Super Critical Fluid Chromatography

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Y.Hima Bindu Sri M.Pharm-I PharmaCEUTICAL ANALYSIS KGRL College of Pharmacy:

Y.Hima Bindu Sri M.Pharm-I PharmaCEUTICAL ANALYSIS KGRL College of Pharmacy SUPER CRITICAL FLUID CHROMATOGRAPHY

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Supercritical Fluid Chromatography 1. What is supercritical fluid 2. Supercritical fluid chromatography (SFC) 3. Theory of SFC 4. Instrumentation 5. Applications

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What is supercritical fluid Supercritical fluid is a state of matter that is intermediate between a gas and liquid in its properties.

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This state formed when a gas or liquid solvent is subjected to temperature and pressure condition exceeding a particular critical point. The temperature and pressure at which this pint Occurs are known as the Critical Temperature and Critical Pressure and are Characteristic of the solvent.

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Beyond this point, the solvent Will be neither a gas or liquid, but will possess properties of both phases.

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Supercritical fluid properties (density, viscosity, and refractive index) vary with T & P

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2. Supercritical fluid chromatography (SFC) SFC is a chromatographic technique in which the mobile phase is a supercritical fluid. SFC is of importance because it permits the separation and determination of a group of compounds that are not conveniently handled by either GC or LC .

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These compounds are either non-volatile or thermally labile so that the GC are in-applicable, and contain no functional groups that make impossible detection by spectroscopic or electrochemical techniques employed in LC.

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The use of a supercritical fluid mobile phase in chromatography was first proposed in 1958 by J. Lovelock. The first actual report use of this in a chromatographic system was in 1962 by Klesper et al, who used it to separate thermally-labile porphyrins .

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3. Theory of SFC Since supercritical fluids have properties between those of gases and liquid, their use as a mobile phase offers several advantages. SFC Mobile Phases:- Advantages of supercritical fluids over carrier gasses and liquid mobile phases are in its solubility properties, physical properties, and detector compatibility.

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SFs have higher densities than gas, so that mobile phase has a greater chance of interacting with the solute than that in GC (i.e., carrier gas). This makes the mobile phase important in determining the retention of solutes on the system and give more flexibility in optimizing the separation. For example, retention of solutes in SFC can be changed by using a different column (i.e. different stationary phases) as in GC, or by changing the mobile phase strength as in LC.

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Generally non-polar compounds with low to moderate critical properties - CO 2 , N 2 O, ethane, pentane Normal phase type separations - non-polar mp and low polarity sp (substrate + amino, diol , or cyano groups) Elution = function of molecular mass & polarity

SFC Separations:-:

SFC Separations:- SFC is a hybrid of gas and liquid chromatography that combines some of the best features of each As in HPLC, variation of the mobile phase composition affects separation. In SFC, mobile phase affinity for the analyte is a function of mobile phase density Density is controlled by pressure controlling system Highly polar samples are not easy to handle (high critical parameters & high reactivity)

SFC Advantages vs HPLC:-:

SFC Advantages vs HPLC:- Supercritical fluids have low viscosities - faster analysis (5 to 10 X faster) - less pressure drop across the column Column lengths from 10 to 20 m are used Can be used with a wide range of sensitive detectors Resolving power is ~5X that of HPLC

SFC Advantages vs GC:-:

SFC Advantages vs GC:- Can analyze non-volatile, polar, or adsorptive solutes without derivatization. Can analyze thermally labile compounds. Can analyze solutes of much higher molecular weight.

4.SFC Instrumentation:

4.SFC Instrumentation Gas Supply or Mobile Phase Pumps Injectors Column Oven SFC Columns Detector Restrictor

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1. Gas supply or Mobile Phase: Cost, interference with chromatographic detectors and physical properties like nontoxic nature, nonflammable, low critical values are considered while selecting a mobile phase. Liquid CO 2 Pump

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Carbon dioxide is the ideal to satisfy all the above properties. Safe to use nontoxic, nonflammable, noncorrosive, inert. Detector compatible & Wide  range The main disadvantage of it is very polar or ionic compounds are not able to be eluted. A modifier fluid is used to over come this problem.

Other SFC Solvents:

Other SFC Solvents Nitrous Oxid e - Similar in solvating and separations properties to CO 2 Alkanes - less safe and not as detector compatible than CO 2 - better solvent characteristics for non-polar solutes Halocarbons, xenon , etc. - specialty applications only More polar solvents for highly polar & high molecular weight compounds

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2. Pumps: 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. Syringe Pump

3.Injectors:

3.Injectors 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.

