SUPER CRITICAL FLUID CHROMATOGRAPHY

Advanced spectral analysis
specialized technique

1. SUPERCRITICAL FLUID CHROMATOGRAPHY Seminar Presentation PRESENTED BY: DARSHAN N U M. PHARM 2nd SEMESTER DEPARTMENT OF PHARMACEUTICAL CHEMISTRY PRESENTED TO: Dr. T. Y UNUS PASHA. HEAD OF THE DEPARTMENT DEPARTMENT OF PHARMACEUTICAL CHEMISTRY 1 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

2. CONTENT Introduction Principle Instrumentation Advantages Applications Previously asked Examination Questions References 2 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

3. HISTORY Use of Super Critical Fluid Chromatography (SFC) Was First Proposed In 1958 By J. Lovelock. First Reported Use Was In 1962 by klesper et.al., Who Separated Thermally Liable Porphyrins SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) 3

4. INTRODUCTION Supercritical fluid chromatography (SFC) is a column chromatographic technique in which a supercritical fluid is used as a mobile phase. A supercritical fluid is a gas or liquid brought to a temperature and a pressure above its critical point. In SFC, the mobile phase is delivered by a high-pressure pump. The sample is usually injected as a solution by means of a high-pressure injection valve. The column may either be a packed column, comparable to a high-performance liquid chromatographic (HPLC) column, or an open capillary column, comparable to a capillary gas chromatographic (GC) column, but with somewhat smaller internal diameters (50-100 μ m). 4 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

5. CONTINUED… Detection is performed either on-line, (i.e., UV-VIS) or after the expansion of the fluid [i.e., flame ionization detection (FID) or mass spectrometry (MS)]. The mobile phase is kept as a supercritical fluid by means of a restrictor until either on-line detection has been performed or just before the expansion into a gas phase detector. 5 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

6. SUPERCRITICAL FLUID SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) 6

7. SUPERCRITICAL FLUID 7 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

8. Phase diagram for Carbon dioxide SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) 8

9. PRINCIPLE It is combination of GC and HPLC, the Principle is based on Adsorption and partition Chromatography. The principle is based on Supercritical Fluid. SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) 9

10. INSTRUMENTATION Mobile phase Stationary Phase Pump Injection System Oven Column Detector 10 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

11. WATERS METHOD STATION II. SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) 11

12. SOFTWARE:THAR SFC SUPERCHROM SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) 12

13. INSTRUMENTATION 13 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

14. Block diagram of an instrument for SFC SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) 14

15. MOBILE PHASE There are a number of possible fluids which may be used in SFC as the mobile phase. Mostly CO 2 is used. The main disadvantage of carbon dioxide is its inability to elute very polar compounds. This can be overcome by adding a small portion of a second fluid called a Modifier fluid. (Alcohols, cyclic ethers, acetonitrile and chloroform) The addition of the modifier fluid improves the solvating ability of the supercritical fluid and sometimes enhances selectivity of the separation. 15 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

16. STATIONARY PHASE Silica/Alumina Useful for non-polar compounds. Lead to irreversible adsorption of some polar solute. 16 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

17. PUMPS Flow controlling is the vital function of pumping systems. In contrast to HPLC pumping systems pressure rather than flow control is necessary and pulseless operation is more important. In general type of high pressure pump used in super critical fluid is determined by column type. For packed columns for easier blending of mobile phase or introduction of modifier fluids reciprocating pumps are generally used. For capillary super critical fluid syringe pumps are most commonly employed. Reciprocating pumps allow easier mixing of mobile phase or introduction of modifier fluids. Syringe pumps provide consistent pressure for neat mobile phase. 17 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

18. OVEN A thermostatic column oven is required for precise temperature control of the mobile phase. Conventional GC or LC ovens are generally used 18 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

19. BACK PRESSURE REGULATOR (BPR) This is a device which is used to maintain desired pressure in column by pressure adjustable diaphragm or controlled nozzle, so that, same column outlet pressure is maintained irrespective of mobile phase pump flow rate. It keeps mobile phase super critical throughout separation. 19 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

20. INJECTORS Injection in SFC is usually achieved by switching of the content of a sample loop into the carrier fluid at the column entrance by means of a suitable valve. For packed column SFC, a conventional HPLC injection system is adequate, but for the capillary column SFC, the sample volume depends on column diameters and small sample volumes must be quickly injected into the column, therefore pneumatically driven valves are used. 20 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

21. COLUMN Once the sample is injected into the supercritical stream it is carried into the analytical column. The column contains a highly viscous liquid (called a stationary phase) into which the analytes can be temporarily adsorbed and then released based on their chemical nature. This temporary retention causes some analytes to remain longer in the column and it allows the separation of the mixture. 21 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

22. COLUMNS There are two types of analytical columns used in SFC, A). Packed columns contain small deactivated particles to which the stationary phases adheres. The columns are made of stainless steel. B). Capillary columns are open tubular columns of narrow internal diameter made of fused silica, with the stationary phase bonded to the wall of the column. 22 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

23. DETECTOR Different types of detectors are used in conjunction with SFC. Some of the commonly used detectors are as follows: Flame ionization Ultraviolet Fluorescence Mass spectrometer 23 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

24. ADVANTAGES Supercritical fluid chromatography has several main advantages over other conventional chromatographic techniques(GC and HPLC). Compared with HPLC, SFC provides rapid separations without the use of organic solvents. With the desire for environmentally conscious technology, the use of organic chemicals as used in HPLC could be reduced with the use of SFC. Because SFC generally uses carbon dioxide collected as a by- product of other chemical reactions or is collected directly from the atmosphere, it contributes no new chemicals to the environment. 24 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

25. ADVANTAGES SFC separations can be done faster than HPLC separations. because the diffusion of solutes in supercritical fluids is about ten times greater than that in liquids (and about three times less than in gases). This results in a decrease in resistance to mass transfer in the column and allows for fast high resolution separations. Compared with GC, capillary SFC can provide high resolution chromatography at much lower temperatures. This allows fast analysis of thermolabile compounds. 25 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

26. APPLICATIONS SFC finds use in industry primarily for separation of chiral molecules, and uses the same columns as standard HPLC systems. SFC is now commonly used for a chiral separations and purifications in the pharmaceutical industry. Fossil Fuels and Hydrocarbons Agrichemicals Drugs and Pharmaceuticals Polymers Explosives and Propellants Separations of Enantiomers 26 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

27. APPLICATIONS 27 Separation of enantiomers Separation of metoprolol by HPLC (a) and SFC (b) on a chiralcel OD stationary phase. In (a) the mobile phase was 20% 2 – propanol in Hexane with 1% diethyl amine; selectivity α = 2.67 and resolution R s = 4.8 in (b), CO 2 was used containing 20% methanol with 0.5% isopropyl amine; α = 2.77 and R s = 12.7 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

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29. PREVIOUSLY ASKED EXAMINATION QUESTIONS. Write a note on supercritical fluid chromatography. RGUHS-2018 Discuss the Phase diagram of Carbon dioxide. 29 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

30. REFERENCES Chromatographic Analysis of Pharmaceuticals; edited by John A. Adomovics Page no: 239 to 269. Super critical fluid chromatography “SKOOG” instrumental analysis P age no: 935 to 940. 30 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

31. 31 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) THANK YOU