ramanspectroscopy-151102123444-lva1-app6891 (1).pptx

Ramanspectroscopy and its types by Abhishek Mahajan

1. Submitted By: Abhishek Mahajan Submitted To: Dr. Vikas Jaitak Reg. No.: 22mphyto13 M. Pharm. Pharmacognosy ASSIGNMENT ON TOPIC: RAMAN SPECTROSCOPY & RADIOIMMUNOASSAY

2. INTRODUCTION Raman spectroscopy was discovered by C. V. Raman in 1928 It is a spectroscopic technique used to observe vibration , rotational , and other low-frequency modes in a system. Raman spectroscopy is commonly used in chemistry to provide a fingerprint by which molecules can be identified. When the radiation pass through the transparent medium the species p r esent sca t t er a f r acti o n of th e bea m i n all di r ecti o n Raman scattering result from the same type of the quantities vibration changed associated with IR spectra The difference in wavelength in between the incident and scattered visible radiation correspond to wave length in mid IR region

3. PRINCIPLE When monochromatic radiation is incident upon a sample then this light will interact with the sample in some fashion. It may be reflected, absorbed or scattered in some manner. It is the scattering of the radiation that occurs which gives information about molecular structure Raman is based on scattering. The sample is irradiated with a coherent source, typically a laser. Most of the radiation is elastically scattered (called the Rayleigh scatter). A small portion is inelastically scattered (Raman scatter, composed of Stokes and anti-Stokes portions). This latter portion is what we are particularly interested in because it contains the information in which we are interested.

4. The spectrum is measured with the laser line as a reference. Hence, the peaks are measured as the shift from the laser line. The peak positions are determined by the vibrational energies associated with the bonds in the molecule(s) of which the sample is composed. Because of this, the spectrum ends up looking very similar t o an IR spe c tru m a n d i s i n t er p r e t ed simila r l y . There is a footnote to this however, as the principle of mutual exclusion applies. That is, peaks that are emphasized in IR (polar bonds with high dipole moments) are de-emphasized somewhat in Raman. The bands that are emphasized in a Raman spectrum are those that are due to highly polarizable bonds such as those with π electrons.

5. The emitted radiation is of three types: Stokes scattering Anti-stokes scattering Rayleigh scattering

6. INSTRUMENTATION

7. INSTRUMENTATION Inst r u me nt at io n fo r moder n Ra m a n s p ect r os c o p y c ons i sts of three components: L aser sou r c e S ample illumination system S uitable spectrometer. 1) Source: The sources used in modern Raman spectrometry are nearly always lasers because their high intensity is necessary to produce Raman scattering of sufficient intensity to be measured with a r easo n able signal- t o- no i se r at i o . Because the intensity of Raman scattering varies as the fourth power of the frequency, argon and krypton ion sources that emit in the blue and green region of the spectrum have and advantage o v er th e oth e r sou r c e s .

8. Source used now a days are laser because high intensity is necessary to produce Raman scattering

9. 2. Sample Illumination System Liquid Samples: A major advantage of sample handling in Raman spectroscopy compared with infrared arises because water is a weak Raman scattered but a strong absorber of infrared radiation. Thus, aqueous solutions can be studied by Raman spectroscopy but not by infrared. This advantage is particularly important for biological and inorganic systems and in studies dealing with water pollution problems. Solid Samples: Raman spectra of solid samples are often acquired by filling a small cavity with the sample after it has been ground to a fine powder. Polymers can usually be examined directly with no sample pretreatment. Gas samples: Gas are normally contain in glass tubes, 1-2 cm in diameter and about 1mm thick. Gases can also be sealed in small capillary tubes

10. 3. Raman Spectrometers Raman spectrometers were similar in design and used the same type of components as the classical ultraviolet/visible dispersing instruments. Most employed double grating systems to minimize the spurious radiation reaching the transducer. Photomultipliers served as transducers. Now Raman spectrometers being marketed are either Fourier transform instruments equipped with cooled germanium transducers or multichannel instruments based upon charge- coupled devices.

