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Separating, purifying or extracting molecules and particles can be achieve though exposing molecules or particles dissolved in a solvent called ”the mobile phase” to a planar or spherical solid or gel called “the stationary phase” at a wide spectrum of pressures and flow rates.
If the aim is to extract say large proteins from a bio-broth no pressure should be applied. On the other side if the aim ist to separate at high speed small molecule we require Ultra High Pressure Chromatography that operates at very high pressures of up to 1200 bars. As competet chromatographers we need to choos those methods and processes that solve our problem effectively and efficiently. This in turn require the knowledge of column and process technology but also how stationary phases sepate molecules.
IOn this page we look at the historical evolution of stationary phase technology . In sub-pages we discuss different types of stationary phases that are being used in Chromatography. The sub pages are headed as follows:
- Phases, the matrix chemistry and physical construction and properties (Under construction)
- The Inter-phase between matrix and mobile phase (Under construction)
- The Interaction mechanism between analytes and inter-phase (Under construction)
- Reverse Phases (Under construction)
- Polar bonded Phases (Under construction)
- Phenyl bonded Phases (Under construction)
- Silica , Silica hydride (Under construction)
- Fluorinated phases (Under construction)
- High Aqueous Phase (Under construction)
- Mixed Mode / Switch Mode Phases (Under construction)
- Wide pore Phases (Under construction)
For the sake of concentration we dicuss only spherical and not planar stationary phases.
The following parameters influence the quality of a spherical stationary phase
- Matrix Chemistry
- Matrix Surface Chemistry
- Matrix Shape
- Matrix Construction features including mechanical stability
- Ligand Chemistry or second phase chemistry in a binary system
Historical evolution of chromatography.
The first application of chromatography was that of the early dye chemists, who tested their dye mixtures by dipping strings or pieces of cloth or filter paper into a dye vat. The dye solution migrated up the inserted material by capillary action, and the dye components produced bands of different colour. The russian botanist Mikhail Tsved was the first to describe and publish this process between 1903 and 1905. Chromatography methods did not change until about 1930 when the search for new analytical and purification technologies started. Between 1938 and 1941 the scientist Martin and Synge packt silica gel into a glas column and to run organic solvents through it to separate amino acids and some organic chemicals and dyestufs. Later on they used alumina particles. During the fifties the first polymerc particles were developed as ion exchanger. The classical LC packings comprised of 40 um or larger particles of totally porous material Thease materials were easily drypacked into columns. Eary sixties one started to decrease size of particles and achieve d better separation results. Mid sixties the so called “pellicular” materials were developed. They comprised of a impervious glas bead that was coated and sintetred with a layer of porous silica. Mid sixties some groups started to develop piston pumps that provided a high pressure. During the late sixties early seventies new finer porous irregular silica particles were developed. At Pittcon 1970 in his paper the late Prof Csaba Horváth coined the term HPLC indicating the fact that high pressure was used to generate the flow required for liquid chromatography in packed columns. In the beginning, pumps only had a pressure capability of 35 bars. The early 1970s saw a tremendous leap in technology development and new HPLC instruments with operating pressures of up to 400 bar of pressure, with reciprocal pistons, incorporated improved injectors, detectors, and steel columns. HPLC really began to take hold in the mid-to late-1970s. At the same time there were manufacture Chemie Uetikon and W.R. Grace and later Merck & Co that manufactured silica gel particlesbased on “Water Glas” that was before used as egg presvative and non-flamable papers and curtain material. At the same time in Universities and in Du Pont researchers were creating new forms of silica gel based chromatography metrials.
