We start with reviewing the key for Test 3 Section 4 ...web.utk.edu/~msepania/Sec 4.pdf · u is the...

1

Section 4 – Analytical Separations

Chp 23Pages 549 551, 553-570Problems 1-3, 15-19, 27, 29-32, 36, 37, 43, 44

Chp 24Pages 579-591Problems 1-3, 6, 8a, 10, 13, 18, 20

Chp 25Pages 608-612, 614-617, 621-623, 627-629, 631-633Problems 1a, 3, 5, 6, 9a, 15, 16, 19, 21-23, 28

Chp 26Pages 641-644, read section on CE methods but mainly use my notesProblems 2-7, 22-26, 29, 31, 33, 39

We start with reviewing the key for Test 3

2

Separations

1. Take a mixture of chemical species and separate it into individual chemical compounds.

2. Analyze the separated components (off-line or on-line).

3. Extractions, precipitations, chromatography, electrophoresis, etc.

3

Extractions (Partition)

[S]2

[S]1

• A solute partitions between two immiscible liquids (2 phases)

m = moles Sq = fraction of S in phase 1V1 = volume of phase 1V2 = volume of phase 2

[ ][ ]

( )1

2

1

2

//1

VqmVmq

SSK −

==

Extractions (Partition)

[S]2

[S]1

[ ][ ]

( )1

2

1

2

//1

VqmVmq

SSK −

==

Rearrange to obtain….

21

1

KVVVq+

=

•n extraction stepsn

n

KVVVq ⎟⎟

⎠

⎞⎜⎜⎝

⎛+

=21

1

4

Extractions Meet Acid-Base Chemistry

[S]2

[S]1

Organic

Aqueous

1. Neutral compounds primarily dissolve in the organic phase.

2. Charged compounds only dissolve in the aqueous phase.

Consider:

B + H2O ⇔ BH+ + OH-

B

BH+

Extractions Meet Acid-Base Chemistry

[S]2

[S]1

Organic

Aqueous

B + H2O ⇔ BH+ + OH-

B

BH+

[ ][ ] [ ]11

2++

=BHB

BD

[ ][ ]1

2

BBK = and

[ ] [ ][ ]1

1+

+

=BH

BHK a

[ ]++⋅

=HKKK

Da

a

5

Chromatography BasicsDefinition – Separation technique based on the differential distribution of introduced solutes between a moving mobile phase and a stationary phase. Important elements of phase equilibria, mass transport, and kinetics are involved

Often performed in columns packed with the stationary phase


Forms

6


Forms

The Chromatogram

HPLC10 pesticidesUV absorbance

(220 nm)

Plot of the response of aDetector at end of column

VersusThe length of time it taketo elute from column

Notice Gaussian profile(many random processesshape the band)

7

Adjusted Retention Time

mrr ttt −=′

1

2

r

r

tt′′

=α

Capacity Factor

m

mr

ttt

k−

=′

K = [ ]stat / [ ]mob & k’ = amtstat / amtmob

k’ = K Vstat / Vmob

8

Separation Efficiency

22/1

2

2

2 55.516w

tw

tN rr ==

NLH =

w1/2 w

Resolution (the main objective)

⎟⎟⎠

⎞⎜⎜⎝

⎛′+

′⎟⎠⎞

⎜⎝⎛

−=

∆=

avav

r

kkN

wtR

1142

αα

w

efficiency selectivity capacity

R = 1.0 goodR = 1.5 excellentR > 2.0 only good

9

Resolution (the main objective)

Note – band width at baseline = 4 σ(recall area under Gaussian)

How to Resolve B and C

⎟⎟⎠

⎞⎜⎜⎝

⎛′+

′⎟⎠⎞

⎜⎝⎛

−=

∆=

avav

r

kkN

wt

R114

2

αα

Increase peak separation through better selectivity; a matter of thermodynamics

10

How to Resolve B and C

⎟⎟⎠

⎞⎜⎜⎝

⎛′+

′⎟⎠⎞

⎜⎝⎛

−=

∆=

avav

r

kkN

wt

R114

2

αα

Increase peak sharpness through efficiency;A matter of kinetics (mass transport)

What Causes Peak Broadening?

