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多孔石墨化碳液相色谱柱的应用
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更新时间：2010-03-26
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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 particular applications area, high temperature LC
Then the properties of PGC will be described and how the analyte shape and polarity affect retention on this stationary phase;
Brief summary of the advantages of using Hypercarb in LC/MS;
A selection of key applications will demonstrated the uniqueness of this phase to solve problem separations.
Firstly a 500Å silica template is taken.
This is impregnated 浸润 with a Phenyl Formaldehyde 苯甲醛 mixture to compley fill the pores
Then the material is “burnt” or carbonised by heating the furnace to 1000oC.
Once this is complete then the original silica that was used is dissolved away using 4M potassium hydroxide氢氧化钾
Then the furnace is taken up to 2500oC to graphatise 石墨化 the material and form the final product that is PGC
No bonding occurs at the surface of the material this is the final product.
You can see from the manufacture process how stable the material will be it has been heated to 2500oC, had 4M KOH put through it and is then packed at high pressure into columns, so it should outlast other parts in the HPLC systems such as seals and filters even with harsh phase systems.
The requirements placed on Hypercarb’s physical properties are similar to other HPLC supports:
It is manufactured using a silica template, to create spherical, porous particles with 250Å pore size （this tight pore size distribution, with a mean around 250A, allows for good mass transfer 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 are essential to the ultimate performance of the phase if good bed uniformity 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 with most silica based stationary phases which tend to be closer to 300m2g-1; however, this doesn’t lead to short retention times; the retention mechanism provided by the PGC surface work quite differently to that of silica, and still provide strong retention.
Because is 100% carbon, Hypercarb is chemically very robust:
it has complete pH stability, stability under extreme conditions of buffer concentration and temperature,
it can be used for normal phase and reversed phase, and shows an incredible column lifetime.
Also, there are no silanols which might otherwise produce secondary 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 phases are very different in their structure:
Silica has a brush type surface with the stationary phase and silanol groups, whereas PGC has a flat surface composed of sheets of hexagonally arranged carbon atoms, where the spacing between sheets is similar to that in a large polycyclic aromatic molecule. This flat surface of the graphite is the key to the ability of Hypercarb to distinguish between 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 silica based stationary phases is pH stability: at low pH （generally below pH 2 ） cleavage of the organosilane bond occurs, whereas at pH above 9 the silica support starts to dissolve. There is no bonded stationary phase to be cleaved on Hypercarb, and thus graphitized carbon 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 of the analyte functional groups at the point of contact with the graphite 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 a ubiquitous degradation product of nonylphenol polyethoxylate （NPE） surfactants, and has been reported to be an endocrine disrupter. In Europe, NPE surfactants have been banned for household use and are being phased out for industrial use. In the US, the use of NPE surfactants is under scrutiny.
In most HPLC analyses nonylphenol elutes as a single, broad peak. Individual isomers of NP have not been separated by nondestructive methods.
This method on Hypercarb can be used to fractionate p-nonylphenol based on structure, and assess the potential for different isomers to act as endocrine disrupters.
Reference is paper published by US EPA （US Environmental Protection Agency）
Retention on Hypercarb also depends on the polarity of the molecule.
This is one of the major strenghts of this stationary phase
How are polar molecules retained on PGC?
Polar molecules have a permanent dipole and thus can induce a dipole on the polarizable surface on the graphite as they approach it; this increases the attraction between the analyte and the graphite surface
极性分子*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 polar compounds such as carbohydrates, and compounds with several hydroxyl, carboxyl and amino groups, which cannot be retained on silica-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上保留的化合物
PGC shows unusual high retention of polar analytes. This phenomenon was denominated Polar Retention Effect on Graphite （PREG）.
The polar retention effect on PGC is demonstrated here, in a paper by M-C H, where the retention （log k） was plotted against the % MeOH in the phase （water/MeOH） for a series of polar compounds （mono, di and tri-substituted benzenes）.
Log P is a physical property used to describe a compound’s hydrophobic properties. The lower the log P the less hydrophobic the compound, ie, the more polar, and thus the less retention is obtained in RP-LC with conventional phases.
[P is the partition coefficient, ie, the ratio of the concentration of compound in octanol to the concentration in water （octanol-water partition coefficient）; thus a low log P means that the compound has a high afinity for water]
On RP resin, the most polar compound （phloroglucinol log P 0.16） elutes first （as expected）, so elution order is phloroglucinol-resorcinol-phenol. Phloroglucinol needs less than 20% organic to be retained.
Conversely, the PREG means that phloroglucinol is the most retained analyte.
In recent years, HPLC column manufacturers have developed RP packings with polar functional groups, in an attempt to promote retention of polar compounds, so important 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 of phloroglucinol on Hypercarb compare with other stationary phases which contain polar functional groups?
Here the retention of the polar molecule phloroglucinol 间苯三酚 on Hypercarb was compared to the retention on a typical C18, and several stationary phases with polar character :
polar embedded （HyPurity Advance ）
C18 with polar endcapping （Aquasil C18）
Perfluorinated 全氟（fluophase pfp）
The capacity factor on Hypercarb is 3 to 6 times higher than on the other phases; it is 3x higher than that observed on the polar embedded phase, which is the most retentive of the other phases tested.
Aquasil C18 is a silica based C18 phase which is polar endcapped to assist the retention of polar compounds.
Purines 嘌呤 and pyrimidines 嘧啶 are retained on Aquasil but peaks 5 & 6 are not resolved.
When these compounds are run on Hypercarb under the same phase conditions retention increases by an average of three fold and the elution order also changes.
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 moderay 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 PQ and DQ of 3 and 20mg/L respectively. Therefore sensitive analytical methods are necessary to 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zui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 analysed by RP-LC with ion pairing; this methodology has the disadvantage of poor sensitivity, requiring several fold preconcentration prior to LC 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 due to the interaction with the electron cloud on the graphite; also, these are flat molecules which can align themselves closely to the surface
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 phase of water / acetonitrile containing 0.05%TFA is used to achieve retention of these ionic species.
SPE Hypercarb could easily be used to extract these quats from water （Ref. : Carneiro, Puignou, Galceran, Analytica Chimica Acta, 408, 2000, 263-269）
Glucosamine sulphate is a very polar molecule, which does not have a chromophore, so alternative detection methods to UV have to be utilised for its analysis; thus phase compatibility becomes an important factor in method development.
Glucosamine is retained on Hypercarb with a phase of 0.1% ammonia in water / acetonitrile （50:50）! at a temperature of 60C,
phase conditions ideal for detection in negative electrospray.
热电Thermo*的多孔石墨化碳黑液相色谱柱的应用（需要资料，可以发邮件给送）
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