09 11 2006 Chromatography

WenHsin Huang

NDMC 1

2006/9/7 WenHsin Huang, NDMC 1

Principles ofChromatography

Introduction toAnalytical Separations

Wen-Hsin HuangPh. D.NDMC


Principles of Chromatography

•Introduction to Analytical Separations–Solvent Extraction–What is Chromatography?–A Plumber’s View of Chromatography–Efficiency of Separation–Why Bands Spread


Simple Separations•Extraction

–transfer of solute from one phase to another–phase can be gas, solid, liquid

•Liquid/liquid extraction–2 immiscible solvents used–typically aqueous solvent and organic solvent

•you know water and oil don’t mix

–organic solvents less dense than water•diethyl ether, toluene, hexane

–organic solvents more dense than water•chloroform, carbon tetrachloride, dichloromethane


Solvent Extraction

shake

add secondimmiscible

solvent

Legend:aqueous solventorganic solventsolute particles

Solute particlesonly have one

choice of solvent

Solute particlesnow have two

choices of solvent

Solute particleschoose preferred

solvent

WenHsin Huang

NDMC 2


Solvent Extraction

•Like dissolves like–solute chooses solvent most like itself–polar compounds (and ionic compounds)

choose water (which is very polar)–nonpolar compounds choose nonpolar organic

solvents


Phase Partitioning

If we have a solute (S) that is partitioned between twophases (1 & 2) then we can write an equilibrium expressionfor this equilibrium.

21 SS

][][

1

2

1

2

SS

A

AK

S

S

Partition Coefficient (K)

•Convention: Organic phase = phase 2


Distribution Coefficient

The distribution coefficient (D) is used in place of thepartition coefficient when dealing with a species that hasmore than one chemical form.

1____2____

phaseinionconcentrattotalphaseinionconcentrattotal

D


Example of Extraction with n-HexaneThe distribution coefficient for compound Z betweenn-hexane and water is 6.25. Calculate the percent Zremaining in 25.0 mL of water that was originally0.0600 M in Z after the extraction with the followingvolumes of n-hexane.

A) One 25.0-mL portion

B) Two 12.5-mL portions

C) Five 5.00-mL portions

D) Ten 2.50-mL portions

13.8%

1.90%

4.99 x 10-3%

2.49 x 10-7%

It is more efficient to do several small extractionsrather than one large extraction.

WenHsin Huang

NDMC 3


Solvent Extraction (pH effects)

•Charges of acidic and basic species changewith pH–neutral species generally more soluble in

organic solvent–charged species generally more soluble in

aqueous solution

•Distribution coefficient (D) describesdistribution of species between two phases–Takes into account all forms of a compound

(i.e. H2A, HA-, A2-)–Different from partition coefficient (K)



•Distribution of HA between two solvents–two equilibria to consider

•Ka equilibrium•D equilibrium

aqueous HA H+ + A-Ka

organic HA H+ + A-



•Distribution of acids and bases between twophases

•Ionic species only found in aqueous layer

D =total conc. in phase 2 (organic)

total conc. in phase 1 (aqueous)

D =[HA]2

[HA]1 + [A-]1D =

[B]2[B]1 + [BH+]1


Solvent Extraction (pH effects)•Distribution of HA between two phases

•Also have K expression for [HA]

•Substitute and rearrange to get

]HA[]A][H[

Ka ]H[]HA[K

]A[ a

1

2

]HA[]HA[

K

aK]H[]H[K

D

Eq’n

WenHsin Huang

NDMC 4


Solvent Extraction (pH effects)D for an acid (HA) D for a base (B)

•Distribution of acids and bases between twophases is pH-dependent

•To find D, need to know–pH of solution–Ka of acid or conjugate acid–K of acid or base

]H[KKK

Da

a

Eq’ns

aK]H[]H[K

D


Solvent Extraction (pH effects)•Adjust pH to effectively extract an acid or

base (and its conjugates) into aqueous layer•Consider base B with a pKa of 9.0

–pH < 9.0, B hydrolyzes so majority of B (in theform BH+) in water layer (low D value)

2 4 6 8 10 12pH

log

D

0

-2

-4

-6

pKa

mainlyB

mainlyBH+

Fig.

Ba

a

aqaq

org KHK

KKBHB

BD

][][][

][

aq

org

B

BK

][

][

aq

aqa BH

BHK

][

]][[


History of ChromatographyMikhail Tswett is credited with theinvention of chromatography.

