Instrumental analysis in research

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Instrumental analysis in research

  1. 1. INSTRUMENTALMETHODS INRESEARCHMETHODOLOGY By Dr. M. Gopikrishna Reader, PG Dept. of Rasashastra SJG Ayurvedic Medical College, Koppal, Karnataka email: rasashastra@rediffmail.com
  2. 2. TO IDENTIFIE NEW THINGS-TO THROUGH MODERN LIGHT ONOLD FACTS-
  3. 3. • 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.
  4. 4. 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.
  5. 5. • -BIO-SYNTHETIC PATHWAY CAN BE ASSESED OF THE ISOLATED CHEMICAL.FOR THIS WE NEED TO-• * QUALITATIVE ANALYSIS.• *QUANTITATIVE ANALYSIS.• SEMIQUALITATIVE AND SEMIQUANTITATIVE EG:-WATER EXTRACTS ETC
  6. 6. METHODS NEEDED FORDOING ALL THESE- 1)INSTRUMENTAL–BY THIS WE CAN KNOWPHYSICOCHEMICAL NATURE. 2)NON INSTRUMENTAL -AN ORGANOLEPTIC EVALUATION.
  7. 7. 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.• 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.
  8. 8. ADVANTAGES:--TO ANALYSE A SAMPLE SMALLAMOUNT OF DRUG IS ENOUGH.-DETERMINATION OF QUALITATIVEAND QUANTITATIVE ANALYSIS INSHORT TIME.-COMPLEX MIXTURES CAN ALSO BEANALYSED WITH OR WITHOUTISOLATION.-IT IS WITH SUFFICIENT RELIABILITYAND ACCURATE.
  9. 9. DISADVANTAGES:--TOO COSTLY.-MAINTANANCE OF INSTRUMENT IS DIFFICULT.-CHEMICAL CLEANLINESS IS ALSO COSTLY.-SENSITIVITY DEPENDS ON ADVANCEMENT OFINSTRUMENT THEREFOR UPGRADE IT REGULARLY.-SPECIALISED TEST FOR HANDLING OR USING ISNEEDED.-FREQUENT NEED OF CHECKING DRUGS ISNECESSARY THEREFORE FREQUENTLY CHECK THEINSTRUMENT.
  10. 10. • EXTRACTION OF DRUGS:-• PHANTA,HIMA,SATWA,ARE EXTRACTS ONLY• -IN MODERN THE METHODS OF EXTACTIONS ARE.• *1)MACERATION• *2)PERCOLATION.• *3)DECOCTION.
  11. 11. *1)MACERATION:-KEEPING THE POWDERED DRUG INSUITABLE SOLVENT FOR FEW HOURSLIKE INWATER,ETHER,METHENOL,WITHOUTAPPLING HEAT TO DISSOLVE ITSSOLUBLE PORTION IN IT CALLED AS“ISOLATION MARC” AND INSOLUBLEPART CALLED AS “MARC”.SUB. NEEDED:-CONICALGLASS,FILTER PAPER,EVOPARATINGDISH,GLASS FUNNEL,HOT WATERBATH,ELECTRIC OVEN,ANALYTICALWEIGHINGBALANCE,PIPPETE,VOLUMETRICFLASK,SOLVENTS,THE TEST DRUG.
  12. 12. PROCEDURE:TAKE 5GMS OF TEST DRUG POWDER I.E.AIRDRIED DRUG POWDER ,ADD 100ML OF WATER TO THIS ANDPLACE IT IN A CONICAL FLASK SHAKE THE MIXTURE NOWAND THEN FOR 18HRS THEN FILTER IT.SEPERATE THERESIDUE.FROM THE SOLVENT COLLECT 20ML AND PLACEIT IN A PORCELINE DISH AND DRY IT ON A HOT WATERBATH AND COLLECT THE RESIDUE AND WEIGH IT ANDCALCULATE THE % OF THE EXTRACTIVE.
