HPLC column. Sunniest. C18, C18-HT, RP-AQUA, C8, PhE, PFP. ChromaNik Technologies Inc.

Transcription

1 HPLC column Sunniest C8, C8-HT, RP-AQUA, C8, PhE, PFP Chromaik Technologies Inc.

2 A ovel Bonding Technique (patent pending) The State of Art trifunctional silyl-reagent was developed as shown in Fig.. This Unique silyl-bonded reagent (HMDTS)can bond with any silanol groups on lica Sorbent surface as shown in Fig..It can expand and contract by itself in Caterpillar manner. This technique can substantially minimise the contribution of residual silanol groups on Reverses phase stationary phase. Finally an end-capping was done with trimethylsilyl-reagent (TMS). X H C CH n X X n:,, or X: Cl, CH, CHCH etc. H H H TMS 化 Fig. Schematic diagram of bonding of novel silylreagent on silica surface Features Fig. ovel C8 reagent Little residual silanol groups by an unique bonding technique Excellent stability, especially under acidic ph conditions Sharp peak shape for acidic, basic and chelating compounds RP-AQUA with C8 bonding offers Performance in 00% aqueous conditions, and shows enhanced retention of polar compounds. Characteristics of Sunniest Particle size (mm) Pore diameter (nm) Specific surface area (m /g) Carbon content (%) Bonded phase ph range and 0 C HT 0 0 C and 0 C8-8 and 0 0 C Sunniest PhE and 0 0 Phenylethyl. - 8 Sunniest PFP 0 0 Pentafluorophenyl - 8

3 Evaluation of End-capping Comparison of plates number () and USP tailing factor (TF) of amitriptyline メタノール CH H, ph7., 0 ºC () =,000 TF() =.0 () =0,00 TF() =.07 Column size:. x 0 mm Particle size: µm Mobile phase: CH H/0mM Phosphate buffer ph7. or.0 =80/0 CH C/0mM Phosphate buffer ph7.0 =0/0 Flow rate:.0 ml/min Temperature: 0 ºC or ºC Sample: = Uracil = Propranolol CH H CH H A 社 C8 B 社 C8 () =8,90 TF() =.7 () =0,000 TF() =.0 = ortriptyline = Amitriptyline アセトニトリル CH C, ph7.0, 0 ºC H CH CH CH () =,00 TF() =.0 メタノール CH H, ph.0, ºC () =9,000 TF() =.09 () =,000 TF() =.09 () =,00 TF() =.0 () =8,000 TF() =.0 () =0,000 TF() =. Sunniest PhE A 社 C8 () =,900 TF() =.9 A 社 C8 () =,000 TF() =. B 社 C8 () =,800 TF() =.8 B 社 C8 () =,00 TF() = Amitriptyline is widely used to evaluate residual silanol groups on the C8 stationary phase. Peak shape of Amitriptyline was compared under kinds of conditions such as methanol/phosphate buffer/0 ºC, methanol/phosphate buffer/ ºC and acetonitrile/phosphate buffer/0 ºC. Majority of the HPLC columns offered good peak shapes under methanol/phosphate buffer/0 ºC conditions. However using Mobile phase of acetonitrile/phosphate buffer/0 ºC, most of the columns(refer column A and B) offered high extent of Tailing unlike Sunniest columns offering a symmetrical peak., RP-AQUA and C8 columns allow to use a wide range of mobile phase without peak tailing because of extremely low content of residual silanol groups on the stationary phase.

