hplc

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Before we begin

Injector Anatomy-the 6 port valve- http://www.shsu.edu/~chm_tgc/sounds/flashfiles/HPLC.swf

Retention Mechanisms and Derivation Techniques in HPLC

Objectives of Lecture

Give brief history and theory of HPLCDescribe column selection for specific molecular groups (proteins, peptides, organics)Column characteristics and retention mechanismSpecial Techniques and TroubleshootingDerivation of functional groups for analysis

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History of HPLC

First seen in 1973 with 10um particle column 30cmX3.9mmAlso in 1973 modification of silica via silanization became commercially feasible73-78 5um particles became available, also batch to batch reproducibility of packing material became better. 78-83 Ion chromatography became standard of inorganic analysis. Kirkland and Glajch formed first framework for rational development for RP chromatography based on solvent selectivity

History of HPLC cont’d

83-present: better understanding of retention mechanisms, mechanical stability of the bed increased, and great advances in HPLC packing chemistry led to improved absorption properties and better batch-to-batch reproducibility.

Theory

• Isocratic and Gradient Chromatography-Isocratic: all conditions and settings of the separation are held constant-Gradient: one or more of the parameters are varied constantly. Typical gradient is continuous variation of the mobile-phase comp. from low elution strength to high. Also can use temp. or flow gradients.

Figure: At the onset of sample introduction, the compounds are initially retained at the inlet of the column. As the solute capacity, or k', for the compound decreases, the compound begins to migrate through the stationary phase. Each of the other compounds in the sample subsequently migrate as their k' values decrease. Compared with isocratic elution, resolution and separation are improved and bandwidths are nearly equal (Snyder 1983)

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Theory cont’d

• Theoretical Plate- one equilibrium between the mobile

and stationary phase; a measure of column efficiency; the more plates, the better the column efficiency

• Height Equivalent to a Theoretical Plate (HETP or H)- value obtained by dividing the column length by the number of theoretical plates; taken as an indication of column quality. A carryover from distillation theory; a measure of a column's efficiency. For a typical HPLC column well-packed with 5-um particles, HETP values are usually between 0.01 and 0.03 mm. HETP = L/N, where L is column length, and N is the number of theoretical plates.

Theory cont’d 2

• Peak Asymmetry -is a common practical measure of the

quality of the column. As columns age, the peak asymmetry usually deteriorates; giving tailing peaks

-Theory assumes a Gaussian shape peak that is symmetrical. The peak asymmetry factor is the ratio (at 10 percent of the peak height) of the distance between the peak apex and the back side of the chromatographic curve to the distance between the peak apex and the front side of the chromatographic curve. A value >1 is a tailing peak, while a value <1 is a fronting peak. Problems associated with peak

tailing-loss of resolution and integration issues. This is due to deterioration of peak asymmetry

The relationship between HETP (height equivalent to a theoretical plate) and velocity is of great importance for the peak width, as the peak width is an impediment to resolution.The HETP is influenced by:

flow dynamicsproperties of the stationary phaseproperties of the sample

The relationship has been described by 3 equations:van Deemter equationGiddings equationKnox equation

Relationship between HETP and velocity

Hydrodynamics of Chromatography

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The HETP is mainly composed of 3 independent contributions:A (size and distribution of the interparticle channels and othernonuniformities in the packed bed), eddy diffusionB/u (inversely proportional to linear velocity,

describes molecular diffusion in axial direction)

Cu (directly proportional to linear velocity,terms related to mass transfer and sorption kinetics)

Einstein equation:

Diffusion of a solute in a liquid is normallydistributed in the form of a Gaussian distribution

Van Deemter and Einstein


An important aspect of column performance is its backpressure. Backpressure is represented by the permeability. The influencingparameters of the permeability equation are:

F is the flow rateη is the viscosityL is the column lengthr is the radius of the column∆p is the pressure drop across the column

The viscosity is an imporant factor for reversed-phase chromatography, as the mobile phase is an aqueous mixture. From the standpoint of permeability, acetonitrile is the preferred mobile-phase modifier in reversed-phase chromatography, as it shows the lowest viscosity.

Permeability


The performance index is a measurement for the overall column performance and depends on the following parameters:interstitial porosity ε of the packed bedflow resistance Фviscosity ηlinear velocity uretention factor k

The performance index also states the overall separation quality of the column.