CuuBAH ++=

A is the Multiple Path Term

Packed (particles) columns result in broadening since different analyte molecules can take different paths down the column

B is the Longitudinal Diffusion Termu is the linear flow rate in the column

A concentrated band of analytes diffuses down the column. The slower the band moves the more it diffuses (u dependence)

C is the Mass Transfer Term

If the analytes move down the column and equilibrium is not established, peaks broaden. The faster the analytes move the less time they have to equilibrate (u dependence)

11

ATerm;eddy

diffusion

BTerm;axial

diffusion

- With smaller particles more decisions averages out- Open tubular columns have no eddy diffusion problem

- Standard deviation of Gaussian band σ = (Dm t)1/2

-Use high flow rates - See Table 23-1



CTerms;

Cm & Cs;Use slow Flow rate

Van Deemter Plot

12

Problems From Chp 23

13



14


Gas Chromatography – Separations based on both analyte volatility andspecific interactions with the stationary phase

INSTUMENTATION

15

Example of a more exotic stationary phase (chiral discrimination)

INSTUMENTATION

Thermal Conductivity

Flameionization

16

Gas Chromatography (GC)

⎟⎟⎠

⎞⎜⎜⎝

⎛′+

′⎟⎠⎞

⎜⎝⎛ −

=∆

=avav

r

kkN

wtR

11

42

αα

Temperature – Analytes must be near or above their boiling points to spend a significant amount of time in the mobile phase (gas). Analytes must be thermally stable for GC analysis!

Adorption/Partition – Analytes move more slowly when they spend more time interacting with the stationary phase. Remember, “Like dissolves like”.

Non-polar stationary phase Polar stationary phase

Above illustrates both boiling pt & stationary phase interactions influence the separations

17

Effect of Column Temperature and T-Programming


⎟⎟⎠

⎞⎜⎜⎝

⎛′+

′⎟⎠⎞

⎜⎝⎛ −

=∆

=avav

r

kkN

wtR

11

42

αα

Interaction with Stationary Phase – The primary way to change α is to change stationary phases. This requires changing the column! Remember that the mobile phase is inert, so changing the gas doesn’t help.

18


CuuBAH ++=

Originally most GC columns had a packed stationary phase (solid particles or particles coated with a polymer).

Today most GC is performed in thin capillaries with the stationary phase coated on the walls of the capillary.

Diffusion in a gas is much faster than diffusion in a liquid; hence, the B-term is very significant in

determining efficiency in GC

19

Chapter 24 Problems

Chapter 24 Problems

20

Why use polymeric phases as GC stationary phases?

Why is OTGC preferred to packed column GC?

To improve H, would you like to increase or decrease the values of the following?Stationary Phase thicknessColumn diameterDiffusion CoefficientFlow rate

Of the two GC detectors discussed which is more sensitive?

Why are internal standards often used in GC analysis?

Doubling the length of a GC column will do what to plate number (N)?

The two most important factors in determining the retention time of a compounds in GC are what?

What is an ideal value for Rs?

The A-term in efficiency pertains to what efficiency factor?

What is a typical length of a OTGC column?

Etc.

HPLC

21

HPLCInstrumentation

Fixed LoopHigh PressureInjector – Typical V = 10 µL

ReciprocatingPiston PumpTypical Flow 1 mL/min

ColumnTypical L = 25 cm

HPLCInstrumentation

22

HPLC The Packing Particles

Silica ParticleTypically 10 µm dia; whopping surf area

Silica particle with bonded“reversed phase” (C-18)

⎟⎟⎠

⎞⎜⎜⎝

⎛′+

′⎟⎠⎞

⎜⎝⎛ −

=∆

=avav

r

kkN

wtR

11

42

αα

Solvent Composition – Solvent polarity is adjusted. Typically a mixture of water and a miscible organic solvent is changed to control solvent polarity

Adsorption/Partition – Analytes move more slowly when they spend more time interacting with the stationary phase. Remember, “Like dissolves like”.

HPLC

Interaction with Stationary Phase – The primary way to change αis to change phases. This requires changing the column or the solvent; the latter is obviously easier.

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• A wide range of detectors are used for HPLC. Absorbance detection is very important.