4 SunniestC8 Stability under acidic and basic ph conditions Relative retention of ethylbenzene (%) Sunniest PhE B company C8 D compnay C Relative plate number of butylbenzene (%) , ph0 0ºC, ph9. 0ºC Time (h)) Time (h) Durable test condition Column size: 0 x. mm Mobile phase: CH C/.0% TFA (ph) = 0/90 Flow rate:.0 ml/min Temperature: 80 ºC Measurement condition Column size: 0 x. mm Mobile phase: CH C/H =0/0 Flow rate:.0 ml/min Temperature: 0 ºC Sample: = Uracil = Ethylbenzene Durable test condition Column:, C8, mm 0 x. mm Mobile phase: C8: CH H/0mM Sodium borate/0mm ah=0//9 (ph0) C8: CH H/0mM Sodium borate (ph9.) =0/70 Flow rate:.0 ml/min Temperature: C8-0 ºC, C8-0 ºC Measurement condition Column:, C8, mm 0 x. mm Mobile phase: CH H/H =7/ Flow rate:.0 ml/min Temperature: 0 ºC Sample: = Butylbenzene Stability under acidic ph conditions 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. -) has kept 90% retention for 00 hours under severe conditions of acetonitrile /% trifluoroacetic acid solution (ph )at 80 deg C. ur Unique HMDTS bonding technique offers significant enhancement of column life, Considering the C8 ligand length the is less stable than. However, Sunniest RP- AQUA C8 column with HMDTS bonding along with end capping offers longer column life in comparison to other RP Aqua columns. ). agae, T. Enami and S. Doshi, LC/GC orth America ctober 00. ) T. Enami and. agae, American Laboratory ctober 00. ) T. Enami and. agae, BUSEKI KAGAKU, (00) 09. Stability under basic ph conditions was evaluated at 0 ºC using methanol/sodium borate buffer ph 0 (0:70) 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. When column is used at 0 ºC, column life becomes,000 hours. Most of the HPLC columns stability data is offered at ambient room temperature alternate ºC at ph -0 units..at temperature of ºC,the column life is sixteen times longer than that at 0 ºC. offers performance at elevated ph and temperature. Regarding stability under basic ph condition, there are very few C8 column like & Hybrid type C8 which can sustain and offer performance under such challenging conditions of high temperature and ph.it is considered that our double end-capping & base deactivation technique leads higher stability. has operational ph Range from. to 0.,Phenyl has operational ph Range. to 9 and Sunniest RP-Aqua and Pentaflorophenyl (PFP) at ph -8..

5 SunniestC8 Relationship between ph and retention of Acidic, Basic and eutral compounds Basic compound (B) B A ph Retention factor (k) eutral compound () A A B B ph7 ph0 Acidic compound (A) ph ph selectivity, ph. ph7 ph0 Column:, mm 0 x. mm Mobile phase: A) 0mM Phosphoric acid ph. A) 0mM Phosphate buffer ph7 A) 0mM Phosphate buffer ph0 B) Acetonitrile Time (min) 0 0 %B (%) Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0 nm Sample: = Thiamine HCl Vitamin B = icotinamide = icotinic acid = Pyridoxine HCl Vitamin B = Folic acid = Riboflavin Vitamin B It is interesting to note that the change in ph of mobile phase can offer different selectivity of ionic compounds. can be used at the ph range from. to 0, so that a suitable analytical method can be developed using Column..

6 Comparison data: Bleeding from column Comparison using Corona CAD Comparison using MS.E+08 F 社 C8 Area: 90,000.E+08.E+08.E+08 B 社 C8 B 社 C8 Area:,0,000 Intensity (cps).e+08.e+08.e+08.e+08 Area: 9,000.E+08.E+07 Column size: 0 x.0 mm Mobile phase: A) 0.% acetic acid B) CH C Gradient: Time: 0min min.min 8min 9min %B: % % 00% 00% % Flow rate: 0. ml/min Temperature: 0 ºC Detection: Corona CAD 0.E Time (min) Column size: 0 x.0 mm Mobile phase: A) 0.% acetic acid B) CH C Gradient: Time: 0min min.min 8min 9min %B: % % 00% 00% % Flow rate: 0. ml/min Temperature: 0 ºC MS: ABI API-000 Ionization: Turboionspray (cation) Measurement mode: Q Scan m/z Separation of antidepressants using Acetonitrile and Ammonium acetate for LC/MS Company E C8 Company F C8 Company G C8 Company H C8 =,00 TF=. =,00 TF=.0 =,000 TF=.8 =8,00 TF=. =,00 TF=. =,00 TF=. =8,00 TF=.0 Column size: 0 x. mm Particle size: mm Mobile phase: CH C/0mM Ammonium acetate ph.8=0/0 Flow rate:.0 ml/min Temperature: 0 ºC Sample: Amitriptyline CH CH 0 8 0

