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Polymer HPLC
Thinking Outside the Silica Framework: Alternate Supports for HPLC and When to Use Them
Hamilton Company – The Syringe and Liquid Handling Company
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Early 1950'sClark Hamilton begins his career at UC Berkeley in the radiation lab
1953Hamilton Company is founded
1955the first 100 µL syringe 710 LTN is produced.
Early 1960'sCommercial Gas Chromatograph production increased by companies.
1962Hamilton introduces the MICROLITER syringe at Pittcon
1962A few of Hamilton's most loyal customers F&M Scientific and Wilkens Aerograph (later became Agilent and Varian).
1979‐1980Polymer HPLC columns (PRP) are introduced.
Is it all just about syringes?
Hamilton Company's Years in Chromatography
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Hamilton Company’s Polymer HPLC Supports
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Pioneer in Polymer HPLC Columns
Hamilton Company has been a pioneer in polymer HPLC column research and manufacturing for more than 30 years.
Unique, polymeric, HPLC columns and applications are a Hamilton specialty. We have developed polymeric HPLC packings for:
• Reversed Phase
• Anion Exchange
• Cation Exchange
• Ion Exclusion
Packings and applications are available for many areas, including:
• Pharmaceuticals
• Herbicides
• Carbohydrates
• Proteins
• And many more
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Silica Support Properties
• Porous silica particles – most common substrate material used for HPLC column packings.
• Can withstand high pressures
• Compatible with most organic and aqueous mobile‐phase solvents
• Come in a wide range of bonded phases
• Silica‐based columns are often used for separations of low molecular weight analytes using mobile phase solvents and samples with a pH range of 2 to 7.5.
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Silica End‐Capping
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• In reversed phase LC, stationary phases based on modified silica are most frequently used
• As this modification is seldom complete, the residual silanol groups may affect the separation
• Surface silanols can interact strongly with electron‐rich atoms in groups such as –C≡N, =NO2, ‐CH2OH.
• A reversed‐phase HPLC column that is end‐capped has gone through a secondary bonding step to cover un‐reacted silanols on the silica surface.
• End‐capping is accomplished with hydrocarbyl silanes having small alkyl (usually methyl) groups
• End‐capping or silylating free silanol groups can minimize interaction for some compounds.
• Even after end‐capping, silica based adsorbents contain almost 50% of their original silanols, but they are mainly not accessible for analytes.
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Silica Structure
Silica structure with bonded C18 chain
and exposed silanol groups
O
ClO
ClSiOSi SiO
SiO
O
SiOHSiHO
Cl
Cl
C18 chain
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Limitations of Silica Substrates
• NaOH treatments that render pH > 10 bring along an intrinsic risk of hydrolyzing siloxane bonds in the silica matrix, which are the backbone of the porous structure.
• Continuous hydrolysis leads to deteriorating column performance
• Species used for surface modification (silanes) will elute from the column and are likely to contaminate product fractions in the case of preparative separations.
• Such leachables from the stationary phase are devastating for preparative chromatography and are gaining more and more attention from e.g. FDA when approving drug manufacturing processes.
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• Silica is normally used as the packing material for HPLC columns because:• It is strong, allowing it to be used at high backpressures, and packed at even higher pressures to give a stable column with sharp peaks.
• There are limitations to silicas:• It dissolves in water, especially at elevated temperature at higher pH, and in higher buffer concentrations.
• Other problems are that the bonded phase can easily be stripped off by pH less than 2 because the Si‐O‐Si bond is hydrolyzed, and the residual silanol sites (SiOH groups) on the surface can cause peak tailing.
Limitations of Silica Substrates cont.
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Introduction to Polymer Supports
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• Various approaches are available to minimize the impact of silica limitations
• The most popular solution is to use a column with a polymer‐based packing material
• For many, this is perceived as an expensive and unknown territory
• However since the columns work comparable to silicas and can last for years, it is actually an easy and cost effective way to solve a lot of problems at once.
• Polymer columns come in a steel or PEEK tube, just like silica‐based columns, but the particles inside are a rigid polymer matrix rather than silica.
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Polymer Properties
• Styrene (vinyl benzene) readily forms a polymer because the vinyl groups link together to form a chain.