  13. 13. PERCOLATION:-IT IS THE METHOD OF EXTRACTIONOF ALCOLOIDS ETC.BY PASSING ALIQUID THROUGH A COARS POWDERDRUG.THE LIQUID IS MADE TO PASS THEDRUG AND THE SOLVENT ISCOLLECTED BY THE OPENING OFTHE STOP CORK,ALSO THE SOLVENTIS MADE TO PASS THE COTTON PLUGAT THE NECK TO FILTER AND TOGAIN ONLY THE SOLLUBLEEXTRACT ONLY.
  14. 14. • 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.
  15. 15. • 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..
  16. 16. SOXLET APPARATUSIS DESIGNED FOR THESAME PURPOSE.
  17. 17. • 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.•
  18. 18. • 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.
  19. 19. The sep funnel on the left is a 60 mL size, theothers are 125 mL. The organic chem teachinglabs are downscaling slowly to this smaller size.
  20. 20. There are two different styles of stopper, too. Somestudents swear by the plastic style, some by theground glass style. The disadvantage of the groundglass style is that it can lodge permanently in thesep funnel if it is not removed and stored separatelyafter use. Whichever style you have, make sure thatthe stopper fits snugly in the top of the flask.
  21. 21. 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!
  22. 22. • 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.
  23. 23. pour in liquid to be extracted . . . pour in the solvent . . . add a stopper
  24. 24. 4. Shake the separatory funnel.Pick up the separatory funnel with the stopper inplace and the stopcock closed, and rock it oncegently. Then, point the stem up and slowly openthe stopcock to release excess pressure. Close thestopcock. Repeat this procedure until only a smallamount of pressure is released when it is vented.
  25. 25. Now, shake the funnel vigorously for a few seconds. Releasethe pressure, then again shake vigorously. About 30 sec totalvigorous shaking is usually sufficient to allow solutes to cometo equilibrium between the two solvents.Vent frequently to prevent pressure buildup, which can cause thestopcock and perhaps hazardous chemicals from blowing out. Takespecial care when washing acidic solutions with bicarbonate or carbonatesince this produces a large volume of CO2 gas.
  26. 26. 5. Separating the layers. Let the funnel rest undisturbed until the layers are clearly separated.While waiting, remove thestopper and place a beaker orflask under the sep funnel.
  27. 27. 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 beremoved from the funnel,remove it by pouring it out of thetop of the funnel.
  28. 28. • 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!
  29. 29. 7. Store your separatory funnel with the cap (stopper)separate from the funnel! Please, remove the stopper before storage!!
  30. 30. • 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.
  31. 31. DISTILLATION:-
  32. 32. SEPERATION OFVOLATILE SUBSTANCESFROM A DRUG IN ADISTILATIONAPPARATUS.
  33. 33. CHROMATOGRAPHY:-IT IS A TECHNIQUE TO SEPARATE INDIVIDUAL COMPONENTS IN A MIXTURE,IN A SPECIFIC MOBILE AND STATIONARY PHASE.CHROMA= COLOUR.GRAPHY= WRITING .
  34. 34. • 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.
  35. 35. • IN 1930-FEW VARITIES OF TLC AND ION EXCHANGE CROMATOGRAPHY WAS INTRODUCED.• IN 1941 PARTITION AND PAPER CHROMATOGRAPHY WAS DEVELOPED.• IN 1952 GAS CHROMATOGRAPHY WAS INTRODUCED.• 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 .
  36. 36. 2 PHASES INCROMATOGAPHY.*STATIONARY PHASE -SOLIDFORM OR A LIQUID IN SOLIDFORM.*MOBILE PHASE- EITHERLIQUID OR GAS TECHNIQUE IN MOBILE PHASE PASSESTHE STATIONARY PHASE TRANSPORTS THE SEPARATECOMPONENT AT DIFFERENT SPEED AT DIRECTION OFFLOW OF MOBILE PHASE.
