HPLC column C18 C8 PFP. SunShell. Core Shell Particle

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1 HPLC column C8 C8 PFP SunShell Core Shell Particle ChromaNik Technologies Inc.

2 SunShell is a core shell silica column made by ChromaNIk Technologies. The next generation to Core Shell particle Features of SunShell *. µm of core and 0. µmof superficially porous silica layer *Same efficiency and high throughput as a Sub µm particle *Same pressure as a µm particle (less than a half then a sub µm particle) *Same chemistry as Sunniest technology (reference figure ) *Good peak shape for all compounds such as basic, acidic and chelating compounds *High stability ( ph range for SunShell C8,. to 0) * Low breeding. µm 0. µm Core 0. µm. µm Core Porous silica Schematic diagram of a core shell silica particle. µm Superficially porous silica H H Final TMS H Schematic diagram of bonding of SunShell C8 H Van Deemter Equation A term : Eddy diffusion(dp is particle diameter) B term : Longitudinal diffusion (Dm is diffusion coefficient) C term : Mass transfer B term A term Van Deemter plot u C term HETP, µm m Comparison of plate height plots Fully porous um Fully porous um 0 0 Mobile phase velocity, mm/sec Fully proous.8 um SunShell. um Fully porous. um C8, 0 x. mm C8 Mobile phase: Acetonitrile/water=(0/0) Temperature: o C Sample : Naphthalene Back pressure, MPa Flow rate, ml/min Sunniest C8-HT.0 um Brand A C8.9 um Brand B C8.8 um Brand C C8. um Brand D C8. um SunShell C8. um Column dimension: 0 x. mm Mobile phase: Acetonitrile/water=(0/0) Temperature: o C Comparison of back pressure for high throughput columns SunShell C8 shows same efficiency as a sub µm C8. In comparison between fully porous. µm and core shell. µm (SunShell), SunShell shows lower values for A term, B term and C term of Van Deemter equation. The core shell structure leads higher performance to compare with the fully porous structure. Furthermore back pressure of SunShell C8 is less than a half to compare with sub- µm C8s.

3 Why does a. µm core shell particle show the same performance as a sub µm particle? Company F, µm D 0 :. µm D 0 :.09 µm D 90 :. µm D 90 /D 0 =.9 Sunniest, µm D 0 :. µm D 0 :.0 µm D 90 :. µm D 90 /D 0 =. SunShell,. µm D 0 :. µm D 0 :. µm D 90 :.8 µm D 90 /D 0 =. Wide particle distribution (Conventional silica gel D 90 /D 0 =.0) Flow of mobile phase Narrow particle distribution (core shell silicad 90 /D 0 =.) 0. (µm) Comparison of Particle ze Distribution Packing state of core shell and fully porous silica The size distribution of a core shell (SunShell) particle is much narrower than that of a conventional fully porous particle, so that the space among particles in the column reduces and efficiency increases by reducing Eddy Diffusion (multi-path diffusion) as the A term in Van Deemter Equation. Comparison of diffusion path As shown in the left figure, a core shell particle has a core so that the diffusion path of samples shortens and mass transfer becomes fast. This means that the C term in Van Deemter Equation reduces. In other words, HETP (theoretical plate) is kept even if flow rate increases. A. µm core shell particle shows as same column efficiency as a fully porous sub- µm particle. The right figure shows that a diffusion width of a sample in a. µm core shell particle and a µm fully porous particle. Both diffusion widths are almost same. The. µm core shell particle is superficially porous, so that the diffusion width becomes narrower than particle size. Same diffusion means same efficiency. Diffusion Ca..0 µm sample Core Shell. µm. µm Sample emanates in a particle after it enters. Diffusion.0 µm sample Totally porous.0 µm Diffusion of sample in core shell and fully porous silica Comparison of Performance by Plate/Pressure Plate Back press. (MPa) Plate/back press. Sunniest C8 HT.0 µm 9, Brand A C8.9 µm,0. 0 Brand B C8.8 µm 0,00 9. Brand C C8. µm,0.0 8 SunShell C8. µm 9, Under a constant back pressure condition, SunShell C8 showed more than times higher performance to compare with fully sub-µm porous C8s. Sunniest C8 HT.0 µm Brand A C8.9 µm Brand B C8.8 µm Brand C C8. µm SunShell C8. µm 0 x. mm C8, Mobile phase: Acetonitrile/water=(0/0), Temperature: o C