Another approach is the separation impedance, which is a purer measurement of column performance because of its independancyon the retention factor and the viscosity.

Performance Index

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The retention factor k for an analyte provides the researcher with the ability to compare and standardize different columns or the samecolumn at different flow rates for the analyte.

k is measured as the retention time of an analyte minus the retention time of an unretained peak divided by the retention time of theunretained peak.

Benefits:the retention factor is the ratio of the time that an analyte spends in the stationary phase, as it spends the same amount of time inthe mobile phase as the unretained peakthe retention factor describes also the ratio of the number of molecules in the mobile phase to the number of molecules in the stationary phase

Thermodynamics of Chromatography

Retention factor and chemical equilibrium

ktR tU

t U

Column Selection by Analyte group

Briefly go over what columns are chosen for certain analyte groups (e.g. polymers, peptides, proteins, nucleic acids, and low molecular weight organics)

Polymer Analysis

The interest or goal in most polymer assays is the determination of molecular-weight distribution of the polymer. So size-exclusion is the main retention mechanism used.-To further determine assay solubility must be taken into consideration. If water soluble use-aqueous size exclusion column based on polar polymers like glycidyl methylacrylate, hydroxyethyl methylacrylate, or polyvinyl alcohol -Can use silica based packings coated with glycidoxypropylsilane for aqSEC but very pH sensitive. Not compatible with basic conditions and have residual silanols that might react with the polymer analyte as well. Very limited in pore size availability as well.

-soluble in an organic solvent then:-Organic SEC-the common packaging used is a highly cross-linked stryrene-divinylbenzene.

Silica based packings are also available that are modified with trimethyl silane. This silica based column still has residual silanol activity and limited pore size range, but still pretty common for use

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Protein Analysis

Can be broken up into two categories: separation for purification and separation for analysis

-Separation for purification: combination of methods, first usually rely on SEC. After this you now have a reasonably uniform MW. Subject the fraction in question to Ion exchange. Usually subject fraction to both to anion to cation exchange. Finally hydrophobic interactions (different than RPC)

-The reason for this separation regime is that these separation techniques are reasonable orthogonal to each other. Meaning the retention mechanisms are characterized by a total lack of correlation. This allows for the maximum separation of the mixture or biological matrix are protein is in

Protein Analysis cont’d

Separation for analysis purposes:

-One can use any of the techniques

-SEC-easiest to use and usually a first choice for analysis. It is most appropriate for things like establishing content uniformity of a dosage form where the protein is the only high-molecular weight ingredient in the formulation. Also used to establish the presence of dimers or higher associations of the parent protein.

-Ion exchange-can use weak or strong anion exchangers or cation exchangers and vary pH and ionic strength of the *gradient* to analysis protein we are wanting to look at.

*Separations of proteins and other high-molecular-weight compounds are-with the exception of SEC-always gradient separations. It is very difficult to obtain constant retention values for macromolecules under isocratic conditions. Making isocratic separations impractical in these cases. Also gradient chromatography has a higher peak capacity than isocratic chromatography, which is an advantage when separating complex mixtures

Sepations for analysis cont’d

Hydrophobic interactions-less versatile than ion exchange. Relies on the interaction of hydrophobic patches on the protein with the stationary phase. Again different than RPRPC-tends to give the sharpest peaks, but plagued with some problems. Some proteins may be only partially recovered giving ghost peaks in blank gradients. If your protein does not exhibit this problem, RP is a great high res. technique that is useful in monitoring impurities in a protein preparation, for example. The only requirmentsfor RP is that the phases are endcapped.

endcapping - an adsorbent is said to be endcapped when a small silylating agent (e.g. trimethylchlorosilane) is used to bond residual silanol groups on a packing surface. Most often used with reversed-phase packings. May cut down on undesirable adsorption of basic or ionic compounds.

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Peptide Analysis

Primary tool for peptide analysis is RPC. Standard procedure employs a gradient from high water content to high ACN with .1% (v/v) TFA in both the water and ACN mobile phases. HCl can be substituted, but both acids have there problems- both yield high reschromatography technique that is useful in peptide mapping. Both 30nm and 10nm pore packackings can be used and up to decapeptides there is no sign of restricted diffusion in 10nm pores.

