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publication further described orientation effects which others have also called ion-pair normal phase or e-HILIC, reflecting retention mechanisms sensitive to a particular ionic portion of the analyte, either attractive or repulsive. ERLIC (eHILIC) separations need not be isocratic, but the net effect is the reduction of the attraction of a particularly strong polar group, which then requires less strong elution conditions, and the enhanced interaction of the remaining polar (opposite charged ionic, or non-ionic) functional groups of the analyte(s).Based on the ERLIC column invented by Andrew Alpert, a new peptide mapping methodology was developed with unique properties of separation of asparagine deamidation and isomerization. This unique properties would be very beneficial for future mass spectrometry based multi-attributes monitoring in biologics quality control.
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molecules. Use of a pH between 1 and 2 pH units will reduce the polarity of two of the three ionizable oxygens of the phosphate group, and thus will allow easy desorption from the (oppositely charged) surface chemistry. It will also reduce the influence of negatively charged carboxyls in the analytes, since they will be protonated at this low a pH value, and thus contribute less overall polarity to the molecule. Any common, positively charged amino groups will be repelled from the column surface chemistry and thus these conditions enhance the role of the phosphate's polarity (as well as other neutral polar groups) in the separation.
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to now allow separation based more on the basic and/or neutral functional groups of these molecules. Modifying the polarity of a weakly ionic group (e.g. carboxyl) on the surface is easily accomplished by adjusting the pH to be within two pH units of that group's pKa. For strongly ionic functional groups of the surface (i.e. sulfates or phosphates) one could instead use a lower amount of buffer so the residual charge is not completely ion paired. An example of this would be the use of a 12.5mM (rather than the recommended >20mM buffer), pH 9.2 mobile phase on a
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42:
225:, creating a liquid/liquid extraction system. The analyte is distributed between these two layers. However, HILIC is more than just simple partitioning and includes hydrogen donor interactions between neutral polar species as well as weak electrostatic mechanisms under the high organic solvent conditions used for retention. This distinguishes HILIC as a mechanism distinct from
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Similarly, the choice of pH affects the polarity of the solutes. However, for column surface chemistries that are strongly ionic, and thus resistant to pH values in the mid-range of the pH scale (pH 3.5–8.5), these separations will be reflective of the polarity of the analytes alone, and thus might be easier to understand when doing methods development.
267:), higher concentrations of buffer (c. 100 mM) are required to ensure that the analyte will be in a single ionic form. Otherwise, asymmetric peak shape, chromatographic tailing, and/or poor recovery from the stationary phase will be observed. For the separation of neutral polar analytes (e.g. carbohydrates), no buffer is necessary.
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In 2008, Alpert coined the term, ERLIC (electrostatic repulsion hydrophilic interaction chromatography), for HILIC separations where an ionic column surface chemistry is used to repel a common ionic polar group on an analyte or within a set of analytes, to facilitate separation by the remaining polar
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functional groups of its surface chemistry over its, slightly diminished (by pH), quaternary amine. Commensurate with this, these analytes will show a reduced retention on the column eluting earlier, and in higher amounts of organic solvent, than if a neutral polar HILIC surface were used. This also
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than neutral polar effects. This allows one to minimize the influence of a common, ionic group within a set of analyte molecules; or to reduce the degree of retention from these more polar functional groups, even enabling isocratic separations in lieu of a gradient in some situations. His subsequent
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For example, one could use a cation exchange (negatively charged) surface chemistry for ERLIC separations to reduce the influence on retention of anionic (negatively charged) groups (the phosphates of nucleotides or of phosphonyl antibiotic mixtures; or sialic acid groups of modified carbohydrates)
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By analogy to the above, one can use an anion exchange (positively charged) column surface chemistry to reduce the influence on retention of cationic (positively charged) functional groups for a set of analytes, such as when selectively isolating phosphorylated peptides or sulfated polysaccharide
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With surface chemistries that are weakly ionic, the choice of pH can affect the ionic nature of the column chemistry. Properly adjusted, the pH can be set to reduce the selectivity toward functional groups with the same charge as the column, or enhance it for oppositely charged functional groups.