• Cross‐linking within the styrene groups occurs with divinyl benzene, which has a second vinyl group (meta or para to the first one)
• Cross‐linking forms a much stronger and more rigid polymer.
• For some industrial applications, very low percentages of DVB are added, but for HPLC much more DVB is used to give a high density of cross‐links ‐one of the most robust polymer supports available for HPLC
• This reaction is precisely controlled, allowing the formation of small spherical particles with a very narrow particle size distribution (within +/‐1 µm).
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What is PS‐DVB?
styrene divinylbenzene
Poly(styrene‐divinylbenzene) Simplification of PS‐DVB
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Polymers in Chromatography
• For reversed phased applications, poly(styrene‐divinylbenzene) columns are used un‐derivatized.
• Columns packed with PS‐DVB:
• Can handle pressures over 5000psi
• Have a usable pH range of 1‐13 (allows the separation of biological samples in
their natural state)
• Polymer columns can be cleaned with 0.1 M sodium hydroxide to remove
strongly retained material.
• Swelling in organic solvents such as THF or chloroform is negligible because of the
cross‐linking
• Can be operated at much higher temperatures as compared to silica based
materials
• Excellent sample recoveries due to the lack of acidic silanol groups
• PS‐DVB resins are similar in retention characteristics to a C18 but do have a
slightly different selectivity in some cases.
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PS‐DVB Resin (under the microscope)
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Reversed Phase
• In reversed phase chromatography, the stationary bed is very nonpolar in nature (e.g., alkane chain, C8 or C18, PS‐DVB), and the mobile phase is polar (such as water).
• The stationary bed is nonpolar (hydrophobic) in nature
• Mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile.
• The more nonpolar the analyte is, the longer it will be retained.
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Reversed Phase Offerings
PS‐DVB PRP‐1 ‐ 100 Å, 5 µm – 75 µm, mw < 3,0005,000 psi PRP‐3 ‐ 300 Å, 10 µm and 12‐20 µm, mw > 10,0005‐85° C PRP‐Infinity ‐ non‐porous, 4 µm, mw > 10,000pH 1‐13
PS‐DVB with bonded C18 PRP‐h1 ‐ 100 Å, 5 µm and 12‐20 µm, mw < 3,0005,000 psi5‐85° CpH 1‐13
PS‐DVB (penta fluorinated) PRP‐h5 ‐ 300 Å, 5 µm and 12‐20 µm, mw > 10,0005,000 psi5‐85° CpH 1‐13
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Reversed Phase Mechanism
PRP‐1 – General purpose pH stable long life column, synthesized DNAPRP‐3 – Gradient protein and peptide separationsPRP‐Infinity – Nonporous support for very fast gradient separation of large proteinsPRP‐h1 – Long life column for LC/MS applications,synthesized DNA, and small moleculePRP‐h5 – Reduced system pressure for protein and peptide separations and enhanced
oligonucleotide recovery
Poly(styrene‐divinylbenzene)
PRP‐1 – 36mer with NaOH, pH 12.7
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PRP‐3 ‐ Protein Separation at pH 12.7
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PRP‐Infinity ‐ Cytochrome c at pH 12.3
PRP‐h1 – Oligos at 75° C
21
22
PRP‐h5 ‐ Proteins at up to 80° C by Reversed Phase
• Note the earlier RT shifts with increasing column temperature
• Resolution of poorly resolved peaks is greatly improved with higher temperatures
23
Ion Exchange
• In ion‐exchange chromatography the stationary bed has an ionically charged surface of opposite charge to the sample ions.• This technique is used almost exclusively with ionic or ionizable samples
• The stronger the charge on the sample, the stronger it will be attracted to the ionic surface, and thus the longer it will take to elute.
• The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.
