chem. 230 – 10/21 lecture. announcements i exam 2 results –ave = 46.5 sd = 8 –similar average,...

Chem. 230 – 10/21 Lecture

Announcements I

• Exam 2 Results– Ave = 46.5 SD = 8– similar average, but

less variability– distribution

• Chem 253 – Environmental Chem offered next semester

Announcements II

• Posted HW Set #3 (original posting had incorrect due date)

• Application Abstract due Today – I should be able to check your and give you an o.k. by next week

• 2D GC references posted on website• Today’s Lecture

– GCXGC Questions– SFC– HPLC (start)

GCGC x GC Questions

1. Why is the second dimension in GC x GC limited in resolution?2. For which column in GC x GC is it important to use columns of

small diameter and thin films? 3. Why is overloading on narrow diameter 2nd dimension columns

less important than when using the same column with 1D GC?4. How many dimensions does a GC x GC x MS chromatogram

have?5. Why does the peak capacity of a two dimensional technique

somewhat over express how many compounds can be resolved?

6. Why are two hot/cold jets needed in the modulator?7. For what type of samples is GC x GC particularly useful?

a) Complex samples with compounds of similar polarityb) Complex samples with a variety of compound polarityc) Complex samples with a small number of analytes (e.g. pesticides

in food)d) Related compounds with different sized alkyl groups R(CH2)nCH3

Supercritical Fluid Chromatography

= SFC• What is a supercritical

fluid?– has properties

intermediate between a liquid and a gas

– Defined by region P – T phase diagram

– Phase boundary between liquid and gas disappears at critical point

– Demonstrated by heating two phase system

– Fluid must operate above critical T and critical P

Temperature

Pre

ssur

e

solid

liquid

gas

liquid + gas

heat

supercritical fluid

critical point

supercritical fluid

initial state

critical T

critical P


• Mobile Phase– Supercritical fluid– Most common fluids are:

• 100% CO2 (31.3°C critical temp. + 71 bar critical temperature)

• Mixture of CO2 and polar modifier (e.g. methanol), where modifier added to adjust retention

– Properties vs. gases and liquids• densities a little below liquids (solute – solvent

interactions matter, unlike ideal gases)• viscosities a little greater than gases – allowing minimal

pressure drop vs. liquids• diffusion coefficients closer to but greater than liquids,

minimizing C-term broadening vs. liquids– Properties generally result in efficient separations that

can be applied to a greater class of compounds than by GC


• Stationary Phase– Since carbon dioxide is non-polar, polar

stationary phase is most common– Both OT columns (smaller diameters since

diffusion is slower than in GC) and packed columns can be used

– Columns can be set up to take advantage of smaller pressure drops of supercritical fluids vs. liquids of using smaller particle sizes (or OT columns) as well as longer column


• Instrumentation– Equipment is somewhat specialized since it requires:

• pumps to reach supercritical pressures• a way to add polar modifiers• heaters to keep temperatures supercritical• a restrictor after the column or detector to ensure pressure

remains high• all fittings must be able to handle higher pressures

– Gradients can be set up by density programming– Both GC type (e.g. FID) and many HPLC detectors (e.g

UV detection) can be used with some modifications). However, FID is limited to 100% CO2 mobile phase

– Fraction collection (which is popular) uses cyclones to trap solids released upon degassing

– The combination of specific equipment requirements and limited market has made instrumentation expensive


• Applications– Analyses where neither GC nor HPLC functions well (e.g.

polymers without chromophores – polyethylene glycol)– Recent interest has been in high value preparative

separations:• SFC uses expensive packing material (e.g. chiral stationary

phases) more efficiently than HPLC• SFC solvent is cheaper than HPLC• Post column solvent removal occurs with chromatography

(as opposed to in additional step)

• Limitations– Expensive equipment (partly a result of limited market)– Limited mobile phases


Questions1. How is the lower viscosity of SFs an advantage

over liquids in terms of chromatography?2. Why are smaller diameter OT columns used in

SFC than in GC?3. A drug manufacturer currently is using

preparative HPLC to isolate a single enantiomer of a chiral mixture. List two advantages and one disadvantage in going to SFC.

4. SFC is being used with 98% CO2/2% methanol to separate polyethylene glycols with a polar stationary phase. The separation of different sized polymers is good but the retention factors are somewhat high causing the separation to take too long. What can be changed to decrease the separation time?

