquid back into the liquid. This serves to “rinse”

the syringe with your sample, ensuring that what you will measure in the GC run is the

composition of your mixture. Repeat the rinse process one or two

times. Then, draw up the plunger slowly again while the needle is in the liquid and carefully fill the syringe with liquid about halfway to the “5”.

It is often hard to see the liquid in the syringe. If the syringe is clogged, the plunger will be in the correct position but the barrel of the syringe will be filled with only air, as in the bottom syringe in the photo to the left. The best thing to do is to carefully examing

the syringe after you think that you have filled it. Hold it up to the light to get a better view.Small air bubbles in the syringe will not affect the GC run (middle syringe in the photo to the left). As long as there is enough liquid in the syringe, the GC run will work fine. If you keep getting bubbles, just pull the plunger up a bit past the "halfway to the 5" mark to compensate. If you have a VERY large air bubble, you will not have enough liquid to show a reading on the GC (e.g., the bottom syringe in the photo).

•

(2) Inject the sample into the injector port.

•

You are need to do two things sequentially and quickly, so  make sure you know where the injection port is and where  the start button on the recorder is.

•

Push the needle of the syringe through the injection port  and immediately press the plunger to inject the sample, 

then immediately press the start button on the recorder.

•

You will feel a bit of resistance from the rubber septum in  the injection port; this is to be expected and you should be  prepared to apply some pressure to the syringe as you 

force the needle into the instrument all the way to the  base of the needle.

Push the needle of the filled syringe through the injector (as far as it will go) and quickly push the plunger.

Remove the syringe immediately . . .

. . . and quickly press the start button on the integrating recorder or the start recording button on the computer

Here's a close-up of the integrating recorder.

•

MECHANISM:-•

Gas is used as a mobile phase and solid or liquid

coated on a solid support is used as a stationary phase. The test mixture is converted to vapor and to this vapor the mobile phase is passed, hence the components through stationary phase with help of mobile phase and separated components which is less soluble in stationary phase travels faster and which is greater soluble capacity in stationary phase. Here with help of some gas , if you heat the test sample it travels and one with higher affinity will react here and one with less affinity will move far, we have detectors and amplifiers and lastly the recorder in different forms.

•

Important criteria in GC-•

1) Mobile phase as gas.

•

2) Test mixture heated and evaporated which should be inert to mobile phase.

•

3) To vaporize mixture if they posses volatile oil or volatile mixture are separated in technique.

•

4) Mobile should not mix with vapor in stationary phase.

•

5) Mixture sample should not change by heat i.e. thermostable, as we heat the mixture.

•

•

Here mobile gas as hydrogen,helium,nitrogen,and

argon are used,

among these helium is expensive.•

Result is in form of curves, individual peaks is of individual components and retention time is the time between injection of sample to the time to get maximum peak of components, retention factor of standard is time of maximum peak.i.e, rf.

•

Compare the test sample curve with standard curve asses it with considering the retention time even.

•

(3) Sit back and wait.•

Observe the recorder. Within several minutes, it should record several peaks.

•

(4) End the GC run.•

When you have seen all of the peaks which you suspect are in the mixture, or when the recorder has shown a flat baseline for a few minutes or so, press stop on the recorder.

•

When you press stop, the recorder will print out the peaks, the retention times, and the areas under the peaks. When it is done printing, you can press “enter”

a couple times to advance the

paper.•

Carefully tear the paper off the recorder. The paper is not perforated, so do not try to pull up and expect it to pop out of

the recorder. Instead, pull it down to start a tear from one edge, and then continue the tear until the paper is cut and free.

Uses:

•

mainly for detecting steroids,food

components    ,identification of various substances.

Study of colors

BEER LAMBERT LAW

MECHANISM

PROCESS INVOLVED

•

When a beam of light pass through a object , this object absorbs some amount of light and transmits the light to some extent.

•

I0 = I0 + IR + IT .•

Incidence of light = incidence of absorbance + incidence of refractence

+ incidence of transmitted.

•

Thickness of the object and concentration is much important for change in transmitted light.i.e., in various frequency depends on thickness.

•

LAMBERTS LAW:-•

When a beam of light is allowed to pass through a transparent media , the rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of light.

•

If intensity is more. The frequency varies or •

If thickness is more. Changes.

Beers law:-Intensity of beam of light decreases with the increase in the concentration of absorbing substance.

1 cm

X                                                      y

x

2 cm

Y1       y

•

Similarly a concentration of solution with the light ray through them , the greater or lesser amount of concentration energy.

»»

•

Standard incidence energy = Is

•

Absorbance                            = As

•

Transmittance                         = Ts•

Suppose 

Is

= 230

As

= 4 of given standard.

Ts

= 6 of a given drug.

Then 

4     ‐

230

6     ‐

?