Developed a technique that separatedvarious plant pigments (e.g.,chlorophylls and xanthophylls) bypassing solutions through glasscolumns filled with finely groundCaCO3.

The separated species appeared ascolored bands on the column.

Hence Greek chroma = color and graphein = writing

M. S. TswettRussian Scientist

(1872-1919)


Tswett’s Experiment

Ground up plant leaves and addedpetroleum ether.

Filled column with chalk.

Method forgotten for many years.

WenHsin Huang

NDMC 5


Tswett’sExperiment


What is Chromatography?

•Method to separate components in amixture based on different distributioncoefficients between the two phases

•Same principle as solvent extraction (likedissolves like), but one phase is “stationary”and one phase is “mobile”–no longer working with organic and aqueous

layers in a separatory funnel–working in a column (which is just a tube)


What is Chromatography?

•Two phases: mobile and stationary•Mobile phase is solvent moving through

column–liquid (Methanol, water, buffer)–gas (He, H2, N2)

•Stationary phase fixed inside column–viscous liquid coated on inside of column–solid particles packed inside column

•Solutes have different affinities for mobilephase and stationary phase


Types of Chromatography

•Adsorption Chromatography•Partition Chromatography•Ion-exchange Chromatography•Molecular Exclusion Chromatography•Affinity Chromatography

WenHsin Huang

NDMC 6


Adsorption ChromatographyStationary phase Mobile phaseSolid Liquid or Gas

How it separates–solute adsorbs on to

stationary phase surface


Partition ChromatographyStationary phase Mobile phaseLiquid on solid support Liquid or Gas

How it separates–solute dissolves into

liquid coating


Ion-Exchange ChromatographyStationary phase Mobile phaseAnions or cations covalently Liquidbound to solid stationary phase

How it separatessolute ions of opposite chargeattracted to stationary phase


Molecular ExclusionChromatography

Stationary phase Mobile phasePorous gel Liquid

How it separates–small molecules

trapped in pores ofstationary phase

Also calledgel filtration,gel permeation,

orsize-exclusion

chromatography

WenHsin Huang

NDMC 7


Affinity Chromatography

Stationary phase Mobile phaseImmobilized molecules on Liquidliquid or solid stationary phase

How it separates–Molecules with specific

shape dock with ligands


Classification Specific Method Stationary Phase Type of equilibriumLiquidchromatography(LC) (Mobilephase a liquid)

Liquid-liquid orpartition

Liquid-bondedphase

Liquid-solid, oradsorption

Ion exchange

Size exclusion ormolecular-exclusion

AffinityChromatography

Liquid adsorbed ontosolid

Organic species bondedto solid surface

Solid

Ion-exchange resin

Interstices of polymericsolid

Covalently bondedmolecule (eg antibody)

Partition between immiscibleliquids

Partition between liquid and bondedsurface

Adsorption

Ion exchange

Sieving

Selective Affinity – (eg specificProtein/antibody bind together)

Gaschromatography(GC) (Mobilephase a gas)

Gas-liquid

Gas-bondedphase

Gas-solid

Liquid adsorbed ontosolid surface

Organic species bondedto solid surface

Solid

Partition between gas and liquid

Partition between gas and bondedliquid

Adsorption


Basic PrinciplesTwo phases considered:

1) Mobile Phase: solvent moving through the column.

2) Stationary Phase: stays in place inside of the column.

COLUMN“Eluent” “Eluate”

Process is called “elution”


Simple Column

Fresh eluent

Initial bandof A and B

ColumnPacking

Porous Disk

WenHsin Huang

NDMC 8


Chromatogram

A chromatogram is agraph showing thedetector response(proportional toconcentration) assolutes emerge from achromato-graphiccolumn as a function oftime or volume.

Detector


Chromatographic Terms

tm = “dead time”which isthe minimum time formobile phase to passthrough the column

tr = “retention time”which is the time requiredfor the solute (analyte) topass through the column.

t’r = “adjusted retentiontime”describes timesolutes spend in stationaryphase

mrr ttt •All solutes spend equal time in mobile phase


Chromatographic Terms

= “relative retention”which is the ratio ofadjusted retention times.

