SunShell 2 μm, 2.6 μm, 3.4μm and 5μm HPLC column

Transcription

1 8, 8-WP, F8-, F8-,RP-AQUA, 8,, PFP, Phenyl, 8-, 8-T, -, -, ILI-Amide, ILI-S and -EP SunShell μm,. μm,.μm and μm PL column ore Shell Particle SunShell - for separation of monoclonal antibody hromaik Technologies Inc.

2 Superficially porous particle SunShell is a core shell silica column made by hromaik Technologies. The next generation to ore Shell particle Superficially porous silica Features of SunShell *. μm,. μm and. μm of core and. μm,. μm and. μm of superficially porous silica layer *Same efficiency and high throughput as a Sub μm and μm particle *Same pressure as a μm and μm particle *Same chemistry as Sunniest technology (reference below figure ) *Good peak shape for all compounds such as basic, acidic and chelating compounds *igh stability ( p range for SunShell8,. to ) * Low breeding

3 Superficially porous particle P-SEM Particle size:. μm Pore diameter: nm Particle size:. μm Pore diameter: nm Particle size:. μm Pore diameter: nm Electron micrograph of core shell silica ore shell silica particles were embedded in resin, cross-section processed by Ar ion milling, s (osmium) vapor deposited for conduction treatment, and observation. You can see the core (fused silica) and the porous layer around it. μm μm Pore diameter. μm μm Pore diameter. μm 9 nm. μm. μm ore. μm ore 9 nm nm nm nm nm 多孔質層 核 μm for routine. μm for protein Pore diameter. μm. μm 9 nm Pore diameter nm. μm. μm ore ore. μm. μm Schematic diagram of a core shell silica particle,.,.,. and. μm

4 Superficially porous particle Why does a. μm core shell particle show the same performance as a sub μm particle? Van Deemter Equation 8 Fully porous um A term : Eddy diffusion(dp is particle diameter) B term : Longitudinal diffusion (Dm is diffusion coefficient) term : Mass transfer ETP, µm Fully porous um Fully proous.8 um SunShell. um Van Deemter plot 8 Fully porous. um B trem A term term olumn: 8, x. mm 8 Acetonitrile/water=(/) Temperature: o Sample : aphthalene Mobile phase velocity, mm/sec u omparison of plate height plots SunShell 8 shows same efficiency as a sub μm 8. In comparison between fully porous. μm and core shell. μm (SunShell), SunShell shows lower values for A term, B term and term of Van Deemter equation. The core shell structure leads higher performance to compare with the fully porous structure. All terms in Van Deemter Equation reduce. A term ompany F, μm D :. µm D :.9 µm D 9 :. µm D 9 /D =.9 Sunniest, μm D :. µm D :. µm D 9 :. µm D 9 /D =. SunShell,. μm D :. µm D :. µm D 9 :.8 µm D 9 /D =. omparison of Particle ze Distribution Wide particle distribution (onventional silica gel D 9 /D =.) Flow of mobile phase arrow particle distribution (core shell silica D 9 /D =.) 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 totally 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. Diffusion of a solute is blocked by the existence of a core, so that a solute diffuses less in a core shell silica column than in a totally porous silica column. onsequently B term in Van Deemter Equation reduces in the core shell silica column. B term Totally porous silica A solute diffuses in a pore as well as outside of particles. ore shell silica A core without pores blocks diffusion of a solute. Difference of longitudinal diffusion Plate height (μm) 8 olumn: SunShell8,. µm x. mm Totally porous µm x. mm Acetonitrile/water=(/) Sample : aphthalene degree ore shell. um degree ore shell. um degree Totally porous um....8 Flow rate (ml/min) Plot of Flow rate and Plates height

5 Superficially porous particle term μm. μm μm omparison 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 term in Van Deemter Equation reduces. In other words, ETP (theoretical plate) is kept even if flow rate increases. A. μm core shell particle shows as same column efficiency as a totally porous sub- μm particle. onsidering diffusion of solute within pore Diffusion a.. μm sample Sample emanates in a particle after it enters. Diffusion. μm sample ore Shell. μm Totally porous. μm. μm Diffusion of sample in core shell and totally porous silica The left figure shows that a diffusion width of a sample in a. μm core shell particle and a μm totally porous particle. Samples or solutes enter into the particle and move by diffusion, then they go out of a particle. In this moment, sample peak width is broadened. This broadening width is statistically same for. μm core shell particle and μm fully porous particle. The. μm core shell particle is superficially porous, so that the diffusion width becomes narrower than particle size. Same diffusion means same efficiency. omparison of Performance by Plate/Pressure Plate Back press. (MPa) Plate/back press. Sunniest 8 T. μm 9,9. 9 Brand A 8.9 μm,. Brand B 8.8 μm, 9. Brand 8. μm,. 8 SunShell 8. μm 9, Sunniest 8 T. μm Brand A 8.9 μm Brand B 8.8 μm Brand 8. μm SunShell 8. μm,, Back pressure and theoretical plate were compared for μm and sub μm 8 and. μm SunShell 8. All columns showed almost the same theoretical plate except for brand A 8.9 μm. owever back pressure was not same. Especially Brand 8.μm showed the highest back pressure. And SunShell 8. μm showed the lowest back pressure. n the comparison of theoretical plate per back pressure, SunShell indicated the largest value. This is a big advantage. olumn: x. mm 8, Acetonitrile/water=(/), Temperature: o

6 Superficially porous particle STATIARY PASE Reversed phase SF 8, 8-WP ( page, page, page) EP ( page) RP-AQUA, ( page, 9 page) 8, 8-, 8-T ( page, page, page) ILI -, - ( page, page) ILI-Amide ( page) R Phenyl ( page) ILI-S ( page) PFP ( page) F F F F F F8-, F8- ( page) *Stationary phase for both SF and ILI was not end-capped. **All revered phases except for PFP was end-capped at high temperature using Sunniest Endcapping technique.