Peptide Analysis cont’d

Endcapped C8 and C18 packings are preferred over shorter chains because of their improved hydrolytic stabilityFor very hydrophilic peptides or glycopeptides Hydrophilic interaction columns can be used instead.

Nucleic Acid Analysis

In general non-porous particles are preferred. The primary separation is ion exchange; stationary phase is non-porous (2-4 microns) with diethylaminoethyl (DEAE) groups or quaternary amino (Q or QMA) groups. Separations are effected by a gradient with increasing ionic strength.

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Low-molecular-weight Analysis

The “your guess is as good as mine” protocol. No one method is exclusively used for analysis. Many choices and best bet is to check literature. Examples of columns one can use is:-SEC (with very small pore size) useful in extract

purification. -RPC-for neutral or ionic compounds. If we are dealing with a mixture of the two Ion-paring RPC can be used.-Hydrophilic interactions-for very polar and neutral compounds.

-Ion exchange- Ionic or ionizable analytes.

Normal Phase Chromatography

Before the development of RP bonded phase NPC was the most popular separation techniqueNPC is still a very powerful tool because of the wide range of solvents available that can be used to fine-tune the selectivity of the separation.Has fallen into disfavor due to some complexities involved. Like reproducibility problems and lengthy equilibrium times.

Retention mechanism

Origin of retention in NPC is the interaction of polar functional groups of the analyte with polar functional groups on the packing. This is mediated by M.P. interactions with the polar functional groups of the packing. Usually the interactions are dipole-dipole or H-bondingStationary phases: classical- Silica and alumina newer- aminopropyl, cyanopropyl and diol phases.

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Retention mechanism cont’d

One can generalize the strength of the interaction of analytes and solvents with the S.P from the last side. Strength of the interaction increases.

- Aliphatic hydrocarbons < olefinic hydrocarbons < aromatic hydrocarbons ~ chlorinated hydrocarbons < sulfides < ethers < ketones ~ aldehydes ~ esters < alcohols < amides.Amines and COOH still larger but depends on the acidity and basicity of the packing. Nuetral diol the are roughly equal, but on silica amines are retained more and acids interact more strongly with aminopropyl.

Retention mechanism cont’d

The retention mechanism is viewed as the competition of analytemolecules with the M.P. molecules that cover the surface. Hydrocarbons low elution strength. Polar high elution strengthMixing solvents with different elution strengths will allow for the adjustment and bring the analyte(s) of interest into the desired retention window.

Elution strengths of solvents

Chart used to determine solvent strengths of solvents used in NPC. Water (not shown) is the strongest eluent for all normal-phase packings.

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Packings- silica

The most commonly used NP packing. Standard particle size is 3, 5, and 10 microns.The active groups on the surface are silanols, and there are several different types. Single vs. geminal, Bridged vs. lone Distribution of these depend on commercial treatment of the silica. High temp removes geminal and bridged silanols, and more single, lone silanols are formed. They are more acidic than the geminal bridged versions and thus are said to be more active.There are no standardized pretreatment procedures for commercially available HPLC silicas. So one can observe retention differences of a set of standards on different silicas.

Silanols

Single v Geminal Bridged v Lone

Silca based packings - aminopropyl

Good substitute for silica or alumina. It replaces alumina in one important application: the class separation of hydrocarbons.More retentive toward acidic samples than rest of SBP. Example-Steroids with phenolic groups are more retained with AP.

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Precautions with AP bonded phase.

NH2 is quite reactive and can form imines with aldehydes and ketones.Can also be oxidized, for example, by peroxidesRetains organic acids quite stronglyIn water a partial hydrolysis occurs as a result of the strong alkaline environment in the pores. The pH in the pores is then about 10 or higher and begins to attack the silica creating silanolswhile washing the aminopropyl groups off the column leaving you with a silica NP column.

Diol bonded phase

Prepared from trimethoxy- or triethoxy-glycidoxypropyl silaneMuch less sensitive to water content of the mobile phase. Also less reactive than AP phases. Can also be washed with water without difficultyFor these reasons diols are considered the best choice for NPC packings.Also seen in packing for aqueous SEC.

