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AN INTERNATIONAL SEPARATION SCIENCE SOCIETY

CASSSPublished by 1

It has been a while since you last read the RetentionTimes®. It may have appeared that CASSS dumped our newslet-

ter, but that is not the case. Not at all. However, CASSS went through dramatic developments and while working on its new image, we did not have enough bandwidth to prepare our Re-tentionTimes®. Please, accept our apologies. As you see, we are back now. Yet, we are still seeking volunteer(s) who would take

full care of RetentionTimes® on a more permanent basis and help with the editing.

So, what happened during the last months? First of all, CASSS settled down in its new office in Emeryville, California. We finally have a full time executive direc-tor, Stephanie Flores, who is running the CASSS “business” like never before and it shows. Our portfolio of meetings that includes the evergreens such as CMC Strategy Forum, the symposia on the Interface of Regulatory and Analytical Sci-ences for Biotechnology Health Products (WCBP), MicroScale Bioseparations (MSB), Practical Applications of Mass Spectrometry in the Biotechnology and Pharmaceutical Industries, and CE in the Biotech & Pharma Industries, is grow-ing steadily. For example, this year we added International Ion Chromatography Symposium (IICS) to our list with the first meeting run by CASSS in September 2008. Since the International Symposium on Capillary Chromatography and Electrophoresis we organized in Albuquerque, New Mexico was such a sheer success, its next issue is coming to Portland, Oregon next year (May 17-23, 2009). This time, its scope will be widened and also includes the 6th GCxGC Confer-ence. In addition, CASSS is already working hard on the major domestic meeting in chromatography: The 35th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC) that will be chaired by Steve Cohen in Boston (June 19-24, 2010). In a slightly longer run, you should know that I will chair this meeting in 2012 in Anaheim (June 16-21, 2012).

In 2006 CASSS’ board of directors adopted an expanded mission to become the leading global society dedicated to providing forums for the dissemination of information and discussions of technology and applications in separation and related sciences. To better meet the needs of our international members, we are no longer organizing meetings only in the United States. Since 2007 CASSS has held meetings in Canada and Europe. In 2009 we will expand to Asia in a collaborative

RetentionTimes®

The Newsletter of Separation Sciences | Fall 2008

Presidents Message In This Issue

Presidents Message..............1

CASSS Names Professor Pat J. Sandra 2008 Scientific Achievement Award Winner...... 1

Electrostatic Repulsion- Hydrophilic Interaction Chromatography...................3

Non-Woven Electrostatic Media For Chromatographic Separation Of Biological Particles..............................6

2009 CASSS Calendar of Events.............................8

CASSS Names Professor Pat J. Sandra 2008 Scientific Achieve-ment Award Winner

CASSS’ Board of Directors and Associate Directors recognized Professor Pat J. San-dra winner of its Scientific Achievement Award in 2008. Each year CASSS bestows the Award on a notable scientist in recog-nition of his or her outstanding contribu-tions to the advancement of separation science. The award will be presented at a ceremony at the E.O. Lawrence Berkeley

Sandra Cont. on page 4Message Cont. on page 2


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effort with Dalian Institute of Chemical Physics to organize the 24th International Symposium on MicroScale Bioseparations (MSB) in Dalian, China (October 18-22, 2009).

But this is still not the end of our activities. As each year, we have also elected winner of the CASSS award for Distinguished Contributions to Separation Science. I am proud to announce that this year winner is a renowned icon of the GC and LC, and a long time friend of CASSS. The award presentation will be held in Berkeley, California on November 20, 2008. Prior to that, on October 22 will be another discussion group meeting in Burlingame, California with a lecture concerning a current hot topic in HPLC, the hydrophilic interaction chromatography or HILIC. And who better to give this talk than Dr. Andy Alpert, father of this technique. Visit www.casss.org for complete details and to register.

However, CASSS is not only meetings. You probably noted that our web site has been upgraded and contains a large number of pages packed with a variety of information, including an on-line member directory. I invite you to visit the site, go to the “Member Login” page and update your member profile. And we continue to seek ways to better meet the needs of members and encourage your comments – about the website…or the organi-zation in general.