7 /C8 Stability of / C8 under 00% aqueous conditions Relative retetion of ethylbenzene (%) Acidic ph test /C Time (h) Relative plate number of thtmine (%) Basic ph test Time (h) Durable test conditions Column:, mm 0 x. mm Mobile phase: 0.% TFA Flow rate:.0 ml/min Temperature: 0 ºC Durable test conditions Column:, mm 0 x. mm Mobile phase: 0mM Phosphate buffer ph8.0 Flow rate:.0 ml/min Temperature: 0 ºC Measurement conditions Column:, mm 0 x. mm Mobile phase: CH H/H =7/ Flow rate:.0 ml/min Temperature: 0 ºC Sample: = Uracil = Amylbenzene Measurement conditions Column:, mm 0 x. mm Mobile phase: 0mM Phosphate buffer ph7.0 Flow rate:.0 ml/min Temperature: 0 ºC Sample: = Thymine It is important that stability is evaluated for RP Aqua columns under 00% aqueous conditions because RP-Aqua column life becomes longer with incremental contents of organic solvent in a mobile phase. /C8 column can be used under 00% aqueous conditions from ph to ph 8. / C8 column can be used under 00% aqueous conditions from ph to ph 8. /C8 is one of the most stable aqua type column. /C8 column with HMDTS bonding along with end capping offers longer column life in comparison to other RP Aqua columns

8 /C8 Reproducibility of retention under 00% aqueous conditions C8 and C8 Rrversed phases exhibit decreased and poorly reproducible retention under more than 98% aqueous conditions as shown in Fig.. This problem traditionally has been explained as being the result of ligand collapse or a matting effect. agae - ascertained, however, that the mobile phase was being expelled from the pores of the packing material under 00% aqueous mobile phase conditions, as Fig. shows. When the surface of packing materials isn t wet by water, water used as a mobile phase expels from the pore of the packing material by capillarity. This is a reason why reproducibility in retention is low under 00% aqueous conditions. Reversely pressure around the packing material makes water permeate into the pore of the packing material to overcome a force worked by capillarity. In other words, the surface of a reversed phase like C8 isn t wet by water anytime even if water permeates into the pore of the packing material or not. So it is wrong that we say dewetting when water expel from the pore. Saying Depermeatiing is more appropriate. /C8 is a reversed stationary phase, so that it is not wet with water. However the contact angle of water on the surface of /C8 is less than that of a conventional C8. Expelling force (pressure) acted by capillarity on /C8 is less than atmospheric pressure. So, /C8 shows reproducible retention under 00% aqueous conditions. lica Pressure 0nm After stopping pump for hour. agae, T. Enami and S. Doshi, LC/GC orth America ctober 00. Fig. Retention behavior of a C8 column under 00% aqueous mobile phase conditions Water Water permeates into the pore. Permeating Stop flow Water expels from the pore by capillarity. Depermenating Fig. Schematic diagram of C8 particle ). agae, T. Enami and S. Doshi, LC/GC orth America ctober 00. ) T. Enami and. agae, American Laboratory ctober 00. ) T. Enami and. agae, BUSEKI KAGAKU, (00) 09. Separation of nucleic acid bases 7 0 ºC ºC Column:,/C8 mm 0 x. mm Mobile phase: 0mM Phosphate buffer ph7.0 Flow rate:.0 ml/min Temperature: 0 ºC and ºC Sample: = Cytosine = Uracil = Thymidine = Uridine = Thymine Relative retention of thymine (%) Change of retention of thymine at 0 ºC (measurement every stop flow for hour) Measurement number ev ery stop flow /C8 showed more than 97% of reproducibility in retention using 00% aqueous buffer as a mobile phase.