Calculate   6  X  230

4 =  345

colorimeter

••

Colorimeter Definition

•

A colorimeter is a device used in the practice of colorimetery, or the science of color. Colorimetry

has also been termed the

quantitative study of color perception. A number of instruments are available to determine the concentration of a solution. The simplest of those instruments is a colorimeter.

•

Function–

A colorimeter is a device used to measure the absorption of a specific wavelength of light by a solution, which can in turn be used to determine the concentration of a solute in a solution.

•

Components–

The critical parts of a colorimeter are a light source (commonly a low-voltage filament lamp), adjustable aperture, colored filters, solution cuvette, a transmitted light detector (such as a photoresistor) and an output display meter. Some colorimeters might also contain a regulator to prevent damage related to the machine from voltage fluctuations as well as a second light path to allow comparison between two solutions.

•

Filters–

A filter is used to select and measure the wavelength of light that the solution absorbs the most. The measurements are done in nanometers (nm). Typically, the wavelength used is between 400nm and 700nm.

•

Results–

Following the reading, the colorimeter will provide data in either an analog or digital formation, depending upon the machine. The data can be shown as transmittance on a linear percentage scale between 0 percent and 100 percent. It can also be shown as absorbance on a logarithmic scale between zero and infinity. Typically, the range of absorbance is from 0 to 2 with the ideal range being between 0 to 1.

•

History–

The colorimeter was invented by Jan Szczepanik

and 

applies the Beer‐Lambert law when determining the  concentration. The Beer‐Lambert law states that 

absorbance is proportional to the concentration of a  solute.

•

Different Parts of a Colorimeter

•

A colorimeter is a tool to measure the ability of a  solution to absorb a specified wavelength of light.  This absorption is used to determine the 

concentration of a substance in the solution.

•

Components–

The basic colorimeter is made up of a low‐voltage light 

source that shines through the solution held in a  cuvette. The cuvette

fits in a small receptacle and, as the 

light shines through it, a detector measures the light  that is passed through. The detector's readings are 

shown on an LCD display.

•

Options–

Colored filters are inserted in front of the cuvette

depending on which wavelength of light you need. This  is determined by knowing which wavelength of light the  solute you are measuring absorbs the best. Field and lab  manuals will have lists of these wavelengths for 

reference.

•

Function–

Once you select and insert the filter for the wavelength 

you need, you must analyze a blank and two to three  standards (minimum) using the colorimeter. The blank is 

typically pure water. Standards are solutions of various  concentrations of the solute you are measuring in your  sample. The readings from the blank and the standards  are used to set up a chart from which you determine 

your sample concentration after you analyze it.

•

Colorimeter Type

•

Colorimeters measure the perception of color. 

•

Colorimetry

is a technique to describe and quantify  the human perception of color by focusing on the 

physical aspects of color. A colorimeter measures  the amount of color from a given medium. Various 

different applications of colorimeters exist today to  quantify color, ranging from laboratories to the 

electronic industry.

•

Tristimulus

Colorimeter

•

Tristimulus

colorimeters are often used in the  application of digital imaging. The tristimulus

colorimeter measures color from light sources such  as lamps, monitors and screens. By taking multiple 

wideband spectral energy readings along the visible  spectrum, this colorimeter can profile and calibrate  specific output devices. The measured quantities 

can approximate tristimulus

values, which are the  three primary colors needed to match a test color

•

Densitometer–

A densitometer measures the density of light passing 

through a given frame. Density can be characterized as  the level of darkness in film or print. When an image is  printed, the ink pigments block light naturally when 

deposited by the printing process. Graphics industry  professionals use densitometers to help control color in 

the various steps of the printing process.

•

Spectroradiometer–

Spectroradiometers

quantify the spectral power 

distribution emitted from a given light source. In other  words, the spectroradiometer

measures the intensity of 

color.

Characteristically similar to spectrophotometers,  spectroradiometers

are used to evaluate lighting for 

sales within manufacturing and for quality control  purposes.

Other applications include confirming a 

customer's light source specifications and calibrating  liquid crystal displays for televisions and laptops.

•

Spectrophotometer–

A spectrophotometer is an analytical tool that measures 

the reflection and transmittance properties of a color  sample. Using functions of light wavelengths, the 

spectrophotometer passes a beam of light through the  sample to record both absorbance and transmittance. 

The instrument does not require human interpretation  and is much more complex than a standard colorimeter. 

Common applications for the spectrophotometer  include color formulation and industry research and 

development.

•

What Is Absorbance When Dealing With a Colorimeter?

•

Absorption is measured by color intensity. 

•

Absorbency is crucial in determining concentration of a  substance in a sample through colorimeter analysis. 

Colorimeters measure the intensity of color and light  transmittance by the sample to achieve the concentration.

