carlopez-khymos-2012 de selectividad columnas en...

Selectividad de Columnas

en HPLC

Humax Mayo 2012carlo

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Carlos Lopez - udea

Criterios Para comprar Columnas de LC?

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Variables Affecting HPLC Column Charactersitics

There are very many characteristics of a stationary phase which affect the quality of a separation, not least of which is the chemical nature of the silica (or polymer etc.) stationary phase support particles and the bonded phase ligand. Below we have listed just some of the parameters which need to be considered when choosing, for example, a C18 column.

One should note that all of the factors listed pertain to the same USP stationary phase classification (L1) and indicate the myriad of combinations which account for the marked differences in retention, selectivity and performance between C18 (L1) columns from different manufacturers:

Bonded phase

• Standard octadecylsilyl alkyl silane

• Polar Embedded C18 (with imide, carbamate etc. spacer) for enhanced retention / selectivity of polar analytes

• Mixture of C18 and shorter alkyl chains to give different selectivity / water wettability etc.

• Nature of the silane substituents (e.g di‐isobutyl silane) used as bulky substituents for pH stability at lower pH

• Carbon loading

• Carbon loading to silica surface area ratio (governs the ‘wetability’ of the phase and its ability to retain and separate polar analytes)

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Nature of the base silica

• Sol or sil‐gel particle

• Type I or Type II silica (governs the surface activity of the silica and the number of available ‘acidic / lone’ silanol groups which ultimately cause peak tailing if not effectively end‐capped)

• Silica metal ion content (effects peak tailing)

• Totally porous, polymeric (effects pH stability) or superficially porous (for increased efficiency) support particles

• Pure silica or organic / inorganic hybrid (to achieve wider pH stability)

• Spherical or irregular silica particle

• Particle size and particle size distribution (including the newer sub 2μm materials)

• Pore size

• Surface area

• Deactivation (end‐capping) post bonding – nature of the end‐capping reagent (volume, polar, non‐polar), end‐capping reaction conditions (determines the extent of end‐capping)

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Bonded phase

• Column Length

• Internal diameter

• Nature of the material used for column construction (typically stainless steel)

• Metal passivation technique

• Interior tubing surface polishing

• Nature of the frit and spreaders used in the column end fittings

• Packing quality (including method of packing, packing pressure, solvents used etc.)

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El ayer..y el hoy….

UHPLC

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Silicas Totalmente porosas vs silicas superficialmente porosas

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SEM Kinetex Core Shellca

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Columnas Core Shellca

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Core Shell – Difusion Eddyca

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Core Shell ‐ Difusiónca

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Columnas Chromolith

Starting Sol

Phase Separationand Gelation

Ageing Drying

Cladding

HPLC

Preparacion

Si(OAlk)4 Polymer

H+

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Carlos Lopez - udea

Caracteristicas de las columnas de silica monolitica

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Carlos Lopez - udea

25 mm10 mm

4.6mm

Columnas de silica Monolitica

3.0 mm

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Carlos Lopez - udea

Chromolith® Performance

p/u curves for Chromolith® and particulate HPLC columns

0

50

100

150

200

250

0 2 4 6 8Flow rate (mL/ min)

Col

umn

back

-pre

ssur

e (b

ar)

Column X: 5mColumn S: 5mColumn Z: 5m

Presión –Columnas de 5µm

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Columna de 1.4 metros…!!!!!!

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USP Designations – Agilent (1)

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Columnas LC – Agilent (2)

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Columnas LC ‐ Agilent (3)

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Columnas LC‐ Agilent (4)

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Columnas LC Aplicaciones Agilent (4)

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Columnas LC Agilent Aplicaciones ‐ Agilent (5)

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USP Designations – Waters (1)

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Columnas Merck

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Como comparar columnas?ca

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USP Columns

http://www.usp.org/USPNF/columns.html

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Chelating Tailing FactorCapacity Factor Amitryptyline

Bonding Density

Tailing Factor Amitryptyline

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Examples of separations of SRM 870 on commercial C18 columns ‐ highly deactivated Type B silica with polar embedded ligand (top), high silanol and metal ion activity phase (bottom).

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Shape selectivity (bonding density)

The term ‘shape selectivity’ is used to denote a chromatographic quality exhibited by certain stationary phases for which enhanced separations of geometric isomers may result based on their molecular structure rather than other physical or chemical properties of the solute. SRM 870 does not characterize shape selectivity, however this property can be assessed by use of other chromatographic test mixes, such as SRM 869a

[http://ts.nist.gov/MeasurementServices/ReferenceMaterials/archived_certificates/869a.pdf], Column Selectivity Test Mixture for Liquid Chromatography. This test uses benzo[a]pyrene (BaP), phenanthro[3,4‐c]phenanthrene(PhPh) and 1,2:3,4:5,6:7,8‐tetrabenzonaphthalene (TBN) as probes and the shape selectivity measurement is the selectivity between TBN and BaP. The structures and space filling models of these compounds are shown in Figure 23.

Testing columns for shape selectivity according to the USP classification model.carlo

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http://www.waters.com/waters/promotionDetail.htm?id=10048475&ev=10058108&locale=en_US

Versión on‐lineca

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Several attempts have been made to produce a “definitive” set of chemical probes and tests to best characterize the huge number of stationary phases available. This number was over 600 in the latest report we could find from 2003, and we speculate that well over 1000 different types are currently available. As yet a harmonized set of test probes and methodologies has not been identified.

An early attempt at producing a generic set of probes for testing HPLC column characteristics was made by Tanka and co‐workers and since then work by the USP Working Group on HPLC Columns, the Impurities Working Group of the PQRI Drug Substance Technical Committee in collaboration with Dr Lloyd Snyder and work carried out by Euerby and Petersson to expand the original probes designed by Tanaka have all undertaken to identify a definitive set of probes which will allow the various important physico‐chemical phase characteristics to be specified. Most of these groups have also combined their data with various chemometric approaches to produce quantitative databases based on principal component analysis (PCA) or tools to visualize the relative groupings of commercially available columns according to their key descriptors.

Spider diagram representing the various characteristics of the stationary phase.

HR ‐ hydrophobic retentionHS ‐ hydrophobic selectivitySS ‐ steric selectivityHBC ‐ hydrogen bonding capacityBA ‐ base activityC ‐ chelationIEX ‐ ion exchange capacity at pH2.6 and 7.6AI ‐ acid integration

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Desarrollo de métodos Automatizados

http://www.molnar-institute.com/carlo

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Desarrollo de métodos Automatizados LC y GC

http://www.acdlabs.com/products/com_iden/meth_dev/lc_sim/carlo

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ChromSword Auto Method Development Software

AgilentAutomated HPLC Method Development: ChemStation with ChromSword

http://www.chromsword.de/

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OSIRISHPLC dvelopment Method Software

http://www.kromatek.co.uk/productlist.asp?CatID=88carlo

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http://www.smatrix.com/carlo

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http://www.chem.agilent.com/en‐US/Events/en‐US/Pages/14736B.aspx

Agilent - eSeminars

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El ayer..y el hoy….

UHPLC

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Carlos Lopez - udea

Ventajas de tamaño de partícula pequeño

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http://gradworks.umi.com/3440078.pdf carlo

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