9 /C8 Separation of water-soluble vitamins Separation of nucleotides =9, Column:,/C8 mm 0 x. mm Mobile phase: 0mM Phosphate buffer ph.8 Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0nm Sample: = icotinic acid, = Pyridoxal = Pyridoxine, = icotinamide Column:, /C8 mm 0 x. mm Mobile phase: 0mM Phosphate buffer ph.0 Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0nm Sample: = -GDP, = -GMP, = -ATP, = -ADP, = -AMP separation of olong tea Separation of water-soluble vitamins using mobile phase for LC/MS Caffeine xalic acid Theanine Guanylic acid Column: /C8, mm 0 x. mm Mobile phase: A) H /H P (99.9:0.) B) CH C Time (min) %B (%) Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0 nm Injection volume: 0 ml Sample: olong tea = (-)-Epigallocatechin = (+)-Catechin = (-)-Epigallocatechin gallate = (-)-Epicatechin = (-)-Epicatechin gallate Column: /C8i mm 0 x. mm Mobile phase: A) 0mM Ammonium acetate Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0 nm Sample: = icotinic acid B) Acetonitrile/ A solution (0:80) Time (min) 0 0 %B (%) = Pyridoxine HCl Vitamin B = icotinamide = Thiamine HCl Vitamin B = Folic acid = Riboflavin Vitamin B 8

10 Separation of standard samples 7 Separation of pyridine and phenol 7 7 a(pyridine/phenol) = 0.8 a(pyridine/phenol) = 0.0, 7 Sunniest PhE a(pyridine/phenol) = 0., Column:, µm.x0 mm, µm.x0mm, µm.x0 mm Sunniest PhE, µm.x0 mm Sunniest PFP, µm.x0 mm Mobile phase: CH H/H =7/ Flow rate:.0 ml/min Temperature: 0 ºC Pressure:. MPa Sample: = Uracil, Hydrophobicity 7 = Caffeine, = Phenol, Sunniest PFP = Butylbenzene, (=,700) = o-terphenyl, (=,000) = Amylbenzene, (=,00) 7 = Triphenylene, (=,00) is plate number of the above. C8 RP-AQUA C8 PhE PFP a(amylbenzene/butylbenzene) Hydrogen bonding capacity a(caffeine/phenol) Steric selectivity a(triphenylene/o-terphenyl) Residual silanol activity a(pyridine/phenol) Sunniest PhE a(pyridine/phenol) = Column:, µm.x0 mm, µm.x0mm, µm.x0 mm Sunniest PhE, µm.x0 mm Mobile phase: CH H/H =0/70 Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0nm Sample: = Uracil = Pyridine = Phenol Separation factor of pyridine and phenol is said to show the amount of residual silanol groups. The lower a value of separation factor, the less an effect of residual silanol groups. All Sunniest columns show one of the lowest value. shows not only high efficiency but also low column pressure. 9

11 Separation of a chelating compound Separation of acidic compounds Sunniest PhE Sunniest PFP Sunniest PhE Column:, µm.x0 mm, µm.x0mm, µm.x0 mm Sunniest PhE, µm.x0 mm Sunniest PFP, µm.x0 mm Mobile phase: CH C/0mM H P =0/90 Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0nm Sample: = 8-Quinolinol = Caffeine, RP-AQUA, C8,PhE and PFP are inert for a metal chelating compound and acidic and basic compounds, so that they show symmetrical peaks of each compound. 0 8 Column:, µm.x0 mm, µm.x0mm, µm.x0 mm Sunniest PhE, µm.x0 mm Mobile phase: CH C/0.% H P =/98 Flow rate:.0 ml/min Temperature: 0 ºC Detection: UV@0nm Sample: = Formic acid = Acetic acid = Propionic Acid Retention comparison between C8 and PFP Column size:. x 0 mm Mobile phase: CH C/0mM Phosphate buffer ph7.0 =0/0 Flow rate:.0 ml/min Sample: =Uracil, =Propranolol, =ortriptyline, =Amitriptyline Sunniest PFP PFP retains a cation such a nortriptyline much longer than a C8. 0

