c18とは違う分離を!chromanik.co.jp/info/wp-content/uploads/2019/04/19...2019/04/19 · glu...
TRANSCRIPT
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C18カラムでは分離できない
異性体 親水性化合物
構造等の類縁物質
カラムを変える 移動相を変える
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標準試料の分離例(全多孔性)
Column: SunriseC30, Sunniest C18, RP-AQUA, 5C8, PhE, PFP, PFP&C18 5 µm 4.6x150 mm Mobile phase: CH3OH/H2O=75/25 Flow rate: 1.0 mL/min Temperature: 40 ºC Pressure: 5.4 MPa Sample: 1 = Uracil, 2 = Caffeine, 3 = Phenol, 4 = Butylbenzene, 5 = o-Terphenyl, 6 = Amylbenzene, 7 = Triphenylene,
Sunniest RP-AQUA
4
5
6
7 1
3
2
Sunniest C18
Retention time/min
1
3
4
5
6
7
Sunniest C8 7
6
5
4 3
2
1
2
Sunniest PhE
6
5
4
2,3 1
7
7
6
5
4
2,3 1
Sunniest PFP
0 5 10 15 20 25 30 35 40
Sunniest PFP&C18
4
1 2,3
7 5 6
4 5 6
7
1
3
Sunrise C30 2
水素結合性 (Caffeine/
Phenol)
疎水性 (Amylbenzene/ Butylbenzene)
立体選択性(Triphenylene/ o-Terphenyl)
C30 0.51 1.59 1.52
C18 0.43 1.56 1.37
RP-QUA 0.49 1.56 1.37
C8 0.33 1.43 1.23
PhE 1.00 1.34 0.92
PFP 1.00 1.29 2.51
PFP&C18 1.00 1.43 2.66
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C30 C28カラム
• 疎水性相互作用が主な相互作用
• 疎水性の高い化合物分離に有用
• 構造異性体分離に有用
トリアコンチル (C30)
CH3
CH3
• 水系の移動相での安定性が高い
• 極性基導入型C18より、耐久性が高い
• 親水性化合物の分離の使用可能
結合密度が高い場合
結合密度が低い場合 (RP-AQUA)
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RP-AQUAとの違い
SunShell C30 SunShell
RP-AQUA
結合密度 1.9 μmol/m2 1.4 μmol/m2
エンド
キャッピング TMS
Sunniest エンドキャップ
細孔径 12 nm 16 nm
pH範囲 2-8 2-8
(2-10)
対象化合物 異性体
低極性化合物 極性化合物
大きな違いは官能基の結合密度
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SunShell RP-Aqua の40 oCでの保持
核酸塩基の分離
緩衝液のみの移動相を用いた場合の保持の再現性を試験しました。毛管作用により,ポンプを停止し充填剤周りの圧力を大気圧にすると,通常のC18カラムは細孔内からの移動相の抜けだしが起こり,保持時間は大幅に減少しますが,SunShell RP-Aquaほとんど細孔内からの抜け出しは起こっておらず,94%以上の再現性を
示しました。
Column: SunShell RP-Aqua, 2.6 μm 75 x 4.6 mm Mobile phase: 10mM Phosphate buffer pH7.0
Flow rate: 1.0 mL/min Temperature: 40 ºC and 25 ºC
Sample: 1 = Cytosine, 2 = Uracil, 3 = Thymidine, 4 = Uridine, 5 = Thymine
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5
Measurement number every stop flow
Rel
ativ
e re
ten
tio
n o
f th
ymin
e
(%)
40℃でのチミンの保持時間の変化 (ポンプ送液1時間停止毎に測定)
5 2
4
1 3 40 ºC
25 ºC
0 1 2 3 4 Retention time/min
5 2
4
1
3
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HILICと逆相クロマトグラフィーの比較
0 1 2 3 4
1
2
3 4
5
Column: SunShell Amide 2.6 mm : 100 x 4.6 mm SunShell RPAqua 2.6 mm : 100 x 4.6 mm Mobile phase: acetonitrile : Amide : 20 mM ammonium acetate(pH4.7) =8:2 RPaqua : 20 mM phosphate buffer(pH7.0) Flow rate: 1.0 mL/min Temperature: 40 oC Detection: UV@250 nm Sample: 1. tymine, 2. uracil, 3. uridine, 4. cytosine, 5. cytidine
SunShell Amide
SunShell RPAQUA
2
5
4
3
1
Retention time/min
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有機酸の分離
Column: SunShell RP-AQUA 2.6 μm, 150 x 4.6 mm, Mobile phase: 0.025 M KH2PO4, pH2.5
Flow rate: 1.0 mL/min, Column pressure: 32 MPa for 1.5mL/min, Temperature: 40 ºC
Detection: UV@210nm, Injection volume: 2 μL
Sample: 1 = Oxalic acid (60 ppm), 2 = Tartaric acid (500 ppm), 3 = Formic acid (1000 ppm),
4 = Malic acid (1000 ppm), 5 = Lactic acid (1000 ppm), 6 = Acetic acid (1000 ppm),
7 = Diglycolic acid (1000 ppm), 8 = Maleic acid (100 ppm), 9 = Citric acid (1000 ppm),
10 = Succinic acid (1000 ppm), 11 = Fumaric acid (10 ppm).