  37. 37. • PRINCIPLE:• SEPERATION OF SINGLE COMPONENT FROM A MIXTURE IN STABLE AND MOBILE PHASE.• 2 PHASES NEEDED-• 1)STATIONARY PHASE -USING SILICA GEL ALSO CELLULOS POWDER ETC.• -2)MOBILE PHASE- ETHANOL,BENZINE,CARBONTETRACHLORIDE,ETC . CAN BE USED.
  38. 38. • 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.• *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.• *ALSO IN BIOCHEMICAL ANALYSIS IN METABOLITES LIKE PLASMA,SERUM ANALYSIS,URINE ANALYSIS.
  39. 39. • ADVANTAGES:• *SIMPLE AND EASY TEST IN STANDERDISATION.• *TIME IS 20-40MIN ,ITS VERY FAST.• *SEPARATE INDIVIDUAL COMPONENTS FROM SMALL AMOUNT OF SAMPLE.• *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.
  40. 40. Procedure for TLC1. Prepare the developing container.The developing container for TLC can bea specially designed chamber, a jar with alid, or a beaker with a watch glass on thetop:In the teaching labs,we use a beaker witha watch glass on top.
  41. 41. Pour solventinto the beakerto a depth ofjust less than0.5 cm.
  42. 42. To aid in thesaturation of theTLC chamber withsolvent vapors, linepart of the inside ofthe beaker withfilter paper.
  43. 43. Cover the beakerwith a watch glass,swirl it gently, andallow it to standwhile you prepareyour TLC plate.
  44. 44. 2. Prepare the TLC plate.TLC plates used in theorganic chem. teachinglabs are purchased as 5 cmx 20 cm sheets. Each largesheet is cut horizontallyinto plates which are 5 cmtall by various widths; themore samples you plan torun on a plate, the wider itneeds to be.
  45. 45. Plates will usually be cut and ready foryou when you come to lab.Handle the plates carefully so that you donot disturb the coating of adsorbent or getthem dirty.
  46. 46. Measure 0.5 cm from thebottom of the plate. Takecare not to press so hard withthe pencil that you disturbthe 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.
  47. 47. Under the line, mark lightly thename of the samples you will spot onthe plate, or mark numbers for timepoints. Leave enough space betweenthe samples so that they do not runtogether, about 4 samples on a 5 cmwide plate is advised.Use a pencil and do not press downso hard that you disturb the surfaceof the plate. A close-up of a platelabeled "1 2 3" is shown to the right.
  48. 48. • 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.
  49. 49. add a few drops of solvent . . . . . . swirl until dissolvedThe solution is applied to the TLC plate with a 1µL microcap.
  50. 50. Microcaps come in plasticvials inside red-and-whiteboxes. If you are opening anew vial, you will need totake off the silver cap,remove the white styrofoamplug, and put the silver capback on. A small hole in thesilver cap allows you toshake out one microcap at atime. Microcaps are verytiny; the arrow points to one,and it is hard to see in thephoto.
  51. 51. • 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
  52. 52. make sure it is filled - hold it up to the light if necessarytouch the filled microcapto TLC plate to spot it -make sure you watch tosee that all the liquid hasdrained from the microcap
  53. 53. rinse the microcap with clean solvent by first filling it . . . do this rinse process 3 times!. . . and then draining it bytouching it to a papertowel
  54. 54. heres the TLC plate, spotted and ready to bedeveloped
  55. 55. • 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).
  56. 56. place the TLC plate in the developingcontainer - make sure the solvent is nottoo deep
  57. 57. 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
  58. 58. Remove the plate from the beaker.quickly mark a line across the plateat 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.
  59. 59. • 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.
  60. 60. this is a UV lamphere are two proper sized spots, viewedunder a UV lamp(you would circle these while viewingthem)
  61. 61. The plate to the left shows three compounds run atthree different concentrations. The middle and rightplate show reasonable spots; the left plate is run tooconcentrated and the spots are running together, makingit difficult to get a good and accurate Rf reading.
  62. 62. Heres what overloaded plates look like compared to well-spotted plates. The plate on the left has a large yellow smear; this smearcontains 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.
  63. 63. • DETECTION OF THE ISOLATED COMPONENT:-• 1)non specific method:-• By colours-florensent phase• Iodine chamber.• H2so4 spray.• u.v chambers.• Color spray reagents can be used.
  64. 64. • 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.• iv)for cardiac glycosides-spray with 3,5dinitro benzoic acid.
  65. 65. 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.
  66. 66. • 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.
  67. 67. These measurements are then taken:The Rf value for each dye is then worked out using the formula:
  68. 68. For example, if the red component travelled 1.7 cm from the baseline while the solvent had travelled 5.0 cm, then the Rf value forthe red dye is:
  69. 69. • 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
  70. 70. • 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.
  71. 71. densitometer
  72. 72. spectrophotometer-instrument
  73. 73. 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.,
  74. 74. 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.
  75. 75. 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.
  76. 76. Adsorption ChromatographyAdsorption chromatographyis probably one of the oldesttypes of chromatographyaround. It utilizes a mobileliquid or gaseous phase thatis adsorbed onto the surfaceof a stationary solid phase.The equilibriation betweenthe mobile and stationaryphase accounts for theseparation of differentsolutes.
  77. 77. 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 equilibriatesbetween the mobile phase and the stationary liquid.
  78. 78. Types ofChromatography- Radial chromatography- Ascendingchromatography- DescendingchromatographyRadial ChromatographyIn this type ofchromatography, as thepigment separates, thedifferent colours moveoutwards.
  79. 79. AscendingChromatographyThe solvent movesupwards on theseparating media
  80. 80. DescendingChromatographyThe solvent movesdownwards on theseparating media.
  81. 81. Ion Exchange ChromatographyIn this type ofchromatography, the use ofa resin (the stationary solidphase) is used to covalentlyattach anions or cationsonto it. Solute ions of theopposite charge in themobile liquid phase areattracted to the resin byelectrostatic forces.
  82. 82. • 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.
  83. 83. (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.
  84. 84. Add dry silica geladsorbent, 230-400mesh -- usually the jaris labeled "for flashchromatography." Oneway to fill the column isto invert it into the jar ofsilica gel and scoop itout . . .
  85. 85. . . . then tamp it down before Another way to fill the column isscooping more out. to pour the gel into the column using a 10 mL beaker.
  86. 86. Whichever method you use to When properly packed, the silicafill the column, you must tamp it gel fills the column to just belowdown on the bench top to pack the indent on the pipet. Thisthe silica gel. You can also use leaves a space of 4–5 cm on topa pipet bulb to force air into the of the adsorbent for the additioncolumn and pack the silica gel. of solvent. Clamp the filled column securely to a ring stand using a small 3-pronged clamp.
  87. 87. • (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.
  88. 88. Monitor the solvent level,both as it flows through thesilica gel and the level atthe top. If you are not in ahurry (or busy doingsomething else), you canlet the top level drop bygravity, but make sure itdoes not go below the topof the silica. Again, thearrow marks how far thesolvent has flowed downthe column.
  89. 89. Speed up the process by usinga pipet bulb to force the solventthrough the silica gel - this putsthe flash in microscale flashchromatography. Place thepipet bulb on top of the column,squeeze the bulb, and thenremove the bulb while it is stillsqueezed. You must be carefulnot to allow the pipet bulb toexpand before you remove itfrom the column, or you willdraw solvent and silica gel intothe bulb.
  90. 90. When the bottomsolvent level is at thebottom of the column,the pre-elution processis completed and thecolumn is ready to load.
  91. 91. If you are not ready to loadyour sample onto thecolumn, it is okay to leavethe column at this point.Just make sure that it doesnot go dry -- keep the topsolvent level above the topof the silica (as shown inthe picture to the left) byadding solvent asnecessary.
