HPLC and GC, Pharmacognosy and phyt.pptx

Pharmacognosy

1. Chromatography Chromatography word was derived from the two greek words chroma and graphy. Chroma means colour and graphy represent the meaning writing and recording . The person who invented the chromatography first was a Russian botanist Mikhail Tswett in 1901. Chromatographic techniques create a basis for analysis and separation of a wide range of physical methods used in complex mixtures.

2. In chromatography, it has two phases: stationary phase and a mobile phase in which the components are distributed. When components pass through the system at different rates they become separated in retention time ( Rt ), affinity , retention factor( Rf ) etc. Each component has a characteristic time of passage through the system, called a retention time . Chromatographic separation is achieved when the retention time of the analyte differs from that of other components in the sample. The difference in rate of travel results in the separation of the individual component . The principle of chromatography differs according to the stationary and mobile phase used.

3. Classification of chromatography Adsorption Chromatography Partition Chromatography: Ion Exchange Chromatography Molecular Exclusion Chromatography Affinity Chromatography Column Chromatography Paper Chromatography (PC) Thin Layer Chromatography (TLC) High-Performance Thin Layer Chromatography(HPTLC) High-Performance Liquid Chromatography(HPLC) Gas Chromatography (GC) Flash Column Chromatography

4. Adsorption Chromatography: Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes.

5. Partition Chromatography: This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid.

6. Column Chromatography In a column chromatography a vertical packed column as a stationery phase. The column may be packed with solid or liquid. If the packing occur with the solid the principle of separation is adsorption while if packing will be done by liquid then the principle of separation will be partition. Generally in the column chromatography the most separation is based on the adsorption principle. The extract which is to be analyzed should be dissolve in the mobile phase and add into the column.

7. The individual constituent in the extract will move with different rates which depend upon their affinities towards the stationery phase or adsorbent. The compounds which have more affinity towards stationery phase will be separated in the last while those have less affinity towards stationery phase will move fast and separated first. The stationary phase which is used to fill the column should have uniform spherical size with high mechanical stability, inert with solute or other components . Mobile phase have free flow, freely available, inexpensive and able to separate the wide variety of compounds

8. Example of weak adsorbent is sucrose, starch, inuline , talc while medium adsorbent is calcium carbonate, magnesium oxide, calcium hydroxide and magnesium carbonate and the example of strong adsorbent is activated alumina , Silica gel , activated charcoal, fuller’s earth etc. The mobile phase may be used in individual or can be used into the mixture depending upon their polarity and the type of constituent to be separated. The column should be made of neutral glass and not affected by acid, alkali, mobile phase or stationery phase. The length and diameter ratio may be 10:1 to 30:1. Sometimes it may be 100:1 ratio.

9. Packing of the Column: The lower portion of the column should be pack with glass wool or cotton wool to above which the adsorbent should be placed. After packing with adsorbent, a paper disc can be placed above the adsorbent layer. So that on introduction of sample or mobile phase will not disturb the adsorbent layer. There are generally two types of column packing techniques 1. Dry packing technique 2. Wet packing technique.

10. Detection of Components: Colored constituents can be detected visually and colored band moving in the column can be collected separately. Colourless compound can be detected by UV technique like UV 254nm UV 366nm, R efractive index detector(RID) etc.

11. Applications : Used to isolate polynuclear aromatics from a petroleum products or triglycerides from lipids. Isolation and purification of vitamins and hormones. Used for separation of Natural oils , Flavours extracts , plant pigments like carotenoids etc. Aflatoxins and other mycotoxins can also be separated by this technique.

12. Paper Chromatography (PC)

13. Paper Chromatography (PC) Paper chromatography is carried out by specially designed filter paper. The principle of separation may be here partition or adsorption. If the filter paper is impregnated with alumina or silica , the adsorption principle will be applied for separation whereas if moisture/water present in the pores of cellulose fibre it works as stationery phase and solvent as mobile phase then the principle of separation will be partition. In general paper chromatography refers to the partition principle.