We are also thinking about future of the separation science. It is clear to all of us that the future is not imaginable without new, young blood. Therefore, CASSS is supporting students to attend our meetings and to present their results there. We believe that this is the right way to make sure that chromatography and related disciplines will thrive in the years to come.

Today’s letter is a little bit longer than usual. Well, I had many issues to tell you about this time. Yet, I don’t think that my letter should be the only means of our communication. I would much better like to see you in person. Our meetings are certainly a good platform for shaking hands and having a chat. Please, come to see me.

Sincerely,

Frantisek Svec

Message Cont. from page 1

2AN INTERNATIONAL SEPARATION SCIENCE SOCIETY

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Hydrophilic interaction and electrostatic effects are inde-pendent forces. Electrostatic repulsion can be exploited to

selectively antagonize the retention of what would normally be the best-retained compounds in HILIC1. As a result, quite het-erogeneous mixtures of compounds can be resolved isocratically. An example is the isocratic separation of all common nucleotides using a cation-exchange column operated in the HILIC mode. ATP, CTP and GTP are retained far better than the other nucleotides in HILIC, but the electrostatic repulsion of their phosphate groups in ERLIC causes them to elute in the same time frame as the other compounds [below].

The same principle can be applied to amino acids, peptides and proteins. In these cases one uses an anion-exchange column and a pH low enough (typically, 2.0-2.2) to uncharge carboxyl- groups, leaving all the compounds with a net positive charge. Unusually ba-sic peptides are retained much better than neutral or acidic peptides in HILIC [TOP], but the electrostatic repulsion in ERLIC causes them to elute in the same time frame as all the other peptides [BOTTOM]. This is convenient for isocratic analysis of mixtures of unknown peptides.

If a peptide has a functional group that retains some negative charge even at pH 2.0-2.2, then that group will be attracted elec-trostatically to the anion-exchange material. With tryptic digests, peptides with phosphate groups are well-retained under conditions where the electrostatic repulsion leads the nonphosphopeptides to elute in or near the void volume [below].2,3

These conditions feature volatile solvents and can be adapted to solid-phase extraction. They also permit the selective isolation of any peptide with a functional group more acidic than n-Asp (pKa ~ 3.7). In addition to peptides containing phosphate (pKa ~ 2.1), this includes glycopeptides containing sialic acid ( pKa ~ 2.6)4 and possibly deamidated peptides that contain an isoAsp residue (pKa ~ 3.1)4. In effect, this is an affinity interaction that is weak compared to those with materials currently used for affinity isolations: titania, IMAC, lectins, etc. The interaction is still as strong as it needs to be, and its comparative weakness is actually an advantage. Since the interaction with the functional group does not completely domi-nate the chromatography, the column is sensitive to other aspects of peptide composition and the peptides within each class can be separated with high resolution. This combining of the affinity and high-resolution steps makes proteomics analyses faster and easier.

REFERENCES:1) A.J. Alpert, Anal. Chem. 80 (2008) 62-76: Electrostatic Repulsion Hydrophilic Interaction

Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides.

2) A.J. Alpert, S.P. Gygi, and A.K. Shukla, Poster MP 438, ASMS Conference, June 2007 (Indianapolis): Desalting Phosphopeptides by Solid-Phase Extraction.

3) A.J. Alpert, G. Mitulovic, and K. Mechtler, Poster P-2412-W, HPLC 2008 Conference, May 2008 (Baltimore): Isolation of Tryptic Phosphopeptides by ERLIC.

4) U. Lewandrowski, K. Lohrig, R.P. Zahedi, D. Wolters, and A. Sickmann, Clin. Proteom. Online 2008: Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography.

Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC):Selective Isolation of Phospho-and Glycopep tides and Gradient Separations Without the Gradientby Andrew Alpert, PolyLC Inc./ Columbia, MD/ [email protected]


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Berkeley National Laboratory in Berkeley, Cali-fornia on November 20, 2008. Prof. Sandra will give a lecture following the ceremony.