• Ion chromatography can employ harsh conditions requiring mobile phases that require extreme pH limits
• Temperatures well above the normal operating conditions where silica materials fail can also be used
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Anion Exchange Offerings
PS‐DVB PRP‐X100 ‐ 100 Å, 5 µm, 10 µm, 12‐20 µmTrimethyl Ammonium PRP‐X110 ‐ 100 Å, 5 µm – 75 µmExchanger RCX‐10 ‐ 100 Å, 7 µm – 75 µm 5,000 psi RCX‐30 ‐ 100 Å, 5 µm – 75 µmpH 1‐13*
Poly(methacryl amidopropyl PRP‐X500 ‐ Superficially porous, 7 µmTrimethyl ammonium chloride)5,000 psipH 1‐13*
Poly (dimethyl amidopropyl PRP‐X600 ‐ Superficially porous, 7 µmmethacrylamide)5,000 psipH 1‐13*
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Anion Exchange Mechanism
+N (CH3)3 E-
A-
+N (CH3)3 A-
+ E-
PRP‐X100 ‐ Anions, inorganic and organic using conductivity or UV detection.PRP‐X110 ‐ lower detection levelsRCX‐10 ‐ Isocratic or gradient separation of carbohydrate oligomers up to DP8RCX‐30 ‐ Gradient separation of complex carbohydrates
SAX – trimethyl ammonium exchanger
Eluent: 4 mM p‐hydroxybenzoic acid at pH 8.5
PRP‐X100 – Eight Anions
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PRP‐X100 – pH 11.6
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PRP‐X100 – Arsenic Speciation
RCX‐10 – NaOH Mobile Phase
RCX‐10 – Sugars in Beer
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Anion Exchange Mechanism – PRP‐X500
WAX ‐Methacrylamido propyl trimethyl ammonium chloride
PRP‐X500 ‐ Gradient separation of large proteins and labeled DNA
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Compatible mobile phases:• Buffered with TRIS, tris(hydroxymethyl)aminomethane, at pH 8 or 9• Phosphate• Borate• Lithium• potassium halides
PRP‐X500 – Protein Standards at pH 9.0
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Anion Exchange Mechanism – PRP‐X600
WAX ‐ weak‐base anion exchange support [Poly (dimethyl amidopropyl methacrylamide)
PRP‐X600 ‐ Gradient separation of labeled and unlabeled DNA based on negative charge
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Mobile phases:
10 mM TRIS, 1 mM EDTA pH 9.0, 1 N Sodium Chloride
PRP‐X600 – Plasmid on a Weak Anion Exchanger
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Cation Exchange
PS‐DVB PRP‐X200 ‐ 100 Å, 10 µm, 12‐20 µmSulfonic Acid Exchanger PRP‐X400 ‐ 7 µm, 12 – 20 µm, 30 – 50 µm5,000 psi 5 – 60° CpH 1‐13
PS‐DVB PRP‐X800 ‐ 100 Å, 5 7 µmItaconate Exchanger5,000 psi5 – 60° CpH 1‐13
PS‐DVB Gel‐Type HC‐40 ‐ 7 µmSulfonic Acid Exchanger400 psi Size Exclusion25‐90° CWater only mobile phase
PS‐DVB Gel‐Type HC‐75 (H+) ‐ 7 µmSulfonic Acid HC‐75 (Ca2+)400 psi HC‐75 (Pb2+)25‐90° C 100% Water 0‐40% Acetonitrile Ligand Exchange
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SO3- H+ SO3
- M++ M+ + H+
Cation Exchange Mechanism
PRP‐X200 for mono and divalent organic and inorganic cationsPRP‐X400 Glyphosate and metabolite in drinking waterHC‐40 Sugar oligomers up to DP8HC‐75 Ca2+ Mono and disaccharides in corn syrupHC‐75 H+ Organic acids and sugarsHC‐75 Pb2+ Sugar alcohols
SCX – sulfonic acid
Eluent: 4 mM HNO3 in 7:3 Water:MeOH
PRP‐X200 ‐ Cations
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PRP‐X400 ‐ Glyphosate
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HC‐75 Ca2+ ‐Milk Product Sugars at 90° C
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HC‐75 H+ ‐ Sulfuric Acid Mobile Phase: Acetylated Amino Sugars
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O
CO- H+
+ M+
O
CO- M+
+ H+
Cation Exchange Mechanism
PRP‐X800 Mono and divalent cations in the same run. Transition metals
SCX – itaconate exchanger
Eluent: 2 mM Copper Sulfate
PRP‐X800 – Mono and Divalent Cations
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• Ion exclusion chromatography is an alternative to ion‐exchange chromatography
• The process in which ionized samples are excluded from the pores of the support and elute first, while the nonionic compounds elute later.
• Mixtures of weak acids like those in fruits and milk products are frequently not very well separated by pure ion‐exchange methods, nor in the reversed phase mode.
Ion Exclusion
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Ion Exclusion
PS‐DVB PRP‐X300 ‐ 100 Å, 7 µm, 12 – 20 µmSulfonic Acid Exchanger5,000 psi5 – 60° CpH 1‐13
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Ion Exclusion – PS‐DVB Structure
(SO3) SO3H
SO3H
SO3H
PS‐DVB Ion Exclusion
PRP‐X300 Organic acids and alcohols
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PRP‐X300 – Alcohols
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PRP‐X300 – Organic Acids and Sugars
4848
Reversed Phase Anion Exchange Cation Exchange Ion Exclusion
Adsorption Ion Exchange Exclusion Chromatography
Separation Modes of Polymers
Polymers open up a wide variety of separation mechanisms
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Separation Mechanisms and Offerings for Hamilton Polymer Supports
Reversed PhasePRP®‐h1 – Long life column for LC/MS applications,synthesized DNA, and small moleculePRP‐h5 – Reduced system pressure for protein and peptide separations and enhanced oligonucleotide recoveryPRP‐1 – General purpose pH stable long life column, synthesized DNAPRP‐3 – Gradient protein and peptide separationsPRP‐Infinity – Nonporous support for very fast gradient separation of large proteins
Anion ExchangePRP‐X100 – Anions, inorganic and organic using conductivity or UV detection. 0 to 100% solvent compatible.PRP‐X110 – Similar to PRP‐X100 but for lower level anions (20 ppb to 20 ppm)PRP‐X500 – Gradient separation of large proteins and labeled DNAPRP‐X600 – Gradient separation of labeled and unlabeled DNARCX‐10 – Isocratic or gradient separation of carbohydrate oligomers up to DP8RCX‐30 – Gradient separation of complex carbohydrates
Cation ExchangePRP‐X200 – Inorganic and organic cations using conductivity or UV detection. Separate mono or divalent cations depending on mobile phase conditions.PRP‐X400 – Glyphosate and metabolite in drinking water. Also unique hydrophilic interaction separationsPRP‐X800 – Mono and divalent cations in the same run. Transition metalsHC‐40 – Sugar oligomers up to DP8. Max pressure HC‐75 Ca2+ – Mono and disaccharides in corn syrup. Max pressureHC‐75 H+ – Organic acids and sugars. Max pressureHC‐75 Pb2+ – Sugar alcohols. Max pressure
Ion ExclusionPRP‐X300 – Organic acids and alcohols
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Recap of Polymers
• No alkane chains to be stripped from the substrate
• No siloxane bonds to hydrolyze
• This leads to a wider range of pH
• This wide range of pH enables the exploitation of selectivity effects of multi‐charged or weakly ionizable solutes
• Higher temperature limits enable better resolution
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What Types of Samples can be Analyzed with Hamilton Polymer HPLC Columns?
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Ion ExclusionOrganic acids
Reversed PhasePeptidesProteinsOligonucleotidesNSAIDs
Anion ExchangeArsenicFuorideChlorideNitrateSulfateSugarsPolysaccharides
Cation ExchangeSodium Potassium Lithium Copper Zinc Glyphosate
459 Applications
Over 236 Published Literature References
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Find an Application and Column for Your Compound
Online, alphabetical compound index for both application chromatograms and literature references make it easy to locate the information you need
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Scale‐up Chromatography
• Initial chromatography separations are performed on an analytical scale
• Scale‐up chromatography is made possible by employing larger column hardware and larger particle size packing materials
• Linear scale‐up of flow rates and sample amounts enable predictability of chromatographic results
• Polymer supports lend themselves to large particle and pore sizes allowing for much higher flow rates
• Hamilton offers a 100 x 250 mm column size for preparative work
• We also offer bulk polymer resin into the mulitple kilogram scale
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Hardware Sizes ‐ comparison
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Hardware sizes ‐ comparison
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Skid System/Fraction Collector
57
Small Scale‐up System
58
Fermentor & Skid System
Column Packing System Included
59
Large Scale