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4. Oven : Conventional GC or LC ovens are used

5.SFC Columns:

5.SFC Columns Open tubular (derived from GC) -Large # theoretical plates ~X500 -Easier to control pressure (low P drop) Packed (derived from HPLC) - Faster analysis - Higher flow rates - Higher sample capacity

Open Tubular Columns:

Open Tubular Columns Capillary columns are open tubular columns made of fused silica which have small internal diameter. Smaller than GC capillary columns, typically 50  m i.d., 10 to 20 m in length Mobile Phase must be more stable due to extreme conditions of supercritical fluids

Packed Columns:

Packed Columns Packed columns contain small deactivated substances to which the stationary phase adheres. These are conventionally stainless steel. Similar to HPLC columns (10, 5, or 3  m porous particles) Silica based chemically bonded phases Typically 10 cm long X 4.6 mm i.d

6.Detectors:

6.Detectors The choice of detectors will depend upon the mobile phase composition, column type, flow rate and ability to withstand the high pressures of SFC.

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Flame ionization detectors and FPD. liquid-phase detectors like RID, ultraviolet-visible spectrophotometric detectors and light scattering detectors have been employed for SFC.

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Photo Iodide Array Detector Fluorescence Detector

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7) Back-Pressure Device or Restrictor: It is used to maintain the desired pressure in the column by a pressure-adjustable diaphragm or controlled nozzle so that the same column-outlet pressure is maintained irrespective of the mobile phase pump flow rate. It keeps the mobile phase supercritical throughout the separation and often must be heated to prevent clogging.

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The pressure restrictor is placed either after the detector or at the end of the column. A typical restrictor for a 50 or 100 µm open tubular column consist of a 2-10 cm length of 5-19 capillary tubing attached to the column. Alternately the restriction may be integral part of the column formed by drawing down the end of the column in the flame.

SFC and Retention:

SFC and Retention Retention dependent on temperature, pressure, mobile phase density, and composition of the stationary and mobile phase. Complex interactions and not easily predictable. For supercritical fluids - solvating properties similar to liquids - viscosity closer to gases Solvating power  density

Temperature/Pressure Effects:

Temperature/Pressure Effects At lower P, > T, < solubility At higher P, > T, > solubility -> T, P v of solute > solute solubility -< fluid density < solubilizing power > T, < solvent  >P, > solvent 

Solvent Programming:

Solvent Programming Programming is very useful in controlling solvent strength. Variations in P (density), T, and mobile phase composition. Density programming is most widely used -> density, > solubility, < retention - Combined T & P programming to control  and thereby solubility and diffusion

Solvent Modifiers:

Solvent Modifiers Add organic modifiers to > solvent strength - methanol - isopropanol - dichloromethane - THF - acetonitrile

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Applications Natural Products To determine the origin of oil and improved possibilities of determination of relations between oil constituents and physical as well as biochemical properties of oil.

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Separation of bile salts 25 and common free bile acids like ursodeoxycholic acid and chenodeoxycholic acid in pharmaceutical preparations has been reported using phenylbonded silica column and SFC-CO 2 modified with methanol . Capillary-SFC has been used for analysis of panaxadiol / panaxatriol in ginseng and its preparations 30 , vegetable carotenoids 31 and pyrrolizidine alkaliods 32 .

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Pesticides Supercritical fluid extraction and chromatography has been used for the analysis of pesticide residues in canned foods, fruits and vegetables wherein pyrethroids, herbicides, fungicides and carbamates have been tested.

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Surfactants Separation of the oligomers in a sample of the nonionic surfactant Triton X100 has been reported where the detection was by measuring the total ion current produced by the chemical ionization mass spectrometer 34 .

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Lipids SFC has also been applied to analyze phospholipids after conversion to diacylglycerol derivatives , fatty acid methyl esters , biosynthetic polyunsaturated fatty acids (PUFA), nonsaponifiable lipids, cholesterol and its esters in human serum, food samples 41 , mono-, di- and triglycerides in pharmaceutical excipients 42 has been carried out by SFC successfully.

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Polymers SFC provides elution of high molecular weight compounds, polymers and large biological molecules from a column at a reasonably low temperature. Separation of the series of dimethyl polysiloxane oligomers and polycyclics aromatic hydrocarbon extracted from carbon black using fluorescence detection.

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Drugs Modern drug substances are commonly nonvolatile and thermally or chemically labile therefore analysis by HPLC is common over GC. In SFC the conditions are mild and no volatilization is required so it is possible to handle such drug substances by SFC.

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Separation of various categories of drugs like antidepressants , phenothiazine antipscychotics , beta blockers , felodipine , a new dihydropyridine drug-clevidipine, methylated betacyclodextrins 57 , vasodialators like isosorbide mononitrate, isosorbide dinitrate, cyclandelate, nimodipine, amlodipine, pentifylline,pentoxifylline, lovastatin ,pyrethrins , impurities have been carried out by SFC. Organometallics Chiral compounds etc.,

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SFC-MS in Pharmaceuticals In pharmaceutical industry, analyte concentrations in the picogram -per-milliliter or lower range are a commonplace. In order to detect the realistic concentration levels, a detector with highest sensitivity, broadest selectivity and best resolution must be used. Currently, the detector that fits all of these criteria is the mass spectrometer .

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