HICHROM. Chromatography Columns and Supplies. LC COLUMNS SeQuant ZIC-HILIC. Catalogue 9. Hichrom Limited

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1 HICHROM Chromatography Columns and Supplies LC COLUMNS SeQuant ZIC-HILIC Catalogue 9 The Markham Centre, Station Road Theale, Reading, Berks, RG7 PE, UK Tel: + (0) Fax: + (0) sales@hichrom.co.uk

2 MERCK MILLIPORE Merck Millipore SeQuant ZIC -HILIC and ZIC -philic Improved separation of hydrophilic and polar compounds Orthogonal selectivity to reversed-phase Compatible with LC-MS Excellent stability Merck SeQuant ZIC -HILIC and ZIC -philic phases have been developed for the separation of polar and hydrophilic compounds in HILIC (Hydrophilic Interaction Liquid Chromatography) mode. Phase Base Material (μm) Surface Area (m /g) ph Stability Max. Temp. ( C) ZIC-HILIC 00Å Spherical silica ZIC-HILIC 00Å Spherical silica.5, ZIC-pHILIC Polymeric The ZIC-HILIC and ZIC-pHILIC phases have a bonded stationary phase consisting of a highly polar, permanent zwitterionic sulphobetaine structure (see Figure ). Separation selectivity is favoured by the : zwitterion charge balance, which makes the column overall neutral, with weak, but important, ionic interactions. The highly hydrophilic suphobetaine structure is very good at establishing an immobilized water-rich layer within the stationary phase. Tuning selectivity of ZIC-HILIC and ZIC-pHILIC columns during method development is facilitated by the ph independent permanent zwitterion. Separations are achieved by hydrophilic partitioning of the analyte into the zwitterionic (hydrophilic) stationary phase. These interactions are typically promoted by using eluents with a high (50-95%) organic solvent (eg. acetonitrile) content. - 0mM ammonium acetate, ph 7 (60:0) for HILIC (5:95) for RP HILIC RPLC. Phe-Gly-Gly-Phe. Leu-Gly-Gly. Gly-Gly-Gly Figure. ZIC-HILIC phase Column: ZIC-HILIC (5µm, 50 x.6mm) - 5mM ammonium acetate, ph 6.8 (70:0) Flow rate: 0.5ml/min Detection: UV at nm. Morphine-6-glucuronide. Morphine--glucuronide. Morphine 0 Mins 5 0 Mins Mins Figure. Comparison of HILIC and RP separations of peptides Figure. Separation of morphine and glucuronated metabolites ZIC -HILIC ZIC-HILIC shows the highest separation efficiency and is suitable for most HILIC applications. ) Orthogonality Polar and hydrophilic compounds that exhibit poor retention and may be affected by wettability problems in reversed-phase HPLC, generally experience stronger retention on ZIC-HILIC columns. This orthogonality is illustrated in Figure by the inverted elution order of peptides on a ZIC-HILIC column compared with a reversed-phase column. Figure shows the separation of morphine and its glucuronated metabolites on a ZIC-HILIC column, using UV detection. The separation mechanism on ZIC-HILIC columns provides a novel selectivity in the analysis of peptides, carbohydrates, protein digests, plant metabolites, pharmaceuticals and various other polar compounds. These compounds are normally characterised by a small or negative log P value and have poor retention on reversed-phase columns. Analyte logp* Examples of polar and hydrophilic compounds with different log P values* that can be separated with ZIC-HILIC. In contrast, two compounds that are too hydrophobic to be retained (toluene and hexane) are also displayed. (*=octanol-water partition coefficient) sales@hichrom.co.uk Tel:

