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الكلية كلية الصيدلة
القسم فرع العقاقير والنباتات الطبية
المرحلة 2
أستاذ المادة سعد علي احسان الاعرجي
5/26/2011 2:56:40 AM
Lecture ( 11 E )
NMR means absorption of radiofrequency radiation by substances held in a magnetic field .NMR important tool for illustration of molecular structure, specially the stereochemistry &configuration
(v)Mass spectrometry :
Is concerned with the electron ionization ,subsequent fragmentation of molecules ,determination of the mass to charge ratio(m/e) & relative abundances of ions which are produced. The application of mass spectrometry is in determination of molecular weight of compounds.
(vi) X-ray diffraction:
Electromagnetic radiations of wave-lengths ,commonly generated by passing high voltage current(about 10,000 volts)through a Coolidge tube; they are able to penetrate most substances to some extent &to affect a photographic plate
(vii)Radioimmuno assays (RIA):
The assay depends on the highly specific reaction of antibodies to certain antigens.
Chromatographic technique
is a physical method of separation in which the components to be separated are distributed between two phases ,one of which is stationary phase while the other the mobile phase moves in a definite direction .
Chromatography represents a group of methods for separating molecular mixtures depend on the differential affinities of the solute between two immiscible phases .
Stationary phase is fixed ,large surface area may be a porous or finely divided solid or liquid as a thin layer on an inert support material , while the mobile phase is a pure liquid or mixture of solution or gas or mixture of gases which moves through or over the surface.
Type of chromatography
1. Thin layer chromatography (TLC) This method based on adsorption chromatography ,the adsorbent silica gel G or C is coated to a thickness of 0.3 mm on clean TLC plate using spreader, the plates are activated at 1050C for 30min & used .The selection of Hmobile depend upon type of constituents to be analyzed .After the development of chromatogram (is the visual output of the chromatograph.) by ascending technique ,the resolved spots are revealed by spraying with suitable detecting agent such as concentrated sulphuric acid.
This technique is useful in analysis of alkaloid ,glycosides , isoprenoids ,lipid component sugars ….etc
Rf = distance travelled by component /solvent front
Rf may varying depend on purity of solvent ,nature of substance to be resolved ,composition of solvent ,% of impurity , adsorbent used ,polarity of solvent ,…..etc. This method is important analytical tool for qualitative &quantitative analysis of natural products.
2.High performance thin layer chromatography (HPTLC)
It s useful in qualitative & quantitive analysis of pharmaceuticals .
The principle is shorter time &better resolution ,the difference between TLC &HPTLC is only particle &pore size of the adsorbent.The plates are similar to TLC ,layers of HPTLC are in the form of pre coats .Silica gel of very fine particle size is widely used as adsorbent in HPTLC, this fine particle helps in greater resolution &sensitive ,3-6cm solvent front migration is sufficient to effect proper separation .HPTLC plates are 4-5um silica gel with an inert binder to form a 200um layer. About 7cm development distance is achieved in about 4minute.Sample preparation in HPTLC needs a high concentrated solution ,the size of the sample spot must not exceed 1mm in diameter.
3.Gas liquid chromatography.
Separation volatile substance by percolating a gas steam over a stationary phase .The bases of separation in GLC is the partition of the sample in &out of the film of liquid ,spreading over an inert solid ,it’s the most selective &versatile form of gas chromatograph. The choice of carrier gas shall determine the efficiency &inertness ;N2,He the most common carrier gas .The important application of GLC include examination of many volatile oil ,plant acids ,alkaloid of opium, tobacco ,conium & belladonna ,the resins of cannabis ,steroidal compound ,cardio active glycoside &aglycones , sugars&amino acid.
4.High performance liquid chromatography(HPLC).
This method takes place with a packed column .The packing material is inert material .A liquid mobile phase is used as the eluent.In HPLC ,the mobile phase is forced through the column under high pressure either autocratic elution or gradient elution .HPLC has become the most versatile ,safest, dependable ,fastest & sensitive chromatography technique for the quality control of drug components .The drugs like morphine ,papaverine , codeine ,emetine antibiotics ,steroids ,ergot alkaloids ,cardiac glycosides, sennosides ,capsaicin ,vitamins ,rhubarb….etc are analyzed by HPLC.
5.Column chromatography.
It is a liquid chromatography in which the mobile phase in the form of liquid passes over the stationary phase packed in a column .The column is either a glass or metallic .The adsorbent agent are used like starch ,calcium carbonate, magnesium , lime ,silica gel ,alumina ,charcoal &to optimum resolution ,various mobile phases are used either single or combination, petroleum ether cyclohexane chloroform ,acetone water , pyridine &organic acid CO2 used for extraction purposes.
6.Gel chromatography.
The separation occurs on the basis of adsorption , partition &on the effective size of solutes present in the solution for the separation purpose , the stationary phases used are cross- linked polymers which give on open network with large number of pores of fairly uniform size. During the flow of mobile phase through such stationary phase ,many large size molecule excluded & travel along with mobile phase , the low molecular size enter freely in to different pores . During the elution the largest molecules in the mobile phase elute first ,followed by molecules with decreasing order in size .
The types of gel used as stationary phase are either soft gel or semi-rigid or rigid gel, soft gel like agarose ,dextran &poly acryamide .The semi-rigid or rigid gel are alkylated dextran , controlled –porosity glass beads. Soft gel are used with aqueous mobile phases (the process called gel filtration ) ,the other gel are used non-aqueous mobile phase like acetone , chloroform ….etc (the process called as gel permeation ).
7.Affinity chromatography.
This technique used for separation of protein , m-RNA ,peptide ,enzymes antigens &antibodies. The adsorbent used is one of the biological substance called receptor a specific affinity for other substances i:e the two substance are biologically interacting . The adsorbent is attached to a porous stationary phase &placed in column , when mixture containing the other complement of the adsorbent is passed through the stationary phase i:e select separation during the elution ,the complementary part adsorbed is collected in pure form by dissociating the interacting pair with help of changing the ionic strength or PH of the column buffer.
Electrophoresis
For the electrophoretic separations a filter paper strip is impregnated with a solution of an electrolyte (usually buffer solution )& supported in the center , its two ends are dipped in to solution in which electrodes are immersed . A spot of the material to be fractionated is placed on the paper .According to the charge on the ions of solute mixture , the solutes will move towards either the anode or cathode ,paper can be replaced by thin layers of gel (gel electrophoresis) ,also there is capillary electrophoresis give efficiencies separation &wih detector systems such as laser-induced fluorescence.
Dr . Ghada Alhusaini
المادة المعروضة اعلاه هي مدخل الى المحاضرة المرفوعة بواسطة استاذ(ة) المادة . وقد تبدو لك غير متكاملة . حيث يضع استاذ المادة في بعض الاحيان فقط الجزء الاول من المحاضرة من اجل الاطلاع على ما ستقوم بتحميله لاحقا . في نظام التعليم الالكتروني نوفر هذه الخدمة لكي نبقيك على اطلاع حول محتوى الملف الذي ستقوم بتحميله .
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