Cyanopropyl bonded phases

Used in both NP and RP chromatographyIt is the least reactive of the silica bonded phase packings, thus considered to be a deactivated NP.Retention times are lower by a factor of ~ 2.This can be very useful in drastically shortening a method for your compound in NPC.

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Shortcomings of Cyanopropyl

Have a nasty habit of collapsing in solvents of intermediate polarity. The instability is mechanical in nature; not chemical-no reactions. In both polar and non-polar solvents the collapse is prevented by the adhesion of particles to each other. If you have to wash column in a intermediate polar solution then be sure to maintain constant pressure to combat collapse. Store in hydrocarbon solution-like hexane.

Optimization of NPC

-Example of flow sheet to optimize the method for an experiment. This is done by creating mixtures with the solvents on the right. * most readily used solvents due to stability and they do not absorb UV over 240nm.

In reversed-phase chromatography the stationary phase is nonpolar, and the mobile phase is polar.

Typical stationary phases are:long-chain hydrocarbons (C2, C8, C18) attached

to a silica supportlong-chain hydrocarbons (C2, C8, C18) attached

to an inert polymer (e.g. monolithic poly-(styrene-co-divinylbenzene) supportTypical mobile phases are:

acetonitrile/water mixtures (low viscosity, electron donator)methanol/water mixtures (potential hydrogen bonding, electron withdrawer)tetrahydrofuran (increases solvent strength)

Retention Mechanisms

Reversed phase chromatography (RPC)

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Surfaces of reversed-phase packings are hydrophobicthe partitioning and adsorption of the analyte between the aqueous mobile phase and the hydrocarbonaceous phase is a competing theorylipophilic parts of the analyte interact with the stationary phase and are retained; by increase in methylene groups, the retention factor kincreases logarithmicallypolar functional groups reduce the retention time in a rather predictable way, but their contribution depends on their position in the molecule and on the solvent

Consequences for the selectivity of the separation:the selectivity of the separation can be influenced significantly by the type of solvent used as the organic modifier of the mobile phasethe amount and activity of free surface silanols or their suppression by modification are significant

Retention MechanismsReversed phase chromatography (RPC)

Advantages of reversed-phase chromatography90% of all low-molecular weight samples are carried out using a reversed-phase methodgood results are obtained for many compounds with few technical complicationsrapid equilibration of the column with aqueous mobile phase

Disadvantages of reversed-phase chromatographyonly small role in analysis of high-molecular weight compoundssecondary interactions (ion interactions) can cause a bad peak shape or non-reproducible retentionspH influences due to free silanol groups (best derivatization only removes about 50% of silanol groups due to steric hindrance)


Comparison of phase / mode and percentage of use

Reversed-Phase 50.6%Normal Phase 24.1%Ion Exchange 14.1%Size Exclusion 6.6%Chiral 3.5%Hydrophobic 1.1%


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If a sample contains weak acids, bases and neutral components, the most sufficient way is to buffer the mobile phase. For ion pairing, reversed-phase chromatography is best working.

The ideal buffer for a reversed-phase chromatography would:be chemically stablehave uniform buffering capacity over a wide pH range (pH 2-7)no UV absorption down to at least 200 nmbe highly soluble in organic solvents (e.g. methanol and acetonitrile)have a rapid rate of proton transfer to reduce kinetic contributionshave the potential to mask free silanol groups on the stationaryphase

Ammonium acetate is the most popular buffer forreversed-phase chromatography (disadvantages: UV cutoff of 220 nm, formation of ion pairs with solutes)

Retention MechanismsIon pairing

Proposed models for the mechanism of retention for ion-pair chromatography:free lipophilic counter-ions are absorbed onto the stationary phase and then interact with the charged ions of the analyte by an ion-exchange processthe counter-ion forms an ion pair with the charged analyte and the uncharged ion pair is then retained by the usual partition chromatography principles

combination of both mechanisms, involving an exchange of the analyte from an ion pair in the mobile phase to a counter-ion retained on the stationary phase

Retention MechanismsIon pairing


Type of counterions:• mainly determined by the nature of the sample ions• anionic counterions form ion pairs with organic bases

B+ + P- ↔ (B±P)• cationic counterions form ion pairs with organic acids

A- + P+ ↔ (A±P)• most frequently used ion-pair reagents

Ion pairing

weakR-NH-SO3HAminosulfuric acid, sodium cyclamate

Very weakNH2R2+Alkylammonium salts, diethylamine

WeakNH+R3Trialkylamine, trioctylamine

StrongRSO3-Alkyl sulfonate, pentane sulfonic acid, hexane sulfonic acid

StrongROSO3-Alkyl sulfate, sodium dodecylsulfate

StrongN+R4Tetraalkylammonium (trioctylmethylammonium salt cetrimide)

WeakN+R4Tetraalkylammonium (tetrabutylammonium salts)

TypeStructurePairing ion

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Influence of the presence of ion pairing reagent in the eluent on the retention of oppositely charged (O); simillarly charged (S); and neutral (N) solutes.

Ion pairing


RP-IPC optimizationeffect related to increasing parameter-value if applicable

Ion pairing

Primary parameters

Secondary parameters

Retention increases with carbon number and content

Type of stationary phase

Retention decreases in most casesTemperature

Retention increasesChain length of reagent

Using stronger reagent, stronger retention is obtained

Type of pairing reagent

Retention decreasingOrganic modifier concentration

Retention decreasingCounterion concentration

Selectivity can be changedNature of organic modifier

Cannot be predictedpH of eluent

Retention increasingIon pair reagent concentration

EffectParameter


Micellar liquid chromatography (MLC)� most of the ion-pair-reagents possess an ionic group and a large

hydrophobic surface, thus they´re known as cationic or anionic surfactants (basically soaps)

� concentration of surfactants must be above the critical micelle concentration in order to obtain stable micelle formation

� distribution of solute between mobile phase, stationary phase, and micelles� micelles can be used as a hydrophobic site of interaction with the solute

instead of an organic modifier

Disadvantages:� loss in separation efficiency compared to RP-IPC (due to slow mass-

transfer from micelle to stationary phase)� this can be optimized by increasing the organic modifier (decreased

polarity) and elevated temperature (faster mass transfer)

Ion pairing

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Development and Validation of a Micellar Liquid Chromatographic Method with UV Detection for Determination of Azithromycin in

Tablets and Capsules

A rapid and simple micellar liquid chromatographic method that does not require use of specific chromatographic columns has been developed and validated for azithromycin determination.

The method uses a Hypersil C18 column at 60 °C, 1-butanol–pH 6.86 phosphate buffer solution–water, 15:25:60 (v/v), containing 0.10 M sodium dodecyl sulfate, as mobile phase,

and UV-detection at 215 nm.Chromatographia, A. U. Kulikov / A. G. Verushkin, 60, July 2004

Micellar liquid chromatography (MLC)� Application example

Ion pairing

RP optimization

Many different ways to “tweek” ones method.Most of the time it can be related to observe and check.Solvents, ion pairing, pH, type of buffer, temp. all can play an important role in getting the desired efficacy from your method.

Varying Selectivity

Snyder and Kirkland, introduction to Modern Liquid Chromatography, Wiley, 1979, p. 287.

30% MeCN

70% Water

45% MeOH

55% Water

30x0.46 cm C-18, 1.5 mL.min,

254 nm, 10 µg each

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Use of Buffers

0.1 pH unit ---> significant effect on retentionBuffer mobile phase for pH reproducibilitypH of buffer should be within 1 pH unit of pKa of acid (best at pH = pKa)Buffers weak (100 mM or less)Check solubility

Common buffers

Buffer pKa Values

Phosphate 2, 7

Acetate 4.75

Citrate 3.08, 4.77, 6.40

Useful buffering between pH 2-8.

RP Example flow sheet

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RP Example flow sheet

Ion exchange Chromatography

Used for the separation of ionic or ionizable compoundsThe separation principle is an exchange of analyte ions with the counterions to the fixed ions of the ion exchanger.Different ions have different affinities to the fixed ions, so those with a higher affinity migrate slowly through the column giving you seperation.Retention is reduced by increasing the concentration of competing ions in the mobile phase.Can have strong or weak cation or anion exchangersCation Strong – sulfonic acid SO3

- , Weak COO-

Anion Strong-Quaternary amino NR3+, Weak 1o, 2o or

3o amines. 3o are most commonly used

Basic principles of Ion exchange

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Influence of pH on IEC

Powerful tool to manipulate retention and selectivity of a separation since for many organic molecules ionic charge is a function of pH. Enolates, organic acids, amides, etc.Classic example being the separation of amino acids driven by pH gradient from acidic to basic.

Silica-based IEC

alkali and alkaline-earth ionssimilar to PEI

cross-linked poly(butadiene-maleicacid) copolymer

peptides and proteins

SCX-poly(2-sulfoethyl aspartamide) WCX-polyaspartic acid

exhibit superior column effciency than the organic counterpartsPolysuccinimide silicas

anion exchanger for peptides anfd proteins

imine absorbed by silica then cross-linking is intiated by epichlorohydrine

polyethylene imine silica (PEI)

to increase exchange capacity simple amino group can be exchanged for ethylendiamine or a diethylenetriamine

direct silianization of aminopropyl silanesand diethylaminopropylsialnes are commonWeak anion exchanger

aliphatic carboxylic acidWeak cation exchanger

amino silane + MeI(quanternized)Strong anion exchanger

silane, or silane w/ phenyl group then chlorosulfonation. Some silane phenyl is lostStrong cation exchanger

Some exchangers (Silica) work well but very few applications found their way into lit.

if analytes are orgainccompounds

Higher packaging density than organic counter part and exibit low hydrophilicitySilica-based

Disadvatagesadvantagesderivization techniquepositivesType of exchangers

Organic based-SDVB

Allows now for SDVB columns for protien analysis

Proprietary process then exchanger is added

changes the resin from hydrophobic to hydrophillicProcessed SDVB

same as above except use ammonia, pri or sec. amines.Weak anion exchanger

SDVB + acrylic acidWeak cation exchanger

SDVB Chloromethylationfollowed by tertiary amineStrong anion exchanger

low temp treatment of SDVB with chlorosulfonic acid with oleum or SO3

controlled rate of sulfonation of benzene rings gives the ability to have high exchange capacity or low exchange capacity (good for ion chrom.)Strong cation exchanger

all organics have the problem of swelling in the presence of aqueous solutions-Hydrophobic resins not good for protein analysis

hydrophobic resin cross-linked with polymethylacrylateor polyvinyl alcohol (hydrophillic)Organic-Styrene based

DisadvatagesadvantagesderivizationtechniquepositivesType of exchangers

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Organic based-MA

peptides and proteins

Hydrophillic resin + DiethylaminoethylchlorideWAE- Diethylaminoethyl (DEAE)

epichlorohydrin pri or sec. amine or ammonia

The choice of the amine can influence the balance of ionic interaction and hydrophobic interactionWeak anion exchanger

epichlorohydrin + Tert. AmineStrong anion exchanger

proteins

Reacting -OH groups on the surface of the hydrophillic beads with sultone-cyclic ester of gamma-hydroxypropylsulfonic acidSulfypropyl (SP) SCX

gets away from problems seen with hydrophobic resins without the use of a proprietary stepMethyl acrylate Hydrophillic resin

Disadvatagesadvantagesderivization techniquepositivesType of exchangers

Things to remember about IEC

Can be a very useful tool for separation, but make sure to remember:If chromatography becomes altered then regeneration of column may be needed. Strong acid- cationexchangers Strong base- Anion exchangers.You are using salts as a way to separate. This means you MUST leave time to wash the system. Any salt residue left in the pump and column can destroy or distort your hardware. Pump pistons are a pain, and if system is old impossible, to replace so take the time to create a cleaning method.

Size exclusion chromatography is the conceptually and mechanistically simplest form of chromatography as the analytes do not interact with the stationary phase, but are separated by their ability topenetrate the pores of the packing.smaller analytes penetrate into smaller pores than larger analytes, thus staying longer in the stationary phase by exploring more pore volumelarger molecules are eluted earlier

Retention MechanismsSize exclusion chromatography (SEC)

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Factors influencing the separation and retention:separation space is constrained by the pore volume and the interstitial volume, which sum up to the total mobile-phase volume. Therefore the plate count is an important factor for the separationhydrodynamic chromatography is the effect of exclusion by largermolecules in the mobile phase changing the velocity in the center of the streamthe retention factor in SEC is defined relative to the elution volume of an excluded peak:

the ratio of pore fraction to the interstitial volume has been used as a measure of quality of a size-exclusion column. It describes the ratio of the useful volume to the unused volume.


ke

VR Ve

Ve

Materials for stationary phase:cross-linked dextrans and polyacrylamide gels (only suitable for aqueous column chromatography)semi-rigid cross-linked polystyrene or controlled-pore-size rigid glasses or silicasselected to be similar to the size of the analyte particle

Mobile phaseplays no role in separation processthe analyte must be soluble in mobile phase


Size Exclusion Calibration Curve Separation Of Standard Protein Mixture (Asahipak GFA-50, 500x7.6mm) Eluent: 0.1 M NaH2PO4 + 0.3 M NaCl, pH 7, Flow: 1.0 mL/min, Temp: 30°C Detector: UV @ 280, Calibration Curve with Chloroform & THF


Size exclusion chromatography (SEC)

1. Glutamate dehydrogenase (MW 290,000) 2. Lactate dehydrogenase (MW 142,000) 3. Enolase (MW 67,000) 4. Adenylate kinase (MW 32,000) 5. Cytochrome C (MW 12,400)

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Application example:



Application of high-performance size exclusion chromatography to the determination of erythropoietin in pharmaceutical preparations.

High performance size exclusion chromatography with fluorimetric detection for the determination of erythropoietin in pharmaceutical preparations

has been developed. The applied chromatographic system has been enabled aquick estimation of the identity and contents of the studied compound.

The method was validated and showed good validation data in terms of linearity,precision and repeatability. The validated method was successfully applied to

the determination of erythropoietin content in commercially available preparations.

Acta Pol Pharm. 2002 Mar-Apr;59(2):83-6

Columns used:

http://www.westernanalytical.com/keystone/SECselection.htm

http://www.islandsci.com/multspin2.htm

http://www.biology.ualberta.ca/facilities/multimedia/?Page=333



Hydrophilic interaction chromatography can be viewed as an extension of normal-phase chromatography:

� aqueous mobile phase (mixture of water or buffer with organic solvents)very hydrophilic, polar stationary phases (silica, polymeric packings, ion exchangers)analytes are very polar (carbohydrates or polar peptides)important tool for analysis of glycopeptides, complex carbohydrates, membrane proteins, lipopeptides, oligonucleotides, and phospholipids

Retention MechanismsHydrophilic interaction chromatography (HILIC)

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� opposite elution like reversed-phase chromatography: least polar compounds elute first, the most polar laststationary phase contains more water than the mobile phase because of strong interactions between water and the packingsalthough only little differences between packings of stationary phase, especially polyamine derivatizations show different selectivitycommon secondary mechanism is ion exchange, that must be put into account when analyzing other then neutral polar compounds (retention can be highly effected)


� polyamine covered columns are very sensitive to acid constituents, therefore a precolumn with the same packaging should be attachedto prevent contamination and rapid degradation

� Buffer concentrations must be selected carefully to avoid reduction in retention (too high concentration) or excessive equilibration times (too low concentrations)


Columns used for HILIC:� PolyHYDROXYETHYL Aspartamide™ column (HEA) will retain

solutes solely through hydrophilic interactions when using mobile phase concentrations in the range of 40-85% acetonitrile. At low pH the column has a slight positive charge, at pH 4.5-5 it is neutral, and at pH 6.5-7.0 there is a slight negative charge

� PolySULFOETHYL Aspartamide™ SCX column or PolyCAT A™column, which perform hydrophilic interaction separations superimposed upon electrostatic effects under HILIC conditions like above

� PolyWAX™ LP , an anion exchange column, provides a net positive surface at pH <7.5 for enhanced selectivity of acidic molecules where the differences are concentrated at the acidic sites of the molecules


Hydrophilic interaction chromatography (HILIC)

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Hydrophilic interaction chromatography (HILIC)

Application of hydrophilic-interaction liquid chromatography to the separation of phosphorylated H1 histones.

A new two-step high-performance liquid chromatography (HPLC) procedure has been developed to separate modified histone H1 subtypes. Reversed-phase (RP) HPLC followed by hydrophilic-interaction liquid chromatography (HILIC) was

used for analytical and semi-preparative scale fractionation of multi-phosphorylated H1histone subtypes into their non-phosphorylated and distinct phosphorylated forms.