3 Merck Millipore ZIC -HILIC (continued) ) LC-MS Since typical eluents for HILIC consist of 0-97% acetonitrile in water or volatile buffer, ZIC -HILIC columns are ideal for LC-MS analyses. By changing from RP to HILIC, a large increase in sensitivity is often observed for hydrophilic analytes. Figure shows the LC-MS separation of N-acetyl-Lcysteine and citrulline on a ZIC-HILIC column. Figure 5 shows the increase in retention time (k) obtained with increasing percentages of acetonitrile for the LC-MS analysis of gamma-hydroxybutyric acid (GHB). Column: ZIC-HILIC (5µm, 00 x.6mm) - 0.M ammonium acetate, ph 5.6 (70:0) Flow rate: 0.5ml/min Detection: ESI-MS (-ve) Citrulline Column: ZIC-HILIC (.5µm, 50 x.mm) - 0.M ammonium formate, ph 6. Flow rate: 0.ml/min Detection: ESI-MS (-ve) m/z 0. k =. 80% CH k =. 75% CH N-Acetyl-L-cysteine k =.7 70% CH Mins 5 Figure. Separation of N-acetyl-L-cysteine and citrulline on ZIC-HILIC 0 Mins Figure 5. Analysis of gamma-hydroxybutyric acid ) Scalability In addition to the standard analytical and semi-preparative columns listed on page 77, the ZIC-HILIC range includes fused silica nano columns and glass-lined stainless steel capillary columns for down-scaling of methods. Methods developed on analytical ZIC-HILIC columns can also be successfully scaled up to preparative dimensions. Figure 6 illustrates that linear scale-up from analytical to preparative dimensions can be easily achieved. This example uses a large injection volume, equivalent to 0% of the column volume. In preparative work, the mass loadability of the column is an important parameter. As exemplified in Figure 7, ZIC-HILIC columns show a comparably high mass loadability. Column: A: ZIC-HILIC (PEEK, 5µm, 50 x.6mm) B: ZIC-HILIC (SS, 5µm, 50 x 0mm) - 0.M ammonium acetate (80:0) Flow rate: A:.0ml/min B: 0ml/min with CH Injection: A: 50µl and B: 5000µl of white wine diluted : with CH Detection: UV at 0nm Column: ZIC-HILIC (5µm, 50 x 0mm) - 5mM ammonium acetate (90:0) Flow rate: 8.6ml/min Injection: 0.5ml (-00mg/ml) in eluent. Ascorbic acid For free technical advice and support technical@hichrom.co.uk B A Mins 5 Figure 6. Separation scale-up on ZIC-HILIC 0 Mins Figure 7. Loadability of nicotinamide on ZIC-HILIC 7

4 Merck Millipore ZIC -HILIC (continued) ) Rapid Resolution and High Throughput A.5μm 0 x.mm PEEK column is available for high throughput analyses. Figure 8 shows an example of the rapid HILIC separation of oseltamavir (Tamiflu ) and oseltamavir carboxylate. Column: ZIC-HILIC (00Å,.5µm, 0 x.mm) - 0mM ammonium acetate, ph.5 Flow rate:.0ml/min Detection: MS (API). Oseltamavir. Oseltamavir carboxylate Secs Figure 8. Analysis of Tamiflu (oseltamavir) Please contact Hichrom for your copy of A Practical Guide to HILIC and other Merck Millipore literature. ZIC -philic The polymer-based ZIC -philic phase comprises the same sulphobetaine type zwitterionic functional group as the ZIC-HILIC phase and offers similar selectivity to the ZIC-HILIC columns. However, it can be used in a wider ph range, thereby extending the classes of analytes that can be separated by HILIC. In addition, the selection of optimum ionisation conditions for MS sensitivity may be enhanced. Figure 9 illustrates how the selectivity of the ZIC-pHILIC material for a mixture of phenolic acids can be enhanced by operating at elevated ph. In this example, the ph increase also results in longer retention and improved peak shape for these analytes. Figure 0 shows the sensitive LC-MS analysis of acetyl-coa and adenosine nucleotides on a ZIC-pHILIC column. Column: ZIC-pHILIC (00 x.6mm) - aqueous buffer (75:5) For ph 6.8, buffer is 7mM NH Ac For ph 9.6, buffer is 7mM NH HCO Column: ZIC-pHILIC (5µm, 50 x.mm) Eluent: A: CH B: 0mM (NH ) CO + 0.% NaOH Gradient: 0-60% B in 5mins Flow rate: 0.ml/min Detection: FTMS, ESI (-ve), scan m/z Courtesy of Okinawa Institute of Science and Technology, Japan 5 6. t 0. Pantothenate. Acetyl-CoA. AMP 5. CoA 6. ADP 7. ATP 7 ph Mins Mins Figure 0. Separation of acetyl-coa on ZIC-pHILIC ph Mins 0 Figure 9. Separation of phenolic acids on ZIC-pHILIC Please see page 77 for ordering information for ZIC-HILIC and ZIC-pHILIC columns. sales@hichrom.co.uk Tel:

