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 Mechanism Mobile Phase Stationary Phase Applications Separation of Ionic or Charged Compounds Ion-Exchange Chromatography Ion Chromatography

Silica or Polymer backbone Illustration of Mechanism of Retention of Solute Using Four Different Types of HPLC (a) Normal Phase Chromatography aka. Adsorption Chromatography or Classically as liquid solid chromatography (LSC) HO Less-polar M.P Polar S.P (a) Adsorption Silica backbone - Solute is adsorbed on the surface of the S.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 Chromatography aka. Partition Chromatography (LLC) (b) Partition More polar M.P Non-polar S.P Silica or Polymer backbone - Solute equilibrate “in” and “out” of the liquid S.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)

Illustration of Mechanism of Retention of Solute Using Four Different Types of HPLC (Continued) Cl- Br- F- SO42- Silica or Polymer Backbone Li+ Na+ K+ Rb+ Cs+ Charged S.P M.P counter ions (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) - Separates molecules based on size, charge and hydration Solute S.P = silica or polymer back bone containing anion-exchange groups [e.g., NH+(CH3)3,] or cation exchange groups [SO3-] bound on its surface M.P = counterions ( inorganic salt buffers e.g., ammonium acetate (CH3COO NH4 )

Silica or Polymer backbone Illustration of Mechanism of Retention of Solute Using Four Different Types of HPLC (Continued) (4)Gel-Permeation Chromatography (GPC) “Small molecules are retained longer than large molecules” separates molecules based on size danalyte > dpore ------no trap danalyte <dpore ----- increase tR Porous S.P Gel permeation chromatography Silica or Polymer backbone Small molecules Large S.P = 10 um particles with uniform pore Silica: rigid- highly porous, stable at high temperature, interacts with solute Polymer: cross-linked, different pore size, (e.g., PSDVB) M.P = Liquid aqueous solvent organic solvent, mixed aqueous/organic solvent Gel filteration: hydrophilic packing, sulfonated divinyl benzenes or polyacrylamide used to separate polar species Gel permeation: hydrophobic packings (PSDVB) used to separate non-polar species

NPC vs. RPC Normal Phase Chromatography Mobile 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 ---- Silica NH2 Diol CN 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-18 Phenyl C-8 C-4 CN --S.P is non-polar The S.P polarity order is shown to the right

Relationship between polarity and elution times for normal-phase and reversed-phase LC. Hexane/ CHCl3 THF Water/MeOH Water/ NPC *Most polar elutes last *Increasing polarity of M.P decrease elution time in NPC RPC *Most polar elutes first *Decreasing polarity of M.P decreases elution time in RPC

“Competition Model is More Generally Accepted” MECHANISM OF SOLUTE RETENTION IN NPC 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 adsorption sites 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”

MECHANISM OF SOLUTE RETENTION IN RPC 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 --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.” “ “Partitioning model is generally accepted in RPC”

the strength of the solvent Elutropic Series (e0) of Some Solvents Used in NPC Higher the value of e0, the greater the strength of the solvent which result in faster elution or lower retention of solutes -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 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”

FA e0A = FB e0B 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 the same 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 e0B 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) of methylene chloride in hexane -- Isoelutropic M.Ps give similar total elution times, but they permit some changes in sample selectivity

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 turn increases the solvent strength-- analytes elutes faster What care must be taken when preparing M.P of mixed solvents? “The Solvents must be miscible”

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 of Methyl 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

DPT = Diphenylphthalate Q.2 Why the elution order of diethyl phthalate or diphenyl phthalate reversed? O C 2 H 5 M e 4 9 methylethanoate (ethyl acetate) methylbutanoate (butyl acetate) d - + DPT = Diphenylphthalate d+ Carbonyl dipole Induce dipole in benzene ring d --Because electron donating groups are different in two solvents, they might induce dipole moment in the benzene ring of phthalates differently.

Disadvantages of NPC a)---Retention times tend to be too long and the chromatographic peak have a tendency to tail b)--Lack of selectivity--.Virtually all compounds are eluted in the same order regardless of column selected Changes in selectivity is only achieved by changing the M.P composition c) Effect of Water Perhaps the biggest disavantage of using NPC (using bare silica column or alumina column) and to lesser extent bonded phase column in NPC is: Water is adsorbed from the atmosphere into the M.P, which in turn when pumped 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 Q. So how to avoid this problem? --Prepare Normal phase eluent (saturated with water) 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.

Benefits of Making Saturated Eluents are: Less variation in sample retention from run to run Higher sample loading Higher column efficiency and peak tailing is reduced Reduced catalytic activity of the adsorbent

Polarity Index (P’)for Reversed Phase Chromatography --Solvent strength in RPC is inversely related to polarity index Higher the value of P’ the weaker the solvent strength and vice- versa -- Water is generally used as a “base-solvent” and M.P strength is increased by increasing the volume of another solvent (organic modifier). --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

SOLVENT STRENGTH EFFECTS IN REVERSED-PHASE CHROMATOGRAPHY --Water is generally used as a base solvent in RPC and the separation is optimized by Increasing the amount of water to the organic solvent. --The following chromatograms shows the effect of solvent strength selectivity on separation of five compounds 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

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

Only difference between 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 RPC k’ can be varied by changing the polarity index (P’) 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 reversed-phase chromatography P1’ and P2 are the initial and final values of solvent polarity k2’ k1’ = 10 (P2’-P1’)/2 k1’ and k2’ are The intial and final values of capacity factor of two solutes Only difference between the two equations is on the exp values Of polarities Relationship between capacity factor (k’) and P’ for normal phase chromatography 10 (P1’-P2’)/2 k2’ k1’ =

Number of digits in mantissa of Logx = number of significant In a RP-HPLC, a solute was found to have a retention time of 31.3 min while an unretained species required 0.48 min for elution when the mobile phase was 30% (v/v) of methanol and 70% (v/v) of water. 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 a value 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’)/2 k2’ k1’ = 5.0 = 10(P2’-8.67)/2 64.2 Log 0.0781 = -1.11 P2’ = -1.11 +4.34 0.5 -1.11 = P2’ -8.67/2.0 -1.11 = 0.5P2’ -4.34 Number of digits in mantissa of Logx = number of significant Figure in x P2’ = 6.46

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