>1 always

then separationbetween two components

k’= “capacity factor”

m

r

tt

mobileintimestationaryintime

k

__

__

1

2

r

r

tt


A Plumber’s View…•kcapacity factor describes how long

solute retained on column

•Higher kindicates solute retained longer•If k= 0, solute unretained•If k= 1, solute spent same amount of time

in stationary phase as in mobile phase

m

mr

ttt

'k

time spent in stationary phasetime spent in mobile phase

=

WenHsin Huang

NDMC 9


A Plumber’s View…•kdescribes time spent in two phases•kalso describes number of moles of solute

partitioned between two phases

•K Partition coefficient comparesconcentration of solute in one phase toconcentration of solute in the other

mm

ss

VCVC

'k

m

s

VV

K'k m

s

1

2

CC

]S[]S[

K

Eq’n

Eq’n


Partition Coefficient Relationship

m

r

tt

mobileinmolesstationaryinmoles

mobileintimestationaryintime

k

__

____

__

m

r

m

s

mm

ss

tt

VV

KVSVS

k

][][

1

2

1

2

1

2

KK

kk

tt

r

r

What this says is that analytes with different partitioncoefficients will have different retention times.


A Plumber’s View…•q fraction of solute in mobile phase

ssmm

mm

ms

m

VCVCVC

molesmolesmoles

q

m

s

mm

ss

VV

K1

1

VCVC

1

1q

'k11

q

fraction in MP

'k1'k

)q1(

fraction in SP2006/9/7 WenHsin Huang, NDMC 36

Review

m

s

CC

K Partition coefficient describesconc. of solute in SP and MP

m

s

m

mr

VV

Kt

tt'k

Capacity factor describestime solute spends (or moles ofsolute) in SP and MP

1

2

1

2

1r

2r

KK

'k'k

't't

Relative retention comparesretention times (or partitioncoefficients or capacity factors)of solute 1 and 2

WenHsin Huang

NDMC 10


Chromatographic Peaks•Solutes elute from a column in a Gaussian peak

shape–w width of peak at base–w1/2 width of peak at half-height

tr

timet0

h

1/2h

w1/2=2.35

w=4

averageretention time

Peak width (w) is defined as the baseline width (4).2006/9/7 WenHsin Huang, NDMC 38

Resolution of peaks

2/)ww()tt(

wt

resolution21

2r1r

av

r

As tr grows,resolutionbetween twopeaksimproves.

R=0.50 R=0.75

R=1.00 R=1.50

Resolution is a measureof how well two peaks areseparated.


Resolution Example

Two solutes have retention times and widths of tr1 =235 s, w1 = 8 sec; tr2 = 250 s, w2 = 10 s.

Resolution = 1.7


Plate Height (H)Bands broaden as they travel through the column.

Plate height (H) relates the amount of broadening tothe linear distance traveled.

xH

2H = plate height

x = distance traveled2 = variance of peak

WenHsin Huang

NDMC 11


Evaluating Separation Efficiency•Plate theory

–Breaks separation up into many discrete stages–Stages represent individual equilibria

•N (Theoretical plate) represents eachequilibrium between MP and SP

•Each time a solute moleculeenters the SP from the mobilephase is a theoretical plate


Evaluating Separation Efficiency•“N”in chromatography is analogous to “n”

in liquid-liquid extractions–each extraction represents a theoretical plate

•Higher N corresponds to better separation–indicates solute molecules enter SP a higher

number of times

[S]1

[S]2


Evaluating Separation Efficiency

•N is dependent on–column–analyte–retention time of analyte–width (at base or at half-height) of peak

2

2r

2

2r

wt16t

N

22/1

2r

wt55.5

N

N is dimensionless someasure tr and w (or w1/2)in same units (i.e. time)


Chromatographic Peaks•Distribution of retention times for a solute•Peak shape represents differences in

behavior of solute molecules duringseparation

tr

timet0

h

1/2h

w1/2=2.35

w=4

averageretention time

WenHsin Huang

NDMC 12


Evaluating Separation Efficiency•Do not want solute band to spread out as it

travels through column•H (Plate height) represents the relationship

between the width of a solute band to thedistance traveled through column

•Lower H corresponds to better separation(higher efficiency)

•H also called HETP–Height Equivalent to a Theoretical Plate–One solute equilibrium between SP and MP is

one theoretical plate (N)–Equilibrium occurs in one HETP



•H related to N and length of column (L)

•Resolution related to N

–R is dimensionless–k2 = capacity factor for solute retained longer

on column (higher tr)

av

2

'k1'k1

4N

R

NL

H Report H in unitsof length (i.e. cm)

Eq’n



•Increase L to increase R

•Increase to increase R

–to change , must change nature of MP and SPto change relative affinity analytes have for SP

1

2

1

2

1r

2r

KK

'k'k

't't

av

2

'k1'k1

4N

R

HL

NNL

H

Eq’n


Number of Theoretical Plates (N)The number of theoretical plates (N) increases asthe efficiency of the separation increases.

