Chromatography

Gas

GSC GLC

Liquid

LSC LLC IEC GPC AC

GC: volatile solutesLC: any mobile phase soluble solutes

Eluent reservoir

inlet filter

pump

pre-column guard-column

injectorvalue

analyticalcolumn

detector Recorder /integrator

HPLC reversed phase

HPLC reversed phase

HPLC reversed phase

IC

HPLC ion pair

HPLC normal phase

HPLC normal phase

HPLC normal phase

SEC gel fitration

HPLC reverse phaseIC

SEC gel fitration

ionic

non-ionic

polar

non-polar

water soluble

organic soluble

water soluble

organic soluble

M<2000

M>2000

sample

Interaction between molecules

solute

M.PS.P

N.P R.Ppacking material polar (silica) non-polar (C18)

sep. interaction polarity polarity & size Eluent weak

strong

(weak to medium polar)

n-hexane/polar solvent

n-hexaneCH2Cl2

Ethyl acetateMeCN

(strong to medium polar)

H2O/organic

H2O

MeOHMeCNTHF

CH2Cl2most rapidly eluented cpd.

low polarity (alkanes)

high polarity (salts)

Pump

• Motor driven syringe• Piston

– Reciprocating – Multiple reciprocating

• Advantage and disadvantage– Solvent reservoir limited?– Pressure pulse produced?– Gradient can be produced?

沖提模式： isocratic elution (A% B% 固定 ) gradient elution

Gradient Elution

Application(1) sample with a wide k’ range(2) sample composed of large molecule(3) samples containing late-eluting interferences(4) maximizing detection sensitivity (& 前濃縮 )

tR

%B100

50

10 20

linear

tR

%B100

50

10 20

curve

tR

%B100

50

10 20

segmented

使用 HPLC須注意：1. 氣泡→壓力、流速不穩 (常為溶液混合所產生 )2. 顆粒→阻塞，壓力上升Eluent考慮 (1) sample

(2) separation mode

Eluent 之選擇α

phy. pro.k’

(1) phy. properties b.p.、 viscosity、 UV cutoff、毒性、純度、價格 UV cutoff：

(2) 1 ≤ k’ ≤ 10 for simple components 0.5 ≤ k’ ≤ 25 for multiple components solute/eluent interaction↑ k’↓solute

M.PS.P

4 major interaction(a) dispersion interaction(b) dipole interaction(c) H-bonding interaction(d) dielectric interaction

dispersion force：(非極性分子 ) x s

Temporacy dipole moment from x will polarize the e in adjacent molecule s (R.I. 值大表示分散力大 )

eluent strength solvent polarity

N.P. solvent polarity ↑ k’↓R.P. solvent polarity ↑ k’↓

2''

1

221

10'' PP

kk

N.P.

2''

1

212

10'' PP

kk

R.P.

P’： polarity

solvent mixture''' bbaa PPP

67..82.107.01.53.0' P

16.82.106.01.54.0 P

8.11010'' 2

5.02

16.867.8

1

2

kk

ba , ： volume ratio lf solvent a,b

30% MeOH 70% H2O

40% MeOH 60% H2O

(3) solvent selectivity proton donor 由 proton acceptor 將 solvent 分類 dipole moment

column由 physical size 分類I.D. L

1. Analytical column： 2.1~5.0 mm 10,15,25 cm

2. microbore column： 1.0~2.0 mm ‘’ ‘’

3. capillary column： ‘’ ‘’同樣濃度， peak大小：A>B>C

A： 1 mm 50 μL/minB： 2.1 mm 0.2 mL/minC： 4.6 mm 1 mL/min

column A： 1 mm i.d. and 50μL/min solvent flowcolumn B： 2.1 mm i.d. and 200μL/min solvent flowcolumn C： 4.6 mm i.d. and 1000μL/min solvent flow

structure of silica gel depicting the various types of bonds and silanol groups present

column 材料bonded-stationary phase

Si OH HOR Si OR+

SiOH + SOCl Si Cl Si

MgBr

Si NH(CH2)2NH2H2N(CH2)2NH2

SiOH + Si

R

R

R

Cl Si O Si

R

R

R

R¡G CH3(H2C)17

C1 , C2 , C4 ,

(CH2)3CN , (CH2)3NH2

, C8 , C18 , C22

N+ R3 Cl-

SO3- H+

anion exchange

cation exchange

C C C C

C C

C C C C C

C C C

+

PS-DVB ( polystyrene – divinyl benzene)

pH=1~13

Inorganic( silica )