•

Function

–

A colorimeter works by shining a white light through an  optical filter into a cuvette

that holds the sample. By 

adding a color reagent to the sample, the substance will  darken in color depending on the amount of 

concentration. The darkening (intensity) of the color is  measured by how much light is absorbed by the sample. 

The higher the intensity, the higher the concentration.

•

Significance

–

Absorbance can be defined as a logarithmic measurement of the 

amount of light at a particular wavelength taken in by sample. 

This measurement is known as the Beer‐Lambert Law or Beer's 

Law. The law states that absorbance is equal to the concentration 

multiplied by the path length of light and the molar extinction 

coefficient (how strongly the solution absorbs color).

•

Color Importance

–

The color of the sample depends on the transmittance of light, 

not absorption. For example, a sample is seen as red because it 

absorbs blue and purple wavelengths. Therefore in that example, 

the wavelength of light used to determine absorbance will be in 

the blue region of the visible color spectrum.

•

How to Use Colorimeters•

A colorimeter is any device that measures the color of a particular substance. It's a generic term that may refer to a range of devices such as a spectrophotometer, which is a specific type of colorimeter typically used to measure a solution's absorbance of a particular wavelength of light. This allows the concentration of a known solute in the solution to be

calculated with considerable accuracy. This type of colorimeter is a standard piece of equipment in analytical chemistry.

•

InstructionsThings You'll Need

•

Colorimeter •

Reference solution

•

Test solution

–

1 •

Determine the wavelength of light that the solution absorbs most strongly. A colorimeter has a set of changeable filters that can show which color of light to examine for the greatest accuracy. Set the colorimeter to this wavelength.–

2

•

Calibrate the colorimeter. Turn the unit on and wait 30 minutes for the colorimeter to warm up before taking any measurements. Remove the container (cuvette) from its chamber.–

3

•

Read the colorimeter. The meter is extremely sensitive, and you must use the correct measurement. Most models have a reflective surface under the needle and you should look at the needle so that you can't see its reflection in the mirror.

–

4 •

Fill the reference cuvette

with the test solution as

indicated by the colorimeter model you're using. Clean the reference cuvette

carefully to ensure it doesn't

have any smudges and place it in the chamber. Read the absorbance and adjust the colorimeter to show an absorbance reading of 0.–

5

•

Put the test solution in the specimen cuvette

and clean the outside of the cuvette. Place it in the chamber and read the absorbance of the test solution. You will typically record this value and use it in calculations to determine the solution's concentration.

•

Colorimeter Analysis•

Colorimeters plot concentration and absorbance on a line graph.

•

Using a colorimeter is one of the fastest and easiest ways to measure unknown concentrations of a sample substance. Measuring absorbency is crucial for colorimetry

analysis since it is in a linear relationship

with concentration.•

Function–

Substances in a sample absorb light, and color pigments also absorb light at different wavelengths. Colorimeters use color and light at different wavelengths to measure the intensity of color and how much light that color absorbs in a sample.

•

Beer-Lambert Law–

To obtain the concentration of a substance in a sample, colorimeters use the Beer-Lambert law to gain results. The mathematical formula states that concentration is equal to the absorbance divided by the path length of light and the molar extinction coefficient (how strong the solution absorbs color).

•

Considerations–

To get the curve for the Beer-Lambert law, standards of known concentration are measured for their absorbance of color and light and then plotted on a graph. This graph uses the y = mx

+ b formula to achieve a straight line with

concentration on the x-axis and absorbance on the y-axis. Unknown samples will then be measured using this curve. For example, if y = .301, then the absorbance (determined by the colorimeter) multiplied by .301 will equal the concentration.

•

Application of Colorimeter

•

Essentially, a colorimeter is a scientific instrument that  measures the amount of light passing through a solution 

relative to the amount that passes through a sample of  pure solvent. Colorimeters have many applications in the 

fields of biology and chemistry.

•

Beer‐Lambert Law

•

The Beer‐Lambert Law states that the concentration of a  dissolved substance, or solute, is proportional to the 

amount of light that it absorbs. A common application of a  colorimeter is therefore to determine the concentration of  a known solute in a given solution

•

Biological Culture–

In biology, a colorimeter can be used to monitor the 

growth of a bacterial or yeast culture. As the culture  grows, the medium in which it is growing becomes 

increasingly cloudy and absorbs more light.

•

Bird Plumage Coloration–

A colorimeter can also be used to eliminate subjectivity, 

or personal opinion, from the assessment of color in bird  plumage. Modern colorimeters provide highly accurate 

results, under standard, repeatable conditions and have  proved to be very reliable.

•

•

What is the Difference Between a Colorimeter and a  Spectrophotometer? 

•

Composed of many different wavelengths and colors,  perceived light can be deconstructed using colorimeters 

and spectrophotometers in different ways. 