11. DIFFERENCE RAMAN It is due to the scattering of light by the vibrating molecules. Th e vi b r at i on i s Raman act i v e i f i t ca u ses a change in polarisability. Th e molec u le nee d n o t possess a permanent dipole moment. W a t er ca n b e use d as a so l v ent. Sample preparation is not very elaborate, it can be in any state. Gives an indication of covalent character in the molecule. Cost of instrumentation is very high INFRA RED I t i s the r esult of abso r pti o n of li g ht b y vibrating molecules. Vibration is IR active if there is change in dipole moment. Th e v ib r ati o n c o n c e r ne d should h a v e a change in dipole moment due to that vibration. W a t er ca n n o t be use d du e t o it s in t ense absorption of IR. Sample preparation is elaborate Gaseous samples can rarely be used. Gives an indication of ionic character in the molecule. Comparatively inexpensive.

12. Raman spectrum Typical Raman spectrum Plot of signal intensity vs Raman shift (Raman shift, in cm-1 = energy of photon in - energy of photon out) Raman shift

13. The Raman instrument can be on the same bench as the FTIR. Of t en, a Y A G: N d3 + laser (10 6 4 nm ) i s us ed t o e x ci t e the sample, so that the excitation energy is lower than the absorption band energies of organic systems. Fluorescence is then minimized. Instruments may be combined with a microscope, or optical fibre, so that scanning over a few (microns)2 of surface area, and Raman mapping is easily performed. Fourier transform Raman spectroscopy

14. Fourier transform Raman spectroscopy

15. APPLICATION Raman Spectra of Inorganic Species The Raman technique is often superior to infrared for spectroscopy investigating inorganic systems because aqueous solutions can be employed. In addition, the vibrational energies of metal-ligand bonds are generally in the range of 100 to 700 cm -1 , a region of the infrared that is experimentally difficult to study. These vibrations are frequently Raman active, however, and peaks with  values in this range are readily observed. Raman studies are potentially useful sources of information concerning the composition, structure, and stability of coordination compounds.

16. Raman Spectra of Organic Species Raman spectra are similar to infrared spectra in that they have regions that are useful for functional group detection and fingerprint regions that permit the identification of specific compounds. Raman spectra yield more information about certain types of organic compounds than do their infrared counterparts. Quantitative applications Raman spectra tend to be less cluttered with peaks than infrared spectra. As a consequence, peak overlap in mixtures is less likely, and quantitative measurements are simpler. In addition, Raman sampling devices are not subject to attack by moisture, and small amounts of water in a sample do not interfere. APPLICATION ( c o n t.)

17. Biological Applications of Raman Spectroscopy Raman spectroscopy has been applied widely for the st u d y o f bi o log i ca l s y s t em s . The advantages of his technique include the small sample requirement, the minimal sensitivity toward interference by water, the spectral detail, and the conformational and environmental sensitivity.

18. METHODS FOR STANDARDIZATION OF ANTIBIOTICS There are mainly three important points are for standardization of antibiotics: FDA (Food Drug Administration) regulations governing all aspects of antibiotics testing are completely detailed and are subject to periodic amendment. FDA regulations need to be referred with regard to prescribed method for the assay of individual antibiotics and their preparations. During the evaluation of potency of antibiotic substances, the actual and apparent measured effect is the degree of inhibition of the growth of a suitable strain of microorganism i.e. the prevention of the multiplication of the test organisms.

19. METHODS FOR STANDARDIZATION OF ANTIBIOTICS Two methods are usually used: Cylinder-plate (or cup-plate) Method Turbidimetric Method The cylinder-plate method depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish. The growth of the added micro- organism is prevented entirely in a zone around the cylinder containing a solution of the antibiotic. The cylinder-plate method depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish. The growth of the added micro- organism is prevented entirely in a zone around the cylinder containing a solution of the antibiotic.

20. Media Preparation: All the required ingredients are dissolved in sufficient water to produce 1000 ml and added sufficient amount of 1 M Sodium hydroxide or 1 M Hydrochloride acid, as required so that after sterilization the pH is between 6.5 to 7.5.

21. Buffer Solution Preparation: Buffer solutions are prepared by dissolving the required quantities of dipotassium hydrogen phosphate Add potassium dihydrogen phosphate in sufficient water to produce 1000 ml and pH adjusted with 8 M phosphoric acid or 10 M potassium hydroxide.