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Column: Sunniest RP-AQUA 5mm, 2.0x 150mm
Mobile phase:
A) 5mM HFBA (Heptafluorobutyric acid)
B) 5mM HFBA in Acetonitorile/water(9/1)
%B 0% to 20% in 20 min
Flow rate: 0.2mL/min
Temperature: 40 oC
Detection: Quattro Micro API (ESI positive) SIM
アミノ酸の分離
0 10 20 30 Retention time/min
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Ala
Arg
Asn
Cys
Asp
Glu
Gln
His
Gly
Ile,Leu
Phe
Pro
Met
Lys
Ser
Tyr
Thr
Trp
Val
0 20 10
Retention time/min
90
175
122
134
133
147
148
76
150
132
156
147
166
116
106
205
118
120
182
アミノ酸のLC/MS
Column: Sunniest RP-AQUA 5mm, 2.0x 150mm
Mobile phase:
A) 5mM HFBA (Heptafluorobutyric acid)
B) 5mM HFBA in Acetonitorile/water(9/1)
%B 0% to 20% in 20 min
Flow rate: 0.2mL/min
Temperature: 40 oC
Detection: Quattro Micro API (ESI positive) SIM
Mass spectrum of Valine
118
MW=117
Structure of Valine
0
20
40
60
80
100
50 65 78 88 102 114 130 140 155 166 181 203 216 231 247
m/z
% m/z
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C30カラムの注意点
トリアコンチル (C30)
CH3
CH3
結合密度が高いほど
異性体分離に優れる
結合密度が高すぎると ピークがテーリングする
結合密度が低いと
分離が不十分になる
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0 5 10 15 20 25 30 35
Retention time / min
結合密度の影響
Company B C30,
5 μm
Company A C30, 5 μm
1
1
1
4
2
2, 3
2 3
3
4
4
Conventional C18, 5 μm
Column dimension, 250 x 4.6 mm;
mobile phase, methanol/water = 97/3;
flow rate, 1.0 mL/min;
temperature, 30 oC;
detection, UV295 nm;
sample,
1 = δ-tocopherol, 2 = γ-tocopherol,
3 = β-tocopherol, 4 = α-tocopherol
非対称性 結合密度 細孔径
1.02 1.9 μmol/m2 13 nm
非対称性 結合密度 細孔径
1.63 3.7 μmol/m2 20 nm
非対称性 結合密度 細孔径
1.04 3.0 μmol/m2 13 nm
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0 5 10 15 20 25 30
35 oC
25 oC
30 oC
15 oC
20 oC
0 2 4 6 8 10
trans
cis
ビタミンK1異性体の分離比較
Column: SunShell C30, 2.6 μm 100 x 2.1 mm
T社 C30, 2.6 μm 100 x 2.1 mm
Mobile phase: CH3OH/H2O= 96/4
Flow rate: 0.35 mL/min,
分離度Rs: 1.23
分離度Rs: 1.94
分離度Rs: 2.88
分離度Rs: 3.79 trans
cis
SunShell C30, 2.6 μm T社 C30, 2.6 μm
分離度Rs: 1.78 trans
cis
分離度Rs: 3.33 分離度Rs: 1.41
分離度Rs: 0.91
Retention time/min Retention time/min
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Column: SunShell C30, 2.6 mm 150 x 3.0 mm Mobile phase: CH3OH/H2O= 97/3 Flow rate: 0.43 mL/min, Temperature: 25 ºC, Pressure: 15.5 MPa
0 5 10 15 20 25 30 35
Column: Sunrise C28, 5 mm 250 x 4.6 mm Mobile phase: CH3OH/H2O= 97/3 Flow rate: 1 mL/min, Temperature: 25 ºC
ビタミンEの分離比較
0 2 4 6 8 10 12 14Retention time/min
Retention time/min
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ペンタフルオロフェニル(PFP)
F
FF
F F
CH3
• 疎水性相互作用
• 水素結合性
• 双極子相互作用
• π-π相互作用
• 静電的相互作用
• 極性化合物の保持が可能
• 立体選択性に優れている
• ハロゲン化合物の分離に有効
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標準試料の分離
Column dimension: 150 x 4.6 mm Mobile phase: CH3OH/H2O=75/25 Flow rate: 1.0 mL/min Temperature: 40 ºC Sample: 1 = Uracil, 2 = Caffeine, 3 = Phenol, 4 = Butylbenzene 5 = o-Terphenyl, 6 = Amylbenzene, 7 = Triphenylene
0 2 4 6 8 10 12 14 16 18 20 22 24
Retention time/min
SunShell PFP
SunShell C18
1
1 2
7 2,3
3
4
4
5
5
6
6
7
1 2
3 4
5
6 7
SunShell RP-Aqua
Hydrogen bonding (Caffeine/
Phenol)
Hydrophobicity (Amylbenzene/ Butylbenzene)
Steric selectivity
(Triphenylene/o-Terphenyl)
RP-Aqua 0.52 1.52 1.30
PFP 1.00 1.31 2.38
C18 0.39 1.60 1.46
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クレゾール異性体の分離
Column:
SunShell C18, 2.6 mm 150 x 4.6 mm
SunShell PFP, 2.7 mm 150 x 4.6 mm
Mobile phase: CH3OH/H2O=40/60
Flow rate: 1.0 mL/min Temperature: 25 ºC
Sample: 1 = p-Cresol
2 = m-Cresol 3 = o-Cresol
SunShell C18
SunShell PFP
1,2
2
3
1 3
C18カラムではp-クレゾールとm-クレゾールは分離不可能
PFP は分離可能
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C18とPFPによる塩基性化合物の保持比較
3 2
4
2,3,4
1
1
SunShell C18
SunShell PFP
Column: SunShell C18, PFP, 2.6μm 150 x 4.6 mm
Mobile phase: CH3CN/10mM Phosphate buffer pH7.0 = 80/20
Flow rate: 1.8 mL/min
Temperature: 25 ºC
Sample: 1 = Uracil, 2 = Propranolol, 3 = Nortriptyline, 4 =Amitriptyline
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CH
3
PFP&C18
F
FF
F F
CH3
• 疎水性相互作用
• 水素結合性
• 双極子相互作用
• π-π相互作用
• 静電的相互作用
• 立体選択性に優れている
• PFPカラムと比べると疎水性相互作用が強い
![Page 20: C18とは違う分離を!chromanik.co.jp/info/wp-content/uploads/2019/04/19...2019/04/19 · Glu Gln His Gly Ile,Leu Phe Mobile Pro Met Lys Ser Tyr Thr Trp Val 0 10 20 Retention](https://reader033.vdocuments.mx/reader033/viewer/2022050109/5f46d36af30831601823dd37/html5/thumbnails/20.jpg)
標準試料の分離例(コアシェル)
Column dimension: 150 x 4.6 mm Mobile phase: CH3OH/H2O=75/25 Flow rate: 1.0 mL/min Temperature: 40 ºC Sample: 1 = Uracil, 2 = Caffeine, 3 = Phenol, 4 = Butylbenzene 5 = o-Terphenyl, 6 = Amylbenzene, 7 = Triphenylene
SunShell PFP (9nm)
SunShell C8 (9nm)
SunShell C18 (9nm)
1
1
1
2
2
7
3
2,3
3
4
4
4
5
5
5
6
6
6
7
7
1
2
3
4 6
5, 7
1 2
3 4
5 6
7
1
4 5
6
7
2,3
SunShell RP-AQUA (16nm)
SunShell Phenyl (9nm)
SunShell C18-WP (16nm)
0 5 10 15 20 25 30
Retention time/min
SunShell PFP&C18 (9nm) 1 2
3 4
5 6 7
水素結合性 (Caffeine/
Phenol)
疎水性 (Amylbenzene/ Butylbenzene)
立体選択性(Triphenylene/ o-Terphenyl)
C18-WP 0.40 1.55 1.35
RP-AQUA 0.52 1.52 1.30
Phenyl 1.00 1.48 1.01
PFP 1.00 1.31 2.38
PFP&C18 1.64 1.41 2.77
C8 0.32 1.46 1.08
C18 0.39 1.60 1.46
5 7
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標準試料の分離例(全多孔性)
Column: SunriseC30, Sunniest C18, RP-AQUA, 5C8, PhE, PFP, PFP&C18 5 µm 4.6x150 mm Mobile phase: CH3OH/H2O=75/25 Flow rate: 1.0 mL/min Temperature: 40 ºC Pressure: 5.4 MPa Sample: 1 = Uracil, 2 = Caffeine, 3 = Phenol, 4 = Butylbenzene, 5 = o-Terphenyl, 6 = Amylbenzene, 7 = Triphenylene,
Sunniest RP-AQUA
4
5
6
7 1
3
2
Sunniest C18
Retention time/min
1
3
4
5
6
7
Sunniest C8 7
6
5
4 3
2
1
2
Sunniest PhE
6
5
4
2,3 1
7
7
6
5
4
2,3 1
Sunniest PFP
0 5 10 15 20 25 30 35 40
Sunniest PFP&C18
4
1 2,3
7 5 6
4 5 6
7
1
3
Sunrise C30 2
水素結合性 (Caffeine/
Phenol)
疎水性 (Amylbenzene/ Butylbenzene)
立体選択性(Triphenylene/ o-Terphenyl)
C30 0.51 1.59 1.52
C18 0.43 1.56 1.37
RP-QUA 0.49 1.56 1.37
C8 0.33 1.43 1.23
PhE 1.00 1.34 0.92
PFP 1.00 1.29 2.51
PFP&C18 1.00 1.43 2.66
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耐久性比較 Test conditions