2

2

2

216

rr tw

tN

or

2

2

2

21

255.5

rr tw

tN

Derived

WenHsin Huang

NDMC 13


Example

A solute with a retention time of 302 s has a width of11 s on a column that is 15 m long. Find both N andH for this separation.

N = 1.2 x 10-4

H = 1.2 mm


Factors Affecting ResolutionThe greater the resolution, the better the separation.The more theoretical plates, the better the separation.

avek

kN1

14

R 2

NR LR 0R 10R 02 k


Principles of Chromatography

•Introduction to Analytical Separations–Solvent Extraction–What is Chromatography?–A Plumber’s View of Chromatography–Efficiency of SeparationWhy Do Bands Spread


Why Do Bands Broaden?

Solute invariably spreads apart as it travels(diffusions) through a chromatographiccolumn.

The observed variance is a sum of variancesfrom all the broadening mechanisms.

22iobs

WenHsin Huang

NDMC 14


Where Does Broadening Occur?

Outside the column:Width of injection plugMixing in detector dead volume

I

Pump

Column Detector

Injector


Where Does Broadening Occur?Inside the column:

Multiple pathsLongitudinal diffusionEquilibration time

I

Pump

Column Detector

Injector


Multiple Paths (A)•Solute molecules can choose many different

paths through packed columns

layer of SPcoated oninside of column

open tubularcolumn

SP particlespacked insidecolumn

packedcolumn

Compare to solutetraveling through

a straw

Compare to solutetraveling through

a bean bag2006/9/7 WenHsin Huang, NDMC 56

Multiple Paths (A)

•Open tubular columns do not provide multiple paths for soluteduring tm (no A term)

time

Band of 3 solutemolecules travelingthrough packed column

Band spreads

Occurs only in packed columns.

Smaller particles reduce the plate height.AH

WenHsin Huang

NDMC 15


Longitudinal Diffusion (B /ux)•S molecules travel through column in a band•[S] varies throughout band

–some solute molecules get ahead of band–some solute molecules lag behind band

start

as bandtravels

H B/ux so,increase ux (flow rate)to reduce problem

Caused by diffusion of solute in the mobile phase.

Faster flow rate means less time, thus less diffusion.


Equilibration Time (Cux)•Finite time required to allow solute molecules to

equilibrate between MP and SP•If flow rate too high, MP will “leave behind”

molecules in SP (i.e., Solute in stationary remains“stuck”while solute in mobile phase moves forward.)

•Keep SP thin to decrease equilibration time

sloweq.

MPSP

Directionof travel

bandwidth

MPSP

bandwidth

H Cux so decrease ux to reduce problem


Van Deemter EquationPutting the three factors together yields the VanDeemter equation that helps predict how thecolumn flow rate will affect the theoretical plateheight.

xx

CuuB

AH

MultiplePaths Longitudinal

Diffusion

EquilibrationTime

•Break equation up into A, B, and C terms

–A H independent of flow rate

–B/ux H inversely proportional to flow rate

–Cux H proportional to flow rate2006/9/7 WenHsin Huang, NDMC 60

Band Spreading

•Three terms in van Deemter equationcorrespond to three sources of bandspreading or broadening

•A term Multiple Paths term(also called Eddy Diffusion)

•B term Longitudinal Diffusion term•C term Equilibration Time term

WenHsin Huang

NDMC 16


van Deemter plot•Plot dependence of H on individual terms•Sum and find optimum ux to use to minimize band

spreading

H

Flowrate

Bux

Cux

A

uopt

Hmin


Column Type Affects H

•Advantages–high resolution (low H)–high sensitivity–low analysis time

•Disadvantages–not useful for large-scale

analyses

•Advantages–useful for large-scale

analyses

•Disadvantages–A term in van Deemter

equation increases H

layer of SPcoated oninside of column

open tubularcolumn

SP particlespacked insidecolumn

packedcolumn


Asymmetric Band Shapes

•Expect Gaussian peak shape from solute Seluting from column, independent of [S]total

•Separation based on K

•In reality, K changes as [S]total changes•If [S] is too high or too low, peak shape

deviates from Gaussian shape

m

s

CC

K


Asymmetric Band Shapes•Overloaded column (high [S])

–so much S enters SP that the SP starts to lookmore like S than it looks like original SP