7 Superficially porous silica particle Superficially porous silica Unique end-capping by new concept This slide shows comparison of hydrophobicity between two 8 stationary phases. We developed silanol activity control technique which was a reaction at extremely high temperature. This technique makes residual silanol groups change to siloxane bond. The upper one is a 8 phase with conventional endcapping and the lower one is a 8 phase with both Sunniest end-capping and silanol activity control. Residual silanol groups contributes as a polar site and makes hydrophobicity of stationary phase decrease. n the other hand siloxane bond in the lower one doesn t make hydrophobicity decrease. onsequently the lower one is more hydrophobic than the upper one. onventional end-capping The whole works as stationary phase. End-capping layer Both Sunniest end-capping and lanol Activity ontrol End-capping layer More ydrophobic State of stationary phase with unique end-capping and comparison of hydrophobicity End-capping method ) Unique end-capping reagent <<examethetyltrisiloxane>> ) Secondly TMS end-capping Final TMS Schematic diagram of bonding of SunShell 8 An end-capping of hexamethyltrisiloxane works as an arm. This arm moves like a Geometrid caterpillar, so that a functional group on the tip of the arm can bond with a silanol group which is located anywhere. Finally TMS reagent is bonded to a remaining silanol group.

8 SunShell8, μm,.μm, μm haracteristics of SunShell 8 Particle size ore shell silica 8 (USP L) Pore diameter Specific surface area arbon content Bonded phase End-capping Maximum operating pressure Available p range SunShell Sunniest endcapping MPa or psi. - SunShell Sunniest endcapping MPaor8, psi. - SunShell Sunniest endcapping MPa or, psi. - ore Shell particle shows. to. times higher plate than fully porous particle. SunShell 8,.μm. x mm Acetonitrile/water=/ Flow rate:. ml/min =9, Fully porous 8, μm. x mm =, min SunShell 8,.μm. x mm Acetonitrile/water=/ Flow rate:. ml/min =,. times higher min Theoretical plate and tailing factor Used columns: SunShell 8 μm, Ascentis Express 8 μm, Kinetex 8.μm, Acquity BE 8.μm, Titan 8.9μm SunShell 8 ompany S ore Shell 8 ompany P ore Shell 8 ompany W 8 olumn:sunshell8, μm x. mm ompany S ore Shell 8, μm x. mm ompany P ore Shell 8,.μm x. mm ompany W 8,.μm x. mm ompany S Monodisperse 8,.9μm x. mm / =/ Flow rate:. ml/min Temperature: º Sample: = Uracil, = Ethylbenzoate, = Acenaphthene, = Butylbenzene Acenaphthene Theoretical plate Tailing factor Pressure (MPa) Theoretical plate /pressure (MPa - ) SunShell 8 9,. 8. ompany S ore Shell 8,.9. ompany S Monodisperse 8 ompany P ore Shell 8, ompany W 8,.. 9 ompany S Monodisperse 8,9..9 *Ascentic Express is a registered trade mark of gma Aldrich. Titan is a registered trade mark of gma Aldrich. omparative separations may not be representative of all applications.

9 omparison of retention and plate using PL SunShell 8 µm 8 MPa SunShell 8. µm MPa MPa =, =, =, olumn size: x. mm / =/ Flow rate:. ml/min Temperature: º Sample: = Uracil = affeine = Phenol = Butylbenzene = o-terphenyl = Amylbenzene = Triphenylene PL: itachi LahromELITE (Tubing,. mm i.d.) Totally porous silica Sunniest 8, μm ore shell silica SunShell 8,. μm ore shell silica SunShell 8, μm Specific surface area m /g m /g 9 m /g Packings weight (x.mm). g. g. g Surface area in a column m /g (%) m /g (9%) 88 m /g (%) Retention time (t R ) Retention factor (k) Retention time (t R ) Retention factor (k) Retention time (t R ) Retention factor (k) ) Uracil... ) Amylbenzene Relative value of Amylbenzene % % 8% % % 8% There is a little difference of k between totally porous and core shell particles. Examples of transfer from a conventional µm column to SunShell column Isocratic separation PL 8 8 ()=,8 SunShell8,. µm x. mm Brand F8, µm x. mm / of analysis time ()=9, ()=,8 olumn: Brand F 8, μm x. mm SunShell8,. μm x. mm /mm Phosphoric acid = / Flow rate:. ml/min,.8 ml/min at the lowest chromatogram Temperature: º Pressure: 9. MPafor Brand F 8 μm. MPafor SunShell8. μm Detection: UV@ nm Sample: = Benzydamine = Ketoprofen = aproxen = Indomethacin = Ibuprofen UPL. ml/min ()=, PL: itachi Lahrom ELITE (Tubing,. mm i.d.) UPL: Jasco X-L.8 ml/min ()=, Analysis time decreases to / to compare with a µm column sized x. mm. 8

