Chromatographic techniques for detection of antimicrobials in biological field
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This document discusses chromatographic techniques for detecting antimicrobials in biological fluids. It provides a brief history of chromatography, explaining that Mikhail Tswett first developed the technique in 1906 to separate plant pigments. The key principles of chromatography are explained, including that it works by allowing molecules in a mixture to distribute between a stationary and mobile phase. Various types of chromatography are classified based on the interaction with the stationary phase and physical state of the mobile phase. Sample preparation techniques for biological fluids like blood, urine and milk are also outlined, including removal of debris, cleanup, and concentration.
Chromatographic techniques for detection of antimicrobials in biological field
- 1. CHROMATOGRAPHIC TECHNIQUES FOR DETECTION OF ANTIMICROBIALS IN BIOLOGICAL FLUIDS SUBMITTED TO DR. SOUMEN CHOUDHARY ASSISTANT PROFESSOR DEPT OF PHARMACOLOGY AND TOXICOLOGY,DUVASU,MATHURA 1 SUBMITTED BY RAKTIM SAIKIA MVSC 1ST YEAR V-1818/17
- 2. HISTORY o Tswett used chromatography to separate plant pigments (1906) o colorful separation of plant pigments was done using a column of calcium carbonate(chalk) o the new technique was called chromatography because the result of the analysis was written in color.(Chroma means color and graphein means to write) Mikhail Tswett, Russian Botanist, 1872-1919 2
- 3. CHROMATOGRAPHY Technique used to separate and identify the components of a mixture PRINCIPLE: Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried along faster. 3
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- 11. Components: mobile phase: a solvent that flows through the supporting medium stationary phase: a layer or coating on the supporting medium that interacts with the analytes supporting medium: a solid surface on which the stationary phase is bound or coated 11
- 12. BASIC TERMS…. Adsorbtion: Interaction of solute molecules with the surface of the stationary phase Eluent: The mobile phase Elution: Motion of the mobile phase through the stationary phase Elution time: The time taken for a solute to pass through the system. A solute with a short elution time travels through the stationary phase rapidly, i.e. it elutes fast Stationary phase: The part of the chromatography system that is fixed in place 12
- 13. Resolution: Degree of separation of different solutes. In principle, resolution can be improved by using a longer stationary phase, finer stationary phase or slower elution. Rf value: distance travelled by solute distance travelled by solvent Rf = retardation factor. The Rf value has to be between 0 and 1, and it is typically reported to two decimal places. 13
- 14. CLASSIFICATION On the basis of interaction of solute to the stationary phase On the basis of chromatographic bed shape Techniques by physical state of mobile phase 14
- 15. On the basis of interaction of solute to the stationary phase Adsorption chromatography Partition chromatography Ion exchange chromatography Molecular exclusion chromatography 15
- 16. On the basis of chromatographic bed shape Column chromatography TLC Paper chromatography 16
- 17. Techniques by physical state of mobile phase Gas chromatography Liquid chromatography Affinity chromatography Supercritical fluid chromatography 17
- 18. • In chromatography, two immiscible phases are brought into contact; one is the stationary (nonmoving) phase and the other is the mobile (moving) phase. • A sample mixture is introduced into the mobile phase and interacts (partitions) with both mobile and stationary phase many times as it moves through the system carried by the mobile phase. • Differences in chemical and physical properties in the sample components influence the rate of migration of each individual component to different degrees, i.e., some components move faster than others. The result is a separation of the components. 18
- 19. • A column is an integral part of contemporary chromatography, and this may be packed or coated with stationary phases from a wide variety of different materials, solids or liquids. 19
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- 21. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 21
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- 24. Normal phase: “Unmodified” stationary phase where POLAR solutes interact strongly and run slowly Reverse phase: “Modified” stationary phase where POLAR solutes run fast i.e. reverse order 24
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- 31. UPLC 31
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- 33. SAMPLES WHOLE BLOOD PLASMA URINE FAECES SEMEN MILK MEAT EGGS…… 33
- 34. Although urine and blood specimens are both commonly tested for antibiotic activity, urine is generally accepted as being more sensitive than serum or plasma because chemical compounds are often concentrated in urine. 34
- 36. 1.REMOVAL OF DEBRIS A large component of crude cell or tissue lysate will consist of debris from insoluble biological material and particulates. Centrifugation at 45, 000 rpm for 30 minute will isolate the bulk of this. Further clarification— even the smallest of particulates can clog pores in any chromatography medium; they can also affect hardware such as HPLC flow lines and rotary injection valves--- achieved by passing the sample through a 0.45- or 0.2-µm filter to remove fine particulates. 36
- 37. 2.CLEANING UP THE SAMPLE… The removal of contaminants may be achieved in a single step via size-exclusion gel filtration, in which proteins are eluted before salts and other small contaminants because they cannot access the interior of the gel matrix. This technique is also known as buffer exchange, and there are several commercially available kits for researchers to use. They provide the advantage of sample elution into a buffer that is appropriate for the next steps in your chromatography workflow. 37
- 38. 3.CONCENTRATION OF SAMPLE Most chromatography experiments require only a small volume of sample; therefore, the samples must have a high protein concentration. For larger volumes, spin columns with appropriate spin concentrator can be used to concentrate the sample. 38