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Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary...

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Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed Phase Chromatography Normal Phase Chromatography Gel Permeation Chromatography Separation of Ionic or Charged Compounds Ion-Exchange Chromatography Ion Chromatography Mechanism Mobile Phase Stationary Phase Applications
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Page 1: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Methodologies in High Performance Liquid Chromatography (HPLC)

Mobile Phases

Mechanism

Stationary Phases

Applications

Separation ofNeutral Compounds Reversed Phase Chromatography

Normal Phase Chromatography

Gel Permeation Chromatography

Separation ofIonic or ChargedCompounds

Ion-Exchange Chromatography

Ion Chromatography

Mechanism

Mobile Phase

Stationary Phase

Applications

Page 2: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Illustration of Mechanism of Retention of Solute Using Four Different Types ofHPLC

(a) Normal Phase Chromatographyaka. Adsorption Chromatography orClassically as liquid solid chromatography (LSC)

- Solute is adsorbed on the surface of theS.P ( polar interactions are dominant)

S.P = Polar (Silica gel or alumina backbone)M.P = Non-polar (hexane mixed with polar solvent)

(b) Reversed Phase Chromatographyaka. Partition Chromatography (LLC)

- Solute equilibrate “in” and “out” of the liquidS.P -(partitioning in and out of the alkane chains)

S.P = Liquid coated on a solid support(silica or polymer backbone)M.P = Polar (water mixed with relatively non-polar solvent)

HO

Less-polarM.P

PolarS.P

HO

HO

HO

HO

(a) Adsorption

Sili

ca b

ackb

on

e

(b) Partition

More polarM.P

Non-polarS.P

Sili

ca o

r P

oly

mer

bac

kbo

ne

Page 3: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Illustration of Mechanism of Retention of Solute Using Four Different Types of HPLC (Continued)

(3) Ion-Exchange Chromatography (IEC) or IC- Solute competes with the M.P counterions (of same charge as the solute) for the fixed charged on the S.P)

S.P = silica or polymer back bone containing anion-exchange groups [e.g., NH+(CH3)3,] or cation exchange groups [SO3

-] bound on its surfaceM.P = counterions ( inorganic salt bufferse.g., ammonium acetate (CH3COO NH4 )

- Separates molecules based on size, charge and hydration

Cl-

Br-

F-

SO42-

Sil

ica

or

Po

lym

er B

ackb

on

e

Li+

Na+

K+

Rb+

Cs+

ChargedS.P

M.P counter ions

So

lute

Page 4: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Illustration of Mechanism of Retention of Solute Using Four Different Types of HPLC (Continued)

(4)Gel-Permeation Chromatography (GPC)“Small molecules are retained longer thanlarge molecules”separates molecules based on sizedanalyte > dpore ------no trapdanalyte <dpore ----- increase tR

S.P = 10 um particles with uniform pore Silica: rigid- highly porous, stable at high temperature, interacts with solutePolymer: cross-linked, different poresize, (e.g., PSDVB)M.P = Liquid aqueous solventorganic solvent, mixed aqueous/organic solvent

Gel filteration: hydrophilic packing, sulfonated divinyl benzenes or polyacrylamideused to separate polar speciesGel permeation: hydrophobic packings (PSDVB) used to separate non-polar species

PorousS.P

Gel permeationchromatography

Sil

ica

or

Po

lym

er b

ackb

on

eSmallmolecules

Large molecules

Page 5: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Normal Phase ChromatographyMobile phase (M.P)

Silica or Polymer support

Polar bonded-phase

--M.P is relatively non-polar(hexane mixed with slightly more polar solvent like isopropanol, ethyl acetate or chloroform)

--S.P is polar. The S.P polarity order is shown to the right ----

SilicaNH2

DiolCN

Polar

Less Polar

Mobile phase (M.P)Silica or Polymer support

Non-polar bonded-phase

Reversed-Phase Chromatography

--M.P is relatively polar(water mixed with acetonitrile)

Non-Polar

More Polar

C-18PhenylC-8C-4CN

--S.P is non-polarThe S.P polarity order is shown to the right

NPC vs. RPC

Page 6: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Relationship between polarity and elution times fornormal-phase and reversed-phase LC.

NPC*Most polar elutes last*Increasing polarity ofM.P decrease elutiontime in NPC

Hexane/CHCl3

Hexane/THF

Water/MeOH

Water/THF

RPC*Most polar elutes first*Decreasing polarity of M.P decreaseselution time in RPC

Page 7: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

MECHANISM OF SOLUTE RETENTION IN NPC

(A)Competition Model:--Assumes that entire S.P is covered by M.P molecules- Retention occurs due to adsorption of process- Competition between solute molecules and M.P molecules for the adsorptionsites on the S.P

(b) Solvent Interaction Model:--Assumes that :- “A bilayer of solvent molecules is formed around S.P particles, which depends on the concentration of polar solvent in the M.P”

“Retention results from the interaction of solutes molecules with the secondary layer of M.P molecules (no interaction with the S. P)”

“Competition Model is More Generally Accepted”

Page 8: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

MECHANISM OF SOLUTE RETENTION IN RPC

(A)Solvophobic Model - “Retention is considered primarily due to hydrophobic interactions between the solute and the M.P.”

