instrumental analysis in research
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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-
•
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.
•
-WATER SOLUBLE MATERIALS WITH MANY COMPOUNDS ARE OBSERVED AND IDENTIFIED.
•
-SEPARATE CHEMICAL AND ISOLATE IT AND ASSES THE DIFFERENT BONDING WITH DIFFERENT ADVANCE TECHNIQUES.
•
-BIO-SYNTHETIC PATHWAY CAN BE ASSESED OF THE ISOLATED CHEMICAL.FOR THIS WE NEED TO-
•
* QUALITATIVE ANALYSIS.•
*QUANTITATIVE ANALYSIS.
•
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.
•
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.
•
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.
•
USE OF PERCOLATE: COLLECT THE PERCOLATE OF 5MIN INTERVAL PF 4-5 BATCHES OF SAME SOLVENT AND ASSES FOR THE RICH ALCOLOID SAMPLE.
•
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.
•
IF IT IS A COMPLEX COMPOUND SEPARATE THE EXTRACTS WITH ANY METHODS ,AS ONE METHOD IS-
•
BATCH EXTRACTION:-•
IT IS A LIQUID LIQUID EXTRACTION,OR EXTRACTION OF EXTRACTIVES FROM THE LIQUIDS.
•
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.
•
•
Separatory Funnel Extraction Procedure•
1. Inspect your separatory funnel.
•
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!
•
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.
•
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.
•
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!!
•
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 .
•
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-
•
1)STATIONARY PHASE -USING SILICA GEL ALSO CELLULOS POWDER ETC.
•
-2)MOBILE PHASE- ETHANOL,BENZINE,CARBONTETRACHLORIDE,ETC . CAN BE USED.
•
APPLICATIONS OF TLC:-•
*IT IS MUCH BENIFICIAL WITH INDIVIDUAL COMPONENTS IN A MIXTURE.
•
*FOR CHECKING PURITY OF THE SAMPLE,ALSO FOR THE PURIFICATION PROCESS.
•
*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.
•
*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.
•
ADVANTAGES:•
*SIMPLE AND EASY TEST IN STANDERDISATION.
•
*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.
•
*LESS EXPENSIVE.•
*VERY EASY FOR DETECTION OF SAMPLE.
•
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.
•
3. Spot the TLC plate•
The sample to be analyzed is added to the plate in a process called "spotting".
•
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.
•
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
•
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.
•
5. Visualize the spots•
If your samples are colored, mark them before they fade by circling them lightly with a pencil.
•
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.
•
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.
•
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.
•
DETECTION OF THE ISOLATED COMPONENT:-
•
1)non specific method:-•
By colours-florensent phase
•
Iodine chamber.•
H2 so4 spray.
•
u.v chambers.•
Color spray reagents can be used.
•
2)specific method:-•
For different types of components-
•
i)phenolic compounds and tannins-ferric chloride spray is advised.
•
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:-
•
QUALITATIVE AND QUANTITATIVE-•
QUALITATIVE ANALYSIS:-
•
-By visual assessment by observing size,density,number of spots with different reagents of components can be identified.
•
-separately measure the spot in mm is directly proportional to substance present in that spot.
•
RETARDATION OR RETENTION FACTOR:‐Rf
• Measuring Rf
values
•
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.
•
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:
•
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
•
QUANTITATIVE ANALYSIS:-•
Carried out in 2 ways-
•
Direct method:-•
a) on the plate i.e., after spray of different reagents.
•
b)by assessing the density of elute.•
c)measurement of spot area in mm is proportion to amount of quantity more in the sample.
•
d)densitometer:- method where intensity of color of substance is measured in chromatogram –in situ method.
•
E)densitometer-method where optical density of separated spots is measured.
•
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.
•
Microanalysis can be performed by colorimeter ,electroporosis.etc.,
HPTLC:-HIGH PERFORMANCE THIN LAYER CROMATOGRAPHY-
•
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.
•
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
-
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.
•
Column Chromatography•
Procedure for Microscale Flash Column Chromatography
•
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.
•
(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.
•
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.
•
Wet loading method
•
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).
•
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).
•
(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
•
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.
•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.
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
•
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.
•
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
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
•
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.
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