14. Various types of filter paper are used for paper chromatography. It may be Whatmann paper of different grade, acid or base wash filter paper, paper modified with glycol, formamide, glass fibre type paper, hydrophobic paper (OH group can be acetylated) or paper can be impregnated with alumina, silica or ion exchange resin. The size of the paper should be suitable for the size of the chamber and apply the sample by using capillary or micro pipette.

15. The sample should be dissolved in the mobile phase and applied with low concentration with small zone. In the mobile phase pure solvent or mixture of solvent or buffer solution can be used. There may be Ascending , Descending , Circular , Two dimensional and ascending descending type different development techniques.

16. Ascending development technique is conventional technique in which the mobile phase moves against the gravity and spot the sample at bottom portion. I n descending development the mobile phase kept at the top and the solvent flow down the paper. Here the samples applied at the top and the development is fast due to gravity assisted solvent flow.

17. In circular or radiant development the sample applied at the centre of the paper and the mobile phase flown through a wick at the centre and spread uniformly in all direction. In two dimensional development techniques the samples is applied in one corner and develop the paper on one axis then dry the paper. After drying turn the paper on ninety degree angle and develop the paper on another axis. This technique is used for more complex sample

18. After development of the chromatogram the isolated compound can be visualized by detecting agents. Detecting agents can be two types: (a) Destructive type and (b) Non destructive type In destructive type the sample cannot be recovered or it will be destroyed due to chemical reaction of spraying reagent with sample. In non destructive method sample can be detected by UV chamber method, densitometric method or iodine chamber method.

19. Paper chromatography can be use for both qualitative and quantitative purpose. For qualitative purpose Rf value can be determined.

20. Applications : Used for separation of mixture having polar and non polar compounds. Used for separation of sugars , amino acids , lipids and inorganic compounds like salts. Used to determine biochemicals in urine. Used in forsenic field for crime scene investigation. The presence of metal ions like Ag+ , Fe3+ , Hg2+ can be analysed using paper chromatography.

21. Thin Layer Chromatography (TLC)

22. Thin Layer Chromatography (TLC) The thin layer chromatography is a widely used, fast technique for the qualitative analysis of a mixture of compounds. The stationary phase consists of a thin layer of adsorbent like silica gel , alumina, or cellulose on a flat carrier like a glass plate, a thick aluminium foil, or a plastic sheet. The principle of TLC is adsorption .

23. The extract of plant to be separated is spotted on the plate and it moves along with mobile phase. The phytoconstituent of extract have different affinity towards the stationary phase and mobile phase. Those constituents have more affinity towards mobile phase separate faster compare to those who have more affinity with coating substances . Silica gel G is one of the most common adsorbent uses for the coating of plate. The other adsorbent which are use in the stationary phase is alumina, cellulose, kiesselguhr , polyamide powder and others.

24. Most often the adsorbent are applied on the glass plate. The dimension of the glass plate may be vary from 20X20 cm, 20X10cm to 20X5 cm. E ven microscopic plates are also used to examine the progress the chemical reaction by TLC method. The glasses are coated with adsorbent with different techniques like: 1. Pouring, 2. Dipping, 3. Spraying, 4. Spreading techniques.

25. Sample can be applied by micro pipette or capillary tube. It should be spotted at least 2 cm above the base of or on such height that spotted area should not immersed in mobile phase. The area of the spotted sample should be minimum with sufficient concentration. Mobile phase and TLC plate should be kept in the development chamber. Chamber should be saturated with mobile phase otherwise edge effect may found in which the solvent front in the middle of plate move faster than the edge. The mobile phase selected on the basis of phytoconstituent which have to be separated and the nature of stationary phase. It may be single or the mixture of the solvent.

26. The polarity of the solvent adjusted such that it easily separated the phytoconstituents. The different development techniques used in the TLC are one dimensional, two dimensional, horizontal and multiple developmental techniques. In one dimensional or vertical technique, the mobile phase flows against the gravity due to the capillary action. Most separations are done by this technique in which one spot is applied at the centre of the plate and the plates are developed in one axis then dried it.