4 Characteristics of SunShell C8 Particle size (µm) Core shell silica Pore diameter (nm) Specific surface area (m /g) Carbon content (%) Bonded phase C8 Maximum operating pressure Available ph range SunShell C C8 0 MPa or 8,0 psi. -0 Comparison of standard samples between core shell C8s Brand D C8 Brand E C8 Brand D C8,. µm 0 x. mm (Core shell type) Brand E C8,. µm 0 x. mm (Core shell type) SunShell C8,. µm 0 x. mm Mobile phase: CH H/H =/ Flow rate:.0 ml/min Temperature: 0 º C Sample: = Uracil, = Caffeine, = Phenol, = Butylbenzene = o-terphenyl, = Amylbenzene, = Triphenylene SunShell C8 (core shell) Hydrogen bonding (Caffeine/Phenol) Hydrophobicity (Amylbenzene/Butylbenzene) Steric selectivity (Triphenylene/o-Terphenyl) Brand D C Brand E C SunShell C Retention of standard samples was compared for three kinds of core shell type C8s. Brand D C8 showed only a half retention to compare with SunShell C8. Steric selectivity becomes large when ligand density on the surface is high. SunShell C8 has the largest steric selectivity so that it has the highest ligand density. This leads the longest retention time. Comparison of pyridine Comparison of xine Comparison of formic acid Brand D C8 Brand D C8 Brand D C8 Brand E C8 Brand E C8 Brand E C8 SunShell C8 (Core Shell) SunShell C8 SunShell C8 0 Brand D C8,. µm 0 x. mm Brand E C8,. µm 0 x. mm SunShell C8,. µm 0 x. mm Mobile phase: CH H/H =0/0 Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0nm Sample: = Uracil = Pyridine = Phenol Brand D C8,. µm 0 x. mm Brand E C8,. µm 0 x. mm SunShell C8,. µm 0 x. mm Mobile phase: CH CN/0mM H P =0/90 Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0nm Sample: = 8-Quinolinol (xine) = Caffeine Brand D C8,. µm 0 x. mm Brand E C8,. µm 0 x. mm SunShell C8,. µm 0 x. mm Mobile phase: CH CN/0.% H P =/98 Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0nm Sample: = Formic acid = Acetic acid = Propionic Acid Residual silanol groups make pyridine be tailing under methanol/water mobile phase condition. nly SunShell C8 shows a sharp peak for pyridine. 8-Quinolinol (xine) is a metal chelating compound. Metal impurities in the core shell particle leads the tailing for oxine peak. Formic acid is used as an indicator for a acidic inertness. SunShell and brand D C8 show a sharp peak.

5 Loading capacity of amitriptyline as a basic compound Amitriptyline overlords much more at acetonitrile/buffer mobile phase than methanol/buffer. Three kinds of core shell C8s were compared loading capacity of amitriptyline at three different mobile phases. Common condition: Column dimension,0 x. mm, flow rate;.0 ml/min, temperature; 0 o C Mobile phase: Acetonitrile/0mM phosphate buffer ph.0=(0:0) Mobile phase: Acetonitrile/0mM acetate ammonium ph.8=(0:0) 00 times 0 times SunShell C8 Brand D C8 Sample: = Uracil (0.0µg) = Propranolol (.µg) = Nortriptyline (0.µg) = Amitriptyline (0.µg) SunShell C8 Brand D C8 Brand E C8 Brand E C Theoretical plate was calculated by σ method using peak width at.% of peak height..% Mobile phase: Acetonitrile/0.% formic acid=(0:0) USP tailing factor..9. times.8. Amitriptyline overloads at low weight when acetonitrile/0.% formic acid mobile phase. A peak is shifted forward under overloading. Brand D C8 overloaded at more than 0.0 µg of amitriptyline while SunShell C8 overloadsed at more than from 0. to µg of amitriptyline. Surprisingly loading capacity of Brand D C8 was only one hundredth to compare with SunShell C8 under acetonitrile/0mm phosphate buffer ph.0=(0:0) mobile phase. Brand E C8 always showed poor peak of amitriptyline.