  92. 92. (3) Load the sample onto thesilica gel column.Two different methods are usedto load the column: the wetmethod and the dry method: wetand dry. Below are illustrationsof both methods of loading acrude sample of ferrocene ontoa column.In the wet method, the sampleto be purified (or separated intocomponents) is dissolved in asmall amount of solvent, suchas hexanes, acetone, or othersolvent. This solution is loadedonto the column.
  93. 93. • 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 its in the column, fresh eluting  solvent is added to the top and you are ready to  begin the elution process (see step 4).
  94. 94. • 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.
  95. 95. Dry loading methodFirst 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). 
  96. 96. • (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.
  97. 97. The photo at the left shows the solvent beingforced through the column with a pipet bulb.The series of 5 photos below show thecolored compound as it moves through thecolumn after successive applications of thepipet bulb process.The last two photos illustrate collection of thecolored sample. Note that the collectionbeaker is changed as soon as the coloredcompound begins to elute.The process is complicated if the compoundis not colored. In such experiments, equalsized fractions are collected sequentially andcarefully labeled for later analysis.
  98. 98. • (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).
  99. 99. • (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.
  100. 100. • 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".
  101. 101. This is a 25 µL glass Hamilton syringe.You only inject 2.5 µL, so it will NOT befilled to the top.
  102. 102. Place the tip of the needle in the liquid. Slowly draw up a small amountof liquid by raising the plunger, then press on the plunger to expel theliquid back into the liquid. This serves to “rinse” the syringe with yoursample, ensuring that what you will measure in the GC run is thecomposition of your mixture. Repeat the rinse process one or two times.Then, draw up the plunger slowly again while the needle is in the liquidand carefully fill the syringe with liquid about halfway to the “5”.
  103. 103. It is often hard to see the liquid in thesyringe. If the syringe is clogged, theplunger will be in the correct position but thebarrel of the syringe will be filled with onlyair, as in the bottom syringe in the photo tothe left.The best thing to do is to carefully examingthe syringe after you think that you havefilled it. Hold it up to the light to get a betterview.Small air bubbles in the syringe will notaffect the GC run (middle syringe in thephoto to the left). As long as there is enoughliquid in the syringe, the GC run will workfine. If you keep getting bubbles, just pullthe plunger up a bit past the "halfway to the5" mark to compensate.If you have a VERY large air bubble, youwill not have enough liquid to show areading on the GC (e.g., the bottom syringein the photo).
  104. 104. • (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.
  105. 105. Push the needle of the filledsyringe through the injector (asfar as it will go) and quicklypush the plunger. Remove the syringe immediately . . .
  106. 106. . . . and quickly press thestart button on theintegrating recorder or thestart recording button onthe computer Heres a close-up of the integrating recorder.
  107. 107. • 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.
  108. 108. • 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.•
  109. 109. • 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.
  110. 110. • (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.
  111. 111. Uses:• mainly for detecting steroids,food components    ,identification of various substances.
  112. 112. Study of colors
  113. 113. BEER LAMBERT LAW
  114. 114. MECHANISM
  115. 115. PROCESS INVOLVED
  116. 116. • 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.
  117. 117. • 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.
  118. 118. Beers law:-Intensity of beam of light decreases withthe increase in the concentration ofabsorbing substance.
  119. 119. 1 cmX                                                      y 2 cmx Y1       y
  120. 120. • Similarly a concentration of solution with the light ray through them , the greater or lesser amount of concentration energy. » »
  121. 121. • 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
  122. 122. colorimeter
  123. 123. •• 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.
  124. 124. • 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.
  125. 125. • 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.
  126. 126. • 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.
  127. 127. • 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 detectors 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.
  128. 128. • 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.
  129. 129. • 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.
  130. 130. • 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
  131. 131. • 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.
  132. 132. • 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  customers light source specifications and calibrating  liquid crystal displays for televisions and laptops.
  133. 133. • 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.
  134. 134. • 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.
  135. 135. • 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 Beers  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.
