High Performance Reversed-phase Columns -Pro Series ODS Column- 現在、医薬品や食品など様々な分野でHPLCが用いられており、 その分離カラムとして各メーカーから高性能HPLCカラムが販売されております。 YMCでも独自のエンドキャッピグ技術により開発しました、高性能HPLCカラムPro C18 を8年ほど前に発売し、その後Proシリーズとして数種類のカラムを発売しております。 今回、Proシリーズの特にODSカラムにつきまして特長などご紹介いたします。 YMC Co., Ltd.

Outline Features of Pro Series ODS Columns Comparison of Separation Characteristics Characteristics of Pro Series Columns Comparison of Various Compounds Introduction of Ultra-fast LC Columns - YMC-UltraHT Pro C18 - Conclusions これからお話しする内容。

YMC HPLC Columns ●Normal phase columns ●Gel filtration columns ●Optical isomer separation columns ●Reversed phase columns ・Conventional reversed phase columns, such as YMC-Pack ODS-A ・High performance reversed phase columns, such as Pro series

High performance reversed phase columns Pro Series Excellent peak shape without tailing High pure silica gel base Unique endcapping technology Stable quality Excellent reproducibility Excellent durability Support validation（2 types of test reports included） Effective for method development 3 types of ODS with different separative characteristics 　　 plus C8, C4 Applicable to a wide range of compounds and mobile phase

Product lineup of Pro Series ODS type 　　Standard type YMC-Pack Pro C18 　　Low carbon type Hydrosphere C18 　　High carbon type YMC-Pack Pro C18 RS 　Other type 　　YMC-Pack Pro C8 　　YMC-Pack Pro C4

YMC Pro Series of ODS Columns Hydrosphere C18 Pro C18 Pro C18 RS Silica ultra pure ultra pure ultra pure Particle size 2,3,5mm 2, 3, 5mm 3,5mm Pore size 120A 120A 80A Carbon content 12% 16% 22% Bonded phase monomeric C18 monomeric C18 polymeric C18 Endcapping completely endcapped completely endcapped completely endcapped Features hydrophilic surface excellent reproducibility high durability

Analysis of Basic Compounds (1) - Pro Series ODS - Pyridine-Phenol Tricyclic antidepressants Antihistamine

Analysis of Basic Compounds (2) - Comparison with Competitors - Pyridine Amitriptyline Diphenhydramine Pro Series ○ Brand B2 △ Brand D4 × Brand F1 Brand I5 Brand J1 ○：Tf≦1.5 △：1.5＜Tf≦2.0 ×：Tf＞2.0 Pro C18 RS Brand J1 Tf(4) 1.16 Tf(4) 6.18 1. Desipramine 2. Diphenyl (I.S.) 3. Imipramine 4. Amitriptyline

Plane recognitive ability Comparison of Separative Selectivity (1) - Hydrophobicity / Plane Recognitive Ability - hydrophobicity K’4 Plane recognitive ability α(5/3) Pro C18 RS 12.50 1.38 Pro C18 6.88 1.29 Hydrosphere C18 5.42 1.19 1 3 2 5 Pro C18 RS 4 Pro C18 1. 2. 3. Uracil Butylbenzene O-Terphenyl 4. 5. Hydrosphere C18 Amylbenzene Triphenylene Column : 150 X4.6 mmI.D. Eluent : methanol/water (80/20) Flow rate : 1.0 mL/min Temperature : 37 ℃ Detection : UV at 254 nm

Eluent : methanol/water (80/20) Comparison of Separative selectivity (2) ー Hydrophobic Selectivity Graph chartー ProシリーズODSカラムと他社カラムの比較を示す。 他社カラムには、親水性化合物分離用カラムから耐酸・耐アルカリ性の高いODSカラムまで 含まれます。 k’（Amylbenzene） Eluent : methanol/water (80/20)

Comparison of Separative Selectivity (3) ー Plane Recognitive Ability Graph chart ー ODSのリガンド密度が高いカラムほど平面認識能は高くなります。 しかし、リガンド密度がさほど高くないカラムでも平面認識能が大きくなる 場合があります。これは、充填剤表面のエンドキャッピングが不十分 な場合、平面認識能が大きくなる傾向にあるためだと考えられます。 α（o-Terphenyl / triphenylene） Eluent : methanol/water (80/20)

