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 500Å silica templateis taken.
This is impregnated浸润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氢氧化钾
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 250Å 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，故其化学性质：具有很好的PH值稳定性，并在极端的PH缓冲液和温度下稳定
可以作为正相或者反相适用，具有难以置信的柱寿命
同时，还没有硅醇基的作用，减少拖尾
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）.
硅胶表面就像一个刷子，布满了硅醇基和固定相，而PGC是由碳原子排布成6角形的平整表面，就像芳环一样。这个平整的表面就是石墨化碳用来分离结构相似化合物的一个重要基础（空间选择性）
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.
硅胶的一个局限就是PH的稳定性，当PH低于2时，键合相流失，当PH高于9，硅胶开始溶剂。而石墨化碳表面没有键合相，故石墨化碳的PH的稳定性非常好，可以在PH 0－14范围。
Hypercarb的保留机制：
-分子的形状（空间结构）
-分子的极性
石墨化碳的作用强度取决于：
分子表面和石墨化碳表面作用;分子越平, 越可以贴近石墨化碳表面, 其作用的机会也就越多 – 因此，保留更强. 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.
壬基酚不是一个单一的化合物，而是几个立体异构体的混合物。Hypercarb就可以降这几个立体异构体分开
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
极性分子永久带电，因此可以引起石墨化碳表面的极性化，这样可以加强分子和石墨化碳之间的作用
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.
这种作用可以很好的保留极性化合物，像碳水化合物，或者有羟基，羧基和氨基，这些难以在C18液相色谱柱上保留的化合物
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.
近年来，HPLC色谱柱生产商已经开始在反相柱上键入极性功能团，以促进对极性化合物的保留
How does retention ofphloroglucinol on Hypercarb compare with other stationary phaseswhich contain polar functional groups?
Here the retention of the polarmolecule phloroglucinol 间苯三酚 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敌草快, paraquat –PQ百草枯） are important cationic herbicides 阳离子除草剂 . 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
这些化合物都是毒性化合物，具有很强的水溶性，在使用后容易被土壤吸收而带入水中
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方法规定饮用水中百草枯和敌草快的最大含量分别为3ug/L和20ug/L，因此，需要建立高灵敏度的分析方法。
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.
这两累化合物在常规RP-LC分析方法中需要应用离子对试剂，这种方法的缺陷就是灵敏度低
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
而百草枯和敌草快在Hypercarb上保留依赖的是电子云和石墨化碳的作用，同时，这个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.
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