Prof. Sandra received his M.S. Degree in Chemistry in 1969 and his Ph.D. in Sciences in 1975 from the Ghent University, Belgium. Since then, he has been on the Faculty of Sciences

at the same University where he is currently Professor in Separa-tion Sciences at the Department of Organic Chemistry. In 1986 he founded the Research Institute for Chromatography (RIC) in Kortrijk, Belgium, a center of excellence for research and education in chromatography, mass spectrometry and capillary electrophore-sis. He is currently also extraordinary professor at the Department of Chemistry at the University of Stellenbosch, South Africa, at the Department of Analytical Chemistry, Evora, Portugal and director of the Pfizer Analytical Research Center at the Ghent University.

Pat Sandra is active in all fields of separation sciences (GC, LC, SFC and CE) and major areas of his research are high-throughput, high-resolution, miniaturization, hyphenation and automation to study chemicals, pharmaceuticals, natural products, food products and pollution. He is the author or co-author of more than 470 scientific papers and wrote books on High Resolution GC, Sample Introduction in Capillary GC, Essential Oil Analysis, Micellar Electrokinetic Chromatography and Water Analysis.

Pat Sandra has been highly recognized for his achievements in research and teaching. Included in his partial list of honors are: 1989 Tswett Award (Russia), the 1994 Dal Nogare Award (USA), the 1994 Martin Gold Medal (Chromatographic Society, UK), the 1995 Golay Award (USA), the 1996 Colacro Medal (Venezuela), the A1 Professor status in South Africa (2000), the 2004 ISEO Award (Sic-ily, Italy), the 2004 Chromatography Award (Buffelspoort, South Africa), the American Chemical Society Chromatography Award 2005 (USA) and EAS Award for Achievements in Separation Sci-ence. He also received the doctor honoris causa title in Pharmacy from the University of Turin, Italy (2004) and in Food Chemistry and Safety from the University of Messina, Italy (2007). He was appointed honorary professor at the Dalian Institute for Chemical Physics, Chinese Academy of Sciences, June 2007.

Other CASSS members who have recently received awards and recognition include Dr. Frantisek Svec of the Lawrence Berkeley National Laboratory and Dr. Ron Majors of Agilent Corporation.

ACS 2008 Chromatography Award goes to Frantisek Svec

The American Chemical Society awarded Frantisek (Frank) Svec its 2008 Award in Chromatography. The purpose of the award is to rec-ognize outstanding contributions to the fields of chromatography. To be eligible a nominee must have made an outstanding contribution to the fields of chromatography with particular consideration given to developments of new methods and be nominated by a peer.

Frank’s career in polymer chemistry has spanned over 40 years. He received both his B.S. in chemistry and Ph.D. degree in poly-mer chemistry from the Institute of Chemical Technology, Prague (Czech Republic) in 1965 and 1969, respectively. After spending time at the University of Karlsruhe (Germany) and the Institute of Chemical Technology, he joined the Institute of Macromolecular Chemistry (IMC) of the Czechoslovak Academy of Sciences. His sci-entific achievements were recognized in 1987 when he was awarded the degree of Doctor of Science from IMC. Frank’s interest in sepa-ration science began at IMC where his research in polymer materials eventually resulted in the introduction of monolithic disc media.

In 1992 Frank joined the faculty at Cornell University, where in col-laboration with Professor Jean Fréchet, he invented the monolithic chromatography column. He continued to work on this technol-ogy while at the same time developing new particulate packings

for HPLC. In 1997, he joined the Department of Chemistry at the University of California, Berke-ley and since 2005 has been at the E.O. Lawrence National Laboratory at Berkeley. He is best known for his research in the area of macroporous polymers in different shapes such as beads, flat sheets, and monoliths, and their use in a variety of applications including supports for solid phase chemistry, gas and liquid chromatography, electrochromatography, enzyme immobilization, and selective resins. A current interest lies in the rapidly developing field of microfluidics.

Dr. Svec is author or co-author of over 300 scientific publications, an extrordinary 60 patents, 30 book chapters and review articles and has edited several books. A recent book - Capillary Electro-chromatography (co-edited by Z. Deyl of the Institute of Physiol-ogy, Academy of Science of the Czech Republic) - was published by Elsevier in 2001.