Mechanically strongHigh efficiencypredictable retention

Tailing peak for baselimited pH rangechem. unstable

organicwide pH rangeno tailingchemical stable

low efficiencyMech. weakunpredictable retention

silica：Si

Si

O Si R

Si

OH

O Si

CH3

CH3

CH3

residual silanol

end-capped

2 < pH < 8 if pH > 8 Si-O-Si →SiO-

pH < 2 Si-R 可能被破壞pre-column 的材料常為 silica

hybrid (silicon-C)：Si

OEt

OEt

OEt

EtO Si

OEt

CH3

OEt

EtO2 + 1

Si O Si

Si OH

Si O Si

polar

polar

(

(

pH=1~12

Si O Si R

Si O Si R

Si O Si

Si R

Si R

Elution order most polar being eluted 1st 靜 相 ： C1， C8， C18

tR ： C1 < C8 < C18

polarity： C1 < C8 < C18

HPLC detectors

• Bulk property detectors– respond to a mobile phase bulk property– refractive index, dielectric constant, or density

• Solute property detectors– respond to some property of solutes– UV absorbance, fluorescence, or diffusion current

Light path in a micro flow cell of a spectrophotometric detector.Cells that have a 0.5-cm pathlength and contain only 8μL of liquid are common.

Refractive Index Detector

A refractive index detector responds to almost every solute, but its detection limit is about 1000 times poorer than that of the ultraviolet detector.Refractive index detectors are useless in gradient elution becauseit is impossible to match exactly the sample and the reference whilethe solvent composition is changing.

Fluorescence detectorsFluorescence detectors excite the eluant with a laser and measure the fluorescence.

These detectors are very sensitive but respond only to the few analytes that fluoresce.

Evaporative Light-Scattering Detector

An evaporative light-scattering

detector responds to any analyte

that is significantly less volatile

than the mobile phase.

LC Detector Commercially Available

Mass LOD (commercial detectors)

Mass LOD (state of the art)

Absorbance Yes 100 pg – 1 ng 1 pgFluorescence Yes 1 – 10 pg 10 fgElectrochemical Yes 10 pg – 1 ng 100 fgRefractive index Yes 100 ng – 1 ng 10 ngConductivity Yes 500 pg – 1 ng 500 pgMass spectrometry Yes 100 pg – 1 ng 1 pg

FT-IR Yes 1μg 100 ngLight scattering Yes 10 μg 500 ngOptical activity No ─ 1 ngElement selective No ─ 10 ng

Photoionization No ─ 1pg – 1 ng

HPLC reversed phase

HPLC reversed phase

HPLC reversed phase

IC

HPLC ion pair

HPLC normal phase

HPLC normal phase

HPLC normal phase

SEC gel fitrationSEC gel fitrationHPLC reverse phaseIC

SEC gel fitrationSEC gel fitration

ionic

non-ionic

polar

non-polar

water soluble

organic soluble

water soluble

organic soluble

M<2000

M>2000

sample

Size-Exclusion Chromatography (SEC)

‧固定相︰ silica or polymer particles with pore size 102-106 Å‧ Type ︰

-Gel Filtration Chromatography (GFC)stationary phase: hydrophilic mobile phase: aqueous適用 : protein, amino acid, peptide.

-Gel Permeation Chromatography (GPC)stationary phase: hydrophobicmobile phase: nonpolar solvent適用 : oligomers

VR = V0 + KVi

Applications:

1. Molecular weight determination2. Group fraction

HPLC reversed phase

HPLC reversed phase

HPLC reversed phase

ICICHPLC ion pair

HPLC normal phase

HPLC normal phase

HPLC normal phase

SEC gel fitration

HPLC reverse phaseIC

SEC gel fitration

ionic

non-ionic

polar

non-polar

water soluble

organic soluble

water soluble

organic soluble

M<2000

M>2000

sample

Ion-exchange chromatography

sample ion： X mobile phase： Ycharged stationary phase： R

X- + R+Y- Y- + R+X-

X+ + R-Y+ Y+ + R-X+

anion exchange

cation exchange

SCX： strong cation exchange –SO3-H+

WCX： weak cation exchange –COOH SAX： strong anion exchange –NR3

+OH-

WAX： weak anion exchange –NH2

SCX

WCXCOOH

COO-

pH

exch.capacity

SAX

WAXpH

exch.capacity

selectivity

SR

SR

NaHHNaNa

HK

R-SO3-H+

(R) + Na+

(S)+ Cl- R-SO3-Na+

(R) + H+

(S)+ Cl-

selectivity coefficient： NaHK

counter ion (M)H+ 1Li+ 0.76Na+ 1.2K+ 1.76

Mg2+ 2.23Cu2+ 3.14Ba2+ 5.66Co2+ 2.46

NaHK

Ba2+ < Pb2+ < Sr2+ < Ca2+ < Ni2+ < Cd2+ < Cu2+ < Zn2+ <Mg2+ < Tl+ < Ag+ < Cs+ < K+ < NH4

+ < Na+ < H+ < L+

R+

OH-

R-

H+

KOH

pump

R-

H+

R+

OH-

HCl

pump

NaCl NaClKCl

HCl

HClNaOH

NaOH

KOH HBr

conductivitycell

R+OH- + HCl → R+Cl- + H2OR+OH- + NaOH → R+Cl- + NaOH

R-H+ + NaOH → R-Na+ + H2OR-H+ + NaCl → Na+ + HCl

separatorSCX

separatorSAX

suppressorSAX

suppressorSCX

HPLC reversed phase

HPLC reversed phase

HPLC reversed phase

IC

HPLC ion pairHPLC ion pairHPLC normal phase

HPLC normal phase

HPLC normal phase

SEC gel fitration

HPLC reverse phaseIC

SEC gel fitration

ionic

non-ionic

polar

non-polar

water soluble

organic soluble

water soluble

organic soluble

M<2000

M>2000

sample

Ion-pair chromatography (IPC)

IPC & RP-HPLC share several features：column and mobile phase and similar

Compounds： weak acids, bases, ionic compound

Concept： solvent extraction

aq： aqueous phase org： organic phase

→Ionized compound Aaq+ in water add suitable counter ion Baq

-

ion-pair A+B- similar to non-ionic compound

→In solvent extraction extraction constant

Aaq- + Baq

- (A+B- )aq A+B-orgion-pair

aqaq

org

BABA

E

aq

aqIP

IP

aq

aqM

s

M

s

aq

org

M

s

Bk

BKABAk

BABAK

BKE

BEVV

VVK

ABA

VVk

'

'

'

capacity (reaction) factor

sample counter ionamine OCl4- , C12OSO3

-

acid N+R4 , acetic acid

pH>7 CH3COO-

buffer 3<pH<7 CH3COO- + CH3COOH

3>pH neutral CH3COOH

pHacid

baseneutral compound

retentiontime

Electrochemical Detector

An electrochemical detector responds to analytes that can be

oxidized or reduced.

The detector is very sensitive to flow rate and temperature changes.

Quantitation method

measurement of signal

peak height (trace analysis) peak area

H Ak’ 1/(1+k’) no changeN N1/2 no changeμ <μ -0.2 1/ μ

A

H

400

300200

20

40

20 30 400 %B

H A

400

300200

20

40

0 μ(mL/min)

H AH

A

(1) External standard calibration

RFAPconcsample

concareapeakfactorresponse

...

.

A

conc.

(2) Internal standard calibration compensate for the change in sample size or conc. due to instrumental variation

.concratiosignalRF

signalratio

conc.(3) Method of standard addition

signal

conc.S0

Δy

... 0

FRSconcoriginal

xyslopeRF

Error

(1) constant systematic error(2) proportional systematic error

S=bc

S=a+bc

S=b’c(1)

(2)

Recovery (sample preparation)

(1)pure reference standard

(2) pure reference standard + sample preparation

(3)

(4) recovery rate

000 xbay

0

0

bayxp

0xbax ppp

%100.0

p

p bxa

RR

xp： mass or conc. of ref. analyte after sample preparation

0pa → constant systematic error

1pb → proportional systematic errorx0

xp