10 omparison between normal and semi-micro PL Flow cell Response Sampling Tubing ID ormal. sec. sec. mm Semi-micro. sec. sec. mm Semi-micro. sec. sec. mm,,99,,,9,,,,, 9,,8 8 9 Theoretical Plate Uracil Toluene Acenaphthene Semi-micro. sec Semi-micro. sec ormal. sec Butylbenzene Peak width/sec Relationship between Peak width and theoretical plate omparison of chromatograms olumn: SunShell8, μm x. mm / = / Flow rate:. ml/min Temperature: º Pressure:. MPa Detection: UV@ nm Sample: = Uracil = Toluene = Acenaphthene = Butylbenzene Semi-micro PL derives near % performance of a core shell column. Even if normal PL is used, it derives PL: itachi Lahrom ELITE 8% performance except for a narrow peak whose width is less than second Effect of inner diameter of tubing Effect of response time of detector Average of theoretical plate (n=).mm.mm.mm (min) Inner diameter of tubing.mm.mm.mm Peak () 9 8 Peak () 9 Peak () 998 Peak () 9 89 Theoretical plate. sec Time (sec) means peak width (δ).. sec. sec Acenaphthene t R. min.8 sec. sec. sec Toluene t R. min. sec Uracil t R. min Response time/sec olumn:sunshell8,. μm x. mm / =/ Flow rate:. ml/min Temperature: Ambient Tube length: cm (Peek, from the column to the flow cell) Instrument: X-L(JAS) Response time:. sec olumn:sunshell8,. μm x. mm / =/ Flow rate:.8 ml/min Temperature: Ambient Sample: Toluene Tube: i.d..mmx cm Peeksil Instrument: X-L(JAS) The above theoretical plate was compared changing the inner diameter of tubing between a column and a flow cell of the detector. A tubing with a large inner diameter has a large dead volume, so that it makes the peak width be wide. As a result, theoretical plate decreases. I recommend to use the tubing with. mm or less than. mm inner diameter for core shell columns. The response time of a detector is important. Regarding uracil, the real peak width is less than.8 sec. When the peak width is less than sec,. sec of response time is needed. Furthermore, the sampling rate of an integrator should be set to be. sec. 9

11 SunShell 8 μm omparison of core shell μmand totally porous sub μm Used columns: SunShell 8 μm, Ascentis Express 8 μm, Kinetex 8.μm, Acquity BE 8.μm, Titan 8.9μm Separation of standard samples SunShell 8 ompany S ore Shell 8 ompany P ore Shell 8 ompany W 8 ompany S Monodisperse 8 k=. k=9. 8 k=. k=. k=. olumn:sunshell8, μm x. mm ompany S ore Shell 8, μm x. mm ompany P ore Shell 8,.μm x. mm ompany W 8,.μm x. mm ompany S Monodisperse 8,.9μm x. mm / =/ Flow rate:. ml/min Temperature: º Sample: = Uracil, = affeine, = Phenol, = Butylbenzene = o-terphenyl, = Amylbenzene, = Triphenylene ydrogen bonding (affeine/phenol) ydrophobicity Steric selectivity (Amylbenzene/Butylbenzene) (Triphenylene/o-Terphenyl) SunShell ompany S ore Shell 8 ompany P ore Shell ompany W 8... ompany S Monodisperse omparison of Pyridine () as a basic compound omparison of xine () as a metal chelating compound omparison of Formic acid () as an acidic compound SunShell 8 =9, TF=.9 SunShell 8 =,9 TF=. SunShell 8 ompany S ore Shell 8 =,8 TF=. ompany S ore Shell 8 =, TF=. ompany S ore Shell 8 ompany P ore Shell 8 = TF=. ompany P ore Shell 8 =, TF=. ompany P ore Shell 8 ompany W 8 =, TF=.8 ompany W 8 =8, TF=. ompany W 8 ompany S Monodisperse 8 =8 TF=.9 ompany S Monodisperse 8 =,8 TF=. ompany S Monodisperse 8 olumn dimension: x. mm / =/ Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = Uracil = Pyridine = Phenol olumn dimension: x. mm /mm P =/9 Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = 8-Quinolinol (xine) = affeine olumn dimension: x. mm /.% P =/98 Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = Formic acid = Acetic acid = Propionic Acid

12 SunShell 8 μm omparison of Amitriptyline () as a strong basic compound =, TF=.8 SunShell 8 =,9 TF=.8 =, TF=. ompany S ore Shell 8 =,8 TF=.8 =, TF=. ompany P ore Shell 8 =,9 TF=.9 =8, TF=.8 ompany W 8 =, TF=. =, TF=.9 ompany S Monodisperse 8 =9, TF=. olumn dimension: x. mm / mmphosphate buffer p.=/ Flow rate:. ml/min Temperature: º Detection: UV@ nm Sample: = Uracil, = Propranolol, = ortriptyline, = Amitriptyline olumn dimension: x. mm / mmammonium acetate p.8=/ Flow rate:. ml/min Temperature: º Detection: UV@ nm Sample: = Uracil = Propranolol = ortriptyline = Amitriptyline Decreasing of theoretical plate due to frictional heating effect Theoretical plate SunShell 8 Totally porous monodisperse 8 Totally porous hybrid Flow rate ml/min ore shell silica has a solid core (non-porous silica), so that thermal conductivity is high in the column. There is no influence of reducing theoretical plate by frictional heating. Regarding totally porous hybrid silica, not only totally porous structure but also including ethylene groups make thermal conductivity be low in the column. It is considered that frictional heating deflects thermal distribution in the column and theoretical plate decreases.. olumn: x. mm / =/ Temperature: º Sample: Acenaphthene,

