多孔石墨化碳液相色譜柱的應

　　This presentation will give an overview of the properties and behaviour of porous graphitic carbon or Hypercarb as a stationary phase in HPLC.

　　It focus on a particularapplications area, high temperature LC

　　Then the properties of PGC willbe described and how the analyte shape and polarity affectretention on this stationary phase;

　　Brief summary of the advantagesof using Hypercarb in LC/MS;

　　A selection of key applicationswill demonstrated the uniqueness of this phase to solve problemseparations.

　　Firstly a 500? silica templateis taken.

　　This is impregnated浸潤with a Phenyl Formaldehyde苯甲醛mixture to completely fill thepores

　　Then the material is“burnt”or carbonised by heating the furnaceto 1000oC.

　　Once this is complete then theoriginal silica that was used is dissolved away using 4M potassiumhydroxide氫氧化鉀

　　Then the furnace is taken up to2500oC to graphatise 石墨化 the material and form. the final product that isPGC

　　No bonding occurs at the surfaceof the material this is the final product.

　　You can see from the manufactureprocess how stable the material will be it has been heated to2500oC, had 4M KOH put through it and is then packed at highpressure into columns, so it should outlast other parts in the HPLCsystems such as seals and filters even with harsh mobile phasesystems.

　　The requirements placed on Hypercarb’s physical properties are similar toother HPLC supports:

　　It is manufactured using a silica template, to createspherical, porous particles with 250? pore size (this tight poresize distribution, with a mean around 250A, allows for good masstransfer of a large number of analyte shapes and sizes)

　　It is available in three particle sizes for chromatography(distribution with mean particle size in the range 3-10um areessential to the ultimate performance of the phase if good beduniformity and a low operating pressures are to be achieved);also available in 30um for SPE and sample preparation.

　　The surface area of PGC is relatively small in comparison withmost silica based stationary phases which tend to be closer to300m2g-1; however, this doesn’t lead to short retention times; theretention mechanism provided by the PGC surface work quitedifferently to that of silica, and still provide strongretention.

　　Because is 100% carbon, Hypercarb is chemically veryrobust:

　　it has complete pH stability, stability under extremeconditions of buffer concentration and temperature,

　　it can be used for normal phase and reversed phase, and showsan incredible column lifetime.

　　Also, there are no silanols which might otherwise producesecondary interactions with analytes.

　　因為是100%的C，故其化學性質：具有很好的PH值穩定性，并在極端的PH緩沖液和溫度下穩定

　　可以作為正相或者反相適用，具有難以置信的柱壽命

　　同時，還沒有硅醇基的作用，減少拖尾

　　Hypercarb meets all conventional operating criteria.

　　PGC and silica stationary phasesare very different in their structure:

　　Silica has a brush type surfacewith the stationary phase and silanol groups, whereas PGC has aflat surface composed of sheets of hexagonally arranged carbonatoms, where the spacing between sheets is similar to that in alarge polycyclic aromatic molecule. This flat surface of thegraphite is the key to the ability of Hypercarb to distinguishbetween compounds with similar structure(stereo-selectivity).

　　硅膠表面就像一個刷子，布滿了硅醇基和固定相，而PGC是由碳原子排布成6角形的平整表面，就像芳環一樣。這個平整的表面就是石墨化碳用來分離結構相似化合物的一個重要基礎(空間選擇性)

　　One of the limitations of silicabased stationary phases is pH stability: at low pH (generally belowpH 2 ) cleavage of the organosilane bond occurs, whereas at pHabove 9 the silica support starts to dissolve. There is no bondedstationary phase to be cleaved on Hypercarb, and thus graphitizedcarbon has complete pH stability.

　　硅膠的一個局限就是PH的穩定性，當PH低于2時，鍵合相流失，當PH高于9，硅膠開始溶劑。而石墨化碳表面沒有鍵合相，故石墨化碳的PH的穩定性非常好，可以在PH 0-14范圍。

　　Hypercarb的保留機制：

　　-分子的形狀(空間結構)

　　-分子的極性

　　石墨化碳的作用強度取決于：

　　分子表面和石墨化碳表面作用;分子越平, 越可以貼近石墨化碳表面, 其作用的機會也就越多 – 因此，保留更強. Retention is reduced for highly structured and rigid moelcules that can contact the surface with only a small part of their surface, compared with planar molecules with the same molecular mass (as schematically illustrated on this slide).

　　- The type and positioning ofthe analyte functional groups at the point of contact with thegraphite surface.

　　Nonylphenol 壬基酚 is not a single compound but a mixture of several isomers due to the branching of the C-9 alkyl group. Here the ability of Hypercarb columns to separate closely related analytes is utilized, and by using the new 3μm particle size, extra resolution is achieved.

　　壬基酚不是一個單一的化合物，而是幾個立體異構體的混合物。Hypercarb就可以降這幾個立體異構體分開

　　p-Nonylphenol is aubiquitous degradation product of nonylphenol polyethoxylate (NPE)surfactants, and has been reported to be an endocrine disrupter. InEurope, NPE surfactants have been banned for household use and arebeing phased out for industrial use. In the US, the use of NPEsurfactants is under scrutiny.

　　In most HPLC analysesnonylphenol elutes as a single, broad peak. Individual isomers ofNP have not been separated by nondestructive methods.

　　This method on Hypercarbcan be used to fractionate p-nonylphenol based on structure, andassess the potential for different isomers to act as endocrinedisrupters.

　　Reference is paperpublished by US EPA (US Environmental ProtectionAgency)

　　Retention on Hypercarb alsodepends on the polarity of the molecule.

　　This is one of the majorstrenghts of this stationary phase

　　How are polar moleculesretained on PGC?

　　Polar molecules have a permanentdipole and thus can induce a dipole on the polarizable surface onthe graphite as they approach it; this increases the attractionbetween the analyte and the graphite surface

　　極性分子永久帶電，因此可以引起石墨化碳表面的極性化，這樣可以加強分子和石墨化碳之間的作用

　　This dipole–dipole interaction results in excellent retention for polarcompounds such as carbohydrates, and compounds with severalhydroxyl, carboxyl and amino groups, which cannot be retained onsilica-based alkyl phases such as C18s.

　　這種作用可以很好的保留極性化合物，像碳水化合物，或者有羥基，羧基和氨基，這些難以在C18液相色譜柱上保留的化合物

　　PGC shows unusual high retentionof polar analytes. This phenomenon was denominated PolarRetention Effect on Graphite (PREG).

　　The polar retention effect onPGC is demonstrated here, in a paper by M-C H, where the retention(log k) was plotted against the % MeOH in the mobile phase(water/MeOH) for a series of polar compounds (mono, di andtri-substituted benzenes).

　　Log P is a physical propertyused to describe a compound’s hydrophobic properties. The lower the log P theless hydrophobic the compound, ie, the more polar, and thus theless retention is obtained in RP-LC with conventionalphases.

　　[P is the partitioncoefficient, ie, the ratio of the concentration of compound inoctanol to the concentration in water (octanol-water partitioncoefficient); thus a low log P means that the compound has a highafinity for water]

　　On RP resin, the most polarcompound (phloroglucinol log P 0.16) elutes first (as expected), soelution order is phloroglucinol-resorcinol-phenol. Phloroglucinolneeds less than 20% organic to be retained.

　　Conversely, the PREG means thatphloroglucinol is the most retained analyte.

　　In recent years, HPLC columnmanufacturers have developed RP packings with polar functionalgroups, in an attempt to promote retention of polar compounds, soimportant in life sciences.

　　近年來，HPLC色譜柱生產商已經開始在反相柱上鍵入極性功能團，以促進對極性化合物的保留

　　How does retention ofphloroglucinol on Hypercarb compare with other stationary phaseswhich contain polar functional groups?

　　Here the retention of the polarmolecule phloroglucinol 間苯三酚 on Hypercarb was compared to the retention on atypical C18, and several stationary phases with polarcharacter :

　　polar embedded (HyPurity Advance)

　　C18 with polar endcapping(Aquasil C18)

　　Perfluorinated全氟(fluophase pfp)

　　The capacity factor onHypercarb is 3 to 6 times higher than on the other phases; it is 3xhigher than that observed on the polar embedded phase, which is themost retentive of the other phases tested.

　　Aquasil C18 is a silicabased C18 phase which is polar endcapped to assist the retention ofpolar compounds.

　　Purines 嘌呤 and pyrimidines 嘧啶 are retained on Aquasil but peaks 5 & 6 are not resolved.

　　When these compounds are runon Hypercarb under the same mobile phase conditions retentionincreases by an average of three fold and the elution order alsochanges.

　　The salts of quaternary ammonium compounds (diquat – DQ敵草快, paraquat –PQ百草枯) are important cationic herbicides 陽離子除草劑 . These compounds are toxic and classified as moderately hazardous; they have high water solubility and low volatility, and after application can be adsorbed by the soil or transported to water by runoff or leaching

　　這些化合物都是毒性化合物，具有很強的水溶性，在使用后容易被土壤吸收而帶入水中

　　EPA established maximum levels in drinking water for PQand DQ of 3 and 20mg/Lrespectively. Therefore sensitive analytical methods are necessaryto monitor the presence of these compounds in drinking water.

　　EPA方法規定飲用水中百草枯和敵草快的最大含量分別為3ug/L和20ug/L，因此，需要建立高靈敏度的分析方法。

　　Quats are ionic species and thus are normally analysedby RP-LC with ion pairing; this methodology has the disadvantage ofpoor sensitivity, requiring several fold preconcentration prior toLC analysis.

　　這兩累化合物在常規RP-LC分析方法中需要應用離子對試劑，這種方法的缺陷就是靈敏度低

　　Retention of cations such as quats on Hypercarb is dueto the interaction with the electron cloud on the graphite; also,these are flat molecules which can align themselves closely to thesurface

　　而百草枯和敵草快在Hypercarb上保留依賴的是電子云和石墨化碳的作用，同時，這個平面分子和以很近的吸附在石墨化碳的表面

　　On Hypercarb a simple mobile phase of water /acetonitrile containing 0.05%TFA is used to achieve retention ofthese ionic species.

　　SPE Hypercarb could easily be used to extract thesequats from water (Ref. : Carneiro, Puignou, Galceran, AnalyticaChimica Acta, 408, 2000, 263-269)

　　Glucosamine sulphate is a verypolar molecule, which does not have a chromophore, so alternativedetection methods to UV have to be utilised for its analysis; thusmobile phase compatibility becomes an important factor in methoddevelopment.

　　Glucosamine is retained onHypercarb with a mobile phase of 0.1% ammonia in water /acetonitrile (50:50)! at a temperature of 60C,

　　Mobile phase conditions idealfor detection in negative electrospray.