27. After development of the chromatogram the isolated compound can be visualized by detecting agents. For colourless compounds visualized under UV 254nm and UV 366nm. Detecting agents can be two types a) Destructive type b) Non destructive type. In destructive type the sample cannot be recovered or it will be destroy due to chemical reaction of spraying reagent with sample. While in non destructive method sample can be recovered. In non destructive method sample can be detected by UV chamber method, densitometric method or iodine chamber method. This chromatography can be use for both qualitative and quantitative purpose. For qualitative purpose Rf value can be determine.

28. High-Performance Thin Laye r Chromatography

29. High-Performance Thin Laye r Chromatography The high-performance thin layer chromatography is a sophisticated and automated form of TLC. It is useful in qualitative and quantitative analysis of natural products. The principle of separation is adsorption (same as that of TLC).

30. In HPTLC, the precoated plates are used and the particle size of stationary phase is less than 10 μ in diameter. There is a wide choice of stationary phases like silica gel(Silica gel G F254) for normal phase and C18, C8, etc., for reverse phase mode. HPTLC provides a higher efficiency than TLC because adsorbents used are small and uniform in size. V ery less amount of sample is spotted on the plate so the sample prepared should be highly concentrated. The size of the sample spot should not be more than 1 mm in diameter.

31. The samples are spotted by various techniques and commonly used method is by semiautomatic linomat V apparatus. New types of development chambers (twin troughchambers ) are used in HPTLC that require less amount of solvents for developing. The plate is placed vertically in development chambers containing solvent, and chromatogram can be developed from the sides. In HPTLC, UV/VIS/fluorescence scanner is used; therefore, it scans the entire chromatogram qualitatively and quantitatively. The scanner is an advanced type of densitometer.

32. Scanning and Documentation: After the development of spot the HPTLC plate are scanned at selected UV regions wavelength and the selected can be measured in the computer in the form of peak and can be compared with standard compound or other constituents. The obtained band can be converted into the peak. The height and area peak of the peak correspond to the concentration of isolated constituents. This document can be stored in the computer for the further references. HPTLC is used for the standardization of herbal extracts and other formulations. By using this technique, the analytical profiles of alkaloids, cardenolides, anthracene glycosides, flavonoids, lipids, steroidal compounds, etc., havebeen developed.

33. High-Performance Liquid Chromatography

34. column detector injector pump solvent Degasser

35. Introduction High-performance liquid chromatography (HPLC; formerly referred to as high-pressure liquid chromatography ) is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column. High-Performance Liquid Chromatography

36. This is a versatile natural product separation technique which works on high pressure (up to 4,000–5,000 psi) is applied to the system to move the mobile phase through the smaller particle sized (2–10 μ m) stationary phase bed.

37. The column is stainless steel to withstand the high pressure. It employs relatively narrow columns about 5 mm diameter for analytical work, operating at ambient temperature or up to about 100°C. Many stationary phases are available and the most widely used is silica based, silanol groups ( SiOH )

38. HPLC run under normal phase mode or reverse phase mode which depends upon the polarity of stationary phase and mobile phase. In normal phase mode the mobile phase is non polar and stationary phase is polar while in the reverse phase mode mobile phase is polar and stationary phase is non polar. The separation technique in HPLC may isocratic or gradient. In the isocratic separation technique the polarity of mobile phase is maintained same throughout the process. while in gradient separation the polarity of mobile phase can be gradually change by changing the ratio of the different solvent present in the mobile phase.

39. Principle The purification takes place in a separation column between a stationary and a mobile phase . The stationary phase is a granular material with very small porous particles in a separation column . The mobile phase on the other hand is a solvent or solvent mixture which is forced at high pressure through the separation column. Via a valve with a connected sample loop,i.e . a small tube or a capillary made of stainless steel. T he sample is injected into the mobile phase flow from the pump to the separation column using a syringe.

40. Subsequently the individual components of the sample separate through the column at different rates because they are retained to a varying degree by interactions with the stationary phase . After leaving the column the individual substances are detected by a suitable detector and passed on as a signal to the HPLC software on the computer. At the end of this operation a chromatogram in the HPLC software on the computer is obtained, which allows the identification and quantification of the different substances .

