C18, C18-WP, HFC18-16, HFC18-30,RP-AQUA, C8, PFP,

Transcription

1 8, 8-WP, F8-, F8-,RP-AQUA, 8, PFP, Phenyl, 8-, -, ILI-Amide and -EP. μm and μm PL column SunShell ore Shell Particle

2 SunShell is a core shell silica column made by hromaik Technologies. The next generation to ore Shell particle Features of SunShell. μm and μm Superficially porous silica *. μm and. μm of core and. 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 figure ) *Good peak shape for all compounds such as basic, acidic and chelating compounds *igh stability ( p range for SunShell 8,. to ) * Low breeding. μm. μm コア. μm Final TMS. μm. μm. μm コア Schematic diagram of bonding of SunShell 8 Schematic diagram of a core shell silica particle,. μm. μm Van Deemter Equation 8 Fully porous um Fully porous um A term : Eddy diffusion(dp is particle diameter) B term : Longitudinal diffusion (Dm is diffusion coefficient) term : Mass transfer ETP, mm 8 Mobile phase velocity, mm/sec omparison of plate height plots Fully proous.8 um SunShell. um Fully porous. um olumn: 8, x. mm 8 Acetonitrile/water=(/) Temperature: o Sample : aphthalene B term A term Van Deemter plot u term 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.

3 Why does a. μm core shell particle show the same performance as a sub μm particle? All terms in Van Deemter Equation reduce. A term ompany F, μm D :. mm D :.9 mm D 9 :. mm D 9 /D =.9 Sunniest, μm D :. μm D :. mm D 9 :. mm D 9 /D =. SunShell,. μm D :. mm D :. mm D 9 :.8 mm 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 ore shell silica A solute diffuses in a pore as well as outside of particles. A core without pores blocks diffusion of a solute. Difference of longitudinal diffusion Plate height (μm) 8 olumn: SunShell 8,. mm x. mm Totally porous mm 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 term 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. mm core shell particle shows as same column efficiency as a totally porous sub- mm particle. The right figure shows that a diffusion width of a sample in a. mm core shell particle and a mm totally porous particle. Both diffusion widths are almost same. The. mm core shell particle is superficially porous, so that the diffusion width becomes narrower than particle size. Same diffusion means same efficiency. Diffusion a.. mm sample ore Shell. mm Sample emanates in a particle after it enters. Diffusion. mm sample Totally porous. mm. mm Diffusion of sample in core shell and totally porous silica omparison of Performance by Plate/Pressure Plate Back press. (MPa) Plate/back press. Sunniest 8 T. mm 9,9. 9 Brand A 8.9 mm,. Brand B 8.8 mm, 9. Brand 8. mm,. 8 SunShell 8. mm 9, Sunniest 8 T. mm Brand A 8.9 mm Brand B 8.8 mm Brand 8. mm SunShell 8. mm,, olumn: x. mm 8, Acetonitrile/water=(/), Temperature: o Under a constant back pressure condition, SunShell 8 showed more than times higher performance to compare with totally sub-mm porous 8s.

4 SunShell 8,. μm, μm haracteristics of SunShell 8 Particle size ore shell silica 8 (USP L) Pore diameter Specific surface area arbon content Bonded phase omparison of retention and plate using PL End-capping Maximum operating pressure Available p range SunShell 8. μm 9 8 Sunniest end-capping MPa or 8, psi. - SunShell 8. μm Sunniest end-capping MPa or 8, psi. - SunShell 8 mm 8 MPa SunShell 8. mm MPa MPa =, =, =, olumn size: x. mm / =/ Flow rate:. ml/min Temperature: º Sample: = Uracil = affeine = Phenol = Butylbenzene = o-terphenyl = Amylbenzene = Triphenylene PL: itachi Lahrom ELITE (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 (tr) 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 mm column to SunShell column Isocratic separation PL 8 8 ()=,8 UPL SunShell 8,. mm x. mm Brand F 8, mm x. mm / of analysis time. ml/min ()=9, ()=,8 ()=, olumn: Brand F 8, μm x. mm SunShell 8,. μm x. mm /mm Phosphoric acid = / Flow rate:. ml/min,.8 ml/min at the lowest chromatogram Temperature: º Pressure: 9. MPa for Brand F 8 μm. MPa for SunShell 8. μm Detection: UV@ nm Sample: = Benzydamine = Ketoprofen = aproxen = Indomethacin = Ibuprofen PL: itachi Lahrom ELITE (Tubing,. mm i.d. ) UPL: Jasco X-L.8 ml/min ()=, Analysis time decreases to / to compare with a mm column sized x. mm.