Comparison of Separative Selectivity (4) - Hydrogen-bonding Interaction - α(1/3) Pro C18 RS 0.067 Pro C18 0.106 Hydrosphere C18 0.142 Pro C18 RS 1. 2. 3. Pro C18 Caffeine Phenol Benzene Column : 150 X4.6 mmI.D. Eluent : methanol/water (30/70) Flow rate : 1.0 mL/min Temperature : 37 ℃ Detection : UV at 254 nm Hydrosphere C18

Eluent : methanol/water (30/70) Comparion of Separative Selectivity (5) ー Hydrogen-bonding Interaction Graph chartー a (caffeine / benzene) Eluent : methanol/water (30/70)

Advantage of YMC packing material Comparison of selectivity balance between k’ and a 0.18 Hydrophobicity:low Hydrogen-bonding capacity:high 0.16 Hydrophobicity:medium Hydrogen-bonding capacity:medium Hydrosphere C18 0.14 Hydrogen-bonding capacity a (caffeine / benzene) 0.12 Hydrophobicity:high Hydrogen-bonding capacity:low Pro C18 (2, 3, 5 mm) 0.10 F3 (2 mm) L4 (1.8 mm) I7 (1.7 mm) それぞれのタイプの説明 Proシリーズの位置の説明 Xterraの説明 0.08 Pro C18 RS 0.06 （■：commercially available ODS columns ） 0.04 2 4 6 8 10 12 14 Hydrophobicity　k’（amylbenzene） Eluent： MeOH/W (80/20) [amylbenzene] MeOH/W (30/70) [caffeine, benzene] 　

Pro C18 -ODS highly applicable to wide range of compounds - Local anesthetic Antioxidants 1. 4. Benzocain Lidocaine 1. n-Propyl gallate(PG) 2. 2,4,5-Trihydroxybutyrophenone(THBP) 3. tert-Butylhydroquinone(TBHQ) 4. Nordihydroguaiaretic acid(NDGA) 5. 4-Hydroxymethyl- 2,6-di-tert-butylphenol(HMBP) 6. n-Octyl gallate(OG) 7. n-Dodecyl gallate(DG) 2. 5. Procaine HCl Tetracaine HCl 3. 6. Cinchonine Butacaine 実例を示します。 Column : YMC-Pack Pro C18(3μm,12nm) 75 X4.6 mmI.D. Eluent : 20mM KH2PO4-K2HPO4(pH6.9) / methanol (40/60) Flow rate : 1.0 mL/min Temperature : 37 ℃ Detection : UV at 254 nm Column : YMC-Pack Pro C18(5μm,12nm) 75 X4.6 mmI.D. Eluent : A) acetonitrile/methanol/5%acetic acid (20/20/60) B) acetonitrile/methanol/5%acetic acid (45/45/10) 0-100%B(0-5min),100%B(5-15min) Flow rate : 1.0 mL/min Temperature : 30 ℃ Detection : UV at 280 nm

Durability for repetitive analysis Features of Pro C18 Conventional standard ODS Suitable as the first choice column Excellent durability as Monomeric type Durability for repetitive analysis 1.Caffeine 2.Sulfamonomethoxine 3.Cortisone 4.Indomethacin 次に、ProシリーズODSカラムについて各々の特長をご説明いたします。 Column : YMC-Pack Pro C18(3μm,12nm) 50 X4.6 mmI.D. Eluent : A) water/formic acid (100/0.05) B) acetonitrile/formic acid(100/0.05) 10-90%B(0-3min), 90%B(3-5min),10%B(5-9min) Flow rate : 1.0 mL/min Temperature : 37 ℃ Detection : UV at 254 nm

Reproducibility of Retention Time under 100% Aqueous Condition 1.Cytosine 2.Uracil 3.Guanine 4.Thymine 5.Adenine Before standing After standing Column　　 ：150×4.6mmI.D. Eluent　　 ：20ｍM KH2PO4-K2HPO4(pH6.9) Flow rate ：1.0mL/min Temperature：37℃ Detection ：UV at 254nm After analysis, columns filled with 100% mobile phase were left to stand overnight. Retention times of nucleic acid bases were analyzed before and after standing and compared.

Strong Retention and Unique Selectivity of Hydrosphere C18: Maltooligosaccharides Competitor LC Competitor WX 1. Glucose (G1) 2. Maltose (G2) 3. Maltotriose (G3) 4. Maltotetraose (G4) 5. Maltopentaose (G5) 6. Maltohexaose (G6) 7. Maltoheptaose (G7) Column : 150 X4.6 mmI.D. Eluent : water Flow rate : 1.0 mL/min Temperature : ambient (26℃) Detection : RI

Strong Retention and Unique Selectivity of Hydrosphere C18: Coenzymes Competitor MY Competitor DA 1. NADP 2. Nicotinic acid 3. NADPH 4. Nicotinamide 5. NAD 6. NADH Column : 150 X4.6 mmI.D. Eluent : 20mM KH2PO4-K2HPO4(pH6.9) / acetonitrile (99/1) Flow rate : 1.0 mL/min Temperature : 37 ℃ Detection : UV at 260 nm

Strong Retention and Unique Selectivity of Hydrosphere C18: Orotic acids Competitor SS Competitor LC Column : 150 X4.6 mmI.D. Eluent : 20mM H3PO4 Flow rate : 1.0 mL/min Temperature : 37 ℃ Detection : UV at 254 nm

Summary Unique endcapping process provides in good peak shapes even for basic compounds Excellent reproducibility of retention time under 100% aqueous conditions vs. conventional ODS columns Hydrophilic compounds can be effectively separated with strong retention under 100% aqueous conditions A natural complex mixture of Cinchona alkaloids including some minor impurities were successfully separated

Characteristics of Pro C18 RSTM High Carbon Content YMC-Pack Pro C18 RSTM is a polymeric-type phase bonded to high surface area silica, which contains the ODS groups at a high density. It shows a higher carbon content (22%) than the other conventional phases. Distinctive Separation Pro C18 RSTM recognizes subtle structural differences in hydrophobic compounds, e.g. structural isomers of terphenyl, to enable excellnet separations. The minimization of residual silanols achieved by the Lewis acid / base chemistry has resulted in less peak tailing of basic compounds. Improved pH-stability The polymeric bonding status and high carbon loading are considered to have resulted in improved pH-stability. It can be used even under highly demanding conditions.

Separation of Structural Isomers e.g., terphenyl analogous Pro C18 RSTM Pro C18TM Hydrosphere C18TM Competitor GI 1. 2. 3. Column : 150×4.6mmI.D. Eluent : methanol / water (85/15) Flow rate : 1.0 mL/min Temperature : 37℃ Detection : UV at 254nm o-Terphenyl m-Terphenyl p-Terphenyl Pro C18 RSTM recognizes subtle structural differences in hydrophobic compounds.

Comparison of Stability under Strongly Acidic Conditions (pH 1, 70℃) % k' remaining (Butyl benzoate) * Recommended pH range: 1-12 Purged with water/acetonitrile/TFA (10/90/1) at 70℃ Pro C18 RSTM is highly stable under acidic conditions.

%N remaining (Methyl Benzoate) Comparison of Stability under Strongly Alkaline Conditions (pH 10, 30℃） %N remaining (Methyl Benzoate) Mobile phase: 20 mM H3BO4-NaOH(pH 9.8)/methanol (50/50) Temperature: 30℃ Life time of Pro C18 RSTM is threefold longer than that of monomeric ODS.

Pro C18 RS - Optimization of Separation by pH (1) - 150×4.6mmI.D.　5μm 1 3, 4, 5 F040716A 5 10 15 20 min 2 pH 3.2 pH 6.8 pH 8.8 2 1 3 4 5 F040714E 10 15 20 min 1 2 3 4 5 F040714C 10 15 20 min Flurbiprofen 1. 2. Methyl benzoate Lidocaine 3. 4. Diltiazem 5. Verapamil Column : YMC-Pack Pro C18 RSTM Eluent : 20mM acetate buffer / methanol （35/65) Flow rate : 1.0 mL/min Temperature : 30 ℃ Detection : UV at 220 nm