6 Evaluation of Stability Accelerated acidic test Alkaline test % TFA 80 ºC ph0 0 ºC Durable test condition Column size: 0 x. mm Mobile phase: CH CN/.0% TFA, ph=0/90 Flow rate: 0. ml/min Temperature: 80 ºC Measurement condition Column size: 0 x. mm Mobile phase: CH CN/H =0/0 Flow rate: 0. ml/min Temperature: 0 ºC Sample: = Uracil (t 0 ) = Butylbenzene Stability under acidic ph condition was evaluated at 80 ºC using acetonitrile/% trifluoroacetic acid solution (0:90) as mobile phase. 00% aqueous mobile phase expels from the pore of packing materials by capillarity and packing materials doesn t deteriorate. 0% acetonitrile in a mobile phase allows an accurate evaluation. -) Sunshell C8 has kept 90% retention for 00 hours under such a severe condition. SunShell C8 is to 0 times more stable than the other core shell C8. ) N. Nagae, T. Enami and S. Doshi, LC/GC North America ctober 00. ) T. Enami and N. Nagae, American Laboratory ctober 00. ) T. Enami and N. Nagae, BUNSEKI KAGAKU, (00) 09. Durable test condition Column ze: 0 x. mm Mobile phase: CH H/0mM Sodium borate/0mm NaH=0//9 (ph0) Flow rate: 0. ml/min Temperature: 0 ºC Measurement condition Column ze: 0 x. mm Mobile phase: CH CN/H =0/0 Flow rate: 0. ml/min Temperature: 0 ºC Sample: = Butylbenzene Stability under basic ph condition was evaluated at 0 ºC using methanol/sodium borate buffer ph 0 (0:0) as mobile phase. Sodium borate is used as a alkaline standard solution for ph meter, so that its buffer capacity is high. Elevated temperature of 0 ºC makes column life be one third. The other company shows stability test at ambient (room temperature). If room temperature is ºC, column life at room temperature ( ºC) is sixteen times longer than that at 0 ºC. SunShell C8 is enough stable even if it is used under ph 0 condition. Regarding stability under basic ph condition, there is little C8 column like SunShell C8 except for hybrid type C8. It is considered that our end-capping technique leads high stability. SunShell C8 can be used at the ph range from. to 0.

7 Particle size (µm) Core shell silica Pore diameter (nm) Specific surface area (m /g) SunShell の基本特性 SunShell C8, PFP(Pentafluorophenyl) PFP Characteristics of SunShell Carbon content (%) Bonded phase Bonded phase Maximum operating pressure Available ph range SunShell C C8 0 MPa or 8,0 psi. -0 SunShellC C8 0 MPa or 8,0 psi. -9 SunShell PFP Pentafluorophenyl 0 MPa or 8,0 psi - 8 Comparison of standard samples, SunShell PFP SunShell C8 SunShell PFP,. µm 0 x. mm SunShell C8,. µm 0 x. mm SunShell C8,. µm 0 x. mm Mobile phase: CH H/H =/ Flow rate:.0 ml/min Temperature: 0 ºC Sample: = Uracil, = Caffeine, = Phenol, = Butylbenzene = o-terphenyl, = Amylbenzene, = Triphenylene SunShell C8 Hydrogen bonding (Caffeine/Phenol) Hydrophobicity (Amylbenzene/Butylbenzene) Steric selectivity (Triphenylene/o-Terphenyl) SunShell PFP SunShell C SunShell C Retention of standard samples was compared for three kinds of phases such as C8, C8 and PFP. C8 showed the highest hydrophobicity and PFP showed both the highest steric selectivity and the highest hydrogen bonding. The feature of PFP phase is to have hydrogen bonding, dipole-dipole interaction, aromatic and pi-pi interactions and hydrophobicity, which causes a different selectivity from a C8 phase. Separation of amitriptyline using C8 Separation of xanthines Separation of isomers of xylene SunShell C8 Water SunShell C8 SunShell PFP Water 0 SunShell C8,. µm 0 x. mm Mobile phase: CH CN/0mM phosphate buffer H.0=0/0 Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0nm Sample: = Uracil = Propranolol = Nortriptyline = Amitriptyline SunShell C8 showed a sharp peak for amitriptyline a well as SunShell C8. SunShell PFP SunShell PFP 0mM phosphate buffer ph 0mM phosphate buffer ph 0 SunShell C8, PFP. µm 0 x. mm Mobile phase: CH H/water or buffer=0/0 Flow rate: 0. ml/min Temperature: ºC Detection: UV@0nm Sample: = Theobromine = Theophyline = Caffeine = Phenol SunShell PFP can retain xanthines more than SunShell C8. The higher the concentration of buffer, the longer the retention time. SunShell PFP 0 SunShellC8, PFP. µm 0 x. mm Mobile phase: CH H/water=: for SunShellC8 CH H/water=0:0 for SunShellPFP Flow rate: 0. ml/min Temperature: ºC Detection: UV@0nm Sample: = o-xylene = m-xylene = p-xylene SunShell PFP showed the different selectivity from SunShell C8.