–most S molecules retained longer on column

Band shape

Majority of S retainedon column longer

Observed peakin chromatogram

Faster-moving (lessretained) S reachesdetector first

WenHsin Huang

NDMC 17


Asymmetric Band Shapes•Underloading (low [S])

–“hot sites”on SP more available when less Smolecules trying to enter SP

–some (minority) S get “stuck”on hot sites–some S retained longer on column

Band shapeObserved peakin chromatogram

Minority of S retainedon column longer

Slower-moving (moreretained) S reachesdetector last 2006/9/7 WenHsin Huang, NDMC 66

Asymmetric Band Shapes•Load less sample to reduce overloading

problem•Protect groups on SP to reduce number of

hot sites to reduce tailing problem

Cs

Cm

Constant K (slope) resultsin ideal peak shape

tailed

overloaded

Fig. 23-19


Chromatographer’s TriangleRelationship

Resolution

Speed Capacity


Quantitative Analysis

•In general, detectors can tell us–“Yes, something is eluting from the column.”

•Use calibration methods to determine how muchof a compound is eluting from column

Finding Peak Area•A = ½ wbaseh•A w½ h•cut-and-weigh•computer integrationconcentration (M)

Pea

kA

rea

x =-bm

WenHsin Huang

NDMC 18


Quantitative Analysis•Generally use an internal standard

–internal standard is a known amount of acompound different from analyte

–compare analytical signal from analyte toanalytical signal from internal standard

–not the same as method of standard additions–internal standards discussed in Ch. 4

]S[A

F]X[

A sx

Ax area of analyte peakAs area of standard peak[X] concentration of analyte[S] concentration of standardF response factor


Example

When 1.06 mmol of 1-pentanol and 1.53 mmolof 1-hexanol were separated by GC, they gaverelative peak areas of 922 and 1570 units,respectively. When 0.57 mmol of pentanol wasadded to an unknown containing hexanol, therelative chromatographic peak areas were843:816 (pentanol:hexanol). How muchhexanol did the unknown contain?


Liquid Chromatography

•Analyte–Soluble in MP (more likely than being volatile)

•Mobile phase–Liquid

•Methanol, Water, Acetonitrile, Hexane

–Plays more active role in separation•Stationary phase

–Liquid (partition) or solid (adsorption)•High Performance Liquid Chromatography(HPLC)

–High pressure used to force solvent through column (asopposed to gravity)


LC Columns

•Constructed of steel or plastic•5 –30 cm length•1 –5 mm i.d.•Easily contaminated and degraded•Guard column

–Contains same SP–Dust, other particles, strongly adsorbed

solutes retained on guard column–Expendable–Extends life of analytical column

ANALYTICAL

GUARD

WenHsin Huang

NDMC 19


LC Columns•Almost exclusively use packed columns•Solutes move much slower through liquids

than through gases–Time needed to diffuse to SP in open tubular

column is too long

S S


LC Columns•Packing particle diameter (dp) crucial

–Typical particle diameters are 3 –10 m–Decrease particle diameter to decrease H

•Provide more uniform flow (low A)•Less time needed for solute to get to particle (low C)

Flow rate (mL/min)

10m

0 2 4 6 8

H(

m)

20

40

60

10

5m

3m

Fig. 21-14


LC –Adsorption Chromatography

•Solid SP –Silica gel–Pure, spherical, microporous

particles•Permeable to solvent•Very high surface area•Use when pH < 8

Silica gel with silanolgroups on surface

Deprotonated silanols(Si –O-) are “hot sites”that can lead to tailing

Si

Si

SiSi

O

O

OO

OSi SiO

SiO

O

Si

O

OHOH OH

HO

OHOH

OHOH

Microporous Silica Particles

Aggregate of Particles Sponge-like Structure


LC –Adsorption Chromatography•Solvent molecules compete with solute molecules

for sites on SP

•Solute is displaced by solvent molecule

SolventSolute

MPFlow SP

Fig. 21-10

WenHsin Huang

NDMC 20


LC –Adsorption Chromatography

•Forcible desolvation of solute by solvent is nearlyindependent of solute identity–Dependent on solvent identity

•Eluent strength (o)–Ability of solvent to displace solute

•Elutropic series–Relative eluent strengths of common solvents–Must know about relative polarity of SP/MP

•Eluent strength increases as MP becomes morelike SP


Isocratic vs. Gradient Elution

•Isocratic elution–One solvent or one constant solvent mixture used as

MP throughout entire separation•Gradient elution

–Adjust solvent mixture through separation–Adjusting eluent strength–May speed up separation process

•Table 21-2 includes o values of solvents foradsorption chromatography on silica–polarity, o