Liquid ChromatographyClassification of Types

• Classification based on pressure:– Low pressure (gravity based, preparative)– Moderate pressure (pressure from compressed gases in

“flash chromatography” or from low pressure pumps, also mainly preparative)

– High pressure (high performance or HPLC)– Ultra-high pressure (UPLC)

• Classification based on separation mechanism:– Normal-phase (polar stationary phase, less polar mobile

phase)– Reversed-phase (non-polar stationary phase, more polar

mobile phase)– Size exclusion chromatography (separation of molecules

based on size)– Ion exchange chromatography (exchange of ions)– Other types (ligand exchange and affinity)

Liquid ChromatographyStationary Phases – Packing Material

Geometry• No geometry comparable

to OT GC• Packed Columns

– Irregularly shaped particles (older technology)

• Larger A term• Less robust due to particles

breaking

– Spherically shaped particles• Smaller particles: decrease H

(smaller A and C terms), but increase back pressure

Advantage: H ~ kdp

Disadvantage: P = k/d2p

(constant column dimensions)

dp (μm) tR (min) N P (bar)

5 30 25,000 19

3 18 42,000 87

1.5 9 83,000 700Ultra performance LC (UPLC) for needed P


Geometry• Packed Columns (cont.)

– Superficially porous spheres• Reduces C term without

increasing P (much)• Less sample capacity

• Monolithic Columns– Single “web” of polymer

(usually silica based) rather than separate particles

– Advantageous because of high efficiency with relatively low back pressure

Dense core

Porous outer shell


Composition• Packing Material Composition

– Silica based packing material– Most commonly used material (many advances tried first

with silica)– Available unbonded (normal phase), a wide variety of

bonded phases and silica hydride (relatively new)– Main disadvantages are stability of silica (in normal

phase) and of Si-OR bonds (limits pH to 2 to 8)

• Polymeric (all hydrocarbon) packing material– Usually styrene-divinylbenzene– Poorer performance (efficiency, maximum pressure)– Common with ion-exchange and size exclusion– “Bonded Phase” contains ion exchange sites


Composition• Other packing material

– Zirconia• More temperature/solvent stable than silica• Both with and without bonded phases

– Porous graphitic carbon• non-bonded reversed phase (similar to phenyl groups)• highly stable

– Silica – polymeric hybrid particles• Silica core with polymer between silica and bonded

phase• Improves stability of silica (possible to run from pH 0 to

14)

Liquid ChromatographySilica-Based Normal Phase

• Historically, normal phase HPLC was the first type regularly used and the stationary phase was (uncoated, non-bonded) silica

• Stationary phase = silica surface• Silica surface is somewhat heterogeneous (can

have Si-OH groups, Si-O- groups, Si-O-Si groups, and adsorbed water)

• Use has decreased due to replacement by bonded phase

• Main disadvantage is slow equilibration (especially with regards to water) and requirement of consistent water% in mobile phase


• Mobile phases– Normal phase requires mobile phases of low

polarity (to be opposite to stationary phase)– Most common with hexane with polar additive

although wide variety of solvents possible (but only low % water)

– Weak solvent = hexane (or other less polar solvent)

– Strong solvent = polar additive (e.g. 2-propanol)

– Increasing strong solvent decreases retention


• Mobile Phase– Other Factors in solvent selection:

• Selectivity (different solvents will have different solvent – analyte interactions; best to choose solvent that emphasizes analyte differences)

• Solvent viscosity (low viscosity means smaller back pressure for given flow rate)

• Solvent miscibility• Detector limitations (e.g. wavelength cut-offs

for UV detection)• Compatibility with column packing and tubing


• Advantages:– Simpler stationary phase– Useful for wide range of analyte

polarities

• Disadvantages:– Long stabilization times– Retention time drifts– Must control % water in solvents

Liquid ChromatographyBonded Phases

• Most common on silica substrate but also common with other substrates

• Bonded layer is stationary phase• Common bonded phases

– Reversed phase:• C18 (most common by far of all bonded phases)• C8 (shorter alkyl chain)• Phenyl (better retention of aromatic groups)• Other groups (e.g. cholesterol)

– Normal phase or hydrophilic interaction (HILIC):• Cyano (most stable)• Diol (glycophase in text)• Amino (least stable)


• Quality of column (particularly for C18) depends on:– % of SiOH bonded to C18

group– % of remaining SiOH left

(many groups “end capped”)– Bulk of side groups

• Normal phase stationary phase has a greater tendency to decompose

Bulk SiO2

Si

O

Si

(CH

2)17

CH

3

R R

Si

O

CH3

Side group

End capped Si

Liquid ChromatographyC18 Phase

• Stationary Phase– Very non-polar group; but base of C18 is polar

(similar in polarity to octanol)– Some columns use imbedded polar groups

• Mobile Phase– Polar mobile phase needed– Most often water with polar additive

(acetonitrile, methanol, and tetrahydrofuran (THF) most common)

– Water is weaker solvent, organic modifier is stronger solvent

– Except for columns with imbedded polar groups, minimum % organic is 10% (needed to “wet” C18 phase)

Liquid ChromatographyPolar Bonded Phase

• Groups like –CN (cyano) provide separations similar to unbonded silica but reach equilibrium faster and have more consistent chromatograms

• Hydrophillic interaction chromatography (HILIC) refers to polar stationary phase with water present in mobile phase (usually in small amounts)– The mechanism in HILIC is somewhat different because

the stationary phase has a higher % water (due to strong attraction to polar stationary phase) than the mobile phase.