22. Standard Preparation: Standard preparation is an authentic sample of the appropriate antibiotic for which the potency has been determined by reference to the appropriate international standard. The potency of the standard preparation is expressed in µg per mg of an international unit of the pure antibiotic. A stock solution is prepared by dissolved a required amount of weighed quantity of the standard preparation of a given antibiotic

23. Preparation of the Sample Solution: Dilutions of sample or unknown samples are prepared at the range of dilutions of standard prepared dilutions. Test Organisms: Some of the test organisms for each antibiotic are listed in

24. Bioassay is defined as the estimation of the potency of an active principle in a unit quantity of preparation. OR Detection and measurement of the concentration of the substance in a preparation using biological methods.

25. Bioassays, as compared to other methods of assay (e.g. chemical or physical assay) is very important. Because it is the only method of assay if : Active principle of drug is unknown or cannot be isolated, e.g. insulin, posterior pituitary extract etc. Chemical method is either not available or if available, it is too complex and insensitive or requires higher dose e.g. insulin, acetylcholine. Chemical composition is not known, e.g. long acting thyroid stimulants. Chemical composition of drug differs but have the same pharmacological action and vice-versa, e.g. cardiac glycosides, catecholamines etc.

26. The b a s i c p r i n c i ple o f b i o a ss ay i s to c o m p a r e the test s u b s t a n ce with t h e International Standard preparation of the same and to find out how much test substance is required to produce the same biological effect, as produced by the standard.

27. ⦿ Mai n ly tw o type s ar e there. Quantal G r ad e d

28. ⦿ Quantal response: the response is in the form of ‘all or none’, i.e. either no response or maximum response. ⦿ Drugs producing quantal effect can be bioassayed by End-point method.

29. Graded response: response is proportional to the dose and response may lie between no response and the maximum response. Types : Bracketing /direct matching Interpolation Multiple point assays Three point assay Four point assay Six point assay Cumulative dose response

30. 1. End- point method The threshold dose producing a predetermined effect is measured Comparison between the results of standard and the test dru g i s do n e. e.g . b i oass a y o f d i g i tal i s i n cats. The cat is anaesthetized with chloralose and its blood pressure i s recorded. The dru g i s sl o w l y i n fuse d i n t o th e an i mal.

31. The moment the heart stops beating and blood pressure falls to zero, the volume of fluid infused is noted down. Two series of such experiments-one using standard digitalis and the other using test preparation of digitalis is done . potenc y i s ca l cu l ate d a s fol l ows: Threshold dose of the Standard Co n c . o f Unk n ow n = X Co n c . o f Std. Thresh o l d dos e o f th e T est

32. 2. Matching and bracketing method A constant dose of the standard is bracketed by varying dose of test sample until an exact matching between the response of std & that of the sample is achieved. Initially, two responses of the standard are taken. Th e dose s ar e adj u ste d suc h tha t on e i s g i vi n g response of ap p roximately 20 % an d othe r 70 % o f th e maximum. The response of unknown which lies between two responses of standar d dos e i s taken.

33. The panel is repeated by increasing or decreasing the dose s of standar d til l a l l th r e e eq u a l respons e s ar e obtain e d. Th e dos e o f tes t sa m p l e i s kep t constant. At the end, a response of the double dose of the standard and tes t wh i c h matc h eac h othe r ar e t aken. These should give equal responses. Concentration of the test sample can be determined as follows: Dose o f t h e Standa r d Co n c . o f Unk n ow n = X Co n c . o f Std. D o s e o f th e T est

34. Example: Bioassay of Histamine on guinea pig ileum.

35. 3. Graphical method This method is based on the assumption of the dose-response relationship. Log-dose-response curve is plotted and the dose of standard producing the same response as produced by the test sample is d i rect l y read f r o m th e g r ap h . In simpler design, 5-6 responses of the graded doses of the standard are taken and then two equiactive responses of the test sampl e ar e taken . The height of contraction is measured and plotted against the log- dose.

36. The characteristic of log-dose response curve is that it is linear in th e midd l e (2 - 80%). Thus, the comparison should be done within this range only. In other words, the response of test sample must lie within this range.