5 Theoretical plate Theoretical Plate 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 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: SunShell 8, 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.mm.mm. sec. sec.8 sec Time (sec) means peak width (δ).. sec Acenaphthene t R. min. sec Average of theoretical plate (n=).mm (min) Inner diameter of tubing.mm.mm.mm Peak () 9 8 Peak () 9 Peak () 998 Peak () sec Toluene t R. min. sec Uracil t R. min Response time/sec olumn: SunShell 8,. 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: SunShell 8,. 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.

6 omparison of core shell columns Used columns. Kinetex 8,. μm omparison of standard samples between core shell 8s SunShell 8.8 MPa k=. k=. ompany A 8. MPa k=. k=9. k=9. ompany B 8. MPa ompany E 8 8. MPa ompany 8. MPa k=. ompany D 8. MPa. Accucore 8,. μm. PoroShell 8 E,. μm. Ascentis Express 8,. μm. ortecs 8,. μm. SunShell 8,. μm 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) SunShell 8,. 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=. TF=.9 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.

7 USP tailing factor Theoretical plate Theoretical plate 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=(:) 8 SunShell 8 ompany 8 Sunniest 8 um ompany A 8 ompany B 8 ompany D 8 ompany E 8 times 8 Sunshell 8 ompany 8 ompany A 8 ompany B 8 ompany D 8 ompany E 8 times Sample weight/μg Sample weight/μg Sample: TF=.8 TF=. SunShell 8 = Uracil (.mg) (core shell) = Propranolol (.mg) SunShell 8 TF=. 8 = ortriptyline (.mg) (core shell) = Amitriptyline (.mg) TF=.89 8 TF=. Sunniest 8 μm (fully porous) TF=. A 8 TF=. A 8 (core shell) TF=. B 8 TF=. B 8 (core shell) TF=. D 8 D 8 TF=.8 (core shell) TF=. E 8 TF=. (core shell) E Theoretical plate was calculated by σ method using peak width at.% of peak height..% 8 9 Acetonitrile/.% formic acid=(:)... ompany D 8 ompany B 8 ompany A 8 ompany E 8 ompany 8 SunShell 8 USP Tailing factor omparison column. Kinetex 8,. μm. Accucore 8,. μm. PoroShell 8 E,. μm. Ascentis Express 8,. μm. ortecs 8. μm. SunShell 8,. μm.... Sample weight/μg Amitriptyline overloads at low weight when acetonitrile/.% formic acid mobile phase. A peak is shifted forward under overloading. 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. mg of amitriptyline while SunShell 8 overloaded at more than from. to mg 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.