5 Merck Millipore SeQuant ZIC -chilic Complementary selectivity for polar hydrophilic compounds Maximum LC-MS compatibility with minimised column bleed Excellent reproducibility and robustness SeQuant ZIC -chilic is a new zwitterionic stationary phase in which phosphorylcholine functional groups are bonded to the silica (see Figure ). This phase is designed for HPLC and LC-MS of polar hydrophilic compounds and offers complementary selectivity to reversed-phase and other HILIC columns. The selectivity features of ZIC-cHILIC are especially beneficial for analysing negatively charged polar compounds such as Figure. ZIC-cHILIC phase nucleosides and organic acids, but are also beneficial for the separation of positively charged hydrophilic molecules, including aminoglycosides and cations. ZIC-cHILIC exhibits weak attraction and repulsion electrostatic interactions as a result of the : charge balance of its zwitterionic bonding. These interactions enable separations to be optimised using low buffer concentrations, without interfering with detection techniques. Selectivity of ZIC-cHILIC versus ZIC-HILIC ZIC-cHILIC and ZIC-HILIC, which have differently oriented zwitterionic functional groups, show different selectivity towards polar hydrophilic compounds. The outermost, more accessible, charged moiety, usually dominates their interaction with solutes. Neutral compounds generally show similar retention on the two zwitterionic columns. Figure demonstrates the similarities and differences in selectivity shown by the two phases for the isocratic separation of positively charged benzyltrimethylamine (BTMA), along with toluene (void marker), uracil and cytosine. SeQuant ZIC-cHILIC Column dimensions: 00 x.6mm 5mM aqueous ammonium acetate, ph 6.8 (80:0) Flow rate: 0.5ml/min Temperature: C Detection: UV, 5nm SeQuant ZIC-HILIC BTMA. Toluene. Uracil. Benzyltrimethylamine. Cytosine Mins Mins 0 Figure. Selectivity of ZIC-cHILIC vs ZIC-HILIC For free technical advice and support technical@hichrom.co.uk LC-MS of Aminoglycoside Antibiotics Aminoglycoside antibiotics contain amino-modified sugar moieties, making them very hydrophilic and multiply positively charged at neutral and acidic ph. As a result, they are difficult to separate by reversed-phase and on most HILIC columns. The high hydrophilicity and weak repulsion electrostatic interactions of ZIC-cHILIC columns enable efficient separations of multiple structurally similar aminoglycosides to be achieved. Figure illustrates the gradient separation of a mixture of five aminoglycoside antibiotics. Please see page 77 for ordering information for SeQuant ZIC-cHILIC columns. Column: ZIC-cHILIC (μm, 00 x.mm) Eluent: A: CH with % formic acid B: 00mM ammonium acetate with % formic acid Gradient: Time (mins) %B Flow rate: 0.ml/min Temperature: 50 C Figure. LC-MS of aminoglycoside antibiotics. Streptomycin. Gentamicin. Paromomycin. Tobramycin 5. Neomycin 0 6 Mins

6 Please contact Hichrom for current prices. Tel: + (0) sales@hichrom.co.uk Merck Millipore Ordering Information - SeQuant ZIC -HILIC, ZIC -philic and ZIC -chilic ZIC -HILIC Nano and Capillary Columns (.5µm and 5µm, 00Å) Column Dimensions (mm) Guard Column 50 x x 0. 0 x x 0. 0 x.0 50 x.0 5 x 0.mm 5 x.0mm.5µm -,50 95, µm,0-8, /pk. 5/pk also available PEEK Glass lined stainless steel ZIC-HILIC Analytical PEEK Columns (.5µm, 00Å) Column Dimensions (mm) 0 x. 50 x. 00 x. 50 x. 50 x. 50 x.6.5µm ZIC-HILIC Analytical PEEK Columns (.5µm and 5µm, 00Å) Column i.d. (mm) Column Length (mm) Guard Column (PEEK).5µm Phase (5/pk) (/pk) 5µm Phase (5/pk) (/pk) Direct connect guard. 5µm, x.0mm for use with.mm i.d. columns Stand alone guard (includes column coupler). 5µm, 0 x.mm for use with.6mm i.d. columns ZIC-HILIC Semi-Preparative and Preparative Columns (Stainless Steel) (5µm, 00Å) Column Dimensions (mm) 50 x x 0 50 x 0 50 x 0 50 x. 50 x. 5µm,0,00,0,600,670, ZIC-pHILIC PEEK Columns (5µm) Column Length (mm) Column i.d. (mm) Guard Column (0 x.mm) 5µm Phase /pk (includes column coupler). /pk also available ZIC-cHILIC Nano and Capillary Columns (µm, 00Å) Column Dimensions (mm) Guard Columns (/pk) 50 x x x.0 5 x 0.mm 5 x.0mm µm PEEK-SIL hardware Glass-lined stainless steel ZIC-cHILIC Analytical PEEK Columns (µm, 00Å) Column i.d. (mm) Column Length (mm) Guard Kit (PEEK) (5µm, 0 x.mm) µm Phase /pk. Kit includes column coupler sales@hichrom.co.uk Tel: