NORMAL AND REVERSE PHASE CHROMATOGRAPHY.ppt

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NORMAL AND REVERSE

PHASE CHROMATOGRAPHY

Hemant A Hemant A IIRBS IIRBS Semester VII Semester VII ID MS 09/05 ID MS 09/05

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 ChromatographyChromatography 

 Separation using ChromatographySeparation using Chromatography 

 Definition of standard chromatographhic terminologyDefinition of standard chromatographhic terminology 

 TTypes of Stationary ypes of Stationary PhasePhase 

 TTypes of Mobile ypes of Mobile PhasePhase 

 Normal Phase HPLCNormal Phase HPLC 

 Reverse Phase HPLCReverse Phase HPLC

Chromatography

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 Three basic ways of Three basic ways of classifying various Separation tecclassifying various Separation techniques :-hniques :-

 1. Displacement Development1. Displacement Development 

 it is effective if the stationary phase is a solid and the solutes are it is effective if the stationary phase is a solid and the solutes are

adsorbed on its

adsorbed on its surface. surface. The solutes adsorb The solutes adsorb sequentially on the sequentially on the dist.dist. surface and array themselves in the order of

surface and array themselves in the order of increasing adsorptionincreasing adsorption strength.

strength. Only a sOnly a substance more ubstance more strongly held than strongly held than thethe

solutes(displacer) can elute the solute out in the order of decreasing solutes(displacer) can elute the solute out in the order of decreasing adsorption strength

adsorption strength

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 2. Frontal Analysis2. Frontal Analysis 

 It separates part of the It separates part of the first compound in a relatively pure state, eachfirst compound in a relatively pure state, each

subsequent component being mixed with those previously eluted. subsequent component being mixed with those previously eluted.

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 Consider a three component mixture, containing solutes (A), (B) andConsider a three component mixture, containing solutes (A), (B) and

(C) as a dilute solution in the mobile phase that is fed continuously onto (C) as a dilute solution in the mobile phase that is fed continuously onto a column. The first component to elute, (A), will

a column. The first component to elute, (A), will be that solute heldbe that solute held least strongly in the stat

least strongly in the stationary phase. Then the second solute, (B), willionary phase. Then the second solute, (B), will elute but it will be mixed with the first solute. Finally, the third solute elute but it will be mixed with the first solute. Finally, the third solute (C), will elute in conjunction with (A) and (B). It is clear that only solute (C), will elute in conjunction with (A) and (B). It is clear that only solute (A) is eluted in a pure form

(A) is eluted in a pure form

Separation using Chromatography

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3. Elution development 3. Elution development

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 It’s a series of adsorption –It’s a series of adsorption – extraction processes which are extraction processes which are

continuous from the time the sample is injected into the continuous from the time the sample is injected into the distribution system until the time the solutes exit from it. distribution system until the time the solutes exit from it.

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:-

 Plate theoryPlate theory 

 Eq b/w mobile and statioary phasesEq b/w mobile and statioary phases 

 Prob b/w solute phase and stationary phase.Prob b/w solute phase and stationary phase.

Theory

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 The baseline is any part of the chromatogram where only mobileThe baseline is any part of the chromatogram where only mobile

phase is emerging from the column. phase is emerging from the column.

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 The injection point is that point when the sample is placed on the The injection point is that point when the sample is placed on the

column. If the sample has a finite volume, then the injection point column. If the sample has a finite volume, then the injection point corresponds the start of the sampling process.

corresponds the start of the sampling process.

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 The deadpoint is the position of the peak maximum of an unretainedThe deadpoint is the position of the peak maximum of an unretained

solute. It is not the initial part of the dead volume peak as this solute. It is not the initial part of the dead volume peak as this represents a retarded portion of the peak that is

represents a retarded portion of the peak that is caused by dispersioncaused by dispersion processes.

processes.

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 The peak maximum is the highest point (the apex) of the peak andThe peak maximum is the highest point (the apex) of the peak and

measurements as dead volume and retention volume. measurements as dead volume and retention volume.

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 The dead time (t0) is the time elapsed between the injection pointThe dead time (t0) is the time elapsed between the injection point

and the dead point. and the dead point.

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 The retention time (tr) is the time elapsed between the injectionThe retention time (tr) is the time elapsed between the injection

point and the peak maximum. Each solute will have a characteristic point and the peak maximum. Each solute will have a characteristic retention time.

retention time.

Chromatogram

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 The retention volume (Vr) is the volume of The retention volume (Vr) is the volume of mobile phase passed through the mobile phase passed through the columncolumn between the injection

between the injection point and the ppoint and the peak maximum. eak maximum. VV_r_r = Qt = Qt_r_r Each solute will also Each solute will also have a characteristic retention

have a characteristic retention volume.volume. 

 The peak height (h) is the distance between the peak maximum and the baselineThe peak height (h) is the distance between the peak maximum and the baseline geometrically produced beneath the peak.

geometrically produced beneath the peak. 

 The peak width (w) is the distance between each side of a peak measured at 0.6065The peak width (w) is the distance between each side of a peak measured at 0.6065 of the peak height.

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 It is the distance b/w 2 neighbouring peaks measured at the base.It is the distance b/w 2 neighbouring peaks measured at the base.

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 wherewhereNN is the plate number, a measure of the performance or the efficiency of is the plate number, a measure of the performance or the efficiency of thethe column.

column. 

 αα is the quotient factor b/w retention times is the quotient factor b/w retention times 

 kkis a measure of the strength of the is a measure of the strength of the interaction of a given compound in a giveninteraction of a given compound in a given chromatographic system.

chromatographic system. 