He is an active participant in separation science affairs both locally and internationally. Frank is President of the CASSS Board of Di-rectors and is active in both the American and Czech Chemical So-cieties. He is a member of the editorial boards of many prestigious

Sandra Cont. from page 1

Past winners include:2007 - Shigeru Terabe

2006 - Pier Giorgio Righetti 2005 - Milton L. Lee

2004 - Milos V. Novotny 2003 - Barry Karger

2002 - Georges Guichon2001 - Fred Regnier


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international journals including the Journal of Separation Science, the Journal of Chromatography, Electrophoresis, Macromolecular Materials and Engineering and Chemical Letters (Prague).

England’s Chromatographic Society Recognizes Ronald Majors

CASSS Board Member Ronald E. (Ron) Majors has been awarded the 2007 Martin Gold Medal by the Chromatographic Society in the United Kingdom for his outstanding contributions to Chromatography.

Ron, who has been with HP/Agilent since 1990, is a Senior Chem-ist for Agilent’s Columns and Supplies Division and has over 250 publications in chromatography, sample preparation, and surface chemistry. In the late ‘60’s and early ‘70’s, he was one of the early researchers in high performance liquid chromatography (HPLC), a separation and analysis technique widely used in the chemical, bio-logical, environmental, and pharmaceutical industries. He has been active in the American Chemical Society’s Sub-Division on Chro-matography and Separations Chemistry, on the editorial boards of several analytical magazines, and was chairman of HPLC 86, one of the largest chromatography meetings ever held. Along with his active participation in CASSS, Ron is active in the Delaware Valley Chromatography Forum. However, he is probably best known for his monthly columns “Column Watch” and “Sample Preparation Perspectives” for LCGC Magazine.

The Chromatographic Society, based in England, is an interna-tional organization devoted to the promotion and dissemination of knowledge on all aspects of chromatography and related separa-tion techniques. It was first established as the Gas Chromatography Discussion Group in 1956 and just celebrated its golden jubilee anniversary year in 2006.

The Martin Gold Medal is the Society’s top award and bears the name of the Nobel Prize winner for chromatography, Archer Martin, who gave his permission for the Society to use his name. It is given to those individuals whose professional as well as scientific accomplishments have fostered progress in chromatography. This award is also recognition of the importance of the input of the key players from the commercial and practical side of separation science as opposed to efforts of the academic/theoretical scientists who often receive such awards.

Ron received this year’s Award based on his early contributions to HPLC column technology, specifically for his definitive work on particle size studies, packing methodologies, and chemi-cally bonded phases. He was the first to efficiently pack 5-micron particles into high performance columns and with the introduc-tion of the first commercial microparticulate column. In his award

announcement, Professor John Lough President, The Chromato-graphic Society and an educator at the University of Sunderland, United Kingdom, stated “In the promotion of separation science your achievements are unsurpassed. Your work for the Internation-al HPLC Series, in particular championing young poster presenters as the Poster Chairperson, has been very valuable for this important series of meetings. More importantly, your scientific writings for LCGC Magazine has been prolific, high quality and very relevant to the needs of practicing separation scientists”. For over 26 years, Ron has been a monthly columnist for LCGC Magazine writing practi-cal articles on column technology and sample preparation.

On receiving notification of this award, Ron said he was “honored to be even considered for the Martin Gold Medal, much less actual-ly receive it.” The Martin Medal has been won by many notable in-dustrial and academic researchers in the chromatography commu-nity. “I want to thank the Chromatographic Society for bestowing this Award on me and for their recognition of my contributions,” added Ron. Dr. Majors was presented the Award at HPLC 2008 in Baltimore this past May and delivered the Plenary Lecture at the Chromatographic Society’s “Recent Developments in Column and Stationary PhaseTechnology” held in the U.K. a week later.


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by Frederick Tepper, Leonid Kaledin and Tatiana Kaledin

Non-Woven Electrostatic Media for Chromatographic Separation Of Biological Particles

Introduction - The filtration and separation of biological par-ticles has been estimated as 40% of the cost in the manufacture

of pharmaceuticals. Liquid chromatography that is the principal method for separating biological particles uses a permanent phase of ultrafine adsorbent particles such as porous silica or resin spheres with particle sizes down to about 1 micron. Speed and resolution are two competing performance factors in conven-tional chromatography using porous media. One feature is often achieved by sacrificing the other. Conventional wisdom focuses on surface area as the defining element for “dynamic capacity” when determining product throughput employing conventional chromatographic media.