13 omparison of core shell.μm columns omparison of standard samples between core shell 8s Used columns. Kinetex8,. μm. Accucore8,. μm. PoroShell8 E,. μm. Ascentis Express 8,. μm. ortecs8,. μm. SunShell8,. μm SunShell 8.8 MPa k=. k=. k=. ompany A 8. MPa k=9. k=9. ompany B 8. MPa ompany E 8 8. MPa ompany 8. MPa k=. ompany D 8. MPa olumn:ompany A 8,. μm x. mm (. Mpa,,8 plate) ompany B 8,. μm x. mm (. MPa,, plate) ompany W 8,.μm x. mm (8. MPa,, plate) ompany 8,.μm x. mm (. MPa,, plate) ompany D 8,. μm x. mm (. MPa,,8 plate) SunShell8,. μm x. mm (.8 MPa,,9 plate) / =/ Flow rate:. ml/min, Temperature: º Sample: = Uracil, = affeine, = Phenol, = Butylbenzene = o-terphenyl, = Amylbenzene, = Triphenylene ydrogen bonding (affeine/phenol) ydrophobicity (Amylbenzene/Butylbenzene) Steric selectivity (Triphenylene/o-Terphenyl) ompany A ompany B 8... ompany E ompany 8... ompany D SunShell Retention of standard samples and back pressure were compared for five kinds of core shell type 8s. ompany A 8 showed only a half retention to compare with SunShell 8. Steric selectivity becomes large when ligand density on the surface is high. SunShell 8 has the largest steric selectivity so that it has the highest ligand density. This leads the longest retention time. omparison of pyridine omparison of xine omparison of formic acid ompany A 8 ompany B 8 ompany E 8 TF=.9 TF=. TF=. ompany A 8 ompany B 8 ompany E 8 TF=. TF=.8 ompany A 8 ompany B 8 TF=.9 ompany EW 8 ompany 8 TF=. ompany 8 TF=. ompany 8 ompany D 8 TF=. ompany D 8 TF=. ompany D 8 SunShell 8 TF=. SunShell 8 TF=. SunShell 8 olumn dimension: x. mm / =/ Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = Uracil = Pyridine = Phenol 8 9 olumn dimension: x. mm /mm P =/9 Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = 8-Quinolinol (xine) = affeine 8 9 olumn dimension: x. mm /.% P =/98 Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = Formic acid = Acetic acid = Propionic Acid Residual silanol groups make pyridine be tailing under methanol/water mobile phase condition. SunShell 8 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 ompany A and 8 show a sharp peak.

14 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 8s were compared loading capacity of amitriptyline at three different mobile phases. ommon condition: olumn dimension, x. mm, flow rate;. ml/min, temperature; o Acetonitrile/mM phosphate buffer p.=(:) Acetonitrile/mM acetate ammonium p.8=(:) Theoretical plate 8 SunShell 8 ompany 8 Sunniest 8 um ompany A 8 ompany B 8 ompany D 8 ompany E 8 times Theoretical plate 8 Sunshell 8 ompany 8 ompany A 8 ompany B 8 ompany D 8 ompany E 8 times... Sample weight/μg TF=.8 TF=. TF=. TF=. TF=. TF=. SunShell 8 (core shell) 8 (core shell) Sample: Sunniest 8 μm (fully porous) A 8 (core shell) B 8 (core shell) D 8 TF=.8 (core shell) E 8 (core shell) = Uracil (.µg) = Propranolol (.µg) = ortriptyline (.µg) = Amitriptyline (.µg)... Sample weight/μg TF=. TF=.89 SunShell 8 TF=. TF=. TF=. TF=. 8 A 8 B 8 D 8 E Theoretical plate was calculated by σ method using peak width at.% of peak height..% 8 9 Physical properties arbon loading (%) Specific surface area a (m /g) Pore volume a (ml) Pore diameter a (nm) SunShell8.() b () b. 8. (9) b AscentisExpress 8 8. () b.8 8. (9) b PoroShell8 E 8.(8) b () b.. () b omparison column. Kinetex8,. μm. Accucore8,. μm. PoroShell8 E,. μm. Ascentis Express 8,. μm. ortecs8. μm. SunShell8,. μm Accucore8 8.8 (9) b () b. 8.9 (8) b ortecs8. (.) b. 9. Kinetex8.9 ( effective) b ( effective) b. 9.() b a. Measured after sintered at degree elsius for 8 hours. b. Value cited in company brochure or literature All columns are core shell type. All columns sized x. mm except for company E show 8, to, plates for a neutral compound. owever regarding a basic compound like amitriptyline, SunShell 8 and company 8 showed a good peak, while ompany A, B and D 8 showed a poor peak. ompany A 8 overloaded at more than. µg of amitriptyline while SunShell 8 overloaded at more than from. to µg of amitriptyline. Surprisingly loading capacity of company A 8 was only one hundredth to compare with SunShell 8 under acetonitrile/mm phosphate buffer p.=(:) mobile phase. ompany D 8 always showed poor peak of amitriptyline.

15 Evaluation of Stability Relative retention/% 8 SunShell 8 ompany D 8 ompany B 8 ompany A 8 ompnay 8 ompany E 8 % TFA 8 º 8 Durable test condition olumn size: x. mm Time/h /.% TFA, p=/9 Flow rate:. ml/min Temperature: 8 º Measurement condition olumn size: x. mm / =/ Flow rate:. ml/min Temperature: º Sample: = Uracil (t ) = Butylbenzene Accelerated acidic test Stability under acidic p condition was evaluated at 8 º using acetonitrile/% trifluoroacetic acid solution (:9). Sunshell 8 has kept 9% retention for hours under such a severe condition. SunShell 8 is to times more stable than the other core shell 8. ontinuous analysis under p9. condition SunShell 8 Relative plate of butylbenzene/% 8 SunShell 8 ompany D 8 ompany A 8 ompany B 8 ompany 8 p º,,,,,, Durable test condition olumn ze: x. mm Elution volume/ml /mm Sodium borate/mm a=//9 (p) Flow rate:. ml/min Temperature: º Measurement condition olumn ze: x. mm / =/ Flow rate:. ml/min Temperature: º Sample: = Butylbenzene Alkaline test Stability under basic p condition was evaluated at º using methanol/sodium borate buffer p (:) as mobile phase. Sodium borate is used as a alkaline standard solution for p meter, so that its buffer capacity is high. Elevated temperature of º makes column life be one third. The other company shows stability test at ambient (room temperature). If room temperature is º, column life at room temperature ( º) is sixteen times longer than that at º. SunShell 8 is enough stable even if it is used under p condition. Regarding stability under basic p condition, there is little 8 column like SunShell 8 except for hybrid type 8. It is considered that our end-capping technique leads high stability. th th omparison of particle size th th th th st olumn: SunShell 8,. μm x. mm A) mm Ammonium bicarbonate p 9. B) Acetonitrile Gradient program: Time (min) %B 9 9. Flow rate:. ml/min Temperature: o Detection: UV@ nm Sample: =Uracil, =Propranolol, = ortriptyline, =Amitriptyline *Measured using Beckman oulter Multisizer after 8 materials were sintered at degree elsius for 8 hours. The measured value of each sintered core shell silica is considered to be different from that of the original core shell silica. a. Median particle size

