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 浸潤(rùn) 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氫氧化鉀
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?yàn)槭?/span>100％的C，故其化學(xué)性質(zhì)：具有很好的PH值穩(wěn)定性，并在的PH緩沖液和溫度下穩(wěn)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(shí)，還沒有硅醇基的作用，減少拖尾
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）.
硅膠表面就像一個(gè)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(huán)一樣。這個(gè)平整的表面就是石墨化碳用來(lái)分離結(jié)構(gòu)相似化合物的一個(gè)重要基礎(chǔ)（空間選擇性）
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.
硅膠的一個(gè)局限就是PH的穩(wěn)定性，當(dāng)PH低于2時(shí)，鍵合相流失，當(dāng)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(wěn)定性非常好，可以在PH 0－14范圍。
Hypercarb的保留機(jī)制：
- 分子的形狀（空間結(jié)構(gòu)）
- 分子的極性
石墨化碳的作用強(qiáng)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(jī)會(huì)也就越多 – 因此，保留更強(qiáng). 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.
壬基酚不是一個(gè)單一的化合物，而是幾個(gè)立體異構(gòu)體的混合物。Hypercarb就可以降這幾個(gè)立體異構(gòu)體分開
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
極性分子*帶電，因此可以引起石墨化碳表面的極性化，這樣可以加強(qiáng)分子和石墨化碳之間的作用
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.
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.
近年來(lái)，HPLC色譜柱生產(chǎn)商已經(jīng)開始在反相柱上鍵入極性功能團(tuán)，以促進(jìn)對(duì)極性化合物的保留
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 間苯三酚 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敵草快, paraquat –PQ百草枯） are important cationic herbicides 陽(yáng)離子除草劑 . 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
這些化合物都是毒性化合物，具有很強(qiáng)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 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方法規(guī)定飲用水中百草枯和敵草快的zui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 analysed by RP-LC with ion pairing; this methodology has the disadvantage of poor sensitivity, requiring several fold preconcentration prior to LC analysis.
這兩累化合物在常規(guī)RP-LC分析方法中需要應(yīng)用離子對(duì)試劑，這種方法的缺陷就是靈敏度低
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(shí)，這個(gè)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.
熱電Thermo*的多孔石墨化碳黑液相色譜柱的應(yīng)用（需要資料，可以發(fā)郵件給送）
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