Start w/ 100% Benzenethen add Acetonitrile gradient

elution

Hexane °=0.01

Acetonitrile °=0.52

isocraticelutions

Fig. 21-18&

Fig. 21-19



•Previous example illustrated balance of goodresolution and reasonable retention times

•Gradient elution may help–Improve resolution–Shorten retention times (and total analysis time)–Improve peak shapes

WenHsin Huang

NDMC 21


LC –Partition Chromatography•Liquid SP coated on solid support

–Often, liquid SP covalently attached to surfaceof silica gel particle

Si O Si R

CH3

CH3

support liquid SP

Common polar R groups Common nonpolar R groups

(CH2)3NH2 (CH2)17CH3 (or C18)

(CH2)3CN (CH2)7CH3 (or C8)(CH2)2OCH2CH(OH)CH2OH (CH2)3C6H5 2006/9/7 WenHsin Huang, NDMC 82

LC Phases•Normal-phase Chromatography

–SP polar–MP weakly polar or non polar

–More polar solvent has higher o

•More attracted to SP and able to displace solute

•Reversed-phase Chromatography–SP nonpolar or weakly polar–MP polar

–Less polar solvent has higher o

•More attracted to SP and able to displace solute


LC Phases

•Normal-phase developed first•Reversed-phase is more common

–Many choices of polar solvents in which solutesare commonly soluble•Water, methanol

–Nonpolar SP less likely to have hot sites•Less peak tailing


HPLC Instrumental Design

•Already covered columns•Focus now on

–Injector–Detectors

I

Pump

Column Detector

Injector

WenHsin Huang

NDMC 22


HPLC 6-way Injection Valve

column

Sample loop Sample loop

waste waste

Fig. 21-17


Detectors

•Questions to ask when evaluating an LCdetector:–Is the detection universal?–Is the detector response linear?–What is the limit of detection (LOD)?–Is the detector useful with gradient elution?

•In other words, is the detector insensitive to solventcomposition?


UV Detector (w/ flow cell)•Common HPLC detector

–Many solutes absorb UV light

Fig. 21-20

Lightsource

Eluate out

Eluate in

Detector

pathlength

Flow cell


UV Detector

•Will cover during spectrophotometry–Any UV spectrophotometers discussed previously are

good LC detectors

•UV cutoff wavelengths–Below given , MP absorbs UV radiation–MP absorbance overwhelms analyte absorbance

WenHsin Huang

NDMC 23


UV Detector

•Applicable for UV chromophoresEssentially linear

–Will discuss deviations from linearity in CH 19

LOD ~ 0.1 ngGood detection method with gradient

elution–Use solvents that do not absorb UV radiation


Fluorescence Detector

•Similar idea to UV detector•Few molecules fluoresce•Derivatize solutes with fluorescent tag

–Derivatize mixture of solutes before separation–Derivatize solutes as they elute from column

before detection•Post-column Derivatization


Fluorescence Detector

Applicable to fluorophoresEssentially linear

–Will discuss deviations from linearity

LOD ~ 0.001 ngFine with gradient elution

–Use solvents that do not fluoresce


Refractive Index Detector•Compare refractive indices of eluate & reference

•Cannot use with gradient elution–reference composition cannot exactly match solvent

composition during experiment

(Reference) (MP solvent & analyte = eluate)

WenHsin Huang

NDMC 24


Refractive Index Detector

Essentially universalLOD ~ 100 ngEssentially non-linear

–Linear range very small

Disaster with gradient elutionFinicky

–Need constant T


Comparison of LC DetectorsType Approx LOD Approx

Linear rangeComments

UV-VIS 10 -10 g 10 4 For light absorbingcompounds

Fluorescence 10-14 g 10 5 For Fluorescentcompounds

MS 10-7-10-9 g 10 5 Universal detector

Electrochemicalconductometric

10-8 g/mL 10 5 Specific detector; forall ions

Electrochemicalamperometric

10- 10-10 –11 g 10 5 Specfic detector;electroactvecompounds


Liquid Chromatography (LC)

Especially high-performance liquid chromatography (HPLC).

The term “high-performance” refers to the use of packed columns with very smallpacking particles (diam. 5-10 m) -giving greatly enhance resolution.

Note: several types of LC. Inaddition to partition (as describedso far) - there are also ion-exchange and size-exclusionchromatography using liquidmobile phases. We will concentrateonly on partition.

09 11 2006 Chromatography

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Transcript of 09 11 2006 Chromatography