The HILIC system utilizes the weak cation-exchange column PolyCAT Aand an increasing sodium perchlorate gradient in a methanephosphonic acid-triethylamine

buffer (pH 3.0) in the presence of 70% (v/v) acetonitrile. The identity and purity of the individual histone subfractions obtained was assayed

by capillary electrophoretic analysis. The results demonstrate that applicationof the combined RP-HPLC-HILIC procedure to the analysis and isolation of modified

H1 histone subtypes provides an innovative and important alternative totraditional separation techniques that will be extremely useful in studying

the biological function of histone phosphorylation.J Chromatogr A. 1997 Oct 3;782(1):55-62

Hydrophobic interaction chromatography can be seen as the extension of reversed-phase chromatography (like HILIC can be viewed as an extension of normal-phase chromatography)pure water functions as the strong eluentretention can be increased by adding salts to the mobile phaseanother name for HIC is “salting-out chromatography”primary application is the separation of proteines as proteines are stable in salt solutions, but denaturate in the presence of organic solventspacking of stationary phase is usually very hydrophilic (silica)salt concentration limits the usefulness of HIC for preparative separations, if other compounds ae degraded

Retention MechanismsHydrophobic interaction chromatography (HIC)

retention is promoted by high salt concentrations, which drives the analyte molecules out of the mobile phase and onto the stationary phaseelution on the other side is achieved by low salt concentrationsgradient methods must be used for the retention of macromolecules instead of isocratic methods

Influencing factors on selectivity:stationary phasetype of salt (both anion and cation), most commonly used is ammonium sulfate because of its very high solubilitytemperaturepHadditives to mobile phase

Retention MechanismsHydrophobic interaction chromatography (HIC)

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Hydrophobic interaction chromatography (HIC)

Simultaneous determination of denatured proteins byhydrophobic interaction chromatography

The quantitation of denatured proteins by hydrophobicinteraction chromatography (HIC) with a mobile phase

containing 8.0 mol urea is proposed. The followingcouples of enzymes, taken as model molecules, have been

examined: glyceraldehyde 3-phosphate dehydrogenase andaldolase from rabbit skeletal muscle, alcohol dehydrogenaseand phosphoglucose isomerase from baker’s yeast. For eachdenatured protein, reproducibility, linearity range, recovery

( > 97%) and determination limit are reported. The feasi-bility of the simultaneous determination of such proteincouples in synthetic mixtures has been tested, and HIC

proved a useful tool for separation of denatured proteins, inparticular those having similar relative molecular mass and/

or charge.

Anal. Communications, 1998, 35, 399-402

Column coupling includes the use of the same type of columns as well as different types of columns connected in a series. Coupling is a standard practice in size-exclusion chromatography, but only rarely used for retention chromatography.

Reasons for column coupling:increase in raw separation power by increasing the plate countcombination of the selectivity of the individual columns to obtain a separation that is not possible using one column type alone

by adding identical columns in a series, the resolution increases proportionally to the square root of the number of columns (precisely the elution volumes and the peak variances are additive)optimum column efficiency is achieved only when the retention volumes of all columns are th same

Special techniquesColumn coupling

Guard columns or precolumns are also a method of column coupling.Reasons for the use of guard columns:

constituents in a sample can be strongly absorbed on the column (proteins in reversed-phase chromatography)if using the same packaging for the guard column like the analytical column, it can be assumed that whatever may strongly absorb to the packaging will now be captured on the guard columnguard columns with the same packaging can be seen as an extension of the analytical column and improve the separation byadding to the plate countremove special contaminants by using different packagings when the analytical column (could increase the band spreading!)

guard columns have to be replaced regularly due to limited capacity

Special techniquesGuard columns

26

The accumulation of strongly retained material on an HPLC columncan cause peak disturbances such as peak splitting and severe peak tailing


Special techniques

Guard columns

The SDS guard cartridges are 5µm, wide-pore hydrophilicmaterials packed into cartridges, respectively. They selectively

remove SDS from peptide mixtures, and are used as guard cartridgeswith reversed-phase columns. When one of these cartridges is used with a RPC

column, conventional TFA/acetonitrile gradients can be used. Accumulated SDS is reportedlywashed off the SDS removal cartridges by levels of acetonitrile higher than 70%, allowing

SDS containing and non-SDS samples to be run sequentially.

hplc

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