--S.P is thought to behave more like a solid than a liquid and retention Is considered primarily due to hydrophobic interaction between solute and M.P

“ “Partitioning model is generally accepted in RPC”

--Because of solvophobic effects solute binds to the surface of the S.P reducing surface area of the analyte exposed to the M.P.

(B) Partitioning Model“Solute is fully embeded in the S.P rather than adsorbed on the surface of the S.P.Therefore ,partitioning of solute occurs between M.P and liquid likd M.P.”

Page 9: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Elutropic Series (e0) of Some Solvents Used in NPC

-e0 in NPC is a measure of adsorption energy/area of solvent and used quantitatively to define sovent strength

Solvent strength in NPC is directly proportional to elutropic series e0 values

Higher the value ofe0, the greater

the strength of the solventwhich result in

fasterelution or

lower retentionof solutes

Which is the strongest and the weakest eluent for NPC?

Water is the strongest eluent--- fast separation (shorter retention times)Pentane/Hexane are the weakest eluent------ long separation time

“A rule of thumb in NPC ---- An increase in e0 by 0.05 units typically decreases k’ by a factor of 3-4”

Page 10: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

ISOELUTROPIC MOBILE PHASES

- Mixtures of different solvents having the same overall polarity are called isoelutropic mixtures.” ----------- These mixtures have same eluent strength

Example:--A 30% (v/v) mixture of chloroform (CHCl3) in hexane has approximately thesame solvent strength as 35% (v/v) mixture of toluene in hexane-so this mixture is classified as isoelutropic mixture

Q. How can you prove this quantitatively?--If the volume fraction (F) and the e0 values of the solvents are known we can prove this using a relationship:

FA e0A = FB e0

B

0.30 x 0.26 = 0.35 x 0.22 0.078 = 0.077

• Similarly 69% (v/v) of ether in hexane has same solvent strength as 99%(v/v) ofmethylene chloride in hexane

-- Isoelutropic M.Ps give similar total elution times, but they permit some changes in sample selectivity

Page 11: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Why an increase in % (v/v) methyltertiary-butyl ether (MTBE) decreases the retention time of aniline and phenol?

--Because an increase in MTBE % (v/v), increases the polarity of M.P, which in turnincreases the solvent strength-- analytes elutes faster

What care must be taken when preparing M.P of mixed solvents?“The Solvents must be miscible”

Page 12: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Chromatogram showing the effect of Solvent strength in NPC--Following chromatogram shows an example of changing the solvent selectivity of a normal phase separation of phthalate esters (plastisizers)

- It can be seen that changing the solvent from methyl ethanoate to methyl butanoate While keeping the ratio same radically changes the selectivity of diethyl/diphenyl phthalate and this pair is better resolved with buty ethanoate

Q. Why the total analysis time increases when we use methyl butanoate instead ofMethyl ethanoate (ethy acetate)?---Methyl butanoate (aka. butyl acetate) is a weaker solvent (e = 0.38) for NPC then methyl ethanoate (e = 0.58), Thus weaker solvent gives longer retention of solutes

Page 13: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Q.2 Why the elution order of diethyl phthalate or diphenyl phthalate reversed?

--Because electron donating groups are different in two solvents, they might induce dipole moment in the benzene ring of phthalates differently.

Carbonyl dipole

Induce dipole in benzene ring

O

O

O

O

OC2H5

MeO OC4H9

MeO

methylethanoate(ethyl acetate)

methylbutanoate(butyl acetate)

d-

d + d+ d-

DPT = Diphenylphthalate

O

O

O

O

OC2H5

MeO OC4H9

MeO

methylethanoate(ethyl acetate)

methylbutanoate(butyl acetate)

d-

d

+d+

d-

DPT = Diphenylphthalate

d

Page 14: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Disadvantages of NPC

a)---Retention times tend to be too long and the chromatographic peak have atendency to tail

b)--Lack of selectivity--.Virtually all compounds are eluted in the same orderregardless of column selectedChanges in selectivity is only achieved by changing the M.P compositionc) Effect of WaterPerhaps the biggest disavantage of using NPC (using bare silica column oralumina column) and to lesser extent bonded phase column in NPC is:Water is adsorbed from the atmosphere into the M.P, which in turn whenpumped through the column is adsorbed on the adsorption sites of the S.P

What is the result of this effect on chromatographic separations?

Solute tr and N continue to decreases and columns becomes deactivated---run times decreases,Rs decreases, the longer the eluent sits around

--Prepare Normal phase eluent (saturated withwater) The Rx-SIL column shows less retention variation and peak height as water is varied but low purity Zorbax takes at least 50% water to achieve some saturation.

