North Jersey Chromatography Group

DoubleTree Hotel

APRIL NJCG Dinner/Seminar

April 8, 2019

Registration:

Professionals: $10

Full-Time Students/Retired/Unemployed: Free

DoubleTree Hotel

200 Atrium Dr.

Somerset, NJ 08873

Hydrophilic Interaction Chromatography with Salts: Hydration State of Counterions is Critical to Retention and Selectivity

The hydration of counterions can have a dramatic impact on retention of charged solutes in HILIC or ERLIC. The use of a well-hydrated cation such as Mg+2 or ethylenediammonium ion (EDA) promotes retention of negatively charged solutes. This was exploited at pH 2.5 to leave peptides with (-) charge only on phosphate, sialic acid, and isoAsp residues, increasing retention of peptides that have these modifications. Poorly hydrated trifluoroacetyl- ion (TFA) was used as the anion to decrease retention of all other peptides. With the use of volatile EDA-TFA salt, retention of a singly phosphorylated tryptic peptide was 2.5-4x greater in HILIC than the nonphosphorylated sequence and 12-18x greater in ERLIC. With phosphopeptides with a tryptic-type sequence, the range of the increase is proportional to the proximity of the phosphate group to the C-terminus, evidence of orientation of the peptide during its migration on the column. This is a ~ 4x increase in selectivity for phosphopeptides over what had been obtained before, making this approach competitive with IMAC and titania-based methods. Unlike those methods, the ERLIC mode permits the separation of phosphopeptides from each other with good selectivity. Glycopeptides are readily identified using the same conditions. Selectivity for peptides with other modifications was increased up to 20x compared with preceding methods.

Andrew Alpert, Ph.D.

President of PolyLC, Inc.

Enhanced Fluidity Liquid Chromatography in Preparative Scale-up

The evolution of supercritical fluid chromatography (SFC) instrumentation, improved detection capability, and expanded modifier range has led to extending the reach of SFC to the analysis of a broader spectrum of analytes beyond enantioselective separations. However, preparative SFC has yet to see the same technological revitalization, especially in regards to the purification of highly polar analytes. Enhanced fluidity liquid chromatography (EFLC) has been demonstrated as one of the ways to extend the applicable range of SFC instrumentation to highly polar analytes such as proteins, carbohydrates, and nucleotides. Despite recent applications of EFLC for challenging mixtures of hydrophilic metabolites and analogs, its viability in preparative purification, which is of great importance to the pharmaceutical industry, remains unknown. Herein, multiple chromatographic parameters that are critical to achieve feasible EFLC purification methods were investigated, including system pressure as a function of modifier composition (for several MeOH:H2O ratios), effect of diluent injection conditions on peak shape, and optimization of mass load with diluent composition. The usage of 50% acetonitrile or methanol diluents provided the most volumetric loading capacity. In the case of sucrose, leveraging higher analyte solubility in water proved to be more favorable than the volumetric loading capacity of diluents with higher organic content. In fact, an 80 mg injection of sucrose was possible on a 2 cm preparative HILIC column with minimal peak shape degradation. The combined information led to the successful demonstration of EFLC for the preparative separation of sugars using readily available MS-directed SFC instrumentation.

Raffeal Bennett, Ph. D.

Postdoctoral Fellow at Merck

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