  136. 136. • How to Use Colorimeters• A colorimeter is any device that measures the color of a particular substance. Its 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 solutions 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.• Instructions Things Youll Need• Colorimeter• Reference solution• Test solution
  137. 137. –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 cant see its reflection in the mirror.
  138. 138. –4• Fill the reference cuvette with the test solution as indicated by the colorimeter model youre using. Clean the reference cuvette carefully to ensure it doesnt 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 solutions concentration.
  139. 139. • 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.
  140. 140. • 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.
  141. 141. • 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
  142. 142. • 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.•
  143. 143. • 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 lEclairage (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.
  144. 144. • Color Measurement – In 1931 the Commission Internationale de lEclairage 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.
  145. 145. • 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.
  146. 146. • Reflectance Spectrophotometers – Similar in the respect that reflectance spectrophotometers can  also be used to determine a samples 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.
  147. 147. • 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.
  148. 148. • 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.
  149. 149. – 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.
  150. 150. • 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.
  151. 151. STUDY OF MULTICOLOR
  152. 152. SPECTROPHOTOMETER
  153. 153. The spectrophotometer isan instrument whichmeasures the amount oflight of a specificedwavelength which passesthrough a medium.According to Beers law, theamount of light absorbed bya medium is proportional tothe concentration of theabsorbing material or solutepresent.
  154. 154. Thus the concentration of acolored solute in a solution maybe determined in the lab bymeasuring the absorbency oflight at a givenwavelength. Wavelength (oftenabbreviated as lambda) ismeasured in nm. Thespectrophotometer allowsselection of a wavelength passthrough the solution. Usually,the wavelength chosen whichcorresponds to the absorptionmaximum of the solute.Absorbency is indicated with acapital A.
  155. 155. To familiarize yourselfwith thespectrophotometer,illustrate and label thefollowing features whichare important to itsproper use. You shouldknow the functionand/or significance ofeach of these featuresbefore you use theinstrument.
  156. 156. At thespectrophotometer, youshould have twocuvettes in a plasticrack. Solutions whichare to be read arepoured into cuvetteswhich are inserted intothe machine. Oneshould be marked "B"forthe blank and one "S"for your sample.
  157. 157. • . 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.
  158. 158. • 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.
  159. 159. WARM-UP:1. Plug in and turn on (left hand frontdial, labeled ZERO in the illustration).Allow about 30 minutes for warm up.
  160. 160. ZERO ADJUST:2. With no cuvette in thechamber, a shutter cuts offall light from passing thoughthe cuvette chamber. Underthis condition therefore, themachine may be adjusted toread infinite absorbance(zero% transmittance) byrotating zero adjust knob(front left on Spectronic 20).
  161. 161. Do not touch this knobagain during the rest ofthe following procedure.
  162. 162. SELECT WAVELENGTH:3. Select the desiredwavelength of light at whichabsorbance will bedetermined by rotatingwavelength selectionknob (top right knob) untilthe desired wavelength innanometers appears in thewindow. A nanometer (nm),formerly millimicron, equals10-9 meter.
  163. 163. BLANK ADJUST:4. Fill the B (blank) cuvette with the solventused to dissolve specimen (often distilledwater). Polish to clean, insert into the cuvettechamber, aligning mark to front. Close chambercover.
  164. 164. 5. Rotate blank adjust knob (front right knob) to adjust absorbance to read zero . 6. Remove blankcuvette, place in plastictest tube rack.
  165. 165. READ SPECIMEN:7. Pour the sample into the S (specimen)cuvette, polish and insert into the chamber,aligning mark to the front.
  166. 166. 8. Note that the scale for absorbance isthe lower scale on the dial, and shouldbe read from R to L .
  167. 167. For all readings of the dial, line up thereflection of the needle in the mirror behindthe dial with the needle itself. Otherwise,parallax error will occur, giving an erroneousreading. The illustration shows the correctlyaligned dial with a reading of 0.116.