 The resolutionThe resolutionRR–– the distance from peak base to peak base the distance from peak base to peak base –– depends only on depends only on 

 the following three factors:the following three factors: •

• the strength of the interaction between the compound and the stationarythe strength of the interaction between the compound and the stationary phase(if the peak comes soon or late), i.e. on the

phase(if the peak comes soon or late), i.e. on thekk _value,_value,

•

• the ability of the cthe ability of the chromatographic system to distinguish between the twohromatographic system to distinguish between the two components

components of interest, i.e. the α value,of interest, i.e. the α value, •

• if the relevant peaks if the relevant peaks are sharp or wide, i.e., the are sharp or wide, i.e., the plate number.plate number.

Resolution

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Improving Resolution

Consequently, to improve resolution, Consequently, to improve resolution, there are in principle only three

there are in principle only three possibilities,

possibilities,

• namely a general increase in the • namely a general increase in the interaction (

interaction (kk _{value increases),}_{value increases),}

• an

• an analyte-specific change in theanalyte-specific change in the interaction (α value increases), or interaction (α value increases), or • an increase in the efficiency of the • an increase in the efficiency of the separation (

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STATIONARY PHASE

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 Non polar hydrophobicNon polar hydrophobic species attached by

species attached by

siloxane or ether bonds siloxane or ether bonds to the surface

to the surface

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 Inorganic silica forms theInorganic silica forms the most common support most common support

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 It’s mechanical strengthIt’s mechanical strength

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 Optimum pH 2-8Optimum pH 2-8

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 Silica pretreatmentSilica pretreatment needed

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 Nominally used to separate non-ionic compounds with highNominally used to separate non-ionic compounds with high

molecular weight. molecular weight.

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 Accounts for 1/5 th of all HPLC separations.Accounts for 1/5 th of all HPLC separations. 

 SP > MPSP > MP 

 Silica used is porous and non crystalline and generally of theSilica used is porous and non crystalline and generally of the

form :- SiO2-x/2(OH)X]n . H20p form :- SiO2-x/2(OH)X]n . H20p

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 Silanols groups are responsible for the polar character ofSilanols groups are responsible for the polar character of

silica packings used in SP. silica packings used in SP.

Normal Phase Chromatography

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 Non polar SPNon polar SP –– Polar MP Polar MP 

 There is a certain non polar component added to the MPThere is a certain non polar component added to the MP

to lower the polarity to lower the polarity

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 The SP can also be varied The SP can also be varied using functional groupsusing functional groups

attached to it to vary factors such as affinity and attached to it to vary factors such as affinity and retention factors.

retention factors.

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 TTo make the column efficieno make the column efficient high pressure is needed.t high pressure is needed. 

 As a result -As a result -> RP == HPLC,UPLC etc…..> RP == HPLC,UPLC etc…..

Reversed Phase Chromatography

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Optimization of RP-HPLC

Our aims must be Our aims must be

:-• to separate better (higher • to separate better (higher resolution),

resolution),

• to separate faster (shorter • to separate faster (shorter retention time),

retention time),

• to see more (lower • to see more (lower detection limit),

detection limit),

• to separate at lower cost • to separate at lower cost (economic effort),

(economic effort),

• to separate more (higher • to separate more (higher throughput).

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VIDEO

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 They are synthesized They are synthesized by silica by silica based based SP by SP by reacting anreacting an

organochlorosilicane with the gel support material in an organochlorosilicane with the gel support material in an appropriate organic solvent.

appropriate organic solvent.

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 TTwo types of such wo types of such phases :-phases

:-1. 1. Monomeric SP :-Monomeric SP :-2. 2. Polymeric SP :-Polymeric SP

:-Chemically bonded SP

Chemically bonded SP

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:-

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 Using a solvent system whose concentration does not varyUsing a solvent system whose concentration does not vary

with time. with time.

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 We use this method when the sample contains analytesWe use this method when the sample contains analytes

that have

that have similar similar properties and properties and the hydrophibicitythe hydrophibicity difference is very small.

difference is very small.

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 Else, The early eluted components may be unresolved andElse, The early eluted components may be unresolved and

the final components elute out over a long period of time. the final components elute out over a long period of time.

Isocratic reverse phase

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 Vary the mobile phase solution continuously in a linearVary the mobile phase solution continuously in a linear

fashion from the beginning to the end of analysis period. fashion from the beginning to the end of analysis period.

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 The strength of The strength of the mobile phase the mobile phase is weak is weak initially andinitially and

increases gradually and then peaks when separation is increases gradually and then peaks when separation is completed

completed

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 This method encourages the use of tertiary and quarternaryThis method encourages the use of tertiary and quarternary

mixtures mixtures

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 The main The main disadvantage of disadvantage of the gradient method the gradient method is that is that thethe

time required to re-equilibriate the column takes few mins to time required to re-equilibriate the column takes few mins to several hours

several hours

Gradient Reverse Phase

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 HPLCHPLC

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 Solvent reservoirs Solvent reservoirs

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 High pressure pumpHigh pressure pump

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 MixerMixer

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 Injection deviceInjection device

  ColumnColumn   DetectorDetector   PCPC

Equipment's needed

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 1. Chromatographic Theory1. Chromatographic Theory –– jack Cazes jack Cazes 

 2. Handbook of HPLC2. Handbook of HPLC –– Elena Katz Elena Katz 

 3. Encyclopaedia of Chromatography3. Encyclopaedia of Chromatography ––Jack CazesJack Cazes 

 4. HPLC Solvent guide4. HPLC Solvent guide –– Paul C Sadek Paul C Sadek 

 5. Video courtesy of the Imad Haidar group at the5. Video courtesy of the Imad Haidar group at the

university of minnesota. university of minnesota.

NORMAL AND REVERSE PHASE CHROMATOGRAPHY.ppt