37. Dose Log dose Response (height) (cm) % response 0.1 -1 0.9 25.71 0.1 -1 0.9 25.71 0.2 -0.69 1.5 42.85 0.4 -0.39 2 57.14 0.6 -0.22 2.8 80 0.8 -0.09 3.5 100 1 2.3 65.71 0.1 -1 0.7 20 0.3 -0.52 1.3 37.14 Observations and calculation For standard For test

38. Maximu m res p on s e (3.5 ) i s con s idere d 10 % res p on s e. For 0.9, % respo n s e = . 9 x 10 = 2 5.71 3.5 100 50 % R E S P O N S E Log dose

39. INTRODUCTION OF ELISA ▶ ELISA(Enzyme Linked ImmunoSorbent assay) is a widely used technique for detection of antigen (Ag) or antibody(Ab). ▶ The technique was developed in 1971 by Peter Perlmann and Eva Engvall at Stockholm University, Sweden. ▶ A technique to prepare something like immunosorbent to fix antibody or antigen to the surface of a container was published by Wide and Jerker Porath in 1966 Eva Engvall Peter Perlmann

40. PRINCIPLE Principle is based on the formation of Ag-Ab complex , which is detected by chromogenic detection using enzyme conjugated secondary antibody. The conjugated enzyme acts on a specific substrate called chromogenic substrate, and generates a coloured reaction product. This product is qualitatively or quantitatively read using an ELISA plate reader. ELIS A micr o plate reader

41. ELISA kits are commercially available, which can be conveniently used for laboratory purpose. Kit from REAGEN Kit from Forsight

42. TYPES OF ELISA Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA

43. 1.DIRECT ELISA ▶ I t i s use d i n th e detecti o n of antigen in the given biological sample. ▶ Microtiter wells are initially coated with antigen to be detected which is followed by an antibody linked to an enzyme conjugate. This follows the addition of substrate which produces colour detected using ELISA detector.

44. 2. INDIRECT ELISA ▶ I t i s use d for dete c t i on of an antibody in the given sample. ▶ Microtiter wells are initially coated with antigen specific for antibody to be detected, followed by the addition of sample. Enzyme conjugated Secondary Antibody is added followed by the substrate which forms a coloured reaction product.

45. 3.SANDWICH ELISA ▶ It is used for detecting an antigen in the given sample. ▶ Microtiter wells are initially coated with monoclonal antibodies(called capture antibody) raised against antigen to be detected, followed by addition of sample. ▶ Any trace of antigen is detected by adding primary antibody (a MAb),followed by enzyme conjugated secondary Ab and a chromogenic substrate; or by directly adding an enzyme conjugated primary Ab.

46. SANDWICH ELISA

47. 4.COMPETITIVE ELISA ▶ This variation of ELISA is used to quantitatively estimate the amount of antigen in the given sample. ▶ Ag and Ab are initially incubated so that they form Ag-Ab complex. This mixture is then added to microtiter wells coated with synthetic analogue of antigen to be detected, any free antibody binds to these antigens . ▶ This complex is estimated by enzyme conjugated secondary antibody by chromogenic detection .More the amount of antigen in the sample, lesser is the antibody available to bind to microtiter wells.

48. COMPETITIVE ELISA

49. APPLICATIONS ▶ Since ELISA can detect both antigen and antibody it is a useful tool for determining serum antibody concentrations . ▶ It has also found applications in the food industry in detecting potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs. ▶ The other uses of ELISA include: D etection of Mycobacterium antibodies in tuberculosis D etection of hepatitis B markers in serum D etection of enterotoxin of E. coli in feces D etection of HIV antibodies in blood samples

50. CONT...

51. SUMMARY Coat plate wit h Ag / Ab wa s h Add blocking buffer w a sh Add test samp le wa s h Add enzyme conjugated Antibody w a sh Add substrate Add stop solution Read absorbance at 450nm.

52. IMMUN O ASS A Y OF INSULIN Purpose and rationale : The first description of an immunoassay of endogenous plasma insulin in man has been given by Yalow and Berson(1959,1960) Insulin activity is important laboratory parameter in the clinical evaluation of several diseases such as diabetes mellitus, states of impaired glucose tolerance and insulin producing tumors, where the insulin secretion released from pancreas beta cells is altered

53. PROCEDURE Semisynthetic or biosynthetic human insulin is used as immunogen and as standard. Guinea pigs weigh- ing 350–450 g are injected subcutaneously with 0.4 ml of an emulsion of 5 mg human insulin dissolved in 1.0 ml 0.01 N HCl and 3.0 ml complete Freund’s adjuvant. For boostering, 0.2 ml of an identically prepared emulsion is injected in monthly intervals. Fourteen days after the third booster injection, the animals are slightly anesthetized and 8–10 ml blood are withdrawn by cardiac puncture. Boosting is continued at monthly intervals and the animals are bled 2 weeks following each booster injection.