8 Efficiency of SunShell C8 UHPLC SunShell C8, 0 x. mm Plate()=0,00 0 SunShell C8, 00 x. mm Plate()=,00 SunShell C8,. µm 0 x. mm Mobile phase: CH CN/H =0/0 Flow rate: 0. ml/min Pressure: MPa Temperature: ºC Sample: = Uracil = Toluene = Acenaphthene = Butylbenzene SunShell C8,. µm 00 x. mm Mobile phase: CH CN/H =0/0 Flow rate: 0. ml/min Pressure:. MPa Temperature: ºC 0 8 SunShell C8,0 x. mm Efficiency=,000 plate/m SunShell C8, 0 x. mm Plate()=8,000 Plate()=,000 HPLC 80% efficiency to compare with UHPLC SunShell C8,. µm 0 x. mm SunShell C8,. µm 00 x. mm Mobile phase: CH CN/H =0/0 Flow rate:.0 ml/min Pressure:.MPa(UHPLC),. MPa(HPLC) for 0 mm 9.MPa(HPLC) for 00 mm Temperature: ºC Sample: = Uracil = Toluene = Acenaphthene = Butylbenzene SunShell C8, 00 x. mm Plate()=0, LaChrom ELITE The same efficiency as µm, 0 x. mm Saving 0% for analytical time and consumption of solvent

9 Examples of transfer from a conventional µm column to SunShell column Isocratic separation HPLC / of analysis time N()=0,8 SunShell C8,. µm 00 x. mm ACEC8, µm 0 x. mm 0 N()=9, N()=0,8 ACE C8, µm 0 x. mm SunShell C8,. µm 00 x. mm Mobile phase: CH CN/0mM Phosphoric acid = / Flow rate:.0 ml/min,.8 ml/min at the lowest chromatogram Temperature: ºC Pressure: 9. MPa for ACE C8 µm. MPa for SunShell C8. µm Detection: UV@0 nm Sample: = Benzydamine = Ketoprofen = Naproxen = Indomethacin = Ibuprofen UHPLC.0 ml/min N()=, HPLC: Hitachi LaChrom ELITE(using 0. mm i.d. tubing) UHPLC: Jasco X-LC.8 ml/min N()=, Analysis time decreases to / to compare with a µm column sized 0 x. mm. 0 Gradient separation HPLC Sunniest C8, µm 0 x. mm SunShell C8,. µm x. mm 8 9 / of Analysis time 8 9 Sunniest C8, µm 0 x. mm SunShell C8,. µm x. mm Mobile phase: A) 0.% Phosphoric acid B) CH CN Gradient program for Sunniest C8 for SunShell C8 Time 0 min min 0 min %B % % % Time 0 min. min 0 min 0 %B % % % Flow rate:.0 ml/min, Temperature: ºC Detection: UV@0 nm 8 9 Sample: olong tea = Gallocatechin, = Epigallocatechin, = Catechin, = Caffeine, = Epicatechin, 0 = Epigallocatechin gallate, = Gallocatechin 8 9 gallate, 8 = Epicatechin gallate, 9 = Catechin gallate UHPLC HPLC: Hitachi LaChrom ELITE (using 0. mm i.d. tubing) UHPLC: Jasco X-LC <<Caution>> There are difference of system time lag between HPLC and UHPLC. UHPLC has much less system time lag than HPLC because of high pressure gradient system for UHPLC and low pressure gradient system for HPLC.