  168. 168. PARALLAX ERROR:Here, the picture was taken of the identicalsolution as in the previous image, but with thepoint of view too far to the right. Note thatthe needle reflection is to the right of theneedle. The apparent reading is 0.120.
  169. 169. PARALLAX ERROR:Here, the picture was taken of the identicalsolution as in the previous image but withthe point of view too far to the left. Notethat the needle reflection is to the left of theneedle. The apparent reading is 0.113.
  170. 170. 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.
  171. 171. • 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.
  172. 172. INFRAREDSPECTROSCOPY INFRARED SPECTROSCOPY
  173. 173. • 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.
  174. 174. • 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.
  175. 175. Symmetrical Antisymmetrical Scissoringstretching stretching Rocking Wagging Twisting
  176. 176. • 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.
  177. 177. • 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).
  178. 178. • 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.
  179. 179. ULTRAVIOLET–VISIBLE SPECTROSCOPY
  180. 180. •Violet: 400 - 420 nm•Indigo: 420 - 440 nm•Blue: 440 - 490 nm•Green: 490 - 570 nm•Yellow: 570 - 585 nm•Orange: 585 - 620 nmRed: 620 - 780 nm
  181. 181. • 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.
  182. 182. • 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.
  183. 183. • 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).
  184. 184. • 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.
  185. 185. • While charge transfer complexes also give rise to  colours, the colours are often too intense to be  used for quantitative measurement. 
  186. 186. Raman spectroscopy
  187. 187. • 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.
  188. 188. • 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.
  189. 189. • 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.
  190. 190. • 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
  191. 191. NMR SPECTROSCOPE
  192. 192. • 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
  193. 193. • 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
  194. 194. • 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 techniques The 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.
  195. 195. • 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
  196. 196. • 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.
  197. 197. • 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
  198. 198. • 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.
  199. 199. • 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
  200. 200. ELECTROPORESIS
  201. 201. Electrophoresis is a molecularseparation technique that involvesthe use of high voltage electriccurrent for inducing the movementof charged molecules---proteins,DNA, nucleic acids---in a supportmedium.The movement of chargedmolecules is called mobility. Themobility of molecules is towardsthe opposite charge, for instance,a protein molecule with a negativecharge moves toward the positivepole of the support medium. Themedium may be a paper, a gel or acapillary tube.
  202. 202. • HOW TO DEFINE ELECTROPHORESIS• Electrophoresis is any process that uses electricity to separate particles in a fluid. Its 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 doesnt refer to a specific process.
  203. 203. • 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."•
  204. 204. • 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 atoms 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.
  205. 205. • 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.
  206. 206. • MICROSCOPIC ELECTROPHORESIS• A technique in which the electrophoresis of individual particles is observed with the aid of a microscope or ultra- microscope.
  207. 207. • 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.
  208. 208. ELECTROPHORESIS IN FREE SOLUTIONMOVING BOUNDARY ELECTROPHORESIS:Arne Tiselius (1937) developed themoving 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
  209. 209. of the applied voltage, the compounds willmigrate at different velocities towards the anodeor the cathode depending on their charges. Thechanges in the refractive index at the boundaryduring migration can be detected at both endson the solution using Schlieren optics.
  210. 210. • 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.
  211. 211. • TYPES:‐ 2 types‐ a) vertical b) horizontal.
  212. 212. Make up the gel which the DNA will be  put into
  213. 213. Dye added to the DNA
  214. 214. Buffer solution added to the tank
  215. 215. DNA samples loaded into wells 
  216. 216. Electrical current applied to the  chamber
  217. 217. DNA is stained using ethidium bromide
  218. 218. • POINTS TO CONSIDER:‐• Selection of buffer• Temperature• Electro osmosis• U.v florescent chamber
  219. 219. • 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
  220. 220. • 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    
  221. 221. • 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.
  222. 222. • 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.)
  223. 223. SCHEMATIC DIAGRAM OF FLUORIMETER
  224. 224. • 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.
  225. 225. LOTS TO READ

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