Column: PFP&C18 5um 2.1x150 mm
PFP 5um 2.1x150 mm
Mobil phase: CH3OH/20mM PB pH7.0=70/30
Flow rate: 0.2 mL/min
Temperature: 40 ºC
Measurement condition
Column: PFP&C18 5um 2.1x150 mm
PFP 5um 2.1x150 mm
Mobil phase: Acetonitrile/water=70/30
Acetonitrile/water=60/40
Flow rate: 0.2 mL/min
Temperature: 40 ºC
Sample: Acenaphthene 0%
20%
40%
60%
80%
100%
120%
0 100 200 300 400 500 600
相対理論段数
通液時間/h
一般的なPFPカラムの3倍の耐久性を実現
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iHILIC-Fusion column by HILICON
SeQuent社でZIC-HILIC充填剤の合成を担当していたWen JiangさんがHILICON社をUMEÅで2014年
に設立
両性イオンとアミドの長所を取り入れた
HILIC-Fusion を開発
Wen Jiang
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24
HILICカラムを設計するうえで・・
1.基材自身の親水性が高い
基材のみでもHILICで使用可能なシリカを選択
コアシェルを使用することで高分離が可能
2.導入されている官能基の構造
官能基が高い水和性を有し,高い密度で導入
可能である
静電的な相互作用が極力低い
官能基の結合部位の疎水性が低いこと
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水10μL注入時のヒリックカラムの
クロマトグラム
移動相:5mM酢酸アンモニウム/アセトニトリル(20:80) 検出:UV@220nm(青),RI (赤)
0 5 10 15 20
TIME (min)
UV
RI 7.1min 6.6min
水 水10mL注入により固定相の状態が乱れている
固定相の平衡化完了
UVとRIのタイムラグ0.5分
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安定な水和層形成のためのイオンとは
ホフマイスターシリーズ (Hofmeister series リオトロピック系列と同意) とは、水を構造化させる能力の順にイオンを配列したもので
ある。(水和を安定させるイオン)
アニオンでは
SO4(2-)>HPO4(2-)>CH3COO(-)~citrate(3-)>tartrate(2-)>HCO3(-)>CrO4(2-)>Cl(-)>NO3(-)>ClO3(-)
カチオンでは
(CH3)4N+>NH4+ > K+ > Na+ > Li+ > Mg2+ > Ca2+
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アミノ酸のHILIC–ESI-MS
LC–MS System: Agilent 1100er LC system and Thermo Fisher LTQ™ equipped with a HESI source, operated in positive ionization mode for analysis of standards. For the dietary supplement, an Orbitrap™ Exactive classic equipped with a HESI source and operated in positive ionization mode. Column: 150 × 2.1 mm, 3.5-μm iHILIC-Fusion(+) Gradient Elution: A) acetonitrile–water–1 M ammonium acetate, pH 5.75 (90:5:5); B) water–acetonitrile–1 M ammonium acetate, pH 5.75 (90:5:5); 0–0.5 min (90:10) A–B; 0.5 to 25 min, gradient elution from (90:10) A–B to (60:40) A–B. Flow Rate: 0.3 mL/min Column Temperature: 40 °C Injection Volume: 5 μL Amino Acids: Arginine, asparagine, aspartic acid, glutamic acid, glutamine, hydroxyl-proline, isoleucine, leucine, lysine, phenylalanine, proline, tryptophan, tyrosine, and valine. 50 μM of each amino acid was dissolved in water–acetonitrile (25:75) solution.
ダイエットサプリ
標準品
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リン脂質のiHILIC-Fusionカラムによる分離
LC–MS system: Thermo Scientifi c Ultimate 3000 HPLC system coupled to a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer, which is equipped with a HESI II source and operated in negative ionization mode (ESI-). Column: 150 × 2.1 mm, 3.5 μm 100 Å iHILIC®-Fusion. Eluent:
A) ammonium acetate solution (35 mM, pH 5.75) and acetonitrile (95:5, v/v); B) acetonitrile. Gradient elution: 0–0.5 min, 97% B; 0.5–26.5 min, from 97% to 75% B; 27–33 min, 60% B; 35–45 min 97% B. Flow rate: 0.3 mL/min Column temperature: 40 °C Injection volume: 10 μL