10 SunShell RP Guard Filter <Cartridge Type, Available as a guard column for reversed phase Bonded with C8 and End-Capped with TMS> Tubing 0.mmID, 0mm length Holder Before Cartridge filter bonded with C8 After The filter is made of porous glass sized mm i.d. and mm thickness. Pore diameter is µm. Low dead volume structure Back pressure on glass filter is ca. 0. MPa at.0 ml/min of flow rate. Upper pressure limit is more than 0 MPa Available for. mm i.d to. mm i.d. column Evaluation of SunShell RP Guard Filter SunShell C8,. µm 0 x. mm SunShell C8,. µm 0 x. mm Without Guard Filter With Guard Filter t R ()=. min N() = 9,9 t R () =. min N() = 8,8 % decrease of plate Mobile phase: CH CN/H =0/0 for. mm i.d. CH CN/H =0/0 for. mm i.d. Flow rate: 0. ml/min for. mm i.d..8 ml/min for. mm i.d. Temperature: ºC Detection: UV@0nm Sample: = Uracil = Toluene = Acenaphthene = Butylbenzene Without Guard Filter With Guard Filter t R ()=. min N() = 9, t R () =. min N() = 8,90 Little change of plate 0 0 Price of SunShell RP Guard Filter Name quantity Part number Photo SunShell RP Guard Filter For exchange pieces CBGAAC SunShell RP Guard Filter Holder piece CBGAAH

11 High-throughput separation SunShell C8 0 x.0 mm. Mobile phase: A) Wtaer, B) Acetonitrile; Gradient (Acetonitorile %), 0.00 min - %, 0.0 min - 00%, 0.80 min - 00%, 0.8 min - %, cycle;.8min, (High-pressure gradient). Flow rate:.0 ml/min. Temperature: 0 ºC. Injection Volume: µl. Wavelength: 00-00nm, CH-9, - 00nm (Max Abs.). Sample: Mixture of ultraviolet absorbers, =,,, -Tetrahydroxybenzophenone, = Ethyl p-aminobenzoate, =, -Dihydroxybenzophenone, =, -Dihydroxy--methoxybenzophenone, =, -Dihydroxy-, -dimethoxybenzophenone, = -Hydroxy--methoxybenzophenone, = -( -Hydroxy- -metylphenyl) benzotriazole, 8 = -tert-butylphenyl salicylate. Courtesy of Jasco. 8 kinds of compounds were separated using SunShell C8 0 x.0 mm column in one minute. A peak width is just one second!! Surface Roughness on Inner Surface of Column Parameter of surface roughness Ra: Average roughness from center line Rz: Roughness calculated from 0 points average ( points of maximum and points of minimum ) Center line Ra Rz Schematic diagram of surface roughness G company Y company S company S company W company ChromaNIk Technologies Ra 0. µm 0. µm 0. µm 0.0 µm 0.0 µm 0.0 µm Rz.88 µm. µm.9 µm 0.9 µm 0.90 µm 0.0 µm It is considered that surface roughness affects column performance. Surface asperity of ChromaNik Technologies column with. mm i.d. and.0 mm i.d. is /0 to /0 to compare with that of GL Sciences, YMC, Shimadzu and Waters columns. ChromaNIk Technologies provides a column with a very smooth surface which is the most suitable for. µm core shell particle packings.. µm particle. µm particle Inner surface of column of A, B, C companies Inner surface of column of ChromaNik Technologies

12 rdering information SunShell C8 Inner diameter(mm)..0. Length(mm) Catalog number Catalog number Catalog number 0 CB9 CB CB 0 CB9 CB CB 0 CB9 CB CB CB9 CB CB 00 CB9 CB CB 0 CB9 CB CB SunShell C8 SunShell PFP Inner diameter(mm)..0. Length(mm) Catalog number Catalog number Catalog number 0 CC9 CC CC 0 CC9 CC CC 0 CC9 CC CC CC9 CC CC 00 CC9 CC CC 0 CC9 CC CC Inner diameter(mm)..0. Length(mm) Catalog number Catalog number Catalog number 0 CF9 CF CF 0 CF9 CF CF 0 CF9 CF CF CF9 CF CF 00 CF9 CF CF 0 CF9 CF CF Next phases, C8-WP ( nm), RP-AQUA, Phenyl and HILIC-Amide Coming soon Global Sales Biotech AB Box, 9 nsala, Sweden TEL: FAX: info@biotech.se URL: Manufacturer ChromaNik Technologies Inc. -- Namiyoke, Minato-ku, saka, -000 Japan TEL: FAX: info@chromanik.co.jp URL:

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