41. HPLC instrumentation Solvent Reservoir Degasser and mixing unit Pump Sample Injector Columns and column heater Detector Data Collection Devices

42. In the mobile phase there may be single solvent or may be more than one solvent. To mix properly more than one solvent there is a mixing unit in HPLC apparatus The solvent pushed into the column under very high pressure (1000 to 5000 psi) ,the particle size of the stationary phase is very small (5-10 μm ) so the resistance force by mobile phase is very high. Pneumatic pumps work under constant pressure with highly compressed air. Flow rate and back pressure is controlled by the check valve.

43. The sample can be introduced into the HPLC by manual method or auto injection method. It can be septum injection, stop flow (on line) injection or loop valve ( Rheodyne injectors). A very important part of the HPLC is the analytical column but before the analytical column one guard column is also provided. Column can be made of polyether ketone, stainless steel, polyethylene or sometime glass. Stainless steel can be most widely used column because it can withstand the high pressure compare to PEER column. The column length can vary from 5-25 cm with 2 to5 mm in diameter using adsorbent particle size 1 μm to 20 μm with uniform spherical porous material.

44. The response of the separated constituents can be recorded by the recorder. Recorder amplifies the response which is detected by the detector. The separated/eluted components are detected in the detectors like UV detectors , Photodiode array detector( PDA) , Flourimetric detector etc. It record the time at which the constituent are separated or retention time. Integrator measures the height and width of the peaks, peak area and percentage of the area.

45. HPLC can be use for the qualitative analysis by measuring the retention time of sample under standard condition but generally it is use for quantitative analysis by direct comparison method, calibration curve method and by internal standard method. Isolation and purification of biologically active natural products. Detection of biogenetic intermediates and enzymes involved. HPLC is used to know the identity , purity and content of the ingredients (drugs , raw materials and pharmaceutical products.

46. Isocratic elution

47. Gradient elution

48. Applications of HPLC: Cleaning validation In process control Impurity profiling Qualitative and Quantitative identification The discovery stage Bioanalytical studies Assay and content uniformity Dissolution studies Active pharmaceutical Ingredient study QC and R&D

49. Gas Chromatography

50. Gas Chromatography Gas chromatography is the most widely used chromatographic technique to analyze volatile compounds. Where those compounds are carried by an inert gas like nitrogen , helium or argon through a heated (50–350°C) stationary bed (silica supported with bonded polar or nonpolar phase). The choice of stationary phase is governed by the temperature at which the column is to operate and the nature of the material to be fractionated. It should be nonvolatile at the operating temperature and should not react with either the stationary and mobile phases or the solutes.

51. Another important part of GLC which effect the separation of the constituent is Column . They are made of either glass or stainless steel. The stainless steel column have long life and easy to handle but sometime it may react the constituent which are not in the case of glass. The column may be Analytical (length 1-1.5 mt, diameter 3-6 mm) or P reparative column (length upto 30 mt, outer diameter 6-9 mm).

52. Depending upon its nature it may be packed column, open tubular or capillary column and support coated open tubular column (SCOT). The volatile compound should be mixed with the carrier gas. The carrier gas may be hydrogen, helium, nitrogen. Hydrogen is a good option for carrier gas because of good thermal conductivity and low density but it reacts with unsaturated compounds. Helium is also good choice but it is expensive. Nitrogen is inexpensive but has reduced sensitivity. Gases are generally stored under high pressure.

53. The sample can be introduced into any form like solid, liquid or gaseous form. Solid samples are generally dissolved in appropriate solvent and then injected through septum. Liquid samples can be dispensed through either loop or septum devices. Though the sample should be converted into the vapour the pre-heaters are required in the GLC which convert the sample into vapour form and mix with the carrier gas.

54. Different type of detector like FID (flame ionization detector), AID (argon ionization detector), ECD (electron captured detector) are used. The most sensitive of them are ECD . Recorders record the response and amplify it. Recorder record the retention time, record base line and record all the peak. Heights, width, area of the individual peak, percentage of area are calculated by integrators.

55. Applications : Separation and identification of lipids , carbohydrates and proteins. Separation and identification of volatile components from plant sources. It helps to check rancidity in dairy products. Analysis of pesticides in plants , soil, water and foods