•

Since the advent of the XYZ color system established by the  Commission Internationale

de l'Eclairage

(CIE) in 1931, 

many devices have been invented to measure and  interpret light ‐‐‐

a science known as spectroscopy. Two of 

these machines ‐‐‐

colorimeters and reflectance  spectrophotometers ‐‐‐

have made homes in laboratories 

and design studios, but their similar quantitative outputs  can be confusing despite their very different functionality  and design.

•

Color Measurement–

In 1931 the Commission Internationale

de l'Eclairage

attempted to quantify the light that humans perceive by matching the three primary colors that make up all colors with three values, called the tristimulus

values ---

x, y and z -

--

which approximately correspond to red, blue and green. Any visible color can be quantified using these three values, and this allowed for objective measuring and comparing of colors.

•

Colorimeters–

Relying on exactly that system laid out by the CIE, colorimeters measure the color of a sample compared to a white control surface and output data for x, y and z values. Used to color-match or color-mix, these relatively simple devices can often be found in the textile, paint and design industries. Colorimeters are generally inexpensive and rugged devices.

•

Colorimeters in the Lab–

Known concentrations of a sample can be tested with a 

colorimeter and plotted on a graph to create a  calibration curve. Using this data, a sample with an 

unknown concentration can be tested and compared  against the graph to ascertain its concentration.

•

Reflectance Spectrophotometers

–

Similar in the respect that reflectance spectrophotometers can 

also be used to determine a sample's color, these more complex 

devices provide a greater amount of data, and are otherwise 

completely different machines. Rather than just one broad 

wavelength analysis, spectrophotometers analyze the intensities 

of 16 or more narrow wavelengths of the sample light by 

diffracting the light into its component wavelengths. Unlike 

colorimeters, spectrophotometers typically include adjustments 

for many variables such as observer angle and illuminant, and are 

usually connected directly to a computer for data analysis. Some

spectrophotometers include a gloss trap to either include or 

exclude the specular

components of reflected light from either 

xenon or tungsten lamps.

•

Spectrophotometers in the Lab–

Reflectance spectrophotometers have a variety of 

applications in many laboratory disciplines. Similar to  colorimeters, spectrophotometers can more accurately 

determine the concentration of solute in a solution  against a graphed curve. But these more expensive  devices can also be used to, for example, measure the 

rate of bacterial growth in a sample or calculate the  color‐changing effect of a chemical reaction in real time.

•

Difference Between Colorimeter & Spectrophotometer

•

Colorimeters and spectrophotometers are used in the medical, research, and scientific industries to provide rapid results of unknown samples through the use of color. Although both the colorimeter and the spectrophotometer use color to analyze samples, they operate differently.

•

Light–

Colorimeters use a colored light beam to measure sample concentration. Spectrophotometers use a white light that is passed through a slit and filter to analyze samples.

–

Wavelengths

–

In colorimetry, colored light passes through an optical  filter to produce a single band of wavelengths. With 

spectrophotometers, the white light is passed through a  special filter that disperses the light into many bands of 

wavelengths.

•

Measurement–

Both of these techniques use the Beer‐Lambert Law to 

determine concentration. The difference is that  colorimeters measure the absorbency of light in a 

sample while spectrophotometers measure the amount  of light that passes through it.

•

Function–

The colorimeter uses psychophysical analysis, comparing color in the same manner as human eye-brain perception. The spectrophotometer uses only physical analysis, using different wavelengths of light to determine the reflection and transmission properties of color.

STUDY OF MULTICOLOR

SPECTROPHOTOMETER

The spectrophotometer

is an instrument which measures the amount of light of a specificed

wavelength which passes through a medium. According to Beer's law, the amount of light absorbed by a medium is proportional to the concentration of the absorbing material or solute present.

Thus the concentration of a colored solute in a solution may be determined in the lab by measuring the absorbency of light at a given wavelength.

Wavelength (often

abbreviated as lambda) is measured in nm.

The

spectrophotometer allows selection of a wavelength pass through the solution.

Usually,

the wavelength chosen which corresponds to the absorption maximum of the solute. Absorbency is indicated with a capital A.

To familiarize yourself with the spectrophotometer, illustrate and label the following features which are important to its proper use. You should know the function and/or significance of each of these features before you use the instrument.

At the spectrophotometer, you should have two cuvettes

in a plastic

rack.

Solutions which are to be read are poured into cuvettes

which are inserted into the machine. One should be marked "B"for

the blank and one "S" for your sample.

•

.

A wipette

should be available to polish them before insertion into the cuvette

chamber.

Cuvettes are carefully manufactured for their optical uniformity and are quite expensive.

They should be handled with

care so that they do not get scratched, and stored separate from standard test tubes.

•

Try not to touch them except at the top of the tube to prevent finger smudges which alter the reading.