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chromatographic surface can be used for HILIC separations. Even non-polar bonded silicas have been used with extremely high organic solvent composition, thanks to the exposed patches of silica in between the bonded ligands on the support, which can affect the interactions. With that exception,
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molecules by differences in polarity. Its utility has increased due to the simplified sample preparation for biological samples, when analyzing for metabolites, since the metabolic process generally results in the addition of polar groups to enhance elimination from the cellular tissue. This
274:), can be used to increase the mobile phase polarity to affect elution These salts are not volatile, so this technique is less useful with a mass spectrometer as the detector. Usually a gradient (to increasing amounts of water) is enough to promote elution.
1078:
Zhen, J., Kim, J., Zhou, Y., Gaidamauskas, E., Subramanian, S., & Feng, P. (2018, October). Antibody characterization using novel ERLIC-MS/MS-based peptide mapping. In MAbs (Vol. 10, No. 7, pp. 951-959). Taylor &
890:
Ahn, Joomi; Bones, Jonathan; Yu, Ying Qing; Rudd, Pauline M.; Gilar, Martin (2010-02-01). "Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 μm sorbent".
259:
and ion strength. In HILIC they can also contribute to the polarity of the analyte, resulting in differential changes in retention. For extremely polar analytes (e.g. aminoglycoside antibiotics (
314:. For the detection of polar compounds with the use of electrospray-ionization mass spectrometry as a chromatographic detector, HILIC can offer a ten fold increase in sensitivity over
569:"Volume and composition of semi-adsorbed stationary phases in hydrophilic interaction liquid chromatography. Comparison of water adsorption in common stationary phases and eluents"
925:
62:
940:"Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides"
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All ions partition into the stationary phase to some degree, so an occasional "wash" with water is required to ensure a reproducible stationary phase.
120:. The name was suggested by Andrew Alpert in his 1990 paper on the subject. He described the chromatographic mechanism for it as liquid-liquid
121:
113:
105:
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211:
861:
530:"Surface silanols in silica-bonded hydrocarbonaceous stationary phases: II. Irregular retention behavior and effect of silanol masking"
667:"Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry"
206:) can be used. Alcohols can also be used, however, their concentration must be higher to achieve the same degree of retention for an
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surface to separate phosphonyl antibiotic mixtures (each containing a phosphate group). This enhances the influence of the column's
80:
810:"Comprehensive evaluation of zwitterionic hydrophilic liquid chromatography stationary phases for oligonucleotide characterization"
862:"Hydrophilic Interaction Chromatography Using Silica Columns for the Retention of Polar Analytes and Enhanced ESI-MS Sensitivity"
628:"Separation of Sorbitol and Mannoheptulose from Fructose, Glucose and Sucrose on Reversed-Phase and Amine-Modified HPLC Columns"
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where analytes elute in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data.
58:
1100:
51:
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Glycosylation analysis by hydrophilic interaction chromatography (HILIC) – N-Glyco mapping of the ZP-domain of murine TGFR-3
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layer than the less polar compounds. Thus, a separation based on a compound's polarity and degree of solvation takes place.
1110:
315:
218:
198:("MeCN", also designated as "ACN") with a small amount of water. However, any aprotic solvent miscible with water (e.g.
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Bajad, Sunil U.; Lu, Wenyun; Kimball, Elizabeth H.; Yuan, Jie; Peterson, Celeste; Rabinowitz, Joshua D. (August 2006).
226:
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627:
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Other salts, such as 100–300 mM sodium perchlorate, that are soluble in high-organic solvent mixtures (c. 70–90%
454:"Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds"
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714:"A rapid method for simultaneous quantification of 13 sugars and sugar alcohols in food products by UPLC-ELSD"
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It is commonly believed that in HILIC, the mobile phase forms a water-rich layer on the surface of the polar
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Koh, Dong-wan; Park, Jae-woong; Lim, Jung-hoon; Yea, Myeong-Jai; Bang, Dae-young (2018).
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Petrus Hemström and Knut Irgum (2006). "Review: Hydrophilic
Interaction Chromatography".
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761:"Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique"
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HILIC phases can be grouped into five categories of neutral polar or ionic surfaces:
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399:"Stationary and mobile phases in hydrophilic interaction chromatography: a review"
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It may require cleanup to comply with
Knowledge's content policies, particularly
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Bij, Klaas E.; Horváth, Csaba; Melander, Wayne R.; Nahum, Avi (1981-01-09).
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increases their detection sensitivity by negative ion mass spectrometry.
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981:"Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography"
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The HILIC mode of separation is used extensively for separation of some
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that partly overlaps with other chromatographic applications such as
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Lardeux, Honorine; Guillarme, Davy; D'Atri, Valentina (2023-02-08).
116:. HILIC uses hydrophilic stationary phases with reversed-phase type
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groups. Electrostatic effects have an order of magnitude stronger
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928:(Application Note TOSOH Biosciences). Retrieved May 23, 2013.
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Redón, Lídia; Subirats, Xavier; Rosés, Martí (2021-10-25).
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relative to an aprotic solvent–water combination. See also
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separation technique is also particularly suitable for
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and ammonium formate, are usually used to control the
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A major contributor to this article appears to have a
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will have a stronger interaction with the stationary
318:because the organic solvent is much more volatile.
860:Eric S. Grumbach; et al. (October 2004).
98:hydrophilic interaction liquid chromatography
18:Hydrophilic interaction liquid chromatography
8:
979:Alpert, Andrew J.; et al. (June 2010).
759:Boguslaw Buszewski and Sylwia Noga (2012).
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81:Learn how and when to remove this message
1028:Ding, W.; et al. (September 2009).
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131:HILIC Partition Technique Useful Range
94:Hydrophilic interaction chromatography
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114:reversed phase liquid chromatography
1034:Molecular & Cellular Proteomics
626:Shaw, P. E.; Wilson, C. W. (1982).
212:Aqueous normal phase chromatography
938:Alpert, Andrew J. (January 2008).
632:Journal of Chromatographic Science
302:analysis and quality assurance of
194:for HILIC chromatography includes
106:normal phase liquid chromatography
25:
61:. Please discuss further on the
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730:10.1016/j.foodchem.2017.07.142
1:
905:10.1016/j.jchromb.2009.12.013
546:10.1016/S0021-9673(00)80282-4
471:10.1016/S0021-9673(00)96972-3
316:reversed-phase chromatography
827:10.1016/j.chroma.2023.463785
683:10.1016/j.chroma.2006.05.019
586:10.1016/j.chroma.2021.462543
893:Journal of Chromatography B
814:Journal of Chromatography A
671:Journal of Chromatography A
573:Journal of Chromatography A
534:Journal of Chromatography A
227:ion exchange chromatography
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1047:10.1074/mcp.M900088-MCP200
452:Alpert, Andrew J. (1990).
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777:10.1007/s00216-011-5308-5
644:10.1093/chromsci/20.5.209
458:Journal of Chromatography
423:10.1016/j.aca.2011.02.047
312:biologic medical products
221:vs. the water-deficient
122:partition chromatography
397:Jandera, Pavel (2011).
149:simple unbonded silica
507:10.1002/jssc.200600199
403:Analytica Chimica Acta
265:adenosine triphosphate
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33:Type of chromatography
1101:Laboratory techniques
153:or diol bonded phases
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59:neutral point of view
1111:Biochemistry methods
765:Anal. Bioanal. Chem
415:2011AcAC..692....1J
248:additives, such as
338:chemical potential
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110:ion chromatography
104:) is a variant of
1106:Molecular biology
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991:(12): 5253–5259.
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192:mobile phase
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186:Mobile phase
179:zwitterionic
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71:October 2015
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229:. The more
1090:Categories
985:Anal. Chem
944:Anal. Chem
876:2008-07-14
820:: 463785.
579:: 462543.
384:References
308:glycoforms
261:gentamicin
190:A typical
27:See also:
836:0021-9673
738:0308-8146
691:0021-9673
652:0021-9665
605:0021-9673
554:0021-9673
540:: 65–84.
431:0003-2670
364:sulfonate
352:polymeric
295:inorganic
293:and some
241:Additives
63:talk page
1079:Francis.
1066:19525481
1015:20481592
966:18027909
913:20036624
844:36641941
795:21879300
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699:16759663
613:34571282
515:16970185
439:21501708
173:cationic
1057:2742440
1006:2884984
786:3249561
480:2324207
411:Bibcode
360:betaine
291:organic
235:aqueous
208:analyte
204:dioxane
161:anionic
151:silanol
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246:Ionic
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157:amino
142:polar
102:HILIC
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909:PMID
840:PMID
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818:1690
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609:PMID
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306:and
140:Any
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