– Analyte interaction can occur with stationary phase or with associated water

Note: Penetration of mobile phase into stationary phase can also occur to some extent with other bonded phases (even with C18), especially with stronger solvents


• Advantages:– Faster equilibration (vs. silica)– Absorption vs. adsorption (less tailing)– Greater stationary phase volume

• Disadvantages:– Often have reduced range of analyte polarities

(very polar analytes are very weakly retained by C18)

– Column bleed (if bonded layer starts degrading)

Liquid ChromatographyGradient Elutions

• Similar to temperature programming in GC

• Program increases % strong solvent during run

• Example for HILIC (Normal Phase) HPLC to right

• Advantageous when wide variety of analyte polarities (plus peak width and S/N advantages)

• Disadvantages:– see those mentioned for GC

plus– Additionally column

equilibration takes time (gradient run took longer when including equilibration time)

– Requires more equipment

Elution of glucose oligosaccharides: glucose (monomer) to maltoheptaose (7 units)

Isocratic run (~62% ACN/38% water)

Gradient run (65% ACN to 50% ACN)

Liquid ChromatographySize Exclusion Chromatography

• Basis for separation:– Analyte inclusion in pores– Smaller analytes fit in pores

and are retained more– Larger analytes pass around

packing material• Used for separation of

polymers based on size• Variety of packing

geometries can be used (best if weaker analyte – stationary phase interactions)

• Pore size is critical and affects retention

large medium

small

Liquid ChromatographySize Exclusion Chromatography

• Applications:– Coarse separations

(monomers from oligomers or oligomers from polymers)

– Determination of polymer/oligomer molecular weight

• When used for determination of molecular weight, standards must also be run

• Standards should be similar and have similar retention

http://www.phenomenex.com/cms400min/litlib/brands/asahipakgfcolumns.pdf

Liquid ChromatographyIon Exchange Chromatography

(IC)• Stationary phase is made of ionic groups attached to

polymeric support• Ionic groups have opposite charge of solute ions being

separated (anion exchange requires cationic groups and cations require anionic groups)

• Anion exchange can be “strong” –NR3+ (permanent charge)

or “weak”, -NH3+ (charged at low pH)

• Cation exchange also uses permanently charged (e.g. –SO3

-) or groups charged at higher pH ( –CO2-)

• Intermolecular forces are very strong (so strong that without an exchanging ion, K is impractically large)

• Mobile phase is aqueous buffer (cations or anions needed to exchange with those on column)

• Strong eluents are higher concentrations and/or more strongly bound ions

Liquid ChromatographyIC

• Strength of ion-ion interaction depends on:– Charge (stronger for more

highly charged)– Ion size (smaller hydrated

size interacts more strongly)

• Cation ranking:Li+ (weak) < H+ < NH4

+ < Mg2+ < Ca2+

• Anion ranking:F- (weak) < OH- < Cl- < NO3

- < SO42-

• pH also affects ion forms and elution

A-

A-

A-

A-

Cl- SO4=

Separation of Ions

A-

A-

A-

Note: cartoon ignores effect of mobile phase counter ions

Liquid ChromatographyOther Stationary Phases

• Ligand Exchange (e.g. sugar separation)– retention based on ligand

sticking to metal (or visa-versa)

• Affinity Chromatography/ Molecular Imprinted Stationary Phases– Stationary phase designed

toward retention of specific groups or compounds

• Zwitterionic Phases– contains both + and - groups

SO3-

SO3-

Ca2+

HO-R

stationary phase

analyte

Liquid Chromatography

1. Why is bonded silica generally the first type of stationary phase considered?

2. What pH range could be used if separating an organic weak acid (RCO2H) with a pKa of 5.2 using a silica based C18 column?

3. What type of reversed-phase stationary phase could be used to separate alkyl amines (bases with conjugate acids of pKa of about 9)?