For experiments in which minor

inprecision

is acceptible, clean, unscratched 13 x 100 mm test tubes may be used.

WARM-UP: 1. Plug in and turn on (left hand front dial, labeled ZERO in the illustration). Allow about 30 minutes for warm up.

ZERO ADJUST: 2. With no cuvette

in the

chamber, a shutter cuts off all light from passing though the cuvette

chamber. Under

this condition therefore, the machine may be adjusted to read infinite absorbance (zero% transmittance)

by

rotating zero adjust knob (front left on Spectronic

20).

Do not touch this knob again during the rest of the following procedure.

SELECT WAVELENGTH: 3. Select the desired wavelength of light at which absorbance will be determined by rotating wavelength selection knob (top right knob) until the desired wavelength in nanometers appears in the window. A nanometer (nm), formerly millimicron, equals 10-9

meter.

BLANK ADJUST:4. Fill the B (blank) cuvette

with the solvent

used to dissolve specimen (often distilled water). Polish to clean, insert into the cuvette

chamber, aligning mark to front. Close chamber cover.

5. Rotate blank adjust  knob

(front right knob) to 

adjust absorbance to read  zero

. 

6. Remove blank cuvette, place in plastic test tube rack.

READ SPECIMEN:7. Pour the sample into the S (specimen) cuvette, polish and insert into the chamber, aligning mark to the front.

8.

Note that the scale for absorbance is the lower scale on the dial, and should be read from R to L .

For all readings of the dial, line up the reflection of the needle in the mirror behind the dial with the needle itself.

Otherwise,

parallax error will occur, giving an erroneous reading.

The illustration shows the correctly

aligned dial with a reading of 0.116.

PARALLAX ERROR: Here, the picture was taken of the identical solution as in the previous image, but with the point of view too far to the right.

Note that

the needle reflection is to the right of the needle.

The apparent reading is 0.120.

PARALLAX ERROR: Here, the picture was taken of the identical solution as in the previous image but with the point of view too far to the left.

Note

that the needle reflection is to the left of the needle.

The apparent reading is 0.113.

9. If you read additional specimens, you  should confirm that the machine is still 

zeroed and blanked out, as in steps 2, 4 and 5  for all readings.

•

CLEAN UP: 10. Remove cuvette

from machine, carefully

wash and store spectrophotometer cuvettes keeping them separate from regular test tubes.

•

Return spectophotometer

to its storage location.

INFRARED SPECTROSCOPY

INFRARED SPECTROSCOPY

•

Infrared spectroscopy exploits the fact that molecules absorb specific frequencies that are characteristic of their structure. These absorptions are resonant frequencies, i.e. the frequency of the absorbed radiation matches the frequency of the bond or group that vibrates. The energies are determined by the shape of the molecular potential energy surfaces, the masses of the atoms, and the associated vibronic

coupling.

•

In particular, in the Born–Oppenheimer

and harmonic approximations, i.e. when the molecular Hamiltonian

corresponding to the

electronic ground state

can be approximated by a harmonic oscillator

in the neighborhood of the

equilibrium molecular geometry, the resonant frequencies are determined by the normal modes

corresponding to the molecular

electronic ground state potential energy surface. Nevertheless, the resonant frequencies can be in a first approach related to the strength of the bond, and the mass of the atoms

at either

end of it. Thus, the frequency of the vibrations can be associated with a particular bond type.

Symmetrical stretching

Antisymmetrical stretching

Scissoring

Rocking Wagging Twisting

http://en.wikipedia.org/wiki/File:IR_spectroscopy_apparatus.svg

•

Uses and applications•

Infrared spectroscopy is a simple and reliable technique widely used in both organic and inorganic chemistry, in research and industry. It is used in quality control, dynamic measurement, and monitoring applications such as the long-term unattended measurement of CO2

concentrations in greenhouses and growth chambers by infrared gas analyzers.

•

It is also used in forensic analysis

in both criminal and civil cases, for example in identifying polymer degradation. It can be used in detecting how much alcohol is in the blood of a suspected drink driver measured as 1/10,000 g/mL

= 100 μg/mL.

•

A useful way of analysing

solid samples without the need for cutting samples uses ATR or attenuated total reflectance

spectroscopy. Using this approach,

samples are pressed against the face of a single crystal. The infrared radiation passes through the crystal and only interacts with the sample at the interface between the two materials.

•

With increasing technology in computer filtering and manipulation of the results, samples in solution can now be measured accurately (water produces a broad absorbance across the range of interest, and thus renders the spectra unreadable without this computer treatment).

•

Infrared spectroscopy has also been successfully utilized in the field of semiconductor microelectronics,for

example,

infrared spectroscopy can be applied to semiconductors like silicon, gallium arsenide, gallium nitride, zinc selenide, amorphous silicon, silicon nitride, etc.