• A wider range of analytes can be analyzed by HPLC compared to GC. Reproducible injection is easier than for GC.

• Plumbing is a significant issue (H) in HPLC.

• Solvent programming means to change the solvent composition during an experiment. This is used to adjust k´continuously during the separation.

HPLC

HPLCThe effect of solvent strength & S-Programming

Reversed phase (C18 stationaryphase) separations of aromatic cmpds.The base solvent is generally water andmodified with a more organic solventlike acetonitrile to increase the eluting strength of the mobile phase.

In a manner analogous toT-programming in GC, SolventProgramming in LC addresses the general elution problem.

24

HPLC

We will focus on “Reversed Phase HPLC” but know whenexclusion and ion exchange are used and the difference betweenreversed and normal phase

HPLCe.g., Ion exchangeStationary phases – PS-DVB modified with charged groups Mobile phases - aqueous and contain acids or basesExchange rxn - Ca 2+ + 2Resin-H+ 2Resin- - Ca 2+ + 2H+

25

HPLC

HPLC Solvent Optimization

22%

30%

40%

26

Chapter 25 Problems

1. (a) The mobile phase competes with the stationaryPhase for interactions with the analytes. CompetitionIs more effective when they become more similar withRegard to polarity ….

2. N = 15 cm / 5.0x10-4 cm = 30,00030,000 = 5.56 (600/W)2

W = 8.2 sec

(b) H = A + B/u + CuC proportional to dp2

And dominates the plot at large u

Chapter 25 Problems

27

What is the most common detector in HPLC and explain why its effective volume must be small?

Why is HPLC sometimes called “high performance” & sometimes “high pressure”?

What are typical flow rates?, injection volumes?, peak volumes?, plate counts?

Define: Bonded Phase, Normal Phase, and Reversed Phase?

In what form of LC is the stationary phase charged?

In HPLC what terms in the VanDeemter (A, B, Cm, Cs) equation are most significant?

What is the equivalent of T-programming in LC?

What is a common solvent mixture used for a mobile phase in reversed phase LC & how does one increase its eluting strength?

What is the principle of size exclusion chromatography?

Draw the stationary phase for a “C-18” packing

Etc.

Capillary ElectrophoresisCapillary Electrophoresis(Last 310 Lecture)(Last 310 Lecture)

•• Capillary Zone Electrophoresis, CZECapillary Zone Electrophoresis, CZE

•• Capillary Electrokinetic ChromatographyCapillary Electrokinetic ChromatographyMicellarMicellar Electrokinetic Capillary Chromatography, MECCElectrokinetic Capillary Chromatography, MECCCyclodextrin Distribution Capillary Cyclodextrin Distribution Capillary ElectrochromatogrElectrochromatogr, CDCE, CDCE

•• SizeSize--Selective Capillary Electrophoresis, SSCESelective Capillary Electrophoresis, SSCE

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Capillary Electrophoresis- Separation technique based on the mobility differences in an

electric field for ions injected into narrow-bore capillaries

- Mobilities scale roughly with charge to mass ratios of the ions

- Running buffer is electrokinetically pumped through the capillary

- Neutrals can be separated based on differential association with charged running buffer additives (e.g., micelles in MECC)

- CDs can be used to selectively modify the effective migration rates of ions or neutrals in CE

- Entangled polymers used for size selective separations

- Attributes of CE include very high efficiency, wide-ranging versatility, low sample and reagent consumption and waste generation, speed

HPLC vs. CE

XSample Variety

XFlexibility

XSpeed

XLimitedSelectivity

XEfficiency

XLarge Sample Size

XSmall Sample Size

CEHPLC

29

CE Instrumentation

Electroosmotic Flow• The bulk flow of the liquid in the capillary and

is a consequence of the surface charge on the capillary wall

• Electromotive pumping action which originates at the wall of the capillary and results in a “plug-like” flow profile.

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EOF Formation

EOF

Tightly adsorbedcations and –O- ionson silica surface

Cation richdiffuse layer

CathodeAnode

Capillary Wall

-+ + + + + + + + + + + +

+

+ + ++ +

--

--

-- -

+

+ +

+ ++

+

+

+

---+

++ +

- - - - - - - - - - - - - - - - - -+

+ -

l = length to the detection windowL = total length of capillary (meters)V = applied voltage (volts)to = retention time of a neutral speciesNote: Veo = µeo E

Separation Mechanism

Velocity = (Velocity = (µµeofeof + + µµee) E : E = V / L) E : E = V / LNote: Note: µµee is roughly proportional to size/chargeis roughly proportional to size/charge

31

* Cartoon of CE separation

CE Characteristics: efficient,fast, versatile, diminutive

Separation MechanismSeparation Mechanism

Efficiency

µµnetnet VVN = N = ----------------

2 D2 D

Equation assumes only axial diffusion Equation assumes only axial diffusion (Einstein(Einstein EqEq. . σσ22 = 2Dt and H = N/L = = 2Dt and H = N/L = σσ22/L are used)/L are used)

Note that there is no length dependenceNote that there is no length dependence

32

CZE Separation of AnionsCZE Separation of Anions

MECC for Separations of NeutralsMECC for Separations of Neutrals

•• Surfactants are polar/charged Surfactants are polar/charged -------- hydrophobichydrophobicmolecules that can aggregate into micellesmolecules that can aggregate into micelles

•• Critical Micelle Concentration (CMC)Critical Micelle Concentration (CMC)

•• Example sodium Example sodium dodecyl sufate dodecyl sufate (SDS)(SDS)C12 C12 –– SOSO33

-- (CMS ~ 8 (CMS ~ 8 mMmM, ~60, ~60SDSsSDSs/micelle)/micelle)

•• Micelles are dynamic entities and can be Micelles are dynamic entities and can be destroyed by organic solventsdestroyed by organic solvents

33


- - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - -

+ -

+++++++++++++++ ++++++++++++ +++

++++++++++++++++++++++++++++++

- --- --- -- --------

-

- --- --- -- --------

- --- --- -- --------

-

-

-

-

-

-Anions Cations

Neutrals

EOF

µneutral=µeof

Observed Mobilities

µeof =µmicelle =µobs(N) =µobs(C) =µobs(A) =


SeparationSeparation

Elution Window (or Range) Elution Window (or Range) –– tto o --toto-- ttm m is very importantis very important

34

Cyclodextrin CharacteristicsCyclodextrin CharacteristicsMacrocylic (small nanometer dimensions) sugar molecules with 6 (alfa), 7 (beta),or 8 (gamma) sugars in the structure.Apolar cavity forms inclusion complexes with analytes based on size, shape, &chemi-physical properties

CM-β-CD-1

Example of GridSearch Docking

The hydroxyl groupscan be derivatizedto influence inclusionor impart a chargefor migration

We have employed molecular mechanics,molecular dynamics, simulated annealing, and various visualization techniquesto study and predict separation behavior.

CDCE for Separations of NeutralsCDCE for Separations of Neutrals

+

- - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - -

-

+++++++++++++++ +++++++++++++++

++++++++++++++++++++++++++++++

NeutralsEOF

-OOC

-OOC

-OOC

-OOC

µCD

µeof=µneutrals=µCD

Capillary Electrophoretic Separations of Neutrals by “CDCE”

Observed Mobilitiesµeof =µCMCD =µobs(N) = depends on inclusionµCD =

Wide windows Wide windows are very desirableare very desirable

migration time

Creates an elution window

µCMCDµeof

35

CDCE for Separations of NeutralsCDCE for Separations of Neutrals

A goal was to to create designer CD cocktails as Running buffer systemsUsing molecular modeling techniques

Figure 2. Separat ion of a mixture n-alky lanthracenecompounds by the CDCE technique.10 The running buffer contained a mixture o f charged and neutral cyclodextrins at low mM concentrations. Excellent resolution of positional isomers of these compounds is shown. Using cyclodextrinsas running buffer additives also permits chiral separations.

Figure 2. Separat ion of a mixture n-alky lanthracenecompounds by the CDCE technique.10 The running buffer contained a mixture o f charged and neutral cyclodextrins at low mM concentrations. Excellent resolution of positional isomers of these compounds is shown. Using cyclodextrinsas running buffer additives also permits chiral separations.

Exceptional separationPower for structurallySimilar compounds

MolCADMolCAD and Molecular Mechanics & Dynamics Experiments and Molecular Mechanics & Dynamics Experiments Guide Creation of a New ReagentsGuide Creation of a New Reagents

Cyclodextrin Reagents Investigated

(HDMS-β-CD) (HDMCM-β-CD)

MolCAD structures with electrostatic potential surfaces

MolCAD structures & MM interaction energy values support efforts to

synthesize new CD reagent

1,8-DNN:-31.3 kcal/mol1,8-DNN:-19.9 kcal/mol

Interaction energies from systematic grid search docking experiment

36

Improved Separation Performance With HDMCMImproved Separation Performance With HDMCM--ββ--CD CD ((dihydroxynaphthalenesdihydroxynaphthalenes))

4 6 8 10 12 Time (min)

1

1-CM-β-CDsmall window

HDMS-β-CDpoor inclusion

1,4-DHN 1,7-DHN

1,6-, 2,6-, 2,7-DHNs

1,3-DHN1,5-DHN 2,3-DHN

HDMCM-β-CDwide windowgood inclusion

HDMCM-β-CDwith β-CD

mixed CDs systems:important for ration-ally designed systems

SSCE for Separations of DNASSCE for Separations of DNA

Polymers likePolymers likehigh MWhigh MWmethyl cellulose methyl cellulose added to RB atadded to RB atconcentrations concentrations above the above the entanglemententanglementthresholdthreshold

37

SSCE for Separations of DNASSCE for Separations of DNA•• Super efficiency (why?)Super efficiency (why?)

•• Example of uses: Example of uses: -- Studies of genetic Studies of genetic

complexitycomplexity-- Studies of ProteinStudies of Protein––DNADNA

interactionsinteractions

•• Notice increasing peak Notice increasing peak heights (why)heights (why)

Methods of Detection in CE

• Absorbance• Laser Induced Fluorescence (LIF)• Surface Enhanced Raman Scattering (SERS)• Others (electrochemical, conductivity, etc)• Usually accomplished on-column by removal of

a section of the polyamide coating on the capillary

38

LIF Detection of DNALIF Detection of DNA

We have We have detected asdetected aslittle aslittle as1010--1919 molesmolesinjected!injected!

SFC DesignSFC Design

39

the problem competing optical process have cross-sections:σfluorescence ~ 10-19 cm2

σRayleigh ~ 10-26 cm2

butbutσ σ RR only ~ 10 only ~ 10 --2929 cmcm22

Anti-Stokes Scatter

Stokes Scatter

Raman Overview

40

Large EM Field Enhancements

Depends on polarizability and field

Chemical and EM models (SERS intensities are proportional to the 4th power of the field at the scattering molecule.)

Potential“Hot Spot”

Closely spaced metal nanospheres (or other shapes) can generatelarge enhancements of incidentfields due to concerted plasmonfield effects.

- Ag colloid in RB for on-capillary approach

- Electro-filament onto moving substrate for off-capillary approach

CE-SERS Detection Approaches

On Ag-colloid sprayed on frosted glass

On Ag - pliable polymer composites

- Integrated µ−fluidic-SERS platformsmolded fromcomposites

41

SERS Spectra Obtained On-the Fly (On-Capillary)

CE separation of rhodamine6G and riboflavinLIF detection

Conditions:Dilor spectrometer;75 µm capillary;~15 mW at 514 nm; 0.5 w/v% Ag;1 sec acquisition

The high S/N spectrum of Rh6G obtained from less than 10-16

moles injected !

SERS detection

CE separation of riboflavin and resorufin

CE-SERS of With EF Transfer and Ag Colloid on Frosted Glass Slide (Off-Capillary)

1 2 3Time, min.

resorufin

riboflavin

LIF detection

riboflavin

resorufin

finding bands actual spectra

SERS detection

We start with reviewing the key for Test 3 Section 4 ...web.utk.edu/~msepania/Sec 4.pdf · u is the...

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Transcript of We start with reviewing the key for Test 3 Section 4 ...web.utk.edu/~msepania/Sec 4.pdf · u is the...