Supercritical Fluid Chromatography

Introduction, Definition, Principle, Instrumentation, Advantages, Disadvantages, Apllications

1. Dr. BASAVARAJAIAH S. M. Assistant Professor and Coordinator P.G. Department of Chemistry Vijaya College Bangalore-560 004 Supercritical Fluid Chromatography

2. Introduction Definition Principle Instrumentation Advantages Disadvantages Applications CONTENTS

3. 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) can be used on an analytical scale. It is a combination of High performance liquid chromatography (HPLC) and Gas chromatography (GC). The most significant difference from HPLC is the replacement of most of the liquid mobile phase with a dense compressed gas, almost always carbon dioxide (CO 2 ). INTRODUCTION

4. At high pressures such as greater than 80 bar, CO 2 acts as a solvent. It is used for the analysis & purification of low moderate molecular weight, thermally liable molecules. It can be used with the universal flame ionization detector. It is important to producing narrower peaks due to rapid diffusion. It is important for the chiral separations and analysis of high-molecular weight hydrocarbons. Supercritical fluids are suitable as a substitute for organic solvents in a range of industrial and laboratory processes .

5. DEFINITION Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase .

6. PRINCIPLE It is combination of GC and HPLC, the Principle is based on Adsorption and partition Chromatography. The principle based on Triple point and Critical point. The principle is based on Supercritical Fluid . However SFC typically utilizes carbon dioxide as the mobile phase; therefore the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a state in which liquid and gas properties converge. Supercritical fluid chromatography is sometimes called "convergence chromatography”.

7. IMPORTANT ERMINOLOGY Critical Temperature – The Constant Temperature. Critical Pressure – The Constant Pressure. SFC Temperature - For every substance there is a temperature above which it can no longer exist as a liquid no matter how much pressure is applied. SFC Pressure - For every substance there is a pressure above which it can no longer exist as a liquid no matter how high temperature is raised . Triple Point – The point in which state of material was changes at constant temperature and pressure.

8. SFC SOLVENTS Carbon Dioxide and water are the most commonly used supercritical fluids, being used for decaffeination and power generation , respectively. Many pressurized gases are actually supercritical fluids. For example, nitrogen has a critical point of 126.2K (- 147 °C) and 3.4 MPa (34 bar). Therefore, nitrogen ( or compressed air) in a gas cylinder above this pressure is actually a supercritical fluid. These are more often known as permanent gases. At room temperature, they are well above their critical temperature, and therefore behave as a gas, similar to CO 2 at 400K above. However , they cannot be liquified by pressure unless cooled below their critical temperature .

9. Properties of Supercritical Fluid There is no surface tension in a supercritical fluid, as there is no liquid/gas phase boundary. Solubility in a supercritical fluid tends to increase with density of the fluid (at constant temperature). Since density increases with pressure, solubility tends to increase with pressure. At constant density, solubility will increase with temperature.

10. Choice of SCFs solvent Good solubilizing and penetrating property Inert to the product Easy separation from the product Cheap Low CP because of economic reasons

11. Advantages of SF’s The solvating power of supercritical fluids is very similar to many conventional organic solvents and much higher than in gases. Diffusion coefficient and viscosity of supercritical fluids are about 5 to 50 times higher than in liquids. High density of SCF able to dissolve large nonvolatile molecules. E.g . CO 2 dissolves, n-alkane (n=5-22), Polycyclic aromatic hydrocarbons. Therefore CO 2 is used for extraction of caffeine and nicotine. Analyte can be easily recovered by allowing the solution to equilibrate at relatively low temp. Diffusivities of solute is higher . Viscosity is lower so very useful for compound which have no functional group, as detection method in HPLC fails.

12. Supercritical Fluid Chromatography INSTRUMENTATION Stationary phase Mobile phase Pumps Injectors Ovens Columns Detectors

13. STATIONARY PHASE Both packed and open tubular columns are used. Packed columns can provide more theoretical plates and handle large volume than open tubular columns. Because of low viscosity of super critical media. The column length is 10 to 20 m . and inside the diameter is 50 to 100 mm common in open tubular columns . For difficult separation column 60 m (or) large have been used. Packed column usually made up of stainless steel,10 to 25 cm. More than 100,000 plates have been achieved in plate column. Many of column coating used in LC have been applied to SFC as well. Typically their are polysiloxane chemically bounded wall of capillary tubing .

14. The mobile phase is composed primarily of super critical carbon dioxide, but since CO 2 on its own is too non-polar to effectively elute many analytes, co-solvents are added to modify the mobile phase polarity. Co-solvents are typically simple alcohols like methanol, ethanol or isopropyl alcohol. Other solvents such as acetonitrile , chloroform or ethyl acetate can be used as modifiers. Modify valves for analytes ,ethane, pentane, diethyl ether, ammonia. Pressure maintained is 72.9 atm, temperature is 35 ºc. MOBILE PHASE

15. Here mainly flow control is necessary so syringe pumps are used for capillary SFC for consistent pressure and for packed columns for easier blending of the mobile phase or introduction of modifier fluids reciprocating pumps are used . PUMPS In capillary SFC small sample should be quickly injected into the column and so pneumatically driven valves are used. For packed SFC a typical injection valve is commonly used. INJECTORS

16. OVENS Conventional GAS chromatography & liquid chromatography ovens are used . COLUMNS Two types of analytical columns are used in SFC i.e. packed and capillary. Packed columns contain small deactivated substances to which the stationary phase adheres. These are conventionally stainless steel. Capillary columns are open tubular columns made of fused silica which have small internal diameter.

17. Flame ionization detectors and flame photometry detector , liquid-phase detectors like refractive index detector, ultraviolet-visible spectrophotometric detectors and light scattering detectors have been employed for SFC. DETECTORS

18. ADVANTAGES SFC is emerging as a separation technique that is superior to both gas chromatography and liquid chromatography for analysis of thermal liable or non volatile compounds. Low viscosity. Lower operating temperature. High diffusion co efficient. High resolution at low temperature.

19. DISADVANTAGES SFC is pressure operating conditions. High-pressure vessels are expensive and bulky. Maintaining pressure in SFC is difficult. Supercritical fluids are highly compressible and their physical properties change with pressure. Cleaning will be time consuming.

20. APPLICATIONS SFC is used in industry primarily for separation of chiral molecules. SFC now commonly used for chiral separation and purification in the pharmaceutical industry . SFC technique has been applied to wide verity of materials , including natural products, drugs, food and polymers etc. In overall ranking of chromatographic techniques it can be judges that SFC falls somewhere between HPLC and GC. In field of pharmaceutical chemistry and bioanalytical applications SFC gained its applications.