•

The instruments are now small, and can be transported, even for use in field trials.

•

Infrared spectroscopy is also useful in measuring the degree of polymerization in polymer

manufacture.

http://en.wikipedia.org/wiki/File:P1020168.JPG

http://en.wikipedia.org/wiki/File:Ir_girl.png

http://en.wikipedia.org/wiki/File:Active-Infrared-Night-Vision.jpg

ULTRAVIOLET–VISIBLE SPECTROSCOPY

•Violet: 400 -

420 nm •Indigo: 420 -

440 nm •Blue: 440 -

490 nm •Green: 490 -

570 nm •Yellow: 570 -

585 nm •Orange: 585 -

620 nm Red: 620 -

780 nm

•

A diagram of the components of a typical  spectrometer are shown in the following diagram. 

The functioning of this instrument is relatively  straightforward. A beam of light from a visible  and/or UV light source (colored red) is separated 

into its component wavelengths by a prism or  diffraction grating. Each monochromatic (single 

wavelength) beam in turn is split into two equal  intensity beams by a half‐mirrored device. One 

beam, the sample beam (colored magenta), passes  through a small transparent container (cuvette) 

containing a solution of the compound being  studied in a transparent solvent.

•

The other beam, the reference (colored blue),  passes through an identical cuvette

containing only 

the solvent. The intensities of these light beams are  then measured by electronic detectors and 

compared. The intensity of the reference beam,  which should have suffered little or no light 

absorption, is defined as I0

. The intensity of the  sample beam is defined as I. Over a short period of 

time, the spectrometer automatically scans all the  component wavelengths in the manner described.  The ultraviolet (UV) region scanned is normally 

from 200 to 400 nm, and the visible portion is from  400 to 800 nm.

•

Applications•

UV/Vis

spectroscopy is routinely used in analytical chemistry

for

the quantitative

determination of different analytes, such as transition metal

ions, highly conjugated

organic compounds,

and biological macromolecules. Determination is usually carried out in solutions.

•

Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons

within the metal atoms can be

excited from one electronic state to another. The colour

of metal ion solutions is strongly affected by the presence of other species, such as certain anions or ligands. For instance, the colour

of a dilute solution of copper sulfate

is a very light blue;

adding ammonia

intensifies the colour

and changes the wavelength of maximum absorption (λmax ).

•

Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water soluble compounds, or ethanol

for organic-

soluble compounds. (Organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths.) Solvent polarity and pH can affect the absorption spectrum of an organic compound. Tyrosine, for example, increases in absorption maxima and molar extinction coefficient when pH increases from 6 to 13 or when solvent polarity decreases.

•

While charge transfer complexes

also give rise to  colours, the colours

are often too intense to be 

used for quantitative measurement. 

http://en.wikipedia.org/wiki/File:UV-vis.png

Raman spectroscopy

•

named after C. V. Raman

is a spectroscopic technique used to study vibrational, rotational,

and other low-frequency modes in a system.[1]

It

relies on inelastic scattering, or Raman scattering, of monochromatic

light, usually from

a laser

in the visible, near infrared, or near ultraviolet

range. The laser light interacts with

molecular vibrations, phonons

or other excitations in the system, resulting in the energy of the laser photons being shifted up or down. The shift in energy gives information about the vibrational

modes in the system.

Infrared spectroscopy

yields similar, but complementary, information.

•

Typically, a sample is illuminated with a laser beam. Light from the illuminated spot is collected with a lens

and sent through a

monochromator. Wavelengths close to the laser line, due to elastic Rayleigh

scattering, are

filtered out while the rest of the collected light is dispersed onto a detector.

•

Spontaneous Raman scattering

is typically very weak, and as a result the main difficulty of Raman spectroscopy is separating the weak inelastically

scattered light from the intense

Rayleigh

scattered laser light.

•

Applications•

Raman spectroscopy is commonly used in chemistry, since vibrational

information is

specific to the chemical bonds

and symmetry of molecules. Therefore, it provides a fingerprint by which the molecule can be identified.

•

Another way that the technique is used to study changes in chemical bonding

•

Raman gas analyzers have many practical applications. For instance, they are used in medicine for real-time monitoring of anaesthetic

and respiratory gas mixtures during surgery.