8 Relative retention/% Relative plate of butylbenzene/% Evaluation of Stability 8 Accelerated acidic test Alkaline test 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 Stability under acidic p condition was evaluated at 8 º using acetonitrile/% trifluoroacetic acid solution (:9) as mobile phase. % aqueous mobile phase expels from the pore of packing materials by capillarity and packing materials doesn t deteriorate. % acetonitrile in a mobile phase allows an accurate evaluation. -) 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. ). agae, T. Enami and S. Doshi, L/G orth America ctober. ) T. Enami and. agae, American Laboratory ctober. ) T. Enami and. agae, BUSEKI KAGAKU, () 9. omparison of particle size 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 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. SunShell 8 can be used at the p range from. to. *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 omparison column. Kinetex 8,. μm (p. to ). Accucore 8,. μm (p to ). PoroShell 8 E,. μm (p to 9). Ascentis Express 8,. μm (p to 9). ortecs 8. μm (p to 8). SunShell 8,. μm (p. to )

9 Ethanbutol l Impurity B SunShell Amino Acids derivatized with PA and FM Ethambutol ydrochloride olumn: SunShell 8,. μm x. mm A) Methanol/water (/ V/V) B) Methanol Flow rate:. ml/min Temperature: o Detection: UV nm Injection volume: μl Time (min) 8 %B 9 9 9, l Ethambutol ydrochloride olumn: SunShell 8. 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, =Glutaminc acid, =Serine, =istidine, =Glycine, =Threonine, =Arginine, 8=Alanine, 9=Tyrosine, =Valine, =Methionine, =Tryptophan, =Pnehylalanine, =Isoleucine, =Leucine, =Lysine, =Proline Dansylated estrogen hormones olong tea olumn: SunShell 8. μm, x. mm A) with.% formic acid. B) with.% formic acid. Gradient program: -. min: % B.. min: - % B. -. min: % B. -. min: - % B. -. min: % B Flow rate:. ml/min. Temperature: º Detection: MS(sim), m/z,.,.,. Samples:. Dansylated estriol,. Dansylated beta-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 Estrone olumn: SunShell 8. 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 Time min. min min %B % % % Epigallocatechin affeine Epigallocatechin gallate l 8 Gallocatechin gallate Epicatechin gallate 9 atechin gallate 8

10 SunShell 8-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. - SunShell 8-WP. μm nm 9 m /g % 8 L Sunniest endcapping MPa. - SunShell RP-AQUA. μm nm 9 m /g % 8 Equivalent to L Sunniest endcapping MPa - 8 a) SunShell 8. μm 9nm m /g.% 8 L Sunniest endcapping MPa. - 9 SunShell Phenyl. μm 9nm m /g % Phenylhexyl L Sunniest endcapping MPa. - 9 SunShell PFP. 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: SunShell 8, 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: SunShell 8-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: SunShell 8,. μm x. mm /mm phosphate buffer.=/ Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = Uracil, = Propranolol, = ortriptyline, = Amitriptyline olumn: SunShell 8,. μm x. mm SunShell PFP,. μm x. mm /mm phosphate buffer p.=8/ Flow rate:.8 ml/min Temperature: º Sample: = Uracil, = Propranolol, = ortriptyline, =Amitriptyline 9

11 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: SunShell 8, PFP,. μm x. mm /water or buffer=/ Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = Theobromine = Theophyline = affeine = Phenol olumn: SunShell 8, PFP,. μm x. mm / =/ Flow rate:. ml/min Temperature: º Detection: UV@nm Sample: = p-resol = m-resol = o-resol olumn: SunShell RP-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, mm x. mm SunShell RP-AQUA,. mm 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: SunShell 8, 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!!

12 Purine analogue rganic acid mm ammonium acetate (p.) Uric acid ypoxanthine S company ore shell 8 AQ,. μm umber and 8 peaks are broaden. 9 8 mm P Xanthine SunShell RP-AQUA,. μm xipurinol 8 9 olumn: SunShell RP-AQUA,. μm x. mm mm P or mm ammonium 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, = Diglycolic acid, 8 = Maleic acid, 9 = itric acid, = Succinic acid, = Fumaric acid. Amino acids (L/MS) Monosaccharides derivatized with L-Tryptphan D-Galactose L-Galactose D-Glucose olumn: SunShell RP-AQUA,. μm, x. mm A) mm FBA, B) mm FBA in / (9/) %B % to % in min (FBA: eptafluorobutyric acid) Flow rate:. ml / min Temperature: o Detection: MS (anofrontier LD) ESI Positive, Extracted ion chromatogram (EI) ourtesy of Dr Takeo Kaneko, okohama ational University L-Mannose L-Glucose D-Mannose olumn: SunShell RP-AQUA,. μm x. mm mm Phosphate and mm tetraborate (p 9.) Flow rate:. ml/min Temperature: º Detection: UV@ nm Sample: Monosaccharides derivatized with L-Tryptphan ourtesy of Dr Shuji Kodama, Tokai University