16 SunShell Amino Acids derivatized with PA and FM Ethambutol ydrochloride 8 Ethanbutol l Impurity B olumn: SunShell8,. μm x. mm A) Methanol/water (/ V/V) B) Methanol Time (min) 8 %B Flow rate:. ml/min Temperature: o Detection: UV nm Injection volume: μl, l Ethambutol ydrochloride olumn: SunShell8. μm, x. mm A) mm a P + mm a B +.mm a (p.8) B) Acetonitrile/Methanol/Water (// %V) Time (min)..8.8 %B Flow rate:. ml/min Temperature: º Detection: UV@8 nm Sample: =Aspartic acid, =Glutamincacid, =Serine, =istidine,=glycine, =Threonine, =Arginine, 8=Alanine, 9=Tyrosine, =Valine, =Methionine, =Tryptophan, =Pnehylalanine, =Isoleucine, =Leucine, =Lysine, =Proline Dansylated estrogen hormones olong tea olumn: SunShell8. μm, x. mm A) with.% formic acid. B) with.% formic acid. Gradient program: -. min: % B.. min: -% B. -. min: % B. -. min: -% B. -. min: % B Estrone Flow rate:. ml/min. Temperature: º Detection: MS(sim), m/z,.,.,. Samples:. Dansylatedestriol,. Dansylatedbeta-estradiol,. Dansylated alpha-estradiol,. Dansylated estrone ourtesy of Department of hemistry & Biochemistry, The University of Texas at Arlington l S (VI) Dansyl chloride Estriol, Beta-estradiol, Alpha-estradiol olumn: SunShell8. μm, x. mm A).% Phosphoric acid B) Gradient program Flow rate:. ml/min, Temperature: º Detection: UV@ nm Sample: olong tea Gallocatechin Epicatechin 8 9 atechin Epigallocatechin affeine Epigallocatechin gallate l Gallocatechin gallate 8 Epicatechin gallate 9 atechin gallate

17 SunShell8-WP, RP-AQUA, 8,Phenyl, PFP,.μm (Pentafluoropheny) haracteristics of SunShell Particle size ore shell silica Pore diameter Specific surface area arbon content Bonding phase Bonded phase USP L line End-capping Maximum operating pressure Available p range SunShell 8. μm 9nm m /g % 8 L Sunniest endcapping MPa. - SunShell8-WP. μm nm 9 m /g % 8 L Sunniest endcapping MPa. - SunShellRP-AQUA. μm nm 9 m /g % 8 Equivalent to L Sunniest endcapping MPa -8 a) SunShell8. μm 9nm m /g.% 8 L Sunniest endcapping MPa. -9 SunShellPhenyl. μm 9nm m /g % Phenylhexyl L Sunniest endcapping MPa. -9 SunShellPFP. μm 9nm m /g.% Pentafluorophenyl L TMS endcapping MPa -8 omparison of standard samples a) Under % aqueous condition,, SunShell 8 SunShell PFP, SunShell Phenyl SunShell 8 SunShell RP-AQUA SunShell 8-WP olumn: SunShell8, 8-WP, RP-AQUA, 8, Phenyl, PFP,. μm x. mm / =/ Flow rate:. ml/min Temperature: º Sample: = Uracil = affeine = Phenol = Butylbenzene = o-terphenyl = Amylbenzene = Triphenylene ydrogen bonding (affeine/phenol) ydrophobicity (Amylbenzene/Butylbenzene) Steric selectivity (Triphenylene/o-Terphenyl) PFP...8 Phenyl RP-AQUA... 8-WP SunShell Separation of peptides Separation of amitriptyline using 8 Separation of basic compounds SunShell 8-WP SunShell 8,, SunShell 8 SunShell PFP olumn: SunShell8-WP,. μm ( nm) x. mm, A).% TFA in Acetonitrile/water(:9) B). % TFA in Acetonitrile Gradient program: %B % % in min Flow rate:. ml/min, Temperature: º, Detection: UV@ nm, Sample: Tryptic digest of BSA olumn: SunShell8,. μm x. mm /mm phosphate buffer.=/ Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = Uracil, = Propranolol, = ortriptyline, = Amitriptyline olumn: SunShell8,. μm x. mm SunShellPFP,. μm x. mm /mm phosphate buffer p.=8/ Flow rate:.8 ml/min Temperature: º Sample: = Uracil, = Propranolol, = ortriptyline, =Amitriptyline

18 Separation of xanthines Separation of cresol isomers Separation of nucleotides SunShell 8 SunShell PFP Water Water SunShell 8, SunShell RP-AQUA ()=,9 ()=, SunShell PFP SunShell PFP mm phosphate buffer p mm phosphate buffer p SunShell PFP olumn: SunShell8, PFP,. μm x. mm /water or buffer=/ Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = Theobromine = Theophyline = affeine = Phenol olumn: SunShell8, PFP,. μm x. mm / =/ Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = p-resol = m-resol = o-resol olumn: SunShellRP-AQUA,. μm x. mm mm Phosphate buffer p. Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = -GDP = -ATP = -ADP = -AMP Separation of nucleic acid bases igh-throughput separation Sunniest RP-AQUA SunShell RP-AQUA olumn: Sunniest RP-AQUA, µm x. mm SunShellRP-AQUA,. µm x. mm mm Phosphate buffer p. Flow rate:. ml/min for Sunniest. ml/min for SunShell Temperature: º Sample: = ytosine, = Uracil, = Thymidine, = Uridine, = Thymine SunShell 8 8 olumn: SunShell8, x. mm. A) Water, B) Acetonitrile; Gradient (Acetonitrile %),. min -%,. min -%,.8 min -%,.8 min -%, cycle;.8min, (igh-pressure gradient). Flow rate:. ml/min. Temperature: º. Injection Volume: µl. Wavelength: -nm, -9, -nm (Max Abs.). Sample: Mixture of ultraviolet absorbers, =,,, -Tetrahydroxybenzophenone, = Ethyl p-aminobenzoate, =, -Dihydroxybenzophenone, =, -Dihydroxy--methoxybenzophenone, =, -Dihydroxy-, -dimethoxybenzophenone, = -ydroxy--methoxybenzophenone, = -( -ydroxy- -metylphenyl) benzotriazole, 8 = -tert-butylphenyl salicylate. ourtesy of Jasco. A peak width is just one second!!

19 Purine analogue rganic acid mmammonium acetate (p.) Uric acid ypoxanthine S company ore shell 8 AQ,. µm umber and 8 peaks are broaden. 9 8 mm P Xanthine xipurinol SunShellRP-AQUA,. µm 9 8 olumn: SunShellRP-AQUA,. μm x. mm mm P or mmammonium acetate (p.) Flow rate:. ml/min Temperature: Ambient Detection: UV@ nm Sample: = Uric acid, = ypoxanthine, = Xanthine, = xipurinol, = Allopurinol Allopurinol olumn dimension: x. mm.% P Flow rate:. ml/min Temperature: o Detection: UV@nm Sample: = xalic acid, = Tartaric acid, = Formic acid, = Malic acid, = Lactic acid, = Acetic acid, = Diglycolicacid, 8 = Maleic acid, 9 = itric acid, = Succinic acid, = Fumaricacid. Amino acids (L/MS) Monosaccharides derivatized with L-Tryptphan D-Galactose L-Galactose D-Glucose olumn: SunShellRP-AQUA,. μm, x. mm A) mmfba, B) mmfba in / (9/) %B % to % in min (FBA: eptafluorobutyric acid) Flow rate:. ml / min Temperature: o Detection: MS (anofrontierld) ESI Positive, Extracted ion chromatogram (EI) ourtesy of Dr Takeo Kaneko, okohama ational University L-Mannose L-Glucose D-Mannose olumn: SunShellRP-AQUA,. μm x. mm mmphosphate and mmtetraborate(p 9.) Flow rate:. ml/min Temperature: º Detection: UV@ nm Sample: Monosaccharides derivatized with L-Tryptphan ourtesy of DrShujiKodama, Tokai University 8

20 SunShell,. μm Specification of SunShell Bonding phase ore shell silica SunShell Particle size (μm) ore size (μm) Pore size (nm) Specific surface area (m/g) arbon loading (%).. 9 Ligand USP L category End-capping Maximum pressure p range L TMS MPa. - 9 Problem of column olumn dimension: x. mm methanol/water = 9/ Flow rate:. ml/min Temperature: o Detection: UV@9 nm Sample, = δ-tocopherol Ligand density =.9 μmol/m α,=.8 Rs,=. Ligand density =. μmol/m α,=. Rs,=.8 = γ-tocopherol = β-tocopherol Ligand density =. μmol/m = α-tocopherol α,=. The higher a ligand density, the larger a separation factor of β-tocopherol and γtocopherol. Too high ligand density causes low theoretical plate and large tailing of a peak. f course 8 columns can t separate β-tocopherol and γ-tocopherol. Separation of tocopherols Fast separation of vitamin K isomers α,=. Rs,=. trans PL PL condition olumn: SunShell,. µm x. mm Methanol Flow rate:.8 ml/min Temperature: o Detection: UV@ nm Sample Vitamin K isomers (trans and cis) SunShell Rs=. cis 8 SF trans trans sub μm 8 column Rs=. olumn: SunShell,. μm x. mm Methanol/water = 9/ Flow rate:. ml/min Temperature: o Detection: UV@9 nm Sample: = δ-tocopherol, = γ-tocopherol, = β-tocopherol, = α-tocopherol. cis cis. Retention time / min. Both PL and SF separations were finished within minute. Regarding resolution of isomers, PL won SF. omparison of isomers separation of Vitamin k SunShell ompany core shell α=. o α=. α=. o α=. α=.9 o α=. olumn: SunShell,. μm x. mm ompany core shell,. μm x. mm methanol/water = 9/ Flow rate:. ml/min Detection: UV@ nm Sample: vitamin K isomers (trans and cis). trans α=. α=. Rs=.9 o trans cis o α=. trans α=.8 Rs=.8 cis Retention time / min cis Retention time / min 9

21 SunShell. μm 8-WP, F8-, F8-, 8-,8-T, 8-T, -, - haracteristics of SunShell Particle size ore shell silica Pore diameter Specific surface area Stationary phase arbon content For separation of peptides and proteins Ligand density Bonding phase End-capping Maximum operating pressure Available p range SunShell 8-WP. μm nm 9 m /g 8 %. μmol/m Sunniest endcapping MPaor8, psi. - L SunShell F8-. μm nm 9 m /g 8.%. μmol/m Sunniest endcapping MPaor8, psi. 9 L SunShell F8-. μm nm m /g 8.%. μmol/m Sunniest endcapping MPa a or8, psi a. -9 L SunShell 8-. μm nm m /g 8.%. μmol/m Sunniest endcapping MPa a or8, psi a. 9 L SunShell 8-T. μm nm m /g 8.%. μmol/m Sunniest endcapping MPa a or8, psi a. 9 L SunShell -. μm nm m /g.9% μmol/m Sunniest endcapping MPa a or8, psi a. 8 L SunShell -. μm nm m /g.% μmol/m Sunniest endcapping MPa a or8, psi a. 8 L USP L line a: MPa, psi for. mm i.d. column What is F8? exa-functional 8 has six functional groups. This F8 is much more stable under acidic condition. Sunniest Bonding Technology examethydichlorotrisiloxan e + Trimethylchlorosilane (TMS) (X: l,, ) Schematic diagram of reagent Schematic diagram of the state of bonding on silica surface Desorption Dv(log d) (cc/g).9 ore shell. um, nm.8 ore shell. um, nm. ore shell. um, nm Pore Diameter (nm) Pore distribution of core shell particle Relative retention (%) 8 9% line.% formic acid, p. º 8 Time (h) Stability under L/MS mobile phase condition Durable test condition olumn : SunShellF8-. μm, x. mm /.% formic acid, p.=/ Flow rate:. ml/min Temperature: º Measurement condition / =/ Flow rate:. ml/min Temperature: º Sample: = Uracil = Butylbenzene Separation of peptides SunShell F8- SunShell 8-WP peaks olumn: SunShellF8-,. μm ( nm) x. mm SunShell8-WP,. μm ( nm) x. mm A).% TFA in Acetonitrile/water(:9) B). % TFA in Acetonitrile Gradient program: Flow rate:. ml/min Temperature: º Detection: UV@ nm Sample: Tryptic digest of cytochrome

22 Superficially porous particle SunShell. μm 8-, 8-T, -, - For separation of peptides and proteins omparison of column temperature omparison of SunShell stationary phase o,min o,min 8 o,min olumn: SunShell 8-,. μm ( nm) x. mm A).% TFA in water B).8 % TFA in acetonitrile Gradient program: Time min min %B % % Flow rate:. ml/min, Temperature: o o or 8 o Detection: UV@ nm, Sample: = ytochrome, = Lysozyme, = BSA, = Myoglobin, = valbumin A macromolecule compound like a protein diffuses very slowly, so that an elevated temperature makes a peak be shaper and improves separation. BSA peak seemed to be tailing at degree elsius. BSA, however, was separated several peaks at 8 degree elsius. olumn dimension: x. mm, A).% TFA in water, B). % TFA in Acetonitrile Gradient program: Time min min %B % % Flow rate:. ml/min, Temperature: 8 o, Detection: UV@ nm, Injection volume:. μl Sample: = ytochrome, = Lysozyme, = BSA, = Myoglobin, = valbumin, UPL instrument: ITAII hromaster omparison of peak width (W., min) ytochrome Lysozyme BSA Myoglobin valbmin T 8-T.μL Sample concentration %.%.%.%.% The above table indicated that - with Å of pore showed a sharper peak than the other. 8-T has a thin porous layer and low surface area, so that low sample loadnig made a peak sharper. Separation of monoclonal antibody olumn dimension: x. mm i.d. A) water/tfa (/.) B) acetonitrile/tfa (/.8) -%B (- min) Flow rate:. ml/min Temperature: 8 º Detection: UV at nm Injection: µl Sample: : Monoclonal antibody purified from cell culture using Protein G ot eluted ore shell8, Å Totally porous, Å ore shell, Å SunShell -, Å Separation usig nanocolumn SunShell - ( x. mm i.d.) Flow rate: µl/min Regarding reversed phase separation of monoclonal antibody (IgG), not only core shell with nm pore showed the better separation than totally porous, but also nm of pore leaded the best separation. ano column showed almost the same separation of IgG as semi-micro column.

23 SunShell-EP,. μm For Supercritical fluid hromatography.μm core shell column shows only one third of back pressure to compare with.μm fully porous column although both show almost same efficiency. By such low back pressure, a difference of density of supercritical fluid between an inlet and an outlet of the column is reduced. onsequently,..μm core shell column performs a superior separation for SF. haracteristics of SunShell -EP Particle size ore shell silica Pore diameter Specific surface area arbon content Bonded phase Endcapping Maximum operating pressure Available p range SunShell-EP. μm 9 nm m /g.% -Ethylpyridine no MPaor 8, psi. omparison between SunShell -EP and. μm fully porous -EP Figure Figure : hromatogram of the separation for he - component mix using the Sun Shell -EP x. mm column. A methanol gradient of < minutes was used on the Agilent Infinity SF system. SF conditions: flow rate:.ml/min; outlet pressure bar; column temperature ⁰. Gradient program:.-.% in. min, then.-% in. min and held at % for. min. Figure : hromatogram of the separation for the - component mix using Acquity UP Viridis -EP x. mm column. of the components were resolved. A methanol gradient of < minutes was used on the Agilent Infinity SF system. SF conditions: flow rate. ml/min; outlet pressure bar; and column temperature ⁰. Gradient program:.-.% in. min,.% for. min, then.- % in. min. Figure ourtesy of Pfizer Inc.

24 SunShellILI-Amide, ILI-S,. μm haracteristics of SunShell ILI-Amide Particle size ore size ore shell silica Amide (USP L8) Pore diameter Specific surface area For ydrophilic Interaction hromatography arbon content Bonded phase Endcapping Maximum operating pressure USP category SunShell ILI-Amide. μm. μm 9 nm m /g % Amide no MPaor 8, psi L -8 SunShell ILI-S. μm. μm 9 nm m /g Bare silica no MPa or 8, psi L - Available p range Stationary phase of ILI-Amide Stationary phase of ILI-S R: ydrophilic group Bare silica Stationary phase of SunShell ILI-Amide consists of AMIDE and YDRPILI GRUP, so that this stationary phase is more polar than an individual group. igh speed separation is leaded by core shell structure that derives high efficiency and fast equilibration. ILI-S is recommended for separation using L/MS. Separation of ucleic acid bases: omparison of the other core shell hilic columns SunShellILI-Amide Sompany ore shell Polyol olumn: SunShellILI-Amide,. μm x. mm, oreshellpolyol,. μm x. mm, ore shell lica,. μm x. mm Acetonitrile/ mmammonium acetate(p.) = 8/ Flow rate:. ml/min Temperature: o Detection: UV@ nm Sample: = Thymine, = Uracil, = Uridine, = ytosine, = ytidine A ompany ore shell lica Regarding retention of cytidine, SunShell ILI-Amide showed % higher retention factor than S core shell polyol. Separation of yanuric acid and Melamine Separation of water- soluble vitamins..... olumn: SunShell ILI-Amide,. μm x. mm Acetonitrile/ mmphosphate Buffer (p.9) =/ Flow rate:. ml/min Temperature: o Detection: UV@ nm, Sample: = yanuricacid, = Melamine olumn: SunShell ILI-Amide,. μm x. mm Acetonitrile/ mmphosphate buffer (p.) =8/ Flow rate:. ml/min Temperature: o Detection: UV@ nm, Sample: = icotinic acid, = Ascorbic acid, = Pyridoxine