Q. So how to avoid this problem?

Page 15: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Benefits of Making Saturated Eluents are:

Less variation in sample retention from run to runHigher sample loadingHigher column efficiency and peak tailing is reducedReduced catalytic activity of the adsorbent

Page 16: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Polarity Index (P’)for Reversed Phase Chromatography

--Solvent strength in RPC isinversely related to polarity indexHigher the value of P’ the weakerthe solvent strength and vice-versa

-- Water is generally used as a“base-solvent” and M.P strengthis increased by increasing the volume of another solvent (organicmodifier).

--Three commonly used modifiers are: Tetrahydrofuran (THF), methanol (MeOH), and acetonitrile (ACN)

Which composition of mobile phase is weaker for RPC?(a) 50/50 mixture of MeOH/H20 or (b) 50/50 mixture of THF/H20

“ A 50/50 mixture of MeOH/H20 is a weaker M.P”A general rule of thumb in RPC ---------- k’ of solute decreases by a factor of 2 or more for every 10% (v/v) addition of organic modifier

Page 17: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

SOLVENT STRENGTH EFFECTS IN REVERSED-PHASE CHROMATOGRAPHY

In RPC the primary purpose of decreasing the %(v/v) organic solvent (increases the % (v/v) of polar solvent such as water) is to increase the Rs, a, and k’

---Resolution of compound pair A/B is poor for M.P of >50% ACN, however, separation Of A/B improves for a decrease in % ACN upto 40% ACN

---Resolution of compound pair C/D first increases upto 50%, then decreases at 40%ACN

--Water is generally used as a base solvent in RPC and the separation is optimized byIncreasing the amount of water to the organic solvent.--The following chromatograms shows the effect of solvent strength selectivity on separation of five compounds

Page 18: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Q. What kind of separation would you observe if the polarity of the M.P and the S.P are similar?

---No separation (interaction of solute with each phase is similar)

MEASURING POLARITY OF MOBILE PHASE IN NORMAL AND REVERSEDPHASE CHROMATOGRAPHY

-In NPC the polarity of M.P is generally expressed in elutropic series (e0)- In RC the polarity of M.P is generally expressed using polarity index (P’)

“When the Rs of one band pair increases and Rs of another band pair decreases with a change in % organic of solvent, best optimization condition is the one whereboth band pair have same Rs at intermediate solvent strength. (i.e., 45% ACN)

Page 19: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Q. What we have learned about the NPC and RPC separation optimization so far?

Easiest way to influence separation in NPC and RPC is by changing the Solvent polarity

Changing the solvent polarity changes k’ in both NPC and RPCk’ can be varied by changing the polarity index (P’)

Q. Suppose we obtained a separation of analyte and we measured its k’ at a certain volume fraction of organic solvents. Can we estimate what volume fraction (%) v/v of organic solvent is needed if we want to increase k’ of the analyte? Yes, we can if we can relate k’ with P’

Relationship between capacity factor (k’) and P’ for normal phase chromatography

Relationship between capacity factor (k’) and P’ for reversed-phase chromatography

k2’k1’

= 10 (P2’-P1’)/2

10 (P1’-P2’)/2k2’

k1’

=

k1’ and k2’ areThe intial and finalvalues of capacity factorof two solutes

P1’ and P2are the initialand final valuesof solvent polarity

Only difference betweenthe two equations

is on the exp valuesOf polarities

Page 20: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

In a RP-HPLC, a solute was found to have a retention time of 31.3 min while anunretained species required 0.48 min for elution when the mobile phase was 30% (v/v) of methanol and 70% (v/v) of water.

(A)What is capacity factor (k’) at this composition of M.P? k’ = tR- t0 = 31.3-0.48 t0 0.48

= 64.20833 ------(Calculator answer)= 64.2

(B) Calculate water/methanol composition that should be required to bring k’ to avalue of about 5.0

P’AB = fA P’A + FB P’B

= 0.30 x 5.1 + 0.70 x 10.2= 1.53 + 7.14

= 8.67

Substituting for reversed phase equation we can write:

10 (P2’-P1’)/2k2’

k1’

= 5.0 = 10(P2’-8.67

)/2

64.2Log 0.0781 = -1.11

Number of digits in mantissa ofLogx = number of significant

Figure in x

-1.11 = P2’ -8.67/2.0-1.11 = 0.5P2’ -4.34

P2’ = -1.11 +4.34

0.5P2’ = 6.46

Page 21: Methodologies in High Performance Liquid Chromatography (HPLC) Mobile Phases Mechanism Stationary Phases Applications Separation of Neutral Compounds Reversed.

Substituting into equation (A) (see above) we get

6.46 = fA X 5.1 + (1-FA)10.2

6.46 = 5.1 fA + 10.2 -10.2fA

fA = 6.46-10.2 = 0.73 or 73% MeOH-27% H2O 5.1

• Note that in RPC higher concentration of methanol means the k’ should decreases compared to the initial conditions of 30% MeOH


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