13 Desorption Dv (log d) (cc/g) Relative retention (%) SunShell. μm F8-, F8-, 8-, - 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 MPa or 8, psi. - L SunShell F8-. μm nm 9 m /g 8.%. μmol/m Sunniest endcapping MPa or 8, psi. 9 L SunShell F8-. μm nm m /g 8.%. μmol/m Sunniest endcapping MPa a or 8, psi a. - 9 L SunShell 8-. μm nm m /g 8.%. μmol/m Sunniest endcapping MPa a or 8, psi a. 9 L SunShell -. μm nm m /g.9% μmol/m Sunniest endcapping MPa a or 8, psi a. 9 L What is F8? exa-functional 8 has six functional groups. This F8 is much more stable under acidic condition. USP L line a: MPa, psi for. mm i.d. column Sunniest Bonding Technology examethydichlorotrisiloxane + Trimethylchlorosilane (TMS) (X: l,, ) Schematic diagram of reagent Schematic diagram of the state of bonding on silica surface.9.8 ore shell nm. ore shell nm Pore Diameter (nm) Pore distribution of core shell particle 8 9% line.% formic acid, p. º 8 Time (h) Stability under L/MS mobile phase condition Durable test condition olumn : SunShell F8-. 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: SunShell F8-,. μm ( nm) x. mm SunShell 8-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

14 SunShell. μm F8-, F8-, 8-, - omparison of column temperature For separation of peptides and proteins omparison of gradient time o,min o,min 8 o,min 8 o, min 8 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. Furthermore separation of proteins was improved by a long gradient time. Although one peak of BSA was obtained under minute gradient elution at degree elsius, more than peaks from BSA were obtained under minute gradient elution at 8 degree elsius. omparison of thickness of porous layer olumn: SunShell 8-,. μm ( nm,. μm layer) or x. mm, Sunshell 8-,. μm ( nm,. μm layer) or x. mm (prototype) A).% TFA in water, B).8 % TFA in acetonitrile Gradient program: Time min or min %B % % Flow rate:. ml/min, Temperature: 8 o, Detection: UV@ nm, Sample: = ytochrome, = Lysozyme, = BSA, = Myoglobin, = valbumin x. mm 8 o, min gradient Prototype. μm (. μm) SunShell 8-. μm (. μm) W. =.8 sec W. =.9 sec W. =8. sec x. mm 8 o, min gradient Prototype. μm (. μm). μm. μm. μm. μm W. =. sec SunShell 8-. μm (. μm) SunShell 8-,. μm Specific surface area: m /g Sunshell 8-,. μm Specific surface area: m /g It has been said that the thin porous layer on a core shell particle had an advantage over separation of macromolecule compounds such as proteins. Indeed regarding high-throughput separation under minute gradient elution, SunShell 8-. μm with. μm thickness porous layer showed shaper peaks than. μm with. μm thickness porous layer. owever, under long gradient elution at 8degree elsius, a reversed phenomenon is observed. The difference of a peak width is bigger than the value due to the difference of a particle size. As a result, the thickness of the porous layer has nothing with the peak width of a protein under such a condition. Regarding retention time, the wider the surface area, the longer the retention time. In other words, the partition interaction on a. μm porous layer material worked for longer time, so that BSA and ovalbumin was separated much better on. μm porous layer material than. μm porous layer material.

15 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 MPa or 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.

16 SunShell ILI-Amide,. μm haracteristics of SunShell ILI-Amide Particle size ore shell silica Amide (USP L8) Pore diameter Specific surface area For ydrophilic Interaction hromatography arbon content Bonded phase End-capping Maximum operating pressure Available p range SunShell ILI-Amide. μm 9 nm m /g % Amide no MPa or 8, psi - 8 Stationary phase of ILI-Amide R: ydrophilic group 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. Separation of ucleic acid bases: omparison of the other core shell hilic columns SunShell ILI-Amide S ompany ore shell Polyol olumn: SunShell ILI-Amide,. μm x. mm, oreshell polyol,. μm x. mm, ore shell lica,. μm x. mm Acetonitrile/ mm ammonium 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/ mm phosphate Buffer (p.9) =/ Flow rate:. ml/min Temperature: o Detection: UV@ nm, Sample: = yanuric acid, = Melamine olumn: SunShell ILI-Amide,. μm x. mm Acetonitrile/ mm phosphate buffer (p.) =8/ Flow rate:. ml/min Temperature: o Detection: UV@ nm, Sample: = icotinic acid, = Ascorbic acid, = Pyridoxine

17 Artificial sweeteners Glycoside. Aspartame -(I) -(I) +(IV) S. Saccharin. elicin. Arbutin. Salitin olumn: SunShell ILI-Amide,. μm, x. mm Acetonitrile: mm phosphate buffer (p.) =8: Flow rate:. ml/min, Temperature: Ambient Detection: nm Sample: = Aspartame, = Saccharin, = Acesulfame K -(I) -(I) +(IV) K S. Acesulfame K olumn: SunShell ILI-Amide,. μm, x. mm Acetonitrile: mm phosphate Ammonium (p.9) =8: Flow rate:. ml/min Temperature: Ambient Detection: UV@ nm Sample: = elicin, = Salicin,,= Arbutin, = Rutin. Rutin

18 SunShell RP Guard Filter <artridge Type, Bonded with 8 and End-apped with TMS> Available as a guard column for reversed phase older Tubing.mmID, mm length Before After exmm artridge filter bonded with 8 The filter is made of porous glass sized mm i.d. and mm thickness. Pore diameter is mm. 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 With Guard Filter t R ()=. min () = 9,9 t R () =. 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 Without Guard Filter With Guard Filter t R ()=. min () = 9, t R () =. min () = 8,9 Little change of plate Price of SunShell RP Guard Filter ame quantity Part number Photo SunShell RP Guard Filter Starter Kit 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

19 rdering information of SunShell Inner diameter (mm).... USP category SunShell 8,. μm SunShell 8, μm SunShell 8,. μm SunShell PFP,. μm SunShell 8-WP,. μm SunShell RP-AQUA,. μm SunShell Phenyl,. μm SunShell ILI-Amide,. μm SunShell -EP,. μm SunShell F8-,. μm SunShell F8-,. μm SunShell 8-,. μm SunShell -,. μm Length (mm) atalog number atalog number atalog number atalog number B9 B B B B9 B B B9 B B B B9 B B B B9 B B B8 B B B B8 B F9 F F F9 F F F9 F F F9 F F F9 F F W9 W W W9 W W W9 W W W9 W W W9 W W R9 R R R R9 R R R9 R R R R9 R R R R9 R R P9 P P P9 P P P9 P P P9 P P P9 P P E9 E E E9 E E E9 E E E9 E E E9 E E G9 G G G9 G G G9 G G L L L L Equivalent to L L L8 L L L L America Sales: Innovations United East th Street Suite J ew York, Y Tel: info@innovationsunited.com Web: 8 Manufacturer: hromaik Technologies Inc. -- amiyoke, Minato-ku, saka, - Japan TEL: FAX: info@chromanik.co.jp URL: