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.

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 )

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

Fig 28-3. Schematic of an apparatus for HPLC

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)

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

Fig. 28-5 . A reciprocating pumps for HPLC.

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.

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

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.

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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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.

Fig. 28-9 254. 280nm line 사용 (from Hg source)

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

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.

5. Electrochemical Detectors (Fig. 28-11) Electrochemical detectors of several types are currently available from instrument manufactures.

Fig. 28-11 Electrochemical Detectors

Fig. 28-12 Amperometric thin-layer detector cell for HPLC

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

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.

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.

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

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.

▪ 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

Fig. 28-15 Effect of chain length on performance of reversed-phase

siloxane columns.

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.

▪ 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

∙ 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..

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.

Fig. 28-19

Chiral Stationary Phase

A typical application if adsorption chromatography of cis- and trans-

pyrazoline.

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

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.

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)

(Cation)

(Anion)

Distribution coefficient.

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

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.

Fig. 28-22 Structure of a cross-linked polystyrene ion-exchange resin.

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Fig. 28-24 Applications of Ion Exchange Chromatography

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.

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.

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.

Eq 28-10 rearranges to K= ( Ve-Vo ) / Vi = cs/cm

Fig. 28-27

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.

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

Fig. 28-29 (a) Ascending-flow developing chamber. (b) Horizontal-flow developing chamber.

Standard 와 RF Value 비교 확인

Fig 28-30. (a) Thin-layer plate after development. (b) Thin-layer chromatogram for sample.

◉ Plate Heights Approximate plate heights can also be determined for a given type of packing by thin-layer chromatographic measurements.

◉ 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

◉ 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.