12 , C8-HT Features Low back pressure and high efficiency by precisely classified particle High pressure packing (0,000 psi) using hard silica gels with high pressure resistant leads long column life without any void. Unique bonding technique for Sunniest The most suitable inner surface of column by special grinding arrow Particle Distribution and Low Back Pressure Measured by Coulter Counter method Conventional particle D0:.7 mm D0:.09 mm D90:. mm D90/D0=.9 (mm) Classified particle D0:.7mm D0:.0mm D90:. mm D90/D0=. arrow particle distribution Pressure, MPa Comparison of back pressure Flow rate, ml/min -HT.0 um Brand B C8.8 um Brand C C8.7 um Conventional mm silica gel particle was classified again. 0% volume was cut off from both sides respectively. Consequently column back pressure reduced more than %. ur mm silica gel particle shows a half pressure to compare with the other sub- mm silica gel particle. Column: Sunniest, Acquity and Zorbax Column dimension: 0 x. mm Mobile phase: Acetonitrile/water=(70/0) Temperature: o C An Unique Modification Leads Good Peak Shape eutral compounds Amitriptyline ( th peak) -HT mm () =,7 pl/m TF() =.9 Pressure =.8 MPa Acquity C8.7mm () = 8,77 pl/m TF() =.7 Pressure =. MPa Leading C8.mm A half of retention () = 7,9 pl/m TF() =.8 Pressure = 0.MPa Column dimension: 0 x. mm Mobile phase: Methanol/water=70/0 for neutral compounds Methanol/mM phosphate buffer (ph.0)=80/0 for antidepressants Flow rate: 0. ml/min Temperature: room temperature Sample: eutral compounds, =Uracil, =Toluene, =Biphenyl, =Penanthrene Antidepressants, =ortriptyline, =Toluene, = Imipramine, =Amitriptyline CH CH It is difficult to end-cap on sub mm or mm silica gel particle as well as mm or mm silica gel particle. Most sub mm or mm C8 columns show smaller plate number and larger tailing factor for a basic compound than -HT. -HT mm shows a good peak shape for amitriptyline under methanol/phosphate buffer mobile phase at room temperature. Furthermore -HT mm shows times longer retention time than the other brand columns.

13 , C8-HT Comparison of Plate umber 0 x. mm ()= 0,07 00 x. mm ()= 9, Retention time (min) Retention time (min) Mobile phase: CH C/H = 0/0 Flow rate: 0. ml/min for. x 0 mm and. x 0 mm, 0. ml/min for. x 7 mm and. x 00 mm Temperature: 0 ºC Detection: UV@0 nm Sample: =Uracil, =Toluene, =Acenaphthene, =Butylbenzene Separation of Analgesics mm, 0 x. mm ()=80,000 plate/m Mobile phase: CH C/0.% Formic acid = 0/80 Flow rate:.0 ml/min for 0 x. mm 0. ml/min for 0 x. mm Temperature: 0 ºC Detection: UV@0 nm Sample: =Acetaminophen, =Antipyrine, =Aspirin, =Ethenzamide CH H H CH CH H H C H CH Separation of Amino Acids derivatized with PA ()=7,000 plate/m High throughput -HT mm, 0 x. mm Retention time (min) 9 7 mm particle allows to reduce retention time because high efficiency is kept under high flow rate conditions. As shown the above chromatograms, analytical time reduced /8 without sacrifices of separation by using mm, 0 x. mm column instead of mm 0 x. mm column. Column: -HT mm, 00 x. mm Mobile phase: A) 0mM ap + 0mM ab7 + 0.mM a B) Acetonitrile/Methanol/Water (//0 %V) Time(min) %B 0 00 Flow rate: 0.7 ml/min Temperature: 0 ºC Detection: UV@8 nm Sample: =Aspartic acid, =Glutaminc acid, =Serine, =Histidine, =Glycine, =Threonine, 7=Arginine, 8=Alanine, 9=Tyrosine, 0=Valine, =Methionine, =Tryptophan, =Pnehylalanine, =Isoleucine, =Leucine, =Lysine, 7=Proline Retention time (min) Characteristics of -HT, mm lica gel support C8 Packings Particle size (mm) Pore diameter nm) Specific surface area (m /g) Carbon content (%) Bonded phase Maximum operating pressure Available ph range -HT.0 (Coulter counter) 0 0 C8 70 MPa or 0,000 psi. - 0 It is very important for mm particle to have a capacity to resist pressure because of high column back pressure. The larger a pore volume of silica gel, the weaker a capacity to resist pressure. The silica gel with 0.8 ml/g of pore volume is used for -HT, mm, so that it have a high capacity to resist pressure and a high operating pressure.

14 Sunniest Guard columns C8, RP-AQUA/C8, C8 PhE & PFP Guard Cartridge (0 x mm) Feature *mple structure *Low dead volume *Available for not only mm column but also mm column Filter Schematic Diagram of Cartridge 0xmm PEEK -ring Photo of Cartridge and Holder Analytical column only Both Guard and Analytical columns Comparison of chromatograms () =0,90 TF() =.0 () =0,770 TF() = Column:, mm 0 x. mm Mobile phase: Guard cartridge 0 x mm CH H/0mM Phosphate buffer ph7. = 80/0 Flow rate:.0 ml/min Temperature: 0 ºC Pressure:.8 MPa,. MPa(+guard) Sample: = Uracil, = Propranolol, = ortriptyline, = Amitriptyline, TF: USP tailing factor Particle size Catalog o., mm Guard cartridge column (-pak + Holder) x 0 mm µm EBAH, mm Guard cartridge column (-pak + Holder) x 0 mm µm ERAH, mm Guard cartridge column (-pak + Holder ) x 0 mm µm ECAH, mm Guard cartridge (-pak) x 0 mm µm EBAC, mm Guard cartridge (-pak) x 0 mm µm ERAC, mm Guard cartridge (-pak) x 0 mm µm ECAC Sunniest Guard cartridge holder x 0 mm --- HLAC

15 , C8-HT Sunniest PhE Sunniest PFP Sunniest rdering information Inner diameter Length, µm, µm Sunniest RP- AQUA, µm Sunniest RP- AQUA, µm, µm, µm [mm] [mm] Catalog o. Catalog o. Catalog o. Catalog o. Catalog o. Catalog o. 0 EB EB ER ER EC EC 7 EB - ER - EC - 00 EB EB ER ER EC EC 0 EB7 EB7 ER7 ER7 EC7 EC7 0 EB8 EB8 ER8 ER8 EC8 EC8 0 EB EB ER ER EC EC 00 EB EB ER ER EC EC 0 EB7 EB7 ER7 ER7 EC7 EC7 0 EB8 EB8 ER8 ER8 EC8 EC8. 0 EB EB ER ER EC EC 0 EB EB ER ER EC EC 7 EB - ER - EC - 00 EB EB ER ER EC EC 0 EB7 EB7 ER7 ER7 EC7 EC7 0 EB8 EB8 ER8 ER8 EC8 EC EB78 - ER78 - EC EB8 - ER8 - EC8 0 - EB87 - ER87 - EC EB88 - ER88 - EC88 Inner diameter Length Sunniest PhE, µm Sunniest PhE, µm Sunniest PFP, µm [mm] [mm] Catalog o. Catalog o. Catalog o..0 0 EP EP - 7 EP EP EP - 0 EP7 EP7-0 EP8 EP EP EP - 00 EP EP - 0 EP7 EP7-0 EP8 EP EP - Inner diameter Length Sunniest C8-HT, µm [mm] [mm] Catalog o.. 0 EB9 0 EB9 0 EB9 7 EB9 00 EB9.0 0 EB 0 EB 0 EB 7 EB 00 EB 0 EP EP EF 7 EP EP EP EF 0 EP7 EP7 EF7 0 EP8 EP8 EF EP EP8-0 - EP EP88 -