EDITOR’S PAGE continued Fast Protein A columns


Protein A is a classic surface chemistry for selective retention of IgGs. It is derived from the protein coat of bacteria and thus is often immunogenic. Daiso (Osaka, Japan) introduced HPLC stationary phases with Protein A covalently bond with epoxy chemistry to porous silica. Daiso introduced ADREPMA™ (advanced recombinant protein for monoclonal antibody) for affinity chromatography. Benchmark studies by Daiso show that ADREPMA binds more quickly with higher capacity and superior recov- ery compared to “agarose material.”


Despite the biospecific affinity of Protein A columns, there are other considerations. Contamination of the product with ligand from the column is a major concern in preparative applications. Ligand leakage is less of a concern with analytical columns, unless they are used for prep. Clean-in-place (CiP) is another issue. Traditionally, preparative columns using agarose for support were cleaned-in-place with washes of 1 molar NaOH. This usually destroyed column packings using silica as a support. The Daiso literature shows that 150 short washes with 0.1M NaOH do not alter performance.


HILIC rages on


It has taken 25 years, but Andrew Alpert’s HILIC, including electrostatic repulsion hydrophilic interaction chromatography (ERLIC), is now firmly entrenched as a very productive separation mode in LC, particularly for biosamples. PolyLC Inc. (Columbia, MD) is Dr. Alpert’s firm that pro- motes the modes and columns. One of the company’s notes compares the peptide coverage for several 2-D LC modes. 2-D LC with ERLIC + RPLC identified 711 peptides which were derived from 123 proteins. The pep- tide count was much more than other modes including SCX-RPLC. With ERLIC in the first dimension, the peptides elute in order of decreasing pI, which is useful in detecting deamidation products.


Another application brief addressed quality control of antibody drug conjugates (ADCs). PolyPROPYL A™ operating in HIC mode was able to rapidly separate homologs of ADCs. Further, the column provided high resolution of oxidation variants.


Antibodix columns Antibodix columns from Sepax Technologies start with nonporous polystyrene/divinylbenzene beads with a choice of 1.7, 3, 5, or 10 μm. A 1-nm-thick hydrophilic polymer is grafted to cover the aromatic sur- face to reduce nonspecific hydrophobic retention. Next, weak cation exchange functional groups are covalently attached to provide a high- capacity ion exchange layer. The columns show good separation of antibodies, including fragments and low multimers.


AS-19 column with 4-μm particles The AS-19 from Dionex, now part of Thermo Fisher Scientific, was rec- ognized for assay of drinking water quality according to U.S. EPA Method 300.0. By reducing the particle size to 4 μm, the new column meets the requirements of U.S. EPA Methods 300.0 and 300.1. The narrow-bore columns scale from 4 mm down to 0.4 mm. Reducing the particle size to 4 μm improves the separation power at all column diameters. Peaks for bromate, etc., stand out from the background.


Solid-phase extraction (SPE) The May issue of American Laboratory reported on new products for


sample processing (http://www.americanlaboratory.com/914-Appli- cation-Notes/160946-Sample-Processing-at-Pittcon-2014/), including SOLA, introduced at Pittcon. But Thermo introduced the SOLAμ at HPLC 2014. Chromatographers at Thermo recognized that Moore’s Law for SPE was suspended. Yes, improved detection limits allowed reduction in sample volume (<100 μL), and the number of samples was increasing. Plus, sample loss during concentration by blow-down wasted time and degraded some analytes.


SPE technology was stuck in a rut. What to do? Thermo’s solution was to adopt a fritless monolith adsorbent bed in each well of a 96-well SPE plate. Thermo introduced the SOLAμ, which is designed to process small sample volumes (<25 μL) and concentrate during the elution stage by as much as 20 times. Sample volumes are so small that blow-down is fast if it is required at all.


Since the SOLAμ is fritless, one applies the sample to the top of the active sorbent monolith. Bed weight is only 2 mg. There is no dead volume. Fabrication of the monolith is more uniform than can often be obtained with packed beds and retaining frits. This shows up as improved consis- tency in replicates.


Update on Chromeleon chromatography software Thermo chose Pittcon® for introduction of Chromeleon CDS, extending


Chromeleon 7.2, which was introduced just the year before. The CDS version supports MS detection for Thermo’s ion, liquid, and gas chro- matographs. The software is fully validated for the company’s single and triple quadrupoles, and benchtop Orbitraps. The staff seemed especially proud of the operational simplicity, including one-click workflow, which reduces training time. CDS is compatible with Microsoft’s “dot.net” archi- tecture, which should assure integration and communication.


Sample loops for UHPLC Optimize Technologies (Oregon City, OR) introduced several acces-


sories to make working with UHPLC less of a challenge. The sample loops are 0.005 in. i.d. for 1 or 2 μL internal volume with 1/16-in. ends. The Pmax rating is 20,000 psi. For Shimadzu’s UHPLC, Optimize offers piston seals and check valves.


Polyolefin characterization Polymer Char (Valencia, Spain) has a singular focus on characterizing


polyolefins (POs). This often includes high-temperature GPC. However, traditional assays of POs use macro assays such as xylene solubles, which is a gravimetric wet chemistry method. This is out of date, plus it is tedious and laborious (~6 hr/sample). Polymer Char introduced the CRYSTEX® QC to replace xylene soluble. With a protocol called Tem- perature Rising Fractionation (TREF), the analyst seals the sample in a long tube and places it in the CRYSTEX. The instrument pumps solvent through the tube as the column temperature increases. Output is ethyl- ene content by infrared, viscosity, plus % amorphous and % crystalline. Run time is reduced from 6 hr to 2, and labor from 6 hr to a few minutes.


AMERICAN LABORATORY • 8 • SEPTEMBER 2014


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52