•

In solid state physics, spontaneous Raman spectroscopy is used to, among other things, characterize materials, measure temperature, and find the crystallographic orientation of a sample

•

Raman spectroscopy is being investigated as a means to detect explosives

for airport security

NMR SPECTROSCOPE

•

Nuclear magnetic resonance (NMR) is a physical  phenomenon

in which magnetic nuclei in a 

magnetic field absorb and re‐emit electromagnetic  radiation. This energy is at a specific resonance

frequency which depends on the strength of the  magnetic field and the magnetic properties of the 

isotope

of the atoms. NMR allows the observation  of specific quantum mechanical

magnetic

properties of the atomic nucleus

•

Many scientific techniques exploit NMR phenomena to study molecular physics, crystals, and non-crystalline materials through NMR spectroscopy. NMR is also routinely used in advanced medical imaging

techniques, such

as in magnetic resonance imaging

(MRI).•

All isotopes that contain an odd number of protons

and/or of neutrons

(see Isotope) have

an intrinsic magnetic moment

and angular momentum, in other words a nonzero spin, while all nuclides

with even numbers of both

have a total spin of zero. The most commonly studied nuclei are 1 H

•

A key feature of NMR is that the resonance

frequency of a particular substance is directly proportional to the strength of the applied magnetic field. It is this feature that is exploited in imaging techniquesThe principle of NMR usually involves two sequential steps:

•

The alignment (polarization) of the magnetic nuclear spins in an applied, constant magnetic field

H0

. •

The perturbation of this alignment of the nuclear spins by employing an electro-magnetic, usually radio frequency (RF) pulse. The required perturbing frequency is dependent upon the static magnetic field (H0

) and the nuclei of observation.

•

History•

Nuclear magnetic resonance was first described and measured in molecular beams by Isidor

Rabi

in 1938,[1]

and in 1944, Rabi was

awarded the Nobel Prize in physics

for this work.[2]

In 1946, Felix Bloch

and Edward Mills

Purcell

expanded the technique for use on liquids and solids, for which they shared the Nobel Prize in Physics

in 1952

•

Theory of nuclear magnetic resonance•

Nuclear spin and magnets

•

All nucleons, that is neutrons

and protons, composing any atomic nucleus, have the intrinsic quantum property of spin. The overall spin of the nucleus is determined by the spin quantum number

S.

•

. It is this magnetic moment that allows the observation of NMR absorption spectra caused by transitions between nuclear spin levels. Most nuclides (with some rare exceptions) that have both even numbers of protons and even numbers of neutrons, also have zero nuclear magnetic moments, and they also have zero magnetic dipole and quadrupole

moments. Hence, such nuclides do not exhibit any NMR absorption spectra.

•

NMR spectroscopy is one of the principal techniques used to obtain physical, chemical, electronic and structural information about molecules

due to either the chemical shift,

Zeeman

effect, or the Knight shift

effect, or a combination of both, on the resonant frequencies of the nuclei present in the sample. It is a powerful technique that can provide detailed information on the topology, dynamics and three-dimensional structure of molecules in solution and the solid state. Additional structural and chemical information may be obtained by performing double-quantum NMR experiments for quadrupolar

nuclei

•

Applications•

Medical MRI

•

The application of nuclear magnetic resonance best known to the general public is magnetic resonance imaging

for medical diagnosis and magnetic

resonance microscopy

in research settings, however, it is also widely used in chemical studies, notably in NMR spectroscopy such as proton NMR, carbon-13 NMR, deuterium NMR and phosphorus-31 NMR. Biochemical information can also be obtained from living tissue (e.g. human brain

tumors) with the

technique known as in vivo magnetic resonance spectroscopy

or chemical shift NMR Microscopy.

http://en.wikipedia.org/wiki/File:MRI-Philips.JPG

•

Chemistry

•

By studying the peaks of nuclear magnetic  resonance spectra, chemists can determine the 

structure of many compounds. It can be a very  selective technique, distinguishing among many 

atoms within a molecule or collection of molecules  of the same type but which differ only in terms of 

their local chemical environment

ELECTROPORESIS

Electrophoresis is a molecular separation technique that involves the use of high voltage electric current for inducing the movement of charged molecules---proteins, DNA, nucleic acids---in a support medium.The movement of charged molecules is called mobility. The mobility of molecules is towards the opposite charge, for instance, a protein molecule with a negative charge moves toward the positive pole of the support medium. The medium may be a paper, a gel or a capillary tube.

•

HOW TO DEFINE ELECTROPHORESIS•

Electrophoresis is any process that uses electricity to separate particles in a fluid. It's a common method of identifying molecules according to some criteria such as molecular weight. Electrophoresis may also be used to prepare a sample for some other scientific technique. There are many different types of electrophoresis and this term doesn't refer to a specific process.

•

ELECTRICAL CHARGE•

Electrical current is placed at one end of the gel, with positive electrodes at the top of the gel --

closest to

the pits with the DNA inside --

and negative electrodes at the bottom. Due to the fact DNA is negatively charged, it will move through the gel toward the bottom and the positive electrodes. The gel will offer resistance for the DNA, so it will take larger strands a longer time to pass through the gel than shorter strands. The process is stopped at a predetermined time, and wherever the DNA is in the gel, that will give an indication of how large each of the strands were. This final image, after the electrophoresis, is the DNA "fingerprint."