Nano alumina media – An electropositive fibrous media has been developed for water filtration that is capable of retaining sub-micron particles at high flow rates. The active component in the filter media is a nano alumina fiber, only 2 nm in diameter and about 250-300 nm long that are attached to a microglass fiber. Figure 1 shows the nanofibers that appear as fuzz on the microglass. The nanoalumina is aluminum oxide monohydrate (AlOOH), also known as the pseudoboehmite.

Figure 1 – Nano alumina uniformly distributed on a microglass fiber

The nano alumina as well as NanoCeram composite have isoelec-tric points at pH 11. Once attached to the microglass and then incorporated into a non-woven structure, the media is highly electropositive, with zeta potential of +50 mV at pH 7.

The nano alumina filter media is also highly effective in filtering out particles such as nucleic acids, proteins, cell debris and endo-toxins, as well as larger particles such as bacteria.

Use as a chromatographic media – We have a work in process de-velopment program to exploit the use of nano alumina media for chromatographic separation of macromolecules. The advantages of such a media are:

1. The charged sites of the nano alumina are exposed to the mov-ing phase. Particles do not have to traverse through pores that are fractions of a micron as in the case of porous silica. This increases the dynamic response for adsorption of particles.

2. Particles as large as hundreds of microns may be separable. Many capsided viruses are about 100 nm or so. They would be too large to separate by conventional HPLC adsorbent spheres.

3. The nano alumina is spatially constrained onto the microglass. No special packing requirements are envisioned.

4. Separations can occur at or near ambient pressure.

5. Separations have been performed at much higher flux as com-pared to packed columns or membrane chromatography devices.

6. The media is produced by low-cost paper-making manufacture. Approximately two tons of filter media have already been produced.

7. Salt would have little effect on separations. Proteins could be separated under salt free conditions

We elected to demonstrate feasibility of chromatographic use by separating two coliphages, MS2 (ATCC 15597-B1) and Alpha 3 (ATCC 13706-B2) that have identical particle size (27 nm). Both are uncapsided. And we also chose to use conventional culture to assay the viruses in eluted fractions. With live viruses, we would be assured that the particle was not affected in transit through the separation process.

We tried two types of eluents. The first was a solution of 3% beef extract, 0.25% glycine and at a pH of 9.3. This solution had been developed earlier for extracting virus from electropositive media. Protocols are being developed by EPA and others for assay of norovirus, adenovirus, polio and bird flu using this or similar eluents. The second eluent was a 0.01 M sodium carbonate solu-tion (pH= 10).

Experimental data to date – NC (“NanoCer-am”®) nano alumina media discs, 25 mm in diameter, were loaded with a mixture of approximately equal quantities of MS2 and alpha 3 viruses at input concentrations from 100 to1000 phage particles/milliliter at a flowrate of 10 mL/min. Elutions were performed by passing 0.5-2.5 ml aliquots of beef extract solution through a stack of 1, 3, 6 and 12 layers of the same filter media at a flowrate of 5 mL/min in the same flow direction as the adsorp-tion step. Results for 1 and 12 layers are shown in Figures 2 and 3.


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Figure 2 - Alpha 3 and MS2 viruses eluted through one NC disc

Figure 3 - Alpha 3 and MS2 viruses eluted through 12 layers of NC discs

Figures 2 and 3 show that the separation of the two viruses improves with increasing thickness of the media. Separation is effected at flow velocities of 1 cm/min and at less than 1 bar that are small fractions of that in HPLC separation. Further separation of viruses would occur if more than 12 layers were used.

Discussion and Conclusions - We have demonstrated feasibility of separation of peaks from viruses and presumably from other bio-logical macromolecules. Separations were effected within minutes rather than tens of minutes or hours when using conventional approaches and at pressures less than 1 bar.

1. Further work is continuing using sodium carbonate as an eluent. That direction include the use of thicker layers (more discs) to further separate MS2 and Alpha 3.

