Chapter 28 Liquid Chromatographybusan2.thecube.kr/bbs/table/board/upload/Chapter28%28%BC... ·...
Transcript of Chapter 28 Liquid Chromatographybusan2.thecube.kr/bbs/table/board/upload/Chapter28%28%BC... ·...
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Chapter 28 Liquid Chromatography
28A Scope of HPLC
Column Chromatography
Glass tube 사용
(diameter:10~50mm)
Particle size
(diameter: 150~200um)
Fig. 28-1. Selection of LC modes.
These include (1) partition, or liquid-liquid, chromatography; (2) adsorption, or liquid-solid, chromatography; (3) ion-exchange, or ion, chromatography; (4) size-exclusion chromatography; (5) affinity chromatography; and (6) chiral chromatography.
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28B Column Efficiency in LC
28B-1 Effects of Particle Size of Packings
The mobile –phase mass-transfer coefficient reveals that C in Eq26-23 is
directly related to the square of the diameter d of the particles making
up a packing.
Fig. 28-2 Particle size 小 → H 小 (유속에 큰 영향 )
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28B-2 Extra-column Band Broadening in LC
In LC, significant band broadening sometimes occurs outside the
column packing itself.
유속 大 H 大 efficiency 小
Effect of relative sample mass
on plate height
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Fig 28-3. Schematic of an apparatus for HPLC
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28C LC Instrumentation
28C-1 Mobile-Phase Reservoirs and Solvent Treatment Systems
Glass or stainless reservoirs 사용
Equipped with removing dissolved gases.
Column 과 Detection에서 bubble 생성 방지
Isocratic elution: single solvent reservoir
Gradient elution: two or more solvent (mixing chamber)
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28C-2 Pumping Systems
At least 1000 psi . 4000~6000 psi
Flow rate at least 3ml/min
a) Reciprocating pumps
b) Displacement pumps
c) Pneumatic pumps
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Fig. 28-5 . A reciprocating pumps for HPLC.
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28C-3 Sample injection systems
The most widely used method of sample introduction in LC is based on
sampling loops, such as that shown in Fig 28-6 and 27-5.
Fig. 28-6. A sampling loop for LC.
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28C-4 Columns for HPLC
a) Analytical columns:
15~150um inside diameter : 2~3mm
① Heavy - walled glass tube : 600psi 이하
② Precision -bore . stainless steel
충전제 : ① Finitely divided . silica gel or alumina &celite
Particle size : 5~10um range.
② Pellicular particles : small beads (40um dia)
Coated with a 1- to 3- um layer of a porous material
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b) Guard columns:
① Contains a packing chemically identical to that in the analytical
column.
② Particle size is much larger
Pressure drop 무시
☆ 용매로부터 불순물제거로 analytical column 의 오염 예방
☆ Mobile phase . stationary phase 로서
Saturate analytical column 의 고정상 stripping 방지
c) Column temperature control
대부분 room temp에서 사용.
항온 필도시 water jacketed column are available.
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28C-6 Detectors
Types of Detectors:
a) Bulk property detectors:
No highly sensitive , universal detector system.
b) Solute property detectors
UV absorption, IR absorption, Fluorometry
Refractive index, Conductometric , Moving wire
Mass spectrometry, Polarography, radioactivity.
28C-5 Columns for HPLC
Two basic types of packing s have been used in LC,
pellicular and porous particle.
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http://www.waters.com/webassets/cms/category/media/other_images/primer_M-1_Columnexamples.jpg http://share.psu.ac.th/system/assets/media/files/000/014/541/original_column-6.JPG?1306827819 http://www.lcresources.com/resources/getstart/p.htm
Columns for HPLC Liquid chromatographic columns are usually constructed from stainless steel tubing, although glass and polymer tubing, such as polyetheretherketone (PEEK), are sometimes used. In addition, stainless steel columns lined with glass or PEEK are also available. Hundreds of packed columns differing in size and packing can be purchased from HPLC suppliers. The cost of standard-sized, nonspecialty columns ranges from $200 to more than $500. Specialized columns, such as chiral columns, can cost more than $1000.
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1. Absorbance Detectors (Fig. 28-8) Fig 28-8 is a schematic of a typical, Z-shape, flowthrough cell for
absorption measurements on eluents form a chromatographic column.
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Fig. 28-9 254. 280nm line 사용 (from Hg source)
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2. Fluorescence Detectors
Fluorescence detector for HPLC are similar in design to the fluorometers
and spectrofluorometers described in Section 15B-2.
3. Refractive-Index Detectors (Fig. 28-10)
Sample or solvent Glass plate 로 분리
General rather than selective
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4. Evaporative Light Scattering Detectors
Moving wire:
Flame ionization detector 에 용해액을 transport 시키기 위해
continuous moving wire loop 사용
① The wire first passes through the elute
② Carries it into an over where the solvent in evaporated.
③ The sample in them passed into a furnace and is paralyzed.
④ The volatile products are detected by the ionization detector.
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5. Electrochemical Detectors (Fig. 28-11) Electrochemical detectors of several types are currently available from instrument manufactures.
Fig. 28-11 Electrochemical Detectors
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Fig. 28-12 Amperometric thin-layer detector cell for HPLC
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6. Mass spectrometric detectors
Stainless
- Steel or polyimide belt to transport the elute to the ion source.
◉ Ion source 에 도달되기 전 IR evaporator 로 Solvent 제거.
* Quadrupole type mass spec.
Detection limit : 0.2~1ng
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28D Partition Chromatography
Origin : Martin & Synge
① Solid supports.
* Silicic acid or silica gel
H2O 강하게 흡착.
Polar solvent : aliphatic alcohols, glycols.
(Stationary phase) Nitromethane 의 단독 또는 H2O 와 혼합.
•Other support media : alumina , diatomaceous, earth, starch, cellulose,
and powdered glass.
(Mobile phase ) Pure or mixture of solvents.
Stationary phase 와 polarity 차 大 Immiscible.
Reverse - phase chromatography
Nonpolar station , polar mobile.
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② Bonded phase packings.
Silica gel 입자에 organic group 을 화학적으로 attach.
R : Octadecyl group
Aliphatic amine , ethers : nitrates. aromatic , hydrocarbons
* Mobile phase 에 의한 stripping
* Limited loading capacities.
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Application.
Closely related substance 의 분리에 주로 사용
ex) Numerous amino acids formed in the
① Hydrolysis of a protein
② Sugar derivatives.
③ Closely related aliphatic alcohol
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28D-1 Columns for Bonded-Phase Chromatography
The support for the majority of bonded-phase packings for
partition chromatography are prepared from rigid silica, or
silica-based compositions.
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▪ Normal-Phase and Reversed-Phased Packings (Fig. 28-14)
Two types of partition chromatorgraphy are distinguishable based on
the relative polarities of the mobile and stationary phases.
Normal Phase Reversed-phase
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Fig. 28-15 Effect of chain length on performance of reversed-phase
siloxane columns.
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28D-2 Method Development in Partition
Chromatography
▪ Column Selection in Partition Chromatographic
Separations
In summary, then, polarities for solute, mobile phase,
and stationary phase must be carefully blended if good
partition chromatographic separations are to be
realized in a reasonable time.
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▪ Mobile Phase Selection in Partition Chromatography
∙ Effect of Solvent Strength on Retention Factors.
Solvents that interact strongly with solutes are often termed
“strong”solvents.
- Normal phase separation
- Reverse phase separation
)228( 'BB
'AA
'AB −+= PPP φφ
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∙ Effect of Mobile Phase on Selectivities.
In many cases, adjusting k to a suitable
level is all that is needed to give a
satisfactory separation..
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28D-3 Applications of Partition Chromatography ▪ Derivative Formation Fig 28-19 illustrates the use of derivatives to reduce polarity and enhance sensitivity. ▪ Ion-Pair Chromatography Fig 28-20 illustrates the separation of ionic and nonionic compounds using alkyl sulfonates of various chain lengths as ion-pairing agents. ▪ Chiral Chromatography Fig 28-21 shows the separation of a racemic mixture of an ester on a chiral stationary phase.
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Fig. 28-19
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Chiral Stationary Phase
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A typical application if adsorption chromatography of cis- and trans-
pyrazoline.
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28E Adsorption Chromatography
Adsorption force 에 의해 retention take places.
* Neutral organic compound 의 분리에 많이 사용
Adsorption isotherm Low concentration에서 적선적
◉ Stationary & mobile phases.
Silica gel , alumina , 10um (8~12um 범위)
k'에 대해 주어진 solvent 의 effect 는 into eluent strength 에 의존
1.2 : Solvent 1,2 K : Partition coefficient
Ax : 용질의 분자 size Table 참조.
Eluent strength of source common solvents with alumina adsorbents.
* High eluent strength 빨리 elute
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Applications.
* Certain functional group 을 갖는 화합물
More strongly held then other.
Tendency to be adsorbed
Acid > alcohol > carbonyl > ester > hydrocarbon.
* 흡착제의 성질로 근거에 영향
The choice of adsorbent & solvent
Trial - and -error.
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28F Ion Chromatography
28F-1 Ion - Exchange Equilibria
Solvent : H2O, ion 의 형태로 separation.
Inorganic chemistry에서 많이 insoluble solid 와 접촉하고 있는 용액속의
like sign 의 이온의 interchange.
Synthetic ion - exchange : high molecular weight polymeric materials
contained large numbers of an ionic functional group per molecular
Cation exchange : Sulfonic acid group
RSO3-H+ (strong)
Carboxylic acid group
K coo H (wear)
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(Cation)
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(Anion)
Distribution coefficient.
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이때 [H+]는 일정 : KD= const.
즉 [H+], [RH] 는 K 값에 영향
KD 는 다른 이온 [H+] 에 관계하는 B+ 이온에 대한 resin 의 affinity.
KD 大 B+ 이온을 reter 할려는 경향 大
☆ Sulfonated cation exchange resin 에 대한 KD 값의 감소 순
Cs+ >Rb+ >K+ >NH4+ >Na+ >H+ >Li+
Bd2+ >Pb2+ >Sr2+ >Cd2+ >Cu2+ >Zn2+ >Mg2+
Fig. Separation example 참조
☆ Amino acid 분석에 많이 이용
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28F-2 Ion – Exchange Packings
Fig 28-22 shows the structure of a strong acid resin.
28F-3 Inorganic-Ion Chromatography
▪ Ion Chromatography Based on Suppressors
Fig 28-23 illustrates a typical micromembrane suppressor.
▪ Single-Column Ion Chromatography
Commercial ion chromatography instrumentation that
requires no suppressor column is also available.
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Fig. 28-22 Structure of a cross-linked polystyrene ion-exchange resin.
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http://www.intechopen.com/books/column-chromatography/affinity-chromatography-and-importance-in-drug-discovery
http://www.sepscience.com/images//Articles/HPLCSol/107/fig1.jpg
http://image.slidesharecdn.com/stationaryandmobilephaseselectionmabphgradientanalysis33974-150324131049-conversion-gate01/95/stationary-and-mobilephaseselectionmabphgradientanalysis33974-10-638.jpg?cb=1427202680
52
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Fig. 28-24 Applications of Ion Exchange Chromatography
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28F-4 Organic and Biochemical Applications of Ion-Exchange
Chromatography
Ion-exchange chromatography has been applied to a variety of
organic and biochemical systems, including drugs and their
metabolites, serums, food preservatives, vitamin mixture, sugars,
and pharmaceutical preparations.
28F-5 Ion-Exclusion Chromatography
Ion-exclusion chromatography is not a form of ion
chromatography because neutral species rather than ions are
being separated.
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28G Gel Chromatography (Size-Exclusion)
Sample 중의 종의 Shape 과 molecular Size 의 fluctuation 에 근거
Including other name, gel-permeation . chro. or
Exclusion . chro. or
Molecular - sieve chro.
Gel 의 pores 內에 속으로 침투
이온 혹은 분자가 Gel pores 보다 더 클 경우
excluding pass quickly through th column
28G-1 Column Packings
Consist of beads of a porous polymeric material .
ex) Polysaccharide electron Sephadex
Table 참조.
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28G-2 Theory of Gel chromatography
H2O에 의해 swelling 된 gel 의 total volume Vt
Vt = Vg +Vi + Vo
Gel 의 solid matrix 에 의한 부패.
Vi = Gel 빈틈에 있는 Solvent volume.
Vo = Exclude 된 성분을 운반하는데 드는 Solvent 의 volume.
Intermediate size 의 분자들 interstitially held solvent 의 some fraction
Kd 로 transfer
이때 elution volume Ve = Vo + KdVi
If size > Gel pores size 인 solute 의 Kd =0
∵Ve = Vo
Applications ; Sephadex G-25. G-50 desalting or removal of Low
molecular weight molecules.
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Eq 28-10 rearranges to K= ( Ve-Vo ) / Vi = cs/cm
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Fig. 28-27
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28G-3. Application of size-exclusion chromatography Gel filtration and gel permeation methods are complementary in that gel filtration is applied to water-soluble samples and gel permeation is used for substance is less-polar organic solvents.
28H Affinity Chromatography Affinity chromatography involves covalently bonding a reagent, called an affinity ligand, to a solid support.
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28I Planar Chromatography(Thin-layer Chromatography)
A sheet or strip of heavy filter paper
1) Sample soln. spotting solvent evaporation
2) Mobile phase 의 flowing 에 의해 developing.
*The flow of a mobile phase
Capillary forces 에 의해 전개
Ascending development
Radial development
Descending development
L-L. L-S. ion exchange
Stationary phase : H2O , or some polar liquid.
The rate of movement of the solute K. 값에 의존
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Fig. 28-29 (a) Ascending-flow developing chamber. (b) Horizontal-flow developing chamber.
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Standard 와 RF Value 비교 확인
Fig 28-30. (a) Thin-layer plate after development. (b) Thin-layer chromatogram for sample.
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◉ Plate Heights Approximate plate heights can also be determined for a given type of packing by thin-layer chromatographic measurements.
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◉ TLC
Stationary Phase : solid absorbents
L-S 의 경우 TLC plate 를 drying
Moisture 흡수 극히 조심
Ion exchange or sephadex ⇒ 사용
◉ Preparation of TLC Plates
Glass or mica plate, microscope slide
☆ A narrow range of particle size
Uniform layer thickness
Separations 의존
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◉ Plate Development
5×20×20 사용
Acid 확인
Ninhydrin pink to purple product
◉ Identification of Species
Iodine or sulfuric acid -spraying
(Organic 확인) dark reaction product
Fluorescence UV 로 확인
◉ Spot area 를 standard 와 비교
Spot에서 analyte 분리
◉ Paper chromatography
Liquid -Liquid Chromatography. Fig 28-31. Two-dimensional thin-layer chromatogram of some amino acids.