25 Artificial sweeteners Glycoside. Aspartame -(I) -(I) +(IV) S. elicin. Arbutin. Salitin. Saccharin -(I) -(I) +(IV) K S olumn: SunShellILI-Amide,. μm, x. mm Acetonitrile: mmphosphate buffer (p.) =8:. Acesulfame K Flow rate:. ml/min, Temperature: Ambient Detection: UV@ nm Sample: = Aspartame, = Saccharin, = Acesulfame K olumn: SunShellILI-Amide,. μm, x. mm Acetonitrile: mmphosphate Ammonium (p.9) =8: Flow rate:. ml/min Temperature: Ambient Detection: UV@ nm Sample: = elicin, = Salicin,,= Arbutin, = Rutin. Rutin ucleic acid base ucleic acid bases... olumn: SunShellILI-S,. μm x. mm mmammonium acetate (p.) /acetonitrile = /9 Flow rate:. ml/min Temperature: o Detection: UV@ nm Sample: = Acenaphthene, = Uridine, = ytosine 8 olumn: SunShellILI-S,. μm x. mm mmammonium acetate (p.) /acetonitrile = /9 Flow rate:. ml/min Temperature: o Detection: UV@ nm Sample: = Thymine, = Uracil, = Uridine, = ytosine, = ytidine ore shell column with. mm I.d. i.d. Separation of proteins using the. mm i.d. column 8 olumn: SunShellRP-AQUA,. μm x. mm A). % trifluoroacetic acid (TFA) in water B).8 % trifluoroaceticacid (TFA) in acetonitrile %B % to % in min Flow rate:. ml / min Temperature: o Detection: UV@ nm Sample: = Gly-Tyr, = Val-Tyr-Val, = Met enkephalin, = Leuenkephalin, = Angiotensin II (PL peptide standard mixture by gma-aldrich)

26 SunShell RP Guard Filter artridge Type, Bonded with 8 and End-apped with TMS Available as a guard column for reversed phase Tubing.mmID, mm length older Before After artridge filter bonded with 8 exmm 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.. MPa at. ml/min of flow rate. Upper pressure limit is more than MPa Available for. mm i.d to. mm i.d. column Evaluation of SunShell RP Guard Filter SunShell 8,. μm x. mm SunShell 8,. μm x. mm Without Guard Filter Without Guard Filter tr()=. min () = 9,9 With Guard Filter tr() =. min () = 8,8 % decrease of plate /=/ for. mm i.d. /=/ for. mm i.d. Flow rate:. ml/min for. mm i.d..8 ml/min for. mm i.d. Temperature: º Detection: UV@nm Sample: = Uracil = Toluene = Acenaphthene = Butylbenzene tr()=. min () = 9, tr() =. min () = 8,9 With Guard Filter Little change of plate Price of SunShell RP Guard Filter ame quantity Part number older: piece, RP Guard filter : piece Tubing: piece, ut: pieces, Ferrule: pieces (pressure Max: 9 psi, MPa) BGAK SunShell RP Guard Filter For exchange pieces BGAA SunShell RP Guard Filter older piece BGAA SunShell RP Guard Filter Starter Kit Photo

27 rdering information of SunShell Inner diameter (mm).... USP category SunShell 8, μm SunShell 8,. μm SunShell 8, μm SunShell 8,. μm SunShell PFP,. μm SunShell 8-WP,. μm SunShell RP-AQUA,. μm SunShell Phenyl,. μm SunShell,. μm SunShell -EP,. μm SunShell ILI-Amide,. μm SunShell ILI-S,. μm Length (mm) atalog number atalog number atalog number atalog number B B B B9 B B B B9 B B B9 B B L B B9 B B B B9 B B B8 B B B B8 B L F9 F F F9 F F F9 F F L F9 F F F9 F F W9 W W W9 W W W9 W W L W9 W W W9 W W R9 R R R R9 R R R9 R R Equivalent to L R R9 R R R R9 R R P9 P P P9 P P P9 P P L P9 P P P9 P P T9 T T9 T T9 T L T9 T T9 T E9 E E E9 E E E9 E E E9 E E E9 E E L U U L U

28 Inner diameter (mm).... USP category SunShell F8-,. μm SunShell F8-,. μm SunShell 8-,. μm SunShell 8-T,.μm SunShell -,. μm SunShell -,.μm Length (mm) atalog number atalog number atalog number atalog number G9 G G G9 G G G9 G G L L L L L L Distributor Technolab-Sorbent AB Biotech AB Inacom dichrom Instruments BioAnalytic Watrex Praha, s.r.o. mkon Kft. brnuta faza j.d.o.o. Espora Lab Innovations United Labquip LabService Analytica S.r.l. acalai USA, Inc. BGB Analytik AG Yair Technologies Ltd Easysci Instruments ompany Limited MZ-AALYSETEIK Ally Friends o., Ltd. hromatech Ltd TWT Scientific Ltd. J Raven Pharma Laboratories Applied hemical and Instrument o., Ltd IT Technologies Pte Ltd Eco -heminc. Prochrome India hromatopak Analytical Allytech orporation Ltd. Smart Labtech Pvt., Ltd., Ramnath Enterprises S&B Analitica Manufacturer 88 hromaik Technologies Inc. -- amiyoke, Minato-ku, saka, - Japan TEL: FAX: info@chromanik.co.jp URL: