Hypercarb Columns Application Notebook

Applications NotebookIssue 1, June 2009Thermo ScientificHypercarb ColumnsA unique solution for difficult separationsPart of Thermo Fisher Scientific

Table of ContentsIntroductionTable of ContentsApplication NotesThermo Scientific Hypercarb ColumnsIntroduction . 4BiochemicalPorous Graphitic Carbon for the Sample Preparationof Hydrophilic Biomolecules. 6Porous Graphitic Carbon for the LC/MS Analysisof Hydrophilic Biomolecules. 7Analysis of Glycopeptides Using Porous Graphite Chromatographyand LTQ Orbitrap XL ETD Hybrid MS.10The Use of Porous Graphitic Carbon LC-MS for the Analysis ofUnderivatised Carbohydrates from Wheat Stems.17Food SafetyQuantitation of Acrylamide in Food Samples on the TSQ Quantum Discoveryby LC/APCI-MS/MS .20EnvironmentalFast LC Separation of Triazine Herbicides at Elevated Temperature .23Analysis of Polar Metabolites of Atrazine in Ground Waters UsingHypercarb as SPE Sorbent Coupled On-Line with Hypercarb LC Column.27Fast and Versatile Analysis of Desphenyl-Chloridazone andMethyl-Desphenyl-Chloridazone in Surface, Ground andDrinking Water Using LC-MS/MS and EQuan.30ClinicalDetermination of Leucine and its Isomers by LC-MS/MS Using aPorous Graphitic Carbon Column .33Determination of Occupational Exposure to Toluene, Xylene and StyreneThrough Metabolite Monitoring Using Isocratic HPLC .35Food and BeveragePorous Graphitic Carbon for Inorganic Ion Analysis in Drinking Water.37Legal Notices2 2009 Thermo Fisher Scientific Inc. All rights reserved. All trademarks not specifically referenced are the property of Thermo Fisher Scientific Inc.and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products.It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.Not all products are available in all countries. Please consult your local sales representative for details.

Applications ReviewReferences.42Application ChromatogramsBiochemical .44Allantoin.44Ceramides .44Cyclic Monophosphates .44L-Carnitine .44Purines and Pyrimidines (UHT-LC) .44RNB-Glycopeptides .44Table of ContentsApplications Reference Guide (Application by Solute Type).40Food Safety .45Aflatoxins.45Methylamines in Fish .45Environmental.45Nonylphenol Isomers .45Quaternary Ammonium Salts .45Water Pollutants .45Clinical .45-46Arginine and Methylated Arginines .45Creatine in Serum .46Pharmaceutical .46Acyclovir.46Fosfomycin .46Glucosamine Sulfate .46Tuberculostatics.46Uracil and Metabolite .46Ordering InformationHypercarb Columns, Hypercarb Drop-in Guard Cartridges, Hypercarb KAPPACapillary Columns, Hypercarb Nanobore Columns, Hypercarb SpecializedColumn Hardware for High Throughput, Hypercarb Preparative Columns,Hypercarb High Temperature Columns .473

Thermo Scientific Hybercarb ColumnsIntroductionIntroductionPhysical and Chemical Properties of PGCPorous graphitic carbon (PGC, Hypercarb) has uniqueproperties as a stationary phase in high performance liquidchromatography (HPLC). Its chemical surface propertiesdistinguish PGC from more conventional LC packings suchas bonded-silica gels and polymers. PGC behaves as astrongly retentive alkyl-bonded silica gel for non-polaranalytes, however its retention and selectivity behaviourtowards polar and structurally related compounds is verydifferent. PGC provides unique retention and separation ofvery polar compounds. Its surface is stereo-selective withthe capability to separate geometric isomers, diastereomersand other closely related compounds. Hypercarb is stablethroughout the entire pH range 0-14, and is not affected byaggressive mobile phases. Its compatibility with all solventsystems enables separation of a wide range of polaritieswithin a single chromatographic run. The selectivity of theHypercarb packing is different from the selectivity of silicaand polymeric phases. Its retention mechanism is differentfrom conventional C18 columns.Reference 1 gives an in-depth review of its HPLCbehaviour and application areas.PGC particles are spherical and fully porous with a porosityof approximately 75%. The surface of PGC is crystallineand highly reproducible and does not contain micropores.At the molecular level, PGC is made up of sheets of hexagonally arranged carbon atoms linked by the same conjugated1.5-order bonds which are present in any large polynucleararomatic hydrocarbon.2 In principle, there are no functionalgroups on the surface since the aromatic carbon atoms havefully satisfied valencies within the graphitic sheets. Table 1lists the more important physical properties of PGC. Therequirements placed on its physical properties are similar toother HPLC supports where factors such as narrow particlesize distribution are essential to the ultimate performance ofthe phase, if good bed uniformity and low operating pressuresare to be achieved. PGC also has a tight pore size distributionwith a mean value around 250 Å, allowing for good masstransfer of a wide range of analyte shapes and sizes. Surfacehomogeneity and absence of highly adsorptive sites areessential for good peak symmetry. PGC meets all the conventional operating criteria of a chromatographic support.PropertySpherical, fully porous120 m2/g250 Å0.7 m3/g3, 5 µm7 µm30 µmNo microporesRetention linearity and loading capacityMass transfer for wide range of analyte’s shapes and sizesPorosity%CMechanical strength75%100% 400 barMass transfer within particlesChemical stabilityOperational particle stability; pressure gradients in packing processTable 1: Physical properties of PGC4To meet requirement ofParticle shapeSpecific surface areaMedian pore diameterPore volumeMean particle diametersAnalytical HPLC columnsPreparative HPLC columnsSPE applications

Retention of Polar Compounds on PGCOn a molecular scale, the surface of the graphite is flat andhighly crystalline unlike that of alkyl-bonded silicas, whichpossess a brush type surface with the bonded phase andresidual silanols. Consequently, the PGC mechanism ofinteraction is very different. The retention by graphite fromaqueous/organic eluents is determined by the balance of twofactors: (1) hydrophobicity, which is primarily a solutioneffect that tends to drive analytes out of solution and (2) theinteraction of polarisable or polarised groups in the analytewith the graphite (these are additional to the normal dispersiveinteractions). The strength of interaction depends on boththe molecular area of an analyte in contact with the graphitesurface and upon the nature and type of functional groupsat the point of interaction with the flat graphite surface.The more planar the analyte, the closer its alignment isto the graphite surface, and so the greater the number ofpoints of interaction possible – hence, maximum retention.Retention is reduced for highly structured, 3-dimensionaland rigid molecules that can contact the surface with only asmall part of their surface, compared with planar moleculeswith the same molecular mass. This is illustrated in Figure 1.In a traditional reversed phase (RP) system, analyte retentionincreases as its hydrophobicity increases. This is due to theincreased dispersive interactions that take place between thestationary phase and the analyte. Conversely, as the polarityof the analyte increases, analyte-solvent interactions begin todominate and retention is reduced. This simple observationholds true for all reversed-phase systems with the exception ofPGC. For Hypercarb columns, it has been observed that insome cases retention increases as the polarity of the analyteincreases. This effect has been called “the polar retention effecton graphite” or PREG. The effect of PREG makes Hypercarbcolumns particularly useful for the separation of highly polarcompounds, such as carbohydrates, and compounds withseveral hydroxyl, carboxyl and amino groups, which aredifficult to retain on conventional alkyl-silica phases.PREG defines the ability of molecules having lone-pairor aromatic-ring electrons to apparently interact throughan electron transfer mechanism to the electronic cloud ofthe graphite. PREG is particularly pronounced when thepolar groups are attached to a benzene ring and other largeraromatic systems. Knox et al.2 have attributed this to sometype of orbital overlap between the conductivity electronsin graphite and lone pair and/or π electrons in analytes.The polarizable properties of the graphite hold the key tounderstanding the mechanism by which polar moleculesare retained at the surface (Figure 2).aIntroductionRetention Mechanisms on PGCabbFigure 1: Effect of the solute shape on the strength of the interaction with thegraphite surface: (a) Good alignment of planar molecule to the flat graphitesurface; (b) Poor alignment of non-planar molecule to the flat graphite surface.Figure 2: Schematic representation of polar analyte retention in which(a) positive charge and (b) negative charges approach the graphite surface,resulting in a charge-induced dipole at the graphite surface.References1. L. Pereira, J. Liq. Chrom. & Rel Technol., 2008, 31, 1687–17312. J. Knox, P.Ross, Advances in Chromatography, 1997, 37, 73-1195

BIOCHEMICALPorous Graphitic Carbon for the Sample Preparation ofHydrophilic BiomoleculesLuisa Pereira, Thermo Fisher Scientific, Runcorn, UKIntroductionApplication NotesMicro-scale solid phase extraction (SPE) can be used as asample purification process to remove contaminants; thistechnique has the advantage of effectively handling limitedsample volumes (low microlitre) to maximise sensitivity. Whenthe analytes are very hydrophilic it is necessary to select asorbent that provides good retention and minimises sample lossthrough breakthrough in the aqueous matrix. Desalting ofhydrophilic peptides can be accomplished by using microscaletips packed with porous graphitic carbon. This enables theoff-line preparation and clean-up of biological samples forfurther analysis and identification by mass spectrometry(MS). In this approach it is important that the sorbent inthe tip is capable of retaining the hydrophilic analytes withno breakthrough in the aqueous matrix, and that goodrecovery of the retained analytes from the tip is achieved.GoalTo demonstrate the capability of porous graphitic carbon(PGC) in micro-scale SPE of hydrophilic peptides.ExperimentalPeptidesArg-Gly-Glu-Ser (RGES) and Asp-Ser-Asp-Pro-Arg(DSDPR).TipsThermo Scientific HyperSep Hypercarb Tips 10-200 µLvolume (part number 60109-212).Micro-scale SPE Protocol:Solvents: A – H2O 0.1%formic acid; B – H2O/ACN(30:70) 0.1% formic acid.Tip conditioning: Aspirate and expel 5 times 20 µL ofsolvent B. Aspirate and expel 5 times 20 µL of solvent A.Sample loading (binding): Aspirate and expel 20 times20 µL of sample.Sample washing: Aspirate and expel 5 times 20 µL ofsolvent A, discarding the expelled solvent each time.Sample elution: Aspirate and expel 20 times 20 µL ofsolvent B, collecting the expelled solution in a clean microcentrifuge tube. Transfer solution to micro-vial for injection.A flow-through fraction from a proteolytic digest wassimulated by diluting a solution containing RGES andDSDPR in Tris buffer (100 mM, pH 8.0) to concentrationsof 0.1 and 0.5 ng/µL respectively.6Figure 1. Recovery from HyperSep Hypercarb Tips for 2 peptides, RGES and DSDPR.Results and DiscussionFollowing the procedure detailed in the experimental section,the recoveries were measured by comparison of the ESI-MSsignal for the tip eluate with the ESI-MS signal for the solutionof the same concentration. Recoveries are between 72 and101%, as shown in Figure 1.ConclusionMicro-scale SPE with porous graphitic carbon packed tipsgives good recovery of small hydrophilic peptides frombuffer matrices.