Andrew J. Alpert

Preparation of a porous microparticulatee anion-exchange chromatography support for proteins

Abstract A hydrophilic, durable anion-exchange material has been developed for high- performance liquid chromatography of proteins. Polyethyleneimine and simpler amines are adsorbed to porous, microparticulate silicas so strongly that the adsorbed coatings may be crosslinked into a stable layer by a wide variety of reagents in organic solution. Epoxy resins, alkyl bromides, and nitro alcohols are the best cross- linkers. The resulting pellicular coating is more stable in aqueous media than the underlying silica. Porous glass, alumina, and titania can also be coated in this manner. The materials are quite reproducible and of high ion-exchange capacity: up to 2.7 mequiv./g. Columns of polyethyleneimine-coated silica resolve proteins quickly and efficiently with excellent recoveries of enzyme activity. Nucleotides are also well resolved. Efficiency does not change during a columns lifetime.

Hydrophilic-interaction chromatography of complex carbohydrates

Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo- and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.

High-performance hydrophobic-interaction chromatography of proteins on a series of poly(alkyl aspart-amide)-silicas

Abstract A series of bonded poly(alkyl aspartamide) coatings was prepared on silica. The products showed a wide range of hydrophobicity, and were evaluated for high-performance hydrophobic-interaction chromatography (HPHIC) of proteins. Poly-(propyl aspartamide)-silica is a good general-purpose material for protein HPHIC; poly(ethyl aspartamide)-silica may be useful for more hydrophobic proteins. Retention of proteins generally increased dramatically with ligand length in the coating, but this was not true of certain apolipoproteins with no well-defined tertiary structure. A correlation was also observed between peak width and conformational lability; the apolipoproteins (an extreme case) were eluted in exceptionally broad peaks. These principles can be used to obtain data on the physical characteristics of new proteins. This was demonstrated with rec A protein.

HILIC at 21: Reflections and perspective

[ [ [ [ [ [ [7] M. Tswett, Izd. Karbasnikov, Warsaw, 1910. Without being named as such, HILIC began in 1975 with the nalysis of sugars by amino-silica columns [1,2]. Its extension to olar solutes in general can be connected in part to Ronald Reaan and Mikhail Gorbachev. In 1987, as a result of their policy of lasnost, Pine Bluff Arsenal in Arkansas was tasked with incinerting the American stocks of 3-quinuclidinyl benzilate, a chemical arfare agent also called Agent BZ. The technical staff discovered hat all of the products of partial pyrolysis coeluted in reversedhase chromatography (RPC), leaving them unable to follow the ourse of the chemical’s destruction. One of them discussed the roblem with an acquaintance who ran an analytical lab down the all from my recently founded company. He in turn asked if I had ny ideas. Since the compound was an alkaloid with a potential ositive charge on the nitrogen atom and our company manufacured a weak cation-exchange column, I decided to try running it y ion-exchange chromatography. The retention was inadequate o resolve the compounds. I systematically varied the salt; the alt concentration; the pH. Nothing helped. Finally I tried varying he % methanol in the mobile phase. This didn’t have much effect ither until the level reached 80%. At that point retention increased ramatically with resolution adequate for the analysis. Pine Bluff rsenal proceeded to order ten PolyCAT A® columns, a decent order n those days, and Agent BZ was successfully incinerated. Months ater I was assessing the variables involved in retention of peptides n our prototype strong cation-exchange material. Retention was tronger at pH 3 than at pH 4, but when the level of methanol in the obile phase exceeded 60%, retention again increased dramatically o matter what the pH [3]. Some force independent of electrostatic ffects seemed to be present. Shortly thereafter, I noticed the same ffect in a paper on analysis of peptides on a neutral size exclusion olumn [4]. By now this behavior seemed more than coincidence. hese observations started the set of experiments that resulted in he paper that promoted the new mode of “Hydrophilic Interaction hromatography”, for separation of polar compounds in general 5]. HILIC moved into labs at a slow but steady pace, with about 80 apers published by 2002. The pace picked up with alacrity in 2003, hen Waters Corp. began to promote its Atlantis® silica columns