Fast separation of glycopeptides demands high selectivity, speed, and run-to-run reproducibility. Reversed-phase chromatography has limited retention and selectivity of hydrophilic peptides, including glycopeptides. Hydrophilic Interaction Chromatography (HILIC) is used for the analysis of hydrophilic samples. If using a 300Å pore size column, the separation of glycopeptides can be achieved.

Now you can achieve fast, high-resolution separation of polar glycopeptides

ZORBAX Rapid Resolution High Defi nition (RRHD) 300-HILIC columns for glycopeptide analysis feature 1.8 µm particles, a 300Å pore size, and are stable to 1200 bar. These enable you to resolve peptide glycoforms for identifi cation of post-translational modifi cations (glycosylation) in a complex peptide map.

• Greater selectivity: HILIC functionality is specifi cally designed to resolve hydrophilic glycopeptide variants.

• Excellent peak shape for polar glycopeptides: These polar compounds have limited retention or resolution by reversed-phase columns.

• Fast analysis and 1200-bar stability: 1.8 µm particles allow ZORBAX RRHD 300-HILIC columns to be used with UHPLC for faster separations without compromising data accuracy.

• An Agilent exclusive: No other manufacturer offers a sub 2-µm, 300Å HILIC column.• More complete sample analysis: Orthogonal to reversed-phase, the HILIC

information complements the separation you get from peptide mapping columns such as the Agilent AdvanceBio Peptide Mapping column.

CONFIRM TARGET PROTEIN IDENTITY

QUICKLY AND CONFIDENTLY

Agilent ZORBAX RRHD 300-HILIC 1.8 µm columns

ZORBAX RRHD 300-HILIC columns are part of the Agilent RRHD family of columns. For additional information visit www.agilent.com/chem/library and enter the publication numbers listed below.

5990-8124EN ZORBAX RRHD 300Å 1.8 μm Brochure

5990-9214ENZORBAX RRHD Family Brochure

5990-9384EN BioHPLC Column Selection Guide

Agilent has HILIC columns available in 1.8 to 3.5 µm particle sizes, including Poroshell 120 HILIC, 2.7 µm for small molecule analysis

Find the right HPLC column and sample prep product for your application with our interactive tool:www.agilent.com/chem/navigator

vitty,y speed, and run-to-runun mittede retention and selectivity ooff lic Intn eraction ChromatographyIf usingg a a 300Å pore size column,

OTTEIN IDENTITY

DENNTLY

µm columns

ZORBAX RRHD 3000-0 HIHILILICC ccollumns are part of the Agilent RRHD family ofcolumns. For additional information visitwww.agilent.com/chem/library and y

enenteterr ththee pupublblicicatatioionn nu bmbers listed below

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This information is subject to change without notice. © Agilent Technologies, Inc., 2013Printed in USA, March 6, 20135991-1435EN

To learn more, visit www.agilent.com/chem/BioHPLC

Or fi nd your local Agilent Representative or Agilent Authorized Distributor at www.agilent.com/chem/contactus

Ordering Information

Part Number Description Dimensions

857750-901 ZORBAX RRHD 300-HILIC, 1.8 µm 2.1 x 50 mm

858750-901 ZORBAX RRHD 300-HILIC, 1.8 µm 2.1 x 100 mm

Gradient tableTime %A %B

0 95 515 0 10015.1 95 525 95 5

Peptide mapping is used for characterization and impurity profi ling of protein biotherapeutics. Reversed-phase UHPLC/HPLC is routinely used but when the digest contains hydrophilic peptides, such as glycopeptides, valuable information may be missed. The ZORBAX RRHD 300-HILIC column retains the hydrophilic glycopeptides, and when coupled with mass spectrometry provides identifi cation of this important group of protein fragments. Figure 1a shows the separation of an erythropoietin (EPO) peptide map using the ZORBAX RRHD 300-HILIC 2.1 x 100 mm column and Figure 1b the extracted compound chromatograms of matched EPO.7 peptides were identifi ed from the HILIC-MS data that were not identifi ed by RP-MS.HILIC is orthogonal to RP and provides additional resolution of the hydrophilic glycopeptides, in a protein enzyme digest.

Column: ZORBAX RRHD 300-HILIC, 2.1 x 100 mm, 1.8 µmMobile phase A: 100% ACNMobile phase B: 50 mM ammonium formate, pH 4.5Sample: Glycopeptide from digested EPO protein (1 mg/mL)Injection: 5 µgFlow rate: 0.4 mL/minDetection: UV@280 nm Instrument: Agilent 1290 Infi nity UHPLC, Agilent 6224 Accurate Mass

TOF-MS, Agilent Dual ESI Source in Positive Ion Mode

2 2 4 4 66 88 10 10 12120

100

200

300

400

500

Identify the glycopeptides in a protein tryptic digestReliably determine changes in the protein glycosylation profi le

Agilent ZORBAX RRHD 300-HILIC columns are part of the AdvanceBio Family of columns, designed for

faster analysis and effi ciency in your lab. To learn more about this column family, visit

www.agilent.com/chem/AdvanceBio

Method

Figure 1a: UV Figure 1b: Mass Spec