a b Chroma Graphein Chromatography

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(1)

分離

抽出

蒸留

ろ過

再結晶

クロマトグラフィー

機器による分離

(2)

クロマトグラフィーの歴史

z

ロシアの植物学者ツウェット

z

植物色素 クロロフィル →

  

クロロフィルa、クロロフィルb、キサンチン、カロチン z

色 Chroma + 記録する Graphein

         → Chromatography

(3)

クロマトグラフィーのしくみ

固定相 (Stationary Phase) 移動相 (Mobile Phase)

(4)

クロマトグラフィー分離において

       利用される性質

z

分子が液体に溶解する傾向 (

溶解性

z

分子が微粉末の固体表面にひきつけられ

る傾向 (

吸着

z

分子が蒸発するか蒸気の状態に入る傾向

  (揮発性)

(5)

クロマトグラフィーの種類

GSC GLC GAS SFC NP RP IEC GPC GFC SEC Column TLC Paper Planar LIQUID CHROMATOGRAPHY

Thin Layer Chromato.

(6)

液体クロマトグラフィー

 固定相:固体

→ 吸着クロマトグラフィー

 固定相:液体

(7)

吸着クロマトグラフィー

        分離メカニズム

30 µ Si - O - H δ− δ+ Normal phase LS Reverse phase LS Silica Gel

(8)

シリカゲルに対する官能基

シリカゲルに対する官能基

       の吸着力の強さ

       の吸着力の強さ

(9)

溶媒の種類

水>メタノール>エタノール>アセトン>エーテル>ク

ロロホルム>ベンゼン>シクロへキサン

Polar solvent

(10)

A + B + C OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO Sample (A+B+C) Column Solid Particles (packing material- stationary phase) Eluant (eluate)

DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY

A B C Solvent(mobile or moving phase) OH OH HO OH HO O HO

(11)
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分配クロマトグラフィー

z

順相 (Normal Phase)クロマトグラフィー

固定相の極性

>移動相の極性

z

逆相 (Reversed Phase)クロマトグラフィー

固定相の極性<

移動相の極性

(13)

O O O | | | −O−Si−O−Si−O−Si−O−H | | | O O O | | | −O−Si−O−Si−O−Si−O−H | | | O O O O O O | | | −O−Si−O−Si−O−Si−O−H | | | O O O | | | −O−Si−O−Si−O−Si−O−H | | | O O O (CH2)17CH3 (CH2)17CH3 (CH2)17CH3 : オクタデシル基 (ODS基)

(14)

M O B I L E P H A S E L I Q U I D L i q u i d - L i q u i d C h r o m a t o g r a p h y ( P a r t i t i o n ) L i q u i d - S o l i d C h r o m a t o g r a p h y ( A d s o r p t i o n ) L i q u i d S o l i d N o r m a l P h a s e R e v e r s e P h a s e N o r m a l P h a s e R e v e r s e P h a s e M o b i l e P h a s e N o n p o l a r S t a t i o n a r y p h a s e P o l a r M o b i l e P h a s e P o l a r S t a t i o n a r y p h a s e N o n p o l a r F O R M A T S T A T I O N A R Y P H A S E

Types of Chromatography

Types of Chromatography

(15)

The 4 basic liquid chromatography modes are named according to the mechanism involved:

1. Liquid/Solid Chromatography (adsorption chromatography) A. Normal Phase LSC

B. Reverse Phase LSC

2. Liquid/Liquid Chromatography (partition chromatography) A. Normal Phase LLC

B. Reverse Phase LLC

3. Ion Exchange Chromatography

4. Gel Permeation Chromatography (exclusion chromatography)

(16)

LIQUID SOLID CHROMATOGRAPHY

Si - OH HEXANE OH C-CH3 CH3 CH3- C CH3 CH3 OH OH CH3 CH3

(17)

LIQUID-LIQUID CHROMATOGRAPHY

ODPN(oxydipropionylnitrile) Normal Phase LLC Reverse Phase LLC NCCH3CH2OCH2CH2CN(Normal) CH3(CH2)16CH3 (Reverse)

The stationary solid surface is coated with a 2nd liquid (the

Stationary Phase) which is immiscible in the solvent (Mobile) phase.

Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation.

(18)

SELECTING AN OPERATING MODE

Sample Type LC Mode

Positional isomers LSC or LLC

Moderate Polarity Molecules LSC or LLC

Compounds with Similar Functionality LSC or LLC

Ionizable Species IEC

Compounds with Differing Solubility LLC

(19)
(20)

1.

1.

Ultraviolet Detector

Ultraviolet Detector

200

200--400nm 400nm 254 nm

254 nm

2.

2.

Reflective Index Detector

Reflective Index Detector

Universal Detector

Universal Detector

Detectors

(21)

High Performance Liquid Chromatography

(22)

High Performance Liquid Chromatography

(23)
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Retention Time

Time required for the sample to travel from the injection port through the column to the detector.

Response Retention Time 5 10 15 20 25 A B C D

(26)

SELECTIVITY (α)

Ratio of Net Retention Time of 2 components.

(Equilibrium Distribution Coefficient)

X

2

-

X

0

X

1

-

X

0

α =

(27)

R e s p o n s e R e t e n t i o n T i m e X X X 1 3 6 2 1 0 – Selectivity

Selectivity

Selectivity

(28)

RESOLUTION EQUATION

V - V

1/2(W + W )

2 2 1 1

R =

Response Volumes W W W W V V1 1 2 2 2 1

(29)

HEIGHT EQUIVALENT TO A THEORETICAL PLATE

Length of a column necessary for the attainment of compound distribution equilibrium (measure the efficiency of the column).

Theoretical plates (N) = 16 ( )X Y

2

X

(30)
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EXAMPLES OF THEORETICAL PLATE, SELECTIVITY AND HEIGHT EQUIVALENT TO A THEORETICAL PLATE

1 2 3 4 V V V V W W W W 2 1 0 1 2 4 3 4 3 V V0 = 1.02(Minutes) V1 = 4.92 V2 = 6.59 V3 = 8.17 V4 = 9.14 W1 = 1.0 (Minutes) W2 =1.0 W3 = 1.0 W4 =1.0

(33)

GENERAL FACTORS INCREASING RESOLUTION

1.

Increase column length

2.

Decrease column diameter

3.

Decrease flow-rate

4.

Pack column uniformly

5.

Use uniform stationary phase (packing material)

6.

Decrease sample size

7.

Select proper stationary phase

8.

Select proper mobile phase

9.

Use proper pressure

(34)

Definition:

Different affinity of these 2 components to stationary

phase causes the separation.

Concentration of component A in stationary phase

Concentration of component A in mobile phase

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参照

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