•

•

Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic

mobility with the use of an applied voltage.

The electrophoretic

mobility is dependent upon the charge

of the molecule, the viscosity, and the atom's radius.

The rate at which the particle moves is directly

proportional to the applied electric field--the greater the field strength, the fast the mobility. Neutral species are not affected, only ions move with the electric field.

•

If two ions are the same size, the one with greater charge will move the fastest.

For ions

of the same charge, the smaller particle has less friction and overall faster migration rate. Capillary electrophoresis is used most predominately because it gives faster results and provides high resolution separation.

It is a

useful technique because there is a large range of detection methods Available.

•

MICROSCOPIC ELECTROPHORESIS•

A technique in which the electrophoresis of individual particles is observedwith the aid of a microscope or ultra-

microscope.

•

The moving boundary method was the first to be used by Tiselius

to demonstrate the efficacy

of the electrophortic

process. The apparatus consisted of a U tube the horizontal lower portion of the U tube being filled with a mixture of the substances under examination dispersed in a suitable buffer. The two vertical limbs were filled solely with buffer and the cathode and anode dipped into the buffer at the top of each limb respectively.

ELECTROPHORESIS IN FREE SOLUTIONMOVING BOUNDARY ELECTROPHORESIS: Arne Tiselius

(1937) developed the

moving boundary technique for the electrophoretic separation of substances,

for which, besides his work on adsorption analysis, hereceived the Nobel prize in 1948. The sample, a mixture

of proteinsfor example, is applied in a U-shaped cell filled with a

buffer solutionand at the end of which electrodes are immersed. Under

the influence

of the applied voltage, the compounds will migrate at different velocities towards the anode or the cathode depending on their charges. The changes in the refractive index at the boundary during migration can be detected at both ends on the solution using Schlieren

optics.

•

ZONE ELECTROPORESIS:-– a sophisticated instrument, which has a chamber ,

two separate containers for buffers, a stage and electric supply.

watt man paper is taken onto the stage and a small quantity of the test solution is placed over it and switch on the instrument wherein due to the electric supply and the capillary action of the substance, the migration of the test solution particles occur and separate bands are formed where in the electrically charged particles can be separated.

•

TYPES:‐2 types‐

a) vertical

b) horizontal.

Make up the gel which the DNA will be  put into

Presenter

Presentation Notes

A comb is put in the gel to create holes which we call wells

Dye added to the DNA

Buffer solution added to the tank

DNA samples loaded into wells 

Electrical current applied to the  chamber

DNA is stained using ethidium

bromide

http://en.wikipedia.org/wiki/File:Agarosegel.jpg

http://en.wikipedia.org/wiki/File:AgarosegelUV.jpg

•

POINTS TO CONSIDER:‐

•

Selection of buffer

•

Temperature

•

Electro osmosis

•

U.v

florescent chamber

•

FLORIMETRIC ANALYSIS:-•

a large number of substances absorb,transmits,reflects

light-during this

process there is a production of heat of varied quantum( less or more ) which form a new wavelength or radiation called as ELECTROMAGNETIC RADIATION .new light is different from absorbed light which emit longer electromagnetic radiation known as LUMINESCENCE.

•

IT IS OF TWO TYPES-FLUORASCENCE-PHOSPHOROSCENCE

•

FLUORESCENCE:‐

•

Means when a beam of light is incident on certain  substance they emit visible light or visible radiation 

and this phenomena is called as fluorescence  substance ,this phenomena is instantaneous and it 

starts immediately after absorption of light and  stops as soon as the incidence light cuts off    

•

PHOSPHOROSCENCE•

Means they emit light continuously even after the incident light is cut off.

•

in florescence the light emit 10-6

to 10-4 seconds

of absorption whereas in phosphorescence the radiation of light re emit in 10-4

to 20 seconds or

longer than that , therefore it takes much time to re emit radiation even after the incident light is cut off.

•

The florescent substance is proportional to the concentration of the incident of light , the devise to analyze such substance of intensity of florescence is florimetry.

•

Fluorimetry

is the instrument to measure the  fluorescence of substance nature , routinely used in 

lab to identifie

the drugs of different fluorescence   substance , hormones , certain protines

, 

components of haemostasis

etc.

•

Types:‐

•

A) fluorimeter

( filters are used.)

•

B) spectrofluorimeter.( prisms and gratings are  used.)

SCHEMATIC DIAGRAM OF FLUORIMETER

•

APPLICATIONS:‐

•

Useful in clinical laboratory to identify specific  proteins and also to determine uranium in salts , 

henceforth used in nuclear research .

•

It is one of the sensitive analysis of many elements  like aluminum in alloys , boron in steel.vit

B1

and  B2,analysis of food products, qualitative and 

quantitative analysis of aromatic substance.

LOTS TO READ