4. Is it possible to run some silica-based columns at extreme pH values?

5. What type of packing material should be chosen for high temperature separations?

6. List two examples of stationary phases that could be used with normal phase HPLC.

7. Which of the following additives could be used to retain ClO4- on

a C18 column?a) CH3NH2 b) (CH3CH2CH2CH2)4N+

c) CH3SO3- d) CH3(CH2)9SO3

-

Liquid ChromatographySome More Questions

8. In a silica based NP-HPLC separation with hexane and ethyl acetate, the % of which solvent should be increased to decrease retention?

9. What can cause tailing in poor quality C18 columns?10. A silica based NP-HPLC separation is using ether/2-

propanol to elute steroids. The retention factors are too small leading to resolution issues even when using 100% ether. Suggest an eluent that would lead to better retention.

11. What are the advantages and disadvantages of using bonded polar phases vs. silica?

12. What types of compounds are least retained in size-exclusion chromatography?

13. What groups are present on the surface of packing material in a cation exchange column?

Liquid ChromatographyInstrumentation – Mobile Phase

Delivery• Mobile Phase Selection

– See slide 18 of lecture for factors influencing selection of mobile phase

– Solvents must meet purity requirements (for column and detector functions)

– Solvent selectivity issue is important because:• Changing solvent affects retention for different

analytes differently• HPLC is less efficient than GC so often more likely to

have overlapping peaks• Changes in pH also are important for acidic/basic

compounds


Delivery

R

CH3CH3 OO

OH

R

CH3O

OH

HPLC-UV Sample 1 (ACN/0.1%TFA)

-500

50100150200250300350

0 5 10 15 20

Time (minutes)

Abs

orba

nce

acetovanilloneacetosyringonecinnamic acidisoeugenolsyringic acid

• Example of solvent changes to affect selectivity:– RP-HPLC Separation of

syringols from guaiacols– Difference is in 2nd MeOH

group– Water/Acetonitrile eluents

produce poor syringol/guaiacol separation factors

– Water/Methanol works better (although greater retention with MeOH of syringol is counter intuitive)

Syringols Guaiacols

HPLC Sample 1 (MeOH/0.1%TFA)

-500

50100150200250300350

0 5 10 15 20

Time (minutes)

Abs

orba

nce

acetosyringoneacetovanillonecinnamic acidisoeugenolsyringic acid

More retention

Less retention


Delivery• Optimization of Mobile

Phase Composition– Separation should be

perfomed on three different water/organic systems

– Then additional separations can be carried out using 3 component mobile phases

– Patterns in retention can be used to optimize mobile phase composition

Acetonitrile (40% in water)

Methanol (50% in water)

THF (30% in water)

20% ACN, 25% MeOH, water


Delivery• Mobile Phase Selection –

pH Buffering– In reversed-phase HPLC,

solute generally must be non-ionized to be retained

– pH is adjusted by adding buffer in water/organic modifier

– pH at pKa means retention factor about half of non-ionized acid retention time

– In ion-exchange chromatography, pH should be in range needed to produce ions

– In ion-pairing RP-HPLC, an ion-pairing reagent is added

O

OH

O

O-

retained unretained

NH2

Benzyl amine (conj. acid pKa = 9.35)

Non-ionized only at high pH

NH3+

O

O

O-

S

CH3

Ion pair reagent = pentane sulfonic acid (sodium salt)


Delivery• Solvent Flow

– HPLC requires high pressures and thus specific pumps

– The solvent also needs low levels of dissolved gases for pumps to function

– For the simplest “dedicated” HPLC, a single solvent reservoir and pump is needed

– For gradients and/or more method development work, switching between different solvents is needed


Delivery• Pumps

– Most pumps use two piston heads 180º out of phase to reduce pressure fluctuations

– Solvents go into and out of piston heads through one-way “check valves”

– Exit check valve closes on “in” stroke and entrance check valve closes on “out” stroke

pistons

Check valves Out Stroke

open

closed

closed

open

In Stroke


Delivery• Example of pump with

non-functioning check valves

• Fluctuation in pressure and signal can occur

• Changes to retention time also will occur

-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

7 7.5 8 8.5 9 9.5 10

Time (min)

Sig

nal

(u

V)

-300

-200

-100

0

100

200

8 8.2 8.4 8.6 8.8 9 9.2

Time (min)

Sig

nal

(u

V)

Bad check valve leaking


Delivery• Solvent Flow (for gradient/greater

flexibility operations)– Dual Pumps (high pressure mixing)– Low Pressure Mixing (stream “open” in proportion to

fraction)

To column

pumps

To column

Mixing chamberpump

chem. 230 – 10/21 lecture. announcements i exam 2 results –ave = 46.5 sd = 8 –similar average,...

Documents

gc x gc x ms chromatogram

week2d gc references

c critical

dimension columns

critical pointdemonstrated

type of samples

packed columns

columns of small diameter