2. Modifying the nano alumina structure by adding a third compo-nent such as nano silica. Media composites have been produced that embody 26 weight percent nano silica (10 nm). The result-ing structure has an even higher zeta potential (+56 mV) than the basic nano alumina (+50 mV). Such variations of the surface of the nano alumina can alter the degree of attachment of dif-ferent molecules, allowing alternative chromatographic process conditions.

3. Alteration of the pore size. Future versions can range in pore size from as low as about 0.5 to as high as tens of microns.

The nano alumina media can be used in the separation of virus, peptides, proteins, oligonucleotides and nano size particles such as virus and plasmid DNA as well as their separation from cell debris and contaminants that have low molecular weight. Specific pro-teins, antigens or antibodies could be attached to the nano alumina to allow affinity chromatography.


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January15th CMC Strategy Forum: Raw Material Control Strate-gies for Bioprocesses (Karen Bertani)January 11, 2009, Intercontinental Hotel, San Francisco, CA USAForum Co-Chairs: John Dougherty, Eli Lilly and Company and Mark Schenerman, MedImmune. Program Co-Chairs: Greg Beck, Eli Lilly and Company, Ruth Cordoba-Rodriguez, CDER, FDA and Christopher Joneckis, CBER, FDA

WCBP 2009: 13th Symposium on the Interface of Regula-tory and Analytical Sciences for Biotechnology Health Products (Karen Bertani)January 12 – 14, 2009, Intercontinental Hotel, San Francisco, CA USASymposium Co-Chairs: Barry Cherney, CDER, FDA, Philip Krause, CBER, FDA, Wassim Nashabeh, Genentech, Inc.

FebruaryMSB 2009: 23rd International Symposium on MicroScale Bioseparations (Erin Kelch)February 1 – 5, 2009, Boston Park Plaza Hotel, Boston, MA USASymposium Chair: Jonathan Sweedler, University of Illinois

AprilCMC Strategy Forum Europe 2009 (Karen Bertani)April 27 – 29, 2009, Sana Hotel, Lisbon, PortugalForum Co-Chairs: TBD

May6th GCxGC SymposiumMay 17-18, 2009 Symposium Co-Chairs: John Dimandja, Spellman College and Philip Marriott, RMIT University

33rd Int’l Symposium on Capillary Chromatography & Electrophoresis (Erin Kelch)May 19 – 21, 2009, Marriott Portland Downtown WaterfrontSymposium Chair: Frantisek Svec, E.O. Lawrence Berkeley Na-tional Laboratory

July16th CMC Strategy Forum (Karen Bertani)July 27-28, 2009, Lister Hill Auditorium, NIH Campus, Bethesda, MD USAForum Co-Chairs: TBD

SeptemberSixth Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology and Pharmaceutical Industries (Erin Kelch)September 15 - 17, 2009, The Ritz-Carlton, Philadelphia, PA USASymposium Co-Chairs: Victor Ling, Genentech, Inc. and Anders Lund, Genzyme Corporation

32nd International Ion Chromatography Symposium (Karen Bertani)September 21-24, 2009, The Grand Hotel, Malahide, IrelandSymposium Co-Chairs: Brett Paull and Mirek Macka, Dublin City University

OctoberCE in the Biotech & Pharma Industries: 11th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules (Erin Kelch)October 9 - 16, 2009, Boston Park Plaza, Boston, USASymposium Co-Chairs: TBD

24th International Symposium on MicroScale Biosepara-tions (Stephanie)October 18 – 22, 2009, Dalian, ChinaSymposium Co-Chairs: Hanfa Zou and Yukui Zhang, Dalian Institute of Chemical Physics, Frantisek Svec, Lawrence Berkeley National Laboratory(Note: Organized by Dalian Institute of Chemical Physics. CASSS will provide marketing, registration and abstract support for non-Chinese attendees.)

NovemberBioassays Conference (Karen Bertani)November, 2009, Lister Hill Auditorium, NIH Campus, Bethesda, MD USAConference Co-Chairs: Chana Fuchs, CDER, FDA; Helena Mad-den, Biogen Idec Inc. and Robert Strouse, MedImmune

2009 CASSS Calendar of Events

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