AKSHITA SHARMA PPT VLPL ABCDEFGHIJK.pptx

GC

1. Gas-Chromatography

2. TABLE OF CONTENTS An Introduction to chromatography Gas Chromatography Types of Gas Chromatography

3. Invention of Chromatography Mikhail Tswett Russian Botanist (1872-1919) Mikhail Tswett invented chromatography in 1901 during his research on plant pigments. He used the technique to separate various plant pigments such as chlorophylls, xanthophylls and carotenoids.

4. Original Chromatography Experiment Later Start : A glass column is filled with powdered limestone (CaCO 3 ). End : A series of colored bands is seen to form, corresponding to the different pigments in the original plant extract. These bands were later determined to be chlorophylls, xanthophylls and carotenoids. An EtOH extract of leaf pigments is applied to the top of the column. EtOH is used to flush the pigments down the column.

5. Chromatography : (Greek = chroma “color” and graphein “writing” ) Tswett named this new technique chromatography based on the fact that it separated the components of a solution by color. Common Types of Chromatography Tswett’s technique is based on Liquid Chromatography. There are now several common chromatographic methods. These include: Paper Chromatography Thin Layer Chromatography (TLC) Liquid Chromatography (LC) High Pressure Liquid Chromatography (HPLC) Ion Chromatography Gas Chromatography (GC)

6. But I will draw you attention towards Gas Chomatography

7. What is Gas Chromatography? Gas chromatography is a term used to describe the group of analytical separation techniques used to analyze volatile substances in the gas phase. The mobile phase is a chemically inert gas that serves to carry the molecules of the analyte through the heated column . Gas chromatography is one of the sole forms of chromatography that does not utilize the mobile phase for interacting with the analyte . The stationary phase is either a solid adsorbant , termed gas-solid chromatography (GSC), or a liquid on an inert support, termed gas-liquid chromatography (GLC).

8. Types of Gas Chromatography? Basically three types of GC instruments are there: GC GC-AUTO LIQUID SAMPLER GC-HEAD SPACE GC-MASS SPECTROSCOPY

9. Instrumentation

10. Detector Unit Inlet unit Column Compartment Gas Supply (H 2 , Zero air & N 2 /He/ Ar ) Gas controllers controlling flow of the required gases Injection Port containing sampler and syrinze Liner Unit containing liner based on our choice like split/ splitless Ferrule nut connector for inlet side of the column Oven controlling Temperature of our oven programming Column of our choice based on required method of analysis Detector based on our requirement Waste unit is also attached along with detector Monitor is attached beside this to get required chromatogram

11. Types of Columns in GC Capillary Columns Closed pack Columns

12. Types of Capillary Columns Non-Polar Columns Polar Columns Mid-Polar Columns

13. Types of Detectors in GC Flame ionization detector (FID) A flame ionization detector works by heating the analyte using a hydrogen flame. A component within the analyte will then become ionized and lose an electron. That makes the flame more electrically conductive, triggering a measurable signal for the detector. Flame photometric detector (FPD) Similar to above, the flame photometric detector involves burning compounds in a flame. In this case, a photomultiplier tube is then used to detect spectral lines as specific elements emit light. Nitrogen Phosphorus Detector (NPD) A nitrogen phosphorus detector is specifically designed to selectively detect the two namesake elements based on the way they alter the function of metal bead. Atomic-emission detector (AED) With an atomic-emission detector, the sample enters a chamber after eluting from the column. It’s then energized to induce a plasma, which itself causes the sample to decompose. That creates an atomic-emission spectra from certain elements, which is then diffracted and detected. Mass spectrometer (MS) One of the most common detectors, a mass spectrometer is used to identify analytes based on their mass spectrum. It can also be used with liquid chromatography, as discussed in the article  ‘Accelerating ADC Development with Mass Spectrometry’ .

14. Types of Detectors in GC Non-destructive gas chromatography detectors On the other hand, you have non-destructive detectors, which measure a property of the eluent directly, making recovery simpler and more effective. They include… Thermal conductivity detector (TCD) Also known as a katharometer , a thermal conductivity detector measures changes in thermal conductivity as a signal to detect compounds within an analyte . Electron capture detector (ECD) Electron capture detectors detect molecules in gas by attaching electrons to analyte molecules and measuring a change in current within a detector chamber – which is proportional to analyte concentration. Photoionization detector (PID) With a photoionization detector, gases are measured instantaneously. Typically used for volatile organic compounds, the detector uses UV rays to ionize molecules, causing them to release an electron and form a positive ion. That’s turned into a parts-per-million meter reading when electrodes collect those ions. Olfactometric detector For this detection method, trained humans are used to smell gases and provide information about the presence of certain odours .  

15. Detector that I worked on FID

16. FID Detector Mechanism

17. Now I will let you know that what I have learnt & done during my training period What I have learnt….?

18. Learnt Things: Some basic concepts of calculations Dillution techniques Calibration of various instruments like pH meter, Balance Calibration & Kar -Fischer Some guidelines and chapters for pharmaceutical work Method development Method validation Some tricks & tips during analysis But my main focus was to learn a chromatographic technique so I have done most of my training in GC-lab.

19. ICH guidelines: I have worked on Q1, Q2, Q3, Q14 Guidelines that I came to know during my work USP chapter: I have studied USP chapter 621 and 1225.

20. There was a molecule named (S)-(+)-1,2-PROPANEDIOL. We had to develop a method for its GC purity. T here were not enough methods available in USP, EP, JP…… monographs.So we have to study this molecule very closely. My work on M ethod Development in GC

21. Continued….. Melting point -59 °C Boiling point 186-188 °C765 mm Hg(lit.) alpha 16 º (c=neat) Density 1.036 g/mL at 20 °C(lit.) refractive index n 20/D 1.432(lit.) Flash point 225 °F storage temp. Keep in dark place,Sealed in dry,Room Temperature solubility Miscible with acetone, chloroform, ethanol (95%), glycerin, and water; soluble at 1 in 6 parts of ether; not miscible with light mineral oil or fixed oils, but will dissolve some essential oils. (S)-(+)-1,2-Propanediol Properties

22. Continued….. Steps involved in method development: As we were doing work in GC we must knew the boiling point of the molecule. Its solubility in various solvents. We selected Methanol as diluent as it was clearly soluble at very good concentration level. This step is most important step. It involves selection of our stationary phase . It only depends upon the nature of compound. As our compound was of polar nature we had to select polar column for sure. So we selected DB-FFAP first. Its stationary phase contains Nitroterephthalic acid modified polyethylene glycol . Designed for the analysis of volatile fatty acids and phenols. So after column selection we have to program a method that involved oven programming, inlet temperature, detector temperature, split ratio, column flow rate and total run time for the injection. After making sure about the column we prepared a solution of 10,000 ppm of respective standard of (S)-(+)-1,2-PROPANEDIOL and then injected it to the system .

23. Continued….. What we got during our first time analysis of this molecule: