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Course content

Biomolecules Overview of the chemical and physical properties of biomolecules – proteins, nucleic acids, carbohydrates, lipids etc.

Mechanism of Separation

Physical forces and their effect on separation, band broadening, resolution, optimization parameters, trouble shooting

HPLC Overview Introduction, instrumentation, pumping systems - types, high pressure vs. low pressure system, gradient elution

Chromatography History of chromatography, basic concepts, classical methods, liquid chromatography on plane surfaces, Chromatographic packing materials- synthesis and design

Detectors Types, non destructive vs. destructive detection

Sample Preparation, quantitative analysis, qualitative analysis, Internal and external standards

Mobile phase Basic characteristics, selectivity, miscibility, buffers and use of additives

Stationary phase General properties, bonded vs. non-bonded phase

Commonly used separation techniques

Size exclusion, Ion exchange, Affinity, Reversed phase, Hydrophobic interaction

Biomolecules

Molecules produced by any living organism.

These molecules may occur as monomers, oligomers or polymers.

Primary metabolites are directly involved in normal growth, development, and reproduction.

Secondary metabolites are not directly involved in normal growth, development, and reproduction but usually have an important ecological function

Types of Biomolecules

A diverse range exists

Amino acids, Peptides, ProteinsCarbohydratesNucleic acids, DNA, RNA Lipids, Phospholipids, Glycolipids, Glycerolipids, SterolsVitaminsHormones, Neurotransmitters

Metabolites arising from degradation of biomolecules

Chromatography

History of chromatography, basic concepts, classical methods, liquid chromatography on plane surfaces, Chromatographic packing materials- synthesis and design

Separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium.

Why separate biomolecules?

• Even molecules with same molecular formula can differ in structure (shape) and show

– different chemical properties

– different biological functions

6 carbons

6 carbons

6 carbons

Form affects function• Structural differences imparts important functional

significance

– amino acid alanine

• L-alanine used in proteins

• but not D-alanine

– medicines

• L-version active

• but not D-version

– sometimes with tragic results…

stereoisomersstereoisomers

The word comes from Greek language

Graphien to write

Chroma Color

and

C h r o m a t o g r a p h y

It is a very powerful and versatile technique

It can separate a mixture into its individual components in a single step process and simultaneously provide an quantitative estimate of each constituent.

Chromatography

Samples may be gaseous, liquid or solid in nature and can range in complexity from a simple blend of two entantiomers to a multi component mixture containing widely differing chemical species.

Moses 2, 15 (23-25)

Removal of bitter taste from waters of mara by addition of specific wood

appr. 1850 Runge’s capillary work with coloured chemicals on paper

1870’s Ion exchange studies by Eichhorn and Boecker

1886 Use of natural and synthetic ion exchangers in sugar production patented

turn of 19th century

Adsorptive capacity of carbon for purification of beet juices

1903Twsett – chromatographic separation of plant pigments explained by adsorptive effects

1904 Wislicenus requests defined materials for adsorptive purposes

1930 Lederer, Kuhn – separation of carotin and zeaxanthin

1934 Standardised aluminumoxides according to Brockmann

1941James and Martin – gas liquid chromatography – trigger for development of chromatographic principles at analytical and preparative levels

since 1970’s Liquid chromatography plays an ever increasing role

1981 1st process scale HPLC system (Kiloprep)

1986 1st preparative chromatography symposium in Paris

1993 1st scaled down simulated moving bed (SMB) units for pharmaceutical applications

1996 1st example of large scale chiral purification process (UCB)

2000 1st 800 mm inner diameter SMB unit for contract purification (Aerojet)

2001 advanced SMB-applications (Multi-Component, VariCol, …)

Development of chromatography over one centuryDevelopment of chromatography over one century

ChromatographyChromatography

Basic conceptsBasic concepts Classical methodsClassical methods High performance liquid chromatography, High performance liquid chromatography, Techniques commonly used in separation of Techniques commonly used in separation of

macromolecules - Size exclusion, ion exchange, macromolecules - Size exclusion, ion exchange, affinity, reversed phaseaffinity, reversed phase

What is Chromatography?What is Chromatography?

Why one needs to make a chromatographic Why one needs to make a chromatographic experiment?experiment?

Physical separation method based on the Physical separation method based on the differential migration of analytes in a mobile phase differential migration of analytes in a mobile phase while moving along a stationary phase.while moving along a stationary phase.

Interested in looking at characteristics of a Interested in looking at characteristics of a particular compoundparticular compound

Principle of Separation

Modes of separation

Elution technique

Scale of separation

Type of analysis

Classification of chromatographyClassification of chromatography

Types of Chromatographic Types of Chromatographic experimentexperimentColumn ChromatographyColumn Chromatography – the stationary phase is held – the stationary phase is held

in a narrow tube through which the mobile phase is in a narrow tube through which the mobile phase is forced under pressure or by gravity.forced under pressure or by gravity.

Planar ChromatographyPlanar Chromatography – the stationary phase is – the stationary phase is supported on a flat plate or the interstices of a paper supported on a flat plate or the interstices of a paper and the mobile phase moves through the stationary and the mobile phase moves through the stationary phase by capillary action or by gravityphase by capillary action or by gravity..

What do we need for performing a column What do we need for performing a column chromatography experiment?chromatography experiment?

ColumnColumnStationary PhaseStationary PhaseMobile Phase Mobile Phase Detection systemDetection systemRecordingRecording

ColumnColumn

GlassGlassPlasticPlasticStainless SteelStainless Steel

Stationary PhaseStationary Phase

CompositionCompositionParticle sizeParticle sizePore sizePore sizeMorphologyMorphology

Stationary PhaseStationary Phase

Most common a) For small molecules - Silica, Alumina, Polyamide etc.b)For large molecules – Sephadex, Sepharose, Cellulose, etc.

Types of ChromatographyTypes of Chromatography: - : - chromatography can be chromatography can be classified based on the type of mobile phase, stationary classified based on the type of mobile phase, stationary phase and support materialphase and support material

Mechanisms of Separation:Mechanisms of Separation:

PartitioningPartitioning AdsorptionAdsorption ExclusionExclusion Ion ExchangeIon Exchange AffinityAffinity

Stationary phase: a layer or coating on the supporting medium that interacts with the analyte

Components

Mobile phase: a solvent that flows through the supporting medium

Supporting medium: a solid surface on which the stationary phase is bound or coated

Paper Chromatography

AscendingDescending

Thin Layer Chromatography – Continuous development

Column Chromatography (low pressure)Column AdsorptionColumn Partition

Gas Chomatography

High Performance Liquid Chromatography

Stationary Phases for chromatography

Relatively weak intermolecular attraction

High discriminating power in recognizing analytes according to their chemical and physical structure ( molecular sensor)

Rapid mass transfer and interaction kinetics

Characteristic features of analyte and stationary phase interactions

Applying molecular sieve type of adsorbents and using pressure swing and temperature variation (Sorbex technology)

Applying selective adsorbents and using elution gradient and displacement techniques

Gas phase separations

Liquid Phase separations

High resolution Fast analysis High mass and bio-recovery High reproducibility Long life time Adaptation to operational conditions (adequate

pressure – flow rate dependency, fast regeneration etc.)

Desired chromatographic properties of stationary phases, packings and columns

Cheap bulk adsorbents Large particle size analytical packing with

identical surface chemistry Tailor made adsorbents specially designed for

isolation purposes

Choices

Theory Practice Originality and Common sense.

Stationary phase

The general approach in stationary phase design involves a carefully balanced combination of

Much of the experience utilized in stationary phase design stems from knowledge accumulated in material and surface science. The key issue is to combine this experience in the most intelligent way.

Phase composition, distribution and transition Type, strength, density and distribution of functional

groups (ligands)

An optimization strategy needs to include both the physical and chemical structure parameters of the support and stationary phase

Particle shape and size Porosity, pore shape and size Surface area

Physical structure parameters

Chemical Structure parameters

Defined surface functionality Surface homogeneity Uniformity of pore size and particle size Variable and reproducible surface chemistry,

particle size and pore size

Primary goals in view of the application

Historical review of column packing development

Phase 1 1930-1960 The Classical era (use of refined technical adsorbents

Phase 2 1970-1980 The discovery of potential of stationary phase selectivity

Phase 3 1980 - Manufacture of tailored stationary phases and packings, modeling

Rigid polymeric organic packing Inorganic packing e.g. Oxides with improved pH

stability (pure silicas, alumina, zirconia) Composites: inorganic core, organic layer or coating

Developments in packing manufacture

Non-porous particles Entirely macroporous or mesoporous particles Particles with a distinct bimodal pore size

distribution (macro + mesoporous)

Novel concepts in particle texture & pore structure

Emulsification polymerization Suspension polymerization Swollen emulsion polymerization (Ugelstad process)

Modes of manufacture of polymer beads

Manufacture of spherical silicas

Dispersion of colloidal silica

Gelling in two phase system dryingSilica hydrogel Silica Xerogel

AgglutinationSilica + polymer burning

Dehydrated Silica

Silica Xerogel

Spray dryingSilica Xerogel

Basic processes

Silicate solution Colloidal Silica particles Aggregates of colloidal silica particles

Manufacture of rigid macro-porous organic supports

Microporous gels Macroporous gels

Cross-linked swelling porosity Pore size in the swollen state -0.5-2.0nm

Highly cross-linkedPermanent porosityPore size >>2 nm

microsphere

Particle morphology and texture

Light and electron microscopy

Texture

Shape of particles

How the particles built up? (agglomerates, aggregates, structures obtained by leaching and dissolution, conversion of liquid droplets into solid spheres)

Method of examination:

Particle size and size distribution

Magnitude of particle size dp (dp= 10,20,40,80 mm) Particle size distribution

How is the size distribution measured, calculated and presented?

Changes of the particle size as a result of pressure and flow

Polydisperse

Monodisperse

Particle size and size distribution effect

Peak profiles

Hydrodynamics and column back pressure

Column efficiency

Column stability

Spray-dried non-porous silica (pd=250nm)

Composite of non-porous silica on polyethylene beads

Spray-dried non-porous silica

Dense packings of non-porous silica

Why do we need particles?

SilicaRODTM

- columns = molithic silica

SEM pictures of SEM pictures of

monolithic SilicaRODmonolithic SilicaRODTMTM

structure structure

Macro-pores: 1.5 - 2 m Meso-pores: 12 nmTotal porosity: > 81 % (65 - 70 % for conventional columns)

SilicaRODSilicaRODTMTM / Conventional Silica / Conventional Silica

Comparison of material properties

SilicaRODTM Conventional silica

particle size - 5-40 µmMacropores 2 - 6 µm -Mesopores 12 nm 12 nmSpecific surface area 350 m²/g 350 m²/gSpecific pore volume 1 ml/g 1.1 ml/gPacking density 0.2 g/l 0.4 g/lModification all silica modifications possible

Starting Sol

Acid, H2OPolymerSi(OCH3)4

Phase Separationand Gelation

Aging andSolvent Exchange

Drying andHeat

Treatment

CladdingChemical Modification

Mesopore Macropore

Synthesis of monolithic silicaSynthesis of monolithic silica

Hydrodynamics (e.g. flow profile)

Particle morphology and texture effect

Packing technology

Operating column pressure range

Column life

Percolation threshold as a measure

Pore size, porosity and pore connectivity

Pore size distribution, (posd)

Porosity of particles

How large should the porosity be?

Pore connectivity

(Assessment by SEC , mercury porosity, nitrogen sorption)

Classification of pores according to the width and function in chromatographic processes

Micropores pd < 2nm High adsorption capacity, slow diffusion

Mesopores 2<pd<50nm

Generates surface areas which are adequate for the retention of low molecular weight analytes

Macropores pd> 50nm Provides sufficient accessibility and retention for high molecular weight analytes

The role of pore size in support design

Non-porous Totally porous (one pore)

Surface functionality

Deposition of a hydrophilic layer

HydrophilicHeterogenousbackbone

Bonding of a hydrophobic moiety

Bonding of a hydrophilic layer

Bonding of a hydrophilic moiety

Bonding of a hydrophobic layer

Choices in varying the surface polarity of silica

Multiple types of ligands Low vs. high ligand density Even vs. uneven ligand distribution Ligand mobility Specific vs. non-specific ligands Leaching of ligands Controlled access of ligands

Variables in tailoring the surface chemistry of packings

Brush structure

Surface homogeneity

Micropore

Pore size effects

Large pore Mesopore

Sandwich structure

RPC IEC PBPC

The multi-functionality of stationary phase is reflected by a bimodal retention pattern typically seen in RPC, IEC and PBPC

Starting Materials Sodium Silicate, Silicone tetrachloride alkoxysilanes

1. Hydrolysis and polycondensationSilica SolColloidal silica particies (5-100 nm) dispersed in the aqueous medium

2. polycondensation, aggregation and gelation (interparticle

bonding) Silica Hydrogel Mass coagulated gel occupying the entire sol volume

3. Further polycondensation, aging, dehydration and grinding

Silica Xerogel Hard porous silica particles (grains) with the desired particle size and porosity

Different steps and intermediates involved in the preparation of irregular or spherical silica particles by the conventional sol-gel process.

Silica production process

Na2SiO3

(water glass)+ H2SO4

Na2SiO3

(water glass)+ H2SO4

Hydrosol(oligosilicic acid,

orthosilicic acid)

Hydrosol(oligosilicic acid,

orthosilicic acid)

HydrogelHydrogel

Xerogel(raw silica powder,particle size 0.5 - 6

mm)

Xerogel(raw silica powder,particle size 0.5 - 6

mm)Ground silica gel(dp 5 - 500 µm)

Ground silica gel(dp 5 - 500 µm)

Classified

silica gels

Classified

silica gels

control oftemperature and

pH

control oftemperature and

pH

temperature control

temperature control

drying temperature control

drying temperature control

washing pH-control,

ion content

washing pH-control,

ion content

Silica Production Plant (1)

Automatically controlled, fully closed production plantAutomatically controlled, fully closed production plant

The biggest chromatographic silica gel plant in the worldThe biggest chromatographic silica gel plant in the world

Silica Production Plant (2)

Storage and packaging in closed systemsStorage and packaging in closed systems

Silica microbeads1960‘s/1970‘s

The introduction of beaded silica supports (silica microbeads) in the late

1960s and early 1970s followed the popularity of beaded organic polymer

supports in chromatography. Accordingly, different suspension systems

initially developed for the production of beaded organic polymer supports

are also the basis of most of the procedures described for the manufacture

of beaded silica.

polydisperse / monodisperseIler (1955), Stober (1968)

Unger et. al., (1985) „Monospher“

Suspension gelationSebastian and Halasz (1975)

Kromasil (1988?)

This procedure is basically similar to the sol-gel process, except

that "gelation" is effected while the silica sol is suspended in the

form of small droplets in an organic liquid (suspension medium).

Following the sol-gel conversion, the resulting "silica hydrogel

beads" are separated, washed, dried and calcined to obtain the

corresponding dry silica microbeads.

Suspension polycondensation(1973, Unger et. al., „old Lichrospher“)

A low-molecular-weight poly-ethoxysilane (PES) is first prepared

by partial hydrolysis of tetraethoxysilane.

The PES oligomer is then stirred in a water-methanol mixture to

form a droplet suspension.

A catalyst (e.g. ammonia) is added to effect polycondensation and

the formation of the corresponding silica micro-beads.

This procedure represents an interesting quasi-suspension system in

which a single liquid apparently serves both as a solvent (monomer

diluent) within the monomer droplets and as a suspension medium

in which the droplets are formed.

Spray drying.

Kiselev et. al., 1972

According to this procedure, small droplets of silica sol are

sprayed into an oven at 400'C. This results in the evaporation of

water and simultancous polycondensation within the droplets.

The semispherical particles obtained are then subjected to a

hydrothermal treatment for porosity adjustment.

MicroencapsulationKirkland(1974); Iller and Mc Queston (1974)

This method involves

a)Entrapment of silica sol particles within an organic polymer

matrix,

b)Sintering and burning of the organic polymer.

For example, formaldehyde and urea are added to a well

dispersed silica sol, followed by adjustment of the pH to effect

the simultaneous polycondensations of silica sol and the organic

monomers.

This is basically a "dispersion polycondensation" process, in

which a network of nanometer-sized silica particles are

encapsulated within a micronmeter-sized network of the

organic polycondensate. The resulting microcapsules are then

subjected to heat treatment , first at 500 °C to burn the organic

polymer, and then at 1000 °C to effect a slight sintering of the

final silica microbeads.

The preparation of monolithic silica packings follows the same chemistry as for particulate materials, even more, historically, preparation was as early as in the mid-1800.

Ebelman, M., Acad. Sci. 25, (1847) 854Graham, T., J. Chem. Soc. 17 (1864) 318

Mix 1Analytical HPLC: SiO2, 5µm

0,68

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Phthalate

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Phenylene

Separation of Polyphenols

Preparative SiO2, 15 µm