54. CONTINUED ….. The percentage binding of insulin is determined for dilutions of antisera The steep- ness of the antisera dilution curve is a measure of the affinity of the antiserum and therefore the potential sensitivity of the radioimmunoassay The selected antisera dilutions are then run in an immunoassay using a full range of standards A reduction in the percent insulin bound to antibody from 50% (in the absence of un- labeled insulin) to 45% (in the presence of unlabeled insulin) (B/Bo = 0.9) is a reasonable measure of assay sensitivity

55. ASSAY PROCEDURE A buffer is prepared from a solution of 8.25 g boric acid and 2.70 g NaOH dissolved in 1 l water. After dissolving 5.0 g of purified bovine serum albumin, pH is adjusted with concentrated HCl to 8.0. In disposable plastic tubes, 10 × 75 mm, the follow- ing volumes are added: 100 μl serum or standard 900 μl buffer 100 μl 1 mU insulin in assay buffer 100 l Guinea pig anti-insulin antiserum diluted in assay buffer (at a concentration to bind 50% of the 125I-insulin in the absence of unlabeled hormone).

56. The mixture is incubated at 4 °C for 72 h. Then, the following solutions are added: 100 μl normal guinea pig serum diluted 1 : 400 in the assay buffer 100 μl rabbit anti-guinea pig globulin serum di- luted in assay buffer The mixture is again incubated at 4 °C for 72 h and then centrifuged at 4 °C and 2 000 g for 20 min. The supernatant is decanted and radioactivity counted in the precipitate for 5 min. CONTINUED…..

57. E V AL U A TION Counts in the non specific binding tubes are subtracted from counts in all other tubes. Micro units insulin in a logarithmic scale are plotted against the ratio bound and unbound on a logit scale. The range of bound to unbound between 0.4 to 0.9 is most suitable for determination of insulin concentration in plasma.

58. RIA OF DIGITALIS Digitalis Commonly known as Foxglove leaves , belonging to the family of Schrophulariaceae . It is also used for drug preparations that contain cardiac glycosides, like Digoxin & Digitoxin extracted from various parts of the plant. It is used to increase cardiac contractility and as an antiarrhythmic agent to control the heart rate, particularly in the irregular atrial fibrillation.

59. The assay is based on the use of 125 -iodine-labelled digoxin and of a gel equilibration technique for the separation of antibody- bound and free digoxin. Digoxin in serum samples competes with radio-labelled (125-1) digoxin derivative for binding sites on the antibody to digoxin. The unbound digoxin is then separated from bound form. It is then quantified by counting radioactivity & concentration of unlabelled digoxin in serum sample is calculated by comparison to digoxin standards. PRINCIPLE OF RIA FOR DIGITALIS

60. Materials Used For Assay Digoxin Standards:-From 0.5- 8.0 ng/ml Anti-serum and Tracer solutions Digoxin antibodies raised in rabbits by subcutaneous injections of digoxin-bovine serum albumin. Phosphate saline buffer D i goxin R a d io Immuno assay kit, w ith 3- - succ i n yl digoxigenin tyrosine 125-1.

61. The assay is carried out using 3-O-succinvl digoxigenin tyrosine 125- Ⅰ . Standard Curve: 1 ml of phosphate buffer solution + 0-50 µl of standard digoxin solution + 10 µl of 3-0-succinyl digoxigenin tyrosine 125- Ⅰ Add 10 µl of digoxin anti-serum, all contents are mixed well. Procedure For Assay

62. Procedure For Unknowns: To 50µl of patient's plasma, + 1 ml of phosphate buffered saline. To this add 10µ of labelled solution, & 10µl of digoxin anti-serum, all contents are mixed well All the tubes are allowed to stand and 0.5ml of charcoal solution is added to all tubes. The tubes are then centrifuged, G amma radioactivity.

63. Application of RIA: Blood banking Detection of presence of Hepatitis B Surface antigen (HBsAg) in donated blood. Diagnosis of allergies Detect inhalant allergens (antibody) Endocrinology Detect physiology of Endocrine functions. Pharmacology Detect of Drug Abuse or Drug poisoning. Study drug kinetics Oncology Det e ct Car c ino e m br y onic Antig e n . Others Narcotic drug detection Tracking of leukemia virus Research with neurotransmitter