Divorced from the silica gel based chromatography development alredy 1940 succeeded the US american chemist Eugene G. Rochow at General Electric a direct synthesis of methylclhorosilane to trigger of the growth of the Silicone industry. During the following 20 years a number of other chemical companies including Bayer, Wacker, Dow Corning and Shin Etsu saw great growth potential in silicone chemistry and to develope new products based on silicone chemistry. Many Universities developed new Silane and Siloxane based monomers. Already in the early sixties started various scientis in USA to develop and to IPR protect porous Polystyrene and Methyl methacrylate polymer bsed particles. 1971 within Bayer Leverkusen a group of chemist under the leaderschip of Dr. Dieter Dieterich fused silica gel with silanes and other monomers to create “inorganic - organic porous plastics”
that required slury packung into steel columns. The commercial HPLC column with 10 um particle was born. Soon the particle became more spherical and ideal to separate small molecules
- Matrix Chemistry
- Silica Gel, Silica hydride, Glas, Titania, Zirconia, PS, PMMA, PS, Agarose, Cellulose etc
- Inertness (little amount of charges, alkaline stability)
- Surface and ligand chemistry
- wanted and unwanted reactive groups,
- Matrix Shape
- Beads vs. powders, monodisperse vs. polydisperse, continuous beds, planr
- Matrix Construction features
- Particle size: nm, um, mm (small diameter for fast mass-transfers)
- Porosity: non-porous, meso-porous, macroporous, flow through beads,
- Pore volume and pore shapes
- Mechanical stability particularly important in uHPLC and SFC
- In various environments (in aromatic solvents,aqueous etc)
- Stand up pressure through the columns
Every stationary phase comprises of a matrix (as indicated with the blue layer) that is either planar or spherically shaped and made of either silica, zirconia, titania or synthesised from a polymers e.g. DVB crosslinked Polystyrene.
The matrix maybe porous or non-porous. The geometry and size of pores depends on production method and varies from brand to brand. Silica is a low cost material that is covered on its surface with three types of reactive silanol groups. Stationary phase manufacturer react to the types of reactive groups ligands of different chemistry and molecular weight. The most common one is the use of chlorodimethylalkyl chain with 18 carbon atoms that form a stale Si-O-Si - Bond. For steric reasons only a fraction of the free silanol groups on the surface (typically about 40 to 50 % resulting in a loading of about 2 - 4 umol/m2). This leaves the remaining silanol groups accessible to small molecules including acids and bases that can diffuse between the alkyl chains and ionise the open silanol groups or to break the silanol-ether bond. This leads to peak tailing and to ligand loss. To minimise silanol interaction Stationary phase manufacturer have developed a number of strategies such as
- 1. Treating the reactive silanol groups with small silanes to fill the gaps between the alkyl chains (This process is called endcapping)
- 2. To reduce and convert all the surface silanol into silica-hydride. Silica hydride forms very stable Si-C ligand bonds
- 3. To coat the particle surface with an thin layer of a silane based polymers leading to hybrid stationary phases.
Silica hydride and Silica hybride based stationary phases have very interesting properties and open up many new possibilities for creative chromatographers.
In the mobile phase we may have either small Molecules ( MW up to 2000) or large Molecules (2 kDa to 60 kDa ) or nanoparticles (MW > 60 kDa). These molecules maybe aromatic or aliphatic, polar or apolar, ionic or non ionic. In turn with the chosen ligand chemistry the surface can be made polar or apolar, aliphatic or aromatic and ionic or non ionic to controll the surface intereaction process with the target molecules. The better we understand this ligand target molecule interaction the more effective and efficient we can design the chromatography process.
Separation, purification and extraction is also about diffusion of molecule into the pores of the stationary phase.
In this section we discuss surface chemistry, physical properties of stationary phases and possible interactions with target molecules as follows:
AQ Phases click here
Reverse Phases (Under Construction)
Wide Pore Phases (Under Construction)
Phenyl Bonded Phases (Under Construction)
Polar Bonded Phases (Under Construction)
HILIC Phases (Under Construction)
Silica and Silica hydride (Under Construction)
Zirconia and Titania Phases (Under Construction)
Polymeric Phases (Under Construction)
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