Pro C18 RS - Optimization of Separation by pH (2) - 150×4.6mmI.D.　5μm 50×4.6mmI.D.　3μm 1 3, 4, 5 F040716A 5 10 15 20 min 2 pH 3.2 pH 6.8 pH 8.8 pH 8.8 5 10 min 12 3 4 F040714D 2 1 3 4 5 F040714E 10 15 20 min 1 2 3 4 5 F040714C 10 15 20 min ダウンサイジング Flurbiprofen 1. 2. Methyl benzoate Lidocaine 3. 4. Diltiazem 5. Verapamil Column : YMC-Pack Pro C18 RSTM Eluent : 20mM acetate buffer / methanol （35/65) Flow rate : 1.0 mL/min Temperature : 30 ℃ Detection : UV at 220 nm

Comparison of Various Compounds Medium Hydrophobic Compounds 　- Parabenzoates Isomers Hydrophilic Compounds - Flavonoid Glycosides -Saikosaponins High Hydrophobic Compounds - Nicardipine Hydrochloride -Methyl Linolenate Isomers Mixture of Hydrophobic and Hydrophilic Compounds

Medium Hydrophobic Compounds ー Parabenzoates Isomers ー Methyl p-hydroxybenzoate Pro C18 RS Ethyl p-hydroxybenzoate Isopropyl p-hydroxybenzoate Pro C18 Propyl p-hydroxybenzoate Isobutyl p-hydroxybenzoate 次に様々な性質の化合物についてODS3種類の分離特性を比較します。 Butyl p-hydroxybenzoate Hydrosphere C18 Column : 150×4.6mmI.D. Eluent : methanol / water (55/45) Flow rate : 1.0mL/min Temperature : 30℃ Detection : UV at 270nm

Hydrophilic Compounds ー Flavonoid Glycosides ー Pro C18 RS Rutin R = rutinose Isoquercitrin R = glucose Quercitrin R = rhamnose Quercetin R = H Pro C18 Hydrosphere C18 Column : 150×4.6mmI.D. Eluent : acetonitrile / 10mM H3PO4 (25/75) Flow rate : 1.0mL/min Temperature : 40℃ Detection : UV at 260nm

Hydrophilic Compounds ー Saikosaponins ー Hydrosphere C18 Pro C18 Saikosaponin c Saikosaponin a Pro C18 RS Saikosaponin b2 Saikosaponin d Brand B2　　Mightysil RP-18GP Aqua Column : 150×4.6mmI.D. Eluent : acetonitrile / water (38/62) Flow rate : 1.0mL/min Temperature : 37℃ Detection : UV at 210nm Brand B2

High Hydrophobic Compounds ーNicardipine Hydrochloride ー Pro C18 RS Pro C18 Hydrosphere C18 Brand D4 1 1 1 1 Rs=3.31 Rs=2.10 Rs=2.48 Rs=1.14 2 2 2 2 Brand D4　　CAPCELL PAK C18 AQ Column : 150×4.6mmI.D. Eluent : 20mM KH2PO4-K2HPO4(pH6.9) / methanol (25/75) Flow rate : 1.0mL/min Temperature : 37℃ Detection : UV at 254nm 1. Nicardipine Hydrochloride 2. The Decomposed product Nicardipine HCl

High Hydrophobic Compounds ー Methyl Linolenate Isomers ー Pro C18 RS Pro C18 Hydrosphere C18 Brand D4 Column : 150×4.6mmI.D. Eluent : acetonitrile / THF / water (35/30/35) Flow rate : 1.0mL/min Temperature : 37℃ Detection : UV at 210nm 1. 2. Methyl linolenate Methyl γ-linolenate

Mixture of Hydrophobic and Hydrophilic Compounds ー Gradient Analysis under 100% aqueous mobile phase conditionー Hydrosphere C18 Pro C18 Eye-drops 5 4 4 5 1 1,2 3 3 6 6 2 Column : 50×4.6mmI.D. Eluent : A) 20mM KH2PO4-H3PO4(pH2.5) B) acetonitrile 0-60%B(0-10min), 60%B(10-15min) Flow rate : 1.0mL/min Temperature : 37℃ Detection : UV at 265nm

YMC designs column for ultra fast LC Functionalized ultra pure 2.0 mm silica gel with 120 Å pore size Same separation selectivity compared to conventional 3 mm, 5 mm Pro C18 and Hydrosphere C18 column Simple scale down applicable without changing eluent conditions Lower pressure than sub-2 mm particle Instruments not only specific LC but ordinary LC available due to lower pressure ・「平衡化」されたところにサンプルをアプライ ・サンプルの物理・化学的性質の違い 　＝移動相とより強い相互作用を示すもの 　＝固定相とより強い相互作用を示すもの

Application data 2 mm YMC Ultra Fast LC column (temporary) vs. 3 mm, 5 mm YMC-Pack Pro C18 and competitors Flavonoids Alkaloids

Flavonoids (1) Problem in usual scale down 0.0 5.0 10.0 15.0 20.0 25.0 min J060303B 50 100 mV Conventional column 5 mm YMC-Pack Pro C18, 150 x 4.6 mmI.D. 1.0 mL/min (1.00 mm/sec) 5 mL Inj. 1 2 N 7 = 79,000 /m Rs (6, 7) = 2.24 3 4 5 6 7 Usual scale down (same particle size and linear velocity) 1. Myricetin 2. Quercetin 3. Apigenin 4. Kaempferol 5. Baicalein 6. Chrysin 7. Acacetin 0.0 5.0 8.0 min J060223A 50 100 150 mV Semi-micro column 5 mm YMC-Pack Pro C18, 50 x 2.0 mmI.D. 2 ・比較的疎水性の大きいニカルジピンの分解性生物との分離例 ・他ではベースライン分離してない ・RSでは疎水性のわずかな差な認識に優れるため分離可能 3 1 ⇒ lower the resolution due to shorten column size 0.2 mL/min (1.06 mm/sec) 1 mL Inj. 4 5 6 N 7 = 80,000 /m Rs (6, 7) = 1.25 7 Eluent : acetonitrile / water / formic acid (35/65/0.1) Temperature : 40℃ Detection : UV at 260nm

Flavonoids (2) Comparison of 5 mm and 2 mm 5 mm YMC-Pack Pro C18 50 x 2.0 mmI.D. 2 mm YMC Ultra Fast LC column 50 x 2.0 mmI.D. J060223A 50 100 150 J060222M 50 100 150 0.0 2.0 4.0 6.0 8.0 min 2 2 3 1 3 N 7 = 80,000 /m Rs (6, 7) = 1.25 N 7 = 149,000 /m Rs (6, 7) = 1.78 1 4 4 5 5 6 6 ・比較的疎水性の大きいニカルジピンの分解性生物との分離例 ・他ではベースライン分離してない ・RSでは疎水性のわずかな差な認識に優れるため分離可能 7 7 0.0 2.0 4.0 6.0 8.0 min Eluent : acetonitrile / water / formic acid　(35/65/0.1) Flow rate : 0.2 mL/min Temperature : 40℃ Detection : UV at 260nm

2 mm YMC Ultra Fast LC column, 50 x 2.0 mmI.D. 1 2 3 4 5 6 7 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min 50 100 150 J060222M mV 1 3 4 5 6 7 0.0 1.0 2.0 3.0 min 50 100 150 2 Flavonoids (3) Method development of ultra fast LC by 2 mm particle 2 mm YMC Ultra Fast LC column, 50 x 2.0 mmI.D. N 7 = 149,000 /m Rs (6, 7) = 1.78 0.2 mL/min (1.06 mm/sec) 1 mL Inj. Flow rate ×3 Eluent : acetonitrile / water / formic acid (35/65/0.1) Temperature : 40℃ Detection : UV at 260 nm ・比較的疎水性の大きいニカルジピンの分解性生物との分離例 ・他ではベースライン分離してない ・RSでは疎水性のわずかな差な認識に優れるため分離可能 N 7 = 143,000 /m Rs (6, 7) = 1.79 0.6 mL/min (3.18 mm/sec) 1 mL Inj. Excellent theoretical plate number and resolution

Flavonoids (4) Conventional LC vs. Ultra fast LC Conventional LC method 5 mm YMC-Pack Pro C18, 150 x 4.6 mmI.D. Eluent : acetonitrile / water / formic acid (35/65/0.1) Temperature : 40℃ Detection : UV at 260 nm 0.0 5.0 10.0 15.0 20.0 25.0 min 50 100 J060303B mV 1.0 mL/min (1.00 mm/sec) 5 mL Inj. 1230 psi N 7 = 79,000 /m Rs (6, 7) = 2.24 1 2 3 4 5 6 7 Ultra Fast LC method 2 mm YMC Ultra Fast LC column, 50 x 2.0 mmI.D. 2 Efficiency 90% ↑ Analysis time 90% ↓ Solvent Vol. 93% ↓ 0.0 1.0 2.0 3.0 min 50 100 50 100 J060223F mV 0.0 5.0 10.0 15.0 20.0 25.0 min 1 3 ・比較的疎水性の大きいニカルジピンの分解性生物との分離例 ・他ではベースライン分離してない ・RSでは疎水性のわずかな差な認識に優れるため分離可能 0.6 mL/min (3.18 mm/sec) 1 mL Inj. 4350 psi 4 5 6 N 7 = 143,000 /m Rs (6, 7) = 1.79 7

Alkaloids (1) Conventional LC vs. Ultra fast LC Conventional LC method 5 mm YMC-Pack Pro C18 150 x 4.6 mmI.D. Ultra Fast LC method 2 mm YMC Ultra Fast LC column 50 x 2.0 mmI.D. 1 2 2.0 0.0 4.0 6.0 8.0 10.0 12.0 14.0 min 20 40 60 mV 3 4 5 F060315F 1 2 3 4 5 0.2 0.0 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 min 20 40 60 mV F060315A 1.0 mL/min (1.00 mm/sec) 5 mL Inj. 1230 psi 0.6 mL/min (3.18 mm/sec) 1 mL Inj. 4350 psi N 5 = 102,000 /m Tf 5 = 1.13 N 5 = 106,000 /m Tf 5 = 1.07 ・比較的疎水性の大きいニカルジピンの分解性生物との分離例 ・他ではベースライン分離してない ・RSでは疎水性のわずかな差な認識に優れるため分離可能 1. Scopolamine 2. Atropine 3. Cinchonine 4. Quinine 5. Dihydroquinine Eluent : 20 mM CH3COOH-CH3COONH4 (pH 4.9) / acetonitrile(80/20) Temperature : 40℃ Detection : UV at 220 nm

Alkaloids (2) YMC 2 mm vs. Competitors sub-2 mm 1 2 3 4 5 0.2 0.0 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 min 20 40 60 mV 2 mm YMC Ultra Fast LC column, 50 x 2.0 mmI.D. N 5 = 106,000 /m Tf 5 = 1.07 4350 psi F060315A F060316D 1 2 3 4 5 0.2 0.0 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 min 20 40 60 mV 1.7 mm competitor WA, 50 x 2.1 mmI.D. N 5 = 89,000 /m Tf 5 = 3.99 7100 psi F060316E 1 2 3 4 5 0.2 0.0 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 min 20 40 60 mV ・比較的疎水性の大きいニカルジピンの分解性生物との分離例 ・他ではベースライン分離してない ・RSでは疎水性のわずかな差な認識に優れるため分離可能 1.8 mm competitor AZ, 50 x 2.1 mmI.D. N 5 = 78,000 /m Tf 5 = 2.35 5360 psi Eluent : 20 mM CH3COOH-CH3COONH4 (pH 4.9) / acetonitrile(80/20) Flow rate : 0.6 mL/min Temperature : 40℃ Detection : UV at 220 nm 1. Scopolamine 2. Atropine 3. Cinchonine 4. Quinine 5. Dihydroquinine

Conclusion ー Selectivity of Pro series ODS ー Pro C18 First choice column in various Fields including pharmaceutical products, agricultural chemicals, foods and natural products, etc UltraHT Pro C18 Ultra fast separation method development Pro C18 RS Strong retention and superior separation of hydrophobic compounds Reduced analysis time of hydrophobic compounds Superior separation of structural isomers Separation under demanding conditions Hydrosphere C18 Strong retention and superior separation of hydrophilic compounds Separation under 100% aqueous condition Reduced analysis time of hydrophilic compounds Changing selectivity greatly