HICHROM. Chromatography Columns and Supplies. LC COLUMNS Vydac. Catalogue 9. Hichrom Limited

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

2 GRACE Vydac Vydac columns, manufactured by Materials Technologies / Discovery Sciences, were developed for bioseparations and became the benchmark for the reversed-phase separation of proteins and peptides. Vydac 00Å wide pore phases are available in a wide range of dimensions from nano and capillary, to micro and analytical, to preparative and process scale. Vydac s Vydac Functional Group Bonding Particle Size (μm) Pore Size (Å) Carbon Load (%) Endcapped 08MS C8 Polymeric, 5, Yes MS C Polymeric, 5, 0 00 Yes 8MS C8 Polymeric, 5, Yes 8MS C8 Monomeric, 5 00 Yes 9MS Diphenyl Polymeric 5 00 Yes 0TP C8 Polymeric, 5, No 0TP C8 Polymeric, 5, No 08TP C8 Polymeric, 5, Yes TP C Polymeric, 5, 0 00 Yes 8TP C8 Polymeric, 5, Yes 9TP Diphenyl Polymeric 5, 0 00 Yes 8TP C8 Monomeric, 5, 0 00 Yes 8EV (Everest) C8 Monomeric 5, Yes 8DE (Denali) C8 Monomeric, 5, Yes Vydac MS s Five reversed-phase chemistries Reduced TFA requirement High protein recovery Vydac MS phases were developed from the original Vydac TP phases, by incorporating a surface treatment and proprietary bonding process. A variety of reversed-phase materials makes this product line suitable for the LC-MS analysis of small peptides to large intact, undenatured proteins. Trifluoroacetic acid (TFA) is frequently used for improving peak shape and symmetry in reversed-phase separations of peptides and proteins. Although its volatility permits easy removal from preparative fractions, it does contribute to the background signal at low UV wavelengths and also has a suppression effect on electrospray MS ion generation. Although a TFA concentration of 0.% is commonly used, changing the TFA concentration can change reversed-phase selectivity of peptides. The Vydac MS phases have undergone proprietary modifications that reduce the dependence on TFA and other ion-pairing reagents. In selected cases, these columns also perform well without the addition of TFA, acetic or formic acid. Figure shows the separation of four peptides on a 8MS column. Peak shapes are maintained down to 0.0% TFA, although selectivity changes.. Neurotensin -8 (fragment). Oxytocin. Angiotensin ll. Neurotensin 0.% TFA 0.05% TFA 0.0% TFA 0.0% TFA Mins Column: Vydac 8MS (5μm, 50 x.6mm) Eluent: A: 5% CH CN in H O with TFA as indicated B: 95% CH CN in H O with TFA as in A Gradient: T(mins) %B Flow rate:.5ml/min Figure. Separation of peptides on Vydac 8MS 0 Everest Columns High resolution for complex peptide digests Unique selectivity for hydrophilic and hydrophobic peptides The Everest phase is a monomerically bonded 00Å phase. A novel, proprietary silica treatment and unique bonding chemistry result in unique selectivity and increased sensitivity for hydrophilic and hydrophobic peptides from complex digests. This improved bonding also leads to increased recovery and the ability to detect trace level peptides. Everest columns are available from nano/capillary to preparative and process scale. Figure (see page 0) shows the analysis of a peptone on Everest C8 (8EV). sales@hichrom.co.uk Tel:

3 Vydac TP s Referenced in a large number of patents and publications Polymeric bonded phases have long column lifetimes The Vydac TP range of phases was the first generation of wide pore media phases developed in the Vydac range and became the industry standard for peptide and protein separations. The large pores of the 00Å TP silica give polypeptide molecules complete access to the interior of the silica pores. Column Selection for Polypeptides The appropriate reversed-phase column for a polypeptide separation should be selected based primarily on the hydrophobicity of the polypeptide and secondly on its molecular weight. Subtle differences in selectivity can be observed between analyses using 8MS or 8TP polymeric and 8MS or 8TP monomeric bonded C8 phases. Figure shows a comparison between the selectivity of the polymeric 8TP and monomeric 8TP phases for a mixture of peptides. Applications for Vydac MS and TP columns are summarised below. 08MS and 08TP Ideal for biomolecules 5-0,000 Da Enzymatic digest fragments Natural and synthetic peptides MS and TP Polypeptides larger than -5,000 Da Hydrophobic polypeptides Glycoproteins Haemoglobin variants Histones Human growth hormone Insulin variants Membrane proteins Column: Everest C8 (8EV) (50 x.6mm) Eluent: A: % TFA in H O B: % TFA in CH CN T(mins) %B Flow rate: ml/min Temperature: 0 C Mins Figure. Peptone analysis on Everest C8 (see page 9) 8MS, 8TP, 8MS and 8TP Small polypeptides less than -5,000 Da Enzymatic digest fragments (< proteins) Natural and synthetic peptides Multi-ring compounds 9MS and 9TP Columns: 50 x.6 mm Eluent: 0.% TFA - CH CN 0-0% linear gradient over 0 mins. Flow rate: ml/min. 8TP , 9 0, 5 6 Polypeptides with aromatic side chains Large, hydrophobic proteins Membrane-spanning peptides Lipid peptides Fusion proteins from inclusion bodies 8TP5, For free technical advice and support technical@hichrom.co.uk 8EV Complex peptide mixtures Tryptic protein digests (> proteins) Peptones Nano/Capillary Columns Ideal for small samples and low flow rates Direct flow to MS without stream splitting Maximise sensitivity 0 Mins. GY. VYV. Neurotensin fragment -8. AcRGGGGLGLGK-NH 5. RGAGGLGLGK-NH 6. AcRGAGGLGLGL-NH. AcRGVGGLGLGK-NH 8. Oxytocin 9. Met-enkephalin 0. Bradykinin. AcRGVVGLGLGK-NH. Neurotensin fragment 8-. Angiotensin ll. Leu-enkephalin 5. Neurotensin 6. Angiotensin l Figure. Separation of synthetic peptides on Vydac 8TP and 8TP phases For low flow applications, the Vydac and 5μm MS phases are available in prepacked capillary columns of 5μm (0.05mm), 50μm (0.5mm), 00μm (0.mm) and 500μm (0.5mm) i.d. and lengths of 50, 00, 50 and 50mm. 0 0

4 Please contact Hichrom for current prices. Tel: + (0) sales@hichrom.co.uk Capillary Columns Column Dimensions (mm) 50 x x x x x x x x MS 08MS MS MS MS MS MS MS MS5.55 MS MS MS5.050 MS5.055 MS5.055 MS5.505 MS5.50 MS5.55 MS5.55 8MS 8MS MS MS MS MS MS5.50 8MS5.55 8MS5.55 8MS 8MS MS MS MS MS MS5.50 8MS5.55 8MS EV 8EV EV EV EV5.50 8EV5.55 8EV5.55 Column Dimensions (mm) 50 x x x x x x x x MS 08MS MS5.0 08MS5.5 08MS5.5 08MS MS MS MS5.55 MS MS5.05 MS5.0 MS5.5 MS5.5 MS5.505 MS5.50 MS5.55 MS5.55 8MS 8MS5.05 8MS5.0 8MS5.5 8MS5.5 8MS MS5.50 8MS5.55 8MS5.55 8MS 8MS5.05 8MS5.0 8MS5.5 8MS5.5 8MS MS5.50 8MS5.55 8MS EV 8EV5.05 8EV5.0 8EV5.5 8EV5.5 8EV EV Prices for these part numbers may vary Microbore, Analytical and Semi-Preparative Columns μm Vydac Analytical Column Dimensions (mm) Guard Cartridges 50 x.6 00 x.6 50 x.6 (For.6mm i.d. Columns) MS - MS0 5 - GDMS 8MS - - 8MS5 50 8GDMS 08TP 08TP TP0 5-08GD TP TP05 TP0 5 - GD 8TP 8TP TP0 5 8TP5 50 8GD 8TP 8TP05 0 8TP0 5 8TP5 50 8GD Microbore Column Dimensions (mm) Guard Cartridges 50 x.0 50 x.0 50 x. 50 x. (For.mm i.d. Columns) 08MS 08MS MS5 5 08MS MS5 5 08GD5MS MS MS MS5 55 MS55 86 MS5 5-8MS 8MS55 5 8MS5 5 8MS55 8 8MS5 5 8GD5MS 8MS 8MS MS5 58 8MS55 9 8MS GD5MS 08TP 08TP TP TP TP GD5 TP TP55 55 TP5 565 TP55 9 TP5 5 GD5 8TP 8TP55 5 8TP5 5 8TP TP5 5 8GD5 5 9TP 9TP TP TP TP5 58 9GD5 8TP 8TP TP TP TP5 58 8GD5 8EV 8EV EV5 66 8EV EV GD5EV 60 Analytical Column Dimensions (mm) Guard Cartridges Semi-Prep Column Guard Cartridges (For. and.6mm Dimensions (mm) (For 0mm 50 x. 50 x. 50 x.6 50 x.6 i.d. Columns) 50 x 0 i.d. Columns) 08MS 08MS55-08MS55 08MS5 5 08GD5MS 5 08MS50,0 - MS - MS5 509 MS55 88 MS5 5 GD5MS MS50,60 GCC50 8MS - 8MS MS55 9 8MS5 56 8GD5MS 5 8MS50,0 8GCC50 5 8MS - - 8MS55 8MS5 5 8GD5MS TP 08TP TP TP TP5 5 08GD5 5 08TP50,0 - TP TP55 89 TP5 58 TP55 98 TP5 5 GD5 TP50,0 GCC50 8TP 8TP TP5 5 8TP TP5 5 8GD5 8TP50,0 8GCC50 5 9TP 9TP TP5 58 9TP55 9 9TP5 50 9GD5 9TP50,50-8TP 8TP TP5 58 8TP TP5 5 8GD5 8TP50,50-8EV - - 8EV55 5 8EV GD5EV 6 8EV50,0 8GCC50EV Other dimensions available /pk. Use with All-Guard TM holder 800 ( 8) includes column coupler /pk. 0 x 0mm. Use with prep guard holder 0 ( ) Please enquire for Vydac TP preparative and process scale columns and for bulk material. sales@hichrom.co.uk Tel:

5 Please contact Hichrom for current prices. Tel: + (0) sales@hichrom.co.uk Vydac 0TP and 0TP C8 Vydac 0TP and 0TP columns were developed specifically for the separation and quantification of polyaromatic hydrocarbons (PAHs) required by environmental regulations. Beyond the 6 EPA priority pollutant PAHs, these columns are used to separate many other PAHs, such as methylated naphthalenes. Vydac 0TP columns also have application in the analysis of carotenoids, retinoids and vitamins. The higher carbon-loaded 0TP phase offers alternative selectivity and is also ideal for derivatized PAH samples or high throughput PAH analyses. Figure shows the analysis of priority pollutant PAHs in accordance with EPA Methods 550, 60 and 80. Vydac Column Dimensions (mm) Guard Cartridges μm 50 x. 50 x. 50 x. 50 x.6 50 x.6 (/pk) 0TP - 0TP GD5T 5μm 0TP 0TP5 5 0TP TP TP5 5 0GD5T 0TP TP55 8-0GD5T Further column dimensions available Use with holder 800 ( 8) includes column coupler 0µm particle size material available Column: Vydac 0TP55 (50 x.6mm) Eluent: H O - CH CN, hold at 50% CH CN for mins, then linear gradient to 00% CH CN in mins. Flow rate:.5ml/min Naphthalene. Acenaphthylene. Acenaphthene. Fluorene 5. Phenanthrene 6. Anthracene. Fluoranthene 8. Pyrene 6 9. Benz(a)anthracene 0. Chrysene. Benzo(b)fluoranthene. Benzo(k)fluoranthene. Benz(a)pyrene. Dibenz(a,h)anthracene 5. Benzo(g,h,i)perylene 6. Indeno(,,,c,d)pyrene Mins Figure. Analysis of priority pollutant PAHs Denali TM Columns High retentivity LC-MS of small molecules Fully scalable from capillary to process 5. Ketoprofen. Naproxen. Fenoprofen. Flurbiprofen 5. Ibuprofen For free technical advice and support technical@hichrom.co.uk The Denali TM phase (Vydac 8DE) is a novel, monomerically bonded 0Å C8 silica with high carbon coverage. It shows excellent reproducibility and peak symmetry for the analysis of both acidic and basic analytes. The phase has applications for small molecule analyses of interest to pharmaceutical and environmental laboratories. Figure 5 shows the separation of five acidic anti-inflammatory compounds on a Denali column. Peaks are well resolved and symmetrical for all analytes Mins Column: Denali C8 (Vydac 8DE) (50 x.6mm) Eluent: CH OH - H O (65:5) with 0mM KH PO, ph.0 Flow rate: ml/min Temperature: Ambient Figure 5. Separation of anti-inflammatories on Denali C8 Column Dimensions (mm) Guard Cartridges 50 x. 50 x. 50 x.6 50 x.6 For.mm i.d. Columns For.6mm i.d. Columns 6 9 Denali C8 8DE55 8DE5 8DE55 8DE5 8GD5DE 8GD5DE Other dimensions and particle sizes available /pk. Use with All-Guard TM holder 800 ( 8) includes column coupler

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